WorldWideScience

Sample records for actin re-organization induced

  1. Actin re-organization induced by Chlamydia trachomatis serovar D--evidence for a critical role of the effector protein CT166 targeting Rac.

    Directory of Open Access Journals (Sweden)

    Jessica Thalmann

    Full Text Available The intracellular bacterium Chlamydia trachomatis causes infections of urogenital tract, eyes or lungs. Alignment reveals homology of CT166, a putative effector protein of urogenital C. trachomatis serovars, with the N-terminal glucosyltransferase domain of clostridial glucosylating toxins (CGTs. CGTs contain an essential DXD-motif and mono-glucosylate GTP-binding proteins of the Rho/Ras families, the master regulators of the actin cytoskeleton. CT166 is preformed in elementary bodies of C. trachomatis D and is detected in the host-cell shortly after infection. Infection with high MOI of C. trachomatis serovar D containing the CT166 ORF induces actin re-organization resulting in cell rounding and a decreased cell diameter. A comparable phenotype was observed in HeLa cells treated with the Rho-GTPase-glucosylating Toxin B from Clostridium difficile (TcdB or HeLa cells ectopically expressing CT166. CT166 with a mutated DXD-motif (CT166-mut exhibited almost unchanged actin dynamics, suggesting that CT166-induced actin re-organization depends on the glucosyltransferase motif of CT166. The cytotoxic necrotizing factor 1 (CNF1 from E. coli deamidates and thereby activates Rho-GTPases and transiently protects them against TcdB-induced glucosylation. CNF1-treated cells were found to be protected from TcdB- and CT166-induced actin re-organization. CNF1 treatment as well as ectopic expression of non-glucosylable Rac1-G12V, but not RhoA-G14A, reverted CT166-induced actin re-organization, suggesting that CT166-induced actin re-organization depends on the glucosylation of Rac1. In accordance, over-expression of CT166-mut diminished TcdB induced cell rounding, suggesting shared substrates. Cell rounding induced by high MOI infection with C. trachomatis D was reduced in cells expressing CT166-mut or Rac1-G12V, and in CNF1 treated cells. These observations indicate that the cytopathic effect of C. trachomatis D is mediated by CT166 induced Rac1 glucosylation

  2. Reactive oxygen species (ROS)-induced actin glutathionylation controls actin dynamics in neutrophils

    Science.gov (United States)

    Sakai, Jiro; Li, Jingyu; Subramanian, Kulandayan K.; Mondal, Subhanjan; Bajrami, Besnik; Hattori, Hidenori; Jia, Yonghui; Dickinson, Bryan C.; Zhong, Jia; Ye, Keqiang; Chang, Christopher J; Ho, Ye-Shih; Zhou, Jun; Luo, Hongbo R.

    2012-01-01

    Summary The regulation of actin dynamics is pivotal for cellular processes such as cell adhesion, migration, and phagocytosis, and thus is crucial for neutrophils to fulfill their roles in innate immunity. Many factors have been implicated in signal-induced actin polymerization, however the essential nature of the potential negative modulators are still poorly understood. Here we report that NADPH oxidase-dependent physiologically generated reactive oxygen species (ROS) negatively regulate actin polymerization in stimulated neutrophils via driving reversible actin glutathionylation. Disruption of glutaredoxin 1 (Grx1), an enzyme that catalyzes actin deglutathionylation, increased actin glutathionylation, attenuated actin polymerization, and consequently impaired neutrophil polarization, chemotaxis, adhesion, and phagocytosis. Consistently, Grx1-deficient murine neutrophils showed impaired in vivo recruitment to sites of inflammation and reduced bactericidal capability. Together, these results present a physiological role for glutaredoxin and ROS- induced reversible actin glutathionylation in regulation of actin dynamics in neutrophils. PMID:23159440

  3. Reactive oxygen species (ROS)-induced actin glutathionylation controls actin dynamics in neutrophils

    OpenAIRE

    Sakai, Jiro; Li, Jingyu; Subramanian, Kulandayan K.; Mondal, Subhanjan; Bajrami, Besnik; Hattori, Hidenori; Jia, Yonghui; Dickinson, Bryan C; Zhong, Jia; Ye, Keqiang; Chang, Christopher J.; Ho, Ye-Shih; Zhou, Jun; Luo, Hongbo R.

    2012-01-01

    The regulation of actin dynamics is pivotal for cellular processes such as cell adhesion, migration, and phagocytosis, and thus is crucial for neutrophils to fulfill their roles in innate immunity. Many factors have been implicated in signal-induced actin polymerization, however the essential nature of the potential negative modulators are still poorly understood. Here we report that NADPH oxidase-dependent physiologically generated reactive oxygen species (ROS) negatively regulate actin poly...

  4. Actin-cytoskeleton rearrangement modulates proton-induced uptake

    Energy Technology Data Exchange (ETDEWEB)

    Ben-Dov, Nadav [Department of Physiology and Pharmacology, Faculty of Medicine, Tel-Aviv University, 69978 Tel-Aviv (Israel); Korenstein, Rafi, E-mail: korens@post.tau.ac.il [Department of Physiology and Pharmacology, Faculty of Medicine, Tel-Aviv University, 69978 Tel-Aviv (Israel)

    2013-04-15

    Recently it has been shown that elevating proton concentration at the cell surface stimulates the formation of membrane invaginations and vesicles accompanied by an enhanced uptake of macromolecules. While the initial induction of inward membrane curvature was rationalized in terms of proton-based increase of charge asymmetry across the membrane, the mechanisms underlying vesicle formation and its scission are still unknown. In light of the critical role of actin in vesicle formation during endocytosis, the present study addresses the involvement of cytoskeletal actin in proton-induced uptake (PIU). The uptake of dextran-FITC is used as a measure for the factual fraction of inward invaginations that undergo scission from the cell's plasma membrane. Our findings show that the rate of PIU in suspended cells is constant, whereas the rate of PIU in adherent cells is gradually increased in time, saturating at the level possessed by suspended cells. This is consistent with pH induced gradual degradation of stress-fibers in adherent cells. Wortmannin and calyculin-A are able to elevate PIU by 25% in adherent cells but not in suspended cells, while cytochalasin-D, rapamycin and latrunculin-A elevate PIU both in adherent and suspended cells. However, extensive actin depolymerization by high concentrations of latrunculin-A is able to inhibit PIU. We conclude that proton-induced membrane vesiculation is restricted by the actin structural resistance to the plasma membrane bending. Nevertheless, a certain degree of cortical actin restructuring is required for the completion of the scission process. - Highlights: ► Acidification of cells' exterior enhances uptake of macromolecules by the cells. ► Disruption of actin stress fibers leads to enhancement of proton induced uptake. ► Extensive depolymerization of cellular actin attenuates proton-induced uptake.

  5. Regulation of water flow by actin-binding protein-induced actin gelatin.

    OpenAIRE

    Ito, T.; Suzuki, A.; Stossel, T. P.

    1992-01-01

    Actin filaments inhibit osmotically driven water flow (Ito, T., K.S. Zaner, and T.P. Stossel. 1987. Biophys. J. 51: 745-753). Here we show that the actin gelation protein, actin-binding protein (ABP), impedes both osmotic shrinkage and swelling of an actin filament solution and reduces markedly the concentration of actin filaments required for this inhibition. These effects depend on actin filament immobilization, because the ABP concentration that causes initial impairment of water flow by a...

  6. Mechanical force-induced polymerization and depolymerization of F-actin at water/solid interfaces

    Science.gov (United States)

    Zhang, Xueqiang; Hu, Xiuyuan; Lei, Haozhi; Hu, Jun; Zhang, Yi

    2016-03-01

    Actin molecules are among the three main cytoskeleton proteins of cells and undergo rapid cycling to regulate critical processes such as endocytosis, cytokinesis, cell polarity, and cell morphogenesis. Although extensive studies have been carried out on the dynamics as well as biological functions of actin polymerization and depolymerization both in vivo and in vitro, the molecular mechanisms by which cells sense and respond to mechanical signals are not fully understood. In particular, little attention has been paid to the effect of a physical force that is exerted directly on the actin cytoskeleton. In this paper, we have explored how the mechanical force affects the actin polymerization and depolymerization behaviors at water/solid interfaces using an atomic force microscope (AFM) operated in liquid. By raster scanning an AFM probe on a substrate surface with a certain load, it was found that actin monomers could polymerize into filaments without the help of actin related proteins (ARPs). Further study indicated that actin monomers were inclined to form filaments only under a small scanning load. The polymerized actin filaments would be depolymerized when the mechanical force was stronger. A possible mechanism has been suggested to explain the mechanical force induced actin polymerization.Actin molecules are among the three main cytoskeleton proteins of cells and undergo rapid cycling to regulate critical processes such as endocytosis, cytokinesis, cell polarity, and cell morphogenesis. Although extensive studies have been carried out on the dynamics as well as biological functions of actin polymerization and depolymerization both in vivo and in vitro, the molecular mechanisms by which cells sense and respond to mechanical signals are not fully understood. In particular, little attention has been paid to the effect of a physical force that is exerted directly on the actin cytoskeleton. In this paper, we have explored how the mechanical force affects the actin

  7. Fullerenol Nanoparticles with Structural Activity Induce Variable Intracellular Actin Filament Morphologies.

    Science.gov (United States)

    Jin, Junjiang; Dong, Ying; Wang, Ying; Xia, Lin; Gu, Weihong; Bai, Xue; Chang, Yanan; Zhang, Mingyi; Chen, Kui; Li, Juan; Zhao, Lina; Xing, Gengmei

    2016-06-01

    Fullerenol nanoparticles are promising for various biological applications; many studies have shown that they induce variable and diverse biological effects including side effects. Separation and purification of two fractions of fullerenols has demonstrated that they have varied chemical structures on the surfaces of their carbon cages. Actin is an important structural protein that is able to transform functional structures under varied physiological conditions. We assessed the abilities of the two fractions of fullerenols to attach to actin and induce variable morphological features in actin filament structures. Specifically the fullerenol fraction with a surface electric charge of -1.913 ± 0.008q (x10(-6) C) has percentages of C-OH and C=O on the carbon cage of 16.14 ± 0.60 and 17.55 ± 0.69. These features allow it to form intermolecular hydrogen bonds with actin at a stoichiometric ratio of four fullerenols per actin subunit. Molecular simulations revealed these specific binding sites and binding modes in atomic details in the interaction between the active fullerenol and actin filament. Conversely, these interactions were not possible for the other fraction of fullerenol with that percentages of C-OH and C=O on the carbon cage were 15.59 ± 0.01 and 1.94 ± 0.11. Neither sample induced appreciable cytotoxicity or acute cell death. After entering cells, active fullerenol binding to actin induces variable morphological features and may transform ATP-actin to ADP-actin. These changes facilitate the binding of ADF/cofilin, allowing cofilin to sever actin filaments to form cofilin/actin/fullerenol rods. Our findings suggest that fullerenol with structural activity binding disturbs actin filament structure, which may inhibit locomotion of cell or induce chronic side effects in to cells. PMID:27319217

  8. Steric effects induce geometric remodeling of actin bundles in filopodia

    CERN Document Server

    Dobramysl, Ulrich; Erban, Radek

    2016-01-01

    Filopodia are ubiquitous fingerlike protrusions, spawned by many eukaryotic cells, to probe and interact with their environments. Polymerization dynamics of actin filaments, comprising the structural core of filopodia, largely determine their instantaneous lengths and overall lifetimes. The polymerization reactions at the filopodial tip require transport of G-actin, which enter the filopodial tube from the filopodial base and diffuse toward the filament barbed ends near the tip. Actin filaments are mechanically coupled into a tight bundle by cross-linker proteins. Interestingly, many of these proteins are relatively short, restricting the free diffusion of cytosolic G-actin throughout the bundle and, in particular, its penetration into the bundle core. To investigate the effect of steric restrictions on G-actin diffusion by the porous structure of filopodial actin filament bundle, we used a particle-based stochastic simulation approach. We discovered that excluded volume interactions result in partial and the...

  9. Cooperative and non-cooperative conformational changes of F-actin induced by cofilin

    International Nuclear Information System (INIS)

    Highlights: •Mobility of MTSL attached to C374 in F-actin became high upon addition of cofilin. •Change of motility of MTSL attached to C374 with cofilin-binding was cooperative. •Mobility of MTSL attached to V43C in F-actin became high upon addition of cofilin. •Change of motility of MTSL attached to V43C with cofilin-binding was linear. -- Abstract: Cofilin is an actin-binding protein that promotes F-actin depolymerization. It is well-known that cofilin-coated F-actin is more twisted than naked F-actin, and that the protomer is more tilted. However, the means by which the local changes induced by the binding of individual cofilin proteins proceed to the global conformational changes of the whole F-actin molecule remain unknown. Here we investigated the cofilin-induced changes in several parts of F-actin, through site-directed spin-label electron paramagnetic resonance spectroscopy analyses of recombinant actins containing single reactive cysteines. We found that the global, cooperative conformational changes induced by cofilin-binding, which were detected by the spin-label attached to the Cys374 residue, occurred without the detachment of the D-loop in subdomain 2 from the neighboring protomer. The two processes of local and global changes do not necessarily proceed in sequence

  10. Actinic keratosis

    Science.gov (United States)

    Solar keratosis; Sun-induced skin changes - keratosis; Keratosis - actinic (solar) ... laser treatment called photodynamic therapy Chemical peels Skin creams such as 5-fluorouracil (5-FU) and imiquimod

  11. Small GTPase Rab21 mediates fibronectin induced actin reorganization in Entamoeba histolytica: implications in pathogen invasion.

    Directory of Open Access Journals (Sweden)

    Merlyn Emmanuel

    2015-03-01

    Full Text Available The protozoan parasite Entamoeba histolytica causes a wide spectrum of intestinal infections. In severe cases, the trophozoites can breach the mucosal barrier, invade the intestinal epithelium and travel via the portal circulation to the liver, where they cause hepatic abscesses, which can prove fatal if left untreated. The host Extra Cellular Matrix (ECM plays a crucial role in amoebic invasion by triggering an array of cellular responses in the parasite, including induction of actin rich adhesion structures. Similar actin rich protrusive structures, known as 'invadosomes', promote chemotactic migration of the metastatic cancer cells and non-transformed cells by remodeling the ECM. Recent studies showed a central role for Rab GTPases, the master regulators of vesicular trafficking, in biogenesis of invadosomes. Here, we showed that fibronectin, a major host ECM component induced actin remodeling in the parasite in a Rab21 dependent manner. The focalized actin structures formed were reminiscent of the mammalian invadosomes. By using various approaches, such as immunofluorescence confocal microscopy and scanning electron microscopy, along with in vitro invasion assay and matrix degradation assay, we show that the fibronectin induced formation of amoebic actin dots depend on the nucleotide status of the GTPase. The ECM components, fibronectin and collagen type I, displayed differential control over the formation of actin dots, with fibronectin positively and collagen type I negatively modulating it. The cell surface adhesion molecule Gal/GalNAc complex was also found to impose additional regulation on this process, which might have implication in collagen type I mediated suppression of actin dots.

  12. Detection of adenosine triphosphate through polymerization-induced aggregation of actin-conjugated gold/silver nanorods

    Science.gov (United States)

    Liao, Yu-Ju; Shiang, Yen-Chun; Chen, Li-Yi; Hsu, Chia-Lun; Huang, Chih-Ching; Chang, Huan-Tsung

    2013-11-01

    We have developed a simple and selective nanosensor for the optical detection of adenosine triphosphate (ATP) using globular actin-conjugated gold/silver nanorods (G-actin-Au/Ag NRs). By simply mixing G-actin and Au/Ag NRs (length ˜56 nm and diameter ˜12 nm), G-actin-Au/Ag NRs were prepared which were stable in physiological solutions (25 mM Tris-HCl, 150 mM NaCl, 5.0 mM KCl, 3.0 mM MgCl2 and 1.0 mM CaCl2; pH 7.4). Introduction of ATP into the G-actin-Au/Ag NR solutions in the presence of excess G-actin induced the formation of filamentous actin-conjugated Au/Ag NR aggregates through ATP-induced polymerization of G-actin. When compared to G-actin-modified spherical Au nanoparticles having a size of 13 nm or 56 nm, G-actin-Au/Ag NRs provided better sensitivity for ATP, mainly because the longitudinal surface plasmon absorbance of the Au/Ag NR has a more sensitive response to aggregation. This G-actin-Au/Ag NR probe provided high sensitivity (limit of detection 25 nM) for ATP with remarkable selectivity (>10-fold) over other adenine nucleotides (adenosine, adenosine monophosphate and adenosine diphosphate) and nucleoside triphosphates (guanosine triphosphate, cytidine triphosphate and uridine triphosphate). It also allowed the determination of ATP concentrations in plasma samples without conducting tedious sample pretreatments; the only necessary step was simple dilution. Our experimental results are in good agreement with those obtained from a commercial luciferin-luciferase bioluminescence assay. Our simple, sensitive and selective approach appears to have a practical potential for the clinical diagnosis of diseases (e.g. cystic fibrosis) associated with changes in ATP concentrations.

  13. Engagement of CD81 induces ezrin tyrosine phosphorylation and its cellular redistribution with filamentous actin

    Energy Technology Data Exchange (ETDEWEB)

    Coffey, Greg P.; Rajapaksa, Ranjani; Liu, Raymond; Sharpe, Orr; Kuo, Chiung-Chi; Wald Krauss, Sharon; Sagi, Yael; Davis, R. Eric; Staudt, Louis M.; Sharman, Jeff P.; Robinson, William H.; Levy, Shoshana

    2009-06-09

    CD81 is a tetraspanin family member involved in diverse cellular interactions in the immune and nervous systems and in cell fusion events. However, the mechanism of action of CD81 and of other tetraspanins has not been defined. We reasoned that identifying signaling molecules downstream of CD81 would provide mechanistic clues. We engaged CD81 on the surface of Blymphocytes and identified the induced tyrosine-phosphorylated proteins by mass spectrometry. This analysis showed that the most prominent tyrosine phosphorylated protein was ezrin, an actin binding protein and a member of the ezrin-radixin-moesin family. We also found that CD81 engagement induces spleen tyrosine kinase (Syk) and that Syk was involved in tyrosine phosphorylation of ezrin. Ezrin colocalized with CD81 and F-actin upon stimulation and this association was disrupted when Syk activation was blocked. Taken together, these studies suggest a model in which CD81 interfaces between the plasma membrane and the cytoskeleton by activating Syk, mobilizing ezrin, and recruiting F-actin to facilitate cytoskeletal reorganization and cell signaling. This may be a mechanism explaining the pleiotropic effects induced in response to stimulating cells by anti-CD81 antibodies or by the hepatitis C virus, which uses this molecule as its key receptor.

  14. Modulating F-actin organization induces organ growth by affecting the Hippo pathway

    OpenAIRE

    Sansores-Garcia, Leticia; Bossuyt, Wouter; Wada, Ken-Ichi; Yonemura, Shigenobu; Tao, Chunyao; Sasaki, Hiroshi; Halder, Georg

    2011-01-01

    This study identifies actin organization as an upstream regulator of the Hippo pathway: F-actin accumulation promotes Yorkie-dependent transcriptional activation. This modulation of Hippo signalling by actin regulators controls organ growth in Drosophila.

  15. Purification from Acanthamoeba castellanii of proteins that induce gelation and syneresis of F-actin.

    Science.gov (United States)

    Maruta, H; Korn, E D

    1977-01-10

    From Acanthamoeba castellanii, we have purified four proteins each of which alone causes a solution of F-actin to gel. The four active proteins have subunit molecular weights of about 23,000, 28,000, 32,000 and 38,000, respectively; the last three may be dimers in their native proteins. Together, these four proteins account for about 97% of the gelation activity of the whole extract; not more than about 3% of the total activity of the unfractionated extract can be due to a 250,000-dalton polypeptide. Another protein fraction, purified by agarose chromatography, induces shrinking (syneresis) of gels formed from F-actin and any of the gelation factors. That fraction contains a high Ca2+-, low (K+,EDTA)-ATPase and a major polypeptide of 170,000 daltons both of which bind to actin in the shrunken gel pellet. The active fraction does not contain the previously described Acanthamoeba myosin (Pollard, T. D., and Korn, E. D. (1973) J. Biol. Chem. 248, 4682-4690).

  16. Septins guide microtubule protrusions induced by actin-depolymerizing toxins like Clostridium difficile transferase (CDT).

    Science.gov (United States)

    Nölke, Thilo; Schwan, Carsten; Lehmann, Friederike; Østevold, Kristine; Pertz, Olivier; Aktories, Klaus

    2016-07-12

    Hypervirulent Clostridium difficile strains, which are associated with increased morbidity and mortality, produce the actin-ADP ribosylating toxin Clostridium difficile transferase (CDT). CDT depolymerizes actin, causes formation of microtubule-based protrusions, and increases pathogen adherence. Here, we show that septins (SEPT) are essential for CDT-induced protrusion formation. SEPT2, -6, -7, and -9 accumulate at predetermined protrusion sites and form collar-like structures at the base of protrusions. The septin inhibitor forchlorfenuron or knockdown of septins inhibits protrusion formation. At protrusion sites, septins colocalize with the GTPase Cdc42 (cell division control protein 42) and its effector Borg (binder of Rho GTPases), which act as up-stream regulators of septin polymerization. Precipitation and surface plasmon resonance studies revealed high-affinity binding of septins to the microtubule plus-end tracking protein EB1, thereby guiding incoming microtubules. The data suggest that CDT usurps conserved regulatory principles involved in microtubule-membrane interaction, depending on septins, Cdc42, Borgs, and restructuring of the actin cytoskeleton. PMID:27339141

  17. Intracellular photoactivation of caged cGMP induces myosin II and actin responses in motile cells.

    Science.gov (United States)

    Pfannes, Eva K B; Anielski, Alexander; Gerhardt, Matthias; Beta, Carsten

    2013-12-01

    Cyclic GMP (cGMP) is a ubiquitous second messenger in eukaryotic cells. It is assumed to regulate the association of myosin II with the cytoskeleton of motile cells. When cells of the social amoeba Dictyostelium discoideum are exposed to chemoattractants or to increased osmotic stress, intracellular cGMP levels rise, preceding the accumulation of myosin II in the cell cortex. To directly investigate the impact of intracellular cGMP on cytoskeletal dynamics in a living cell, we released cGMP inside the cell by laser-induced photo-cleavage of a caged precursor. With this approach, we could directly show in a live cell experiment that an increase in intracellular cGMP indeed induces myosin II to accumulate in the cortex. Unexpectedly, we observed for the first time that also the amount of filamentous actin in the cell cortex increases upon a rise in the cGMP concentration, independently of cAMP receptor activation and signaling. We discuss our results in the light of recent work on the cGMP signaling pathway and suggest possible links between cGMP signaling and the actin system. PMID:24136144

  18. TIMP-1 Induces α-Smooth Muscle Actin in Fibroblasts to Promote Urethral Scar Formation

    Directory of Open Access Journals (Sweden)

    Yinglong Sa

    2015-04-01

    Full Text Available Background/Aims: Tissue inhibitor of metalloproteinases-1 (TIMP-1 has been reported to upregulate in urethral scar. However, the underlying molecular mechanisms remain undefined. Methods: Here, we studied levels of TIMP-1 and α-smooth muscle actin (α-SMA in the fibroblasts isolated from urethral scar tissues, compared to the fibroblasts isolated from normal urethra. Then we either overexpressed TIMP-1, or inhibited TIMP-1 by lentiviruses carrying a transgene or a short hairpin small interfering RNA for TIMP-1 in human fibroblasts. We examined the effects of modulation of TIMP-1 on α-SMA, and on epithelial-mesenchymal transition (EMT-related genes. We also studied the underlying mechanisms. Results: We detected significantly higher levels of TIMP-1 and α-smooth muscle actin (α-SMA in the fibroblasts isolated from urethral scar tissues, compared to the fibroblasts isolated from normal urethra. Moreover, the levels of TIMP-1 and α-SMA strongly correlated. Moreover, we found that TIMP-1 significantly increased levels of α-SMA, transforming growth factor β 1 (TGFβ1, Collagen I and some other key factors related to an enhanced EMT, suggesting that TIMP-1 may induce transformation of fibroblasts into myofibroblasts to promote tissue EMT to enhance the formation of urethral scar. Moreover, increases in TIMP-1 also induced an increase in fibroblast cell growth and cell invasion, in an ERK/MAPK-signaling-dependent manner. Conclusion: Our study thus highlights a pivotal role of TIMP-1 in urethral scar formation.

  19. Role of nitric oxide in actin depolymerization and programmed cell death induced by fusicoccin in sycamore (Acer pseudoplatanus) cultured cells.

    Science.gov (United States)

    Malerba, Massimo; Contran, Nicla; Tonelli, Mariagrazia; Crosti, Paolo; Cerana, Raffaella

    2008-06-01

    Programmed cell death (PCD) plays a vital role in plant development and is involved in defence mechanisms against biotic and abiotic stresses. Different forms of PCD have been described in plants on the basis of the cell organelle first involved. In sycamore (Acer pseudoplatanus L.) cultured cells, the phytotoxin fusicoccin (FC) induces cell death. However, only a fraction of the dead cells shows the typical hallmarks of animal apoptosis, including cell shrinkage, chromatin condensation, DNA fragmentation and release of cytochrome c from the mitochondrion. In this work, we show that the scavenging of nitric oxide (NO), produced in the presence of FC, by 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) and rutin inhibits cell death without affecting DNA fragmentation and cytochrome c release. In addition, we show that FC induces a massive depolymerization of actin filaments that is prevented by the NO scavengers. Finally, the addition of actin-depolymerizing drugs induces PCD in control cells and overcomes the inhibiting effect of cPTIO on FC-induced cell death. Vice versa, the addition of actin-stabilizing drugs to FC-treated cells partially inhibits the phytotoxin-induced PCD. These results suggest that besides an apoptotic-like form of PCD involving the release of cytochrome c, FC induces at least another form of cell death, likely mediated by NO and independent of cytochrome c release, and they make it tempting to speculate that changes in actin cytoskeleton are involved in this form of PCD.

  20. Blue-light-induced reorganization of the actin cytoskeleton and the avoidance response of chloroplasts in epidermal cells of Vallisneria gigantea.

    Science.gov (United States)

    Sakurai, Nami; Domoto, Kikuko; Takagi, Shingo

    2005-04-01

    In leaf epidermal cells of the aquatic angiosperm Vallisneria gigantea Graebner, high-intensity blue light induces the actin-dependent avoidance response of chloroplasts. By semi-quantitative motion analysis and phalloidin staining, time courses of the blue-light-induced changes in the mode of movement of individual chloroplasts and in the configuration of actin filaments were examined in the presence and absence of a flavoprotein inhibitor, diphenylene iodonium. In dark-adapted cells, short, thick actin bundles seemed to surround each chloroplast, which was kept motionless in the outer periclinal cytoplasm of the cells. After 10 min of irradiation with high-intensity blue light, a rapid, unidirectional movement of chloroplasts was induced, concomitant with the appearance of aggregated, straight actin bundles stretched over the outer periclinal cytoplasm. Diphenylene iodonium inhibited the avoidance response of chloroplasts, apparently by delaying a change in the mode of chloroplast movement from random sway to unidirectional migration, by suppressing the appearance of aggregated, straight actin bundles. In partially irradiated individual cells, redistribution of chloroplasts and reorganization of actin filaments occurred only in the areas exposed to blue light. From the results, we propose that the short, thick actin bundles in the vicinity of chloroplasts function to anchor the chloroplasts in dark-adapted cells, and that the aggregated, straight actin bundles organized under blue-light irradiation provide tracks for unidirectional movement of chloroplasts. PMID:15843965

  1. Hyperosmotic stress induces Rho/Rho kinase/LIM kinase-mediated cofilin phosphorylation in tubular cells: key role in the osmotically triggered F-actin response

    DEFF Research Database (Denmark)

    Thirone, Ana C P; Speight, Pam; Zulys, Matthew;

    2009-01-01

    treatment. Inhibition of cofilin phosphorylation by Y-27632 prevented the hyperosmolarity-provoked F-actin increase. Taken together, cofilin is necessary for maintaining the osmotic responsiveness of the cytoskeleton in tubular cells, and the Rho/ROCK/LIMK-mediated cofilin phosphorylation is a key mechanism......Hyperosmotic stress induces cytoskeleton reorganization and a net increase in cellular F-actin, but the underlying mechanisms are incompletely understood. While de novo F-actin polymerization likely contributes to the actin response, the role of F-actin severing is unknown. To address this problem...... cofilin. Here we show that hyperosmolarity induced rapid, sustained and reversible phosphorylation of cofilin in kidney tubular (LLC-PK1 and MDCK) cells. Hyperosmolarity-provoked cofilin phosphorylation was mediated by the Rho/Rho kinase (ROCK)/LIM Kinase (LIMK) but not the Rac/PAK/LIMK pathway, because a...

  2. Confined diffusion of transmembrane proteins and lipids induced by the same actin meshwork lining the plasma membrane.

    Science.gov (United States)

    Fujiwara, Takahiro K; Iwasawa, Kokoro; Kalay, Ziya; Tsunoyama, Taka A; Watanabe, Yusuke; Umemura, Yasuhiro M; Murakoshi, Hideji; Suzuki, Kenichi G N; Nemoto, Yuri L; Morone, Nobuhiro; Kusumi, Akihiro

    2016-04-01

    The mechanisms by which the diffusion rate in the plasma membrane (PM) is regulated remain unresolved, despite their importance in spatially regulating the reaction rates in the PM. Proposed models include entrapment in nanoscale noncontiguous domains found in PtK2 cells, slow diffusion due to crowding, and actin-induced compartmentalization. Here, by applying single-particle tracking at high time resolutions, mainly to the PtK2-cell PM, we found confined diffusion plus hop movements (termed "hop diffusion") for both a nonraft phospholipid and a transmembrane protein, transferrin receptor, and equal compartment sizes for these two molecules in all five of the cell lines used here (actual sizes were cell dependent), even after treatment with actin-modulating drugs. The cross-section size and the cytoplasmic domain size both affected the hop frequency. Electron tomography identified the actin-based membrane skeleton (MSK) located within 8.8 nm from the PM cytoplasmic surface of PtK2 cells and demonstrated that the MSK mesh size was the same as the compartment size for PM molecular diffusion. The extracellular matrix and extracellular domains of membrane proteins were not involved in hop diffusion. These results support a model of anchored TM-protein pickets lining actin-based MSK as a major mechanism for regulating diffusion. PMID:26864625

  3. Confined diffusion of transmembrane proteins and lipids induced by the same actin meshwork lining the plasma membrane.

    Science.gov (United States)

    Fujiwara, Takahiro K; Iwasawa, Kokoro; Kalay, Ziya; Tsunoyama, Taka A; Watanabe, Yusuke; Umemura, Yasuhiro M; Murakoshi, Hideji; Suzuki, Kenichi G N; Nemoto, Yuri L; Morone, Nobuhiro; Kusumi, Akihiro

    2016-04-01

    The mechanisms by which the diffusion rate in the plasma membrane (PM) is regulated remain unresolved, despite their importance in spatially regulating the reaction rates in the PM. Proposed models include entrapment in nanoscale noncontiguous domains found in PtK2 cells, slow diffusion due to crowding, and actin-induced compartmentalization. Here, by applying single-particle tracking at high time resolutions, mainly to the PtK2-cell PM, we found confined diffusion plus hop movements (termed "hop diffusion") for both a nonraft phospholipid and a transmembrane protein, transferrin receptor, and equal compartment sizes for these two molecules in all five of the cell lines used here (actual sizes were cell dependent), even after treatment with actin-modulating drugs. The cross-section size and the cytoplasmic domain size both affected the hop frequency. Electron tomography identified the actin-based membrane skeleton (MSK) located within 8.8 nm from the PM cytoplasmic surface of PtK2 cells and demonstrated that the MSK mesh size was the same as the compartment size for PM molecular diffusion. The extracellular matrix and extracellular domains of membrane proteins were not involved in hop diffusion. These results support a model of anchored TM-protein pickets lining actin-based MSK as a major mechanism for regulating diffusion.

  4. Near-ultraviolet radiation-induced damage using an actinic reticuloid strain as a possible sensitive model

    Energy Technology Data Exchange (ETDEWEB)

    Kralli, A.

    1987-01-01

    The introduction to this thesis consists of a review of current concepts regarding the effects of ultraviolet radiation on living cells. Actinic reticuloid, a disease condition for which a near-ultraviolet radiation cellular sensitivity has been proposed as an underlying cause, is described. The experimental work, the broad aim of which is to expand existing knowledge of the effects of near-ultraviolet radiation that may lead to cell lethality, has centred upon the irradiation of a normal human skin fibroblast strain, GM730, and a strain derived from an actinic reticuloid patient, AR6LO. Parts 1 and 2 examine the effects of the irradiation on both normal and actinic fibroblast sensitivities to a range of ultraviolet wavelengths. The next two sections include observations on the protective effect of Trolox-C, a vitamin E analogue and the sensitization resulting from the replacement of the irradiation medium by a deuterated one, using both normal and actinic reticuloid fibroblasts. The final part examines broad-band near- and far-ultraviolet radiation induced membrane damage by the use of radioactively labelled rubidium as a potassium analogue.

  5. Actinic Cheilitis

    Science.gov (United States)

    ... actinic cheilitis. Overview Actinic cheilitis, sometimes known as "farmer's lip" or "sailor's lip," is a precancerous condition ... Last Updated: 22 Dec 2008 Information for other ages: Table of Contents: Overview Who's At Risk Signs ...

  6. Confined diffusion of transmembrane proteins and lipids induced by the same actin meshwork lining the plasma membrane

    OpenAIRE

    Fujiwara, Takahiro K.; Iwasawa, Kokoro; Kalay, Ziya; Tsunoyama, Taka A.; Watanabe, Yusuke; Yasuhiro M Umemura; Murakoshi, Hideji; Suzuki, Kenichi G. N.; Nemoto, Yuri L.; Morone, Nobuhiro; Kusumi, Akihiro

    2016-01-01

    The mechanisms by which the diffusion rate in the plasma membrane (PM) is regulated remain unresolved, despite their importance in spatially regulating the reaction rates in the PM. Proposed models include entrapment in nanoscale noncontiguous domains found in PtK2 cells, slow diffusion due to crowding, and actin-induced compartmentalization. Here, by applying single-particle tracking at high time resolutions, mainly to the PtK2-cell PM, we found confined diffusion plus hop movements (termed ...

  7. Rat alveolar myofibroblasts acquire alpha-smooth muscle actin expression during bleomycin-induced pulmonary fibrosis.

    OpenAIRE

    Vyalov, S. L.; Gabbiani, G.; Kapanci, Y.

    1993-01-01

    The majority of fibroblasts in alveolar septa are characterized by the presence of cytoplasmic bundles of microfilaments that contain cytoplasmic actin isoforms; these cells have been named contractile interstitial cells or V-type myofibroblasts. In the rat, they express desmin as intermediate filament protein. In this study, we explored the possibility that modulation and replication of such septal fibroblasts result in the appearance of alpha-smooth muscle (alpha-SM) actin-positive myofibro...

  8. Rheological characterization of the bundling transition in F-actin solutions induced by methylcellulose.

    Directory of Open Access Journals (Sweden)

    Simone Köhler

    Full Text Available In many in vitro experiments Brownian motion hampers quantitative data analysis. Therefore, additives are widely used to increase the solvent viscosity. For this purpose, methylcellulose (MC has been proven highly effective as already small concentrations can significantly slow down diffusive processes. Beside this advantage, it has already been reported that high MC concentrations can alter the microstructure of polymer solutions such as filamentous actin. However, it remains to be shown to what extent the mechanical properties of a composite actin/MC gel depend on the MC concentration. In particular, significant alterations might occur even if the microstructure seems unaffected. Indeed, we find that the viscoelastic response of entangled F-actin solutions depends sensitively on the amount of MC added. At concentrations higher than 0.2% (w/v MC, actin filaments are reorganized into bundles which drastically changes the viscoelastic response. At small MC concentrations the impact of MC is more subtle: the two constituents, actin and MC, contribute in an additive way to the mechanical response of the composite material. As a consequence, the effect of methylcellulose on actin solutions has to be considered very carefully when MC is used in biochemical experiments.

  9. Actin-dependent vacuolar occupancy of the cell determines auxin-induced growth repression

    Science.gov (United States)

    Scheuring, David; Löfke, Christian; Krüger, Falco; Kittelmann, Maike; Eisa, Ahmed; Hughes, Louise; Smith, Richard S.; Hawes, Chris; Schumacher, Karin; Kleine-Vehn, Jürgen

    2016-01-01

    The cytoskeleton is an early attribute of cellular life, and its main components are composed of conserved proteins. The actin cytoskeleton has a direct impact on the control of cell size in animal cells, but its mechanistic contribution to cellular growth in plants remains largely elusive. Here, we reveal a role of actin in regulating cell size in plants. The actin cytoskeleton shows proximity to vacuoles, and the phytohormone auxin not only controls the organization of actin filaments but also impacts vacuolar morphogenesis in an actin-dependent manner. Pharmacological and genetic interference with the actin–myosin system abolishes the effect of auxin on vacuoles and thus disrupts its negative influence on cellular growth. SEM-based 3D nanometer-resolution imaging of the vacuoles revealed that auxin controls the constriction and luminal size of the vacuole. We show that this actin-dependent mechanism controls the relative vacuolar occupancy of the cell, thus suggesting an unanticipated mechanism for cytosol homeostasis during cellular growth. PMID:26715743

  10. The Pseudomonas syringae Type III Effector HopG1 Induces Actin Remodeling to Promote Symptom Development and Susceptibility during Infection.

    Science.gov (United States)

    Shimono, Masaki; Lu, Yi-Ju; Porter, Katie; Kvitko, Brian H; Henty-Ridilla, Jessica; Creason, Allison; He, Sheng Yang; Chang, Jeff H; Staiger, Christopher J; Day, Brad

    2016-07-01

    The plant cytoskeleton underpins the function of a multitude of cellular mechanisms, including those associated with developmental- and stress-associated signaling processes. In recent years, the actin cytoskeleton has been demonstrated to play a key role in plant immune signaling, including a recent demonstration that pathogens target actin filaments to block plant defense and immunity. Herein, we quantified spatial changes in host actin filament organization after infection with Pseudomonas syringae pv. tomato DC3000 (Pst DC3000), demonstrating that the type-III effector HopG1 is required for pathogen-induced changes to actin filament architecture and host disease symptom development during infection. Using a suite of pathogen effector deletion constructs, coupled with high-resolution microscopy, we found that deletion of hopG1 from Pst DC3000 resulted in a reduction in actin bundling and a concomitant increase in the density of filament arrays in Arabidopsis, both of which correlate with host disease symptom development. As a mechanism underpinning this activity, we further show that the HopG1 effector interacts with an Arabidopsis mitochondrial-localized kinesin motor protein. Kinesin mutant plants show reduced disease symptoms after pathogen infection, which can be complemented by actin-modifying agents. In total, our results support a model in which HopG1 induces changes in the organization of the actin cytoskeleton as part of its virulence function in promoting disease symptom development. PMID:27217495

  11. Berberine induces dedifferentiation by actin cytoskeleton reorganization via phosphoinositide 3-kinase/Akt and p38 kinase pathways in rabbit articular chondrocytes.

    Science.gov (United States)

    Yu, Seon-Mi; Cho, Hongsik; Kim, Gwang-Hoon; Chung, Ki-Wha; Seo, Sung-Yum; Kim, Song-Ja

    2016-04-01

    Osteoarthritis is a nonrheumatologic joint disease characterized by progressive degeneration of the cartilage extracellular matrix. Berberine (BBR) is an isoquinoline alkaloid used in traditional Chinese medicine, the majority of which is extracted from Huang Lian (Coptis chinensis). Although numerous studies have revealed the anticancer activity of BBR, its effects on normal cells, such as chondrocytes, and the molecular mechanisms underlying its actions remain elusive. Therefore, we examined the effects of BBR on rabbit articular chondrocytes, and the underlying molecular mechanisms, focusing on actin cytoskeletal reorganization. BBR induced dedifferentiation by inhibiting activation of phosphoinositide-3(PI3)-kinase/Akt and p38 kinase. Furthermore, inhibition of p38 kinase and PI3-kinase/Akt with SB203580 and LY294002, respectively, accelerated the BBR-induced dedifferentiation. BBR also caused actin cytoskeletal architecture reorganization and, therefore, we investigated if these effects were involved in the dedifferentiation. Disruption of the actin cytoskeleton by cytochalasin D reversed the BBR-induced dedifferentiation by activating PI3-kinase/Akt and p38 kinase. In contrast, the induction of actin filament aggregation by jasplakinolide accelerated the BBR-induced dedifferentiation via PI3-kinase/Akt inhibition and p38 kinase activation. Taken together, these data suggest that BBR strongly induces dedifferentiation, and actin cytoskeletal reorganization is a crucial requirement for this effect. Furthermore, the dedifferentiation activity of BBR appears to be mediated via PI3-kinase/Akt and p38 kinase pathways in rabbit articular chondrocytes. PMID:26851252

  12. Protein Kinases Possibly Mediate Hypergravity-Induced Changes in F-Actin Expression by Endothelial Cells

    Science.gov (United States)

    Love, Felisha D.; Melhado, Caroline D.; Bosah, Francis N.; Harris-Hooker, Sandra A.; Sanford, Gary L.

    1998-01-01

    Basic cellular functions such as electrolyte concentration, cell growth rate, glucose utilization, bone formation, response to growth stimulation, and exocytosis are modified in microgravity. These studies indicate that microgravity affects a number of physiological systems and included in this are cell signaling mechanisms. Rijken and coworkers performed growth factor studies that showed PKC signaling and actin microfilament organization appears to be sensitive to microgravity, suggesting that the inhibition of signal transduction by microgravity may be related to alterations in actin microfilament organization. However, similar studies have not been done for vascular cells. Vascular endothelial cells play critical roles in providing nutrients to organ and tissues and in wound repair. The major deterrent to ground-based microgravity studies is that it is impossible to achieved true microgravity for longer than a few minutes on earth. Hence, it has not been possible to conduct prolonged microgravity studies except for two models that simulate certain aspects of microgravity. However, hypergravity is quite easily achieved. Several researchers have shown that hypergravity will increase the proliferation of several different cell lines while decreasing cell motility and slowing liver regeneration following partial hepatectomy, These studies indicate the hypergravity also alters the behavior of most cells. Several investigators have shown that hypergravity affects the activation of several protein kinases (PKs) in cells. In this study, we investigated whether hypergravity alters the expression of f-actin by bovine aortic endothelial cells (BAECs) and the role of PK's (calmodulin 11 dependent, PKA and PKC) as mediators of these effects.

  13. Actin cytoskeleton-dependent pathways for ADMA-induced NF-κB activation and TGF-β high expression in human renal glomerular endothelial cells

    Institute of Scientific and Technical Information of China (English)

    Liyan Wang; Dongliang Zhang; Junfang Zheng; Yiduo Feng; Yu Zhang; Wenhu Liu

    2012-01-01

    Asymmetric dimethylarginine (ADMA),an endogenous nitric oxide synthase inhibitor,is considered to be an independent risk factor in the progression of chronic kidney diseases (CKD).It can induce kidney fibrosis by increasing transforming growth factor (TGF)-β1 expression,but its molecular mechanism is unclear.The aim of the present study was to investigate the role of actin cytoskeleton in ADMA-induced TGF-β1 high expression in human renal glomerular endothelial cells (HRGECs).The structure of stress fibers was visualized by immunofluorescence,nuclear factor-κB (NF-κB) DNA-binding activity was assessed by an electrophoretic mobility shift assay and TGF-β1 expression was assessed by western blot analysis.Results showed that ADMA induced the assembly of stress fibers,DNA binding of NF-κB,and increasing expression of TGF-β1.When the dynamics of actin cytoskeleton was perturbed by the actin-depolymerizing agent cytochalasin D and the actin-stabilizing agent jasplakinolide,or ablation of stress fiber bundles by the nicotineamide adenine dinucleotide phosphate oxidase inhibitor apocynin and p38 mitogen-activated protein kinase inhibitor SB203580,ADMA-induced DNA binding of NF-κB and TGF-β1 expression were inhibited.These results revealed an actin cytoskeleton-dependent mechanism in ADMA-induced NF-κB activation and TGF-β1 high expression in HRGECs.The specific targeting of the actin cytoskeleton may be a useful strategy to prevent ADMA-activated kidney fibrosis in CKD.

  14. Cross-modal re-organization in adults with early stage hearing loss.

    Science.gov (United States)

    Campbell, Julia; Sharma, Anu

    2014-01-01

    Cortical cross-modal re-organization, or recruitment of auditory cortical areas for visual processing, has been well-documented in deafness. However, the degree of sensory deprivation necessary to induce such cortical plasticity remains unclear. We recorded visual evoked potentials (VEP) using high-density electroencephalography in nine persons with adult-onset mild-moderate hearing loss and eight normal hearing control subjects. Behavioral auditory performance was quantified using a clinical measure of speech perception-in-noise. Relative to normal hearing controls, adults with hearing loss showed significantly larger P1, N1, and P2 VEP amplitudes, decreased N1 latency, and a novel positive component (P2') following the P2 VEP. Current source density reconstruction of VEPs revealed a shift toward ventral stream processing including activation of auditory temporal cortex in hearing-impaired adults. The hearing loss group showed worse than normal speech perception performance in noise, which was strongly correlated with a decrease in the N1 VEP latency. Overall, our findings provide the first evidence that visual cross-modal re-organization not only begins in the early stages of hearing impairment, but may also be an important factor in determining behavioral outcomes for listeners with hearing loss, a finding which demands further investigation.

  15. Cross-modal re-organization in adults with early stage hearing loss.

    Directory of Open Access Journals (Sweden)

    Julia Campbell

    Full Text Available Cortical cross-modal re-organization, or recruitment of auditory cortical areas for visual processing, has been well-documented in deafness. However, the degree of sensory deprivation necessary to induce such cortical plasticity remains unclear. We recorded visual evoked potentials (VEP using high-density electroencephalography in nine persons with adult-onset mild-moderate hearing loss and eight normal hearing control subjects. Behavioral auditory performance was quantified using a clinical measure of speech perception-in-noise. Relative to normal hearing controls, adults with hearing loss showed significantly larger P1, N1, and P2 VEP amplitudes, decreased N1 latency, and a novel positive component (P2' following the P2 VEP. Current source density reconstruction of VEPs revealed a shift toward ventral stream processing including activation of auditory temporal cortex in hearing-impaired adults. The hearing loss group showed worse than normal speech perception performance in noise, which was strongly correlated with a decrease in the N1 VEP latency. Overall, our findings provide the first evidence that visual cross-modal re-organization not only begins in the early stages of hearing impairment, but may also be an important factor in determining behavioral outcomes for listeners with hearing loss, a finding which demands further investigation.

  16. Amyloid beta dimers/trimers potently induce cofilin-actin rods that are inhibited by maintaining cofilin-phosphorylation

    Directory of Open Access Journals (Sweden)

    Podlisny Marcia

    2011-01-01

    Full Text Available Abstract Background Previously we reported 1 μM synthetic human amyloid beta1-42 oligomers induced cofilin dephosphorylation (activation and formation of cofilin-actin rods within rat hippocampal neurons primarily localized to the dentate gyrus. Results Here we demonstrate that a gel filtration fraction of 7PA2 cell-secreted SDS-stable human Aβ dimers and trimers (Aβd/t induces maximal neuronal rod response at ~250 pM. This is 4,000-fold more active than traditionally prepared human Aβ oligomers, which contain SDS-stable trimers and tetramers, but are devoid of dimers. When incubated under tyrosine oxidizing conditions, synthetic human but not rodent Aβ1-42, the latter lacking tyrosine, acquires a marked increase (620 fold for EC50 in rod-inducing activity. Gel filtration of this preparation yielded two fractions containing SDS-stable dimers, trimers and tetramers. One, eluting at a similar volume to 7PA2 Aβd/t, had maximum activity at ~5 nM, whereas the other, eluting at the void volume (high-n state, lacked rod inducing activity at the same concentration. Fractions from 7PA2 medium containing Aβ monomers are not active, suggesting oxidized SDS-stable Aβ1-42 dimers in a low-n state are the most active rod-inducing species. Aβd/t-induced rods are predominantly localized to the dentate gyrus and mossy fiber tract, reach significance over controls within 2 h of treatment, and are reversible, disappearing by 24 h after Aβd/t washout. Overexpression of cofilin phosphatases increase rod formation when expressed alone and exacerbate rod formation when coupled with Aβd/t, whereas overexpression of a cofilin kinase inhibits Aβd/t-induced rod formation. Conclusions Together these data support a mechanism by which Aβd/t alters the actin cytoskeleton via effects on cofilin in neurons critical to learning and memory.

  17. Yersinia effector YopO uses actin as bait to phosphorylate proteins that regulate actin polymerization.

    Science.gov (United States)

    Lee, Wei Lin; Grimes, Jonathan M; Robinson, Robert C

    2015-03-01

    Pathogenic Yersinia species evade host immune systems through the injection of Yersinia outer proteins (Yops) into phagocytic cells. One Yop, YopO, also known as YpkA, induces actin-filament disruption, impairing phagocytosis. Here we describe the X-ray structure of Yersinia enterocolitica YopO in complex with actin, which reveals that YopO binds to an actin monomer in a manner that blocks polymerization yet allows the bound actin to interact with host actin-regulating proteins. SILAC-MS and biochemical analyses confirm that actin-polymerization regulators such as VASP, EVL, WASP, gelsolin and the formin diaphanous 1 are directly sequestered and phosphorylated by YopO through formation of ternary complexes with actin. This leads to a model in which YopO at the membrane sequesters actin from polymerization while using the bound actin as bait to recruit, phosphorylate and misregulate host actin-regulating proteins to disrupt phagocytosis.

  18. Actinic reticuloid

    Energy Technology Data Exchange (ETDEWEB)

    Marx, J.L.; Vale, M.; Dermer, P.; Ragaz, A.; Michaelides, P.; Gladstein, A.H.

    1982-09-01

    A 58-year-old man has his condition diagnosed as actinic reticuloid on the basis of clinical and histologic findings and phototesting data. He had clinical features resembling mycosis fungoides in light-exposed areas. Histologic findings disclosed a bandlike infiltrate with atypical mononuclear cells in the dermis and scattered atypical cells in the epidermis. Electron microscopy disclosed mononuclear cells with bizarre, convoluted nuclei, resembling cerebriform cells of Lutzner. Phototesting disclosed a diminished minimal erythemal threshold to UV-B and UV-A. Microscopic changes resembling actinic reticuloid were reproduced in this patient 24 and 72 hours after exposure to 15 minimal erythemal doses of UV-B.

  19. Parvin overexpression uncovers tissue-specific genetic pathways and disrupts F-actin to induce apoptosis in the developing epithelia in Drosophila.

    Directory of Open Access Journals (Sweden)

    Maria Chountala

    Full Text Available Parvin is a putative F-actin binding protein important for integrin-mediated cell adhesion. Here we used overexpression of Drosophila Parvin to uncover its functions in different tissues in vivo. Parvin overexpression caused major defects reminiscent of metastatic cancer cells in developing epithelia, including apoptosis, alterations in cell shape, basal extrusion and invasion. These defects were closely correlated with abnormalities in the organization of F-actin at the basal epithelial surface and of integrin-matrix adhesion sites. In wing epithelium, overexpressed Parvin triggered increased Rho1 protein levels, predominantly at the basal side, whereas in the developing eye it caused a rough eye phenotype and severely disrupted F-actin filaments at the retina floor of pigment cells. We identified genes that suppressed these Parvin-induced dominant effects, depending on the cell type. Co-expression of both ILK and the apoptosis inhibitor DIAP1 blocked Parvin-induced lethality and apoptosis and partially ameliorated cell delamination in epithelia, but did not rescue the elevated Rho1 levels, the abnormal organization of F-actin in the wing and the assembly of integrin-matrix adhesion sites. The rough eye phenotype was suppressed by coexpression of either PTEN or Wech, or by knock-down of Xrp1. Two main conclusions can be drawn from our studies: (1, high levels of cytoplasmic Parvin are toxic in epithelial cells; (2 Parvin in a dose dependent manner affects the organization of actin cytoskeleton in both wing and eye epithelia, independently of its role as a structural component of the ILK-PINCH-Parvin complex that mediates the integrin-actin link. Thus, distinct genetic interactions of Parvin occur in different cell types and second site modifier screens are required to uncover such genetic circuits.

  20. Activation of the cAMP Pathway Induces RACK1-Dependent Binding of β-Actin to BDNF Promoter

    Science.gov (United States)

    Neasta, Jeremie; Fiorenza, Anna; He, Dao-Yao; Phamluong, Khanhky; Kiely, Patrick A.; Ron, Dorit

    2016-01-01

    RACK1 is a scaffolding protein that contributes to the specificity and propagation of several signaling cascades including the cAMP pathway. As such, RACK1 participates in numerous cellular functions ranging from cell migration and morphology to gene transcription. To obtain further insights on the mechanisms whereby RACK1 regulates cAMP-dependent processes, we set out to identify new binding partners of RACK1 during activation of the cAMP signaling using a proteomics strategy. We identified β-actin as a direct RACK1 binding partner and found that the association between β-actin and RACK1 is increased in response to the activation of the cAMP pathway. Furthermore, we show that cAMP-dependent increase in BDNF expression requires filamentous actin. We further report that β-actin associates with the BDNF promoter IV upon the activation of the cAMP pathway and present data to suggest that the association of β-actin with BDNF promoter IV is RACK1-dependent. Taken together, our data suggest that β-actin is a new RACK1 binding partner and that the RACK1 and β-actin association participate in the cAMP-dependent regulation of BDNF transcription. PMID:27505161

  1. Regulation of the actin cytoskeleton in Helicobacter pylori-induced migration and invasive growth of gastric epithelial cells

    Directory of Open Access Journals (Sweden)

    Rieder Gabriele

    2011-11-01

    Full Text Available Abstract Dynamic rearrangement of the actin cytoskeleton is a significant hallmark of Helicobacter pylori (H. pylori infected gastric epithelial cells leading to cell migration and invasive growth. Considering the cellular mechanisms, the type IV secretion system (T4SS and the effector protein cytotoxin-associated gene A (CagA of H. pylori are well-studied initiators of distinct signal transduction pathways in host cells targeting kinases, adaptor proteins, GTPases, actin binding and other proteins involved in the regulation of the actin lattice. In this review, we summarize recent findings of how H. pylori functionally interacts with the complex signaling network that controls the actin cytoskeleton of motile and invasive gastric epithelial cells.

  2. Transgenic overexpression of γ-cytoplasmic actin protects against eccentric contraction-induced force loss in mdx mice

    Directory of Open Access Journals (Sweden)

    Baltgalvis Kristen A

    2011-10-01

    Full Text Available Abstract Background γ-cytoplasmic (γ-cyto actin levels are elevated in dystrophin-deficient mdx mouse skeletal muscle. The purpose of this study was to determine whether further elevation of γ-cyto actin levels improve or exacerbate the dystrophic phenotype of mdx mice. Methods We transgenically overexpressed γ-cyto actin, specifically in skeletal muscle of mdx mice (mdx-TG, and compared skeletal muscle pathology and force-generating capacity between mdx and mdx-TG mice at different ages. We investigated the mechanism by which γ-cyto actin provides protection from force loss by studying the role of calcium channels and stretch-activated channels in isolated skeletal muscles and muscle fibers. Analysis of variance or independent t-tests were used to detect statistical differences between groups. Results Levels of γ-cyto actin in mdx-TG skeletal muscle were elevated 200-fold compared to mdx skeletal muscle and incorporated into thin filaments. Overexpression of γ-cyto actin had little effect on most parameters of mdx muscle pathology. However, γ-cyto actin provided statistically significant protection against force loss during eccentric contractions. Store-operated calcium entry across the sarcolemma did not differ between mdx fibers compared to wild-type fibers. Additionally, the omission of extracellular calcium or the addition of streptomycin to block stretch-activated channels did not improve the force-generating capacity of isolated extensor digitorum longus muscles from mdx mice during eccentric contractions. Conclusions The data presented in this study indicate that upregulation of γ-cyto actin in dystrophic skeletal muscle can attenuate force loss during eccentric contractions and that the mechanism is independent of activation of stretch-activated channels and the accumulation of extracellular calcium.

  3. A magnetically actuated cellular strain assessment tool for quantitative analysis of strain induced cellular reorientation and actin alignment

    Science.gov (United States)

    Khademolhosseini, F.; Liu, C.-C.; Lim, C. J.; Chiao, M.

    2016-08-01

    Commercially available cell strain tools, such as pneumatically actuated elastomer substrates, require special culture plates, pumps, and incubator setups. In this work, we present a magnetically actuated cellular strain assessment tool (MACSAT) that can be implemented using off-the-shelf components and conventional incubators. We determine the strain field on the MACSAT elastomer substrate using numerical models and experimental measurements and show that a specific region of the elastomer substrate undergoes a quasi-uniaxial 2D stretch, and that cells confined to this region of the MACSAT elastomer substrate undergo tensile, compressive, or zero axial strain depending on their angle of orientation. Using the MACSAT to apply cyclic strain on endothelial cells, we demonstrate that actin filaments within the cells reorient away from the stretching direction, towards the directions of minimum axial strain. We show that the final actin orientation angles in strained cells are spread over a region of compressive axial strain, confirming previous findings on the existence of a varied pre-tension in the actin filaments of the cytoskeleton. We also demonstrate that strained cells exhibit distinctly different values of actin alignment coherency compared to unstrained cells and therefore propose that this parameter, i.e., the coherency of actin alignment, can be used as a new readout to determine the occurrence/extent of actin alignment in cell strain experiments. The tools and methods demonstrated in this study are simple and accessible and can be easily replicated by other researchers to study the strain response of other adherent cells.

  4. Chondramides, novel cyclodepsipeptides from myxobacteria, influence cell development and induce actin filament polymerization in the green alga Micrasterias.

    Science.gov (United States)

    Holzinger, A; Lütz-Meindl, U

    2001-02-01

    The effects of chondramides A-D, new actin targeting cyclodepsipeptides from the myxobacterium Chondromyces crocatus, are probed on the unicellular green alga Micrasterias denticulata, a model organism for studies on cytomorphogenesis. All four chondramides readily enter the cells and cause severe shape malformations when applied during growth. However, the four derivatives have different lowest effective concentrations. Chondramide A: 20 microM, chondramide B: 15 microM, chondramide C: 5 microM chondramide D: 10 microM. At the ultrastructural level, chondramide C, the most effective drug, causes the appearance of abnormal, dense F-actin bundles, and a substantial increase in ER, which covers large parts of the developing semicell. Also the secondary cell wall is malformed by the drug. When chondramide C effects are investigated by means of indirect immunofluorescence, alterations of the F-actin system are also visible. Instead of the cortical F-actin network of untreated controls, distinct parts of the cell are covered by abundant F-actin aggregations. Phalloidin staining of chondramide C treated cells results in a decreased fluorescence in a time-dependent manner due to binding competitions between these drugs. F-actin polymerizing and bundling capacities of chondramides A-D are presented in Micrasterias for the first time, and may in future make this substances a useful tool for cell biological research. PMID:11169761

  5. Filopodia-like actin cables position nuclei in association with perinuclear actin in Drosophila nurse cells.

    Science.gov (United States)

    Huelsmann, Sven; Ylänne, Jari; Brown, Nicholas H

    2013-09-30

    Controlling the position of the nucleus is vital for a number of cellular processes from yeast to humans. In Drosophila nurse cells, nuclear positioning is crucial during dumping, when nurse cells contract and expel their contents into the oocyte. We provide evidence that in nurse cells, continuous filopodia-like actin cables, growing from the plasma membrane and extending to the nucleus, achieve nuclear positioning. These actin cables move nuclei away from ring canals. When nurse cells contract, actin cables associate laterally with the nuclei, in some cases inducing nuclear turning so that actin cables become partially wound around the nuclei. Our data suggest that a perinuclear actin meshwork connects actin cables to nuclei via actin-crosslinking proteins such as the filamin Cheerio. We provide a revised model for how actin structures position nuclei in nurse cells, employing evolutionary conserved machinery.

  6. Formin-induced actin cables are required for polarized recruitment of the Ste5 scaffold and high level activation of MAPK Fus3.

    Science.gov (United States)

    Qi, Maosong; Elion, Elaine A

    2005-07-01

    Little is known about how a mitogen-activated protein kinase (MAPK) cascade is targeted to specific sites at the plasma membrane during receptor stimulation. In budding yeast, the Ste5 scaffold is recruited to a receptor-coupled G protein during mating pheromone stimulation, allowing the tethered MAPK cascade to be activated by Ste20, a Cdc42-anchored kinase. Here we show that stable recruitment of Ste5 at cortical sites requires the formin Bni1, Bni1-induced actin cables, Rho1 and Myo2. Rho1 directs recruitment of Bni1 via the Rho-binding domain, and Bni1 mediates localization of Ste5 through actin cables and Myo2, which co-immunoprecipitates with Ste5 during receptor stimulation. Bni1 is also required for polarized recruitment and full activation of MAPK Fus3, which must bind Ste5 to be activated, and polarized recruitment of Cdc24, the guanine exchange factor that binds Ste5 and promotes its recruitment to the G protein. In contrast, Bni1 is not important for activation of MAPK Kss1, which can be activated while not bound to Ste5 and does not accumulate at cortical sites. These findings reveal that Bni1 mediates the formation of a Ste5 scaffold/Fus3 MAPK signaling complex at polarized sites, and suggests that a pool of Ste5 may translocate along formin-induced actin cables to the cell cortex. PMID:15961405

  7. A myosin activator improves actin assembly and sarcomere function of human-induced pluripotent stem cell-derived cardiomyocytes with a troponin T point mutation.

    Science.gov (United States)

    Broughton, K M; Li, J; Sarmah, E; Warren, C M; Lin, Y-H; Henze, M P; Sanchez-Freire, V; Solaro, R J; Russell, B

    2016-07-01

    We have investigated cardiac myocytes derived from human-induced pluripotent stem cells (iPSC-CMs) from two normal control and two family members expressing a mutant cardiac troponin T (cTnT-R173W) linked to dilated cardiomyopathy (DCM). cTnT is a regulatory protein of the sarcomeric thin filament. The loss of this basic charge, which is strategically located to control tension, has consequences leading to progressive DCM. iPSC-CMs serve as a valuable platform for understanding clinically relevant mutations in sarcomeric proteins; however, there are important questions to be addressed with regard to myocyte adaptation that we model here by plating iPSC-CMs on softer substrates (100 kPa) to create a more physiologic environment during recovery and maturation of iPSC-CMs after thawing from cryopreservation. During the first week of culture of the iPSC-CMs, we have determined structural and functional characteristics as well as actin assembly dynamics. Shortening, actin content, and actin assembly dynamics were depressed in CMs from the severely affected mutant at 1 wk of culture, but by 2 wk differences were less apparent. Sarcomeric troponin and myosin isoform composition were fetal/neonatal. Furthermore, the troponin complex, reconstituted with wild-type cTnT or recombinant cTnT-R173W, depressed the entry of cross-bridges into the force-generating state, which can be reversed by the myosin activator omecamtiv mecarbil. Therapeutic doses of this drug increased both contractility and the content of F-actin in the mutant iPSC-CMs. Collectively, our data suggest the use of a myosin activation reagent to restore function within patient-specific iPSC-CMs may aid in understanding and treating this familial DCM. PMID:27199119

  8. γ-H2AX induced by linear alkylbenzene sulfonates is due to deoxyribonuclease-1 translocation to the nucleus via actin disruption

    Energy Technology Data Exchange (ETDEWEB)

    Zhao, Xiaoxu; Toyooka, Tatsushi; Kubota, Toru; Yang, Guang; Ibuki, Yuko, E-mail: ibuki@u-shizuoka-ken.ac.jp

    2015-07-15

    Graphical abstract: - Highlights: • Non-genotoxic linear alkylbenzene sulfonates (LAS) generated γ-H2AX. • The γ-H2AX was not induced through direct LAS-induced DNA damage. • LAS weakened interactions between actin and DNase I. • Released DNase I translocated to nucleus and broke DNA strands, generating γ-H2AX. • This is a novel pathway for chemically induced γ-H2AX. - Abstract: Phosphorylation of histone H2AX (γ-H2AX) occurs following formation of DNA double strand breaks (DSBs). Other types of DNA damage also generate DSBs through DNA replication and repair, leading to the production of γ-H2AX. In the present study, we demonstrated that linear alkylbenzene sulfonates (LAS), the most widely used and non-genotoxic anionic surfactants, could generate γ-H2AX via a novel pathway. Breast adenocarcinoma MCF-7 cells were treated with five kinds of LAS with alkyl chains ranging from 10 to 14 carbon units (C{sub 10}–C{sub 14}LAS). The generation of DSBs and subsequent production of γ-H2AX increased in a manner that depended on the number of carbon units in LAS. γ-H2AX could also be generated with non-cytotoxic doses of LAS and was independent of the cell cycle, indicating the non-apoptotic and DNA replication-independent formation of DSBs. The generation of γ-H2AX could be attenuated by EGTA and ZnCl{sub 2}, deoxyribonuclease-1 (DNase I) inhibitors, as well as by the knockdown of DNase I. LAS weakened the interaction between DNase I and actin, and the enhanced release of DNase I was dependent on the number of carbon units in LAS. DNase I released by the LAS treatment translocated to the nucleus, in which DNase I attacked DNA and generated γ-H2AX. These results suggested that the LAS-induced generation of γ-H2AX could be attributed to the translocation of DNase I to the nucleus through the disruption of actin, and not to LAS-induced DNA damage.

  9. CNS myelin wrapping is driven by actin disassembly.

    Science.gov (United States)

    Zuchero, J Bradley; Fu, Meng-Meng; Sloan, Steven A; Ibrahim, Adiljan; Olson, Andrew; Zaremba, Anita; Dugas, Jason C; Wienbar, Sophia; Caprariello, Andrew V; Kantor, Christopher; Leonoudakis, Dmitri; Leonoudakus, Dmitri; Lariosa-Willingham, Karen; Kronenberg, Golo; Gertz, Karen; Soderling, Scott H; Miller, Robert H; Barres, Ben A

    2015-07-27

    Myelin is essential in vertebrates for the rapid propagation of action potentials, but the molecular mechanisms driving its formation remain largely unknown. Here we show that the initial stage of process extension and axon ensheathment by oligodendrocytes requires dynamic actin filament assembly by the Arp2/3 complex. Unexpectedly, subsequent myelin wrapping coincides with the upregulation of actin disassembly proteins and rapid disassembly of the oligodendrocyte actin cytoskeleton and does not require Arp2/3. Inducing loss of actin filaments drives oligodendrocyte membrane spreading and myelin wrapping in vivo, and the actin disassembly factor gelsolin is required for normal wrapping. We show that myelin basic protein, a protein essential for CNS myelin wrapping whose role has been unclear, is required for actin disassembly, and its loss phenocopies loss of actin disassembly proteins. Together, these findings provide insight into the molecular mechanism of myelin wrapping and identify it as an actin-independent form of mammalian cell motility.

  10. Visual Cross-Modal Re-Organization in Children with Cochlear Implants.

    Directory of Open Access Journals (Sweden)

    Julia Campbell

    Full Text Available Visual cross-modal re-organization is a neurophysiological process that occurs in deafness. The intact sensory modality of vision recruits cortical areas from the deprived sensory modality of audition. Such compensatory plasticity is documented in deaf adults and animals, and is related to deficits in speech perception performance in cochlear-implanted adults. However, it is unclear whether visual cross-modal re-organization takes place in cochlear-implanted children and whether it may be a source of variability contributing to speech and language outcomes. Thus, the aim of this study was to determine if visual cross-modal re-organization occurs in cochlear-implanted children, and whether it is related to deficits in speech perception performance.Visual evoked potentials (VEPs were recorded via high-density EEG in 41 normal hearing children and 14 cochlear-implanted children, aged 5-15 years, in response to apparent motion and form change. Comparisons of VEP amplitude and latency, as well as source localization results, were conducted between the groups in order to view evidence of visual cross-modal re-organization. Finally, speech perception in background noise performance was correlated to the visual response in the implanted children.Distinct VEP morphological patterns were observed in both the normal hearing and cochlear-implanted children. However, the cochlear-implanted children demonstrated larger VEP amplitudes and earlier latency, concurrent with activation of right temporal cortex including auditory regions, suggestive of visual cross-modal re-organization. The VEP N1 latency was negatively related to speech perception in background noise for children with cochlear implants.Our results are among the first to describe cross modal re-organization of auditory cortex by the visual modality in deaf children fitted with cochlear implants. Our findings suggest that, as a group, children with cochlear implants show evidence of visual cross

  11. Myotonic dystrophy protein kinase (DMPK) induces actin cytoskeletal reorganization and apoptotic-like blebbing in lens cells

    Science.gov (United States)

    Jin, S.; Shimizu, M.; Balasubramanyam, A.; Epstein, H. F.

    2000-01-01

    DMPK, the product of the DM locus, is a member of the same family of serine-threonine protein kinases as the Rho-associated enzymes. In DM, membrane inclusions accumulate in lens fiber cells producing cataracts. Overexpression of DMPK in cultured lens epithelial cells led to apoptotic-like blebbing of the plasma membrane and reorganization of the actin cytoskeleton. Enzymatically active DMPK was necessary for both effects; inactive mutant DMPK protein did not produce either effect. Active RhoA but not constitutive GDP-state mutant protein produced similar effects as DMPK. The similar actions of DMPK and RhoA suggest that they may function in the same regulatory network. The observed effects of DMPK may be relevant to the removal of membrane organelles during normal lens differentiation and the retention of intracellular membranes in DM lenses. Copyright 2000 Wiley-Liss, Inc.

  12. G protein-coupled receptors engage the mammalian Hippo pathway through F-actin: F-Actin, assembled in response to Galpha12/13 induced RhoA-GTP, promotes dephosphorylation and activation of the YAP oncogene.

    Science.gov (United States)

    Regué, Laura; Mou, Fan; Avruch, Joseph

    2013-05-01

    The Hippo pathway, a cascade of protein kinases that inhibits the oncogenic transcriptional coactivators YAP and TAZ, was discovered in Drosophila as a major determinant of organ size in development. Known modes of regulation involve surface proteins that mediate cell-cell contact or determine epithelial cell polarity which, in a tissue-specific manner, use intracellular complexes containing FERM domain and actin-binding proteins to modulate the kinase activities or directly sequester YAP. Unexpectedly, recent work demonstrates that GPCRs, especially those signaling through Galpha12/13 such as the protease activated receptor PAR1, cause potent YAP dephosphorylation and activation. This response requires active RhoA GTPase and increased assembly of filamentous (F-)actin. Morever, cell architectures that promote F-actin assembly per se also activate YAP by kinase-dependent and independent mechanisms. These findings unveil the ability of GPCRs to activate the YAP oncogene through a newly recognized signaling function of the actin cytoskeleton, likely to be especially important for normal and cancerous stem cells.

  13. Calmodulin-Dependent Protein Kinase mediates Hypergravity-Induced Changes in F-Actin Expression by Endothelial Cells

    Science.gov (United States)

    Love, Felisha D.; Melhado, Caroline; Bosah, Francis; Harris-Hooker, Sandra A.; Sanford, Gary L.

    1997-01-01

    A number of basic cellular functions, e.g., electrolyte concentration cell growth rate, glucose utilization, bone formation, response to growth stimulation and exocytosis are modified by microgravity or during spaceflight. Studies with intact animal during spaceflights have found lipid accumulations within the lumen of the vasculature and degeneration of the vascular wall. Capillary alterations with extensive endothelial invaginations were also seen. Hemodynamic studies have shown that there is a redistribution of blood from the lower extremities to the upper part of the body; this will alter vascular permeability, resulting in leakage into surrounding tissues. These studies indicate that changes in gravity will affect a number of physiological systems, including the vasculature. However, few studies have addressed the effect of microgravity on vascular cell function and metabolism. A major problem with ground based studies is that achieving a true microgravity hand, environment for prolonged period is not possible. On the other increasing gravity (i.e., hypergravity) is easily achieved. Several researchers have shown that hypergravity will increase the proliferation of several different cell limes (e.g., chick embryo fibroblasts) while decreasing cell motility and slowing liver regeneration following partial hepatectomy. These studies suggest that hypergravity will alter the behavior of most cells. Several investigators have shown that hypergravity affects the expression of the early response genes (c-fos and c-myc) and the activation of several protein kinases (PK's) in cells (10,11). In this study we investigated whether hypergravity alters the expression of f-actin by aortic endothelial cells, and the possible role of protein kinases (calmodulin(II)-dependent and PKA) as mediators of these effects.

  14. Optogenetics to target actin-mediated synaptic loss in Alzheimer's

    Science.gov (United States)

    Zahedi, Atena; DeFea, Kathryn; Ethell, Iryna

    2013-03-01

    Numerous studies in Alzheimer's Disease (AD) animal models show that overproduction of Aβ peptides and their oligomerization can distort dendrites, damage synapses, and decrease the number of dendritic spines and synapses. Aβ may trigger synapse loss by modulating activity of actin-regulating proteins, such as Rac1 and cofilin. Indeed, Aβ1-42 oligomers can activate actin severing protein cofilin through calcineurin-mediated activation of phosphatase slingshot and inhibit an opposing pathway that suppresses cofilin phosphorylation through Rac-mediated activation of LIMK1. Excessive activation of actin-severing protein cofilin triggers the formation of a non-dynamic actin bundles, called rods that are found in AD brains and cause loss of synapses. Hence, regulation of these actin-regulating proteins in dendritic spines could potentially provide useful tools for preventing the synapse/spine loss associated with earlier stages of AD neuropathology. However, lack of spatiotemporal control over their activity is a key limitation. Recently, optogenetic advancements have provided researchers with convenient light-activating proteins such as photoactivatable Rac (PARac). Here, we transfected cultured primary hippocampal neurons and human embryonic kidney (HEK) cells with a PARac/ mCherry-containing plasmid and the mCherry-positive cells were identified and imaged using an inverted fluorescence microscope. Rac1 activation was achieved by irradiation with blue light (480nm) and live changes in dendritic spine morphology were observed using mCherry (587nm). Rac activation was confirmed by immunostaining for phosphorylated form of effector proteinP21 protein-activated kinase 1 (PAK1) and reorganization of actin. Thus, our studies confirm the feasibility of using the PA-Rac construct to trigger actin re-organization in the dendritic spines.

  15. Sensing actin dynamics: Structural basis for G-actin-sensitive nuclear import of MAL

    Energy Technology Data Exchange (ETDEWEB)

    Hirano, Hidemi; Matsuura, Yoshiyuki, E-mail: matsuura.yoshiyuki@d.mbox.nagoya-u.ac.jp

    2011-10-22

    Highlights: {yields} MAL has a bipartite NLS that binds to Imp{alpha} in an extended conformation. {yields} Mutational analyses verified the functional significance of MAL-Imp{alpha} interactions. {yields} Induced folding and NLS-masking by G-actins inhibit nuclear import of MAL. -- Abstract: The coordination of cytoskeletal actin dynamics with gene expression reprogramming is emerging as a crucial mechanism to control diverse cellular processes, including cell migration, differentiation and neuronal circuit assembly. The actin-binding transcriptional coactivator MAL (also known as MRTF-A/MKL1/BSAC) senses G-actin concentration and transduces Rho GTPase signals to serum response factor (SRF). MAL rapidly shuttles between the cytoplasm and the nucleus in unstimulated cells but Rho-induced depletion of G-actin leads to MAL nuclear accumulation and activation of transcription of SRF:MAL-target genes. Although the molecular and structural basis of actin-regulated nucleocytoplasmic shuttling of MAL is not understood fully, it is proposed that nuclear import of MAL is mediated by importin {alpha}/{beta} heterodimer, and that G-actin competes with importin {alpha}/{beta} for the binding to MAL. Here we present structural, biochemical and cell biological evidence that MAL has a classical bipartite nuclear localization signal (NLS) in the N-terminal 'RPEL' domain containing Arg-Pro-X-X-X-Glu-Leu (RPEL) motifs. The NLS residues of MAL adopt an extended conformation and bind along the surface groove of importin-{alpha}, interacting with the major- and minor-NLS binding sites. We also present a crystal structure of wild-type MAL RPEL domain in complex with five G-actins. Comparison of the importin-{alpha}- and actin-complexes revealed that the binding of G-actins to MAL is associated with folding of NLS residues into a helical conformation that is inappropriate for importin-{alpha} recognition.

  16. The Protrusive Phase and Full Development of Integrin-Dependent Adhesions in Colon Epithelial Cells Require FAK- and ERKMediated Actin Spike Formation: Deregulation in Cancer Cells

    Directory of Open Access Journals (Sweden)

    Valerie G. Brunton

    2001-01-01

    Full Text Available Integrins play an important role in tumour progression by influencing cellular responses and matrix-dependent adhesion. However, the regulation of matrix-dependent adhesion assembly in epithelial cells is poorly understood. We have investigated the integrin and signalling requirements of cell-matrix adhesion assembly in colon carcinoma cells after plating on fibronectin. Adhesion assembly in these, and in the adenoma cells from which they were derived, was largely dependent on αvβ6 integrin and required phosphorylation of FAK on tyrosine-397. The rate of fibronectin-induced adhesion assembly and the expression of both αvβ6 integrin and FAK were increased during the adenoma-to-carcinoma transition. The matrix-dependent adhesion assembly process, particularly the final stages of complex protrusion that is required for optimal cell spreading, required the activity of extracellular signal-regulated kinase (ERK. Furthermore, phosphorylated ERK was targeted to newly forming cell-matrix adhesions in the carcinoma cells but not the adenoma cells, and inhibition of FAK-tyrosine-397 phosphorylation or MEK suppressed the appearance of phosphorylated ERK at peripheral sites. In addition, inhibition of MEK-ERK activation blocked the formation of peripheral actin microspikes that were necessary for the protrusive phase of cell-matrix adhesion assembly. Thus, MEK-ERK-dependent peripheral actin re-organization is required for the full development of integrin-induced adhesions and this pathway is stimulated in an in vitro model of colon cancer progression.

  17. Computational Study of the Binding Mechanism of Actin-Depolymerizing Factor 1 with Actin in Arabidopsis thaliana.

    Science.gov (United States)

    Du, Juan; Wang, Xue; Dong, Chun-Hai; Yang, Jian Ming; Yao, Xiao Jun

    2016-01-01

    Actin is a highly conserved protein. It plays important roles in cellular function and exists either in the monomeric (G-actin) or polymeric form (F-actin). Members of the actin-depolymerizing factor (ADF)/cofilin protein family bind to both G-actin and F-actin and play vital roles in actin dynamics by manipulating the rates of filament polymerization and depolymerization. It has been reported that the S6D and R98A/K100A mutants of actin-depolymerizing factor 1 (ADF1) in Arabidopsis thaliana decreased the binding affinity of ADF for the actin monomer. To investigate the binding mechanism and dynamic behavior of the ADF1-actin complex, we constructed a homology model of the AtADF1-actin complex based on the crystal structure of AtADF1 and the twinfilin C-terminal ADF-H domain in a complex with a mouse actin monomer. The model was then refined for subsequent molecular dynamics simulations. Increased binding energy of the mutated system was observed using the Molecular Mechanics Generalized Born Surface Area and Poisson-Boltzmann Surface Area (MM-GB/PBSA) methods. To determine the residues that make decisive contributions to the ADF1 actin-binding affinity, per-residue decomposition and computational alanine scanning analyses were performed, which provided more detailed information on the binding mechanism. Root-mean-square fluctuation and principal component analyses confirmed that the S6D and R98A/K100A mutants induced an increased conformational flexibility. The comprehensive molecular insight gained from this study is of great importance for understanding the binding mechanism of ADF1 and G-actin. PMID:27414648

  18. The role of actin and myosin in PtK2 spindle length changes induced by laser microbeam irradiations across the spindle.

    Science.gov (United States)

    Sheykhani, Rozhan; Baker, Norman; Gomez-Godinez, Veronica; Liaw, Lih-Huei; Shah, Jagesh; Berns, Michael W; Forer, Arthur

    2013-05-01

    This study investigates spindle biomechanical properties to better understand how spindles function. In this report, laser microbeam cutting across mitotic spindles resulted in movement of spindle poles toward the spindle equator. The pole on the cut side moved first, the other pole moved later, resulting in a shorter but symmetric spindle. Intervening spindle microtubules bent and buckled during the equatorial movement of the poles. Because of this and because there were no detectable microtubules within the ablation zone, other cytoskeletal elements would seem to be involved in the equatorial movement of the poles. One possibility is actin and myosin since pharmacological poisoning of the actin-myosin system altered the equatorial movements of both irradiated and unirradiated poles. Immunofluorescence microscopy confirmed that actin, myosin and monophosphorylated myosin are associated with spindle fibers and showed that some actin and monophosphorylated myosin remained in the irradiated regions. Overall, our experiments suggest that actin, myosin and microtubules interact to control spindle length. We suggest that actin and myosin, possibly in conjunction with the spindle matrix, cause the irradiated pole to move toward the equator and that cross-talk between the two half spindles causes the unirradiated pole to move toward the equator until a balanced length is obtained. PMID:23475753

  19. Necessity of Re-Organization of Turkish Agricultural Higher Education System

    Directory of Open Access Journals (Sweden)

    P. Ulger

    2006-01-01

    Full Text Available Education in Agricultural Faculties has been changing from beginning to these days in Turkey. However, education in agriculture always contains all area of agriculture and students are awarded the same diploma which is “Agricultural Engineer” after four years. By means of science and technology has been developing, agricultural applications also have been changing recently. This resulted in increasing of requirements from agricultural engineer in respect of both wide and deep knowledge about agriculture. In this study it was aimed that clarification of history of agricultural higher education from the beginning till now in Turkey and agricultural higher education systems in some developed countries. Necessity of re-organization of Turkish agricultural higher education system was also discussed and some recommendations about this theme were given.

  20. Actin-Dynamics in Plant Cells: The Function of Actin Perturbing Substances Jasplakinolide, Chondramides, Phalloidin, Cytochalasins, and Latrunculins

    Science.gov (United States)

    Holzinger, Andreas; Blaas, Kathrin

    2016-01-01

    This chapter will give an overview of the most common F-actin perturbing substances, that are used to study actin dynamics in living plant cells in studies on morphogenesis, motility, organelle movement or when apoptosis has to be induced. These substances can be divided into two major subclasses – F-actin stabilizing and polymerizing substances like jasplakinolide, chondramides and F-actin severing compounds like chytochalasins and latrunculins. Jasplakinolide was originally isolated form a marine sponge, and can now be synthesized and has become commercially available, which is responsible for its wide distribution as membrane permeable F-actin stabilizing and polymerizing agent, which may even have anti-cancer activities. Cytochalasins, derived from fungi show an F-actin severing function and many derivatives are commercially available (A, B, C, D, E, H, J), also making it a widely used compound for F-actin disruption. The same can be stated for latrunculins (A, B), derived from red sea sponges, however the mode of action is different by binding to G-actin and inhibiting incorporation into the filament. In the case of swinholide a stable complex with actin dimers is formed resulting also in severing of F-actin. For influencing F-actin dynamics in plant cells only membrane permeable drugs are useful in a broad range. We however introduce also the phallotoxins and synthetic derivatives, as they are widely used to visualize F-actin in fixed cells. A particular uptake mechanism has been shown for hepatocytes, but has also been described in siphonal giant algae. In the present chapter the focus is set on F-actin dynamics in plant cells where alterations in cytoplasmic streaming can be particularly well studied; however methods by fluorescence applications including phalloidin- and antibody staining as well as immunofluorescence-localization of the inhibitor drugs are given. PMID:26498789

  1. The actin binding protein adseverin regulates osteoclastogenesis.

    Science.gov (United States)

    Hassanpour, Siavash; Jiang, Hongwei; Wang, Yongqiang; Kuiper, Johannes W P; Glogauer, Michael

    2014-01-01

    Adseverin (Ads), a member of the Gelsolin superfamily of actin binding proteins, regulates the actin cytoskeleton architecture by severing and capping existing filamentous actin (F-actin) strands and nucleating the assembly of new F-actin filaments. Ads has been implicated in cellular secretion, exocytosis and has also been shown to regulate chondrogenesis and megakaryoblastic leukemia cell differentiation. Here we report for the first time that Ads is involved in regulating osteoclastogenesis (OCG). Ads is induced during OCG downstream of RANK-ligand (RANKL) stimulation and is highly expressed in mature osteoclasts. The D5 isoform of Ads is not involved in regulating OCG, as its expression is not induced in response to RANKL. Three clonal Ads knockdown RAW264.7 (RAW) macrophage cell lines with varying degrees of Ads expression and OCG deficiency were generated. The most drastic OCG defect was noted in the clonal cell line with the greatest degree of Ads knockdown as indicated by a lack of TRAcP staining and multinucleation. RNAi mediated knockdown of Ads in osteoclast precursors resulted in distinct morphological changes characterized by altered F-actin distribution and increased filopodia formation. Ads knockdown precursor cells experienced enhanced migration while fusion of knockdown precursors cells was limited. Transient reintroduction of de novo Ads back into the knockdown system was capable of rescuing TRAcP expression but not osteoclast multinucleation most likely due to the transient nature of Ads expression. This preliminary study allows us to conclude that Ads is a RANKL induced early regulator of OCG with a potential role in pre-osteoclast differentiation and fusion. PMID:25275604

  2. The actin binding protein adseverin regulates osteoclastogenesis.

    Directory of Open Access Journals (Sweden)

    Siavash Hassanpour

    Full Text Available Adseverin (Ads, a member of the Gelsolin superfamily of actin binding proteins, regulates the actin cytoskeleton architecture by severing and capping existing filamentous actin (F-actin strands and nucleating the assembly of new F-actin filaments. Ads has been implicated in cellular secretion, exocytosis and has also been shown to regulate chondrogenesis and megakaryoblastic leukemia cell differentiation. Here we report for the first time that Ads is involved in regulating osteoclastogenesis (OCG. Ads is induced during OCG downstream of RANK-ligand (RANKL stimulation and is highly expressed in mature osteoclasts. The D5 isoform of Ads is not involved in regulating OCG, as its expression is not induced in response to RANKL. Three clonal Ads knockdown RAW264.7 (RAW macrophage cell lines with varying degrees of Ads expression and OCG deficiency were generated. The most drastic OCG defect was noted in the clonal cell line with the greatest degree of Ads knockdown as indicated by a lack of TRAcP staining and multinucleation. RNAi mediated knockdown of Ads in osteoclast precursors resulted in distinct morphological changes characterized by altered F-actin distribution and increased filopodia formation. Ads knockdown precursor cells experienced enhanced migration while fusion of knockdown precursors cells was limited. Transient reintroduction of de novo Ads back into the knockdown system was capable of rescuing TRAcP expression but not osteoclast multinucleation most likely due to the transient nature of Ads expression. This preliminary study allows us to conclude that Ads is a RANKL induced early regulator of OCG with a potential role in pre-osteoclast differentiation and fusion.

  3. Correlative nanoscale imaging of actin filaments and their complexes.

    Science.gov (United States)

    Sharma, Shivani; Zhu, Huanqi; Grintsevich, Elena E; Reisler, Emil; Gimzewski, James K

    2013-07-01

    Actin remodeling is an area of interest in biology in which correlative microscopy can bring a new way to analyze protein complexes at the nanoscale. Advances in EM, X-ray diffraction, fluorescence, and single molecule techniques have provided a wealth of information about the modulation of the F-actin structure and its regulation by actin binding proteins (ABPs). Yet, there are technological limitations of these approaches to achieving quantitative molecular level information on the structural and biophysical changes resulting from ABPs interaction with F-actin. Fundamental questions about the actin structure and dynamics and how these determine the function of ABPs remain unanswered. Specifically, how local and long-range structural and conformational changes result in ABPs induced remodeling of F-actin needs to be addressed at the single filament level. Advanced, sensitive and accurate experimental tools for detailed understanding of ABP-actin interactions are much needed. This article discusses the current understanding of nanoscale structural and mechanical modulation of F-actin by ABPs at the single filament level using several correlative microscopic techniques, focusing mainly on results obtained by Atomic Force Microscopy (AFM) analysis of ABP-actin complexes.

  4. The tail domain of myosin M catalyses nucleotide exchange on Rac1 GTPases and can induce actin-driven surface protrusions.

    Science.gov (United States)

    Geissler, H; Ullmann, R; Soldati, T

    2000-05-01

    Members of the myosin superfamily play crucial roles in cellular processes including management of the cortical cytoskeleton, organelle transport and signal transduction. GTPases of the Rho family act as key control elements in the reorganization of the actin cytoskeleton in response to growth factors, and other functions such as membrane trafficking, transcriptional regulation, growth control and development. Here, we describe a novel unconventional myosin from Dictyostelium discoideum, MyoM. Primary sequence analysis revealed that it has the appearance of a natural chimera between a myosin motor domain and a guanine nucleotide exchange factor (GEF) domain for Rho GTPases. The functionality of both domains was established. Binding of the motor domain to F-actin was ATP-dependent and potentially regulated by phosphorylation. The GEF domain displayed selective activity on Rac1-related GTPases. Overexpression, rather than absence of MyoM, affected the cell morphology and viability. Particularly in response to hypo-osmotic stress, cells overexpressing the MyoM tail domain extended massive actin-driven protrusions. The GEF was enriched at the tip of growing protuberances, probably through its pleckstrin homology domain. MyoM is the first unconventional myosin containing an active Rac-GEF domain, suggesting a role at the interface of Rac-mediated signal transduction and remodeling of the actin cytoskeleton. PMID:11208126

  5. Bulkiness or aromatic nature of tyrosine-143 of actin is important for the weak binding between F-actin and myosin-ADP-phosphate

    Energy Technology Data Exchange (ETDEWEB)

    Gomibuchi, Yuki [Graduate School of Science and Engineering, Teikyo University, Toyosatodai 1-1, Utsunomiya 320-8551 (Japan); Uyeda, Taro Q.P. [Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology, AIST Tsukuba Central 4, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8562 (Japan); Wakabayashi, Takeyuki, E-mail: tw007@nasu.bio.teikyo-u.ac.jp [Graduate School of Science and Engineering, Teikyo University, Toyosatodai 1-1, Utsunomiya 320-8551 (Japan); Department of Judo Therapy, Faculty of Medical Technology, Teikyo University, Toyosatodai 1-1, Utsunomiya 320-8551 (Japan)

    2013-11-29

    Highlights: •The effect of mutation of Tyr143 that becomes more exposed on assembly was examined. •Mutation of tyrosine-143 of Dictyostelium actin changed actin polymerizability. •The bulkiness or aromatic nature of Tyr143 is important for the weak binding. •The weak interaction between myosin and actin strengthened by Tyr143Trp mutation. -- Abstract: Actin filaments (F-actin) interact with myosin and activate its ATPase to support force generation. By comparing crystal structures of G-actin and the quasi-atomic model of F-actin based on high-resolution cryo-electron microscopy, the tyrosine-143 was found to be exposed more than 60 Å{sup 2} to the solvent in F-actin. Because tyrosine-143 flanks the hydrophobic cleft near the hydrophobic helix that binds to myosin, the mutant actins, of which the tyrosine-143 was replaced with tryptophan, phenylalanine, or isoleucine, were generated using the Dictyostelium expression system. It polymerized significantly poorly when induced by NaCl, but almost normally by KCl. In the presence of phalloidin and KCl, the extents of the polymerization of all the mutant actins were comparable to that of the wild-type actin so that the actin-activated myosin ATPase activity could be reliably compared. The affinity of skeletal heavy meromyosin to F-actin and the maximum ATPase activity (V{sub max}) were estimated by a double reciprocal plot. The Tyr143Trp-actin showed the higher affinity (smaller K{sub app}) than that of the wild-type actin, with the V{sub max} being almost unchanged. The K{sub app} and V{sub max} of the Tyr143Phe-actin were similar to those of the wild-type actin. However, the activation by Tyr143Ile-actin was much smaller than the wild-type actin and the accurate determination of K{sub app} was difficult. Comparison of the myosin ATPase activated by the various mutant actins at the same concentration of F-actin showed that the extent of activation correlates well with the solvent-accessible surface areas (ASA

  6. The fin-to-limb transition as the re-organization of a Turing pattern.

    Science.gov (United States)

    Onimaru, Koh; Marcon, Luciano; Musy, Marco; Tanaka, Mikiko; Sharpe, James

    2016-01-01

    A Turing mechanism implemented by BMP, SOX9 and WNT has been proposed to control mouse digit patterning. However, its generality and contribution to the morphological diversity of fins and limbs has not been explored. Here we provide evidence that the skeletal patterning of the catshark Scyliorhinus canicula pectoral fin is likely driven by a deeply conserved Bmp-Sox9-Wnt Turing network. In catshark fins, the distal nodular elements arise from a periodic spot pattern of Sox9 expression, in contrast to the stripe pattern in mouse digit patterning. However, our computer model shows that the Bmp-Sox9-Wnt network with altered spatial modulation can explain the Sox9 expression in catshark fins. Finally, experimental perturbation of Bmp or Wnt signalling in catshark embryos produces skeletal alterations which match in silico predictions. Together, our results suggest that the broad morphological diversity of the distal fin and limb elements arose from the spatial re-organization of a deeply conserved Turing mechanism. PMID:27211489

  7. The fin-to-limb transition as the re-organization of a Turing pattern

    Science.gov (United States)

    Onimaru, Koh; Marcon, Luciano; Musy, Marco; Tanaka, Mikiko; Sharpe, James

    2016-01-01

    A Turing mechanism implemented by BMP, SOX9 and WNT has been proposed to control mouse digit patterning. However, its generality and contribution to the morphological diversity of fins and limbs has not been explored. Here we provide evidence that the skeletal patterning of the catshark Scyliorhinus canicula pectoral fin is likely driven by a deeply conserved Bmp–Sox9–Wnt Turing network. In catshark fins, the distal nodular elements arise from a periodic spot pattern of Sox9 expression, in contrast to the stripe pattern in mouse digit patterning. However, our computer model shows that the Bmp–Sox9–Wnt network with altered spatial modulation can explain the Sox9 expression in catshark fins. Finally, experimental perturbation of Bmp or Wnt signalling in catshark embryos produces skeletal alterations which match in silico predictions. Together, our results suggest that the broad morphological diversity of the distal fin and limb elements arose from the spatial re-organization of a deeply conserved Turing mechanism. PMID:27211489

  8. Cooperation between actin-binding proteins of invasive Salmonella: SipA potentiates SipC nucleation and bundling of actin

    OpenAIRE

    Emma J McGhie; Hayward, Richard D.; Koronakis, Vassilis

    2001-01-01

    Pathogen-induced remodelling of the host cell actin cytoskeleton drives internalization of invasive Salmon ella by non-phagocytic intestinal epithelial cells. Two Salmonella actin-binding proteins are involved in internalization: SipC is essential for the process, while SipA enhances its efficiency. Using purified SipC and SipA proteins in in vitro assays of actin dynamics and F-actin bundling, we demonstrate that SipA stimulates substantially SipC-mediated nucleation of actin polymerization....

  9. Gamma-actin is involved in regulating centrosome function and mitotic progression in cancer cells.

    Science.gov (United States)

    Po'uha, Sela T; Kavallaris, Maria

    2015-01-01

    Reorganization of the actin cytoskeleton during mitosis is crucial for regulating cell division. A functional role for γ-actin in mitotic arrest induced by the microtubule-targeted agent, paclitaxel, has recently been demonstrated. We hypothesized that γ-actin plays a role in mitosis. Herein, we investigated the effect of γ-actin in mitosis and demonstrated that γ-actin is important in the distribution of β-actin and formation of actin-rich retraction fibers during mitosis. The reduced ability of paclitaxel to induce mitotic arrest as a result of γ-actin depletion was replicated with a range of mitotic inhibitors, suggesting that γ-actin loss reduces the ability of broad classes of anti-mitotic agents to induce mitotic arrest. In addition, partial depletion of γ-actin enhanced centrosome amplification in cancer cells and caused a significant delay in prometaphase/metaphase. This prolonged prometaphase/metaphase arrest was due to mitotic defects such as uncongressed and missegregated chromosomes, and correlated with an increased presence of mitotic spindle abnormalities in the γ-actin depleted cells. Collectively, these results demonstrate a previously unknown role for γ-actin in regulating centrosome function, chromosome alignment and maintenance of mitotic spindle integrity.

  10. Control of actin-based motility through localized actin binding

    International Nuclear Information System (INIS)

    A wide variety of cell biological and biomimetic systems use actin polymerization to drive motility. It has been suggested that an object such as a bacterium can propel itself by self-assembling a high concentration of actin behind it, if it is repelled by actin. However, it is also known that it is essential for the moving object to bind actin. Therefore, a key question is how the actin tail can propel an object when it both binds and repels the object. We present a physically consistent Brownian dynamics model for actin-based motility that includes the minimal components of the dendritic nucleation model and allows for both attractive and repulsive interactions between actin and a moveable disc. We find that the concentration gradient of filamentous actin generated by polymerization is sufficient to propel the object, even with moderately strong binding interactions. Additionally, actin binding can act as a biophysical cap, and may directly control motility through modulation of network growth. Overall, this mechanism is robust in that it can drive motility against a load up to a stall pressure that depends on the Young’s modulus of the actin network and can explain several aspects of actin-based motility. (paper)

  11. Structure of a pentavalent G-actin*MRTF-A complex reveals how G-actin controls nucleocytoplasmic shuttling of a transcriptional coactivator.

    Science.gov (United States)

    Mouilleron, Stéphane; Langer, Carola A; Guettler, Sebastian; McDonald, Neil Q; Treisman, Richard

    2011-06-14

    Subcellular localization of the actin-binding transcriptional coactivator MRTF-A is controlled by its interaction with monomeric actin (G-actin). Signal-induced decreases in G-actin concentration reduce MRTF-A nuclear export, leading to its nuclear accumulation, whereas artificial increases in G-actin concentration in resting cells block MRTF-A nuclear import, retaining it in the cytoplasm. This regulation is dependent on three actin-binding RPEL motifs in the regulatory domain of MRTF-A. We describe the structures of pentavalent and trivalent G-actin•RPEL domain complexes. In the pentavalent complex, each RPEL motif and the two intervening spacer sequences bound an actin monomer, forming a compact assembly. In contrast, the trivalent complex lacked the C-terminal spacer- and RPEL-actins, both of which bound only weakly in the pentavalent complex. Cytoplasmic localization of MRTF-A in unstimulated fibroblasts also required binding of G-actin to the spacer sequences. The bipartite MRTF-A nuclear localization sequence was buried in the pentameric assembly, explaining how increases in G-actin concentration prevent nuclear import of MRTF-A. Analyses of the pentavalent and trivalent complexes show how actin loads onto the RPEL domain and reveal a molecular mechanism by which actin can control the activity of one of its binding partners.

  12. Wnt Signalling Promotes Actin Dynamics during Axon Remodelling through the Actin-Binding Protein Eps8.

    Directory of Open Access Journals (Sweden)

    Eleanna Stamatakou

    Full Text Available Upon arrival at their synaptic targets, axons slow down their growth and extensively remodel before the assembly of presynaptic boutons. Wnt proteins are target-derived secreted factors that promote axonal remodelling and synaptic assembly. In the developing spinal cord, Wnts secreted by motor neurons promote axonal remodelling of NT-3 responsive dorsal root ganglia neurons. Axon remodelling induced by Wnts is characterised by growth cone pausing and enlargement, processes that depend on the re-organisation of microtubules. However, the contribution of the actin cytoskeleton has remained unexplored. Here, we demonstrate that Wnt3a regulates the actin cytoskeleton by rapidly inducing F-actin accumulation in growth cones from rodent DRG neurons through the scaffold protein Dishevelled-1 (Dvl1 and the serine-threonine kinase Gsk3β. Importantly, these changes in actin cytoskeleton occurs before enlargement of the growth cones is evident. Time-lapse imaging shows that Wnt3a increases lamellar protrusion and filopodia velocity. In addition, pharmacological inhibition of actin assembly demonstrates that Wnt3a increases actin dynamics. Through a yeast-two hybrid screen, we identified the actin-binding protein Eps8 as a direct interactor of Dvl1, a scaffold protein crucial for the Wnt signalling pathway. Gain of function of Eps8 mimics Wnt-mediated axon remodelling, whereas Eps8 silencing blocks the axon remodelling activity of Wnt3a. Importantly, blockade of the Dvl1-Eps8 interaction completely abolishes Wnt3a-mediated axonal remodelling. These findings demonstrate a novel role for Wnt-Dvl1 signalling through Eps8 in the regulation of axonal remodeling.

  13. Pattern formation in polymerising actin flocks: spirals, spots and waves without nonlinear chemistry

    CERN Document Server

    Goff, Thomas Le; Marenduzzo, Davide

    2016-01-01

    We propose a model solely based on actin treadmilling and polymerisation which describes many characteristic states of actin wave formation: spots, spirals and travelling waves. In our model, as in experiments on cell recovering motility following actin depolymerisation, we choose an isotropic low density initial condition; polymerisation of actin filaments then raises the density towards the Onsager threshold where they align. We show that this alignment, in turn, destabilizes the isotropic phase and generically induces transient actin spots or spirals as part of the dynamical pathway towards a polarized phase which can either be uniform or consist of a series of actin-wave trains (flocks). Our results uncover a universal route to actin wave formation in the absence of any system specific nonlinear biochemistry, and it may help understand the mechanism underlying the observation of actin spots and waves in vivo. They also suggest a minimal setup to design similar patterns in vitro.

  14. Regulation of actin catch-slip bonds with a RhoA-formin module

    Science.gov (United States)

    Lee, Cho-Yin; Lou, Jizhong; Wen, Kuo-Kuang; McKane, Melissa; Eskin, Suzanne G.; Rubenstein, Peter A.; Chien, Shu; Ono, Shoichiro; Zhu, Cheng; McIntire, Larry V.

    2016-10-01

    The dynamic turnover of the actin cytoskeleton is regulated cooperatively by force and biochemical signaling. We previously demonstrated that actin depolymerization under force is governed by catch-slip bonds mediated by force-induced K113:E195 salt-bridges. Yet, the biochemical regulation as well as the functional significance of actin catch bonds has not been elucidated. Using AFM force-clamp experiments, we show that formin controlled by RhoA switches the actin catch-slip bonds to slip-only bonds. SMD simulations reveal that the force does not induce the K113:E195 interaction when formin binds to actin K118 and E117 residues located at the helical segment extending to K113. Actin catch-slip bonds are suppressed by single residue replacements K113E and E195K that interrupt the force-induced K113:E195 interaction; and this suppression is rescued by a K113E/E195K double mutant (E/K) restoring the interaction in the opposite orientation. These results support the biological significance of actin catch bonds, as they corroborate reported observations that RhoA and formin switch force-induced actin cytoskeleton alignment and that either K113E or E195K induces yeast cell growth defects rescued by E/K. Our study demonstrates how the mechano-regulation of actin dynamics is modulated by biochemical signaling molecules, and suggests that actin catch bonds may be important in cell functions.

  15. Viscoelastic properties of actin-coated membranes

    Science.gov (United States)

    Helfer, E.; Harlepp, S.; Bourdieu, L.; Robert, J.; Mackintosh, F. C.; Chatenay, D.

    2001-02-01

    In living cells, cytoskeletal filaments interact with the plasma membrane to form structures that play a key role in cell shape and mechanical properties. To study the interaction between these basic components, we designed an in vitro self-assembled network of actin filaments attached to the outer surface of giant unilamellar vesicles. Optical tweezers and single-particle tracking experiments are used to study the rich dynamics of these actin-coated membranes (ACM). We show that microrheology studies can be carried out on such an individual microscopic object. The principle of the experiment consists in measuring the thermally excited position fluctuations of a probe bead attached biochemically to the membrane. We propose a model that relates the power spectrum of these thermal fluctuations to the viscoelastic properties of the membrane. The presence of the actin network modifies strongly the membrane dynamics with respect to a fluid, lipid bilayer one. It induces first a finite (ω=0) two-dimensional (2D) shear modulus G02D~0.5 to 5 μN/m in the membrane plane. Moreover, the frequency dependence at high frequency of the shear modulus [G'2D(f )~f0.85+/-0.07] and of the bending modulus (κACM(f)~f0.55+/-0.21) demonstrate the viscoelastic behavior of the composite membrane. These results are consistent with a common exponent of 0.75 for both moduli as expected from our model and from prior measurements on actin solutions.

  16. Regulation of actin cytoskeleton architecture by Eps8 and Abi1

    Directory of Open Access Journals (Sweden)

    Miller Jeffrey R

    2005-10-01

    Full Text Available Abstract Background The actin cytoskeleton participates in many fundamental processes including the regulation of cell shape, motility, and adhesion. The remodeling of the actin cytoskeleton is dependent on actin binding proteins, which organize actin filaments into specific structures that allow them to perform various specialized functions. The Eps8 family of proteins is implicated in the regulation of actin cytoskeleton remodeling during cell migration, yet the precise mechanism by which Eps8 regulates actin organization and remodeling remains elusive. Results Here, we show that Eps8 promotes the assembly of actin rich filopodia-like structures and actin cables in cultured mammalian cells and Xenopus embryos, respectively. The morphology of actin structures induced by Eps8 was modulated by interactions with Abi1, which stimulated formation of actin cables in cultured cells and star-like structures in Xenopus. The actin stars observed in Xenopus animal cap cells assembled at the apical surface of epithelial cells in a Rac-independent manner and their formation was accompanied by recruitment of N-WASP, suggesting that the Eps8/Abi1 complex is capable of regulating the localization and/or activity of actin nucleators. We also found that Eps8 recruits Dishevelled to the plasma membrane and actin filaments suggesting that Eps8 might participate in non-canonical Wnt/Polarity signaling. Consistent with this idea, mis-expression of Eps8 in dorsal regions of Xenopus embryos resulted in gastrulation defects. Conclusion Together, these results suggest that Eps8 plays multiple roles in modulating actin filament organization, possibly through its interaction with distinct sets of actin regulatory complexes. Furthermore, the finding that Eps8 interacts with Dsh and induced gastrulation defects provides evidence that Eps8 might participate in non-canonical Wnt signaling to control cell movements during vertebrate development.

  17. Actin cytoskeleton regulates Hippo signaling.

    Directory of Open Access Journals (Sweden)

    Pradeep Reddy

    Full Text Available Hippo pathway controls the organ size by modulating cell proliferation and apoptosis. However, the upstream regulation of hippo signaling by actin cytoskeleton is not clear. To elucidate the role of actin as an upstream regulator of Hippo signaling, the levels of F (filamentous-actin in cells were elevated using jasplakinolide, an actin-stabilizing drug. Induction of F-actin formation in HeLa cells resulted in decreased phosphorylation of YAP, a key effector molecule for Hippo signaling. The activated YAP is localized to the cell nucleus and YAP increase was associated with increased expression of downstream CCN growth factors CCN1/CYR61 and CCN2/CTGF. The effect of the actin-stabilizing drug was blocked when YAP levels were suppressed in YAP "knock-down" cells. In summary, using an actin-stabilizing drug we show that actin cytoskeleton is one of the upstream regulators of Hippo signaling capable of activating YAP and increasing its downstream CCN growth factors.

  18. Role of actin in auxin transport and transduction of gravity

    Science.gov (United States)

    Hu, S.; Basu, S.; Brady, S.; Muday, G.

    Transport of the plant hormone auxin is polar and the direction of the hormone movement appears to be controlled by asymmetric distribution of auxin transport protein complexes. Changes in the direction of auxin transport are believed to drive asymmetric growth in response to changes in the gravity vector. To test the possibility that asymmetric distribution of the auxin transport protein complex is mediated by attachment to the actin cytoskeleton, a variety of experimental approaches have been used. The most direct demonstration of the role of the actin cytoskeleton in localization of the protein complex is the ability of one protein in this complex to bind to affinity columns containing actin filaments. Additionally, treatments of plant tissues with drugs that fragment the actin c toskeleton reducey polar transport. In order to explore this actin interaction and the affect of gravity on auxin transport and developmental polarity, embryos of the brown alga, Fucus have been examined. Fucus zygotes are initially symmetrical, but develop asymmetry in response to environmental gradients, with light gradients being the best- characterized signal. Gravity will polarize these embryos and gravity-induced polarity is randomized by clinorotation. Auxin transport also appears necessary for environmental controls of polarity, since auxin efflux inhibitors perturb both photo- and gravity-polarization at a very discrete temporal window within six hours after fertilization. The actin cytoskeleton has previously been shown to reorganize after fertilization of Fucus embryos leading to formation of an actin patch at the site of polar outgrowth. These actin patches still form in Fucus embryos treated with auxin efflux inhibitors, yet the position of these patches is randomized. Together, these results suggest that there are connections between the actin cytoskeleton, auxin transport, and gravity oriented growth and development. (Supported by NASA Grant: NAG2-1203)

  19. Transgelin is a TGFβ-inducible gene that regulates osteoblastic and adipogenic differentiation of human skeletal stem cells through actin cytoskeleston organization

    DEFF Research Database (Denmark)

    Elsafadi, E; Manikandan, M; Dawud, RA;

    2016-01-01

    bone marrow-derived stromal (skeletal) stem cells (hMSC). siRNA-mediated gene silencing of TAGLN impaired lineage differentiation into osteoblasts and adipocytes but enhanced cell proliferation. Additional functional studies revealed that TAGLN deficiency impaired hMSC cell motility and in vitro...... transwell cell migration. On the other hand, TAGLN overexpression reduced hMSC cell proliferation, but enhanced cell migration, osteoblastic and adipocytic differentiation, and in vivo bone formation. In addition, deficiency or overexpression of TAGLN in hMSC was associated with significant changes...... in cellular and nuclear morphology and cytoplasmic organelle composition as demonstrated by high content imaging and transmission electron microscopy that revealed pronounced alterations in the distribution of the actin filament and changes in cytoskeletal organization. Molecular signature of TAGLN...

  20. Possible association of actin filaments with chloroplasts of spinach mesophyll cells in vivo and in vitro.

    Science.gov (United States)

    Kumatani, T; Sakurai-Ozato, N; Miyawaki, N; Yokota, E; Shimmen, T; Terashima, I; Takagi, S

    2006-11-01

    In palisade mesophyll cells of spinach (Spinacia oleracea L.) kept under low-intensity white light, chloroplasts were apparently immobile and seemed to be surrounded by fine bundles of actin filaments. High-intensity blue light induced actin-dependent chloroplast movement concomitant with the appearance of a couple of long, straight bundles of actin filaments in each cell, whereas high-intensity red light was essentially ineffective in inducing these responses. The actin organization observed under low-intensity white light has been postulated to function in anchoring chloroplasts at proper intracellular positions through direct interaction with the chloroplasts. Intact chloroplasts, which retained their outer envelopes, were isolated after homogenization of leaves and Percoll centrifugation. No endogenous actin was detected by immunoblotting in the final intact-chloroplast fraction prepared from the leaves kept under low-intensity white light or in darkness. In cosedimentation assays with exogenously added skeletal muscle filamentous actin, however, actin was detected in the intact-chloroplast fraction precipitated after low-speed centrifugation. The association of actin with chloroplasts was apparently dependent on incubation time and chloroplast density. After partial disruption of the outer envelope of isolated chloroplasts by treatment with trypsin, actin was no longer coprecipitated. The results suggest that chloroplasts in spinach leaves can directly interact with actin, and that this interaction may be involved in the regulation of intracellular positioning of chloroplasts.

  1. Roles of Asp179 and Glu270 in ADP-Ribosylation of Actin by Clostridium perfringens Iota Toxin.

    Directory of Open Access Journals (Sweden)

    Alexander Belyy

    Full Text Available Clostridium perfringens iota toxin is a binary toxin composed of the enzymatically active component Ia and receptor binding component Ib. Ia is an ADP-ribosyltransferase, which modifies Arg177 of actin. The previously determined crystal structure of the actin-Ia complex suggested involvement of Asp179 of actin in the ADP-ribosylation reaction. To gain more insights into the structural requirements of actin to serve as a substrate for toxin-catalyzed ADP-ribosylation, we engineered Saccharomyces cerevisiae strains, in which wild type actin was replaced by actin variants with substitutions in residues located on the Ia-actin interface. Expression of the actin mutant Arg177Lys resulted in complete resistance towards Ia. Actin mutation of Asp179 did not change Ia-induced ADP-ribosylation and growth inhibition of S. cerevisiae. By contrast, substitution of Glu270 of actin inhibited the toxic action of Ia and the ADP-ribosylation of actin. In vitro transcribed/translated human β-actin confirmed the crucial role of Glu270 in ADP-ribosylation of actin by Ia.

  2. Regulation of myosin IIA and filamentous actin during insulin-stimulated glucose uptake in 3T3-L1 adipocytes

    International Nuclear Information System (INIS)

    Insulin stimulated glucose uptake requires the colocalization of myosin IIA (MyoIIA) and the insulin-responsive glucose transporter 4 (GLUT4) at the plasma membrane for proper GLUT4 fusion. MyoIIA facilitates filamentous actin (F-actin) reorganization in various cell types. In adipocytes F-actin reorganization is required for insulin-stimulated glucose uptake. What is not known is whether MyoIIA interacts with F-actin to regulate insulin-induced GLUT4 fusion at the plasma membrane. To elucidate the relationship between MyoIIA and F-actin, we examined the colocalization of MyoIIA and F-actin at the plasma membrane upon insulin stimulation as well as the regulation of this interaction. Our findings demonstrated that MyoIIA and F-actin colocalized at the site of GLUT4 fusion with the plasma membrane upon insulin stimulation. Furthermore, inhibition of MyoII with blebbistatin impaired F-actin localization at the plasma membrane. Next we examined the regulatory role of calcium in MyoIIA-F-actin colocalization. Reduced calcium or calmodulin levels decreased colocalization of MyoIIA and F-actin at the plasma membrane. While calcium alone can translocate MyoIIA it did not stimulate F-actin accumulation at the plasma membrane. Taken together, we established that while MyoIIA activity is required for F-actin localization at the plasma membrane, it alone is insufficient to localize F-actin to the plasma membrane. - Highlights: • Insulin induces colocalization of MyoIIA and F-actin at the cortex in adipocytes. • MyoIIA is necessary but not sufficient to localize F-actin at the cell cortex. • MyoIIA-F-actin colocalization is regulated by calcium and calmodulin

  3. Progresses in studies of nuclear actin

    Institute of Scientific and Technical Information of China (English)

    ZHU Xiaojuan; ZENG Xianlu; SONG Zhaoxia; HAO Shui

    2004-01-01

    Actin is a protein abundant in cells. Recently, it has been proved to be universally existent in the nuclei of many cell types. Actin and actin-binding proteins, as well as actin-related proteins, are necessary for the mediation of the conformation and function of nuclear actin, including the transformation of actin between unpolymerized and polymerized, chroinatin remodeling, regulation of gene expression and RNA processing as well as RNA transportation. In this paper, we summarized the progresses in the research of nu clear actin.

  4. The Actin Binding Protein Adseverin Regulates Osteoclastogenesis

    OpenAIRE

    Hassanpour, Siavash; Jiang, Hongwei; Wang, Yongqiang; Kuiper, Johannes W. P.; Glogauer, Michael

    2014-01-01

    Adseverin (Ads), a member of the Gelsolin superfamily of actin binding proteins, regulates the actin cytoskeleton architecture by severing and capping existing filamentous actin (F-actin) strands and nucleating the assembly of new F-actin filaments. Ads has been implicated in cellular secretion, exocytosis and has also been shown to regulate chondrogenesis and megakaryoblastic leukemia cell differentiation. Here we report for the first time that Ads is involved in regulating osteoclastogenesi...

  5. Cofilin-mediated actin dynamics promotes actin bundle formation during Drosophila bristle development.

    Science.gov (United States)

    Wu, Jing; Wang, Heng; Guo, Xuan; Chen, Jiong

    2016-08-15

    The actin bundle is an array of linear actin filaments cross-linked by actin-bundling proteins, but its assembly and dynamics are not as well understood as those of the branched actin network. Here we used the Drosophila bristle as a model system to study actin bundle formation. We found that cofilin, a major actin disassembly factor of the branched actin network, promotes the formation and positioning of actin bundles in the developing bristles. Loss of function of cofilin or AIP1, a cofactor of cofilin, each resulted in increased F-actin levels and severe defects in actin bundle organization, with the defects from cofilin deficiency being more severe. Further analyses revealed that cofilin likely regulates actin bundle formation and positioning by the following means. First, cofilin promotes a large G-actin pool both locally and globally, likely ensuring rapid actin polymerization for bundle initiation and growth. Second, cofilin limits the size of a nonbundled actin-myosin network to regulate the positioning of actin bundles. Third, cofilin prevents incorrect assembly of branched and myosin-associated actin filament into bundles. Together these results demonstrate that the interaction between the dynamic dendritic actin network and the assembling actin bundles is critical for actin bundle formation and needs to be closely regulated.

  6. F-actin distribution and function during sexual differentiation in Schizosaccharomyces pombe

    DEFF Research Database (Denmark)

    Petersen, J; Nielsen, O; Egel, R;

    1998-01-01

    Sexual differentiation in Schizosaccharomyces pombe is induced from the G1 phase of the cell cycle by nitrogen starvation and the presence of mating pheromones. We describe the distribution of F-actin during sexual differentiation. Cortical F-actin dots have previously been shown to be restricted...

  7. Boolean gates on actin filaments

    Science.gov (United States)

    Siccardi, Stefano; Tuszynski, Jack A.; Adamatzky, Andrew

    2016-01-01

    Actin is a globular protein which forms long polar filaments in the eukaryotic cytoskeleton. Actin networks play a key role in cell mechanics and cell motility. They have also been implicated in information transmission and processing, memory and learning in neuronal cells. The actin filaments have been shown to support propagation of voltage pulses. Here we apply a coupled nonlinear transmission line model of actin filaments to study interactions between voltage pulses. To represent digital information we assign a logical TRUTH value to the presence of a voltage pulse in a given location of the actin filament, and FALSE to the pulse's absence, so that information flows along the filament with pulse transmission. When two pulses, representing Boolean values of input variables, interact, then they can facilitate or inhibit further propagation of each other. We explore this phenomenon to construct Boolean logical gates and a one-bit half-adder with interacting voltage pulses. We discuss implications of these findings on cellular process and technological applications.

  8. Technical advance: identification of plant actin-binding proteins by F-actin affinity chromatography

    Science.gov (United States)

    Hu, S.; Brady, S. R.; Kovar, D. R.; Staiger, C. J.; Clark, G. B.; Roux, S. J.; Muday, G. K.

    2000-01-01

    Proteins that interact with the actin cytoskeleton often modulate the dynamics or organization of the cytoskeleton or use the cytoskeleton to control their localization. In plants, very few actin-binding proteins have been identified and most are thought to modulate cytoskeleton function. To identify actin-binding proteins that are unique to plants, the development of new biochemical procedures will be critical. Affinity columns using actin monomers (globular actin, G-actin) or actin filaments (filamentous actin, F-actin) have been used to identify actin-binding proteins from a wide variety of organisms. Monomeric actin from zucchini (Cucurbita pepo L.) hypocotyl tissue was purified to electrophoretic homogeneity and shown to be native and competent for polymerization to actin filaments. G-actin, F-actin and bovine serum albumin affinity columns were prepared and used to separate samples enriched in either soluble or membrane-associated actin-binding proteins. Extracts of soluble actin-binding proteins yield distinct patterns when eluted from the G-actin and F-actin columns, respectively, leading to the identification of a putative F-actin-binding protein of approximately 40 kDa. When plasma membrane-associated proteins were applied to these columns, two abundant polypeptides eluted selectively from the F-actin column and cross-reacted with antiserum against pea annexins. Additionally, a protein that binds auxin transport inhibitors, the naphthylphthalamic acid binding protein, which has been previously suggested to associate with the actin cytoskeleton, was eluted in a single peak from the F-actin column. These experiments provide a new approach that may help to identify novel actin-binding proteins from plants.

  9. Allyl Isothiocyanate Inhibits Actin-Dependent Intracellular Transport in Arabidopsis thaliana

    Directory of Open Access Journals (Sweden)

    Bjørnar Sporsheim

    2015-12-01

    Full Text Available Volatile allyl isothiocyanate (AITC derives from the biodegradation of the glucosinolate sinigrin and has been associated with growth inhibition in several plants, including the model plant Arabidopsis thaliana. However, the underlying cellular mechanisms of this feature remain scarcely investigated in plants. In this study, we present evidence of an AITC-induced inhibition of actin-dependent intracellular transport in A. thaliana. A transgenic line of A. thaliana expressing yellow fluorescent protein (YFP-tagged actin filaments was used to show attenuation of actin filament movement by AITC. This appeared gradually in a time- and dose-dependent manner and resulted in actin filaments appearing close to static. Further, we employed four transgenic lines with YFP-fusion proteins labeling the Golgi apparatus, endoplasmic reticulum (ER, vacuoles and peroxisomes to demonstrate an AITC-induced inhibition of actin-dependent intracellular transport of or, in these structures, consistent with the decline in actin filament movement. Furthermore, the morphologies of actin filaments, ER and vacuoles appeared aberrant following AITC-exposure. However, AITC-treated seedlings of all transgenic lines tested displayed morphologies and intracellular movements similar to that of the corresponding untreated and control-treated plants, following overnight incubation in an AITC-absent environment, indicating that AITC-induced decline in actin-related movements is a reversible process. These findings provide novel insights into the cellular events in plant cells following exposure to AITC, which may further expose clues to the physiological significance of the glucosinolate-myrosinase system.

  10. Fascin regulates nuclear actin during Drosophila oogenesis.

    Science.gov (United States)

    Kelpsch, Daniel J; Groen, Christopher M; Fagan, Tiffany N; Sudhir, Sweta; Tootle, Tina L

    2016-10-01

    Drosophila oogenesis provides a developmental system with which to study nuclear actin. During Stages 5-9, nuclear actin levels are high in the oocyte and exhibit variation within the nurse cells. Cofilin and Profilin, which regulate the nuclear import and export of actin, also localize to the nuclei. Expression of GFP-tagged Actin results in nuclear actin rod formation. These findings indicate that nuclear actin must be tightly regulated during oogenesis. One factor mediating this regulation is Fascin. Overexpression of Fascin enhances nuclear GFP-Actin rod formation, and Fascin colocalizes with the rods. Loss of Fascin reduces, whereas overexpression of Fascin increases, the frequency of nurse cells with high levels of nuclear actin, but neither alters the overall nuclear level of actin within the ovary. These data suggest that Fascin regulates the ability of specific cells to accumulate nuclear actin. Evidence indicates that Fascin positively regulates nuclear actin through Cofilin. Loss of Fascin results in decreased nuclear Cofilin. In addition, Fascin and Cofilin genetically interact, as double heterozygotes exhibit a reduction in the number of nurse cells with high nuclear actin levels. These findings are likely applicable beyond Drosophila follicle development, as the localization and functions of Fascin and the mechanisms regulating nuclear actin are widely conserved.

  11. Oral nicotinamide and actinic keratosis: a supplement success story.

    Science.gov (United States)

    Kim, Burcu; Halliday, Gary M; Damian, Diona L

    2015-01-01

    Nicotinamide has shown potential as a safe and effective intervention for the prevention of malignant and premalignant skin lesions. Recent studies have shown that nicotinamide, in both oral and topical forms, is able to prevent ultraviolet-induced immunosuppression in humans [1,2,3] and mice [4,5]. Immunosuppression is a known factor for the progression of premalignant lesions, such as actinic keratosis [6]. Murine studies have shown that nicotinamide is also able to protect against photocarcinogenesis [4,5]. Preliminary human studies suggest that nicotinamide may help prevent skin cancers and enhance the regression of actinic keratoses.

  12. Oral nicotinamide and actinic keratosis: a supplement success story.

    Science.gov (United States)

    Kim, Burcu; Halliday, Gary M; Damian, Diona L

    2015-01-01

    Nicotinamide has shown potential as a safe and effective intervention for the prevention of malignant and premalignant skin lesions. Recent studies have shown that nicotinamide, in both oral and topical forms, is able to prevent ultraviolet-induced immunosuppression in humans [1,2,3] and mice [4,5]. Immunosuppression is a known factor for the progression of premalignant lesions, such as actinic keratosis [6]. Murine studies have shown that nicotinamide is also able to protect against photocarcinogenesis [4,5]. Preliminary human studies suggest that nicotinamide may help prevent skin cancers and enhance the regression of actinic keratoses. PMID:25561219

  13. Cellular Levels of Signaling Factors Are Sensed by β-actin Alleles to Modulate Transcriptional Pulse Intensity

    Directory of Open Access Journals (Sweden)

    Alon Kalo

    2015-04-01

    Full Text Available The transcriptional response of β-actin to extra-cellular stimuli is a paradigm for transcription factor complex assembly and regulation. Serum induction leads to a precisely timed pulse of β-actin transcription in the cell population. Actin protein is proposed to be involved in this response, but it is not known whether cellular actin levels affect nuclear β-actin transcription. We perturbed the levels of key signaling factors and examined the effect on the induced transcriptional pulse by following endogenous β-actin alleles in single living cells. Lowering serum response factor (SRF protein levels leads to loss of pulse integrity, whereas reducing actin protein levels reveals positive feedback regulation, resulting in elevated gene activation and a prolonged transcriptional response. Thus, transcriptional pulse fidelity requires regulated amounts of signaling proteins, and perturbations in factor levels eliminate the physiological response, resulting in either tuning down or exaggeration of the transcriptional pulse.

  14. Actin induction during PMA and cAMP-dependent signal pathway activation in Entamoeba histolytica trophozoites.

    Science.gov (United States)

    Ortiz, D; del Carmen Dominguez-Robles, M; Villegas-Sepúlveda, N; Meza, I

    2000-10-01

    Activation of PKC or cAMP-dependent signalling pathways in Entamoeba histolytica triggers the phosphorylation of proteins involved in actin rearrangements necessary for adhesion and locomotion. Analogous motifs to SRE and CRE sequences--known to respond to PMA and cAMP--were identified within the 5' regulatory region (5'RR) of one of the parasite actin genes. These sequences could be involved in the actin transcriptional upregulation reported during signalling. To test this hypothesis, a plasmid containing the 5'RR of the actin gene fused to the bacterial neomycin gene (neo) was used for stable transfection. Expression of neo and endogenous actin was measured after stimulation of transfected amoebae by PMA and dcAMP. It was found that both compounds induced neo and actin expression and showed a co-operative effect in the induction of neo. Induction by PMA or dcAMP failed if the directing amoebic 5'RR lacked SRE and CRE motifs. Transfection of amoebae with plasmid constructs, containing either progressive deletions of the actin 5'RR or site-directed mutations of the SRE and CRE-like motifs, corroborated that these sequences and a co-ordinated participation of PKC- and PKA-activated transcription factors are responsible for the increments in neo and actin mRNAs. In vivo, these PMA and cAMP-response elements could play an important role in regulating actin expression and organization in signalling processes activated during tissue invasion.

  15. Double localization of F-actin in chemoattractant-stimulated polymorphonuclear leucocytes.

    Science.gov (United States)

    Lepidi, H; Benoliel, A M; Mege, J L; Bongrand, P; Capo, C

    1992-09-01

    Uniform concentrations of chemoattractants such as formylpeptides induced a morphological polarization of human polymorphonuclear leucocytes (PMNs) and a concentration of F-actin at the cell front. They also induced a transient increase in filamentous actin (F-actin) which preceded the cell shape change. We combined fluorescence microscopy and image analysis to study the localization of F-actin, as revealed by a specific probe (bodipyTM phallacidin) in suspended PMNs stimulated by chemoattractants. F-actin exhibited remarkable concentration in focal points after a 30 s exposure to 10(-8) M formylmethionyl-leucyl-phenylalanine (fMet-Leu-Phe), although no shape change of PMNs was detectable. A 10-min incubation with formylpeptide (10(-6) to 10(-9) M) induced the morphological polarization of PMNs and the appearance of a principal focus of F-actin in the cell head region and a secondary focus in the cell posterior end. The distribution of F-actin-associated fluorescence in 2D images of polarized PMNs might be due to an actual concentration of F-actin in privileged areas, to a local concentration of plasma membrane drawing filamentous actin or to variations in the cell volume. Then, we studied the distribution of a cytoplasmic marker, fluorescein diacetate and a membrane probe, TMA-DPH, in unstimulated rounded PMNs and in spherical and morphologically polarized PMNs stimulated by formylpeptide. The distribution of neither of these probes was correlated with F-actin distribution, especially in rounded PMNs stimulated 30 s with 10(-8) M fMet-Leu-Phe, suggesting that F-actin was concentrated in two foci located in the cell head region and in the cell posterior end. In addition, zymosan-activated serum induced the morphological polarization of PMNs and the appearance of two foci of filamentous actin, demonstrating that binding of formylpeptide to its specific receptor was not required for F-actin reorganization. We conclude that the accumulation of F-actin probably

  16. Drosophila actin-Capping Protein limits JNK activation by the Src proto-oncogene.

    Science.gov (United States)

    Fernández, B G; Jezowska, B; Janody, F

    2014-04-17

    The Src family kinases c-Src, and its downstream effectors, the Rho family of small GTPases RhoA and Jun N-terminal kinase (JNK) have a significant role in tumorigenesis. In this report, using the Drosophila wing disc epithelium as a model system, we demonstrate that the actin-Capping Protein (CP) αβ heterodimer, which regulates actin filament (F-actin) polymerization, limits Src-induced apoptosis or tissue overgrowth by restricting JNK activation. We show that overexpressing Src64B drives JNK-independent loss of epithelial integrity and JNK-dependent apoptosis via Btk29A, p120ctn and Rho1. However, when cells are kept alive with the Caspase inhibitor P35, JNK acts as a potent inducer of proliferation via activation of the Yorkie oncogene. Reducing CP levels direct apoptosis of overgrowing Src64B-overexpressing tissues. Conversely, overexpressing capping protein inhibits Src64B and Rho1, but not Rac1-induced JNK signaling. CP requires the actin-binding domain of the α-subunit to limit Src64B-induced apoptosis, arguing that the control of F-actin mediates this effect. In turn, JNK directs F-actin accumulation. Moreover, overexpressing capping protein also prevents apoptosis induced by ectopic JNK expression. Our data are consistent with a model in which the control of F-actin by CP limits Src-induced apoptosis or tissue overgrowth by acting downstream of Btk29A, p120ctn and Rho1, but upstream of JNK. In turn, JNK may counteract the effect of CP on F-actin, providing a positive feedback, which amplifies JNK activation. We propose that cytoskeletal changes triggered by misregulation of F-actin modulators may have a significant role in Src-mediated malignant phenotypes during the early stages of cellular transformation.

  17. Disruption of microtubule network rescues aberrant actin comets in dynamin2-depleted cells.

    Directory of Open Access Journals (Sweden)

    Yuji Henmi

    Full Text Available A large GTPase dynamin, which is required for endocytic vesicle formation, regulates the actin cytoskeleton through its interaction with cortactin. Dynamin2 mutants impair the formation of actin comets, which are induced by Listeria monocytogenes or phosphatidylinositol-4-phosphate 5-kinase. However, the role of dynamin2 in the regulation of the actin comet is still unclear. Here we show that aberrant actin comets in dynamin2-depleted cells were rescued by disrupting of microtubule networks. Depletion of dynamin2, but not cortactin, significantly reduced the length and the speed of actin comets induced by Listeria. This implies that dynamin2 may regulate the actin comet in a cortactin-independent manner. As dynamin regulates microtubules, we investigated whether perturbation of microtubules would rescue actin comet formation in dynamin2-depleted cells. Treatment with taxol or colchicine created a microtubule-free space in the cytoplasm, and made no difference between control and dynamin2 siRNA cells. This suggests that the alteration of microtubules by dynamin2 depletion reduced the length and the speed of the actin comet.

  18. Actin phosphorylation correlates with actin sequestration in ATP-depleted abbit renal proximal tubules

    Institute of Scientific and Technical Information of China (English)

    Yingchun Li; Yingbin Ge; Jun Du; Rong Zhou; Jin Chen; Luo Gu

    2005-01-01

    Objective: To demonstrate the relationship between actin phosphorylation and actin sequestration in ATP-depleted rabbit renal proximal tubules. Methods: Using two-dimensional electrophoreses and Western blotting to analyze the phosphorylation state of the sequestered actin in rabbit renal proximal tubules. Results: The analytical result of the sequestered actin indicated that nearly half of the actin was phosphorylated on serine residue(s). Conclusion: Result suggested a close correlation between actin sequestration and actin phosphorylation in ATP-depleted rabbit renal proximal tubules.

  19. PIP2: choreographer of actin-adaptor proteins in the HIV-1 dance

    Science.gov (United States)

    Rocha-Perugini, Vera; Gordon-Alonso, Mónica; Sánchez-Madrid, Francisco

    2014-01-01

    The actin cytoskeleton plays a key role during the replication cycle of human immunodeficiency virus-1 (HIV-1). HIV-1 infection is affected by cellular proteins that influence the clustering of viral receptors or the subcortical actin cytoskeleton. Several of these actin-adaptor proteins are controlled by the second messenger phosphatidylinositol 4,5-biphosphate (PIP2), an important regulator of actin organization. PIP2 production is induced by HIV-1 attachment and facilitates viral infection. However, the importance of PIP2 in regulating cytoskeletal proteins and thus HIV-1 infection has been overlooked. This review examines recent reports describing the roles played by actin-adaptor proteins during HIV-1 infection of CD4+ T cells, highlighting the influence of the signaling lipid PIP2 in this process. PMID:24768560

  20. Non-lytic, actin-based exit of intracellular parasites from C. elegans intestinal cells.

    Directory of Open Access Journals (Sweden)

    Kathleen A Estes

    2011-09-01

    Full Text Available The intestine is a common site for invasion by intracellular pathogens, but little is known about how pathogens restructure and exit intestinal cells in vivo. The natural microsporidian parasite N. parisii invades intestinal cells of the nematode C. elegans, progresses through its life cycle, and then exits cells in a transmissible spore form. Here we show that N. parisii causes rearrangements of host actin inside intestinal cells as part of a novel parasite exit strategy. First, we show that N. parisii infection causes ectopic localization of the normally apical-restricted actin to the basolateral side of intestinal cells, where it often forms network-like structures. Soon after this actin relocalization, we find that gaps appear in the terminal web, a conserved cytoskeletal structure that could present a barrier to exit. Reducing actin expression creates terminal web gaps in the absence of infection, suggesting that infection-induced actin relocalization triggers gap formation. We show that terminal web gaps form at a distinct stage of infection, precisely timed to precede spore exit, and that all contagious animals exhibit gaps. Interestingly, we find that while perturbations in actin can create these gaps, actin is not required for infection progression or spore formation, but actin is required for spore exit. Finally, we show that despite large numbers of spores exiting intestinal cells, this exit does not cause cell lysis. These results provide insight into parasite manipulation of the host cytoskeleton and non-lytic escape from intestinal cells in vivo.

  1. Proneural proteins Achaete and Scute associate with nuclear actin to promote formation of external sensory organs.

    Science.gov (United States)

    Hsiao, Yun-Ling; Chen, Yu-Ju; Chang, Yi-Jie; Yeh, Hsiao-Fong; Huang, Yi-Chun; Pi, Haiwei

    2014-01-01

    Basic helix-loop-helix (bHLH) proneural proteins promote neurogenesis through transcriptional regulation. Although much is known about the tissue-specific regulation of proneural gene expression, how proneural proteins interact with transcriptional machinery to activate downstream target genes is less clear. Drosophila proneural proteins Achaete (Ac) and Scute (Sc) induce external sensory organ formation by activating neural precursor gene expression. Through co-immunoprecipitation and mass spectrometric analyses, we found that nuclear but not cytoplasmic actin associated with the Ac and Sc proteins in Drosophila S2 cells. Daughterless (Da), the common heterodimeric partner of Drosophila bHLH proteins, was observed to associate with nuclear actin through proneural proteins. A yeast two-hybrid assay revealed that the binding specificity between actin and Ac or Sc was conserved in yeast nuclei without the presence of additional Drosophila factors. We further show that actin is required in external sensory organ formation. Reduction in actin gene activity impaired proneural-protein-dependent expression of the neural precursor genes, as well as formation of neural precursors. Furthermore, increased nuclear actin levels, obtained by expression of nucleus-localized actin, elevated Ac-Da-dependent gene transcription as well as Ac-mediated external sensory organ formation. Taken together, our in vivo and in vitro observations suggest a novel link for actin in proneural-protein-mediated transcriptional activation and neural precursor differentiation.

  2. Kindlin-2 directly binds actin and regulates integrin outside-in signaling.

    Science.gov (United States)

    Bledzka, Kamila; Bialkowska, Katarzyna; Sossey-Alaoui, Khalid; Vaynberg, Julia; Pluskota, Elzbieta; Qin, Jun; Plow, Edward F

    2016-04-11

    Reduced levels of kindlin-2 (K2) in endothelial cells derived from K2(+/-)mice or C2C12 myoblastoid cells treated with K2 siRNA showed disorganization of their actin cytoskeleton and decreased spreading. These marked changes led us to examine direct binding between K2 and actin. Purified K2 interacts with F-actin in cosedimentation and surface plasmon resonance analyses and induces actin aggregation. We further find that the F0 domain of K2 binds actin. A mutation, LK(47)/AA, within a predicted actin binding site (ABS) of F0 diminishes its interaction with actin by approximately fivefold. Wild-type K2 and K2 bearing the LK(47)/AA mutation were equivalent in their ability to coactivate integrin αIIbβ3 in a CHO cell system when coexpressed with talin. However, K2-LK(47)/AA exhibited a diminished ability to support cell spreading and actin organization compared with wild-type K2. The presence of an ABS in F0 of K2 that influences outside-in signaling across integrins establishes a new foundation for considering how kindlins might regulate cellular responses. PMID:27044892

  3. Actinic Keratoses: A Comprehensive Update

    OpenAIRE

    Ibrahim, Sherrif F.; Brown, Marc D.

    2009-01-01

    Actinic keratoses are common intra-epidermal neoplasms that lie on a continuum with squamous cell carcinoma. Tightly linked to ultraviolet irradiation, they occur in areas of chronic sun exposure, and early treatment of these lesions may prevent their progression to invasive disease. A large variety of effective treatment modalities exist, and the optimal therapeutic choice is dependent on a variety of patient- and physician-associated variables. Many established and more recent approaches ar...

  4. Actin organization, bristle morphology, and viability are affected by actin capping protein mutations in Drosophila

    OpenAIRE

    1996-01-01

    Regulation of actin filament length and orientation is important in many actin-based cellular processes. This regulation is postulated to occur through the action of actin-binding proteins. Many actin-binding proteins that modify actin in vitro have been identified, but in many cases, it is not known if this activity is physiologically relevant. Capping protein (CP) is an actin-binding protein that has been demonstrated to control filament length in vitro by binding to the barbed ends and pre...

  5. Mesoscopic model of actin-based propulsion.

    Directory of Open Access Journals (Sweden)

    Jie Zhu

    Full Text Available Two theoretical models dominate current understanding of actin-based propulsion: microscopic polymerization ratchet model predicts that growing and writhing actin filaments generate forces and movements, while macroscopic elastic propulsion model suggests that deformation and stress of growing actin gel are responsible for the propulsion. We examine both experimentally and computationally the 2D movement of ellipsoidal beads propelled by actin tails and show that neither of the two models can explain the observed bistability of the orientation of the beads. To explain the data, we develop a 2D hybrid mesoscopic model by reconciling these two models such that individual actin filaments undergoing nucleation, elongation, attachment, detachment and capping are embedded into the boundary of a node-spring viscoelastic network representing the macroscopic actin gel. Stochastic simulations of this 'in silico' actin network show that the combined effects of the macroscopic elastic deformation and microscopic ratchets can explain the observed bistable orientation of the actin-propelled ellipsoidal beads. To test the theory further, we analyze observed distribution of the curvatures of the trajectories and show that the hybrid model's predictions fit the data. Finally, we demonstrate that the model can explain both concave-up and concave-down force-velocity relations for growing actin networks depending on the characteristic time scale and network recoil. To summarize, we propose that both microscopic polymerization ratchets and macroscopic stresses of the deformable actin network are responsible for the force and movement generation.

  6. From pollen actin to crop male sterility

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Actin plays an important role in the life activity of animal and plant cells. Pollen cells have plenty of actin whose structure and characteristics are very similar to the animal actin. The nucleotide sequence and amino acid sequence of plant actin gene are very similar to those of the animal gene. The content of pollen actin from male sterile plants is much more lower than that from its maintainer plants. The expression of actin gene is organ-specific during the plant development. The expression quantity of actin gene in pollen is much more higher than those from root, stem and leaf. The expression plasmid of the anti-sense actin gene was constructed, transferred to the protoplasts of wheat and tomato to inhibit the expression of actin gene in pollen and thus the male sterile plants of wheat and tomato were obtained. The actin in pollens from the transgenic plants was reduced significantly, whereas the pistil was not affected. This study might pave a new way to breeding male sterile lines for the application of hybrid vigor of wheat and tomato.

  7. Wdr1-Dependent Actin Reorganization in Platelet Activation.

    Science.gov (United States)

    Dasgupta, Swapan K; Le, Anhquyen; Da, Qi; Cruz, Miguel; Rumbaut, Rolando E; Thiagarajan, Perumal

    2016-01-01

    In resting platelets, the integrin αIIbβ3 is present in a low-affinity "bent" state. During platelet aggregation, intracytoplasmic signals induce conformational changes (inside-out signaling) that result in a "swung-out" conformation competent to bind ligands such as fibrinogen. The cytoskeleton plays an essential role in αIIbβ3 activation. We investigated the role of the actin interacting protein Wdr1 in αIIbβ3 activation. Wdr1-hypomorphic mice had a prolonged bleeding time (> 10 minutes) compared to that of wild-type mice (2.1 ± 0.7 minutes). Their platelets had impaired aggregation to collagen and thrombin. In a FeCl3 induced carotid artery thrombosis model, vessel occlusion in Wdr1-hypomorphic mice was prolonged significantly compared to wild-type mice (9.0 ± 10.5 minutes versus 5.8 ± 12.6 minutes (p = 0.041). Activation-induced binding of JON/A (a conformation-specific antibody to activated αIIbβ3) was significantly less in Wdr1-hypomorphic platelets at various concentrations of collagen, indicating impaired inside-out activation of αIIbβ3, despite a normal calcium response. Actin turnover, assessed by measuring F-actin and G-actin ratios during collagen- and thrombin-induced platelet aggregation, was highly impaired in Wdr1-hypomorphic platelets. Furthermore, talin failed to redistribute and translocate to the cytoskeleton following activation in Wdr1-hypomorphic platelets. These studies show that Wdr1 is essential for talin-induced activation of αIIbβ3 during platelet activation. PMID:27627652

  8. Changes of the content and distribution of cochlear actin iron-deficient growing rats

    Institute of Scientific and Technical Information of China (English)

    YUAN Jun; SUN Ai-hua; WANG Jin-ling; FAN Rong; JIAO Xi-ying; LIN Shun-zhang

    2001-01-01

    To observe the changes of the content and distribution of cochlear actin in growing rats at different periods of iron deficiency (ID) and to explore the pathogenetic mechanism of ID-induced sensorineuralhear ing loss. Methods: Actin of cochlear membranous tissue was separated and determined with SDS-PAGE and West em blot. The relative content and distribution of cochlear actin in both ID and normal rats in week 8 and 12 were studied by TLC-densitometric determination and immunohistochemical procedures. Results: A marked reduction of relative content and immunohistochemical reaction product of cochlear actin was found in ID rats with sensorineural hearing loss in comparison with normal controls. There was no significant difference of cochlea actin among differ ent groups of ID rats with hearing loss. Conclusion: ID-induced sensorineural hearing loss is probably associated with reduction of actin in the cochlea. It is suggested that the experimental ID period of time have no considerable effect on the relative content and distribution of cochlear actin.

  9. Multiple actin binding domains of Ena/VASP proteins determine actin network stiffening.

    Science.gov (United States)

    Gentry, Brian S; van der Meulen, Stef; Noguera, Philippe; Alonso-Latorre, Baldomero; Plastino, Julie; Koenderink, Gijsje H

    2012-11-01

    Vasodilator-stimulated phosphoprotein (Ena/VASP) is an actin binding protein, important for actin dynamics in motile cells and developing organisms. Though VASP's main activity is the promotion of barbed end growth, it has an F-actin binding site and can form tetramers, and so could additionally play a role in actin crosslinking and bundling in the cell. To test this activity, we performed rheology of reconstituted actin networks in the presence of wild-type VASP or mutants lacking the ability to tetramerize or to bind G-actin and/or F-actin. We show that increasing amounts of wild-type VASP increase network stiffness up to a certain point, beyond which stiffness actually decreases with increasing VASP concentration. The maximum stiffness is 10-fold higher than for pure actin networks. Confocal microscopy shows that VASP forms clustered actin filament bundles, explaining the reduction in network elasticity at high VASP concentration. Removal of the tetramerization site results in significantly reduced bundling and bundle clustering, indicating that VASP's flexible tetrameric structure causes clustering. Removing either the F-actin or the G-actin binding site diminishes VASP's effect on elasticity, but does not eliminate it. Mutating the F-actin and G-actin binding site together, or mutating the F-actin binding site and saturating the G-actin binding site with monomeric actin, eliminates VASP's ability to increase network stiffness. We propose that, in the cell, VASP crosslinking confers only moderate increases in linear network elasticity, and unlike other crosslinkers, VASP's network stiffening activity may be tuned by the local concentration of monomeric actin.

  10. Regulation of Actin Dynamics in Pollen Tubes: Control of Actin Polymer Level

    Institute of Scientific and Technical Information of China (English)

    Naizhi Chen; Xiaolu Qu; Youjun Wu; Shanjin Huang

    2009-01-01

    Actin cytoskeleton undergoes rapid reorganization In response to internal and external cues. How the dynamics of actin cytoskeleton are regulated, and how its dynamics relate to its function are fundamental questions inplant cell biology. The pollen tube is a well characterized actin-based call morphogenesis in plants. One of the striking features of actin cytoskeleton characterized in the pollen tube is its surprisingly low level of actin polymer. This special phenomenon might relate to the function of actin cytoskeleton in pollen tubes. Understanding the molecular mechanism underlying this special phenomenon requires careful analysis of actin-binding proteins that modulate actin dynamics directly. Recent biochemical and biophysical analyses of several highly conserved plant actin-binding proteins reveal unusual and un-expected properties, which emphasizes the importance of carefully analyzing their action mechanism and cellular activity. In this review, we highlight an actin monomer sequestering protein, a barbed end capping protein and an F-actin severing and dynamizing protein in plant. We propose that these proteins function in harmony to regulate actin dynamics and maintain the low level of actin polymer in pollen tubes.

  11. TIRF microscopy analysis of human Cof1, Cof2, and ADF effects on actin filament severing and turnover.

    Science.gov (United States)

    Chin, Samantha M; Jansen, Silvia; Goode, Bruce L

    2016-04-24

    Dynamic remodeling and turnover of cellular actin networks requires actin filament severing by actin-depolymerizing factor (ADF)/Cofilin proteins. Mammals express three different ADF/Cofilins (Cof1, Cof2, and ADF), and genetic studies suggest that in vivo they perform both overlapping and unique functions. To gain mechanistic insights into their different roles, we directly compared their G-actin and F-actin binding affinities, and quantified the actin filament severing activities of human Cof1, Cof2, and ADF using in vitro total internal reflection fluorescence microscopy. All three ADF/Cofilins had similar affinities for G-actin and F-actin. However, Cof2 and ADF severed filaments much more efficiently than Cof1 at both lower and higher concentrations and using either muscle or platelet actin. Furthermore, Cof2 and ADF were more effective than Cof1 in promoting "enhanced disassembly" when combined with actin disassembly co-factors Coronin-1B and actin-interacting protein 1 (AIP1), and these differences were observed on both preformed and actively growing filaments. To probe the mechanism underlying these differences, we used multi-wavelength total internal reflection fluorescence microscopy to directly observe Cy3-Cof1 and Cy3-Cof2 interacting with actin filaments in real time during severing. Cof1 and Cof2 each bound to filaments with similar kinetics, yet Cof2 induced severing much more rapidly than Cof1, decreasing the time interval between initial binding on a filament and severing at the same location. These differences in ADF/Cofilin activities and mechanisms may be used in cells to tune filament turnover rates, which can vary widely for different actin structures.

  12. Actin gene family in Branchiostoma belched

    Institute of Scientific and Technical Information of China (English)

    2016-01-01

    Actin is a highly conserved cytoskeletal protein that is found in essentially all eukaryotic cells,which plays a paramount role in several basic functions of the organism, such as the maintenance of cellshape, cell division, cell mobility and muscle contraction. However, little is known about actin gene family inChinese amphioxus (Branchiostoma belcheri). Here we systemically analyzed the actin genes family inBranchiostoma belched and found that amphioxus contains 33 actin genes. These genes have undergoneextensive expansion through tandem duplications by phylogenetic analysis. In addition, we also providedevidence indicating that actin genes have divergent functions by specializing their EST data in both Bran-chiostoma belched and Branchiostoma florida. Our results provided an alternative explanation for the evolu-tion of actin genes, and gave new insights into their functional roles.

  13. The proline-rich domain of tau plays a role in interactions with actin

    Directory of Open Access Journals (Sweden)

    Wang Dong

    2009-11-01

    Full Text Available Abstract Background The microtubule-associated protein tau is able to interact with actin and serves as a cross-linker between the microtubule and actin networks. The microtubule-binding domain of tau is known to be involved in its interaction with actin. Here, we address the question of whether the other domains of tau also interact with actin. Results Several tau truncation and deletion mutants were constructed, namely N-terminal region (tauN, proline-rich domain (tauPRD, microtubule binding domain (tauMTBD and C-terminal region (tauC truncation mutants, and microtubule binding domain (tauΔMTBD and proline-rich domain/microtubule binding domain (tauΔPRD&MTBD deletion mutants. The proline-rich domain truncation mutant (tauPRD and the microtubule binding domain deletion mutant (tauΔMTBD promoted the formation of actin filaments. However, actin assembly was not observed in the presence of the N-terminal and C-terminal truncation mutants. These results indicate that the proline-rich domain is involved in the association of tau with G-actin. Furthermore, results from co-sedimentation, solid phase assays and electron microscopy showed that the proline-rich domain is also capable of binding to F-actin and inducing F-actin bundles. Using solid phase assays to analyze apparent dissociation constants for the binding of tau and its mutants to F-actin resulted in a sequence of affinity for F-actin: tau >> microtubule binding domain > proline-rich domain. Moreover, we observed that the proline-rich domain was able to associate with and bundle F-actin at physiological ionic strength. Conclusion The proline-rich domain is a functional structure playing a role in the association of tau with actin. This suggests that the proline-rich domain and the microtubule-binding domain of tau are both involved in binding to and bundling F-actin.

  14. Packaging of actin into Ebola virus VLPs

    Directory of Open Access Journals (Sweden)

    Harty Ronald N

    2005-12-01

    Full Text Available Abstract The actin cytoskeleton has been implicated in playing an important role assembly and budding of several RNA virus families including retroviruses and paramyxoviruses. In this report, we sought to determine whether actin is incorporated into Ebola VLPs, and thus may play a role in assembly and/or budding of Ebola virus. Our results indicated that actin and Ebola virus VP40 strongly co-localized in transfected cells as determined by confocal microscopy. In addition, actin was packaged into budding VP40 VLPs as determined by a functional budding assay and protease protection assay. Co-expression of a membrane-anchored form of Ebola virus GP enhanced the release of both VP40 and actin in VLPs. Lastly, disruption of the actin cytoskeleton with latrunculin-A suggests that actin may play a functional role in budding of VP40/GP VLPs. These data suggest that VP40 may interact with cellular actin, and that actin may play a role in assembly and/or budding of Ebola VLPs.

  15. The pros and cons of common actin labeling tools for visualizing actin dynamics during Drosophila oogenesis

    OpenAIRE

    Spracklen, Andrew J.; Fagan, Tiffany N.; Lovander, Kaylee E.; Tootle, Tina L.

    2014-01-01

    Dynamic remodeling of the actin cytoskeleton is required for both development and tissue homeostasis. While fixed image analysis has provided significant insight into such events, a complete understanding of cytoskeletal dynamics requires live imaging. Numerous tools for the live imaging of actin have been generated by fusing the actin-binding domain from an actin-interacting protein to a fluorescent protein. Here we comparatively assess the utility of three such tools – Utrophin, Lifeact, an...

  16. The vascular smooth muscle alpha-actin gene is reactivated during cardiac hypertrophy provoked by load.

    OpenAIRE

    Black, F M; Packer, S E; Parker, T G; Michael, L H; Roberts, R; R J Schwartz; Schneider, M D

    1991-01-01

    Cardiac hypertrophy triggered by mechanical load possesses features in common with growth factor signal transduction. A hemodynamic load provokes rapid expression of the growth factor-inducible nuclear oncogene, c-fos, and certain peptide growth factors specifically stimulate the "fetal" cardiac genes associated with hypertrophy, even in the absence of load. These include the gene encoding vascular smooth muscle alpha-actin, the earliest alpha-actin expressed during cardiac myogenesis; howeve...

  17. The pros and cons of common actin labeling tools for visualizing actin dynamics during Drosophila oogenesis.

    Science.gov (United States)

    Spracklen, Andrew J; Fagan, Tiffany N; Lovander, Kaylee E; Tootle, Tina L

    2014-09-15

    Dynamic remodeling of the actin cytoskeleton is required for both development and tissue homeostasis. While fixed image analysis has provided significant insight into such events, a complete understanding of cytoskeletal dynamics requires live imaging. Numerous tools for the live imaging of actin have been generated by fusing the actin-binding domain from an actin-interacting protein to a fluorescent protein. Here we comparatively assess the utility of three such tools--Utrophin, Lifeact, and F-tractin--for characterizing the actin remodeling events occurring within the germline-derived nurse cells during Drosophila mid-oogenesis or follicle development. Specifically, we used the UAS/GAL4 system to express these tools at different levels and in different cells, and analyzed these tools for effects on fertility, alterations in the actin cytoskeleton, and ability to label filamentous actin (F-actin) structures by both fixed and live imaging. While both Utrophin and Lifeact robustly label F-actin structures within the Drosophila germline, when strongly expressed they cause sterility and severe actin defects including cortical actin breakdown resulting in multi-nucleate nurse cells, early F-actin filament and aggregate formation during stage 9 (S9), and disorganized parallel actin filament bundles during stage 10B (S10B). However, by using a weaker germline GAL4 driver in combination with a higher temperature, Utrophin can label F-actin with minimal defects. Additionally, strong Utrophin expression within the germline causes F-actin formation in the nurse cell nuclei and germinal vesicle during mid-oogenesis. Similarly, Lifeact expression results in nuclear F-actin only within the germinal vesicle. F-tractin expresses at a lower level than the other two labeling tools, but labels cytoplasmic F-actin structures well without causing sterility or striking actin defects. Together these studies reveal how critical it is to evaluate the utility of each actin labeling tool

  18. Effects of F/G-actin ratio and actin turn-over rate on NADPH oxidase activity in microglia

    DEFF Research Database (Denmark)

    Rasmussen, Izabela; Pedersen, Line Hjortshøj; Byg, Luise;

    2010-01-01

    Most in vivo studies that have addressed the role of actin dynamics in NADPH oxidase function in phagocytes have used toxins to modulate the polymerization state of actin and mostly effects on actin has been evaluated by end point measurements of filamentous actin, which says little about actin d...

  19. Actin filaments are involved in the maintenance of Golgi cisternae morphology and intra-Golgi pH.

    Science.gov (United States)

    Lázaro-Diéguez, Francisco; Jiménez, Nuria; Barth, Holger; Koster, Abraham J; Renau-Piqueras, Jaime; Llopis, Juan L; Burger, Koert N J; Egea, Gustavo

    2006-12-01

    Here we examine the contribution of actin dynamics to the architecture and pH of the Golgi complex. To this end, we have used toxins that depolymerize (cytochalasin D, latrunculin B, mycalolide B, and Clostridium botulinum C2 toxin) or stabilize (jasplakinolide) filamentous actin. When various clonal cell lines were examined by epifluorescence microscopy, all of these actin toxins induced compaction of the Golgi complex. However, ultrastructural analysis by transmission electron microscopy and electron tomography/three-dimensional modelling of the Golgi complex showed that F-actin depolymerization first induces perforation/fragmentation and severe swelling of Golgi cisternae, which leads to a completely disorganized structure. In contrast, F-actin stabilization results only in cisternae perforation/fragmentation. Concomitantly to actin depolymerization-induced cisternae swelling and disorganization, the intra-Golgi pH significantly increased. Similar ultrastructural and Golgi pH alkalinization were observed in cells treated with the vacuolar H+ -ATPases inhibitors bafilomycin A1 and concanamycin A. Overall, these results suggest that actin filaments are implicated in the preservation of the flattened shape of Golgi cisternae. This maintenance seems to be mediated by the regulation of the state of F-actin assembly on the Golgi pH homeostasis.

  20. Actin remodeling confers BRAF inhibitor resistance to melanoma cells through YAP/TAZ activation.

    Science.gov (United States)

    Kim, Min Hwan; Kim, Jongshin; Hong, Hyowon; Lee, Si-Hyung; Lee, June-Koo; Jung, Eunji; Kim, Joon

    2016-03-01

    The activation of transcriptional coactivators YAP and its paralog TAZ has been shown to promote resistance to anti-cancer therapies. YAP/TAZ activity is tightly coupled to actin cytoskeleton architecture. However, the influence of actin remodeling on cancer drug resistance remains largely unexplored. Here, we report a pivotal role of actin remodeling in YAP/TAZ-dependent BRAF inhibitor resistance in BRAF V600E mutant melanoma cells. Melanoma cells resistant to the BRAF inhibitor PLX4032 exhibit an increase in actin stress fiber formation, which appears to promote the nuclear accumulation of YAP/TAZ. Knockdown of YAP/TAZ reduces the viability of resistant melanoma cells, whereas overexpression of constitutively active YAP induces resistance. Moreover, inhibition of actin polymerization and actomyosin tension in melanoma cells suppresses both YAP/TAZ activation and PLX4032 resistance. Our siRNA library screening identifies actin dynamics regulator TESK1 as a novel vulnerable point of the YAP/TAZ-dependent resistance pathway. These results suggest that inhibition of actin remodeling is a potential strategy to suppress resistance in BRAF inhibitor therapies.

  1. A prophage-encoded actin-like protein required for efficient viral DNA replication in bacteria.

    Science.gov (United States)

    Donovan, Catriona; Heyer, Antonia; Pfeifer, Eugen; Polen, Tino; Wittmann, Anja; Krämer, Reinhard; Frunzke, Julia; Bramkamp, Marc

    2015-05-26

    In host cells, viral replication is localized at specific subcellular sites. Viruses that infect eukaryotic and prokaryotic cells often use host-derived cytoskeletal structures, such as the actin skeleton, for intracellular positioning. Here, we describe that a prophage, CGP3, integrated into the genome of Corynebacterium glutamicum encodes an actin-like protein, AlpC. Biochemical characterization confirms that AlpC is a bona fide actin-like protein and cell biological analysis shows that AlpC forms filamentous structures upon prophage induction. The co-transcribed adaptor protein, AlpA, binds to a consensus sequence in the upstream promoter region of the alpAC operon and also interacts with AlpC, thus connecting circular phage DNA to the actin-like filaments. Transcriptome analysis revealed that alpA and alpC are among the early induced genes upon excision of the CGP3 prophage. Furthermore, qPCR analysis of mutant strains revealed that both AlpA and AlpC are required for efficient phage replication. Altogether, these data emphasize that AlpAC are crucial for the spatio-temporal organization of efficient viral replication. This is remarkably similar to actin-assisted membrane localization of eukaryotic viruses that use the actin cytoskeleton to concentrate virus particles at the egress sites and provides a link of evolutionary conserved interactions between intracellular virus transport and actin. PMID:25916847

  2. Actin filament attachments for sustained motility in vitro are maintained by filament bundling.

    Directory of Open Access Journals (Sweden)

    Xiaohua Hu

    Full Text Available We reconstructed cellular motility in vitro from individual proteins to investigate how actin filaments are organized at the leading edge. Using total internal reflection fluorescence microscopy of actin filaments, we tested how profilin, Arp2/3, and capping protein (CP function together to propel thin glass nanofibers or beads coated with N-WASP WCA domains. Thin nanofibers produced wide comet tails that showed more structural variation in actin filament organization than did bead substrates. During sustained motility, physiological concentrations of Mg(2+ generated actin filament bundles that processively attached to the nanofiber. Reduction of total Mg(2+ abolished particle motility and actin attachment to the particle surface without affecting actin polymerization, Arp2/3 nucleation, or filament capping. Analysis of similar motility of microspheres showed that loss of filament bundling did not affect actin shell formation or symmetry breaking but eliminated sustained attachments between the comet tail and the particle surface. Addition of Mg(2+, Lys-Lys(2+, or fascin restored both comet tail attachment and sustained particle motility in low Mg(2+ buffers. TIRF microscopic analysis of filaments captured by WCA-coated beads in the absence of Arp2/3, profilin, and CP showed that filament bundling by polycation or fascin addition increased barbed end capture by WCA domains. We propose a model in which CP directs barbed ends toward the leading edge and polycation-induced filament bundling sustains processive barbed end attachment to the leading edge.

  3. AQP2 is necessary for vasopressin- and forskolin-mediated filamentous actin depolymerization in renal epithelial cells

    Directory of Open Access Journals (Sweden)

    Naofumi Yui

    2012-02-01

    Remodeling of the actin cytoskeleton is required for vasopressin (VP-induced aquaporin 2 (AQP2 trafficking. Here, we asked whether VP and forskolin (FK-mediated F-actin depolymerization depends on AQP2 expression. Using various MDCK and LLC-PK1 cell lines with different AQP2 expression levels, we performed F-actin quantification and immunofluorescence staining after VP/FK treatment. In MDCK cells, in which AQP2 is delivered apically, VP/FK mediated F-actin depolymerization was significantly correlated with AQP2 expression levels. A decrease of apical membrane associated F-actin was observed upon VP/FK treatment in AQP2 transfected, but not in untransfected cells. There was no change in basolateral actin staining under these conditions. In LLC-PK1 cells, which deliver AQP2 basolaterally, a significant VP/FK mediated decrease in F-actin was also detected only in AQP2 transfected cells. This depolymerization response to VP/FK was significantly reduced by siRNA knockdown of AQP2. By immunofluorescence, an inverse relationship between plasma membrane AQP2 and membrane-associated F-actin was observed after VP/FK treatment again only in AQP2 transfected cells. This is the first report showing that VP/FK mediated F-actin depolymerization is dependent on AQP2 protein expression in renal epithelial cells, and that this is not dependent on the polarity of AQP2 membrane insertion.

  4. A method for rapidly screening functionality of actin mutants and tagged actins

    Directory of Open Access Journals (Sweden)

    Rommelaere Heidi

    2004-01-01

    Full Text Available Recombinant production and biochemical analysis of actin mutants has been hampered by the fact that actin has an absolute requirement for the eukaryotic chaperone CCT to reach its native state. We therefore have developed a method to rapidly screen the folding capacity and functionality of actin variants, by combining in vitro expression of labelled actin with analysis on native gels, band shift assays or copolymerization tests. Additionally, we monitor, using immuno-fluorescence, incorporation of actin variants in cytoskeletal structures in transfected cells. We illustrate the method by two examples. In one we show that tagged versions of actin do not always behave native-like and in the other we study some of the molecular defects of three &bgr;-actin mutants that have been associated with diseases.

  5. Xenopus egg cytoplasm with intact actin.

    Science.gov (United States)

    Field, Christine M; Nguyen, Phuong A; Ishihara, Keisuke; Groen, Aaron C; Mitchison, Timothy J

    2014-01-01

    We report optimized methods for preparing Xenopus egg extracts without cytochalasin D, that we term "actin-intact egg extract." These are undiluted egg cytoplasm that contains abundant organelles, and glycogen which supplies energy, and represents the least perturbed cell-free cytoplasm preparation we know of. We used this system to probe cell cycle regulation of actin and myosin-II dynamics (Field et al., 2011), and to reconstitute the large, interphase asters that organize early Xenopus embryos (Mitchison et al., 2012; Wühr, Tan, Parker, Detrich, & Mitchison, 2010). Actin-intact Xenopus egg extracts are useful for analysis of actin dynamics, and interaction of actin with other cytoplasmic systems, in a cell-free system that closely mimics egg physiology, and more generally for probing the biochemistry and biophysics of the egg, zygote, and early embryo. Detailed protocols are provided along with assays used to check cell cycle state and tips for handling and storing undiluted egg extracts.

  6. Load fluctuations drive actin network growth

    CERN Document Server

    Shaevitz, Joshua W

    2007-01-01

    The growth of actin filament networks is a fundamental biological process that drives a variety of cellular and intracellular motions. During motility, eukaryotic cells and intracellular pathogens are propelled by actin networks organized by nucleation-promoting factors, which trigger the formation of nascent filaments off the side of existing filaments in the network. A Brownian ratchet (BR) mechanism has been proposed to couple actin polymerization to cellular movements, whereby thermal motions are rectified by the addition of actin monomers at the end of growing filaments. Here, by following actin--propelled microspheres using three--dimensional laser tracking, we find that beads adhered to the growing network move via an object--fluctuating BR. Velocity varies with the amplitude of thermal fluctuation and inversely with viscosity as predicted for a BR. In addition, motion is saltatory with a broad distribution of step sizes that is correlated in time. These data point to a model in which thermal fluctuati...

  7. Dynamic Actin Controls Polarity Induction de novo in Protoplasts

    Institute of Scientific and Technical Information of China (English)

    Beatrix Zaban; Jan Maisch; Peter Nick

    2013-01-01

    Cell polarity and axes are central for plant morphogenesis.To study how polarity and axes are induced de novo,we investigated protoplasts of tobacco Nicotiana tabacum cv.BY-2 expressing fluorescentlytagged cytoskeletal markers.We standardized the system to such a degree that we were able to generate quantitative data on the temporal patterns of regeneration stages.The synthesis of a new cell wall marks the transition to the first stage of regeneration,and proceeds after a long preparatory phase within a few minutes.During this preparatory phase,the nucleus migrates actively,and cytoplasmic strands remodel vigorously.We probed this system for the effect of anti-cytoskeletal compounds,inducible bundling of actin,RGD-peptides,and temperature.Suppression of actin dynamics at an early stage leads to aberrant tripolar cells,whereas suppression of microtubule dynamics produces aberrant sausagelike cells with asymmetric cell walls.We integrated these data into a model,where the microtubular cytoskeleton conveys positional information between the nucleus and the membrane controlling the release or activation of components required for cell wall synthesis.Cell wall formation is followed by the induction of a new cell pole requiring dynamic actin filaments,and the new cell axis is manifested as elongation growth perpendicular to the orientation of the aligned cortical microtubules.

  8. Dynamic actin controls polarity induction de novo in protoplasts.

    Science.gov (United States)

    Zaban, Beatrix; Maisch, Jan; Nick, Peter

    2013-02-01

    Cell polarity and axes are central for plant morphogenesis. To study how polarity and axes are induced de novo, we investigated protoplasts of tobacco Nicotiana tabacum cv. BY-2 expressing fluorescently-tagged cytoskeletal markers. We standardized the system to such a degree that we were able to generate quantitative data on the temporal patterns of regeneration stages. The synthesis of a new cell wall marks the transition to the first stage of regeneration, and proceeds after a long preparatory phase within a few minutes. During this preparatory phase, the nucleus migrates actively, and cytoplasmic strands remodel vigorously. We probed this system for the effect of anti-cytoskeletal compounds, inducible bundling of actin, RGD-peptides, and temperature. Suppression of actin dynamics at an early stage leads to aberrant tripolar cells, whereas suppression of microtubule dynamics produces aberrant sausage-like cells with asymmetric cell walls. We integrated these data into a model, where the microtubular cytoskeleton conveys positional information between the nucleus and the membrane controlling the release or activation of components required for cell wall synthesis. Cell wall formation is followed by the induction of a new cell pole requiring dynamic actin filaments, and the new cell axis is manifested as elongation growth perpendicular to the orientation of the aligned cortical microtubules.

  9. Crystal structure of an archaeal actin homolog.

    Science.gov (United States)

    Roeben, Annette; Kofler, Christine; Nagy, István; Nickell, Stephan; Hartl, F Ulrich; Bracher, Andreas

    2006-04-21

    Prokaryotic homologs of the eukaryotic structural protein actin, such as MreB and ParM, have been implicated in determination of bacterial cell shape, and in the segregation of genomic and plasmid DNA. In contrast to these bacterial actin homologs, little is known about the archaeal counterparts. As a first step, we expressed a predicted actin homolog of the thermophilic archaeon Thermoplasma acidophilum, Ta0583, and determined its crystal structure at 2.1A resolution. Ta0583 is expressed as a soluble protein in T.acidophilum and is an active ATPase at physiological temperature. In vitro, Ta0583 forms sheets with spacings resembling the crystal lattice, indicating an inherent propensity to form filamentous structures. The fold of Ta0583 contains the core structure of actin and clearly belongs to the actin/Hsp70 superfamily of ATPases. Ta0583 is approximately equidistant from actin and MreB on the structural level, and combines features from both eubacterial actin homologs, MreB and ParM. The structure of Ta0583 co-crystallized with ADP indicates that the nucleotide binds at the interface between the subdomains of Ta0583 in a manner similar to that of actin. However, the conformation of the nucleotide observed in complex with Ta0583 clearly differs from that in complex with actin, but closely resembles the conformation of ParM-bound nucleotide. On the basis of sequence and structural homology, we suggest that Ta0583 derives from a ParM-like actin homolog that was once encoded by a plasmid and was transferred into a common ancestor of Thermoplasma and Ferroplasma. Intriguingly, both genera are characterized by the lack of a cell wall, and therefore Ta0583 could have a function in cellular organization.

  10. Actin as deathly switch? How auxin can suppress cell-death related defence.

    Directory of Open Access Journals (Sweden)

    Xiaoli Chang

    Full Text Available Plant innate immunity is composed of two layers--a basal immunity, and a specific effector-triggered immunity, which is often accompanied by hypersensitive cell death. Initiation of cell death depends on a complex network of signalling pathways. The phytohormone auxin as central regulator of plant growth and development represents an important component for the modulation of plant defence. In our previous work, we showed that cell death is heralded by detachment of actin from the membrane. Both, actin response and cell death, are triggered by the bacterial elicitor harpin in grapevine cells. In this study we investigated, whether harpin-triggered actin bundling is necessary for harpin-triggered cell death. Since actin organisation is dependent upon auxin, we used different auxins to suppress actin bundling. Extracellular alkalinisation and transcription of defence genes as the basal immunity were examined as well as cell death. Furthermore, organisation of actin was observed in response to pharmacological manipulation of reactive oxygen species and phospholipase D. We find that induction of defence genes is independent of auxin. However, auxin can suppress harpin-induced cell death and also counteract actin bundling. We integrate our findings into a model, where harpin interferes with an auxin dependent pathway that sustains dynamic cortical actin through the activity of phospholipase D. The antagonism between growth and defence is explained by mutual competition for signal molecules such as superoxide and phosphatidic acid. Perturbations of the auxin-actin pathway might be used to detect disturbed integrity of the plasma membrane and channel defence signalling towards programmed cell death.

  11. Simvastatin enhances Rho/actin/cell rigidity pathway contributing to mesenchymal stem cells' osteogenic differentiation.

    Science.gov (United States)

    Tai, I-Chun; Wang, Yao-Hsien; Chen, Chung-Hwan; Chuang, Shu-Chun; Chang, Je-Ken; Ho, Mei-Ling

    2015-01-01

    Recent studies have indicated that statins induce osteogenic differentiation both in vitro and in vivo. The molecular mechanism of statin-stimulated osteogenesis is unknown. Activation of RhoA signaling increases cytoskeletal tension, which plays a crucial role in the osteogenic differentiation of mesenchymal stem cells. We thus hypothesized that RhoA signaling is involved in simvastatin-induced osteogenesis in bone marrow mesenchymal stem cells. We found that although treatment with simvastatin shifts localization of RhoA protein from the membrane to the cytosol, the treatment still activates RhoA dose-dependently because it reduces the association with RhoGDIα. Simvastatin also increased the expression of osteogenic proteins, density of actin filament, the number of focal adhesions, and cellular tension. Furthermore, disrupting actin cytoskeleton or decreasing cell rigidity by using chemical agents reduced simvastatin-induced osteogenic differentiation. In vivo study also confirms that density of actin filament is increased in simvastatin-induced ectopic bone formation. Our study is the first to demonstrate that maintaining intact actin cytoskeletons and enhancing cell rigidity are crucial in simvastatin-induced osteogenesis. The results suggested that simvastatin, which is an osteoinductive factor and acts by increasing actin filament organization and cell rigidity combined with osteoconductive biomaterials, may benefit stem-cell-based bone regeneration. PMID:26451103

  12. Cytosolic pressure provides a propulsive force comparable to actin polymerization during lamellipod protrusion

    Science.gov (United States)

    Manoussaki, Daphne; Shin, William D.; Waterman, Clare M.; Chadwick, Richard S.

    2015-07-01

    Does cytosolic pressure facilitate f-actin polymerization to push the leading edge of a cell forward during self-propelled motion? AFM force-distance (f-d) curves obtained from lamellipodia of live cells often exhibit a signal from which the tension, bending modulus, elastic modulus and thickness in the membrane-cortex complex can be estimated close to the contact point. These measurements permit an estimate of the cytosolic pressure via the canonical Laplace force balance. The deeper portion of the f-d curve allows estimation of the bulk modulus of the cytoskeleton after removal of the bottom effect artifact. These estimates of tension, pressure, cortex thickness and elastic moduli imply that cytosolic pressure both pushes the membrane forward and compresses the actin cortex rearward to facilitate f-actin polymerization. We also estimate that cytosolic pressure fluctuations, most likely induced by myosin, provide a propulsive force comparable to that provided by f-actin polymerization in a lamellipod.

  13. Redundant mechanisms recruit actin into the contractile ring in silkworm spermatocytes.

    Directory of Open Access Journals (Sweden)

    Wei Chen

    2008-09-01

    Full Text Available Cytokinesis is powered by the contraction of actomyosin filaments within the newly assembled contractile ring. Microtubules are a spindle component that is essential for the induction of cytokinesis. This induction could use central spindle and/or astral microtubules to stimulate cortical contraction around the spindle equator (equatorial stimulation. Alternatively, or in addition, induction could rely on astral microtubules to relax the polar cortex (polar relaxation. To investigate the relationship between microtubules, cortical stiffness, and contractile ring assembly, we used different configurations of microtubules to manipulate the distribution of actin in living silkworm spermatocytes. Mechanically repositioned, noninterdigitating microtubules can induce redistribution of actin at any region of the cortex by locally excluding cortical actin filaments. This cortical flow of actin promotes regional relaxation while increasing tension elsewhere (normally at the equatorial cortex. In contrast, repositioned interdigitating microtubule bundles use a novel mechanism to induce local stimulation of contractility anywhere within the cortex; at the antiparallel plus ends of central spindle microtubules, actin aggregates are rapidly assembled de novo and transported laterally to the equatorial cortex. Relaxation depends on microtubule dynamics but not on RhoA activity, whereas stimulation depends on RhoA activity but is largely independent of microtubule dynamics. We conclude that polar relaxation and equatorial stimulation mechanisms redundantly supply actin for contractile ring assembly, thus increasing the fidelity of cleavage.

  14. Erbium laser resurfacing for actinic cheilitis.

    Science.gov (United States)

    Cohen, Joel L

    2013-11-01

    Actinic cheilitis is a precancerous condition characterized by grayish-whitish area(s) of discoloration on the mucosal lip, often blunting the demarcation between mucosa and cutaneous lip. Actinic cheilitis is considered to be an early part of the spectrum of squamous cell carcinoma. Squamous cell carcinoma specifically of the lip has a high rate of recurrence and metastasis through the oral cavity leading to a poor overall survival. Risk factors for the development of actinic cheilitis include chronic solar irradiation, increasing age, male gender, light skin complexion, immunosuppression, and possibly tobacco and alcohol consumption. Treatment options include topical pharmacotherapy (eg, fluorouracil, imiquimod) or procedural interventions (eg, cryotherapy, electrosurgery, surgical vermillionectomy, laser resurfacing), each with their known advantages and disadvantages. There is little consensus as to which treatment options offer the most clinical utility given the paucity of comparative clinical data. In my practice, laser resurfacing has become an important tool for the treatment of actinic cheilitis owing to its ease of use and overall safety, tolerability, and cosmetic acceptability. Herein the use of erbium laser resurfacing is described for three actinic cheilitis presentations for which I find it particularly useful: clinically prominent actinic cheilitis, biopsy-proven actinic cheilitis, and treatment of the entire lip following complete tumor excision of squamous cell carcinoma. All patients were treated with a 2940-nm erbium laser (Sciton Profile Contour Tunable Resurfacing Laser [TRL], Sciton, Inc., Palo Alto, CA). PMID:24196339

  15. Dynamin2 organizes lamellipodial actin networks to orchestrate lamellar actomyosin.

    Directory of Open Access Journals (Sweden)

    Manisha Menon

    Full Text Available Actin networks in migrating cells exist as several interdependent structures: sheet-like networks of branched actin filaments in lamellipodia; arrays of bundled actin filaments co-assembled with myosin II in lamellae; and actin filaments that engage focal adhesions. How these dynamic networks are integrated and coordinated to maintain a coherent actin cytoskeleton in migrating cells is not known. We show that the large GTPase dynamin2 is enriched in the distal lamellipod where it regulates lamellipodial actin networks as they form and flow in U2-OS cells. Within lamellipodia, dynamin2 regulated the spatiotemporal distributions of α-actinin and cortactin, two actin-binding proteins that specify actin network architecture. Dynamin2's action on lamellipodial F-actin influenced the formation and retrograde flow of lamellar actomyosin via direct and indirect interactions with actin filaments and a finely tuned GTP hydrolysis activity. Expression in dynamin2-depleted cells of a mutant dynamin2 protein that restores endocytic activity, but not activities that remodel actin filaments, demonstrated that actin filament remodeling by dynamin2 did not depend of its functions in endocytosis. Thus, dynamin2 acts within lamellipodia to organize actin filaments and regulate assembly and flow of lamellar actomyosin. We hypothesize that through its actions on lamellipodial F-actin, dynamin2 generates F-actin structures that give rise to lamellar actomyosin and for efficient coupling of F-actin at focal adhesions. In this way, dynamin2 orchestrates the global actin cytoskeleton.

  16. Actinic Granuloma with Focal Segmental Glomerulosclerosis

    Directory of Open Access Journals (Sweden)

    Ruedee Phasukthaworn

    2016-02-01

    Full Text Available Actinic granuloma is an uncommon granulomatous disease, characterized by annular erythematous plaque with central clearing predominately located on sun-damaged skin. The pathogenesis is not well understood, ultraviolet radiation is recognized as precipitating factor. We report a case of a 52-year-old woman who presented with asymptomatic annular erythematous plaques on the forehead and both cheeks persisting for 2 years. The clinical presentation and histopathologic findings support the diagnosis of actinic granuloma. During that period of time, she also developed focal segmental glomerulosclerosis. The association between actinic granuloma and focal segmental glomerulosclerosis needs to be clarified by further studies.

  17. Simulated microgravity inhibits osteogenic differentiation of mesenchymal stem cells via depolymerizing F-actin to impede TAZ nuclear translocation

    Science.gov (United States)

    Chen, Zhe; Luo, Qing; Lin, Chuanchuan; Kuang, Dongdong; Song, Guanbin

    2016-01-01

    Microgravity induces observed bone loss in space flight, and reduced osteogenesis of bone mesenchymal stem cells (BMSCs) partly contributes to this phenomenon. Abnormal regulation or functioning of the actin cytoskeleton induced by microgravity may cause the inhibited osteogenesis of BMSCs, but the underlying mechanism remains obscure. In this study, we demonstrated that actin cytoskeletal changes regulate nuclear aggregation of the transcriptional coactivator with PDZ-binding motif (TAZ), which is indispensable for osteogenesis of bone mesenchymal stem cells (BMSCs). Moreover, we utilized a clinostat to model simulated microgravity (SMG) and demonstrated that SMG obviously depolymerized F-actin and hindered TAZ nuclear translocation. Interestingly, stabilizing the actin cytoskeleton induced by Jasplakinolide (Jasp) significantly rescued TAZ nuclear translocation and recovered the osteogenic differentiation of BMSCs in SMG, independently of large tumor suppressor 1(LATS1, an upstream kinase of TAZ). Furthermore, lysophosphatidic acid (LPA) also significantly recovered the osteogenic differentiation of BMSCs in SMG through the F-actin-TAZ pathway. Taken together, we propose that the depolymerized actin cytoskeleton inhibits osteogenic differentiation of BMSCs through impeding nuclear aggregation of TAZ, which provides a novel connection between F-actin cytoskeleton and osteogenesis of BMSCs and has important implications in bone loss caused by microgravity. PMID:27444891

  18. Correlation between polymerizability and conformation in scallop beta-like actin and rabbit skeletal muscle alpha-actin.

    Science.gov (United States)

    Khaitlina, S; Antropova, O; Kuznetsova, I; Turoverov, K; Collins, J H

    1999-08-01

    In order to investigate the structural basis for functional differences among actin isoforms, we have compared the polymerization properties and conformations of scallop adductor muscle beta-like actin and rabbit skeletal muscle alpha-actin. Polymerization of scallop Ca(2+)-actin was slower than that of skeletal muscle Ca(2+)-actin. Cleavage of the actin polypeptide chain between Gly-42 and Val-43 with Escherichia coli protease ECP 32 impaired the polymerization of scallop Mg(2+)-actin to a greater extent than skeletal muscle Mg(2+)-actin. When monomeric scallop and skeletal muscle Ca(2+)-actins were subjected to limited proteolysis with trypsin, subtilisin, or ECP 32, no differences in the conformation of actin subdomain 2 were detected. At the same time, local differences in the conformations of scallop and skeletal muscle actin subdomains 1 were revealed as intrinsic fluorescence differences. Replacement of tightly bound Ca(2+) with Mg(2+) resulted in more extensive proteolysis of segment 61-69 of scallop actin than in the case of skeletal muscle actin. Furthermore, segment 61-69 was more accessible to proteolysis with subtilisin in polymerized scallop Ca(2+)-actin than in polymerized skeletal muscle Ca(2+)-actin, indicating that, in the polymeric form, the nucleotide-containing cleft is in a more open conformation in beta-like scallop actin than in skeletal muscle alpha-actin. We suggest that this difference between scallop and skeletal muscle actins is due to a less efficient shift of scallop actin subdomain 2 to the position it has in the polymer. The possible consequences of amino acid substitutions in actin subdomain 1 in the allosteric regulation of the actin cleft, and hence in the different stabilities of polymers formed by different actins, are discussed. PMID:10415117

  19. Actinic review of EUV masks

    Science.gov (United States)

    Feldmann, Heiko; Ruoff, Johannes; Harnisch, Wolfgang; Kaiser, Winfried

    2010-04-01

    Management of mask defects is a major challenge for the introduction of EUV for HVM production. Once a defect has been detected, its printing impact needs to be predicted. Potentially the defect requires some repair, the success of which needs to be proven. This defect review has to be done with an actinic inspection system that matches the imaging conditions of an EUV scanner. During recent years, several concepts for such an aerial image metrology system (AIMS™) have been proposed. However, until now no commercial solution exists for EUV. Today, advances in EUV optics technology allow envisioning a solution that has been discarded before as unrealistic. We present this concept and its technical cornerstones.While the power requirement for the EUV source is less demanding than for HVM lithography tools, radiance, floor space, and stability are the main criteria for source selection. The requirement to emulate several generations of EUV scanners demands a large flexibility for the ilumination and imaging systems. New critical specifications to the EUV mirrors in the projection microscope can be satisfied using our expertise from lithographic mirrors. In summary, an EUV AIMS™ meeting production requirements seems to be feasible.

  20. The dynamin inhibitor dynasore inhibits bone resorption by rapidly disrupting actin rings of osteoclasts.

    Science.gov (United States)

    Thirukonda, Gnanasagar J; Uehara, Shunsuke; Nakayama, Takahiro; Yamashita, Teruhito; Nakamura, Yukio; Mizoguchi, Toshihide; Takahashi, Naoyuki; Yagami, Kimitoshi; Udagawa, Nobuyuki; Kobayashi, Yasuhiro

    2016-07-01

    The cytoskeletal organization of osteoclasts is required for bone resorption. Binding of dynamin with guanosine triphosphate (GTP) was previously suggested to be required for the organization of the actin cytoskeleton. However, the role of the GTPase activity of dynamin in the organization of the actin cytoskeleton as well as in the bone-resorbing activity of osteoclasts remains unclear. This study investigated the effects of dynasore, an inhibitor of the GTPase activity of dynamin, on the bone-resorbing activity of and actin ring formation in mouse osteoclasts in vitro and in vivo. Dynasore inhibited the formation of resorption pits in osteoclast cultures by suppressing actin ring formation and rapidly disrupting actin rings in osteoclasts. A time-lapse image analysis showed that dynasore shrank actin rings in osteoclasts within 30 min. The intraperitoneal administration of dynasore inhibited receptor activator of nuclear factor κB ligand (RANKL)-induced trabecular bone loss in mouse femurs. These in vitro and in vivo results suggest that the GTPase activity of dynamin is critical for the bone-resorbing activity of osteoclasts and that dynasore is a seed for the development of novel anti-resorbing agents. PMID:26063501

  1. Fascin links Btl/FGFR signalling to the actin cytoskeleton during Drosophila tracheal morphogenesis.

    Science.gov (United States)

    Okenve-Ramos, Pilar; Llimargas, Marta

    2014-02-01

    A key challenge in normal development and in disease is to elucidate the mechanisms of cell migration. Here we approach this question using the tracheal system of Drosophila as a model. Tracheal cell migration requires the Breathless/FGFR pathway; however, how the pathway induces migration remains poorly understood. We find that the Breathless pathway upregulates singed at the tip of tracheal branches, and that this regulation is functionally relevant. singed encodes Drosophila Fascin, which belongs to a conserved family of actin-bundling proteins involved in cancer progression and metastasis upon misregulation. We show that singed is required for filopodia stiffness and proper morphology of tracheal tip cells, defects that correlate with an abnormal actin organisation. We propose that singed-regulated filopodia and cell fronts are required for timely and guided branch migration and for terminal branching and branch fusion. We find that singed requirements rely on its actin-bundling activity controlled by phosphorylation, and that active Singed can promote tip cell features. Furthermore, we find that singed acts in concert with forked, another actin cross-linker. The absence of both cross-linkers further stresses the relevance of tip cell morphology and filopodia for tracheal development. In summary, our results on the one hand reveal a previously undescribed role for forked in the organisation of transient actin structures such as filopodia, and on the other hand identify singed as a new target of Breathless signal, establishing a link between guidance cues, the actin cytoskeleton and tracheal morphogenesis.

  2. Actin: its cumbersome pilgrimage through cellular compartments.

    Science.gov (United States)

    Schleicher, Michael; Jockusch, Brigitte M

    2008-06-01

    In this article, we follow the history of one of the most abundant, most intensely studied proteins of the eukaryotic cells: actin. We report on hallmarks of its discovery, its structural and functional characterization and localization over time, and point to present days' knowledge on its position as a member of a large family. We focus on the rather puzzling number of diverse functions as proposed for actin as a dual compartment protein. Finally, we venture on some speculations as to its origin.

  3. PLEKHG3 enhances polarized cell migration by activating actin filaments at the cell front.

    Science.gov (United States)

    Nguyen, Trang Thi Thu; Park, Wei Sun; Park, Byung Ouk; Kim, Cha Yeon; Oh, Yohan; Kim, Jin Man; Choi, Hana; Kyung, Taeyoon; Kim, Cheol-Hee; Lee, Gabsang; Hahn, Klaus M; Meyer, Tobias; Heo, Won Do

    2016-09-01

    Cells migrate by directing Ras-related C3 botulinum toxin substrate 1 (Rac1) and cell division control protein 42 (Cdc42) activities and by polymerizing actin toward the leading edge of the cell. Previous studies have proposed that this polarization process requires a local positive feedback in the leading edge involving Rac small GTPase and actin polymerization with PI3K likely playing a coordinating role. Here, we show that the pleckstrin homology and RhoGEF domain containing G3 (PLEKHG3) is a PI3K-regulated Rho guanine nucleotide exchange factor (RhoGEF) for Rac1 and Cdc42 that selectively binds to newly polymerized actin at the leading edge of migrating fibroblasts. Optogenetic inactivation of PLEKHG3 showed that PLEKHG3 is indispensable both for inducing and for maintaining cell polarity. By selectively binding to newly polymerized actin, PLEKHG3 promotes local Rac1/Cdc42 activation to induce more local actin polymerization, which in turn promotes the recruitment of more PLEKHG3 to induce and maintain cell front. Thus, autocatalytic reinforcement of PLEKHG3 localization to the leading edge of the cell provides a molecular basis for the proposed positive feedback loop that is required for cell polarization and directed migration.

  4. A Role for Nuclear F-Actin Induction in Human Cytomegalovirus Nuclear Egress

    Science.gov (United States)

    Wilkie, Adrian R.; Lawler, Jessica L.

    2016-01-01

    ABSTRACT Herpesviruses, which include important pathogens, remodel the host cell nucleus to facilitate infection. This remodeling includes the formation of structures called replication compartments (RCs) in which herpesviruses replicate their DNA. During infection with the betaherpesvirus, human cytomegalovirus (HCMV), viral DNA synthesis occurs at the periphery of RCs within the nuclear interior, after which assembled capsids must reach the inner nuclear membrane (INM) for translocation to the cytoplasm (nuclear egress). The processes that facilitate movement of HCMV capsids to the INM during nuclear egress are unknown. Although an actin-based mechanism of alphaherpesvirus capsid trafficking to the INM has been proposed, it is controversial. Here, using a fluorescently-tagged, nucleus-localized actin-binding peptide, we show that HCMV, but not herpes simplex virus 1, strongly induced nuclear actin filaments (F-actin) in human fibroblasts. Based on studies using UV inactivation and inhibitors, this induction depended on viral gene expression. Interestingly, by 24 h postinfection, nuclear F-actin formed thicker structures that appeared by super-resolution microscopy to be bundles of filaments. Later in infection, nuclear F-actin primarily localized along the RC periphery and between the RC periphery and the nuclear rim. Importantly, a drug that depolymerized nuclear F-actin caused defects in production of infectious virus, capsid accumulation in the cytoplasm, and capsid localization near the nuclear rim, without decreasing capsid accumulation in the nucleus. Thus, our results suggest that for at least one herpesvirus, nuclear F-actin promotes capsid movement to the nuclear periphery and nuclear egress. We discuss our results in terms of competing models for these processes. PMID:27555312

  5. Early events of fertilization in sea urchin eggs are sensitive to actin-binding organic molecules.

    Science.gov (United States)

    Chun, Jong T; Limatola, Nunzia; Vasilev, Filip; Santella, Luigia

    2014-08-01

    We previously demonstrated that many aspects of the intracellular Ca(2+) increase in fertilized eggs of starfish are significantly influenced by the state of the actin cytoskeleton. In addition, the actin cytoskeleton appeared to play comprehensive roles in modulating cortical granules exocytosis and sperm entry during the early phase of fertilization. In the present communication, we have extended our work to sea urchin which is believed to have bifurcated from the common ancestor in the phylogenetic tree some 500 million years ago. To corroborate our earlier findings in starfish, we have tested how the early events of fertilization in sea urchin eggs are influenced by four different actin-binding drugs that promote either depolymerization or stabilization of actin filaments. We found that all the actin drugs commonly blocked sperm entry in high doses and significantly reduced the speed of the Ca(2+) wave. At low doses, however, cytochalasin B and phalloidin increased the rate of polyspermy. Overall, certain aspects of Ca(2+) signaling in these eggs were in line with the morphological changes induced by the actin drugs. That is, the time interval between the cortical flash and the first Ca(2+) spot at the sperm interaction site (the latent period) was significantly prolonged in the eggs pretreated with cytochalasin B or latrunculin A, whereas the Ca(2+) decay kinetics after the peak was specifically attenuated in the eggs pretreated with jasplakinolide or phalloidin. In addition, the sperm interacting with the eggs pretreated with actin drugs often generated multiple Ca(2+) waves, but tended to fail to enter the egg. Thus, our results indicated that generation of massive Ca(2+) waves is neither indicative of sperm entry nor sufficient for cortical granules exocytosis in the inseminated sea urchin eggs, whereas the structure and functionality of the actin cytoskeleton are the major determining factors in the two processes.

  6. Sarcomeric Pattern Formation by Actin Cluster Coalescence

    Science.gov (United States)

    Friedrich, Benjamin M.; Fischer-Friedrich, Elisabeth; Gov, Nir S.; Safran, Samuel A.

    2012-01-01

    Contractile function of striated muscle cells depends crucially on the almost crystalline order of actin and myosin filaments in myofibrils, but the physical mechanisms that lead to myofibril assembly remains ill-defined. Passive diffusive sorting of actin filaments into sarcomeric order is kinetically impossible, suggesting a pivotal role of active processes in sarcomeric pattern formation. Using a one-dimensional computational model of an initially unstriated actin bundle, we show that actin filament treadmilling in the presence of processive plus-end crosslinking provides a simple and robust mechanism for the polarity sorting of actin filaments as well as for the correct localization of myosin filaments. We propose that the coalescence of crosslinked actin clusters could be key for sarcomeric pattern formation. In our simulations, sarcomere spacing is set by filament length prompting tight length control already at early stages of pattern formation. The proposed mechanism could be generic and apply both to premyofibrils and nascent myofibrils in developing muscle cells as well as possibly to striated stress-fibers in non-muscle cells. PMID:22685394

  7. Sarcomeric pattern formation by actin cluster coalescence.

    Directory of Open Access Journals (Sweden)

    Benjamin M Friedrich

    Full Text Available Contractile function of striated muscle cells depends crucially on the almost crystalline order of actin and myosin filaments in myofibrils, but the physical mechanisms that lead to myofibril assembly remains ill-defined. Passive diffusive sorting of actin filaments into sarcomeric order is kinetically impossible, suggesting a pivotal role of active processes in sarcomeric pattern formation. Using a one-dimensional computational model of an initially unstriated actin bundle, we show that actin filament treadmilling in the presence of processive plus-end crosslinking provides a simple and robust mechanism for the polarity sorting of actin filaments as well as for the correct localization of myosin filaments. We propose that the coalescence of crosslinked actin clusters could be key for sarcomeric pattern formation. In our simulations, sarcomere spacing is set by filament length prompting tight length control already at early stages of pattern formation. The proposed mechanism could be generic and apply both to premyofibrils and nascent myofibrils in developing muscle cells as well as possibly to striated stress-fibers in non-muscle cells.

  8. Prostaglandins temporally regulate cytoplasmic actin bundle formation during Drosophila oogenesis

    OpenAIRE

    Spracklen, Andrew J.; Kelpsch, Daniel J.; Chen, Xiang; Spracklen, Cassandra N.; Tootle, Tina L.

    2014-01-01

    Prostaglandins (PGs)—lipid signals produced downstream of cyclooxygenase (COX) enzymes—regulate actin dynamics in cell culture and platelets, but their roles during development are largely unknown. Here we define a new role for Pxt, the Drosophila COX-like enzyme, in regulating the actin cytoskeleton—temporal restriction of actin remodeling during oogenesis. PGs are required for actin filament bundle formation during stage 10B (S10B). In addition, loss of Pxt results in extensive early actin ...

  9. Resemblance of actin-binding protein/actin gels to covalently crosslinked networks

    Science.gov (United States)

    Janmey, Paul A.; Hvidt, Søren; Lamb, Jennifer; Stossel, Thomas P.

    1990-05-01

    THE maintainance of the shape of cells is often due to their surface elasticity, which arises mainly from an actin-rich cytoplasmic cortex1,2. On locomotion, phagocytosis or fission, however, these cells become partially fluid-like. The finding of proteins that can bind to actin and control the assembly of, or crosslink, actin filaments, and of intracellular messages that regulate the activities of some of these actin-binding proteins, indicates that such 'gel sol' transformations result from the rearrangement of cortical actin-rich networks3. Alternatively, on the basis of a study of the mechanical properties of mixtures of actin filaments and an Acanthamoeba actin-binding protein, α-actinin, it has been proposed that these transformations can be accounted for by rapid exchange of crosslinks between actin filaments4: the cortical network would be solid when the deformation rate is greater than the rate of crosslink exchange, but would deform or 'creep' when deformation is slow enough to permit crosslinker molecules to rearrange. Here we report, however, that mixtures of actin filaments and actin-binding protein (ABP), an actin crosslinking protein of many higher eukaryotes, form gels Theologically equivalent to covalently crosslinked networks. These gels do not creep in response to applied stress on a time scale compatible with most cell-surface movements. These findings support a more complex and controlled mechanism underlying the dynamic mechanical properties of cortical cytoplasm, and can explain why cells do not collapse under the constant shear forces that often exist in tissues.

  10. Effects of chondroitin sulfate on alteration of actin cytoskeleton in rats with acute necrotizing pancreatitis

    Institute of Scientific and Technical Information of China (English)

    Zhong-Ye He; Ren-Xuan Guo

    2007-01-01

    BACKGROUND: In experimental acute pancreatitis, a large amount of reactive oxygen species are produced, and in turn cytoskeletal changes may be induced in pancreatic tissue. These changes contribute to an imbalance of digestive enzyme segregation, transport, exocytosis and activation, resulting in cell injury. In this study, we assessed the effects of chondroitin sulfate (CS) on attenuation of oxidative damage and protection of F-actin in rats with acute necrotizing pancreatitis (ANP). METHODS:Ninety male Wistar rats were divided randomly into three groups. Group A was infused with 5% sodium taurocholate; group B was treated with CS;and group C served as control. Rats from the three groups were killed at 1, 3 or 8 hours. The levels were measured of malonyl dialdehyde (MDA), total superoxide dismutase (SOD), glutathione synthetase (GSH), serum amylase (SAM) and adenosine triphosphate (ATP). F-actin immunostained with rhodamine-phalloidin was analyzed using a confocal laser scanning system and the content of F-actin protein was determined. RESULTS: The levels of SAM increased in groups A and B, whereas the levels of GSH, SOD and ATP in group A decreased markedly during pancreatitis, and MDA increased signiifcantly. The levels of GSH, SOD and ATP in group B were higher than those in group A, but the level of MDA was lower than in group A. At the same time, ANP resulted in early disruption of the cytoskeleton with dramatic changes and a loss of F-actin. Administration of CS moderated the damage to the actin cytoskeleton. CONCLUSIONS:Retrograde infusion of sodium taurocholate via the pancreatic duct may produce pancreatic necrosis and a marked increase in serum amylase activity, induce a severe depletion of ATP level, prime lipid peroxidation, and damage F-actin. Treatment with CS can ameliorate pancreatic cell conditions, limit cell membrane peroxidation, protect F-actin, and attenuate pancreatitis.

  11. Topical treatment of actinic keratoses with potassium dobesilate 5% cream. a preliminary open-label study

    Directory of Open Access Journals (Sweden)

    Cuevas P

    2011-02-01

    Full Text Available Abstract Background Fibroblast growth factor (FGF is involved in skin tumorigenesis: it promotes cell viability, induces angiogenesis and stimulates invasiveness. Dobesilate is a drug that blocks the activity of FGF. The primary objective was to evaluate the efficacy and tolerability of potassium dobesilate 5% cream in the treatment of actinic keratoses. Methods Potassium dobesilate 5% cream was applied twice daily for 16 weeks to actinic keratosis lesions in 30 patients. The lesions were evaluated clinically at an initial baseline visit, at intermediate visits, and at 16 weeks of treatment. Results The use of potassium dobesilate 5% cream for 16 weeks induced complete regression in 70% of evaluated actinic keratoses, corresponding to grade I, II and III clinical variants, and a partial response (at least 75% reduction of lesions in 20% of the cases. Conclusion Our preliminary trial shows that potassium dobesilate exerts anti-tumorigenic effects and may play a useful role in the chemoprevention of skin cancers.

  12. Separation of actin-dependent and actin-independent lipid rafts

    NARCIS (Netherlands)

    Klappe, Karin; Hummel, Ina; Kok, Jan Willem

    2013-01-01

    Lipid rafts have been isolated on the basis of their resistance to various detergents and more recently by using detergent-free procedures. The actin cytoskeleton is now recognized as a dynamic regulator of lipid raft stability. We carefully analyzed the effects of the cortical actin-disrupting agen

  13. Effects of polymerization and nucleotide identity on the conformational dynamics of the bacterial actin homolog MreB

    OpenAIRE

    Colavin, Alexandre; Hsin, Jen; Huang, Kerwyn Casey

    2014-01-01

    Cytoskeletal filaments drive many dynamic cellular processes, such as the regulation of shape by actin networks in eukaryotes and by the actin homolog MreB in rod-shaped bacteria. Here, we use all-atom molecular dynamics simulations to demonstrate close parallels between the conformational dynamics of actin and MreB, in which polymerization induces flattening of MreB subunits that restructures the ATP binding pocket to promote hydrolysis. We also find that ATP-bound MreB filaments are substan...

  14. Myofibrillogenesis regulator-1 induces hypertrophy by promoting sarcomere organization in neonatal rat cardiomyocytes%肌纤生成调节因子-1促肌节 F-actin 组装引起新生乳大鼠心肌细胞肥大

    Institute of Scientific and Technical Information of China (English)

    王晓礽; 刘秀华; 王松; 栾康

    2015-01-01

    Objective Exploration of myofibrillogenesis reguiator-1 ( MR-1) by regulating the generation mechanism of causing myofibrillar hyperlrophy of the cardigmyocytes .Methods Af-ter intervention of MR-1 expressions in neonatal rat cardiomyocytes , we measured several hyper-trophic index e .g.the mRNA levels of the atrial natriuretic factor ( ANF) and brain natriuretic pep-tide ( BNP ) , the cell size , and the velocity of protein synthesis .The sarcomeric F-actin organization were detacted by staining with phalloidin-fluorescein isothiocyanate ( FITC);sublocation of myome-sin-1 and myosin regulatory light chain ( MLC-2) were assessed by semi-quantitative reverse tran-scription-polymerase chain reaction ( RT-PCR), western blot, and immunocytofluorescent assays . Results MR-1 overexpression induced cardiomyocyte hypertrophy , promoted the sarcomeric F-actin organization , increased the expression of MLC-2 and myomesin-1 ( P<0.05 ) , as well as the myome-sin-1 translocation from nucleus to cytoplasm .Those results of myomesin-1 translocation and F-actin organization were similar to the small ubiquitin modifier-1 ( SUMO-1) overexpression , MR-1 inhibi-tion by RNA interference did not induce the translocation of myomesin-1, even decrease the SUMO-1 overexpression induced myomesin-1 translocation , as well as the promoted F-actin organization . Conclusion MR-1 induces cardiomyocytes hypertrophy by promoting the MLC-2 and myomesin-1 induced myofibrillogenesis .%目的:探索肌纤生成调节因子-1( myofibrillogenesis regulator-1,MR-1)通过调节肌原纤维生成引起心肌肥大的机制。方法干预MR-1在体外培养的新生乳大鼠心肌细胞中的表达,检测心肌细胞表面积、心房利钠因子和脑钠肽水平及蛋白合成速率3种心肌肥大指标的变化,以鬼笔环肽-异硫氰酸荧光素标志并观察肌节F-actin组装,以半定量反转录-聚合酶链反应、免疫印迹和细胞免疫荧光术

  15. Differential effects of LifeAct-GFP and actin-GFP on cell mechanics assessed using micropipette aspiration.

    Science.gov (United States)

    Sliogeryte, Kristina; Thorpe, Stephen D; Wang, Zhao; Thompson, Clare L; Gavara, Nuria; Knight, Martin M

    2016-01-25

    The actin cytoskeleton forms a dynamic structure involved in many fundamental cellular processes including the control of cell morphology, migration and biomechanics. Recently LifeAct-GFP (green fluorescent protein) has been proposed for visualising actin structure and dynamics in live cells as an alternative to actin-GFP which has been shown to affect cell mechanics. Here we compare the two approaches in terms of their effect on cellular mechanical behaviour. Human mesenchymal stem cells (hMSCs) were analysed using micropipette aspiration and the effective cellular equilibrium and instantaneous moduli calculated using the standard linear solid model. We show that LifeAct-GFP provides clearer visualisation of F-actin organisation and dynamics. Furthermore, LifeAct-GFP does not alter effective cellular mechanical properties whereas actin-GFP expression causes an increase in the cell modulus. Interestingly, LifeAct-GFP expression did produce a small (~10%) increase in the percentage of cells exhibiting aspiration-induced membrane bleb formation, whilst actin-GFP expression reduced blebbing. Further studies examined the influence of LifeAct-GFP in other cell types, namely chondrogenically differentiated hMSCs and murine chondrocytes. LifeAct-GFP also had no effect on the moduli of these non-blebbing cells for which mechanical properties are largely dependent on the actin cortex. In conclusion we show that LifeAct-GFP enables clearer visualisation of actin organisation and dynamics without disruption of the biomechanical properties of either the whole cell or the actin cortex. Thus the study provides new evidence supporting the use of LifeAct-GFP rather than actin-GFP for live cell microscopy and the study of cellular mechanobiology. PMID:26792287

  16. Lamellipodin promotes actin assembly by clustering Ena/VASP proteins and tethering them to actin filaments.

    Science.gov (United States)

    Hansen, Scott D; Mullins, R Dyche

    2015-01-01

    Enabled/Vasodilator (Ena/VASP) proteins promote actin filament assembly at multiple locations, including: leading edge membranes, focal adhesions, and the surface of intracellular pathogens. One important Ena/VASP regulator is the mig-10/Lamellipodin/RIAM family of adaptors that promote lamellipod formation in fibroblasts and drive neurite outgrowth and axon guidance in neurons. To better understand how MRL proteins promote actin network formation we studied the interactions between Lamellipodin (Lpd), actin, and VASP, both in vivo and in vitro. We find that Lpd binds directly to actin filaments and that this interaction regulates its subcellular localization and enhances its effect on VASP polymerase activity. We propose that Lpd delivers Ena/VASP proteins to growing barbed ends and increases their polymerase activity by tethering them to filaments. This interaction represents one more pathway by which growing actin filaments produce positive feedback to control localization and activity of proteins that regulate their assembly.

  17. Septins promote F-actin ring formation by crosslinking actin filaments into curved bundles.

    Science.gov (United States)

    Mavrakis, Manos; Azou-Gros, Yannick; Tsai, Feng-Ching; Alvarado, José; Bertin, Aurélie; Iv, Francois; Kress, Alla; Brasselet, Sophie; Koenderink, Gijsje H; Lecuit, Thomas

    2014-04-01

    Animal cell cytokinesis requires a contractile ring of crosslinked actin filaments and myosin motors. How contractile rings form and are stabilized in dividing cells remains unclear. We address this problem by focusing on septins, highly conserved proteins in eukaryotes whose precise contribution to cytokinesis remains elusive. We use the cleavage of the Drosophila melanogaster embryo as a model system, where contractile actin rings drive constriction of invaginating membranes to produce an epithelium in a manner akin to cell division. In vivo functional studies show that septins are required for generating curved and tightly packed actin filament networks. In vitro reconstitution assays show that septins alone bundle actin filaments into rings, accounting for the defects in actin ring formation in septin mutants. The bundling and bending activities are conserved for human septins, and highlight unique functions of septins in the organization of contractile actomyosin rings.

  18. Green fluorescent protein-mtalin causes defects in actin organization and cell expansion in Arabidopsis and inhibits actin depolymerizing factor's actin depolymerizing activity in vitro

    NARCIS (Netherlands)

    Ketelaar, T.; Anthony, R.G.; Hussey, P.J.

    2004-01-01

    Expression of green fluorescent protein (GFP) linked to an actin binding domain is a commonly used method for live cell imaging of the actin cytoskeleton. One of these chimeric proteins is GFP-mTalin (GFP fused to the actin binding domain of mouse talin). Although it has been demonstrated that GFP-m

  19. Structural Basis of Actin Filament Nucleation by Tandem W Domains

    Directory of Open Access Journals (Sweden)

    Xiaorui Chen

    2013-06-01

    Full Text Available Spontaneous nucleation of actin is very inefficient in cells. To overcome this barrier, cells have evolved a set of actin filament nucleators to promote rapid nucleation and polymerization in response to specific stimuli. However, the molecular mechanism of actin nucleation remains poorly understood. This is hindered largely by the fact that actin nucleus, once formed, rapidly polymerizes into filament, thus making it impossible to capture stable multisubunit actin nucleus. Here, we report an effective double-mutant strategy to stabilize actin nucleus by preventing further polymerization. Employing this strategy, we solved the crystal structure of AMPPNP-actin in complex with the first two tandem W domains of Cordon-bleu (Cobl, a potent actin filament nucleator. Further sequence comparison and functional studies suggest that the nucleation mechanism of Cobl is probably shared by the p53 cofactor JMY, but not Spire. Moreover, the double-mutant strategy opens the way for atomic mechanistic study of actin nucleation and polymerization.

  20. Glutamyl phosphate is an activated intermediate in actin crosslinking by actin crosslinking domain (ACD toxin.

    Directory of Open Access Journals (Sweden)

    Elena Kudryashova

    Full Text Available Actin Crosslinking Domain (ACD is produced by several life-threatening Gram-negative pathogenic bacteria as part of larger toxins and delivered into the cytoplasm of eukaryotic host cells via Type I or Type VI secretion systems. Upon delivery, ACD disrupts the actin cytoskeleton by catalyzing intermolecular amide bond formation between E270 and K50 residues of actin, leading to the formation of polymerization-deficient actin oligomers. Ultimately, accumulation of the crosslinked oligomers results in structural and functional failure of the actin cytoskeleton in affected cells. In the present work, we advanced in our understanding of the ACD catalytic mechanism by discovering that the enzyme transfers the gamma-phosphoryl group of ATP to the E270 actin residue, resulting in the formation of an activated acyl phosphate intermediate. This intermediate is further hydrolyzed and the energy of hydrolysis is utilized for the formation of the amide bond between actin subunits. We also determined the pH optimum for the reaction and the kinetic parameters of ACD catalysis for its substrates, ATP and actin. ACD showed sigmoidal, non-Michaelis-Menten kinetics for actin (K(0.5 = 30 µM reflecting involvement of two actin molecules in a single crosslinking event. We established that ACD can also utilize Mg(2+-GTP to support crosslinking, but the kinetic parameters (K(M = 8 µM and 50 µM for ATP and GTP, respectively suggest that ATP is the primary substrate of ACD in vivo. The optimal pH for ACD activity was in the range of 7.0-9.0. The elucidated kinetic mechanism of ACD toxicity adds to understanding of complex network of host-pathogen interactions.

  1. Actin-dependent mechanisms in AMPA receptor trafficking

    Directory of Open Access Journals (Sweden)

    Jonathan G Hanley

    2014-11-01

    Full Text Available The precise regulation of AMPA receptor (AMPAR number and subtype at the synapse is crucial for the regulation of excitatory neurotransmission, synaptic plasticity and the consequent formation of appropriate neural circuits during learning and memory. AMPAR trafficking involves the dynamic processes of exocytosis, endocytosis and endosomal recycling, all of which involve the actin cytoskeleton. The actin cytoskeleton is highly dynamic and highly regulated by an abundance of actin-binding proteins and upstream signalling pathways that modulate actin polymerization and depolymerisation. Actin dynamics generate forces that manipulate membranes in the process of vesicle biogenesis, and also for propelling vesicles through the cytoplasm to reach their destination. In addition, trafficking mechanisms exploit more stable aspects of the actin cytoskeleton by using actin-based motor proteins to traffic vesicular cargo along actin filaments. Numerous studies have shown that actin dynamics are critical for AMPAR localization and function. The identification of actin-binding proteins that physically interact with AMPAR subunits, and research into their mode of action is starting to shed light on the mechanisms involved. Such proteins either regulate actin dynamics to modulate mechanical forces exerted on AMPAR-containing membranes, or associate with actin filaments to target or transport AMPAR-containing vesicles to specific subcellular regions. In addition, actin-regulatory proteins that do not physically interact with AMPARs may influence AMPAR trafficking by regulating the local actin environment in the dendritic spine.

  2. Incorporation of mammalian actin into microfilaments in plant cell nucleus

    Directory of Open Access Journals (Sweden)

    Paves Heiti

    2004-04-01

    Full Text Available Abstract Background Actin is an ancient molecule that shows more than 90% amino acid homology between mammalian and plant actins. The regions of the actin molecule that are involved in F-actin assembly are largely conserved, and it is likely that mammalian actin is able to incorporate into microfilaments in plant cells but there is no experimental evidence until now. Results Visualization of microfilaments in onion bulb scale epidermis cells by different techniques revealed that rhodamine-phalloidin stained F-actin besides cytoplasm also in the nuclei whereas GFP-mouse talin hybrid protein did not enter the nuclei. Microinjection of fluorescently labeled actin was applied to study the presence of nuclear microfilaments in plant cells. Ratio imaging of injected fluorescent rabbit skeletal muscle actin and phalloidin staining of the microinjected cells showed that mammalian actin was able to incorporate into plant F-actin. The incorporation occurred preferentially in the nucleus and in the perinuclear region of plant cells whereas part of plant microfilaments, mostly in the periphery of cytoplasm, did not incorporate mammalian actin. Conclusions Microinjected mammalian actin is able to enter plant cell's nucleus, whereas incorporation of mammalian actin into plant F-actin occurs preferentially in the nucleus and perinuclear area.

  3. The actinome of Dictyostelium discoideum in comparison to actins and actin-related proteins from other organisms.

    Directory of Open Access Journals (Sweden)

    Jayabalan M Joseph

    Full Text Available Actin belongs to the most abundant proteins in eukaryotic cells which harbor usually many conventional actin isoforms as well as actin-related proteins (Arps. To get an overview over the sometimes confusing multitude of actins and Arps, we analyzed the Dictyostelium discoideum actinome in detail and compared it with the genomes from other model organisms. The D. discoideum actinome comprises 41 actins and actin-related proteins. The genome contains 17 actin genes which most likely arose from consecutive gene duplications, are all active, in some cases developmentally regulated and coding for identical proteins (Act8-group. According to published data, the actin fraction in a D. discoideum cell consists of more than 95% of these Act8-type proteins. The other 16 actin isoforms contain a conventional actin motif profile as well but differ in their protein sequences. Seven actin genes are potential pseudogenes. A homology search of the human genome using the most typical D. discoideum actin (Act8 as query sequence finds the major actin isoforms such as cytoplasmic beta-actin as best hit. This suggests that the Act8-group represents a nearly perfect actin throughout evolution. Interestingly, limited data from D. fasciculatum, a more ancient member among the social amoebae, show different relationships between conventional actins. The Act8-type isoform is most conserved throughout evolution. Modeling of the putative structures suggests that the majority of the actin-related proteins is functionally unrelated to canonical actin. The data suggest that the other actin variants are not necessary for the cytoskeleton itself but rather regulators of its dynamical features or subunits in larger protein complexes.

  4. The interaction between actin and FA fragment of diphtheria toxin

    OpenAIRE

    Ünlü, A.; Bektaş, M.; Şener, S.; Nurten, R.

    2012-01-01

    Actin protein has many other cellular functions such as movement, chemotaxis, secretion and cytodiaresis. Besides, it have structural function. Actin is a motor protein that it has an important role in the movement process of toxin in the cell. It is known that F-actin gives carriage support during the endosomal process. Actin is found in globular (G) and filamentous (F) structure in the cell. The helix of actin occurs as a result of polymerisation of monomeric G-actin molecules through seque...

  5. Spontaneous actin dynamics in contractile rings

    Science.gov (United States)

    Kruse, Karsten; Wollrab, Viktoria; Thiagarajan, Raghavan; Wald, Anne; Riveline, Daniel

    Networks of polymerizing actin filaments are known to be capable to self-organize into a variety of structures. For example, spontaneous actin polymerization waves have been observed in living cells in a number of circumstances, notably, in crawling neutrophils and slime molds. During later stages of cell division, they can also spontaneously form a contractile ring that will eventually cleave the cell into two daughter cells. We present a framework for describing networks of polymerizing actin filaments, where assembly is regulated by various proteins. It can also include the effects of molecular motors. We show that the molecular processes driven by these proteins can generate various structures that have been observed in contractile rings of fission yeast and mammalian cells. We discuss a possible functional role of each of these patterns. The work was supported by Agence Nationale de la Recherche, France, (ANR-10-LABX-0030-INRT) and by Deutsche Forschungsgemeinschaft through SFB1027.

  6. Vacuole formation in mast cells responding to osmotic stress and to F-actin disassembly

    DEFF Research Database (Denmark)

    Koffer, Anna; Williams, Mark; Johansen, Torben

    2002-01-01

    Fluorescent probes were used to visualize the morphology of membranes and of F-actin in rat peritoneal mast cells, exposed to hyperosmotic medium and consequently reversed to isotonicity. Hypertonicity induced cell shrinkage followed by a regulatory volume increase, and cell alkalinization...

  7. Isoflurane reversibly destabilizes hippocampal dendritic spines by an actin-dependent mechanism.

    Directory of Open Access Journals (Sweden)

    Jimcy Platholi

    Full Text Available General anesthetics produce a reversible coma-like state through modulation of excitatory and inhibitory synaptic transmission. Recent evidence suggests that anesthetic exposure can also lead to sustained cognitive dysfunction. However, the subcellular effects of anesthetics on the structure of established synapses are not known. We investigated effects of the widely used volatile anesthetic isoflurane on the structural stability of hippocampal dendritic spines, a postsynaptic structure critical to excitatory synaptic transmission in learning and memory. Exposure to clinical concentrations of isoflurane induced rapid and non-uniform shrinkage and loss of dendritic spines in mature cultured rat hippocampal neurons. Spine shrinkage was associated with a reduction in spine F-actin concentration. Spine loss was prevented by either jasplakinolide or cytochalasin D, drugs that prevent F-actin disassembly. Isoflurane-induced spine shrinkage and loss were reversible upon isoflurane elimination. Thus, isoflurane destabilizes spine F-actin, resulting in changes to dendritic spine morphology and number. These findings support an actin-based mechanism for isoflurane-induced alterations of synaptic structure in the hippocampus. These reversible alterations in dendritic spine structure have important implications for acute anesthetic effects on excitatory synaptic transmission and synaptic stability in the hippocampus, a locus for anesthetic-induced amnesia, and have important implications for anesthetic effects on synaptic plasticity.

  8. Antiepileptic teratogen valproic acid (VPA) modulates organisation and dynamics of the actin cytoskeleton

    DEFF Research Database (Denmark)

    Walmod, P S; Skladchikova, G; Kawa, A;

    1999-01-01

    of control cells and cells treated with VPA, indicating that VPA affected the cytoskeletal determinants of cell morphology. Furthermore, VPA treatment induced an increase of F-actin, and of FAK, paxillin, vinculin, and phosphotyrosine in focal adhesion complexes. These changes were accompanied by increased...

  9. Two-Photon Correlation Spectroscopy in Single Dendritic Spines Reveals Fast Actin Filament Reorganization during Activity-Dependent Growth.

    Directory of Open Access Journals (Sweden)

    Jian-Hua Chen

    Full Text Available Two-photon fluorescence correlation spectroscopy (2P-FCS within single dendritic spines of living hippocampal pyramidal neurons was used to resolve various subpopulations of mobile F-actin during activity-dependent structural changes such as potentiation induced spine head growth. Two major classes of mobile F-actin were discovered: very dynamic and about a hundred times less dynamic F-actin. Spine head enlargement upon application of Tetraethylammonium (TEA, a protocol previously used for the chemical induction of long-term potentiation (cLTP strictly correlated to changes in the dynamics and filament numbers in the different actin filament fractions. Our observations suggest that spine enlargement is governed by a mechanism in which longer filaments are first cut into smaller filaments that cooperate with the second, increasingly dynamic shorter actin filament population to quickly reorganize and expand the actin cytoskeleton within the spine head. This process would allow a fast and efficient spine head enlargement using a major fraction of the actin filament population that was already present before spine head growth.

  10. The actin family protein ARP6 contributes to the structure and the function of the nucleolus

    Energy Technology Data Exchange (ETDEWEB)

    Kitamura, Hiroshi [Laboratory of Molecular Biology, Graduate School of Agricultural Science, Tohoku University, Tsutsumidori-Amamiyamachi 1-1, Aoka-ku, Sendai 981-8555 (Japan); Matsumori, Haruka [Department of Medical Cell Biology, Institute of Molecular Embryology and Genetics, Kumamoto University, 2-2-1 Honjo, Chuo-ku, Kumamoto 860-0811 (Japan); Kalendova, Alzbeta; Hozak, Pavel [Department of Biology of the Cell Nucleus, Institute of Molecular Genetics of the Academy of Sciences of the Czech Republic, v.v.i., Vídeňská 1083, 142 20 Prague (Czech Republic); Goldberg, Ilya G. [Image Informatics and Computational Biology Unit, Laboratory of Genetics, National Institute on Aging, National Institutes of Health, 251 Bayview Boulevard, Suite 100, Baltimore, MD 21224 (United States); Nakao, Mitsuyoshi [Department of Medical Cell Biology, Institute of Molecular Embryology and Genetics, Kumamoto University, 2-2-1 Honjo, Chuo-ku, Kumamoto 860-0811 (Japan); Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency, Tokyo 102-0076 (Japan); Saitoh, Noriko [Department of Medical Cell Biology, Institute of Molecular Embryology and Genetics, Kumamoto University, 2-2-1 Honjo, Chuo-ku, Kumamoto 860-0811 (Japan); Harata, Masahiko, E-mail: mharata@biochem.tohoku.ac.jp [Laboratory of Molecular Biology, Graduate School of Agricultural Science, Tohoku University, Tsutsumidori-Amamiyamachi 1-1, Aoka-ku, Sendai 981-8555 (Japan)

    2015-08-21

    The actin family members, consisting of actin and actin-related proteins (ARPs), are essential components of chromatin remodeling complexes. ARP6, one of the nuclear ARPs, is part of the Snf-2-related CREB-binding protein activator protein (SRCAP) chromatin remodeling complex, which promotes the deposition of the histone variant H2A.Z into the chromatin. In this study, we showed that ARP6 influences the structure and the function of the nucleolus. ARP6 is localized in the central region of the nucleolus, and its knockdown induced a morphological change in the nucleolus. We also found that in the presence of high concentrations of glucose ARP6 contributed to the maintenance of active ribosomal DNA (rDNA) transcription by placing H2A.Z into the chromatin. In contrast, under starvation, ARP6 was required for cell survival through the repression of rDNA transcription independently of H2A.Z. These findings reveal novel pleiotropic roles for the actin family in nuclear organization and metabolic homeostasis. - Highlights: • ARP6, an actin related protein, is important for nucleolar function and structure. • A population of ARP6 is localized in the center of nucleolus. • Depletion of ARP6 resulted in aberrant shape of the nucleolus. • ARP6 maintains the active rDNA transcription under high glucose. • ARP6 is required for the repression of rDNA transcription under starvation.

  11. Regulation of bone mass and osteoclast function depend on the F-actin modulator SWAP-70.

    Science.gov (United States)

    Garbe, Annette I; Roscher, Anne; Schüler, Christiane; Lutter, Anne-Helen; Glösmann, Martin; Bernhardt, Ricardo; Chopin, Michael; Hempel, Ute; Hofbauer, Lorenz C; Rammelt, Stefan; Egerbacher, Monika; Erben, Reinhold G; Jessberger, Rolf

    2012-10-01

    Bone remodeling involves tightly regulated bone-resorbing osteoclasts and bone-forming osteoblasts. Determining osteoclast function is central to understanding bone diseases such as osteoporosis and osteopetrosis. Here, we report a novel function of the F-actin binding and regulatory protein SWAP-70 in osteoclast biology. F-actin ring formation, cell morphology, and bone resorption are impaired in Swap-70(-/-) osteoclasts, whereas the expression of osteoclast differentiation markers induced in vitro by macrophage colony-stimulating factor (M-CSF) and receptor activator of NF-κB ligand (RANKL) remains unaffected. Swap-70(-/-) mice develop osteopetrosis with increased bone mass, abnormally dense bone, and impaired osteoclast function. Ectopic expression of SWAP-70 in Swap-70(-/-) osteoclasts in vitro rescues their deficiencies in bone resorption and F-actin ring formation. Rescue requires a functional pleckstrin homology (PH) domain, known to support membrane localization of SWAP-70, and the F-actin binding domain. Transplantation of SWAP-70-proficient bone marrow into Swap-70(-/-) mice restores osteoclast resorption capacity in vivo. The identification of the role of SWAP-70 in promoting osteoclast function through modulating membrane-proximal F-actin rearrangements reveals a new pathway to control osteoclasts and bone homeostasis.

  12. The actin family protein ARP6 contributes to the structure and the function of the nucleolus

    International Nuclear Information System (INIS)

    The actin family members, consisting of actin and actin-related proteins (ARPs), are essential components of chromatin remodeling complexes. ARP6, one of the nuclear ARPs, is part of the Snf-2-related CREB-binding protein activator protein (SRCAP) chromatin remodeling complex, which promotes the deposition of the histone variant H2A.Z into the chromatin. In this study, we showed that ARP6 influences the structure and the function of the nucleolus. ARP6 is localized in the central region of the nucleolus, and its knockdown induced a morphological change in the nucleolus. We also found that in the presence of high concentrations of glucose ARP6 contributed to the maintenance of active ribosomal DNA (rDNA) transcription by placing H2A.Z into the chromatin. In contrast, under starvation, ARP6 was required for cell survival through the repression of rDNA transcription independently of H2A.Z. These findings reveal novel pleiotropic roles for the actin family in nuclear organization and metabolic homeostasis. - Highlights: • ARP6, an actin related protein, is important for nucleolar function and structure. • A population of ARP6 is localized in the center of nucleolus. • Depletion of ARP6 resulted in aberrant shape of the nucleolus. • ARP6 maintains the active rDNA transcription under high glucose. • ARP6 is required for the repression of rDNA transcription under starvation

  13. A new mechanism for nuclear import by actin-based propulsion used by a baculovirus nucleocapsid.

    Science.gov (United States)

    Au, Shelly; Wu, Wei; Zhou, Lixin; Theilmann, David A; Panté, Nelly

    2016-08-01

    The transport of macromolecules into the nucleus is mediated by soluble cellular receptors of the importin β superfamily and requires the Ran-GTPase cycle. Several studies have provided evidence that there are exceptions to this canonical nuclear import pathway. Here, we report a new unconventional nuclear import mechanism exploited by the baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV). We found that AcMNPV nucleocapsids entered the nucleus of digitonin-permeabilized cells in the absence of exogenous cytosol or under conditions that blocked the Ran-GTPase cycle. AcMNPV contains a protein that activates the Arp2/3 complex and induces actin polymerization at one end of the rod-shaped nucleocapsid. We show that inhibitors of Arp2/3 blocked nuclear import of nucleocapsids in semi-permeabilized cells. Nuclear import of nucleocapsids was also reconstituted in purified nuclei supplemented with G-actin and Arp2/3 under actin polymerization conditions. Thus, we propose that actin polymerization drives not only migration of baculovirus through the cytoplasm but also pushes the nucleocapsid through the nuclear pore complex to enter the cell nucleus. Our findings point to a very distinct role of actin-based motility during the baculovirus infection cycle. PMID:27284005

  14. Rearrangement of actin cytoskeleton mediates invasion of Lotus japonicus roots by Mesorhizobium loti.

    Science.gov (United States)

    Yokota, Keisuke; Fukai, Eigo; Madsen, Lene H; Jurkiewicz, Anna; Rueda, Paloma; Radutoiu, Simona; Held, Mark; Hossain, Md Shakhawat; Szczyglowski, Krzysztof; Morieri, Giulia; Oldroyd, Giles E D; Downie, J Allan; Nielsen, Mette W; Rusek, Anna Maria; Sato, Shusei; Tabata, Satoshi; James, Euan K; Oyaizu, Hiroshi; Sandal, Niels; Stougaard, Jens

    2009-01-01

    Infection thread-dependent invasion of legume roots by rhizobia leads to internalization of bacteria into the plant cells, which is one of the salient features of root nodule symbiosis. We found that two genes, Nap1 (for Nck-associated protein 1) and Pir1 (for 121F-specific p53 inducible RNA), involved in actin rearrangements were essential for infection thread formation and colonization of Lotus japonicus roots by its natural microsymbiont, Mesorhizobium loti. nap1 and pir1 mutants developed an excess of uncolonized nodule primordia, indicating that these two genes were not essential for the initiation of nodule organogenesis per se. However, both the formation and subsequent progression of infection threads into the root cortex were significantly impaired in these mutants. We demonstrate that these infection defects were due to disturbed actin cytoskeleton organization. Short root hairs of the mutants had mostly transverse or web-like actin filaments, while bundles of actin filaments in wild-type root hairs were predominantly longitudinal. Corroborating these observations, temporal and spatial differences in actin filament organization between wild-type and mutant root hairs were also observed after Nod factor treatment, while calcium influx and spiking appeared unperturbed. Together with various effects on plant growth and seed formation, the nap1 and pir1 alleles also conferred a characteristic distorted trichome phenotype, suggesting a more general role for Nap1 and Pir1 in processes establishing cell polarity or polar growth in L. japonicus.

  15. Reversibility and Viscoelastic Properties of Micropillar Supported and Oriented Magnesium Bundled F-Actin.

    Directory of Open Access Journals (Sweden)

    Timo Maier

    Full Text Available Filamentous actin is one of the most important cytoskeletal elements. Not only is it responsible for the elastic properties of many cell types, but it also plays a vital role in cellular adhesion and motility. Understanding the bundling kinetics of actin filaments is important in the formation of various cytoskeletal structures, such as filopodia and stress fibers. Utilizing a unique pillar-structured microfluidic device, we investigated the time dependence of bundling kinetics of pillar supported free-standing actin filaments. Microparticles attached to the filaments allowed the measurement of thermal motion, and we found that bundling takes place at lower concentrations than previously found in 3-dimensional actin gels, i.e. actin filaments formed bundles in the presence of 5-12 mM of magnesium chloride in a time-dependent manner. The filaments also displayed long term stability for up to hours after removing the magnesium ions from the buffer, which suggests that there is an extensive hysteresis between cation induced crosslinking and decrosslinking.

  16. Actin filaments target the oligomeric maturation of the dynamin GTPase Drp1 to mitochondrial fission sites.

    Science.gov (United States)

    Ji, Wei-ke; Hatch, Anna L; Merrill, Ronald A; Strack, Stefan; Higgs, Henry N

    2015-01-01

    While the dynamin GTPase Drp1 plays a critical role during mitochondrial fission, mechanisms controlling its recruitment to fission sites are unclear. A current assumption is that cytosolic Drp1 is recruited directly to fission sites immediately prior to fission. Using live-cell microscopy, we find evidence for a different model, progressive maturation of Drp1 oligomers on mitochondria through incorporation of smaller mitochondrially-bound Drp1 units. Maturation of a stable Drp1 oligomer does not forcibly lead to fission. Drp1 oligomers also translocate directionally along mitochondria. Ionomycin, a calcium ionophore, causes rapid mitochondrial accumulation of actin filaments followed by Drp1 accumulation at the fission site, and increases fission rate. Inhibiting actin polymerization, myosin IIA, or the formin INF2 reduces both un-stimulated and ionomycin-induced Drp1 accumulation and mitochondrial fission. Actin filaments bind purified Drp1 and increase GTPase activity in a manner that is synergistic with the mitochondrial protein Mff, suggesting a role for direct Drp1/actin interaction. We propose that Drp1 is in dynamic equilibrium on mitochondria in a fission-independent manner, and that fission factors such as actin filaments target productive oligomerization to fission sites. PMID:26609810

  17. Actin dynamics and the elasticity of cytoskeletal networks

    Directory of Open Access Journals (Sweden)

    2009-09-01

    Full Text Available The structural integrity of a cell depends on its cytoskeleton, which includes an actin network. This network is transient and depends upon the continual polymerization and depolymerization of actin. The degradation of an actin network, and a corresponding reduction in cell stiffness, can indicate the presence of disease. Numerical simulations will be invaluable for understanding the physics of these systems and the correlation between actin dynamics and elasticity. Here we develop a model that is capable of generating actin network structures. In particular, we develop a model of actin dynamics which considers the polymerization, depolymerization, nucleation, severing, and capping of actin filaments. The structures obtained are then fed directly into a mechanical model. This allows us to qualitatively assess the effects of changing various parameters associated with actin dynamics on the elasticity of the material.

  18. Microrheology and micromechanics of actin-coated membranes

    Science.gov (United States)

    Bourdieu, Laurent

    2002-03-01

    To study the interaction between cytoskeletal filaments and the plasma membrane, we designed composite membranes obtained by self-assembly of actin filaments on the outer leaflet of giant unilamellar fluid vesicles. Their rich dynamics is studied by micromanipulation with optical tweezers and by single particle tracking experiments. We first show that microrheology study can be carried out on such an individual microscopic object by measuring the thermally excited position fluctuations of a probed bead bound biochemically to the membrane. We propose a model that relates the power spectrum of these thermal fluctuations to the viscoelastic properties of the membrane. The presence of the actin filaments network first induces a finite 2D shear modulus of the order of 1 microN/m. Moreover, these membranes exhibit a clear viscoelastic behavior at high frequency: above a few tens of Hz, both the shear and the bending moduli exhibit the same frequency dependence, a power law of exponent 0.75. These results are consistent in the framework of our model with previous measurements on actin solutions. We show moreover that these complexes exhibit typical mechanical features of a solid shell. For example, a buckling instability is observed when a localized force of the order of 0.5 picoNewton is applied perpendicular to the membrane plane. Although predicted for polymerized vesicles, this is the first evidence of such an instability. This instability is a striking example of the coupling between in-plane stretch and shear and out-of-plane bending, which takes place for curves shells when it becomes more favorable energetically to concentrate the in-plane stress due to the bending within a narrow ring, centered on the force application point.

  19. Actin cytoskeleton regulation of epithelial mesenchymal transition in metastatic cancer cells.

    Directory of Open Access Journals (Sweden)

    Jay Shankar

    Full Text Available Epithelial-mesenchymal transition (EMT is associated with loss of the cell-cell adhesion molecule E-cadherin and disruption of cell-cell junctions as well as with acquisition of migratory properties including reorganization of the actin cytoskeleton and activation of the RhoA GTPase. Here we show that depolymerization of the actin cytoskeleton of various metastatic cancer cell lines with Cytochalasin D (Cyt D reduces cell size and F-actin levels and induces E-cadherin expression at both the protein and mRNA level. Induction of E-cadherin was dose dependent and paralleled loss of the mesenchymal markers N-cadherin and vimentin. E-cadherin levels increased 2 hours after addition of Cyt D in cells showing an E-cadherin mRNA response but only after 10-12 hours in HT-1080 fibrosarcoma and MDA-MB-231 cells in which E-cadherin mRNA level were only minimally affected by Cyt D. Cyt D treatment induced the nuclear-cytoplasmic translocation of EMT-associated SNAI 1 and SMAD1/2/3 transcription factors. In non-metastatic MCF-7 breast cancer cells, that express E-cadherin and represent a cancer cell model for EMT, actin depolymerization with Cyt D induced elevated E-cadherin while actin stabilization with Jasplakinolide reduced E-cadherin levels. Elevated E-cadherin levels due to Cyt D were associated with reduced activation of Rho A. Expression of dominant-negative Rho A mutant increased and dominant-active Rho A mutant decreased E-cadherin levels and also prevented Cyt D induction of E-cadherin. Reduced Rho A activation downstream of actin remodelling therefore induces E-cadherin and reverses EMT in cancer cells. Cyt D treatment inhibited migration and, at higher concentrations, induced cytotoxicity of both HT-1080 fibrosarcoma cells and normal Hs27 fibroblasts, but only induced mesenchymal-epithelial transition in HT-1080 cancer cells. Our studies suggest that actin remodelling is an upstream regulator of EMT in metastatic cancer cells.

  20. Inhibition of keratinocyte proliferation by phospholipid-conjugates of a TLR7 ligand in a Myc-induced hyperplastic actinic keratosis model in the absence of systemic side effects

    Science.gov (United States)

    CRAIN, Brian; YAO, Shiyin; KEOPHILAONE, Vina; PROMESSI, Victor; KANG, McNancy; BARBERIS, Alcide; MAJ, Roberto; MURA, Emanuela; PASSINI, Nadia; HOLLDACK, Johanna; OCHOA, Ricardo; COTTAM, Howard B.; CARSON, Dennis A.; HAYASHI, Tomoko

    2014-01-01

    Background The Toll-like receptor 7 (TLR7) activator imiquimod (IMQ) is safe and effective in treating actinic keratosis; however, an intermittent treatment regimen is necessary because of excessive local reactions. Objectives To evaluate in vitro potency, pharmacodynamics/pharmacokinetics, toxicity and efficacy in vivo of the newly developed TLR7 ligand-phospholipid conjugate, TMX-202, in a gel formulation. Material and Methods The effects of TMX-202 were assessed both in vitro on a murine macrophage cell line and in primary bone marrow-derived dendritic cells and in vivo on mice (C57BL/6-wild type, Myd88−/− and Tlr7−/−). Results TMX-202 was more potent than IMQ in vitro using murine and human cells. In contrast, in vivo it showed less systemic pro-inflammatory activity and better safety than IMQ. Moreover, the TMX-202 gel formulation exhibited at least comparable efficacy to Aldara in a mouse model for skin proliferative diseases. Conclusion TMX-202 is safe and efficacious without causing excessive adverse effects, suggesting that it may be an alternative to Aldara for the treatment of proliferative skin conditions. PMID:24225049

  1. Actin protofilament orientation in deformation of the erythrocyte membrane skeleton.

    OpenAIRE

    Picart, C.; Dalhaimer, P.; Discher, D. E.

    2000-01-01

    The red cell's spectrin-actin network is known to sustain local states of shear, dilation, and condensation, and yet the short actin filaments are found to maintain membrane-tangent and near-random azimuthal orientations. When calibrated with polarization results for single actin filaments, imaging of micropipette-deformed red cell ghosts has allowed an assessment of actin orientations and possible reorientations in the network. At the hemispherical cap of the aspirated projection, where the ...

  2. Dendritic Actin Filament Nucleation Causes Traveling Waves and Patches

    CERN Document Server

    Carlsson, Anders E

    2010-01-01

    The polymerization of actin via branching at a cell membrane containing nucleation-promoting factors is simulated using a stochastic-growth methodology. The polymerized-actin distribution displays three types of behavior: a) traveling waves, b) moving patches, and c) random fluctuations. Increasing actin concentration causes a transition from patches to waves. The waves and patches move by a treadmilling mechanism which does not require myosin II. The effects of downregulation of key proteins on actin wave behavior are evaluated.

  3. Dendritic Actin Filament Nucleation Causes Traveling Waves and Patches

    Science.gov (United States)

    Carlsson, Anders E.

    2010-06-01

    The polymerization of actin via branching at a cell membrane containing nucleation-promoting factors is simulated using a stochastic-growth methodology. The polymerized-actin distribution displays three types of behavior: (a) traveling waves, (b) moving patches, and (c) random fluctuations. Increasing actin concentration causes a transition from patches to waves. The waves and patches move by a treadmilling mechanism not involving myosin II. The effects of downregulation of key proteins on actin wave behavior are evaluated.

  4. Actin of Beta vulgaris seedlings under the clinorotation

    Science.gov (United States)

    Kozeko, L. Ye.

    We study the influence of altered gravity on actin expression in roots of Beta vulguris seedlings grown on the horizontal clinostat (2 rpm) from seed germination for three days. It is shown that the total actin quantity was not influenced. Three actin isoforms are revealed; a relative protein quantity of these isoforms was similar both in clinorotated seedlings and in ones grown in norm. This point to stable expression of actin under the altered gravity conditions.

  5. Competition of two distinct actin networks for actin defines a bistable switch for cell polarization

    Science.gov (United States)

    Lomakin, Alexis J.; Lee, Kun-Chun; Han, Sangyoon J.; Bui, D A.; Davidson, Michael; Mogilner, Alex; Danuser, Gaudenz

    2015-01-01

    Symmetry-breaking polarization enables functional plasticity of cells and tissues and is yet not well understood. Here we show that epithelial cells, hard-wired to maintain a static morphology and to preserve tissue organization, can spontaneously switch to a migratory polarized phenotype upon relaxation of the actomyosin cytoskeleton. We find that myosin-II engages actin in the formation of cortical actomyosin bundles and thus makes it unavailable for deployment in the process of dendritic growth normally driving cell motility. At low contractility regimes epithelial cells polarize in a front-back manner due to emergence of actin retrograde flows powered by dendritic polymerization of actin. Coupled to cell movement, the flows transport myosin-II from the front to the back of the cell, where the motor locally “locks” actin in contractile bundles. This polarization mechanism could be employed by embryonic and cancer epithelial cells in microenvironments where high contractility-driven cell motion is inefficient. PMID:26414403

  6. The spatial response of nonlinear strain propagation in response to actively driven microspheres through entangled actin networks

    Science.gov (United States)

    Falzone, Tobias; Blair, Savanna; Robertson-Anderson, Rae

    2015-03-01

    The semiflexible biopolymer actin, a ubiquitous component of nearly all biological organisms, plays an important role in many mechanically-driven processes such as muscle contraction, cancer invasion and cell motility. As such, entangled actin networks, which possess unique and complex viscoelastic properties, have been the subject of much theoretical and experimental work. However, due to this viscoelastic complexity, much is still unknown regarding the correlation of the applied stress on actin networks to the induced filament strain at the molecular and micro scale. Here, we use simultaneous optical trapping and fluorescence microscopy to characterize the link between applied microscopic forces and strain propagation as a function of strain rate and concentration. Specifically, we track fiduciary markers on entangled actin filaments before, during and after actively driving embedded microspheres through the network. These measurements provide much needed insight into the molecular-level dynamics connecting stress and strain in semiflexible polymer networks.

  7. Actin-organising properties of the muscular dystrophy protein myotilin.

    Science.gov (United States)

    von Nandelstadh, Pernilla; Grönholm, Mikaela; Moza, Monica; Lamberg, Arja; Savilahti, Harri; Carpén, Olli

    2005-10-15

    Myotilin is a sarcomeric Z-disc protein that binds F-actin directly and bundles actin filaments, although it does not contain a conventional actin-binding domain. Expression of mutant myotilin leads to sarcomeric alterations in the dominantly inherited limb-girdle muscular dystrophy 1A and in myofibrillar myopathy/desmin-related myopathy. Together, with previous in vitro studies, this indicates that myotilin has an important function in the assembly and maintenance of Z-discs. This study characterises further the interaction between myotilin and actin. Functionally important regions in myotilin were identified by actin pull-down and yeast two-hybrid assays and with a novel strategy that combines in vitro DNA transposition-based peptide insertion mutagenesis with phenotype analysis in yeast cells. The shortest fragment to bind actin was the second Ig domain together with a short C-terminal sequence. Concerted action of the first and second Ig domain was, however, necessary for the functional activity of myotilin, as verified by analysis of transposon mutants, actin binding and phenotypic effect in mammalian cells. Furthermore, the Ig domains flanked with N- and C-terminal regions were needed for actin-bundling, indicating that the mere actin-binding sequence was insufficient for the actin-regulating activity. None of the four known disease-associated mutations altered the actin-organising ability. These results, together with previous studies in titin and kettin, identify the Ig domain as an actin-binding unit.

  8. Dynamics and Regulation of Actin Cytoskeleton in Plant Cells

    Institute of Scientific and Technical Information of China (English)

    Ren Haiyun

    2007-01-01

    @@ The actin cytoskeleton constituted of globular actin (G-actin) is a ubiquitous component of eukaryotic cells and plays crucial roles in diverse physiological processes in plant cells, such as cytoplasmic streaming, organelle and nucleus positioning, cell morphogenesis, cell division, tip growth, etc.

  9. Prostaglandins temporally regulate cytoplasmic actin bundle formation during Drosophila oogenesis.

    Science.gov (United States)

    Spracklen, Andrew J; Kelpsch, Daniel J; Chen, Xiang; Spracklen, Cassandra N; Tootle, Tina L

    2014-02-01

    Prostaglandins (PGs)--lipid signals produced downstream of cyclooxygenase (COX) enzymes--regulate actin dynamics in cell culture and platelets, but their roles during development are largely unknown. Here we define a new role for Pxt, the Drosophila COX-like enzyme, in regulating the actin cytoskeleton--temporal restriction of actin remodeling during oogenesis. PGs are required for actin filament bundle formation during stage 10B (S10B). In addition, loss of Pxt results in extensive early actin remodeling, including actin filaments and aggregates, within the posterior nurse cells of S9 follicles; wild-type follicles exhibit similar structures at a low frequency. Hu li tai shao (Hts-RC) and Villin (Quail), an actin bundler, localize to all early actin structures, whereas Enabled (Ena), an actin elongation factor, preferentially localizes to those in pxt mutants. Reduced Ena levels strongly suppress early actin remodeling in pxt mutants. Furthermore, loss of Pxt results in reduced Ena localization to the sites of bundle formation during S10B. Together these data lead to a model in which PGs temporally regulate actin remodeling during Drosophila oogenesis by controlling Ena localization/activity, such that in S9, PG signaling inhibits, whereas at S10B, it promotes Ena-dependent actin remodeling.

  10. Filopodia-like actin cables position nuclei in association with perinuclear actin in Drosophila nurse cells

    OpenAIRE

    Huelsmann, Sven; Ylänne, Jari; Brown, Nicholas H

    2013-01-01

    Summary Controlling the position of the nucleus is vital for a number of cellular processes from yeast to humans. In Drosophila nurse cells, nuclear positioning is crucial during dumping, when nurse cells contract and expel their contents into the oocyte. We provide evidence that in nurse cells, continuous filopodia-like actin cables, growing from the plasma membrane and extending to the nucleus, achieve nuclear positioning. These actin cables move nuclei away from ring canals. When nurse cel...

  11. Role of cyclic nucleotide-dependent actin cytoskeletal dynamics:Ca(2+](i and force suppression in forskolin-pretreated porcine coronary arteries.

    Directory of Open Access Journals (Sweden)

    Kyle M Hocking

    Full Text Available Initiation of force generation during vascular smooth muscle contraction involves a rise in intracellular calcium ([Ca(2+]i and phosphorylation of myosin light chains (MLC. However, reversal of these two processes alone does not account for the force inhibition that occurs during relaxation or inhibition of contraction, implicating that other mechanisms, such as actin cytoskeletal rearrangement, play a role in the suppression of force. In this study, we hypothesize that forskolin-induced force suppression is dependent upon changes in actin cytoskeletal dynamics. To focus on the actin cytoskeletal changes, a physiological model was developed in which forskolin treatment of intact porcine coronary arteries (PCA prior to treatment with a contractile agonist resulted in complete suppression of force. Pretreatment of PCA with forskolin suppressed histamine-induced force generation but did not abolish [Ca(2+]i rise or MLC phosphorylation. Additionally, forskolin pretreatment reduced filamentous actin in histamine-treated tissues, and prevented histamine-induced changes in the phosphorylation of the actin-regulatory proteins HSP20, VASP, cofilin, and paxillin. Taken together, these results suggest that forskolin-induced complete force suppression is dependent upon the actin cytoskeletal regulation initiated by the phosphorylation changes of the actin regulatory proteins and not on the MLC dephosphorylation. This model of complete force suppression can be employed to further elucidate the mechanisms responsible for smooth muscle tone, and may offer cues to pathological situations, such as hypertension and vasospasm.

  12. Tailor-made ezrin actin binding domain to probe its interaction with actin in-vitro.

    Directory of Open Access Journals (Sweden)

    Rohini Shrivastava

    Full Text Available Ezrin, a member of the ERM (Ezrin/Radixin/Moesin protein family, is an Actin-plasma membrane linker protein mediating cellular integrity and function. In-vivo study of such interactions is a complex task due to the presence of a large number of endogenous binding partners for both Ezrin and Actin. Further, C-terminal actin binding capacity of the full length Ezrin is naturally shielded by its N-terminal, and only rendered active in the presence of Phosphatidylinositol bisphosphate (PIP2 or phosphorylation at the C-terminal threonine. Here, we demonstrate a strategy for the design, expression and purification of constructs, combining the Ezrin C-terminal actin binding domain, with functional elements such as fusion tags and fluorescence tags to facilitate purification and fluorescence microscopy based studies. For the first time, internal His tag was employed for purification of Ezrin actin binding domain based on in-silico modeling. The functionality (Ezrin-actin interaction of these constructs was successfully demonstrated by using Total Internal Reflection Fluorescence Microscopy. This design can be extended to other members of the ERM family as well.

  13. Possible interrelationship between changes in F-actin and myosin II, protein phosphorylation, and cell volume regulation in Ehrlich ascites tumor cells

    DEFF Research Database (Denmark)

    Pedersen, S F; Hoffmann, E K

    2002-01-01

    Osmotic shrinkage of Ehrlich ascites tumor cells (EATC) elicited translocation of myosin II from the cytosol to the cortical region, and swelling elicits concentration of myosin II in the Golgi region. Rho kinase and p38 both appeared to be involved in shrinkage-induced myosin II reorganization. In...... effects on F-actin. The subsequent F-actin depolymerization, however, appeared MLCK- and PKC-dependent, and the initial swelling-induced F-actin depolymerization was MLCK-dependent; both effects were apparently secondary to kinase-mediated effects on cell volume changes. NHE1 in EATC is activated both by...

  14. Reorganized actin filaments anchor chloroplasts along the anticlinal walls of Vallisneria epidermal cells under high-intensity blue light.

    Science.gov (United States)

    Sakai, Yuuki; Takagi, Shingo

    2005-08-01

    In epidermal cells of the aquatic angiosperm Vallisneria gigantea Graebner, high-intensity blue light (BL) induces the avoidance response of chloroplasts. We examined simultaneous BL-induced changes in the configuration of actin filaments in the cytoplasmic layers that face the outer periclinal wall (P side) and the anticlinal wall (A side). The results clearly showed that dynamic reorganization of the actin cytoskeleton occurs on both sides. Upon BL irradiation, thick, long bundles of actin filaments appeared, concomitant with the directed migration of chloroplasts from the P side to the A side. After 15-20 min of BL irradiation, fine actin bundles on only the A side appeared to associate with chloroplasts that had migrated from the P side. To examine the role of the fine actin bundles, we evaluated the anchorage of chloroplasts by centrifuging living cells. Upon BL irradiation, the resistance of chloroplasts on both the P and A sides to the centrifugal force decreased remarkably. After 20 min of BL irradiation, the resistance of chloroplasts on the A side increased again, but chloroplasts on the P side could still be displaced. The BL-induced recovery of resistance of chloroplasts on the A side was sensitive to photosynthesis inhibitors but insensitive to an inhibitor of flavoproteins. The photosynthesis inhibitors also prevented the fine actin bundles from appearing on the A side under BL irradiation. These results strongly suggest that the BL-induced avoidance response of chloroplasts includes photosynthesis-dependent and actin-dependent anchorage of chloroplasts on the A side of epidermal cells. PMID:15809866

  15. The Structural Basis of Actin Organization by Vinculin and Metavinculin.

    Science.gov (United States)

    Kim, Laura Y; Thompson, Peter M; Lee, Hyunna T; Pershad, Mihir; Campbell, Sharon L; Alushin, Gregory M

    2016-01-16

    Vinculin is an essential adhesion protein that links membrane-bound integrin and cadherin receptors through their intracellular binding partners to filamentous actin, facilitating mechanotransduction. Here we present an 8.5-Å-resolution cryo-electron microscopy reconstruction and pseudo-atomic model of the vinculin tail (Vt) domain bound to F-actin. Upon actin engagement, the N-terminal "strap" and helix 1 are displaced from the Vt helical bundle to mediate actin bundling. We find that an analogous conformational change also occurs in the H1' helix of the tail domain of metavinculin (MVt) upon actin binding, a muscle-specific splice isoform that suppresses actin bundling by Vt. These data support a model in which metavinculin tunes the actin bundling activity of vinculin in a tissue-specific manner, providing a mechanistic framework for understanding metavinculin mutations associated with hereditary cardiomyopathies. PMID:26493222

  16. Chorein Sensitivity of Actin Polymerization, Cell Shape and Mechanical Stiffness of Vascular Endothelial Cells

    Directory of Open Access Journals (Sweden)

    Ioana Alesutan

    2013-09-01

    Full Text Available Background/Aims: Endothelial cell stiffness plays a key role in endothelium-dependent control of vascular tone and arterial blood pressure. Actin polymerization and distribution of microfilaments is essential for mechanical cell stiffness. Chorein, a protein encoded by the VPS13A gene, defective in chorea-acanthocytosis (ChAc, is involved in neuronal cell survival as well as cortical actin polymerization of erythrocytes and blood platelets. Chorein is expressed in a wide variety of further cells, yet nothing is known about the impact of chorein on cells other than neurons, erythrocytes and platelets. The present study explored whether chorein is expressed in human umbilical vein endothelial cells (HUVECs and addressed the putative role of chorein in the regulation of cytoskeletal architecture, stiffness and survival of those cells. Methods: In HUVECs with or without silencing of the VPS13A gene, VPS13A mRNA expression was determined utilizing quantitative RT-PCR, cytoskeletal organization visualized by confocal microscopy, G/F actin ratio and phosphorylation status of focal adhesion kinase quantified by western blotting, cell death determined by flow cytometry, mechanical properties studied by atomic force microscopy (AFM and cell morphology analysed by scanning ion conductance microscopy (SICM. Results: VPS13A mRNA expression was detectable in HUVECs. Silencing of the VPS13A gene attenuated the filamentous actin network, decreased the ratio of soluble G-actin over filamentous F-actin, reduced cell stiffness and changed cell morphology as compared to HUVECs silenced with negative control siRNA. These effects were paralleled by a significant decrease in FAK phosphorylation following VPS13A silencing. Moreover, silencing of the VPS13A gene increased caspase 3 activity and induced necrosis in HUVECs. Conclusions: Chorein is a novel regulator of cytoskeletal architecture, cell shape, mechanical stiffness and survival of vascular endothelial cells.

  17. Arabidopsis Actin-Depolymerizing Factor-4 links pathogen perception, defense activation and transcription to cytoskeletal dynamics.

    Directory of Open Access Journals (Sweden)

    Katie Porter

    Full Text Available The primary role of Actin-Depolymerizing Factors (ADFs is to sever filamentous actin, generating pointed ends, which in turn are incorporated into newly formed filaments, thus supporting stochastic actin dynamics. Arabidopsis ADF4 was recently shown to be required for the activation of resistance in Arabidopsis following infection with the phytopathogenic bacterium Pseudomonas syringae pv. tomato DC3000 (Pst expressing the effector protein AvrPphB. Herein, we demonstrate that the expression of RPS5, the cognate resistance protein of AvrPphB, was dramatically reduced in the adf4 mutant, suggesting a link between actin cytoskeletal dynamics and the transcriptional regulation of R-protein activation. By examining the PTI (PAMP Triggered Immunity response in the adf4 mutant when challenged with Pst expressing AvrPphB, we observed a significant reduction in the expression of the PTI-specific target gene FRK1 (Flg22-Induced Receptor Kinase 1. These data are in agreement with recent observations demonstrating a requirement for RPS5 in PTI-signaling in the presence of AvrPphB. Furthermore, MAPK (Mitogen-Activated Protein Kinase-signaling was significantly reduced in the adf4 mutant, while no such reduction was observed in the rps5-1 point mutation under similar conditions. Isoelectric focusing confirmed phosphorylation of ADF4 at serine-6, and additional in planta analyses of ADF4's role in immune signaling demonstrates that nuclear localization is phosphorylation independent, while localization to the actin cytoskeleton is linked to ADF4 phosphorylation. Taken together, these data suggest a novel role for ADF4 in controlling gene-for-gene resistance activation, as well as MAPK-signaling, via the coordinated regulation of actin cytoskeletal dynamics and R-gene transcription.

  18. The Nebivolol action on vascular tone is dependent on actin cytoskeleton polymerization and Rho-A activity into ECs and SMCs.

    Science.gov (United States)

    Kadi, A; de Isla, N; Moby, V; Lacolley, P; Labrude, P; Stoltz, J F; Menu, P

    2014-01-01

    Nitric oxide is implicated in the target action of Nebivolol, a selective β1 adrenoceptor blocker used in hypertension treatment. As the Nitric Oxide (NO) production and the actin cytoskeleton are linked, the aim of this work was to study the involvement of actin cytoskeleton on mechanism of action of Nebivolol in cultured endothelial cells. We studied the effect of Nebivolol (200 μM) on actin filaments remodeling and its impact on NO production and eNOS activation. Results showed that Nebivolol perturbs actin filaments polymerization, increases NO production and eNOS activity between 30 minutes and 1 h. Stabilization of actin filaments with phalloïdine (50 μM) abolishes Nebivolol effects on eNOS activation and NO production. Furthermore, Rho-kinase activity decreased during the first hour of Nebivolol treatment, then increased after 3 h, while actin filaments repolymerized, eNOS activation and NO production decreased. In SMCs, Nebivolol induced a decrease in the Rho-kinase activity from 1 h until 24 h of incubation. In conclusion, we suggest that Nebivolol induced NO production in Endothelial Cells (ECs) via complementary actions between actin cytoskeleton remodeling inducing eNOS activation and Rho-kinase implication. The effect of Nebivolol on ECs occurs during the first hour, this effect on SMCs seems to be maintained until 24 h, explaining persisted action of Nebivolol observed in vivo.

  19. Bleb formation is induced by alkaline but not acidic pH in estrogen receptor silenced breast cancer cells.

    Science.gov (United States)

    Khajah, Maitham A; Mathew, Princy M; Alam-Eldin, Nada S; Luqmani, Yunus A

    2015-04-01

    De novo and acquired resistance to endocrine-based therapies in breast cancer occurs in parallel with epithelial to mesenchymal transition (EMT), which is associated with enhanced proliferative and metastatic potential, and poor clinical outcome. We have established several endocrine insensitive breast cancer lines by shRNA-induced depletion of estrogen receptor (ER) by transfection of MCF7 cells. All of these exhibit EMT. We have previously reported that brief exposure of specifically ER- breast cancer cells, to extracellular alkaline pH, results in cell rounding and segregation, and leads to enhanced invasive potential. In this study we describe more detailed morphological changes and compare these with cell exposure to acidic pH. Morphological changes and localization of various molecules critical for cell adhesion and motility, associated with pH effects, were assessed by live cell microscopy, electron microscopy, and immunofluorescence. Exposure of either ER- or ER+ breast cancer cells to extracellular acidic pH did not induce significant changes in morphological appearance. Conversely, brief exposure of specifically ER silenced cells, to alkaline pH, resulted in cell contractolation and formation of bleb-like actin-rich structures which were evenly distributed on the outer membrane. Integrin α2, FAK, and JAM-1 were found in the cytoplasm streaming into the newly formed blebs. These blebs appear to be related to cell polarity and movement. Pre-treatment with cytochalasin-D or inhibitors of Rho or MLCK prevented both contractolation and bleb formation. Our data suggest that the effect of pH on the microenvironment of endocrine resistant breast cancer cells needs to be more extensively investigated. Alkaline, rather than acidic pH, appears to induce dramatic morphological changes, and enhances their invasive capabilities, through re-organization of cortical actin. PMID:25672508

  20. Distinct functional interactions between actin isoforms and nonsarcomeric myosins.

    Directory of Open Access Journals (Sweden)

    Mirco Müller

    Full Text Available Despite their near sequence identity, actin isoforms cannot completely replace each other in vivo and show marked differences in their tissue-specific and subcellular localization. Little is known about isoform-specific differences in their interactions with myosin motors and other actin-binding proteins. Mammalian cytoplasmic β- and γ-actin interact with nonsarcomeric conventional myosins such as the members of the nonmuscle myosin-2 family and myosin-7A. These interactions support a wide range of cellular processes including cytokinesis, maintenance of cell polarity, cell adhesion, migration, and mechano-electrical transduction. To elucidate differences in the ability of isoactins to bind and stimulate the enzymatic activity of individual myosin isoforms, we characterized the interactions of human skeletal muscle α-actin, cytoplasmic β-actin, and cytoplasmic γ-actin with human myosin-7A and nonmuscle myosins-2A, -2B and -2C1. In the case of nonmuscle myosins-2A and -2B, the interaction with either cytoplasmic actin isoform results in 4-fold greater stimulation of myosin ATPase activity than was observed in the presence of α-skeletal muscle actin. Nonmuscle myosin-2C1 is most potently activated by β-actin and myosin-7A by γ-actin. Our results indicate that β- and γ-actin isoforms contribute to the modulation of nonmuscle myosin-2 and myosin-7A activity and thereby to the spatial and temporal regulation of cytoskeletal dynamics. FRET-based analyses show efficient copolymerization abilities for the actin isoforms in vitro. Experiments with hybrid actin filaments show that the extent of actomyosin coupling efficiency can be regulated by the isoform composition of actin filaments.

  1. Cdc42 and actin control polarized expression of TI-VAMP vesicles to neuronal growth cones and their fusion with the plasma membrane.

    Science.gov (United States)

    Alberts, Philipp; Rudge, Rachel; Irinopoulou, Theano; Danglot, Lydia; Gauthier-Rouvière, Cécile; Galli, Thierry

    2006-03-01

    Tetanus neurotoxin-insensitive vesicle-associated membrane protein (TI-VAMP)-mediated fusion of intracellular vesicles with the plasma membrane is crucial for neurite outgrowth, a pathway not requiring synaptobrevin-dependent exocytosis. Yet, it is not known how the TI-VAMP membrane trafficking pathway is regulated or how it is coordinated with cytoskeletal dynamics within the growth cone that guide neurite outgrowth. Here, we demonstrate that TI-VAMP, but not synaptobrevin 2, concentrates in the peripheral, F-actin-rich region of the growth cones of hippocampal neurons in primary culture. Its accumulation correlates with and depends upon the presence of F-actin. Moreover, acute stimulation of actin remodeling by homophilic activation of the adhesion molecule L1 induces a site-directed, actin-dependent recruitment of the TI-VAMP compartment. Expression of a dominant-positive mutant of Cdc42, a key regulator of cell polarity, stimulates formation of F-actin- and TI-VAMP-rich filopodia outside the growth cone. Furthermore, we report that Cdc42 activates exocytosis of pHLuorin tagged TI-VAMP in an actin-dependent manner. Collectively, our data suggest that Cdc42 and regulated assembly of the F-actin network control the accumulation and exocytosis of TI-VAMP-containing membrane vesicles in growth cones to coordinate membrane trafficking and actin remodeling during neurite outgrowth.

  2. Cdc42 and Actin Control Polarized Expression of TI-VAMP Vesicles to Neuronal Growth Cones and Their Fusion with the Plasma MembraneV⃞

    Science.gov (United States)

    Alberts, Philipp; Rudge, Rachel; Irinopoulou, Theano; Danglot, Lydia; Gauthier-Rouvière, Cécile; Galli, Thierry

    2006-01-01

    Tetanus neurotoxin-insensitive vesicle-associated membrane protein (TI-VAMP)-mediated fusion of intracellular vesicles with the plasma membrane is crucial for neurite outgrowth, a pathway not requiring synaptobrevin-dependent exocytosis. Yet, it is not known how the TI-VAMP membrane trafficking pathway is regulated or how it is coordinated with cytoskeletal dynamics within the growth cone that guide neurite outgrowth. Here, we demonstrate that TI-VAMP, but not synaptobrevin 2, concentrates in the peripheral, F-actin-rich region of the growth cones of hippocampal neurons in primary culture. Its accumulation correlates with and depends upon the presence of F-actin. Moreover, acute stimulation of actin remodeling by homophilic activation of the adhesion molecule L1 induces a site-directed, actin-dependent recruitment of the TI-VAMP compartment. Expression of a dominant-positive mutant of Cdc42, a key regulator of cell polarity, stimulates formation of F-actin- and TI-VAMP-rich filopodia outside the growth cone. Furthermore, we report that Cdc42 activates exocytosis of pHLuorin tagged TI-VAMP in an actin-dependent manner. Collectively, our data suggest that Cdc42 and regulated assembly of the F-actin network control the accumulation and exocytosis of TI-VAMP-containing membrane vesicles in growth cones to coordinate membrane trafficking and actin remodeling during neurite outgrowth. PMID:16381811

  3. Reduction of exportin 6 activity leads to actin accumulation via failure of RanGTP restoration and NTF2 sequestration in the nuclei of senescent cells

    International Nuclear Information System (INIS)

    We have previously reported that G-actin accumulation in nuclei is a universal phenomenon of cellular senescence. By employing primary culture of human diploid fibroblast (HDF) and stress-induced premature senescence (SIPS), we explored whether the failure of actin export to cytoplasm is responsible for actin accumulation in nuclei of senescent cells. Expression of exportin 6 (Exp6) and small G-protein, Ran, was significantly reduced in the replicative senescence, but not yet in SIPS, whereas nuclear import of actin by cofilin was already increased in SIPS. After treatment of young HDF cells with H2O2, rapid reduction of nuclear RanGTP was observed along with cytoplasmic increase of RanGDP. Furthermore, significantly reduced interaction of Exp6 with RanGTP was found by GST-Exp6 pull-down analysis. Failure of RanGTP restoration was accompanied with inhibition of ATP synthesis and NTF2 sequestration in the nuclei along with accordant change of senescence morphology. Indeed, knockdown of Exp6 expression significantly increased actin molecule in the nuclei of young HDF cells. Therefore, actin accumulation in nuclei of senescent cells is most likely due to the failure of RanGTP restoration with ATP deficiency and NTF2 accumulation in nuclei, which result in the decrease of actin export via Exp6 inactivation, in addition to actin import by cofilin activation.

  4. From filaments to function:The role of the plant actin cytoskeleton in pathogen perception, signaling and immunity

    Institute of Scientific and Technical Information of China (English)

    Katie Porter; Brad Day

    2016-01-01

    The eukaryotic actin cytoskeleton is required for numerous cellular processes, including cell shape, develop-ment and movement, gene expression and signal transduc-tion, and response to biotic and abiotic stress. In recent years, research in both plants and animal systems have described a function for actin as the ideal surveillance platform, linking the function and activity of primary physiological processes to the immune system. In this review, we will highlight recent advances that have defined the regulation and breadth of function of the actin cytoskeleton as a network required for defense signaling following pathogen infection. Coupled with an overview of recent work demonstrating specific targeting of the plant actin cytoskeleton by a diversity of pathogens, including bacteria, fungi and viruses, we will highlight the importance of actin as a key signaling hub in plants, one that mediates surveillance of cellular homeostasis and the activa-tion of specific signaling responses following pathogen perception. B4ased on the studies highlighted herein, we propose a working model that posits changes in actin filament organization is in and of itself a highly specific signal, which induces, regulates and physically directs stimulus-specific signaling processes, most importantly, those associated with response to pathogens.

  5. Virulent Burkholderia species mimic host actin polymerases to drive actin-based motility

    Science.gov (United States)

    Benanti, Erin L.; Nguyen, Catherine M.; Welch, Matthew D.

    2015-01-01

    Summary Burkholderia pseudomallei and B. mallei are bacterial pathogens that cause melioidosis and glanders, while their close relative B. thailandensis is nonpathogenic. All use the trimeric autotransporter BimA to facilitate actin-based motility, host cell fusion and dissemination. Here, we show that BimA orthologs mimic different host actin-polymerizing proteins. B. thailandensis BimA activates the host Arp2/3 complex. In contrast, B. pseudomallei and B. mallei BimA mimic host Ena/VASP actin polymerases in their ability to nucleate, elongate and bundle filaments by associating with barbed ends, as well as in their use of WH2 motifs and oligomerization for activity. Mechanistic differences among BimA orthologs resulted in distinct actin filament organization and motility parameters, which affected the efficiency of cell fusion during infection. Our results identify bacterial Ena/VASP mimics and reveal that pathogens imitate the full spectrum of host actin-polymerizing pathways, suggesting that mimicry of different polymerization mechanisms influences key parameters of infection. PMID:25860613

  6. The actin-binding protein capulet genetically interacts with the microtubule motor kinesin to maintain neuronal dendrite homeostasis.

    Directory of Open Access Journals (Sweden)

    Paul M B Medina

    Full Text Available BACKGROUND: Neurons require precise cytoskeletal regulation within neurites, containing microtubule tracks for cargo transport in axons and dendrites or within synapses containing organized actin. Due to the unique architecture and specialized function of neurons, neurons are particularly susceptible to perturbation of the cytoskeleton. Numerous actin-binding proteins help maintain proper cytoskeletal regulation. METHODOLOGY/PRINCIPAL FINDINGS: From a Drosophila forward genetic screen, we identified a mutation in capulet--encoding a conserved actin-binding protein--that causes abnormal aggregates of actin within dendrites. Through interaction studies, we demonstrate that simultaneous genetic inactivation of capulet and kinesin heavy chain, a microtubule motor protein, produces elongate cofilin-actin rods within dendrites but not axons. These rods resemble actin-rich structures induced in both mammalian neurodegenerative and Drosophila Alzheimer's models, but have not previously been identified by loss of function mutations in vivo. We further demonstrate that mitochondria, which are transported by Kinesin, have impaired distribution along dendrites in a capulet mutant. While Capulet and Cofilin may biochemically cooperate in certain circumstances, in neuronal dendrites they genetically antagonize each other. CONCLUSIONS/SIGNIFICANCE: The present study is the first molecularly defined loss of function demonstration of actin-cofilin rods in vivo. This study suggests that simultaneous, seemingly minor perturbations in neuronal dendrites can synergize producing severe abnormalities affecting actin, microtubules and mitochondria/energy availability in dendrites. Additionally, as >90% of Alzheimer's and Parkinson's cases are sporadic this study suggests mechanisms by which multiple mutations together may contribute to neurodegeneration instead of reliance on single mutations to produce disease.

  7. The neuronal and actin commitment: Why do neurons need rings?

    Science.gov (United States)

    Leite, Sérgio Carvalho; Sousa, Mónica Mendes

    2016-09-01

    The role of the actin cytoskeleton in neurons has been extensively studied in actin-enriched compartments such as the growth cone and dendritic spines. The recent discovery of actin rings in the axon shaft and in dendrites, together with the identification of axon actin trails, has advanced our understanding on actin organization and dynamics in neurons. However, specifically in the case of actin rings, the mechanisms regulating their nucleation and assembly, and the functions that they may exert in axons and dendrites remain largely unexplored. Here we discuss the possible structural, mechanistic and functional properties of the subcortical neuronal cytoskeleton putting the current knowledge in perspective with the information available on actin rings formed in other biological contexts, and with the organization of actin-spectrin lattices in other cell types. The detailed analysis of these novel neuronal actin ring structures, together with the elucidation of the function of actin-binding proteins in neuron biology, has a large potential to uncover new mechanisms of neuronal function under normal conditions that may have impact in our understanding of axon degeneration and regeneration. © 2016 Wiley Periodicals, Inc.

  8. Tropomyosin - master regulator of actin filament function in the cytoskeleton.

    Science.gov (United States)

    Gunning, Peter W; Hardeman, Edna C; Lappalainen, Pekka; Mulvihill, Daniel P

    2015-08-15

    Tropomyosin (Tpm) isoforms are the master regulators of the functions of individual actin filaments in fungi and metazoans. Tpms are coiled-coil parallel dimers that form a head-to-tail polymer along the length of actin filaments. Yeast only has two Tpm isoforms, whereas mammals have over 40. Each cytoskeletal actin filament contains a homopolymer of Tpm homodimers, resulting in a filament of uniform Tpm composition along its length. Evidence for this 'master regulator' role is based on four core sets of observation. First, spatially and functionally distinct actin filaments contain different Tpm isoforms, and recent data suggest that members of the formin family of actin filament nucleators can specify which Tpm isoform is added to the growing actin filament. Second, Tpms regulate whole-organism physiology in terms of morphogenesis, cell proliferation, vesicle trafficking, biomechanics, glucose metabolism and organ size in an isoform-specific manner. Third, Tpms achieve these functional outputs by regulating the interaction of actin filaments with myosin motors and actin-binding proteins in an isoform-specific manner. Last, the assembly of complex structures, such as stress fibers and podosomes involves the collaboration of multiple types of actin filament specified by their Tpm composition. This allows the cell to specify actin filament function in time and space by simply specifying their Tpm isoform composition. PMID:26240174

  9. Gelsolin mediates calcium-dependent disassembly of Listeria actin tails

    Science.gov (United States)

    Larson, Laura; Arnaudeau, Serge; Gibson, Bruce; Li, Wei; Krause, Ryoko; Hao, Binghua; Bamburg, James R.; Lew, Daniel P.; Demaurex, Nicolas; Southwick, Frederick

    2005-01-01

    The role of intracellular Ca2+ in the regulation of actin filament assembly and disassembly has not been clearly defined. We show that reduction of intracellular free Ca2+ concentration ([Ca2+]i) to <40 nM in Listeria monocytogenes-infected, EGFP–actin-transfected Madin–Darby canine kidney cells results in a 3-fold lengthening of actin filament tails. This increase in tail length is the consequence of marked slowing of the actin filament disassembly rate, without a significant change in assembly rate. The Ca2+-sensitive actin-severing protein gelsolin concentrates in the Listeria rocket tails at normal resting [Ca2+]i and disassociates from the tails when [Ca2+]i is lowered. Reduction in [Ca2+]i also blocks the severing activity of gelsolin, but not actin-depolymerizing factor (ADF)/cofilin microinjected into Listeria-infected cells. In Xenopus extracts, Listeria tail lengths are also calcium-sensitive, markedly shortening on addition of calcium. Immunodepletion of gelsolin, but not Xenopus ADF/cofilin, eliminates calcium-sensitive actin-filament shortening. Listeria tail length is also calcium-insensitive in gelsolin-null mouse embryo fibroblasts. We conclude that gelsolin is the primary Ca2+-sensitive actin filament recycling protein in the cell and is capable of enhancing Listeria actin tail disassembly at normal resting [Ca2+]i (145 nM). These experiments illustrate the unique and complementary functions of gelsolin and ADF/cofilin in the recycling of actin filaments. PMID:15671163

  10. Tropomyosin - master regulator of actin filament function in the cytoskeleton.

    Science.gov (United States)

    Gunning, Peter W; Hardeman, Edna C; Lappalainen, Pekka; Mulvihill, Daniel P

    2015-08-15

    Tropomyosin (Tpm) isoforms are the master regulators of the functions of individual actin filaments in fungi and metazoans. Tpms are coiled-coil parallel dimers that form a head-to-tail polymer along the length of actin filaments. Yeast only has two Tpm isoforms, whereas mammals have over 40. Each cytoskeletal actin filament contains a homopolymer of Tpm homodimers, resulting in a filament of uniform Tpm composition along its length. Evidence for this 'master regulator' role is based on four core sets of observation. First, spatially and functionally distinct actin filaments contain different Tpm isoforms, and recent data suggest that members of the formin family of actin filament nucleators can specify which Tpm isoform is added to the growing actin filament. Second, Tpms regulate whole-organism physiology in terms of morphogenesis, cell proliferation, vesicle trafficking, biomechanics, glucose metabolism and organ size in an isoform-specific manner. Third, Tpms achieve these functional outputs by regulating the interaction of actin filaments with myosin motors and actin-binding proteins in an isoform-specific manner. Last, the assembly of complex structures, such as stress fibers and podosomes involves the collaboration of multiple types of actin filament specified by their Tpm composition. This allows the cell to specify actin filament function in time and space by simply specifying their Tpm isoform composition.

  11. Actin is required for IFT regulation in Chlamydomonas reinhardtii.

    Science.gov (United States)

    Avasthi, Prachee; Onishi, Masayuki; Karpiak, Joel; Yamamoto, Ryosuke; Mackinder, Luke; Jonikas, Martin C; Sale, Winfield S; Shoichet, Brian; Pringle, John R; Marshall, Wallace F

    2014-09-01

    Assembly of cilia and flagella requires intraflagellar transport (IFT), a highly regulated kinesin-based transport system that moves cargo from the basal body to the tip of flagella [1]. The recruitment of IFT components to basal bodies is a function of flagellar length, with increased recruitment in rapidly growing short flagella [2]. The molecular pathways regulating IFT are largely a mystery. Because actin network disruption leads to changes in ciliary length and number, actin has been proposed to have a role in ciliary assembly. However, the mechanisms involved are unknown. In Chlamydomonas reinhardtii, conventional actin is found in both the cell body and the inner dynein arm complexes within flagella [3, 4]. Previous work showed that treating Chlamydomonas cells with the actin-depolymerizing compound cytochalasin D resulted in reversible flagellar shortening [5], but how actin is related to flagellar length or assembly remains unknown. Here we utilize small-molecule inhibitors and genetic mutants to analyze the role of actin dynamics in flagellar assembly in Chlamydomonas reinhardtii. We demonstrate that actin plays a role in IFT recruitment to basal bodies during flagellar elongation and that when actin is perturbed, the normal dependence of IFT recruitment on flagellar length is lost. We also find that actin is required for sufficient entry of IFT material into flagella during assembly. These same effects are recapitulated with a myosin inhibitor, suggesting that actin may act via myosin in a pathway by which flagellar assembly is regulated by flagellar length.

  12. Actin turnover is required to prevent axon retraction driven by endogenous actomyosin contractility

    OpenAIRE

    Gallo, Gianluca; Yee, Hal F.; Letourneau, Paul C.

    2002-01-01

    Growth cone motility and guidance depend on the dynamic reorganization of filamentous actin (F-actin). In the growth cone, F-actin undergoes turnover, which is the exchange of actin subunits from existing filaments. However, the function of F-actin turnover is not clear. We used jasplakinolide (jasp), a cell-permeable macrocyclic peptide that inhibits F-actin turnover, to study the role of F-actin turnover in axon extension. Treatment with jasp caused axon retraction, demonstrating that axon ...

  13. TaADF3, an actin-depolymerizing factor, negatively modulates wheat resistance against Puccinia striiformis

    Directory of Open Access Journals (Sweden)

    Chunlei eTang

    2016-01-01

    Full Text Available The actin cytoskeleton has been implicated in plant defense against pathogenic fungi, oomycetes and bacteria. Actin depolymerizing factors (ADF are stimulus responsive actin cytoskeleton modulators. However, there is limited evidence linking ADFs with plant defense against pathogens. In this study, we have isolated and functionally characterized a stress-responsive ADF gene (TaADF3 from wheat, which was detectable in all examined wheat tissues. TaADF3 is a three-copy gene located on chromosomes 5AL, 5BL and 5DL. A particle bombardment assay in onion epidermal cells revealed the cytoplasmic and nuclear localization of TaADF3. The expression of TaADF3 was inducible by abscisic acid (ABA, as well as various abiotic stresses (drought and cold and virulent Puccinia striiformis f. sp. tritici (Pst but was down regulated in response to avirulent Pst. Virus-induced silencing of TaADF3 copies enhanced wheat resistance to avirulent Pst, with decreased reactive oxygen species (ROS accumulation and hypersensitive response (HR. Upon treatment with virulent Pst, TaADF3-knockdown plants exhibited reduced susceptibility, which was accompanied by increased ROS production and HR. Interestingly, the silencing of TaADF3 resulted in hindered pathogen penetration and haustoria formation for both avirulent and virulent Pst. Moreover, the array and distribution of actin filaments was transformed in TaADF3-knockdown epidermal cells, which possibly facilitated attenuating the fungus penetration. Thus, our findings suggest that TaADF3 positively regulates wheat tolerance to abiotic stresses and negatively regulates wheat resistance to Pst in an ROS-dependent manner, possibly underlying the mechanism of impeding fungal penetration dependent on the actin architecture dynamics.

  14. TaADF3, an Actin-Depolymerizing Factor, Negatively Modulates Wheat Resistance Against Puccinia striiformis.

    Science.gov (United States)

    Tang, Chunlei; Deng, Lin; Chang, Dan; Chen, Shuntao; Wang, Xiaojie; Kang, Zhensheng

    2015-01-01

    The actin cytoskeleton has been implicated in plant defense against pathogenic fungi, oomycetes, and bacteria. Actin depolymerizing factors (ADFs) are stimulus responsive actin cytoskeleton modulators. However, there is limited evidence linking ADFs with plant defense against pathogens. In this study, we have isolated and functionally characterized a stress-responsive ADF gene (TaADF3) from wheat, which was detectable in all examined wheat tissues. TaADF3 is a three-copy gene located on chromosomes 5AL, 5BL, and 5DL. A particle bombardment assay in onion epidermal cells revealed the cytoplasmic and nuclear localization of TaADF3. The expression of TaADF3 was inducible by abscisic acid (ABA), as well as various abiotic stresses (drought and cold) and virulent Puccinia striiformis f. sp. tritici (Pst) but was down regulated in response to avirulent Pst. Virus-induced silencing of TaADF3 copies enhanced wheat resistance to avirulent Pst, with decreased reactive oxygen species (ROS) accumulation and hypersensitive response (HR). Upon treatment with virulent Pst, TaADF3-knockdown plants exhibited reduced susceptibility, which was accompanied by increased ROS production and HR. Interestingly, the silencing of TaADF3 resulted in hindered pathogen penetration and haustoria formation for both avirulent and virulent Pst. Moreover, the array and distribution of actin filaments was transformed in TaADF3-knockdown epidermal cells, which possibly facilitated attenuating the fungus penetration. Thus, our findings suggest that TaADF3 positively regulates wheat tolerance to abiotic stresses and negatively regulates wheat resistance to Pst in an ROS-dependent manner, possibly underlying the mechanism of impeding fungal penetration dependent on the actin architecture dynamics. PMID:26834758

  15. Adhesive F-actin waves: a novel integrin-mediated adhesion complex coupled to ventral actin polymerization.

    Directory of Open Access Journals (Sweden)

    Lindsay B Case

    Full Text Available At the leading lamellipodium of migrating cells, protrusion of an Arp2/3-nucleated actin network is coupled to formation of integrin-based adhesions, suggesting that Arp2/3-mediated actin polymerization and integrin-dependent adhesion may be mechanistically linked. Arp2/3 also mediates actin polymerization in structures distinct from the lamellipodium, in "ventral F-actin waves" that propagate as spots and wavefronts along the ventral plasma membrane. Here we show that integrins engage the extracellular matrix downstream of ventral F-actin waves in several mammalian cell lines as well as in primary mouse embryonic fibroblasts. These "adhesive F-actin waves" require a cycle of integrin engagement and disengagement to the extracellular matrix for their formation and propagation, and exhibit morphometry and a hierarchical assembly and disassembly mechanism distinct from other integrin-containing structures. After Arp2/3-mediated actin polymerization, zyxin and VASP are co-recruited to adhesive F-actin waves, followed by paxillin and vinculin, and finally talin and integrin. Adhesive F-actin waves thus represent a previously uncharacterized integrin-based adhesion complex associated with Arp2/3-mediated actin polymerization.

  16. Novel regulation of Ski protein stability and endosomal sorting by actin cytoskeleton dynamics in hepatocytes.

    Science.gov (United States)

    Vázquez-Victorio, Genaro; Caligaris, Cassandre; Del Valle-Espinosa, Eugenio; Sosa-Garrocho, Marcela; González-Arenas, Nelly R; Reyes-Cruz, Guadalupe; Briones-Orta, Marco A; Macías-Silva, Marina

    2015-02-13

    TGF-β-induced antimitotic signals are highly regulated during cell proliferation under normal and pathological conditions, such as liver regeneration and cancer. Up-regulation of the transcriptional cofactors Ski and SnoN during liver regeneration may favor hepatocyte proliferation by inhibiting TGF-β signals. In this study, we found a novel mechanism that regulates Ski protein stability through TGF-β and G protein-coupled receptor (GPCR) signaling. Ski protein is distributed between the nucleus and cytoplasm of normal hepatocytes, and the molecular mechanisms controlling Ski protein stability involve the participation of actin cytoskeleton dynamics. Cytoplasmic Ski is partially associated with actin and localized in cholesterol-rich vesicles. Ski protein stability is decreased by TGF-β/Smads, GPCR/Rho signals, and actin polymerization, whereas GPCR/cAMP signals and actin depolymerization promote Ski protein stability. In conclusion, TGF-β and GPCR signals differentially regulate Ski protein stability and sorting in hepatocytes, and this cross-talk may occur during liver regeneration.

  17. Actin and DNA Protect Histones from Degradation by Bacterial Proteases but Inhibit Their Antimicrobial Activity.

    Science.gov (United States)

    Sol, Asaf; Skvirsky, Yaniv; Blotnick, Edna; Bachrach, Gilad; Muhlrad, Andras

    2016-01-01

    Histones are small polycationic proteins located in the cell nucleus. Together, DNA and histones are integral constituents of the nucleosomes. Upon apoptosis, necrosis, and infection - induced cell death, histones are released from the cell. The extracellular histones have strong antimicrobial activity but are also cytotoxic and thought as mediators of cell death in sepsis. The antimicrobial activity of the cationic extracellular histones is inhibited by the polyanionic DNA and F-actin, which also become extracellular upon cell death. DNA and F-actin protect histones from degradation by the proteases of Pseudomonas aeruginosa and Porphyromonas gingivalis. However, though the integrity of the histones is protected, the activity of histones as antibacterial agents is lost. The inhibition of the histone's antibacterial activity and their protection from proteolysis by DNA and F-actin indicate a tight electrostatic interaction between the positively charged histones and negatively charged DNA and F-actin, which may have physiological significance in maintaining the equilibrium between the beneficial antimicrobial activity of extracellular histones and their cytotoxic effects. PMID:27555840

  18. The actin family protein ARP6 contributes to the structure and the function of the nucleolus.

    Science.gov (United States)

    Kitamura, Hiroshi; Matsumori, Haruka; Kalendova, Alzbeta; Hozak, Pavel; Goldberg, Ilya G; Nakao, Mitsuyoshi; Saitoh, Noriko; Harata, Masahiko

    2015-08-21

    The actin family members, consisting of actin and actin-related proteins (ARPs), are essential components of chromatin remodeling complexes. ARP6, one of the nuclear ARPs, is part of the Snf-2-related CREB-binding protein activator protein (SRCAP) chromatin remodeling complex, which promotes the deposition of the histone variant H2A.Z into the chromatin. In this study, we showed that ARP6 influences the structure and the function of the nucleolus. ARP6 is localized in the central region of the nucleolus, and its knockdown induced a morphological change in the nucleolus. We also found that in the presence of high concentrations of glucose ARP6 contributed to the maintenance of active ribosomal DNA (rDNA) transcription by placing H2A.Z into the chromatin. In contrast, under starvation, ARP6 was required for cell survival through the repression of rDNA transcription independently of H2A.Z. These findings reveal novel pleiotropic roles for the actin family in nuclear organization and metabolic homeostasis.

  19. Interaction of formin FH2 with skeletal muscle actin. EPR and DSC studies.

    Science.gov (United States)

    Kupi, Tünde; Gróf, Pál; Nyitrai, Miklós; Belágyi, József

    2013-10-01

    Formins are highly conserved proteins that are essential in the formation and regulation of the actin cytoskeleton. The formin homology 2 (FH2) domain is responsible for actin binding and acts as an important nucleating factor in eukaryotic cells. In this work EPR and DSC were used to investigate the properties of the mDia1-FH2 formin fragment and its interaction with actin. MDia1-FH2 was labeled with a maleimide spin probe (MSL). EPR results suggested that the MSL was attached to a single SH group in the FH2. In DSC and temperature-dependent EPR experiments we observed that mDia1-FH2 has a flexible structure and observed a major temperature-induced conformational change at 41 °C. The results also confirmed the previous observation obtained by fluorescence methods that formin binding can destabilize the structure of actin filaments. In the EPR experiments the intermolecular connection between the monomers of formin dimers proved to be flexible. Considering the complex molecular mechanisms underlying the cellular roles of formins this internal flexibility of the dimers is probably important for manifestation of their biological functions.

  20. Immune-mediated changes in actinic keratosis following topical treatment with imiquimod 5% cream

    Directory of Open Access Journals (Sweden)

    Raghavan Shalini

    2007-01-01

    Full Text Available Abstract Background The objective of this study was to identify the molecular processes responsible for the anti-lesional activity of imiquimod in subjects with actinic keratosis using global gene expression profiling. Methods A double-blind, placebo-controlled, randomized study was conducted to evaluate gene expression changes in actinic keratosis treated with imiquimod 5% cream. Male subjects (N = 17 with ≥ 5 actinic keratosis on the scalp applied placebo cream or imiquimod 3 times a week on nonconsecutive days for 4 weeks. To elucidate the molecular processes involved in actinic keratosis lesion regression by imiquimod, gene expression analysis using oligonucleotide arrays and real time reverse transcriptase polymerase chain reaction were performed on shave biopsies of lesions taken before and after treatment. Results Imiquimod modulated the expression of a large number of genes important in both the innate and adaptive immune response, including increased expression of interferon-inducible genes with known antiviral, anti-proliferative and immune modulatory activity, as well as various Toll-like receptors. In addition, imiquimod increased the expression of genes associated with activation of macrophages, dendritic cells, cytotoxic T cells, and natural killer cells, as well as activation of apoptotic pathways. Conclusion Data suggest that topical application of imiquimod stimulates cells in the skin to secrete cytokines and chemokines that lead to inflammatory cell influx into the lesions and subsequent apoptotic and immune cell-mediated destruction of lesions.

  1. Identification of sucrose synthase as an actin-binding protein

    Science.gov (United States)

    Winter, H.; Huber, J. L.; Huber, S. C.; Davies, E. (Principal Investigator)

    1998-01-01

    Several lines of evidence indicate that sucrose synthase (SuSy) binds both G- and F-actin: (i) presence of SuSy in the Triton X-100-insoluble fraction of microsomal membranes (i.e. crude cytoskeleton fraction); (ii) co-immunoprecipitation of actin with anti-SuSy monoclonal antibodies; (iii) association of SuSy with in situ phalloidin-stabilized F-actin filaments; and (iv) direct binding to F-actin, polymerized in vitro. Aldolase, well known to interact with F-actin, interfered with binding of SuSy, suggesting that a common or overlapping binding site may be involved. We postulate that some of the soluble SuSy in the cytosol may be associated with the actin cytoskeleton in vivo.

  2. The effect of Cytochalasin D on F-Actin behavior of single-cell electroendocytosis using multi-chamber micro cell chip

    KAUST Repository

    Lin, Ran

    2012-03-01

    Electroendocytosis (EED) is a pulsed-electric-field (PEF) induced endocytosis, facilitating cells uptake molecules through nanometer-sized EED vesicles. We herein investigate the effect of a chemical inhibitor, Cytochalasin D (CD) on the actin-filaments (F-Actin) behavior of single-cell EED. The CD concentration (C CD) can control the depolymerization of F-actin. A multi-chamber micro cell chip was fabricated to study the EED under different conditions. Large-scale single-cell data demonstrated EED highly depends on both electric field and C CD. © 2012 IEEE.

  3. EFFECTS OF ESTETROL ON MIGRATION AND INVASION IN T47-D BREAST CANCER CELLS THROUGH THE ACTIN CYTOSKELETON

    Directory of Open Access Journals (Sweden)

    Maria Silvia eGiretti

    2014-05-01

    Full Text Available Estetrol (E4 is a natural human estrogen present at high concentrations during pregnancy. Due to its high oral bioavailability and long plasma half-life, E4 is particularly suitable for therapeutic applications. E4 acts as a selective estrogen receptor modulator, exerting estrogenic actions on the endometrium or the central nervous system, while antagonizing the actions of estradiol in the breast. We tested the effects of E4 on its own or in the presence of 17β-estradiol (E2 on T47-D estrogen receptor (ER positive breast cancer cell migration and invasion of three-dimensional matrices. E4 administration to T47-D cells weakly stimulated migration and invasion. However, E4 decreased the extent of movement and invasion induced by E2. Breast cancer cell movement requires a remodeling of the actin cytoskeleton. During exposure to E4, a weak, concentration-dependent, redistribution of actin fibers towards the cell membrane was observed. However, when E4 was added to E2, a inhibition of actin remodeling induced by E2 was seen. Estrogens stimulate ER+ breast cancer cell movement through the ezrin-radixin-moesin (ERM family of actin regulatory proteins, inducing actin and cell membrane remodeling. E4 was a weak inducer of moesin phosphorylation on Thr558, which accounts for its functional activation. In co-treatment with E2, E4 blocked the activation of this actin controller in a concentration-related fashion. These effects were obtained through recruitment of ERα. In conclusion, E4 acted as a weak estrogen on breast cancer cell cytoskeleton remodeling and movement. However, when E2 was present, E4 counteracted the stimulatory actions of E2. This contributes to the emerging hypothesis that E4 may be a naturally occurring estrogen receptor modulator in the breast.

  4. Effects of Estetrol on Migration and Invasion in T47-D Breast Cancer Cells through the Actin Cytoskeleton.

    Science.gov (United States)

    Giretti, Maria Silvia; Montt Guevara, Maria Magdalena; Cecchi, Elena; Mannella, Paolo; Palla, Giulia; Spina, Stefania; Bernacchi, Guja; Di Bello, Silvia; Genazzani, Andrea Riccardo; Genazzani, Alessandro D; Simoncini, Tommaso

    2014-01-01

    Estetrol (E4) is a natural human estrogen present at high concentrations during pregnancy. Due to its high oral bioavailability and long plasma half-life, E4 is particularly suitable for therapeutic applications. E4 acts as a selective estrogen receptor (ER) modulator, exerting estrogenic actions on the endometrium or the central nervous system, while antagonizing the actions of estradiol in the breast. We tested the effects of E4 on its own or in the presence of 17β-estradiol (E2) on T47-D ER+ breast cancer cell migration and invasion of three-dimensional matrices. E4 administration to T47-D cells weakly stimulated migration and invasion. However, E4 decreased the extent of movement and invasion induced by E2. Breast cancer cell movement requires a remodeling of the actin cytoskeleton. During exposure to E4, a weak, concentration-dependent, re-distribution of actin fibers toward the cell membrane was observed. However, when E4 was added to E2, an inhibition of actin remodeling induced by E2 was seen. Estrogens stimulate ER+ breast cancer cell movement through the ezrin-radixin-moesin family of actin regulatory proteins, inducing actin and cell membrane remodeling. E4 was a weak inducer of moesin phosphorylation on Thr(558), which accounts for its functional activation. In co-treatment with E2, E4 blocked the activation of this actin controller in a concentration-related fashion. These effects were obtained through recruitment of estrogen receptor-α. In conclusion, E4 acted as a weak estrogen on breast cancer cell cytoskeleton remodeling and movement. However, when E2 was present, E4 counteracted the stimulatory actions of E2. This contributes to the emerging hypothesis that E4 may be a naturally occurring ER modulator in the breast. PMID:24904530

  5. Reverse actin sliding triggers strong myosin binding that moves tropomyosin

    OpenAIRE

    Bekyarova, T. I.; Reedy, M C; Baumann, B. A. J.; Tregear, R T; Ward, A; Krzic, U.; Prince, K.M.; Perz-Edwards, R. J.; Reconditi, M.; Gore, D.; Irving, T C; Reedy, M K

    2008-01-01

    Actin/myosin interactions in vertebrate striated muscles are believed to be regulated by the “steric blocking” mechanism whereby the binding of calcium to the troponin complex allows tropomyosin (TM) to change position on actin, acting as a molecular switch that blocks or allows myosin heads to interact with actin. Movement of TM during activation is initiated by interaction of Ca2+ with troponin, then completed by further displacement by strong binding cross-bridges. We report x-ray evidence...

  6. A Robust Actin Filaments Image Analysis Framework.

    Science.gov (United States)

    Alioscha-Perez, Mitchel; Benadiba, Carine; Goossens, Katty; Kasas, Sandor; Dietler, Giovanni; Willaert, Ronnie; Sahli, Hichem

    2016-08-01

    The cytoskeleton is a highly dynamical protein network that plays a central role in numerous cellular physiological processes, and is traditionally divided into three components according to its chemical composition, i.e. actin, tubulin and intermediate filament cytoskeletons. Understanding the cytoskeleton dynamics is of prime importance to unveil mechanisms involved in cell adaptation to any stress type. Fluorescence imaging of cytoskeleton structures allows analyzing the impact of mechanical stimulation in the cytoskeleton, but it also imposes additional challenges in the image processing stage, such as the presence of imaging-related artifacts and heavy blurring introduced by (high-throughput) automated scans. However, although there exists a considerable number of image-based analytical tools to address the image processing and analysis, most of them are unfit to cope with the aforementioned challenges. Filamentous structures in images can be considered as a piecewise composition of quasi-straight segments (at least in some finer or coarser scale). Based on this observation, we propose a three-steps actin filaments extraction methodology: (i) first the input image is decomposed into a 'cartoon' part corresponding to the filament structures in the image, and a noise/texture part, (ii) on the 'cartoon' image, we apply a multi-scale line detector coupled with a (iii) quasi-straight filaments merging algorithm for fiber extraction. The proposed robust actin filaments image analysis framework allows extracting individual filaments in the presence of noise, artifacts and heavy blurring. Moreover, it provides numerous parameters such as filaments orientation, position and length, useful for further analysis. Cell image decomposition is relatively under-exploited in biological images processing, and our study shows the benefits it provides when addressing such tasks. Experimental validation was conducted using publicly available datasets, and in osteoblasts grown in

  7. A Robust Actin Filaments Image Analysis Framework

    Science.gov (United States)

    Alioscha-Perez, Mitchel; Benadiba, Carine; Goossens, Katty; Kasas, Sandor; Dietler, Giovanni; Willaert, Ronnie; Sahli, Hichem

    2016-01-01

    The cytoskeleton is a highly dynamical protein network that plays a central role in numerous cellular physiological processes, and is traditionally divided into three components according to its chemical composition, i.e. actin, tubulin and intermediate filament cytoskeletons. Understanding the cytoskeleton dynamics is of prime importance to unveil mechanisms involved in cell adaptation to any stress type. Fluorescence imaging of cytoskeleton structures allows analyzing the impact of mechanical stimulation in the cytoskeleton, but it also imposes additional challenges in the image processing stage, such as the presence of imaging-related artifacts and heavy blurring introduced by (high-throughput) automated scans. However, although there exists a considerable number of image-based analytical tools to address the image processing and analysis, most of them are unfit to cope with the aforementioned challenges. Filamentous structures in images can be considered as a piecewise composition of quasi-straight segments (at least in some finer or coarser scale). Based on this observation, we propose a three-steps actin filaments extraction methodology: (i) first the input image is decomposed into a ‘cartoon’ part corresponding to the filament structures in the image, and a noise/texture part, (ii) on the ‘cartoon’ image, we apply a multi-scale line detector coupled with a (iii) quasi-straight filaments merging algorithm for fiber extraction. The proposed robust actin filaments image analysis framework allows extracting individual filaments in the presence of noise, artifacts and heavy blurring. Moreover, it provides numerous parameters such as filaments orientation, position and length, useful for further analysis. Cell image decomposition is relatively under-exploited in biological images processing, and our study shows the benefits it provides when addressing such tasks. Experimental validation was conducted using publicly available datasets, and in osteoblasts

  8. Formins: Bringing new insights to the organization of actin cytoskeleton

    Institute of Scientific and Technical Information of China (English)

    GUO Chunqing; REN Haiyun

    2006-01-01

    The actin cytoskeleton is an important component of eukaryotic cell cytoskeleton and is temporally and spatially controlled by a series of actin binding proteins (ABPs). Among ABPs, formin family proteins have attracted much attention as they can nucleate unbranched actin filament from the profilin bound actin pool in vivo. In recent years, a number of formin family members from different organisms have been reported, and their characteristics are known more clearly, although some questions are still to be clarified. Here, we summarize the structures, functions and nucleation mechanisms of different formin family proteins, intending to compare them and give some new clues to the study of formins.

  9. Immunocytochemical identification of actin in mitochondria of Physarum polycephalum

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    Mitochondria isolated from the plasmodia of Physarum polycephalum Schw. are reacted with rabbit anti-actin antibody, and detected with FITC-conjugated sheep anti-rabbit IgG antibody. The results of indirect immunofluorescence show that actin exists in the mitochondria. Western blot analysis confirms the existence of actin in the protein preparation of the mitochondria. The indirect immunoelectron microscopic observation using the same antibodies verifies further that actin is the constituents of mitochondria, and it is dispersively distributed in the mitochondria of P. polycephalum.

  10. An unconventional form of actin in protozoan hemoflagellate, Leishmania.

    Science.gov (United States)

    Kapoor, Prabodh; Sahasrabuddhe, Amogh A; Kumar, Ashutosh; Mitra, Kalyan; Siddiqi, Mohammad Imran; Gupta, Chhitar M

    2008-08-15

    Leishmania actin was cloned, overexpressed in baculovirus-insect cell system, and purified to homogeneity. The purified protein polymerized optimally in the presence of Mg2+ and ATP, but differed from conventional actins in its following properties: (i) it did not polymerize in the presence of Mg2+ alone, (ii) it polymerized in a restricted range of pH 7.0-8.5, (iii) its critical concentration for polymerization was found to be 3-4-fold lower than of muscle actin, (iv) it predominantly formed bundles rather than single filaments at pH 8.0, (v) it displayed considerably higher ATPase activity during polymerization, (vi) it did not inhibit DNase-I activity, and (vii) it did not bind the F-actin-binding toxin phalloidin or the actin polymerization disrupting agent Latrunculin B. Computational and molecular modeling studies revealed that the observed unconventional behavior of Leishmania actin is related to the diverged amino acid stretches in its sequence, which may lead to changes in the overall charge distribution on its solvent-exposed surface, ATP binding cleft, Mg2+ binding sites, and the hydrophobic loop that is involved in monomer-monomer interactions. Phylogenetically, it is related to ciliate actins, but to the best of our knowledge, no other actin with such unconventional properties has been reported to date. It is therefore suggested that actin in Leishmania may serve as a novel target for design of new antileishmanial drugs. PMID:18539603

  11. Evolutionary conservation of the WASH complex, an actin polymerization machine involved in endosomal fission

    OpenAIRE

    Derivery, Emmanuel; Gautreau, Alexis

    2010-01-01

    WASH is the Arp2/3 activating protein that is localized at the surface of endosomes, where it induces the formation of branched actin networks. This activity of WASH favors, in collaboration with dynamin, the fission of transport intermediates from endosomes, and hence regulates endosomal trafficking of several cargos. We have purified a novel stable multiprotein complex containing WASH, the WASH complex, and we examine here the evolutionary conservation of its seven subunits across diverse e...

  12. Inverse relationship between TCTP/RhoA and p53/ /cyclin A/actin expression in ovarian cancer cells Inverse relationship between TCTP/RhoA and p53/ /cyclin A/actin expression in ovarian cancer cells

    Directory of Open Access Journals (Sweden)

    Malgorzata Kloc

    2012-10-01

    Full Text Available The translationally controlled tumor protein (TCTP plays a role in cell growth, cell cycle and cancer
    progression. TCTP controls negatively the stability of the p53 tumor suppressor protein and interacts with the
    cellular cytoskeleton. The deregulation of the actin and cytokeratin cytoskeleton is responsible for the increased
    migratory activity of tumor cells and is linked with poor patient outcome. Recent studies indicate that cyclin A,
    a key regulator of cell cycle, controls actin organization and negatively regulates cell motility via regulation of RhoA
    expression. We studied the organization of actin and cytokeratin cytoskeleton and the expression of TCTP, p53,
    cyclin A, RhoA and actin in HIO180 non-transformed ovarian epithelial cells, and OVCAR3 and SKOV3 (expressing
    low level of inducible p53 ovarian epithelial cancer cells with different metastatic potential. Immunostaining
    and ultrastructural analyses illustrated a dramatic difference in the organization of the cytokeratin and actin
    filaments in non-transformed versus cancer cell lines. We also determined that there is an inverse relationship between
    the level of TCTP/RhoA and actin/p53/cyclin A expression in ovarian cancer cell lines. This previously unidentified
    negative relationship between TCTP/RhoA and actin/p53/cyclin A may suggest that this interaction is linked
    with the high aggressiveness of ovarian cancers.The translationally controlled tumor protein (TCTP plays a role in cell growth, cell cycle and cancer
    progression. TCTP controls negatively the stability of the p53 tumor suppressor protein and interacts with the
    cellular cytoskeleton. The deregulation of the actin and cytokeratin cytoskeleton is responsible for the increased
    migratory activity of tumor cells and is linked with poor patient outcome. Recent studies indicate that cyclin A,
    a key regulator of cell cycle, controls actin organization

  13. Dynamic buckling of actin within filopodia

    DEFF Research Database (Denmark)

    Leijnse, Natascha; Oddershede, Lene B; Bendix, Pól Martin

    2015-01-01

    Filopodia are active tubular structures protruding from the cell surface which allow the cell to sense and interact with the surrounding environment through repetitive elongation-retraction cycles. The mechanical behavior of filopodia has been studied by measuring the traction forces exerted on...... external substrates.(1) These studies have revealed that internal actin flow can transduce a force across the cell surface through transmembrane linkers like integrins. In addition to the elongation-retraction behavior filopodia also exhibit a buckling and rotational behavior. Filopodial buckling in...

  14. Visualization of Actin Cytoskeletal Dynamics in Fixed and Live Drosophila Egg Chambers.

    Science.gov (United States)

    Groen, Christopher M; Tootle, Tina L

    2015-01-01

    Visualization of actin cytoskeletal dynamics is critical for understanding the spatial and temporal regulation of actin remodeling. Drosophila oogenesis provides an excellent model system for visualizing the actin cytoskeleton. Here, we present methods for imaging the actin cytoskeleton in Drosophila egg chambers in both fixed samples by phalloidin staining and in live egg chambers using transgenic actin labeling tools.

  15. Interactions between the Yeast SM22 Homologue Scp1 and Actin Demonstrate the Importance of Actin Bundling in Endocytosis* S⃞

    OpenAIRE

    Gheorghe, Dana M.; Aghamohammadzadeh, Soheil; Rooij, Iwona I. Smaczynska-de; Allwood, Ellen G.; Winder, Steve J.; Ayscough, Kathryn R.

    2008-01-01

    The yeast SM22 homologue Scp1 has previously been shown to act as an actin-bundling protein in vitro. In cells, Scp1 localizes to the cortical actin patches that form as part of the invagination process during endocytosis, and its function overlaps with that of the well characterized yeast fimbrin homologue Sac6p. In this work we have used live cell imaging to demonstrate the importance of key residues in the Scp1 actin interface. We have defined two actin binding domains within Scp1 that all...

  16. Megakaryocytes regulate expression of Pyk2 isoforms and caspase-mediated cleavage of actin in osteoblasts.

    Science.gov (United States)

    Kacena, Melissa A; Eleniste, Pierre P; Cheng, Ying-Hua; Huang, Su; Shivanna, Mahesh; Meijome, Tomas E; Mayo, Lindsey D; Bruzzaniti, Angela

    2012-05-18

    The proliferation and differentiation of osteoblast (OB) precursors are essential for elaborating the bone-forming activity of mature OBs. However, the mechanisms regulating OB proliferation and function are largely unknown. We reported that OB proliferation is enhanced by megakaryocytes (MKs) via a process that is regulated in part by integrin signaling. The tyrosine kinase Pyk2 has been shown to regulate cell proliferation and survival in a variety of cells. Pyk2 is also activated by integrin signaling and regulates actin remodeling in bone-resorbing osteoclasts. In this study, we examined the role of Pyk2 and actin in the MK-mediated increase in OB proliferation. Calvarial OBs were cultured in the presence of MKs for various times, and Pyk2 signaling cascades in OBs were examined by Western blotting, subcellular fractionation, and microscopy. We found that MKs regulate the temporal expression of Pyk2 and its subcellular localization. We also found that MKs regulate the expression of two alternatively spliced isoforms of Pyk2 in OBs, which may regulate OB differentiation and proliferation. MKs also induced cytoskeletal reorganization in OBs, which was associated with the caspase-mediated cleavage of actin, an increase in focal adhesions, and the formation of apical membrane ruffles. Moreover, BrdU incorporation in MK-stimulated OBs was blocked by the actin-polymerizing agent, jasplakinolide. Collectively, our studies reveal that Pyk2 and actin play an important role in MK-regulated signaling cascades that control OB proliferation and may be important for therapeutic interventions aimed at increasing bone formation in metabolic diseases of the skeleton. PMID:22447931

  17. Megakaryocytes Regulate Expression of Pyk2 Isoforms and Caspase-mediated Cleavage of Actin in Osteoblasts*

    Science.gov (United States)

    Kacena, Melissa A.; Eleniste, Pierre P.; Cheng, Ying-Hua; Huang, Su; Shivanna, Mahesh; Meijome, Tomas E.; Mayo, Lindsey D.; Bruzzaniti, Angela

    2012-01-01

    The proliferation and differentiation of osteoblast (OB) precursors are essential for elaborating the bone-forming activity of mature OBs. However, the mechanisms regulating OB proliferation and function are largely unknown. We reported that OB proliferation is enhanced by megakaryocytes (MKs) via a process that is regulated in part by integrin signaling. The tyrosine kinase Pyk2 has been shown to regulate cell proliferation and survival in a variety of cells. Pyk2 is also activated by integrin signaling and regulates actin remodeling in bone-resorbing osteoclasts. In this study, we examined the role of Pyk2 and actin in the MK-mediated increase in OB proliferation. Calvarial OBs were cultured in the presence of MKs for various times, and Pyk2 signaling cascades in OBs were examined by Western blotting, subcellular fractionation, and microscopy. We found that MKs regulate the temporal expression of Pyk2 and its subcellular localization. We also found that MKs regulate the expression of two alternatively spliced isoforms of Pyk2 in OBs, which may regulate OB differentiation and proliferation. MKs also induced cytoskeletal reorganization in OBs, which was associated with the caspase-mediated cleavage of actin, an increase in focal adhesions, and the formation of apical membrane ruffles. Moreover, BrdU incorporation in MK-stimulated OBs was blocked by the actin-polymerizing agent, jasplakinolide. Collectively, our studies reveal that Pyk2 and actin play an important role in MK-regulated signaling cascades that control OB proliferation and may be important for therapeutic interventions aimed at increasing bone formation in metabolic diseases of the skeleton. PMID:22447931

  18. Hem-1 complexes are essential for Rac activation, actin polymerization, and myosin regulation during neutrophil chemotaxis.

    Directory of Open Access Journals (Sweden)

    Orion D Weiner

    2006-02-01

    Full Text Available Migrating cells need to make different actin assemblies at the cell's leading and trailing edges and to maintain physical separation of signals for these assemblies. This asymmetric control of activities represents one important form of cell polarity. There are significant gaps in our understanding of the components involved in generating and maintaining polarity during chemotaxis. Here we characterize a family of complexes (which we term leading edge complexes, scaffolded by hematopoietic protein 1 (Hem-1, that organize the neutrophil's leading edge. The Wiskott-Aldrich syndrome protein family Verprolin-homologous protein (WAVE2 complex, which mediates activation of actin polymerization by Rac, is only one member of this family. A subset of these leading edge complexes are biochemically separable from the WAVE2 complex and contain a diverse set of potential polarity-regulating proteins. RNA interference-mediated knockdown of Hem-1-containing complexes in neutrophil-like cells: (a dramatically impairs attractant-induced actin polymerization, polarity, and chemotaxis; (b substantially weakens Rac activation and phosphatidylinositol-(3,4,5-tris-phosphate production, disrupting the (phosphatidylinositol-(3,4,5-tris-phosphate/Rac/F-actin-mediated feedback circuit that organizes the leading edge; and (c prevents exclusion of activated myosin from the leading edge, perhaps by misregulating leading edge complexes that contain inhibitors of the Rho-actomyosin pathway. Taken together, these observations show that versatile Hem-1-containing complexes coordinate diverse regulatory signals at the leading edge of polarized neutrophils, including but not confined to those involving WAVE2-dependent actin polymerization.

  19. Deafness and espin-actin self-organization in stereocilia

    Science.gov (United States)

    Wong, Gerard C. L.

    2009-03-01

    Espins are F-actin-bundling proteins associated with large parallel actin bundles found in hair cell stereocilia in the ear, as well as brush border microvilli and Sertoli cell junctions. We examine actin bundle structures formed by different wild-type espin isoforms, fragments, and naturally-occurring human espin mutants linked to deafness and/or vestibular dysfunction. The espin-actin bundle structure consisted of a hexagonal arrangement of parallel actin filaments in a non-native twist state. We delineate the structural consequences caused by mutations in espin's actin-bundling module. For espin mutation with a severely damaged actin-bundling module, which are implicated in deafness in mice and humans, oriented nematic-like actin filament structures, which strongly impinges on bundle mechanical stiffness. Finally, we examine what makes espin different, via a comparative study of bundles formed by espin and those formed by fascin, a prototypical bundling protein found in functionally different regions of the cell, such as filopodia.

  20. Filament assembly by Spire: key residues and concerted actin binding.

    Science.gov (United States)

    Rasson, Amy S; Bois, Justin S; Pham, Duy Stephen L; Yoo, Haneul; Quinlan, Margot E

    2015-02-27

    The most recently identified class of actin nucleators, WASp homology domain 2 (WH2) nucleators, use tandem repeats of monomeric actin-binding WH2 domains to facilitate actin nucleation. WH2 domains are involved in a wide variety of actin regulatory activities. Structurally, they are expected to clash with interprotomer contacts within the actin filament. Thus, the discovery of their role in nucleation was surprising. Here we use Drosophila Spire (Spir) as a model system to investigate both how tandem WH2 domains can nucleate actin and what differentiates nucleating WH2-containing proteins from their non-nucleating counterparts. We found that the third WH2 domain in Spir (Spir-C or SC) plays a unique role. In the context of a short nucleation construct (containing only two WH2 domains), placement of SC in the N-terminal position was required for the most potent nucleation. We found that the native organization of the WH2 domains with respect to each other is necessary for binding to actin with positive cooperativity. We identified two residues within SC that are critical for its activity. Using this information, we were able to convert a weak synthetic nucleator into one with activity equal to a native Spir construct. Lastly, we found evidence that SC binds actin filaments, in addition to monomers.

  1. Actin puts the squeeze on Drosophila glue secretion.

    Science.gov (United States)

    Merrifield, Christien J

    2016-02-01

    An actin filament coat promotes cargo expulsion from large exocytosing vesicles, but the mechanisms of coat formation and force generation have been poorly characterized. Elegant imaging studies of the Drosophila melanogaster salivary gland now reveal how actin and myosin are recruited, and show that myosin II forms a contractile 'cage' that facilitates exocytosis.

  2. G-actin guides p53 nuclear transport: potential contribution of monomeric actin in altered localization of mutant p53

    Science.gov (United States)

    Saha, Taniya; Guha, Deblina; Manna, Argha; Panda, Abir Kumar; Bhat, Jyotsna; Chatterjee, Subhrangsu; Sa, Gaurisankar

    2016-01-01

    p53 preserves genomic integrity by restricting anomaly at the gene level. Till date, limited information is available for cytosol to nuclear shuttling of p53; except microtubule-based trafficking route, which utilizes minus-end directed motor dynein. The present study suggests that monomeric actin (G-actin) guides p53 traffic towards the nucleus. Histidine-tag pull-down assay using purified p53(1–393)-His and G-actin confirms direct physical association between p53 and monomeric G-actin. Co-immunoprecipitation data supports the same. Confocal imaging explores intense perinuclear colocalization between p53 and G-actin. To address atomistic details of the complex, constraint-based docked model of p53:G-actin complex was generated based on crystal structures. MD simulation reveals that p53 DNA-binding domain arrests very well the G-actin protein. Docking benchmark studies have been carried out for a known crystal structure, 1YCS (complex between p53DBD and BP2), which validates the docking protocol we adopted. Co-immunoprecipitation study using “hot-spot” p53 mutants suggested reduced G-actin association with cancer-associated p53 conformational mutants (R175H and R249S). Considering these findings, we hypothesized that point mutation in p53 structure, which diminishes p53:G-actin complexation results in mutant p53 altered subcellular localization. Our model suggests p53Arg249 form polar-contact with Arg357 of G-actin, which upon mutation, destabilizes p53:G-actin interaction and results in cytoplasmic retention of p53R249S. PMID:27601274

  3. G-actin guides p53 nuclear transport: potential contribution of monomeric actin in altered localization of mutant p53.

    Science.gov (United States)

    Saha, Taniya; Guha, Deblina; Manna, Argha; Panda, Abir Kumar; Bhat, Jyotsna; Chatterjee, Subhrangsu; Sa, Gaurisankar

    2016-01-01

    p53 preserves genomic integrity by restricting anomaly at the gene level. Till date, limited information is available for cytosol to nuclear shuttling of p53; except microtubule-based trafficking route, which utilizes minus-end directed motor dynein. The present study suggests that monomeric actin (G-actin) guides p53 traffic towards the nucleus. Histidine-tag pull-down assay using purified p53(1-393)-His and G-actin confirms direct physical association between p53 and monomeric G-actin. Co-immunoprecipitation data supports the same. Confocal imaging explores intense perinuclear colocalization between p53 and G-actin. To address atomistic details of the complex, constraint-based docked model of p53:G-actin complex was generated based on crystal structures. MD simulation reveals that p53 DNA-binding domain arrests very well the G-actin protein. Docking benchmark studies have been carried out for a known crystal structure, 1YCS (complex between p53DBD and BP2), which validates the docking protocol we adopted. Co-immunoprecipitation study using "hot-spot" p53 mutants suggested reduced G-actin association with cancer-associated p53 conformational mutants (R175H and R249S). Considering these findings, we hypothesized that point mutation in p53 structure, which diminishes p53:G-actin complexation results in mutant p53 altered subcellular localization. Our model suggests p53Arg249 form polar-contact with Arg357 of G-actin, which upon mutation, destabilizes p53:G-actin interaction and results in cytoplasmic retention of p53R249S. PMID:27601274

  4. Actin-Based Feedback Circuits in Cell Migration and Endocytosis

    Science.gov (United States)

    Wang, Xinxin

    In this thesis, we study the switch and pulse functions of actin during two important cellular processes, cell migration and endocytosis. Actin is an abundant protein that can polymerize to form a dendritic network. The actin network can exert force to push or bend the cell membrane. During cell migration, the actin network behaves like a switch, assembling mostly at one end or at the other end. The end with the majority of the actin network is the leading edge, following which the cell can persistently move in the same direction. The other end, with the minority of the actin network, is the trailing edge, which is dragged by the cell as it moves forward. When subjected to large fluctuations or external stimuli, the leading edge and the trailing edge can interchange and change the direction of motion, like a motion switch. Our model of the actin network in a cell reveals that mechanical force is crucial for forming the motion switch. We find a transition from single state symmetric behavior to switch behavior, when tuning parameters such as the force. The model is studied by both stochastic simulations, and a set of rate equations that are consistent with the simulations. Endocytosis is a process by which cells engulf extracellular substances and recycle the cell membrane. In yeast cells, the actin network is transiently needed to overcome the pressure difference across the cell membrane caused by turgor pressure. The actin network behaves like a pulse, which assembles and then disassembles within about 30 seconds. Using a stochastic model, we reproduce the pulse behaviors of the actin network and one of its regulatory proteins, Las17. The model matches green fluorescence protein (GFP) experiments for wild-type cells. The model also predicts some phenotypes that modify or diminish the pulse behavior. The phenotypes are verified with both experiments performed at Washington University and with other groups' experiments. We find that several feedback mechanisms are

  5. Actin related protein complex subunit 1b controls sperm release, barrier integrity and cell division during adult rat spermatogenesis.

    Science.gov (United States)

    Kumar, Anita; Dumasia, Kushaan; Deshpande, Sharvari; Gaonkar, Reshma; Balasinor, N H

    2016-08-01

    Actin remodeling is a vital process for signaling, movement and survival in all cells. In the testes, extensive actin reorganization occurs at spermatid-Sertoli cell junctions during sperm release (spermiation) and at inter Sertoli cell junctions during restructuring of the blood testis barrier (BTB). During spermiation, tubulobulbar complexes (TBCs), rich in branched actin networks, ensure recycling of spermatid-Sertoli cell junctional molecules. Similar recycling occurs during BTB restructuring around the same time as spermiation occurs. Actin related protein 2/3 complex is an essential actin nucleation and branching protein. One of its subunits, Arpc1b, was earlier found to be down-regulated in an estrogen-induced rat model of spermiation failure. Also, Arpc1b was found to be estrogen responsive through estrogen receptor beta in seminiferous tubule culture. Here, knockdown of Arpc1b by siRNA in adult rat testis led to defects in spermiation caused by failure in TBC formation. Knockdown also compromised BTB integrity and caused polarity defects of mature spermatids. Apart from these effects pertaining to Sertoli cells, Arpc1b reduction perturbed ability of germ cells to enter G2/M phase thus hindering cell division. In summary, Arpc1b, an estrogen responsive gene, is a regulator of spermiation, mature spermatid polarity, BTB integrity and cell division during adult spermatogenesis. PMID:27113856

  6. Exploring the Possible Role of Lysine Acetylation on Entamoeba histolytica Virulence: A Focus on the Dynamics of the Actin Cytoskeleton

    Directory of Open Access Journals (Sweden)

    L. López-Contreras

    2013-01-01

    Full Text Available Cytoskeleton remodeling can be regulated, among other mechanisms, by lysine acetylation. The role of acetylation on cytoskeletal and other proteins of Entamoeba histolytica has been poorly studied. Dynamic rearrangements of the actin cytoskeleton are crucial for amebic motility and capping formation, processes that may be effective means of evading the host immune response. Here we report the possible effect of acetylation on the actin cytoskeleton dynamics and in vivo virulence of E. histolytica. Using western blot, immunoprecipitation, microscopy assays, and in silico analysis, we show results that strongly suggest that the increase in Aspirin-induced cytoplasm proteins acetylation reduced cell movement and capping formation, likely as a consequence of alterations in the structuration of the actin cytoskeleton. Additionally, intrahepatic inoculation of Aspirin-treated trophozoites in hamsters resulted in severe impairment of the amebic virulence. Taken together, these results suggest an important role for lysine acetylation in amebic invasiveness and virulence.

  7. Exploring the possible role of lysine acetylation on Entamoeba histolytica virulence: a focus on the dynamics of the actin cytoskeleton.

    Science.gov (United States)

    López-Contreras, L; Hernández-Ramírez, V I; Lagunes-Guillén, A E; Montaño, Sarita; Chávez-Munguía, B; Sánchez-Ramírez, B; Talamás-Rohana, P

    2013-01-01

    Cytoskeleton remodeling can be regulated, among other mechanisms, by lysine acetylation. The role of acetylation on cytoskeletal and other proteins of Entamoeba histolytica has been poorly studied. Dynamic rearrangements of the actin cytoskeleton are crucial for amebic motility and capping formation, processes that may be effective means of evading the host immune response. Here we report the possible effect of acetylation on the actin cytoskeleton dynamics and in vivo virulence of E. histolytica. Using western blot, immunoprecipitation, microscopy assays, and in silico analysis, we show results that strongly suggest that the increase in Aspirin-induced cytoplasm proteins acetylation reduced cell movement and capping formation, likely as a consequence of alterations in the structuration of the actin cytoskeleton. Additionally, intrahepatic inoculation of Aspirin-treated trophozoites in hamsters resulted in severe impairment of the amebic virulence. Taken together, these results suggest an important role for lysine acetylation in amebic invasiveness and virulence.

  8. Structural Modeling and Molecular Dynamics Simulation of the Actin Filament

    Energy Technology Data Exchange (ETDEWEB)

    Splettstoesser, Thomas [University of Heidelberg; Holmes, Kenneth [Max Planck Institute, Heidelberg, Germany; Noe, Frank [DFG Research Center Matheon, FU Berlin, Germany; Smith, Jeremy C [ORNL

    2011-01-01

    Actin is a major structural protein of the eukaryotic cytoskeleton and enables cell motility. Here, we present a model of the actin filament (F-actin) that not only incorporates the global structure of the recently published model by Oda et al. but also conserves internal stereochemistry. A comparison is made using molecular dynamics simulation of the model with other recent F-actin models. A number of structural determents such as the protomer propeller angle, the number of hydrogen bonds, and the structural variation among the protomers are analyzed. The MD comparison is found to reflect the evolution in quality of actin models over the last 6 years. In addition, simulations of the model are carried out in states with both ADP or ATP bound and local hydrogen-bonding differences characterized.

  9. A36-dependent actin filament nucleation promotes release of vaccinia virus.

    Directory of Open Access Journals (Sweden)

    Jacquelyn Horsington

    2013-03-01

    Full Text Available Cell-to-cell transmission of vaccinia virus can be mediated by enveloped virions that remain attached to the outer surface of the cell or those released into the medium. During egress, the outer membrane of the double-enveloped virus fuses with the plasma membrane leaving extracellular virus attached to the cell surface via viral envelope proteins. Here we report that F-actin nucleation by the viral protein A36 promotes the disengagement of virus attachment and release of enveloped virus. Cells infected with the A36(YdF virus, which has mutations at two critical tyrosine residues abrogating localised actin nucleation, displayed a 10-fold reduction in virus release. We examined A36(YdF infected cells by transmission electron microscopy and observed that during release, virus appeared trapped in small invaginations at the plasma membrane. To further characterise the mechanism by which actin nucleation drives the dissociation of enveloped virus from the cell surface, we examined recombinant viruses by super-resolution microscopy. Fluorescently-tagged A36 was visualised at sub-viral resolution to image cell-virus attachment in mutant and parental backgrounds. We confirmed that A36(YdF extracellular virus remained closely associated to the plasma membrane in small membrane pits. Virus-induced actin nucleation reduced the extent of association, thereby promoting the untethering of virus from the cell surface. Virus release can be enhanced via a point mutation in the luminal region of B5 (P189S, another virus envelope protein. We found that the B5(P189S mutation led to reduced contact between extracellular virus and the host membrane during release, even in the absence of virus-induced actin nucleation. Our results posit that during release virus is tightly tethered to the host cell through interactions mediated by viral envelope proteins. Untethering of virus into the surrounding extracellular space requires these interactions be relieved, either

  10. Force Generation, Polymerization Dynamics and Nucleation of Actin Filaments

    Science.gov (United States)

    Wang, Ruizhe

    We study force generation and actin filament dynamics using stochastic and deterministic methods. First, we treat force generation of bundled actin filaments by polymerization via molecular-level stochastic simulations. In the widely-used Brownian Ratchet model, actin filaments grow freely whenever the tip-obstacle gap created by thermal fluctuation exceeds the monomer size. We name this model the Perfect Brownian Ratchet (PBR) model. In the PBR model, actin monomer diffusion is treated implicitly. We perform a series of simulations based on the PBR, in which obstacle motion is treated explicitly; in most previous studies, obstacle motion has been treated implicitly. We find that the cooperativity of filaments is generally weak in the PBR model, meaning that more filaments would grow more slowly given the same force per filament. Closed-form formulas are also developed, which match the simulation results. These portable and accurate formulas provide guidance for experiments and upper and lower bounds for theoretical analyses. We also studied a variation of the PBR, called the Diffusing Brownian Ratchet (DBR) model, in which both actin monomer and obstacle diffusion are treated explicitly. We find that the growth rate of multiple filaments is even lower, compared with that in PBR. This finding challenges the widely-accepted PBR assumption and suggests that pushing the study of actin dynamics down to the sub-nanometer level yields new insights. We subsequently used a rate equation approach to model the effect of local depletion of actin monomers on the nucleation of actin filaments on biomimetic beads, and how the effect is regulated by capping protein (CP). We find that near the bead surface, a higher CP concentration increases local actin concentration, which leads to an enhanced activities of actin filaments' nucleation. Our model analysis matches the experimental results and lends support to an important but undervalued hypothesis proposed by Carlier and

  11. Pearling instability of membrane tubes driven by curved proteins and actin polymerization

    Science.gov (United States)

    Jelerčič, U.; Gov, N. S.

    2015-12-01

    Membrane deformation inside living cells is crucial for the proper shaping of various intracellular organelles and is necessary during the fission/fusion processes that allow membrane recycling and transport (e.g. endocytosis). Proteins that induce membrane curvature play a key role in such processes, mostly by adsorbing to the membrane and forming a scaffold that deforms the membrane according to the curvature of the proteins. In this paper we explore the possibility of membrane tube destabilization through a pearling mechanism enabled by the combined effects of the adsorbed curved proteins and the actin polymerization that they recruit. The pearling instability can serve as the initiation for fission of the tube into vesicles. We find that adsorbed curved proteins are more likely to stabilize the tubes, while the actin polymerization can provide the additional constrictive force needed for the robust instability. We discuss the relevance of the theoretical results to in vivo and in vitro experiments.

  12. Pearling instability of membrane tubes driven by curved proteins and actin polymerization

    CERN Document Server

    Jelerčič, Urška

    2014-01-01

    Membrane deformation inside living cells is crucial for the proper shaping of various intracellular organelles and is necessary during the fission/fusion processes that allow membrane recycling and transport (e.g. endocytosis). Proteins that induce membrane curvature play a key role in such processes, mostly by adsorbing to the membrane and forming a scaffold that deforms the membrane according to the curvature of the proteins. In this paper we explore the possibility of membrane tube destabilisation through a pearling mechanism enabled by the combined effects of the adsorbed curved proteins and the actin polymerization they may recruit. The pearling instability can furthermore serve as the initiation for fission of the tube into vesicles. We find that adsorbed proteins are more likely to stabilise the tubes, while the actin polymerization can provide the additional constrictive force needed for the robust instability. We discuss the relevance of the theoretical results to in-vivo and in-vitro experiments.

  13. Mechanical properties of branched actin filaments

    CERN Document Server

    Razbin, Mohammadhosein; Benetatos, Panayotis; Zippelius, Annette

    2015-01-01

    Cells moving on a two dimensional substrate generate motion by polymerizing actin filament networks inside a flat membrane protrusion. New filaments are generated by branching off existing ones, giving rise to branched network structures. We investigate the force-extension relation of branched filaments, grafted on an elastic structure at one end and pushing with the free ends against the leading edge cell membrane. Single filaments are modeled as worm-like chains, whose thermal bending fluctuations are restricted by the leading edge cell membrane, resulting in an effective force. Branching can increase the stiffness considerably; however the effect depends on branch point position and filament orientation, being most pronounced for intermediate tilt angles and intermediate branch point positions. We describe filament networks without cross-linkers to focus on the effect of branching. We use randomly positioned branch points, as generated in the process of treadmilling, and orientation distributions as measur...

  14. Titin-Actin Interaction: PEVK-Actin-Based Viscosity in a Large Animal

    Directory of Open Access Journals (Sweden)

    Charles S. Chung

    2011-01-01

    Full Text Available Titin exhibits an interaction between its PEVK segment and the actin filament resulting in viscosity, a speed dependent resistive force, which significantly influences diastolic filling in mice. While diastolic disease is clinically pervasive, humans express a more compliant titin (N2BA:N2B ratio ~0.5–1.0 than mice (N2BA:N2B ratio ~0.2. To examine PEVK-actin based viscosity in compliant titin-tissues, we used pig cardiac tissue that expresses titin isoforms similar to that in humans. Stretch-hold experiments were performed at speeds from 0.1 to 10 lengths/s from slack sarcomere lengths (SL to SL of 2.15 μm. Viscosity was calculated from the slope of stress-relaxation vs stretch speed. Recombinant PEVK was added to compete off native interactions and this found to reduce the slope by 35%, suggesting that PEVK-actin interactions are a strong contributor of viscosity. Frequency sweeps were performed at frequencies of 0.1–400 Hz and recombinant protein reduced viscous moduli by 40% at 2.15 μm and by 50% at 2.25 μm, suggesting a SL-dependent nature of viscosity that might prevent SL ``overshoot’’ at long diastolic SLs. This study is the first to show that viscosity is present at physiologic speeds in the pig and supports the physiologic relevance of PEVK-actin interactions in humans in both health and disease.

  15. Cloning and characterization of an actin gene of Chlamys farreri and the phylogenetic analysis of mollusk actins

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    An actin gene (CfACT1) was cloned by using RT-PCR, 3' and 5'RACE from hemocytes of the sea scallop Chlamys farreri. The full length of the transcript is 1535 bp, which contains a long 3' un-translated region of 436bp and 59bp of a 5' un-translated sequence. The open reading frame encodes a polypeptide of 376 amino acids. Sequence comparisons indicated that CfACT1 is more closely related to vertebrate cytoplasmic actins than muscle types. Phylogenetic analysis showed that molluscan actins could be generally divided into two categories: muscle and cytoplasmic, although both are similar to vertebrate cytoplasmic actins. It was also inferred that different isotypes existed in muscle or cytoplasma in mollusks. The genomic sequence of CfACT1 was cloned and sequenced. Only one intron was detected:it was located between codons 42 and 43 and different from vertebrate actin genes.

  16. ARF6 promotes the formation of Rac1 and WAVE-dependent ventral F-actin rosettes in breast cancer cells in response to epidermal growth factor.

    Directory of Open Access Journals (Sweden)

    Valentina Marchesin

    Full Text Available Coordination between actin cytoskeleton assembly and localized polarization of intracellular trafficking routes is crucial for cancer cell migration. ARF6 has been implicated in the endocytic recycling of surface receptors and membrane components and in actin cytoskeleton remodeling. Here we show that overexpression of an ARF6 fast-cycling mutant in MDA-MB-231 breast cancer-derived cells to mimick ARF6 hyperactivation observed in invasive breast tumors induced a striking rearrangement of the actin cytoskeleton at the ventral cell surface. This phenotype consisted in the formation of dynamic actin-based podosome rosette-like structures expanding outward as wave positive for F-actin and actin cytoskeleton regulatory components including cortactin, Arp2/3 and SCAR/WAVE complexes and upstream Rac1 regulator. Ventral rosette-like structures were similarly induced in MDA-MB-231 cells in response to epidermal growth factor (EGF stimulation and to Rac1 hyperactivation. In addition, interference with ARF6 expression attenuated activation and plasma membrane targeting of Rac1 in response to EGF treatment. Our data suggest a role for ARF6 in linking EGF-receptor signaling to Rac1 recruitment and activation at the plasma membrane to promote breast cancer cell directed migration.

  17. Concentration profiles of actin-binding molecules in lamellipodia

    Science.gov (United States)

    Falcke, Martin

    2016-04-01

    Motile cells form lamellipodia in the direction of motion, which are flat membrane protrusions containing an actin filament network. The network flows rearward relative to the leading edge of the lamellipodium due to actin polymerization at the front. Thus, actin binding molecules are subject to transport towards the rear of the cell in the bound state and diffuse freely in the unbound state. We analyze this reaction-diffusion-advection process with respect to the concentration profiles of these species and provide an analytic approximation for them. Network flow may cause a depletion zone of actin binding molecules close to the leading edge. The existence of such zone depends on the free molecule concentration in the cell body, on the ratio of the diffusion length to the distance bound molecules travel rearward with the flow before dissociating, and the ratio of the diffusion length to the width of the region with network flow and actin binding. Our calculations suggest the existence of depletion zones for the F-actin cross-linkers filamin and α-actinin in fish keratocytes (and other cell types), which is in line with the small elastic moduli of the F-actin network close to the leading edge found in measurements of the force motile cells are able to exert.

  18. Interconnection between actin cytoskeleton and plant defense signaling.

    Science.gov (United States)

    Janda, Martin; Matoušková, Jindřiška; Burketová, Lenka; Valentová, Olga

    2014-01-01

    Actin cytoskeleton is the fundamental structural component of eukaryotic cells. It has a role in numerous elementary cellular processes such as reproduction, development and also in response to abiotic and biotic stimuli. Remarkably, the role of actin cytoskeleton in plant response to pathogens is getting to be under magnifying glass. Based on microscopic studies, most of the data showed, that actin plays an important role in formation of physiological barrier in the site of infection. Actin dynamics is involved in the transport of antimicrobial compounds and cell wall fortifying components (e.g. callose) to the site of infection. Also the role in PTI (pathogen triggered immunity) and ETI (effector triggered immunity) was recently indicated. On the other hand much less is known about the transcriptome reprogramming upon changes in actin dynamics. Our recently published results showed that drugs inhibiting actin polymerization (latrunculin B, cytochalasin E) cause the induction of genes which are involved in salicylic acid (SA) signaling pathway. In this addendum we would like to highlight in more details current state of knowledge concerning the involvement of actin dynamics in plant defense signaling.

  19. Drebrin attenuates the interaction between actin and myosin-V.

    Science.gov (United States)

    Ishikawa, Ryoki; Katoh, Kaoru; Takahashi, Ayumi; Xie, Ce; Oseki, Koushi; Watanabe, Michitoshi; Igarashi, Michihiro; Nakamura, Akio; Kohama, Kazuhiro

    2007-07-27

    Drebrin-A is an actin-binding protein localized in the dendritic spines of mature neurons, and has been suggested to affect spine morphology [K. Hayashi, T. Shirao, Change in the shape of dendritic spines caused by overexpression of drebrin in cultured cortical neurons, J. Neurosci. 19 (1999) 3918-3925]. However, no biochemical analysis of drebrin-A has yet been reported. In this study, we purified drebrin-A using a bacterial expression system, and characterized it in vitro. Drebrin-A bound to actin filaments with a stoichiometry of one drebrin molecule to 5-6 actin molecules. Furthermore, drebrin-A decreased the Mg-ATPase activity of myosin V. In vitro motility assay revealed that the attachment of F-actin to glass surface coated with myosin-V was decreased by drebrin-A, but once F-actin attached to the surface, the sliding speed of F-actin was unaffected by the presence of drebrin A. These findings suggest that drebrin-A may affect spine dynamics, vesicle transport, and other myosin-V-driven motility in neurons through attenuating the interaction between actin and myosin-V.

  20. Visualization of endothelial actin cytoskeleton in the mouse retina.

    Directory of Open Access Journals (Sweden)

    Alessia Fraccaroli

    Full Text Available Angiogenesis requires coordinated changes in cell shape of endothelial cells (ECs, orchestrated by the actin cytoskeleton. The mechanisms that regulate this rearrangement in vivo are poorly understood - largely because of the difficulty to visualize filamentous actin (F-actin structures with sufficient resolution. Here, we use transgenic mice expressing Lifeact-EGFP to visualize F-actin in ECs. We show that in the retina, Lifeact-EGFP expression is largely restricted to ECs allowing detailed visualization of F-actin in ECs in situ. Lifeact-EGFP labels actin associated with cell-cell junctions, apical and basal membranes and highlights actin-based structures such as filopodia and stress fiber-like cytoplasmic bundles. We also show that in the skin and the skeletal muscle, Lifeact-EGFP is highly expressed in vascular mural cells (vMCs, enabling vMC imaging. In summary, our results indicate that the Lifeact-EGFP transgenic mouse in combination with the postnatal retinal angiogenic model constitutes an excellent system for vascular cell biology research. Our approach is ideally suited to address structural and mechanistic details of angiogenic processes, such as endothelial tip cell migration and fusion, EC polarization or lumen formation.

  1. Analysis of actinic flux profiles measured from an ozonesonde balloon

    Science.gov (United States)

    Wang, P.; Allaart, M.; Knap, W. H.; Stammes, P.

    2015-04-01

    A green light sensor has been developed at KNMI to measure actinic flux profiles using an ozonesonde balloon. In total, 63 launches with ascending and descending profiles were performed between 2006 and 2010. The measured uncalibrated actinic flux profiles are analysed using the Doubling-Adding KNMI (DAK) radiative transfer model. Values of the cloud optical thickness (COT) along the flight track were taken from the Spinning Enhanced Visible and Infrared Imager (SEVIRI) Cloud Physical Properties (CPP) product. The impact of clouds on the actinic flux profile is evaluated on the basis of the cloud modification factor (CMF) at the cloud top and cloud base, which is the ratio between the actinic fluxes for cloudy and clear-sky scenes. The impact of clouds on the actinic flux is clearly detected: the largest enhancement occurs at the cloud top due to multiple scattering. The actinic flux decreases almost linearly from cloud top to cloud base. Above the cloud top the actinic flux also increases compared to clear-sky scenes. We find that clouds can increase the actinic flux to 2.3 times the clear-sky value at cloud top and decrease it to about 0.05 at cloud base. The relationship between CMF and COT agrees well with DAK simulations, except for a few outliers. Good agreement is found between the DAK-simulated actinic flux profiles and the observations for single-layer clouds in fully overcast scenes. The instrument is suitable for operational balloon measurements because of its simplicity and low cost. It is worth further developing the instrument and launching it together with atmospheric chemistry composition sensors.

  2. Actin-based dynamics during spermatogenesis and its significance

    Institute of Scientific and Technical Information of China (English)

    XIAO Xiang; YANG Wan-xi

    2007-01-01

    Actin can be found in all kinds ofeukaryotic cells, maintaining their shapes and motilities, while its dynamics in sperm cells is understood less than their nonmuscle somatic cell counterparts. Spermatogenesis is a complicated process, resulting in the production of mature sperm from primordial germ cell. Significant structural and biochemical changes take place in the seminiferous epithelium of the adult testis during spermatogenesis. It was proved that all mammalian sperm contain actin, and that F-actin may play an important role during spermatogenesis, especially in nuclear shaping. Recently a new model for sperm head elongation based on the acrosome-acroplaxome-manchette complex has been proposed. In Drosophila, F-actin assembly is supposed to be very crucial during individualization. In this mini-review, we provide an overview of the structure, function, and regulation characteristics of actin cytoskeleton, and a summary of the current status of research of actin-based structure and movement is also provided, with emphasis on the role of actins in sperm head shaping during spermiogenesis and the cell junction dynamics in the testis. Research of the Sertoli ectoplasmic specialization is in the spotlight, which is a testis-specific actin-based junction very important for the movement of germ cells across the epithelium. Study of the molecular architecture and the regulating mechanism of the Sertoli ectoplasmic specialization has become an intriguing field. All this may lead to a new strategy for male infertility and,at the same time, a novel idea may result in devising much safer contraception with high efficiency. It is hoped that the advances listed in this review would give developmental and morphological researchers a favorable investigating outline and could help to enlarge the view of new strategies and models for actin dynamics during spermatogenesis.

  3. Pathogenic microbes manipulate cofilin activity to subvert actin cytoskeleton.

    Science.gov (United States)

    Zheng, Kai; Kitazato, Kaio; Wang, Yifei; He, Zhendan

    2016-09-01

    Actin-depolymerizing factor (ADF)/cofilin proteins are key players in controlling the temporal and spatial extent of actin dynamics, which is crucial for mediating host-pathogen interactions. Pathogenic microbes have evolved molecular mechanisms to manipulate cofilin activity to subvert the actin cytoskeletal system in host cells, promoting their internalization into the target cells, modifying the replication niche and facilitating their intracellular and intercellular dissemination. The study of how these pathogens exploit cofilin pathways is crucial for understanding infectious disease and providing potential targets for drug therapies. PMID:25853495

  4. Estrogen and Resveratrol Regulate Rac and Cdc42 Signaling to the Actin Cytoskeleton of Metastatic Breast Cancer Cells

    Directory of Open Access Journals (Sweden)

    Nicolas G. Azios

    2007-02-01

    Full Text Available Estrogen and structurally related molecules play critical roles in breast cancer. We reported that resveratrol (50 µM, an estrogen-like phytosterol from grapes, acts in an antiestrogenic manner in breast cancer cells to reduce cell migration and to induce a global and sustained extension of actin structures called filopodia. Herein, we report that resveratrol-induced filopodia formation is time-dependent and concentration-dependent. In contrast to resveratrol at 50 µM, resveratrol at 5 µM acts in a manner similar to estrogen by increasing lamellipodia, as well as cell migration and invasion. Because Rho GTPases regulate the extension of actin structures, we investigated a role for Rac and Cdc42 in estrogen and resveratrol signaling. Our results demonstrate that 50 µM resveratrol decreases Rac and Cdc42 activity, whereas estrogen and 5 µM resveratrol increase Rac activity in breast cancer cells. MDA-MB-231 cells expressing dominant-negative Cdc42 or dominantnegative Rac retain filopodia response to 50 µM resveratrol. Lamellipodia response to 5 µM resveratrol, estrogen, or epidermal growth factor is inhibited in cells expressing dominant-negative Rac, indicating that Rac regulates estrogen and resveratrol (5 µM signaling to the actin cytoskeleton. These results indicate that signaling to the actin cytoskeleton by low and high concentrations of resveratrol may be differentially regulated by Rac and Cdc42.

  5. Live cell imaging of the assembly, disassembly, and actin cable–dependent movement of endosomes and actin patches in the budding yeast, Saccharomyces cerevisiae

    OpenAIRE

    Huckaba, Thomas M.; Gay, Anna Card; Pantalena, Luiz Fernando; Yang, Hyeong-Cheol; Liza A Pon

    2004-01-01

    Using FM4-64 to label endosomes and Abp1p-GFP or Sac6p-GFP to label actin patches, we find that (1) endosomes colocalize with actin patches as they assemble at the bud cortex; (2) endosomes colocalize with actin patches as they undergo linear, retrograde movement from buds toward mother cells; and (3) actin patches interact with and disassemble at FM4-64–labeled internal compartments. We also show that retrograde flow of actin cables mediates retrograde actin patch movement. An Arp2/3 complex...

  6. Dexmedetomidine-induced Contraction Involves Phosphorylation of Caldesmon by JNK in Endothelium-denuded Rat Aortas

    OpenAIRE

    Baik, Jiseok; Ok, Seong-Ho; Cho, Hyunhoo; Yu, Jongsun; Kim, Woochan; Nam, In-Koo; Choi, Mun-Jeoung; Lee, Heon-Keun; Sohn, Ju-Tae

    2014-01-01

    Caldesmon, an inhibitory actin binding protein, binds to actin and inhibits actin-myosin interactions, whereas caldesmon phosphorylation reverses the inhibitory effect of caldesmon on actin-myosin interactions, potentially leading to enhanced contraction. The goal of this study was to investigate the cellular signaling pathway responsible for caldesmon phosphorylation, which is involved in the regulation of the contraction induced by dexmedetomidine (DMT), an alpha-2 adrenoceptor agonist, in ...

  7. Comparison of three light doses in the photodynamic treatment of actinic keratosis using mathematical modeling.

    Science.gov (United States)

    Vignion-Dewalle, Anne-Sophie; Betrouni, Nacim; Tylcz, Jean-Baptiste; Vermandel, Maximilien; Mortier, Laurent; Mordon, Serge

    2015-05-01

    Photodynamic therapy (PDT) is an emerging treatment modality for various diseases, especially for cancer therapy. Although high efficacy is demonstrated for PDT using standardized protocols in nonhyperkeratotic actinic keratoses, alternative light doses expected to increase efficiency, to reduce adverse effects or to expand the use of PDT, are still being evaluated and refined. We propose a comparison of the three most common light doses in the treatment of actinic keratosis with 5-aminolevulinic acid PDT through mathematical modeling. The proposed model is based on an iterative procedure that involves determination of the local fluence rate, updating of the local optical properties, and estimation of the local damage induced by the therapy. This model was applied on a simplified skin sample model including an actinic keratosis lesion, with three different light doses (red light dose, 37 J∕cm2, 75 mW∕cm2, 500 s; blue light dose, 10 J∕cm2, 10 mW∕cm2, 1000 s; and daylight dose, 9000 s). Results analysis shows that the three studied light doses, although all efficient, lead to variable local damage. Defining reference damage enables the nonoptimal parameters for the current light doses to be refined and the treatment to be more suitable.

  8. ERK reinforces actin polymerization to power persistent edge protrusion during motility.

    Science.gov (United States)

    Mendoza, Michelle C; Vilela, Marco; Juarez, Jesus E; Blenis, John; Danuser, Gaudenz

    2015-05-19

    Cells move through perpetual protrusion and retraction cycles at the leading edge. These cycles are coordinated with substrate adhesion and retraction of the cell rear. We tracked spatial and temporal fluctuations in the molecular activities of individual moving cells to elucidate how extracellular signal-regulated kinase (ERK) signaling controlled the dynamics of protrusion and retraction cycles. ERK is activated by many cell surface receptors, and we found that ERK signaling specifically reinforced cellular protrusions so that they translated into rapid, sustained forward motion of the leading edge. Using quantitative fluorescent speckle microscopy and cross-correlation analysis, we showed that ERK controlled the rate and timing of actin polymerization by promoting the recruitment of the actin nucleator Arp2/3 to the leading edge. These findings support a model in which surges in ERK activity induced by extracellular cues enhance Arp2/3-mediated actin polymerization to generate protrusion power phases with enough force to counteract increasing membrane tension and to promote sustained motility.

  9. Androgens Regulate T47D Cells Motility and Invasion through Actin Cytoskeleton Remodeling

    Science.gov (United States)

    Montt-Guevara, Maria Magdalena; Shortrede, Jorge Eduardo; Giretti, Maria Silvia; Giannini, Andrea; Mannella, Paolo; Russo, Eleonora; Genazzani, Alessandro David; Simoncini, Tommaso

    2016-01-01

    The relationship between androgens and breast cancer is controversial. Androgens have complex effects on breast cancer progression and metastasis. Moreover, androgen receptor (AR) is expressed in approximately 70 to 90% of invasive breast carcinomas, which has prognostic relevance in basal-like cancers and in triple-negative breast cancers. Recent studies have associated the actin-binding proteins of the ezrin–radixin–moesin (ERM) family with metastasis in endocrine-sensitive cancers. We studied on T47D breast cancer cells whether androgens with different characteristics, such as testosterone (T), dihydrotestosterone (DHT), and dehydroepiandrosterone (DHEA) may regulate breast cancer cell motility and invasion through the control of actin remodeling. We demonstrate that androgens promote migration and invasion in T47D via Moesin activation. We show that T and DHEA exert their actions via the AR and estrogen receptor (ER), while the non-aromatizable androgen – DHT – only recruits AR. We further report that androgen induced significant changes in actin organization with pseudopodia along with membrane ruffles formation, and this process is mediated by Moesin. Our work identifies novel mechanisms of action of androgens on breast cancer cells. Through the modulation of Moesin, androgens alter the architecture of cytoskeleton in T47D breast cancer cell and promote cell migration and invasion. These results could help to understand the biological actions of androgens on breast cancer and, eventually, to develop new strategies for breast cancer treatment. PMID:27746764

  10. Aurora A kinase modulates actin cytoskeleton through phosphorylation of Cofilin: Implication in the mitotic process.

    Science.gov (United States)

    Ritchey, Lisa; Chakrabarti, Ratna

    2014-11-01

    Aurora A kinase regulates early mitotic events through phosphorylation and activation of a variety of proteins. Specifically, Aur-A is involved in centrosomal separation and formation of mitotic spindles in early prophase. The effect of Aur-A on mitotic spindles is mediated by the modulation of microtubule dynamics and association with microtubule binding proteins. In this study we show that Aur-A exerts its effects on spindle organization through the regulation of the actin cytoskeleton. Aurora A phosphorylates Cofilin at multiple sites including S(3) resulting in the inactivation of its actin depolymerizing function. Aur-A interacts with Cofilin in early mitotic phases and regulates its phosphorylation status. Cofilin phosphorylation follows a dynamic pattern during the progression of prophase to metaphase. Inhibition of Aur-A activity induced a delay in the progression of prophase to metaphase. Aur-A inhibitor also disturbed the pattern of Cofilin phosphorylation, which correlated with the mitotic delay. Our results establish a novel function of Aur-A in the regulation of actin cytoskeleton reorganization, through Cofilin phosphorylation during early mitotic stages.

  11. Stress-dependent proteolytic processing of the actin assembly protein Lsb1 modulates a yeast prion.

    Science.gov (United States)

    Ali, Moiez; Chernova, Tatiana A; Newnam, Gary P; Yin, Luming; Shanks, John; Karpova, Tatiana S; Lee, Andrew; Laur, Oskar; Subramanian, Sindhu; Kim, Dami; McNally, James G; Seyfried, Nicholas T; Chernoff, Yury O; Wilkinson, Keith D

    2014-10-01

    Yeast prions are self-propagating amyloid-like aggregates of Q/N-rich protein that confer heritable traits and provide a model of mammalian amyloidoses. [PSI(+)] is a prion isoform of the translation termination factor Sup35. Propagation of [PSI(+)] during cell division under normal conditions and during the recovery from damaging environmental stress depends on cellular chaperones and is influenced by ubiquitin proteolysis and the actin cytoskeleton. The paralogous yeast proteins Lsb1 and Lsb2 bind the actin assembly protein Las17 (a yeast homolog of human Wiskott-Aldrich syndrome protein) and participate in the endocytic pathway. Lsb2 was shown to modulate maintenance of [PSI(+)] during and after heat shock. Here, we demonstrate that Lsb1 also regulates maintenance of the Sup35 prion during and after heat shock. These data point to the involvement of Lsb proteins in the partitioning of protein aggregates in stressed cells. Lsb1 abundance and cycling between actin patches, endoplasmic reticulum, and cytosol is regulated by the Guided Entry of Tail-anchored proteins pathway and Rsp5-dependent ubiquitination. Heat shock-induced proteolytic processing of Lsb1 is crucial for prion maintenance during stress. Our findings identify Lsb1 as another component of a tightly regulated pathway controlling protein aggregation in changing environments. PMID:25143386

  12. Effect of the ulcerogenic agents ethanol, acetylsalicylic acid and taurocholate on actin cytoskeleton and cell motility in cultured rat gastric mucosal cells

    Institute of Scientific and Technical Information of China (English)

    Siamak Bidel; Harri Mustonen; Giti Khalighi-Sikaroudi; Eero Lehtonen; Pauli Puolakkainen; Tuula Kiviluoto; Eero Kivilaakso

    2005-01-01

    AIM: To assess the effects of ulcerogenic agents on actin cytoskeleton and cell motility and the contribution of oxidative stress.METHODS: Rat gastric mucosal cell monolayers were cultured on coverslips. The cells were exposed, with or without allopurinol (2 mmol/L), for 15 min to ethanol (10-150 mL/L), ASA (1-20 mmol/L) or taurocholate (1-20 mmol/L), then the cells were processed for actin and vinculin staining. Cell migration after wounding was also measured.RESULTS: Exposure to 10 mL/L ethanol caused divergence of zonula adherens-associated actin bundles of adjacent cells and decreased rate of migration. These actions were opposed by xanthine oxidase inhibitor allopurinol. Exposure to 50 mL/L ethanol induced degradation and divergence of zonula adherens-associated vinculin from adjacent cells,which was, again, partially reverted by allopurinol. With 1 mmol/L ASA actin filaments became shorter and thicker.However, higher concentrations (10, 20 mmol/L) of ASA returned microfilaments thinner and longer, and decreased rate of migration. Zonula adherens-associated actin bundles were moderately distorted with 10 mmol/L ASA and with 10 mmol/L taurocholate. Exposure to taurocholate provoked changes resembling those of ASA. Taurocholate 5-20 mmol/L decreased the rate of migration dose dependently. The effects of ASA and taurocholate were not prevented by allopurinol.CONCLUSION: All ulcerogenic agents decreased the rate of migration dose dependently and induced divergence of zonula adherens-associated actin bundles of adjacent cells.In addition, ethanol and ASA caused degradation of actin cytoskeleton. Oxidative stress seems to underlie ethanol,but not ASA or taurocholate, induced cytoskeletal damage.

  13. Antenna mechanism of length control of actin cables

    CERN Document Server

    Mohapatra, Lishibanya; Kondev, Jane

    2014-01-01

    Actin cables are linear cytoskeletal structures that serve as tracks for myosin-based intracellular transport of vesicles and organelles in both yeast and mammalian cells. In a yeast cell undergoing budding, cables are in constant dynamic turnover yet some cables grow from the bud neck toward the back of the mother cell until their length roughly equals the diameter of the mother cell. This raises the question: how is the length of these cables controlled? Here we describe a novel molecular mechanism for cable length control inspired by recent experimental observations in cells. This antenna mechanism involves three key proteins: formins, which polymerize actin, Smy1 proteins, which bind formins and inhibit actin polymerization, and myosin motors, which deliver Smy1 to formins, leading to a length-dependent actin polymerization rate. We compute the probability distribution of cable lengths as a function of several experimentally tuneable parameters such as the formin-binding affinity of Smy1 and the concentra...

  14. Differential requirements for actin during yeast and mammalian endocytosis.

    Science.gov (United States)

    Aghamohammadzadeh, Soheil; Ayscough, Kathryn R

    2009-08-01

    Key features of clathrin-mediated endocytosis have been conserved across evolution. However, endocytosis in Saccharomyces cerevisiae is completely dependent on a functional actin cytoskeleton, whereas actin appears to be less critical in mammalian cell endocytosis. We reveal that the fundamental requirement for actin in the early stages of yeast endocytosis is to provide a strong framework to support the force generation needed to direct the invaginating plasma membrane into the cell against turgor pressure. By providing osmotic support, pressure differences across the plasma membrane were removed and this reduced the requirement for actin-bundling proteins in normal endocytosis. Conversely, increased turgor pressure in specific yeast mutants correlated with a decreased rate of endocytic patch invagination. PMID:19597484

  15. Nanosecond electric pulses trigger actin responses in plant cells

    International Nuclear Information System (INIS)

    We have analyzed the cellular effects of nanosecond pulsed electrical fields on plant cells using fluorescently tagged marker lines in the tobacco cell line BY-2 and confocal laser scanning microscopy. We observe a disintegration of the cytoskeleton in the cell cortex, followed by contraction of actin filaments towards the nucleus, and disintegration of the nuclear envelope. These responses are accompanied by irreversible permeabilization of the plasma membrane manifest as uptake of Trypan Blue. By pretreatment with the actin-stabilizing drug phalloidin, the detachment of transvacuolar actin from the cell periphery can be suppressed, and this treatment can also suppress the irreversible perforation of the plasma membrane. We discuss these findings in terms of a model, where nanosecond pulsed electric fields trigger actin responses that are key events in the plant-specific form of programmed cell death.

  16. Roles of Cortactin, an Actin Polymerization Mediator, in Cell Endocytosis

    Institute of Scientific and Technical Information of China (English)

    Li CHEN; Zhi-Wei WANG; Jian-wei ZHU; Xi ZHAN

    2006-01-01

    Cortactin, an actin-binding protein and a substrate of Src, is encoded by the EMS 1 oncogene.Cortactin is known to activate Arp2/3 complex-mediated actin polymerization and interact with dynamin, a large GTPase and proline rich domain-containing protein. Transferrin endocytosis was significantly reduced in cells by knock-down of cortactin expression as well as in vivo introduction of cortactin immunoreagents.Cortactin-dynamin interaction displayed morphologically dynamic co-distribution with a change in the endocytosis level in cells treated with an actin depolymerization reagent, cytochalasin D. In an in vitro beads assay, a branched actin network was recruited onto dynamin-coated beads in a cortactin Src homology domain 3 (SH3)-dependent manner. In addition, cortactin was found to function in the late stage of clathrin coated vesicle formation.Taken together, cortactin is required for optimal clathrin mediated endocytosis in a dynamin directed manner.

  17. Tracing myoblast fusion in Drosophila embryos by fluorescent actin probes.

    Science.gov (United States)

    Haralalka, Shruti; Abmayr, Susan M

    2015-01-01

    Myoblast fusion in the Drosophila embryo is a highly elaborate process that is initiated by Founder Cells and Fusion-Competent Myoblasts (FCMs). It occurs through an asymmetric event in which actin foci assemble in the FCMs at points of cell-cell contact and direct the formation of membrane protrusions that drive fusion. Herein, we describe the approach that we have used to image in living embryos the highly dynamic actin foci and actin-rich projections that precede myoblast fusion. We discuss resources currently available for imaging actin and myogenesis, and our experience with these resources if available. This technical report is not intended to be comprehensive on providing instruction on standard microscopy practices or software utilization. However, we discuss microscope parameters that we have used in data collection, and our experience with image processing tools in data analysis.

  18. A model actin comet tail disassembling by severing

    International Nuclear Information System (INIS)

    We use a numerical simulation to model an actin comet tail as it grows from the surface of a small object (a bead) and disassembles by severing. We explore the dependence of macroscopic properties such as the local tail radius and tail length on several controllable properties, namely the bead diameter, the bead velocity, the severing rate per unit length, and the actin gel mesh size. The model predicts an F-actin density with an initial exponential decay followed by an abrupt decay at the edge of the tail, and predicts that the comet tail diameter is constant along the length of the tail. The simulation results are used to fit a formula relating the comet tail length to the control parameters, and it is proposed that this formula offers a means to extract quantitative information on the actin gel mesh size and severing kinetics from simple macroscopic measurements

  19. Computational defect review for actinic mask inspections

    Science.gov (United States)

    Morgan, Paul; Rost, Daniel; Price, Daniel; Corcoran, Noel; Satake, Masaki; Hu, Peter; Peng, Danping; Yonenaga, Dean; Tolani, Vikram

    2013-04-01

    As optical lithography continues to extend into low-k1 regime, resolution of mask patterns continues to diminish. The limitation of 1.35 NA posed by water-based lithography has led to the application of various resolution enhancement techniques (RET), for example, use of strong phase-shifting masks, aggressive OPC and sub-resolution assist features, customized illuminators, etc. The adoption of these RET techniques combined with the requirements to detect even smaller defects on masks due to increasing MEEF, poses considerable challenges for a mask inspection engineer. Inspecting masks under their actinic-aerial image conditions would detect defects that are more likely to print under those exposure conditions. However, this also makes reviewing such defects in their low-contrast aerial images very challenging. On the other hand, inspecting masks under higher resolution inspection optics would allow for better viewing of defects post-inspection. However, such inspections generally would also detect many more defects, including printable and nuisance, thereby making it difficult to judge which are of real concern for printability on wafer. Often, an inspection engineer may choose to use Aerial and/or high resolution inspection modes depending on where in the process flow the mask is and the specific device-layer characteristics of the mask. Hence, a comprehensive approach is needed in handling defects both post-aerial and post-high resolution inspections. This analysis system is designed for the Applied Materials Aera™ mask inspection platform, all data reported was collected using the Aera.

  20. Adenosine Diphosphate Ribosylation Factor-GTPaseActivating Protein Stimulates the Transport of AUX1Endosome, Which Relies on Actin Cytoskeletal Organization in Rice Root DevelopmentF

    Institute of Scientific and Technical Information of China (English)

    Cheng Du; Yunyuan XU; Yingdian Wang; Kang Chong

    2011-01-01

    Polar auxin transport,which depends on polarized subcellular distribution of AUXIN RESISTANT 1/LIKE AUX1 (AUX1/LAX) influx carriers and PIN-FORMED (PIN) efflux carriers,mediates various processes of plant growth and development.Endosomal recycling of PIN1 is mediated by an adenosine diphosphate (ADP)ribosylation factor (ARF)-GTPase exchange factor protein,GNOM.However,the mediation of auxin influx carrier recycling is poorly understood.Here,we report that overexpression of OsAGAP,an ARF-GTPase-activating protein in rice,stimulates vesicle transport from the plasma membrane to the Golgi apparatus in protoplasts and transgenic plants and induces the accumulation of early endosomes and AUX1.AUX1 endosomes could partially colocalize with FM4-64 labeled early endosome after actin disruption.Furthermore,OsAGAP is involved in actin cytoskeletal organization,and its overexpression tends to reduce the thickness and bundling of actin filaments.Fluorescence recovery after photobleaching analysis revealed exocytosis of the AUX1 recycling endosome was not affected in the OsAGAP overexpression cells,and was only slightly promoted when the actin filaments were completely disrupted by Lat B.Thus,we propose that AUX1 accumulation in the OsAGAP overexpression and actin disrupted cells may be due to the fact that endocytosis of the auxin influx carrier AUX1 early endosome was greatly promoted by actin cytoskeleton disruption.

  1. Polymerization of fluorescent analogue of plant actin in vitro and in vivo

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Maize pollen actin has been labeled with Oregon Green 488 iodoacetamide. A yield of 3 mg fluorescent actin analogue has been obtained from 10 mg of maize pollen actin, which is 99% in purity and the dye/protein ratio is 72%. In the presence of Mg2+ and K+, the fluorescent actin analogue polymerized into filaments in vitro. Green fluorescent filaments were observed when the fluorescent actin was introduced into living plant cells by microinjection, indicating that the fluorescent actin analogue functions similarly to the native actin.

  2. The core and conserved role of MAL is homeostatic regulation of actin levels.

    Science.gov (United States)

    Salvany, Lara; Muller, Julius; Guccione, Ernesto; Rørth, Pernille

    2014-05-15

    The transcription cofactor MAL is regulated by free actin levels and thus by actin dynamics. MAL, together with its DNA-binding partner, SRF, is required for invasive cell migration and in experimental metastasis. Although MAL/SRF has many targets, we provide genetic evidence in both Drosophila and human cellular models that actin is the key target that must be regulated by MAL/SRF for invasive cell migration. By regulating MAL/SRF activity, actin protein feeds back on production of actin mRNA to ensure sufficient supply of actin. This constitutes a dedicated homeostatic feedback system that provides a foundation for cellular actin dynamics.

  3. Computational Analysis of the Transcriptional Regulation of the Actin Family

    Institute of Scientific and Technical Information of China (English)

    郑家顺; 吴加金; 孙之荣

    2002-01-01

    Transcriptional regulation is a very important regulatory step in the regulation of gene expression. Transcription factors (TFs) play an important role in controlling the temporal special specificity of gene expression. The regulation area of actin genes was analyzed statistically to predict the transcription factor binding sites in the regulatory area. A group of transcription factors located in most of the sequences is believed to play an important role in co-regulating the expression of actin genes.

  4. Course 6: Physics of Composite Cell Membrane and Actin Based Cytoskeleton

    Science.gov (United States)

    Sackmann, E.; Bausch, A. R.; Vonna, L.

    1 Architecture of composite cell membranes 1.1 The lipid/protein bilayer is a multicomponent smectic phase with mosaic like architecture 1.2 The spectrin/actin cytoskeleton as hyperelastic cell stabilizer 1.3 The actin cortex: Architecture and function 2 Physics of the actin based cytoskeleton 2.1 Actin is a living semiflexible polymer 2.2 Actin network as viscoelastic body 2.3 Correlation between macroscopic viscoelasticity and molecular 3 Heterogeneous actin gels in cells and biological function 3.1 Manipulation of actin gels 3.2 Control of organization and function of actin cortex by cell signalling 4 Micromechanics and microrheometry of cells 5 Activation of endothelial cells: On the possibility of formation of stress fibers as phase transition of actin-network triggered by cell signalling pathways 6 On cells as adaptive viscoplastic bodies 7 Controll of cellular protrusions controlled by actin/myosin cortex

  5. Competition for actin between two distinct F-actin networks defines a bistable switch for cell polarization.

    Science.gov (United States)

    Lomakin, Alexis J; Lee, Kun-Chun; Han, Sangyoon J; Bui, Duyen A; Davidson, Michael; Mogilner, Alex; Danuser, Gaudenz

    2015-11-01

    Symmetry-breaking polarization enables functional plasticity of cells and tissues and is yet not well understood. Here we show that epithelial cells, hard-wired to maintain a static morphology and to preserve tissue organization, can spontaneously switch to a migratory polarized phenotype after relaxation of the actomyosin cytoskeleton. We find that myosin II engages actin in the formation of cortical actomyosin bundles and thus makes it unavailable for deployment in the process of dendritic growth normally driving cell motility. Under low-contractility regimes, epithelial cells polarize in a front-back manner owing to the emergence of actin retrograde flows powered by dendritic polymerization of actin. Coupled to cell movement, the flows transport myosin II from the front to the back of the cell, where the motor locally 'locks' actin in contractile bundles. This polarization mechanism could be employed by embryonic and cancer epithelial cells in microenvironments where high-contractility-driven cell motion is inefficient.

  6. Human endothelial actin-binding protein (ABP-280, nonmuscle filamin): a molecular leaf spring

    OpenAIRE

    1990-01-01

    Actin-binding protein (ABP-280, nonmuscle filamin) is a ubiquitous dimeric actin cross-linking phosphoprotein of peripheral cytoplasm, where it promotes orthogonal branching of actin filaments and links actin filaments to membrane glycoproteins. The complete nucleotide sequence of human endothelial cell ABP cDNA predicts a polypeptide subunit chain of 2,647 amino acids, corresponding to 280 kD, also the mass derived from physical measurements of the native protein. The actin-binding domain is...

  7. Intracellular Theileria annulata promote invasive cell motility through kinase regulation of the host actin cytoskeleton.

    Directory of Open Access Journals (Sweden)

    Min Ma

    2014-03-01

    Full Text Available The intracellular, protozoan Theileria species parasites are the only eukaryotes known to transform another eukaryotic cell. One consequence of this parasite-dependent transformation is the acquisition of motile and invasive properties of parasitized cells in vitro and their metastatic dissemination in the animal, which causes East Coast Fever (T. parva or Tropical Theileriosis (T. annulata. These motile and invasive properties of infected host cells are enabled by parasite-dependent, poorly understood F-actin dynamics that control host cell membrane protrusions. Herein, we dissected functional and structural alterations that cause acquired motility and invasiveness of T. annulata-infected cells, to understand the molecular basis driving cell dissemination in Tropical Theileriosis. We found that chronic induction of TNFα by the parasite contributes to motility and invasiveness of parasitized host cells. We show that TNFα does so by specifically targeting expression and function of the host proto-oncogenic ser/thr kinase MAP4K4. Blocking either TNFα secretion or MAP4K4 expression dampens the formation of polar, F-actin-rich invasion structures and impairs cell motility in 3D. We identified the F-actin binding ERM family proteins as MAP4K4 downstream effectors in this process because TNFα-induced ERM activation and cell invasiveness are sensitive to MAP4K4 depletion. MAP4K4 expression in infected cells is induced by TNFα-JNK signalling and maintained by the inhibition of translational repression, whereby both effects are parasite dependent. Thus, parasite-induced TNFα promotes invasive motility of infected cells through the activation of MAP4K4, an evolutionary conserved kinase that controls cytoskeleton dynamics and cell motility. Hence, MAP4K4 couples inflammatory signaling to morphodynamic processes and cell motility, a process exploited by the intracellular Theileria parasite to increase its host cell's dissemination capabilities.

  8. N-terminal Slit2 inhibits HIV-1 replication by regulating the actin cytoskeleton

    Directory of Open Access Journals (Sweden)

    Anand Appakkudal R

    2013-01-01

    Full Text Available Abstract Background Slit2 is a ~ 200 kDa secreted glycoprotein that has been recently shown to regulate immune functions. However, not much is known about its role in HIV (human immunodeficiency virus-1 pathogenesis. Results In the present study, we have shown that the N-terminal fragment of Slit2 (Slit2N (~120 kDa inhibits replication of both CXCR4 and CCR5-tropic HIV-1 viruses in T-cell lines and peripheral blood T-cells. Furthermore, we demonstrated inhibition of HIV-1 infection in resting CD4+ T-cells. In addition, we showed that Slit2N blocks cell-to-cell transmission of HIV-1. We have shown that Slit2N inhibits HIV-1 infection by blocking viral entry into T-cells. We also ruled out Slit2N-mediated inhibition of various other steps in the life cycle including binding, integration and viral transcription. Elucidation of the molecular mechanism revealed that Slit2N mediates its functional effects by binding to Robo1 receptor. Furthermore, we found that Slit2N inhibited Gp120-induced Robo1-actin association suggesting that Slit2N may inhibit cytoskeletal rearrangements facilitating HIV-1 entry. Studies into the mechanism of inhibition of HIV-1 revealed that Slit2N abrogated HIV-1 envelope-induced actin cytoskeletal dynamics in both T-cell lines and primary T-cells. We further showed that Slit2N specifically attenuated the HIV-1 envelope-induced signaling pathway consisting of Rac1, LIMK and cofilin that regulates actin polymerization. Conclusions Taken together, our results show that Slit2N inhibits HIV-1 replication through novel mechanisms involving modulation of cytoskeletal dynamics. Our study, thus, provides insights into the role of Slit2N in HIV-1 infection and underscores its potential in limiting viral replication in T-cells.

  9. Identification and Characterization of a Candidate Wolbachia pipientis Type IV Effector That Interacts with the Actin Cytoskeleton

    Directory of Open Access Journals (Sweden)

    Kathy B. Sheehan

    2016-07-01

    Full Text Available Many bacteria live as intracellular symbionts, causing persistent infections within insects. One extraordinarily common infection is that of Wolbachia pipientis, which infects 40% of insect species and induces reproductive effects. The bacteria are passed from generation to generation both vertically (through the oocyte and horizontally (by environmental transmission. Maintenance of the infection within Drosophila melanogaster is sensitive to the regulation of actin, as Wolbachia inefficiently colonizes strains hemizygous for the profilin or villin genes. Therefore, we hypothesized that Wolbachia must depend on the host actin cytoskeleton. In this study, we identify and characterize a Wolbachia protein (WD0830 that is predicted to be secreted by the bacterial parasite. Expression of WD0830 in a model eukaryote (the yeast Saccharomyces cerevisiae induces a growth defect associated with the appearance of aberrant, filamentous structures which colocalize with rhodamine-phalloidin-stained actin. Purified WD0830 bundles actin in vitro and cosediments with actin filaments, suggesting a direct interaction of the two proteins. We characterized the expression of WD0830 throughout Drosophila development and found it to be upregulated in third-instar larvae, peaking in early pupation, during the critical formation of adult tissues, including the reproductive system. In transgenic flies, heterologously expressed WD0830 localizes to the developing oocyte. Additionally, overexpression of WD0830 results in increased Wolbachia titers in whole flies, in stage 9 and 10 oocytes, and in embryos, compared to controls, suggesting that the protein may facilitate Wolbachia’s replication or transmission. Therefore, this candidate secreted effector may play a role in Wolbachia’s infection of and persistence within host niches.

  10. Foci of cyclin A2 interact with actin and RhoA in mitosis

    OpenAIRE

    Abdelhalim Loukil; Fanny Izard; Mariya Georgieva; Shaereh Mashayekhan; Jean-Marie Blanchard; Andrea Parmeggiani; Marion Peter

    2016-01-01

    Cyclin A2 is a key player in the regulation of the cell cycle. Its degradation in mid-mitosis depends primarily on the ubiquitin-proteasome system (UPS), while autophagy also contributes. However, a fraction of cyclin A2 persists beyond metaphase. In this work, we focus on cyclin A2-rich foci detected in mitosis by high resolution imaging and analyse their movements. We demonstrate that cyclin A2 interacts with actin and RhoA during mitosis, and that cyclin A2 depletion induces a dramatic dec...

  11. Ion Implantation Hampers Pollen Tube Growth and Disrupts Actin Cytoskeleton Organization in Pollen Tubes of Pinus thunbergii

    Institute of Scientific and Technical Information of China (English)

    LI Guoping; HUANG Qunce; YANG Lusheng; QIN Guangyong

    2008-01-01

    Pollen grains of Pinus thunbergii Parl. (Japanese black pine) were implanted with 30 keV nitrogen ion beams and the effects of nitrogen ion implantation on pollen tube growth in vitro and the organization of actin cytoskeleton in the pollen tube cell were investigated using a confocal laser scanning microscope after fluorescence labeling. Treatment with ion implanta-tion significantly blocked pollen tube growth. Confocal microscopy showed that ion implantation disrupted actin filament cytoskeleton organization in the pollen tube. It was found that there was a distinct correlation between the inhibition of pollen tube growth and the disruption of actin cytoskeleton organization, indicating that an intact actin cytoskeleton is essential for con-tinuous pollen tube elongation in Pinus thunbergii. Although the detailed mechanism for the ion-implantation-induced bioeffect still remains to be elucidated, the present study assumes that the cytoskeleton system in pollen grains may provide a key target in response to ion beam im-plantation and is involved in mediating certain subsequent cytological changes.

  12. Host-cell-dependent role of actin cytoskeleton during the replication of a human strain of influenza A virus.

    Science.gov (United States)

    Arcangeletti, M C; De Conto, F; Ferraglia, F; Pinardi, F; Gatti, R; Orlandini, G; Covan, S; Motta, F; Rodighiero, I; Dettori, G; Chezzi, C

    2008-01-01

    This study was aimed at investigating the possible involvement of the actin cytoskeleton in the modulation of host permissiveness to A/NWS/33 human influenza virus infection in two mammalian (MDCK and LLC-MK2) cell lines in vitro. During the early stages of infection, no appreciable association between incoming NWS/33 virions and cortical actin was detectable in the permissive MDCK model by confocal microscopy, while extensive colocalization and a slower infection progression were observed in LLC-MK2 cells. In the latter model, we also demonstrated the inability of the virus to carry out multiple replication cycles, irrespective of the presence of cleaved HA subunits in the released virions. Treatment with the actin-depolymerizing agent cytochalasin D significantly increased the infection efficiency in LLC-MK2 cells, while a detrimental effect was observed in the MDCK cell line. Our data suggest a selective role of the actin network in inducing a restriction to influenza virus replication, mostly depending on its molecular organization, the host cell type and virus replication phase. PMID:18488136

  13. Mechanics of Biomimetic Liposomes Encapsulating an Actin Shell.

    Science.gov (United States)

    Guevorkian, Karine; Manzi, John; Pontani, Léa-Lætitia; Brochard-Wyart, Françoise; Sykes, Cécile

    2015-12-15

    Cell-shape changes are insured by a thin, dynamic, cortical layer of cytoskeleton underneath the plasma membrane. How this thin cortical structure impacts the mechanical properties of the whole cell is not fully understood. Here, we study the mechanics of liposomes or giant unilamellar vesicles, when a biomimetic actin cortex is grown at the inner layer of the lipid membrane via actin-nucleation-promoting factors. Using a hydrodynamic tube-pulling technique, we show that tube dynamics is clearly affected by the presence of an actin shell anchored to the lipid bilayer. The same force pulls much shorter tubes in the presence of the actin shell compared to bare membranes. However, in both cases, we observe that the dynamics of tube extrusion has two distinct features characteristic of viscoelastic materials: rapid elastic elongation, followed by a slower elongation phase at a constant rate. We interpret the initial elastic regime by an increase of membrane tension due to the loss of lipids into the tube. Tube length is considerably shorter for cortex liposomes at comparable pulling forces, resulting in a higher spring constant. The presence of the actin shell seems to restrict lipid mobility, as is observed in the corral effect in cells. The viscous regime for bare liposomes corresponds to a leakout of the internal liquid at constant membrane tension. The presence of the actin shell leads to a larger friction coefficient. As the tube is pulled from a patchy surface, membrane tension increases locally, leading to a Marangoni flow of lipids. As a conclusion, the presence of an actin shell is revealed by its action that alters membrane mechanics.

  14. Arabidopsis AtADF1 is Functionally Affected by Mutations on Actin Binding Sites

    Institute of Scientific and Technical Information of China (English)

    Chun-Hai Dong; Wei-Ping Tang; Jia-Yao Liu

    2013-01-01

    The plant actin depolymerizing factor (ADF) binds to both monomeric and filamentous actin,and is directly involved in the depolymerization of actin filaments.To better understand the actin binding sites of the Arabidopsis thaliana L.AtADF1,we generated mutants of AtADF1 and investigated their functions in vitro and in vivo.Analysis of mutants harboring amino acid substitutions revealed that charged residues (Arg98 and Lys100) located at the α-helix 3 and forming an actin binding site together with the N-terminus are essential for both G-and F-actin binding.The basic residues on the β-strand 5 (K82/A) and the α-helix 4 (R135/A,R137/A) form another actin binding site that is important for F-actin binding.Using transient expression of CFP-tagged AtADF1 mutant proteins in onion (Allium cepa) peel epidermal cells and transgenic Arabidopsis thaliana L.plants overexpressing these mutants,we analyzed how these mutant proteins regulate actin organization and affect seedling growth.Our results show that the ADF mutants with a lower affinity for actin filament binding can still be functional,unless the affinity foractin monomers is also affected.The G-actin binding activity of the ADF plays an essential role in actin binding,depolymerization of actin polymers,and therefore in the control of actin organization.

  15. Dynamic Regulation of Activated Leukocyte Cell Adhesion Molecule–mediated Homotypic Cell Adhesion through the Actin CytoskeletonV⃞

    OpenAIRE

    Nelissen, Judith M. D. T.; Peters, Inge M.; de Grooth, Bart G.; Van Kooyk, Yvette; Figdor, Carl G.

    2000-01-01

    Restricted expression of activated leukocyte cell adhesion molecule (ALCAM) by hematopoietic cells suggests an important role in the immune system and hematopoiesis. To get insight into the mechanisms that control ALCAM-mediated adhesion we have investigated homotypic ALCAM–ALCAM interactions. Here, we demonstrate that the cytoskeleton regulates ALCAM-mediated cell adhesion because inhibition of actin polymerization by cytochalasin D (CytD) strongly induces homotypic ALCAM–ALCAM interactions....

  16. Effect of actin microfilament on potassium current in guinea pig gastric myocytes

    Institute of Scientific and Technical Information of China (English)

    Xiang-Lan Li; Hai-Feng Zheng; Zherg-Yuan Jin; Meng Yang; Zai-Liu Li; Wen-Xie Xu

    2004-01-01

    AIM: To investigate the effect of actin microfilament on potassium current and hyposmotic membrane stretch-induced increase of potassium current in gastric antral circular myocytes of guinea pig.METHODS: Whole-cell patch clamp technique was used to record potassium current in isolated gastric myocyes.RESULTS: When the membrane potential was clamped at-60 mV, an actin microfilament disruptor, cytochanlasin-B(Cyt-B, 20 μmol/L in pipette) increased calcium-activated potassium current (IK(Ca)) and delayed rectifier potassium current (IK(V))to 138.4±14.3% and 142.1±13.1%respectively at +60 mV. In the same condition, an actin microfilament stabilizer phalloidin(20 μmol/L in pipette)inhibited IK(Ca) and IK(V) to 74.2±7.1% and 75.4±9.9%respectively. At the holding potential of -60 mV, hyposmotic membrane stretch increased IK(Ca)and IK(V) by 50.6±9.7%and 24.9±3.3% at +60 mV respectively. In the presence of cytochalasin-B and phalloidin (20 μmol/L, in the pipette)condition, hyposmotic membrane stretch also increased IK(Ca)by 44.5±7.9% and 55.7±9.8% at +60 mV respectively. In the same condition, cytochalasin-B and phalloidin also increased IK(V) by 23.0±5.5% and 30.3±4.5% respectively. However,Cyt-B and phalloidin did not affect the amplitude of hyposmotic membrane stretch-induced increase of IK(Ca)and Ik(V).CONCLUSION: Actin microfilaments regulate the activities of potassium channels, but they are not involved in the process of hyposmotic membrane stretch-induced increase of potassium currents in gastric antral circular myocytes of guinea pig.

  17. Interactions between the yeast SM22 homologue Scp1 and actin demonstrate the importance of actin bundling in endocytosis.

    Science.gov (United States)

    Gheorghe, Dana M; Aghamohammadzadeh, Soheil; Smaczynska-de Rooij, Iwona I; Allwood, Ellen G; Winder, Steve J; Ayscough, Kathryn R

    2008-05-30

    The yeast SM22 homologue Scp1 has previously been shown to act as an actin-bundling protein in vitro. In cells, Scp1 localizes to the cortical actin patches that form as part of the invagination process during endocytosis, and its function overlaps with that of the well characterized yeast fimbrin homologue Sac6p. In this work we have used live cell imaging to demonstrate the importance of key residues in the Scp1 actin interface. We have defined two actin binding domains within Scp1 that allow the protein to both bind and bundle actin without the need for dimerization. Green fluorescent protein-tagged mutants of Scp1 also indicate that actin localization does not require the putative phosphorylation site Ser-185 to be functional. Deletion of SCP1 has few discernable effects on cell growth and morphology. However, we reveal that scp1 deletion is compensated for by up-regulation of Sac6. Furthermore, Scp1 levels are increased in the absence of sac6. The presence of compensatory pathways to up-regulate Sac6 or Scp1 levels in the absence of the other suggest that maintenance of sufficient bundling activity is critical within the cell. Analysis of cortical patch assembly and movement during endocytosis reveals a previously undetected role for Scp1 in movement of patches away from the plasma membrane. Additionally, we observe a dramatic increase in patch lifetime in a strain lacking both sac6 and scp1, demonstrating the central role played by actin-bundling proteins in the endocytic process. PMID:18400761

  18. State transitions of actin cortices in vitro and in vivo

    Science.gov (United States)

    Tan, Tzer Han; Keren, Kinneret; Mackintosh, Fred; Schmidt, Christoph; Fakhri, Nikta

    Most animal cells are enveloped by a thin layer of actin cortex which governs the cell mechanics. A functional cortex must be rigid to provide mechanical support while being flexible to allow for rapid restructuring events such as cell division. To satisfy these requirements, the actin cortex is highly dynamic with fast actin turnover and myosin-driven contractility. The regulatory mechanism responsible for the transition between a mechanically stable state and a restructuring state is not well understood. Here, we develop a technique to map the dynamics of reconstituted actin cortices in emulsion droplets using IR fluorescent single-walled carbon nanotubes (SWNTs). By increasing crosslinker concentration, we find that a homogeneous cortex transitions to an intermediate state with broken rotational symmetry and a globally contractile state which further breaks translational symmetry. We apply this new dynamic mapping technique to cortices of live starfish oocytes in various developmental stages. To identify the regulatory mechanism for steady state transitions, we subject the oocytes to actin and myosin disrupting drugs.

  19. Cortactin promotes exosome secretion by controlling branched actin dynamics.

    Science.gov (United States)

    Sinha, Seema; Hoshino, Daisuke; Hong, Nan Hyung; Kirkbride, Kellye C; Grega-Larson, Nathan E; Seiki, Motoharu; Tyska, Matthew J; Weaver, Alissa M

    2016-07-18

    Exosomes are extracellular vesicles that influence cellular behavior and enhance cancer aggressiveness by carrying bioactive molecules. The mechanisms that regulate exosome secretion are poorly understood. Here, we show that the actin cytoskeletal regulatory protein cortactin promotes exosome secretion. Knockdown or overexpression of cortactin in cancer cells leads to a respective decrease or increase in exosome secretion, without altering exosome cargo content. Live-cell imaging revealed that cortactin controls both trafficking and plasma membrane docking of multivesicular late endosomes (MVEs). Regulation of exosome secretion by cortactin requires binding to the branched actin nucleating Arp2/3 complex and to actin filaments. Furthermore, cortactin, Rab27a, and coronin 1b coordinately control stability of cortical actin MVE docking sites and exosome secretion. Functionally, the addition of purified exosomes to cortactin-knockdown cells rescued defects of those cells in serum-independent growth and invasion. These data suggest a model in which cortactin promotes exosome secretion by stabilizing cortical actin-rich MVE docking sites. PMID:27402952

  20. Cortactin Adopts a Globular Conformation and Bundles Actin into Sheets

    Energy Technology Data Exchange (ETDEWEB)

    Cowieson, Nathan P.; King, Gordon; Cookson, David; Ross, Ian; Huber, Thomas; Hume, David A.; Kobe, Bostjan; Martin, Jennifer L. (Queensland); (Aust. Synch.)

    2008-08-21

    Cortactin is a filamentous actin-binding protein that plays a pivotal role in translating environmental signals into coordinated rearrangement of the cytoskeleton. The dynamic reorganization of actin in the cytoskeleton drives processes including changes in cell morphology, cell migration, and phagocytosis. In general, structural proteins of the cytoskeleton bind in the N-terminal region of cortactin and regulatory proteins in the C-terminal region. Previous structural studies have reported an extended conformation for cortactin. It is therefore unclear how cortactin facilitates cross-talk between structural proteins and their regulators. In the study presented here, circular dichroism, chemical cross-linking, and small angle x-ray scattering are used to demonstrate that cortactin adopts a globular conformation, thereby bringing distant parts of the molecule into close proximity. In addition, the actin bundling activity of cortactin is characterized, showing that fully polymerized actin filaments are bundled into sheet-like structures. We present a low resolution structure that suggests how the various domains of cortactin interact to coordinate its array of binding partners at sites of actin branching.

  1. Antenna Mechanism of Length Control of Actin Cables.

    Directory of Open Access Journals (Sweden)

    Lishibanya Mohapatra

    2015-06-01

    Full Text Available Actin cables are linear cytoskeletal structures that serve as tracks for myosin-based intracellular transport of vesicles and organelles in both yeast and mammalian cells. In a yeast cell undergoing budding, cables are in constant dynamic turnover yet some cables grow from the bud neck toward the back of the mother cell until their length roughly equals the diameter of the mother cell. This raises the question: how is the length of these cables controlled? Here we describe a novel molecular mechanism for cable length control inspired by recent experimental observations in cells. This "antenna mechanism" involves three key proteins: formins, which polymerize actin, Smy1 proteins, which bind formins and inhibit actin polymerization, and myosin motors, which deliver Smy1 to formins, leading to a length-dependent actin polymerization rate. We compute the probability distribution of cable lengths as a function of several experimentally tuneable parameters such as the formin-binding affinity of Smy1 and the concentration of myosin motors delivering Smy1. These results provide testable predictions of the antenna mechanism of actin-cable length control.

  2. Emerging roles of actin cytoskeleton regulating enzymes in drug addiction: Actin or reactin’?

    Science.gov (United States)

    Rothenfluh, Adrian; Cowan, Christopher W.

    2013-01-01

    Neurons rely on their cytoskeleton to give them shape and stability, and on cytoskeletal dynamics for growth and synaptic plasticity. Because drug addiction is increasingly seen as the inappropriate learning of strongly reinforcing stimuli, the role of the cytoskeleton in shaping drug memories has been of increasing interest in recent years. Does the cytoskeleton have an active role in shaping these memories, and to what extent do alterations in the cytoskeleton reflect the acute actions of drug exposure, or homeostatic reactions to the chronic exposure to drugs of abuse? Here we will review recent advances in understanding the role of the cytoskeleton in the development of drug addiction, with a focus on actin filaments, as they have been studied in greater detail. PMID:23428655

  3. Tetraspanin CD82 inhibits protrusion and retraction in cell movement by attenuating the plasma membrane-dependent actin organization.

    Directory of Open Access Journals (Sweden)

    Wei M Liu

    Full Text Available To determine how tetraspanin KAI1/CD82, a tumor metastasis suppressor, inhibits cell migration, we assessed which cellular events critical for motility are altered by KAI1/CD82 and how KAI1/CD82 regulates these events. We found that KAI1/CD82-expressing cells typically exhibited elongated cellular tails and diminished lamellipodia. Live imaging demonstrated that the polarized protrusion and retraction of the plasma membrane became deficient upon KAI1/CD82 expression. The deficiency in developing these motility-related cellular events was caused by poor formations of actin cortical network and stress fiber and by aberrant dynamics in actin organization. Rac1 activity was reduced by KAI1/CD82, consistent with the diminution of lamellipodia and actin cortical network; while the growth factor-stimulated RhoA activity was blocked by KAI1/CD82, consistent with the loss of stress fiber and attenuation in cellular retraction. Upon KAI1/CD82 expression, Rac effector cofilin was not enriched at the cell periphery to facilitate lamellipodia formation while Rho kinase exhibited a significantly lower activity leading to less retraction. Phosphatidylinositol 4, 5-biphosphate, which initiates actin polymerization from the plasma membrane, became less detectable at the cell periphery in KAI1/CD82-expressing cells. Moreover, KAI1/CD82-induced phenotypes likely resulted from the suppression of multiple signaling pathways such as integrin and growth factor signaling. In summary, at the cellular level KAI1/CD82 inhibited polarized protrusion and retraction events by disrupting actin reorganization; at the molecular level, KAI1/CD82 deregulated Rac1, RhoA, and their effectors cofilin and Rho kinase by perturbing the plasma membrane lipids.

  4. New Insights into Dynamic Actin-Based Chloroplast Photorelocation Movement

    Institute of Scientific and Technical Information of China (English)

    Sam-Geun Kong; Masamitsu Wada

    2011-01-01

    Chloroplast movement is essential for plants to survive under various environmental light conditions.Phototropins-plant-specific blue-light-activated receptor kinases-mediate the response by perceiving light intensity and direction.Recently,novel chloroplast actin (cp-actin) filaments have been identified as playing a pivotal role in the directional chloroplast photorelocation movement.Encouraging progress has recently been made in this field of research through molecular genetics and cell biological analyses.This review describes factors that have been identified as being involved in chloroplast movement and their roles in the regulation of cp-actin filaments,thus providing a basis for reflection on their biochemical activities and functions.

  5. Dissecting principles governing actin assembly using yeast extracts.

    Science.gov (United States)

    Michelot, Alphée; Drubin, David G

    2014-01-01

    In this chapter, we describe recent protocols that we have developed to trigger actin assembly and actin-based motility in yeast cell extracts. Our method allows for the fast preparation of yeast extracts that are competent in dynamic assembly of distinct actin filament structures of biologically appropriate protein composition. Compared to previous extract-based systems using other eukaryotic cell types, yeast provides a unique advantage for combining reconstituted assays with the preparation of extracts from genetically modified yeast strains. We present a global strategy for dissecting the functions of individual proteins, where the activities of the proteins are analyzed in systems of variable complexity, ranging from simple mixtures of pure proteins to the full complexity of a cell's cytoplasm.

  6. Formation of actin networks in microfluidic concentration gradients

    Science.gov (United States)

    Strelnikova, Natalja; Herren, Florian; Schoenenberger, Cora-Ann; Pfohl, Thomas

    2016-05-01

    The physical properties of cytoskeletal networks are contributors in a number of mechanical responses of cells including cellular deformation and locomotion, and are crucial for the proper action of living cells. Local chemical gradients modulate cytoskeletal functionality including the interactions of the cytoskeleton with other cellular components. Actin is a major constituent of the cytoskeleton. Introducing a microfluidic-based platform, we explored the impact of concentration gradients on the formation and structural properties of actin networks. Microfluidics-controlled flow-free steady state experimental conditions allow for the generation of chemical gradients of different profiles, such as linear or step-like. We discovered specific features of actin networks emerging in defined gradients. In particular, we analyzed the effects of spatial conditions on network properties, bending rigidities of network links, and the network elasticity.

  7. Signaling of the p21-activated kinase (PAK1) coordinates insulin-stimulated actin remodeling and glucose uptake in skeletal muscle cells.

    Science.gov (United States)

    Tunduguru, Ragadeepthi; Chiu, Tim T; Ramalingam, Latha; Elmendorf, Jeffrey S; Klip, Amira; Thurmond, Debbie C

    2014-11-15

    Skeletal muscle accounts for ∼ 80% of postprandial glucose clearance, and skeletal muscle glucose clearance is crucial for maintaining insulin sensitivity and euglycemia. Insulin-stimulated glucose clearance/uptake entails recruitment of glucose transporter 4 (GLUT4) to the plasma membrane (PM) in a process that requires cortical F-actin remodeling; this process is dysregulated in Type 2 Diabetes. Recent studies have implicated PAK1 as a required element in GLUT4 recruitment in mouse skeletal muscle in vivo, although its underlying mechanism of action and requirement in glucose uptake remains undetermined. Toward this, we have employed the PAK1 inhibitor, IPA3, in studies using L6-GLUT4-myc muscle cells. IPA3 fully ablated insulin-stimulated GLUT4 translocation to the PM, corroborating the observation of ablated insulin-stimulated GLUT4 accumulation in the PM of skeletal muscle from PAK1(-/-) knockout mice. IPA3-treatment also abolished insulin-stimulated glucose uptake into skeletal myotubes. Mechanistically, live-cell imaging of myoblasts expressing the F-actin biosensor LifeAct-GFP treated with IPA3 showed blunting of the normal insulin-induced cortical actin remodeling. This blunting was underpinned by a loss of normal insulin-stimulated cofilin dephosphorylation in IPA3-treated myoblasts. These findings expand upon the existing model of actin remodeling in glucose uptake, by placing insulin-stimulated PAK1 signaling as a required upstream step to facilitate actin remodeling and subsequent cofilin dephosphorylation. Active, dephosphorylated cofilin then provides the G-actin substrate for continued F-actin remodeling to facilitate GLUT4 vesicle translocation for glucose uptake into the skeletal muscle cell.

  8. Identification of Actin-Binding Proteins from Maize Pollen

    Energy Technology Data Exchange (ETDEWEB)

    Staiger, C.J.

    2004-01-13

    Specific Aims--The goal of this project was to gain an understanding of how actin filament organization and dynamics are controlled in flowering plants. Specifically, we proposed to identify unique proteins with novel functions by investigating biochemical strategies for the isolation and characterization of actin-binding proteins (ABPs). In particular, our hunt was designed to identify capping proteins and nucleation factors. The specific aims included: (1) to use F-actin affinity chromatography (FAAC) as a general strategy to isolate pollen ABPs (2) to produce polyclonal antisera and perform subcellular localization in pollen tubes (3) to isolate cDNA clones for the most promising ABPs (4) to further purify and characterize ABP interactions with actin in vitro. Summary of Progress By employing affinity chromatography on F-actin or DNase I columns, we have identified at least two novel ABPs from pollen, PrABP80 (gelsolin-like) and ZmABP30, We have also cloned and expressed recombinant protein, as well as generated polyclonal antisera, for 6 interesting ABPs from Arabidopsis (fimbrin AtFIM1, capping protein a/b (AtCP), adenylyl cyclase-associated protein (AtCAP), AtCapG & AtVLN1). We performed quantitative analyses of the biochemical properties for two of these previously uncharacterized ABPs (fimbrin and capping protein). Our studies provide the first evidence for fimbrin activity in plants, demonstrate the existence of barbed-end capping factors and a gelsolin-like severing activity, and provide the quantitative data necessary to establish and test models of F-actin organization and dynamics in plant cells.

  9. Filament attachment dynamics in actin-based propulsion

    CERN Document Server

    Katz, J I

    2005-01-01

    Theory and experiment have established that F-actin filaments are strongly attached to the intracellular parasites (such as Listeria) they propel with ``comet tails''. We consider the implications of these observations for propulsion. By calculating the motion produced in various models of attachment and comparing to experiment we demonstrate that the attachment must be sliding rather than hinged. By modeling experiments on ActA-coated spheres we draw conclusions regarding the interaction between F-actin and their surfaces that may also be applicable to living systems.

  10. Modelling phagosomal lipid networks that regulate actin assembly

    Directory of Open Access Journals (Sweden)

    Schwarz Roland

    2008-12-01

    Full Text Available Abstract Background When purified phagosomes are incubated in the presence of actin under appropriate conditions, microfilaments start growing from the membrane in a process that is affected by ATP and the lipid composition of the membrane. Isolated phagosomes are metabolically active organelles that contain enzymes and metabolites necessary for lipid interconversion. Hence, addition of ATP, lipids, and actin to the system alter the steady-state composition of the phagosomal membrane at the same time that the actin nucleation is initiated. Our aim was to model all these processes in parallel. Results We compiled detailed experimental data on the effects of different lipids and ATP on actin nucleation and we investigated experimentally lipid interconversion and ATP metabolism in phagosomes by using suitable radioactive compounds. In a first step, a complex lipid network interconnected by chemical reactions catalyzed by known enzymes was modelled in COPASI (Complex Pathway Simulator. However, several lines of experimental evidence indicated that only the phosphatidylinositol branch of the network was active, an observation that dramatically reduced the number of parameters in the model. The results also indicated that a lipid network-independent ATP-consuming activity should be included in the model. When this activity was introduced, the set of differential equations satisfactorily reproduced the experimental data. On the other hand, a molecular mechanism connecting membrane lipids, ATP, and the actin nucleation process is still missing. We therefore adopted a phenomenological (black-box approach to represent the empirical observations. We proposed that lipids and ATP influence the dynamic interconversion between active and inactive actin nucleation sites. With this simple model, all the experimental data were satisfactorily fitted with a single positive parameter per lipid and ATP. Conclusion By establishing an active 'dialogue' between an

  11. Health related quality of life in patients with actinic keratosis

    DEFF Research Database (Denmark)

    Tennvall, Gunnel Ragnarson; Norlin, J M; Malmberg, I;

    2015-01-01

    BACKGROUND: Actinic keratosis (AK) is a common skin condition that may progress to non-melanoma skin cancer (NMSC). The disease may influence Health Related Quality of Life (HRQoL), but studies of HRQoL in patients with AK are limited. The purpose of the study was to analyze HRQoL in patients......-center setting. Dermatologists assessed AK severity and patients completed: Actinic Keratosis Quality of Life Questionnaire (AKQoL), Dermatology Life Quality Index (DLQI), and EQ-5D-5 L including EQ-VAS. Differences between categorical subgroups were tested with Wilcoxon rank-sum test. The relationship between...

  12. Actin and Arp2/3 localize at the centrosome of interphase cells

    Energy Technology Data Exchange (ETDEWEB)

    Hubert, Thomas; Vandekerckhove, Joel; Gettemans, Jan, E-mail: jan.gettemans@vib-ugent.be

    2011-01-07

    Research highlights: {yields} Actin was detected at the centrosome with the anti-actin antibody 1C7 that recognizes antiparallel ('lower dimer') actin dimers. {yields} Centrosomal actin was found in interphase but not mitotic MDA-MB-231 cells. {yields} Neither the anti-actin antibody C4 that binds to globular, monomer actin, nor the anti-actin antibody 2G2 that recognizes the nuclear conformation of actin detect actin at the centrosome. {yields} The Arp2/3 complex transiently localizes at the pericentriolar matrix but not at the centrioles of interphase HEK 293T cells. -- Abstract: Although many actin binding proteins such as cortactin and the Arp2/3 activator WASH localize at the centrosome, the presence and conformation of actin at the centrosome has remained elusive. Here, we report the localization of actin at the centrosome in interphase but not in mitotic MDA-MB-231 cells. Centrosomal actin was detected with the anti-actin antibody 1C7 that recognizes antiparallel ('lower dimer') actin dimers. In addition, we report the transient presence of the Arp2/3 complex at the pericentriolar matrix but not at the centrioles of interphase HEK 293T cells. Overexpression of an Arp2/3 component resulted in expansion of the pericentriolar matrix and selective accumulation of the Arp2/3 component in the pericentriolar matrix. Altogether, we hypothesize that the centrosome transiently recruits Arp2/3 to perform processes such as centrosome separation prior to mitotic entry, whereas the observed constitutive centrosomal actin staining in interphase cells reinforces the current model of actin-based centrosome reorientation toward the leading edge in migrating cells.

  13. Glutathionylation of beta-actin via a cysteinyl sulfenic acid intermediary

    Directory of Open Access Journals (Sweden)

    Lundberg Mathias

    2007-12-01

    Full Text Available Abstract Background Cysteinyl residues in actin are glutathionylated, ie. form a mixed disulfide with glutathione, even in the absence of exogenous oxidative stress. Glutathionylation inhibits actin polymerization and reversible actin glutathionylation is a redox dependent mechanism for regulation of the cytoskeleton structure. The molecular mechanism that mediates actin glutathionylation in vivo is unclear. Results We have studied glutathionylation of α- and β-actin in vitro using an enzyme-linked immunosorbant assay with a monoclonal anti-glutathione antibody. α- and β-actin were both glutathionylated when incubated with reduced glutathione (GSH combined with diamide as a thiol oxidant. However, β-actin was also glutathionylated by both glutathione disulfide (GSSG and GSH in the absence of diamide whereas α-actin was poorly glutathionylated by GSH or GSSG. Glutathionylation of β-actin by GSSG is likely to be mediated by a thiol-exchange mechanism whereas glutathionylation by GSH requires thiol oxidation. β-actin glutathionylation by GSH was inhibited by arsenite and dimedone suggesting that the mechanism involved formation of a cysteinyl sulfenic acid residue in β-actin. Conclusion We conclude that glutathionylation of β-actin may occur via spontaneous oxidation of a cysteinyl residue to a sulfenic acid that readily reacts with GSH to form a mixed disulfide. We also show that the reactivity and oxidation to a reactive protein thiol intermediary differ between different actin isoforms.

  14. Cdc42 and phosphoinositide 3-kinase drive Rac-mediated actin polymerization downstream of c-Met in distinct and common pathways

    DEFF Research Database (Denmark)

    Bosse, Tanja; Ehinger, Julia; Czuchra, Aleksandra;

    2007-01-01

    Activation of c-Met, the hepatocyte growth factor (HGF)/scatter factor receptor induces reorganization of the actin cytoskeleton, which drives epithelial cell scattering and motility and is exploited by pathogenic Listeria monocytogenes to invade nonepithelial cells. However, the precise...... required the simultaneous inactivation of both Cdc42 and PI3-kinase signaling. Moreover, Cdc42 activation was fully independent of PI3-kinase activity, whereas the latter partly depended on Cdc42. Finally, Cdc42 function did not require its interaction with the actin nucleation-promoting factor N...

  15. Intranasal immunisation with recombinant Toxoplasma gondii actin partly protects mice against toxoplasmosis.

    Directory of Open Access Journals (Sweden)

    Li-Tian Yin

    Full Text Available Toxoplasma gondii is a ubiquitous protozoan intracellular parasite, the causative agent of toxoplasmosis, and a worldwide zoonosis for which an effective vaccine is needed. Actin is a highly conserved microfilament protein that plays an important role in the invasion of host cells by T. gondii. This study investigated the immune responses elicited by BALB/c mice after nasal immunisation with a recombinant T. gondii actin (rTgACT and the subsequent protection against chronic and lethal T. gondii infections. We evaluated the systemic response by proliferation, cytokine and antibody measurements, and we assessed the mucosal response by examining the levels of TgACT-specific secretory IgA (SIgA in nasal, vaginal and intestinal washes. Parasite load was assessed in the liver and brain, and the survival of mice challenged with a virulent strain was determined. The results showed that the mice immunised with rTgACT developed high levels of specific anti-rTgACT IgG titres and a mixed IgG1/IgG2a response with a predominance of IgG2a. The systemic immune response was associated with increased production of Th1 (IFN-γ and IL-2, Th2 (IL-4 and Treg (IL-10 cytokines, indicating that not only Th1-type response was induced, but also Th2- and Treg-types responses were induced, and the splenocyte stimulation index (SI was increased in the mice immunised with rTgACT. Nasal immunisation with rTgACT led to strong mucosal immune responses, as seen by the increased secretion of SIgA in nasal, vaginal and intestinal washes. The vaccinated mice displayed significant protection against lethal infection with the virulent RH strain (survival increased by 50%, while the mice chronically infected with RH exhibited lower liver and brain parasite loads (60.05% and 49.75%, respectively than the controls. Our data demonstrate, for the first time, that actin triggers a strong systemic and mucosal response against T. gondii. Therefore, actin may be a promising vaccine candidate

  16. Simvastatin enhances Rho/actin/cell rigidity pathway contributing to mesenchymal stem cells’ osteogenic differentiation

    Directory of Open Access Journals (Sweden)

    Tai IC

    2015-09-01

    Full Text Available I-Chun Tai,1–3 Yao-Hsien Wang,3 Chung-Hwan Chen,3,4 Shu-Chun Chuang,3 Je-Ken Chang,3–5 Mei-Ling Ho1–3,6 1Graduate Institute of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan; 2Department of Physiology, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan; 3Orthopaedic Research Center, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan; 4Department of Orthopedics, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan; 5Department of Orthopedics, Kaohsiung Municipal Ta-Tung Hospital, Kaohsiung, Taiwan; 6Department of Marine Biotechnology and Resources, National Sun Yat-sen UniVersity, Kaohsiung, Taiwan Abstract: Recent studies have indicated that statins induce osteogenic differentiation both in vitro and in vivo. The molecular mechanism of statin-stimulated osteogenesis is unknown. Activation of RhoA signaling increases cytoskeletal tension, which plays a crucial role in the osteogenic differentiation of mesenchymal stem cells. We thus hypothesized that RhoA signaling is involved in simvastatin-induced osteogenesis in bone marrow mesenchymal stem cells. We found that although treatment with simvastatin shifts localization of RhoA protein from the membrane to the cytosol, the treatment still activates RhoA dose-dependently because it reduces the association with RhoGDIα. Simvastatin also increased the expression of osteogenic proteins, density of actin filament, the number of focal adhesions, and cellular tension. Furthermore, disrupting actin cytoskeleton or decreasing cell rigidity by using chemical agents reduced simvastatin-induced osteogenic differentiation. In vivo study also confirms that density of actin filament is increased in simvastatin-induced ectopic bone formation. Our study is the first to demonstrate that maintaining intact actin cytoskeletons and enhancing cell rigidity are crucial in simvastatin-induced osteogenesis. The results suggested that simvastatin, which is an

  17. Identification of regions within the Legionella pneumophila VipA effector protein involved in actin binding and polymerization and in interference with eukaryotic organelle trafficking.

    Science.gov (United States)

    Bugalhão, Joana N; Mota, Luís Jaime; Franco, Irina S

    2016-02-01

    The Legionella pneumophila effector protein VipA is an actin nucleator that co-localizes with actin filaments and early endosomes in infected macrophages and which interferes with organelle trafficking when expressed in yeast. To identify the regions of VipA involved in its subcellular localization and functions, we ectopically expressed specific VipA mutant proteins in eukaryotic cells. This indicated that the characteristic punctate distribution of VipA depends on its NH2 -terminal (amino acid residues 1-133) and central coiled-coil (amino acid residues 133-206) regions, and suggested a role for the COOH-terminal (amino acid residues 206-339) region in association with actin filaments and for the NH2 -terminal in co-localization with early endosomes. Co-immunoprecipitation and in vitro assays showed that the COOH-terminal region of VipA is necessary and sufficient to mediate actin binding, and is essential but insufficient to induce microfilament formation. Assays in yeast revealed that the NH2 and the COOH-terminal regions, and possibly an NPY motif within the NH2 region of VipA, are necessary for interference with organelle trafficking. Overall, this suggests that subversion of eukaryotic vesicular trafficking by VipA involves both its ability to associate with early endosomes via its NH2 -terminal region and its capacity to bind and polymerize actin through its COOH-terminal region. PMID:26626407

  18. Analysis of cytoskeleton dynamics and cell migration in drosophila ovaries using GFP-actin and E-cadherin-GFP fusion molecules

    Science.gov (United States)

    Verkhusha, Vladyslav V.; Tsukita, Shoichiro; Oda, Hiroki

    1999-06-01

    Coordination of cell migration and adhesion is essential for movement of tissues during morphogenesis. During Drosophila oogenesis so called border cells (BCs) break from an anterior epithelium of egg chamber, acquire a mesenchymal-like morphology, and migrate posteriorly between nurse cells to oocyte. The confocal microscopic observation of BCs has revealed well-developed forepart lamellipodium stained with Drosophila E-cadherin (DE-cadherin), PS2 integrin, cytoplasmic myosin and F-actin. To investigate mechanism of BC migration in vivo we have constructed a DE-cadherin-GFP and a GFP-actin fusion proteins and induced their expression BCs utilizing the UAS/GAL4 system. The DE-cadherin-GFP signal as well as immunostaining of PS2 integrin visualized a track of migrating BCs providing an evidence that adhesive molecules are pulled out and left behind on the surface of nurse cells. Our data suggest that two distinct adhesive systems, DE-cadherins and PS2 integrins simultaneously mediate the migration of BCs. Release of adhesive contacts in the tail region is a rate- limited event in BC migration. The spatial-temporal sequence of actin-based events visualized by the GFP-actin suggest a treadmilling model for actin behavior in BC lamellipodium. BC migration can be considered as simultaneous reiterating processes of lamellipodium extension and adhesive attachment, cytoskeletal contraction, and rear detachment.

  19. Loss of cofilin 1 disturbs actin dynamics, adhesion between enveloping and deep cell layers and cell movements during gastrulation in zebrafish.

    Directory of Open Access Journals (Sweden)

    Chun-Wei Lin

    Full Text Available During gastrulation, cohesive migration drives associated cell layers to the completion of epiboly in zebrafish. The association of different layers relies on E-cadherin based cellular junctions, whose stability can be affected by actin turnover. Here, we examined the effect of malfunctioning actin turnover on the epibolic movement by knocking down an actin depolymerizing factor, cofilin 1, using antisense morpholino oligos (MO. Knockdown of cfl1 interfered with epibolic movement of deep cell layer (DEL but not in the enveloping layer (EVL and the defect could be specifically rescued by overexpression of cfl1. It appeared that the uncoordinated movements of DEL and EVL were regulated by the differential expression of cfl1 in the DEL, but not EVL as shown by in situ hybridization. The dissociation of DEL and EVL was further evident by the loss of adhesion between layers by using transmission electronic and confocal microscopy analyses. cfl1 morphants also exhibited abnormal convergent extension, cellular migration and actin filaments, but not involution of hypoblast. The cfl1 MO-induced cell migration defect was found to be cell-autonomous in cell transplantation assays. These results suggest that proper actin turnover mediated by Cfl1 is essential for adhesion between DEL and EVL and cell movements during gastrulation in zebrafish.

  20. Fimbrin phosphorylation by metaphase Cdk1 regulates actin cable dynamics in budding yeast.

    Science.gov (United States)

    Miao, Yansong; Han, Xuemei; Zheng, Liangzhen; Xie, Ying; Mu, Yuguang; Yates, John R; Drubin, David G

    2016-01-01

    Actin cables, composed of actin filament bundles nucleated by formins, mediate intracellular transport for cell polarity establishment and maintenance. We previously observed that metaphase cells preferentially promote actin cable assembly through cyclin-dependent kinase 1 (Cdk1) activity. However, the relevant metaphase Cdk1 targets were not known. Here we show that the highly conserved actin filament crosslinking protein fimbrin is a critical Cdk1 target for actin cable assembly regulation in budding yeast. Fimbrin is specifically phosphorylated on threonine 103 by the metaphase cyclin-Cdk1 complex, in vivo and in vitro. On the basis of conformational simulations, we suggest that this phosphorylation stabilizes fimbrin's N-terminal domain, and modulates actin filament binding to regulate actin cable assembly and stability in cells. Overall, this work identifies fimbrin as a key target for cell cycle regulation of actin cable assembly in budding yeast, and suggests an underlying mechanism.

  1. N-Acylhomoserine lactones are potent neutrophil chemoattractants that act via calcium mobilization and actin remodeling.

    Science.gov (United States)

    Karlsson, Thommie; Musse, Farah; Magnusson, Karl-Eric; Vikström, Elena

    2012-01-01

    In gram-negative bacteria, cell-cell communication based on HSL QS molecules is known to coordinate the production of virulence factors and biofilms. These bacterial signals can also modulate human immune cell behavior. Using a Transwell migration assay, we found that human primary neutrophils are strongly stimulated by 3O-C(12)-HSL and -C(10)-HSL but not C(4)-HSL in a concentration-dependent manner. Moreover, 3O-C(12)-HSL and -C(10)-HSL activate PLCγ1 but not -γ2, mobilize intracellular calcium, and up-regulate IP(3)R. These changes were paralleled by F-actin accumulation, primarily in the leading edge of neutrophils, as evidenced by phalloidin staining and confocal microscopy. F- and G-actin isolation and quantification by immunoblotting revealed that the F/G-actin ratio was increased significantly after treatment with all three HSLs. Furthemore, 3O-C(12)-HSL- and 3O-C(10)-HSL treatment resulted in phosphorylation of Rac1 and Cdc42. In contrast, C(4)-HSL had negligible influence on the phosphorylation status of PLC and Rac1/Cdc42 and failed to attract neutrophils and induce calcium release. The calcium inhibitor thapsigargin, which blocks ER calcium uptake, strongly prevented neutrophil migration toward 3O-C(12)-HSL and -C(10)-HSL. These findings show that the bacterial QS molecules 3O-C(12)-HSL and -C(10)-HSL may attract human neutrophils to the sites of bacterial infection and developing biofilms. Indeed, recognition of HSL QS signals by neutrophils may play a critical role in their recruitment during infections.

  2. Wolbachia utilize host actin for efficient maternal transmission in Drosophila melanogaster.

    Directory of Open Access Journals (Sweden)

    Irene L G Newton

    2015-04-01

    Full Text Available Wolbachia pipientis is a ubiquitous, maternally transmitted bacterium that infects the germline of insect hosts. Estimates are that Wolbachia infect nearly 40% of insect species on the planet, making it the most prevalent infection on Earth. The bacterium, infamous for the reproductive phenotypes it induces in arthropod hosts, has risen to recent prominence due to its use in vector control. Wolbachia infection prevents the colonization of vectors by RNA viruses, including Drosophila C virus and important human pathogens such as Dengue and Chikungunya. Here we present data indicating that Wolbachia utilize the host actin cytoskeleton during oogenesis for persistence within and transmission between Drosophila melanogaster generations. We show that phenotypically wild type flies heterozygous for cytoskeletal mutations in Drosophila profilin (chic(221/+ and chic(1320/+ or villin (qua(6-396/+ either clear a Wolbachia infection, or result in significantly reduced infection levels. This reduction of Wolbachia is supported by PCR evidence, Western blot results and cytological examination. This phenotype is unlikely to be the result of maternal loading defects, defects in oocyte polarization, or germline stem cell proliferation, as the flies are phenotypically wild type in egg size, shape, and number. Importantly, however, heterozygous mutant flies exhibit decreased total G-actin in the ovary, compared to control flies and chic(221 heterozygous mutants exhibit decreased expression of profilin. Additionally, RNAi knockdown of profilin during development decreases Wolbachia titers. We analyze evidence in support of alternative theories to explain this Wolbachia phenotype and conclude that our results support the hypothesis that Wolbachia utilize the actin skeleton for efficient transmission and maintenance within Drosophila.

  3. Wolbachia utilize host actin for efficient maternal transmission in Drosophila melanogaster.

    Science.gov (United States)

    Newton, Irene L G; Savytskyy, Oleksandr; Sheehan, Kathy B

    2015-04-01

    Wolbachia pipientis is a ubiquitous, maternally transmitted bacterium that infects the germline of insect hosts. Estimates are that Wolbachia infect nearly 40% of insect species on the planet, making it the most prevalent infection on Earth. The bacterium, infamous for the reproductive phenotypes it induces in arthropod hosts, has risen to recent prominence due to its use in vector control. Wolbachia infection prevents the colonization of vectors by RNA viruses, including Drosophila C virus and important human pathogens such as Dengue and Chikungunya. Here we present data indicating that Wolbachia utilize the host actin cytoskeleton during oogenesis for persistence within and transmission between Drosophila melanogaster generations. We show that phenotypically wild type flies heterozygous for cytoskeletal mutations in Drosophila profilin (chic(221/+) and chic(1320/+)) or villin (qua(6-396/+)) either clear a Wolbachia infection, or result in significantly reduced infection levels. This reduction of Wolbachia is supported by PCR evidence, Western blot results and cytological examination. This phenotype is unlikely to be the result of maternal loading defects, defects in oocyte polarization, or germline stem cell proliferation, as the flies are phenotypically wild type in egg size, shape, and number. Importantly, however, heterozygous mutant flies exhibit decreased total G-actin in the ovary, compared to control flies and chic(221) heterozygous mutants exhibit decreased expression of profilin. Additionally, RNAi knockdown of profilin during development decreases Wolbachia titers. We analyze evidence in support of alternative theories to explain this Wolbachia phenotype and conclude that our results support the hypothesis that Wolbachia utilize the actin skeleton for efficient transmission and maintenance within Drosophila.

  4. Midgut epithelial responses of different mosquito–Plasmodium combinations: The actin cone zipper repair mechanism in Aedes aegypti

    Science.gov (United States)

    Gupta, Lalita; Kumar, Sanjeev; Han, Yeon Soo; Pimenta, Paulo F. P.; Barillas-Mury, Carolina

    2005-01-01

    In vivo responses of midgut epithelial cells to ookinete invasion of three different vector–parasite combinations, Aedes aegypti–Plasmodium gallinaceum, Anopheles stephensi–Plasmodium berghei, and A. stephensi–P. gallinaceum, were directly compared by using enzymatic markers and immunofluorescence stainings. Our studies indicate that, in A. aegypti and A. stephensi ookinetes traverse the midgut via an intracellular route and inflict irreversible damage to the invaded cells. These two mosquito species differ, however, in their mechanisms of epithelial repair. A. stephensi detaches damaged cells by an actin-mediated budding-off mechanism when invaded by either P. berghei or P. gallinaceum. In A. aegypti, the midgut epithelium is repaired by a unique actin cone zipper mechanism that involves the formation of a cone-shaped actin aggregate at the base of the cell that closes sequentially, expelling the cellular contents into the midgut lumen as it brings together healthy neighboring cells. Invasion of A. stephensi by P. berghei induced expression of nitric oxide synthase and peroxidase activities, which mediate tyrosine nitration. These enzymes and nitrotyrosine, however, were not induced in the other two vector–parasite combinations examined. These studies indicate that the epithelial responses of different mosquito–parasite combinations are not universal. The implications of these observations to validate animal experimental systems that reflect the biology of natural vectors of human malarias are discussed. PMID:15753303

  5. Midgut epithelial responses of different mosquito-Plasmodium combinations: the actin cone zipper repair mechanism in Aedes aegypti.

    Science.gov (United States)

    Gupta, Lalita; Kumar, Sanjeev; Han, Yeon Soo; Pimenta, Paulo F P; Barillas-Mury, Carolina

    2005-03-15

    In vivo responses of midgut epithelial cells to ookinete invasion of three different vector-parasite combinations, Aedes aegypti-Plasmodium gallinaceum, Anopheles stephensi-Plasmodium berghei, and A. stephensi-P. gallinaceum, were directly compared by using enzymatic markers and immunofluorescence stainings. Our studies indicate that, in A. aegypti and A. stephensi ookinetes traverse the midgut via an intracellular route and inflict irreversible damage to the invaded cells. These two mosquito species differ, however, in their mechanisms of epithelial repair. A. stephensi detaches damaged cells by an actin-mediated budding-off mechanism when invaded by either P. berghei or P. gallinaceum. In A. aegypti, the midgut epithelium is repaired by a unique actin cone zipper mechanism that involves the formation of a cone-shaped actin aggregate at the base of the cell that closes sequentially, expelling the cellular contents into the midgut lumen as it brings together healthy neighboring cells. Invasion of A. stephensi by P. berghei induced expression of nitric oxide synthase and peroxidase activities, which mediate tyrosine nitration. These enzymes and nitrotyrosine, however, were not induced in the other two vector-parasite combinations examined. These studies indicate that the epithelial responses of different mosquito-parasite combinations are not universal. The implications of these observations to validate animal experimental systems that reflect the biology of natural vectors of human malarias are discussed. PMID:15753303

  6. Actin based processes that could determine the cytoplasmic architecture of plant cells

    OpenAIRE

    Honing; Emons, A.M.C.; Ketelaar, M.J.

    2007-01-01

    Actin polymerisation can generate forces that are necessary for cell movement, such as the propulsion of a class of bacteria, including Listeria, and the protrusion of migrating animal cells. Force generation by the actin cytoskeleton in plant cells has not been studied. One process in plant cells that is likely to depend on actin-based force generation is the organisation of the cytoplasm. We compare the function of actin binding proteins of three well-studied mammalian models that depend on...

  7. Maternal effect mutations of the sponge locus affect actin cytoskeletal rearrangements in Drosophila melanogaster embryos

    OpenAIRE

    1992-01-01

    In the syncytial blastoderm stage of Drosophila embryogenesis, dome- shaped actin "caps" are observed above the interphase nuclei. During mitosis, this actin rearranges to participate in the formation of pseudocleavage furrows, transient membranous invaginations between dividing nuclei. Embryos laid by homozygous sponge mothers lack these characteristic actin structures, but retain other actin associated structures and processes. Our results indicate that the sponge product is specifically re...

  8. Nuclear actin and protein 4.1: Essential interactions during nuclear assembly in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Krauss, Sharon Wald; Chen, Cynthia; Penman, Sheldon; Heald, Rebecca

    2003-06-11

    Structural protein 4.1, which has crucial interactions within the spectin-actin lattice of the human red cell membrane skeleton, also is widely distributed at diverse intracellular sites in nucleated cells. We previously showed that 4.1 is essential for assembly of functional nuclei in vitro and that the capacity of 4.1 to bind actin is required. Here we report that 4.1 and actin colocalize in mammalian cell nuclei using fluorescence microscopy and, by higher resolution cell whole mount electron microscopy, are associated on nuclear filaments. We also devised a cell-free assay using Xenopus egg extract containing fluorescent actin to follow actin during nuclear assembly. By directly imaging actin under non-perturbing conditions, the total nuclear actin population is retained and is visualized in situ relative to intact chromatin. We detected actin initially when chromatin and nuclear pores began assembling. As the nuclear lamina assembled, but preceding DNA synthesis, a discrete actin network formed throughout the nucleus. Protein 4.1 epitopes also were detected when actin began to accumulate in nuclei, producing a diffuse coincident pattern. As nuclei matured, actin was detected both coincident with and also independent of 4.1 epitopes. To test whether acquisition of nuclear actin is required for nuclear assembly, the actin inhibitor latrunculin A was added to Xenopus egg extracts during nuclear assembly. Latrunculin A strongly perturbed nuclear assembly and produced distorted nuclear structures containing neither actin nor protein 4.1. Our results suggest that actin as well as 4.1 is necessary for nuclear assembly and that 4.1-actin interactions may be critical.

  9. Drosophila Fascin is a novel downstream target of prostaglandin signaling during actin remodeling

    OpenAIRE

    Groen, Christopher M.; Spracklen, Andrew J.; Fagan, Tiffany N.; Tootle, Tina L.

    2012-01-01

    Although prostaglandins (PGs)—lipid signals produced downstream of cyclooxygenase (COX) enzymes—regulate actin cytoskeletal dynamics, their mechanisms of action are unknown. We previously established Drosophila oogenesis, in particular nurse cell dumping, as a new model to determine how PGs regulate actin remodeling. PGs, and thus the Drosophila COX-like enzyme Pxt, are required for both the parallel actin filament bundle formation and the cortical actin strengthening required for dumping. He...

  10. Histamine activates p38 MAP kinase and alters local lamellipodia dynamics, reducing endothelial barrier integrity and eliciting central movement of actin fibers.

    Science.gov (United States)

    Adderley, Shaquria P; Lawrence, Curtis; Madonia, Eyong; Olubadewo, Joseph O; Breslin, Jerome W

    2015-07-01

    The role of the actin cytoskeleton in endothelial barrier function has been debated for nearly four decades. Our previous investigation revealed spontaneous local lamellipodia in confluent endothelial monolayers that appear to increase overlap at intercellular junctions. We tested the hypothesis that the barrier-disrupting agent histamine would reduce local lamellipodia protrusions and investigated the potential involvement of p38 mitogen-activated protein (MAP) kinase activation and actin stress fiber formation. Confluent monolayers of human umbilical vein endothelial cells (HUVEC) expressing green fluorescent protein-actin were studied using time-lapse fluorescence microscopy. The protrusion and withdrawal characteristics of local lamellipodia were assessed before and after addition of histamine. Changes in barrier function were determined using electrical cell-substrate impedance sensing. Histamine initially decreased barrier function, lamellipodia protrusion frequency, and lamellipodia protrusion distance. A longer time for lamellipodia withdrawal and reduced withdrawal distance and velocity accompanied barrier recovery. After barrier recovery, a significant number of cortical fibers migrated centrally, eventually resembling actin stress fibers. The p38 MAP kinase inhibitor SB203580 attenuated the histamine-induced decreases in barrier function and lamellipodia protrusion frequency. SB203580 also inhibited the histamine-induced decreases in withdrawal distance and velocity, and the subsequent actin fiber migration. These data suggest that histamine can reduce local lamellipodia protrusion activity through activation of p38 MAP kinase. The findings also suggest that local lamellipodia have a role in maintaining endothelial barrier integrity. Furthermore, we provide evidence that actin stress fiber formation may be a reaction to, rather than a cause of, reduced endothelial barrier integrity. PMID:25948734

  11. Transportation of Nanoscale Cargoes by Myosin Propelled Actin Filaments

    NARCIS (Netherlands)

    Persson, Malin; Gullberg, Maria; Tolf, Conny; Lindberg, A. Michael; Mansson, Alf; Kocer, Armagan

    2013-01-01

    Myosin II propelled actin filaments move ten times faster than kinesin driven microtubules and are thus attractive candidates as cargo-transporting shuttles in motor driven lab-on-a-chip devices. In addition, actomyosin-based transportation of nanoparticles is useful in various fundamental studies.

  12. The actin Cytoskeleton in Root Hairs: a cell elongation device

    NARCIS (Netherlands)

    Ketelaar, T.; Emons, A.M.C.

    2009-01-01

    The actin cytoskeleton plays an important role in root hair development. It is involved in both the delivery of growth materials to the expanding tip of root hairs and the regulation of the area of tip growth. This review starts with a discussion of the techniques that are available to visualize the

  13. Photodynamic therapy for actinic keratosis in organ transplant patients

    DEFF Research Database (Denmark)

    Basset-Seguin, N; Baumann Conzett, K; Gerritsen, M J P;

    2013-01-01

    Background The incidence of actinic keratoses (AK) and non-melanoma skin cancer (NMSC) in organ transplant recipients (OTRs) is significantly higher than in immunocompetent patients. Rates of progression and recurrence following treatment are higher too, in part due to the effects...

  14. Evaluation of actinic cheilitis using fluorescence lifetime spectroscopy

    Science.gov (United States)

    Saito Nogueira, Marcelo; Cosci, Alessandro; Pratavieira, Sebastião.; Takahama, Ademar; Souza Azevedo, Rebeca; Kurachi, Cristina

    2016-03-01

    Actinic cheilitis is a potentially malignant disorder that mostly affects the vermilion border of the lower lip and can lead to squamous cell carcinoma. Because of its heterogeneous clinical aspect, it is difficult to indicate representative biopsy area. Late diagnosis is a limiting factor of therapeutic possibilities available to treat oral cancer. The diagnosis of actinic cheilitis is mainly based on clinical and histopathological analysis and it is a time consuming procedure to get the results. Information about the organization and chemical composition of the tissues can be obtained using fluorescence lifetime spectroscopy techniques without the need for biopsy. The main targeted fluorophores are NADH (nicotinamide adenine dinucleotide) and FAD (flavin adenine dinucleotide), which have free and bound states, each one with different average lifetimes. The average lifetimes for free and bound NADH and FAD change according to tissue metabolic alterations and allow a quick and non-invasive clinical investigation of injuries and to help clinicians with the early diagnosis of actinic cheilitis. This study aims to evaluate the fluorescence lifetime parameters at the discrimination of three degrees of epithelial dysplasia, the most important predictor of malignant development, described in up to 100% of actinic cheilitis cases.

  15. Mical links semaphorins to F-actin disassembly

    NARCIS (Netherlands)

    Hung, R.J.; Yazdani, U.; Yoon, J.; Wu, H.; Yang, T.; Gupta, N.; Huang, Z.; Berkel, van W.J.H.; Terman, J.R.

    2010-01-01

    How instructive cues present on the cell surface have their precise effects on the actin cytoskeleton is poorly understood. Semaphorins are one of the largest families of these instructive cues and are widely studied for their effects on cell movement, navigation, angiogenesis, immunology and cancer

  16. Actin-Dependent Alterations of Dendritic Spine Morphology in Shankopathies

    Science.gov (United States)

    Sarowar, Tasnuva

    2016-01-01

    Shank proteins (Shank1, Shank2, and Shank3) act as scaffolding molecules in the postsynaptic density of many excitatory neurons. Mutations in SHANK genes, in particular SHANK2 and SHANK3, lead to autism spectrum disorders (ASD) in both human and mouse models. Shank3 proteins are made of several domains—the Shank/ProSAP N-terminal (SPN) domain, ankyrin repeats, SH3 domain, PDZ domain, a proline-rich region, and the sterile alpha motif (SAM) domain. Via various binding partners of these domains, Shank3 is able to bind and interact with a wide range of proteins including modulators of small GTPases such as RICH2, a RhoGAP protein, and βPIX, a RhoGEF protein for Rac1 and Cdc42, actin binding proteins and actin modulators. Dysregulation of all isoforms of Shank proteins, but especially Shank3, leads to alterations in spine morphogenesis, shape, and activity of the synapse via altering actin dynamics. Therefore, here, we highlight the role of Shank proteins as modulators of small GTPases and, ultimately, actin dynamics, as found in multiple in vitro and in vivo models. The failure to mediate this regulatory role might present a shared mechanism in the pathophysiology of autism-associated mutations, which leads to dysregulation of spine morphogenesis and synaptic signaling.

  17. Multidrug Resistance-Related Protein 1 (MRP1) Function and Localization Depend on Cortical Actin

    NARCIS (Netherlands)

    Hummel, Ina; Klappe, Karin; Ercan, Cigdem; Kok, Jan Willem

    2011-01-01

    MRP1 (ABCC1) is known to be localized in lipid rafts. Here we show in two different cell lines that localization of Mrp1/MRP1 (Abcc1/ABCC1) in lipid rafts and its function as an efflux pump are dependent on cortical actin. Latrunculin B disrupts both cortical actin and actin stress fibers. This resu

  18. Dynamic organization of actin cytoskeleton during the polarity formation and germination of pollen protoplasts

    Institute of Scientific and Technical Information of China (English)

    XU Xia; Zl Huijun; SUN Yina; REN Haiyun

    2004-01-01

    The formation of the polarity of pollen protoplast and the dynamics of actin cytoskeleton were observed by non-fixation, Alexa-Phalloidin probing and confocal laser scanning microscopy. Our results showed that the protoplast obtained from stored pollen contained numerous crystalline fusiform bodies to constitute a storage form of actin. When dormant pollen was hydrated, the actin cytoskeleton forms a fine network spreading uniformly in the protoplast. In the process of polarity formation and germination of pollen protoplast, actin filaments marshaled slowly to the brim, and then formed multilayer continuous actin filament bundles surrounding the cortical of the protoplast. When the protoplast was exposed to actin filament-disrupting drugs, such as Latrunculin A and Cytochalasin D, continuously arranged actin bundles were disturbed and in this condition, the protoplast could not germinate. But when exposed to actin filament stabiling drug-phalliodin, the dynamics of actin filaments in the protoplasts behaved normally and the protoplasts could germinate normally. These results were also confirmed by the pharmacology experiments on pollen grains. And when Latrunculin A or Cytochalasin D was washed off, the ratio of pollen germination was resumed partly. All the results above show that the dynamic organization of the actin cytoskeleton are critical in the cell polarity formation and germination of pollen protoplast, and that the reorganization of actin cytoskeleton is mainly due to the rearrangement of actin filament arrays.

  19. Probing cytoplasmic organization and the actin cytoskeleton of plant cells with optical tweezers

    NARCIS (Netherlands)

    Ketelaar, T.; Honing, van der H.S.; Emons, A.M.C.

    2010-01-01

    In interphase plant cells, the actin cytoskeleton is essential for intracellular transport and organization. To fully understand how the actin cytoskeleton functions as the structural basis for cytoplasmic organization, both molecular and physical aspects of the actin organization have to be conside

  20. Cysteine-rich protein 1 (CRP1 regulates actin filament bundling

    Directory of Open Access Journals (Sweden)

    Fraley Tamara S

    2005-12-01

    Full Text Available Abstract Background Cysteine-rich protein 1 (CRP1 is a LIM domain containing protein localized to the nucleus and the actin cytoskeleton. CRP1 has been demonstrated to bind the actin-bundling protein α-actinin and proposed to modulate the actin cytoskeleton; however, specific regulatory mechanisms have not been identified. Results CRP1 expression increased actin bundling in rat embryonic fibroblasts. Although CRP1 did not affect the bundling activity of α-actinin, CRP1 was found to stabilize the interaction of α-actinin with actin bundles and to directly bundle actin microfilaments. Using confocal and photobleaching fluorescence resonance energy transfer (FRET microscopy, we demonstrate that there are two populations of CRP1 localized along actin stress fibers, one associated through interaction with α-actinin and one that appears to bind the actin filaments directly. Consistent with a role in regulating actin filament cross-linking, CRP1 also localized to the membrane ruffles of spreading and PDGF treated fibroblasts. Conclusion CRP1 regulates actin filament bundling by directly cross-linking actin filaments and stabilizing the interaction of α-actinin with actin filament bundles.

  1. A function for filamentous alpha-smooth muscle actin: Retardation of motility in human breast fibroblasts

    DEFF Research Database (Denmark)

    Rønnov-Jessen, Lone; Petersen, Ole William

    1996-01-01

    reactions. Here, we show that the presence of alpha-sm actin is a signal for retardation of migratory behavior in fibroblasts. Comparison in a migration assay of fibroblast cell strains with and without alpha-sm actin revealed migratory restraint in alpha-sm actin-positive fibroblasts. Electroporation...

  2. Reverse actin sliding triggers strong myosin binding that moves tropomyosin

    Energy Technology Data Exchange (ETDEWEB)

    Bekyarova, T.I.; Reedy, M.C.; Baumann, B.A.J.; Tregear, R.T.; Ward, A.; Krzic, U.; Prince, K.M.; Perz-Edwards, R.J.; Reconditi, M.; Gore, D.; Irving, T.C.; Reedy, M.K. (IIT); (EMBL); (Scripps); (Duke); (Prince); (FSU); (MRC); (U. Florence)

    2008-09-03

    Actin/myosin interactions in vertebrate striated muscles are believed to be regulated by the 'steric blocking' mechanism whereby the binding of calcium to the troponin complex allows tropomyosin (TM) to change position on actin, acting as a molecular switch that blocks or allows myosin heads to interact with actin. Movement of TM during activation is initiated by interaction of Ca{sup 2+} with troponin, then completed by further displacement by strong binding cross-bridges. We report x-ray evidence that TM in insect flight muscle (IFM) moves in a manner consistent with the steric blocking mechanism. We find that both isometric contraction, at high [Ca{sup 2+}], and stretch activation, at lower [Ca{sup 2+}], develop similarly high x-ray intensities on the IFM fourth actin layer line because of TM movement, coinciding with x-ray signals of strong-binding cross-bridge attachment to helically favored 'actin target zones.' Vanadate (Vi), a phosphate analog that inhibits active cross-bridge cycling, abolishes all active force in IFM, allowing high [Ca{sup 2+}] to elicit initial TM movement without cross-bridge attachment or other changes from relaxed structure. However, when stretched in high [Ca{sup 2+}], Vi-'paralyzed' fibers produce force substantially above passive response at pCa {approx} 9, concurrent with full conversion from resting to active x-ray pattern, including x-ray signals of cross-bridge strong-binding and TM movement. This argues that myosin heads can be recruited as strong-binding 'brakes' by backward-sliding, calcium-activated thin filaments, and are as effective in moving TM as actively force-producing cross-bridges. Such recruitment of myosin as brakes may be the major mechanism resisting extension during lengthening contractions.

  3. Statistics of actin-propelled trajectories in noisy environments.

    Science.gov (United States)

    Wen, Fu-Lai; Chen, Hsuan-Yi; Leung, Kwan-Tai

    2016-06-01

    Actin polymerization is ubiquitously utilized to power the locomotion of eukaryotic cells and pathogenic bacteria in living systems. Inevitably, actin polymerization and depolymerization proceed in a fluctuating environment that renders the locomotion stochastic. Previously, we have introduced a deterministic model that manages to reproduce actin-propelled trajectories in experiments, but not to address fluctuations around them. To remedy this, here we supplement the deterministic model with noise terms. It enables us to compute the effects of fluctuating actin density and forces on the trajectories. Specifically, the mean-squared displacement (MSD) of the trajectories is computed and found to show a super-ballistic scaling with an exponent 3 in the early stage, followed by a crossover to a normal, diffusive scaling of exponent 1 in the late stage. For open-end trajectories such as straights and S-shaped curves, the time of crossover matches the decay time of orientational order of the velocities along trajectories, suggesting that it is the spreading of velocities that leads to the crossover. We show that the super-ballistic scaling of MSD arises from the initial, linearly increasing correlation of velocities, before time translational symmetry is established. When the spreading of velocities reaches a steady state in the long-time limit, short-range correlation then yields a diffusive scaling in MSD. In contrast, close-loop trajectories like circles exhibit localized periodic motion, which inhibits spreading. The initial super-ballistic scaling of MSD arises from velocity correlation that both linearly increases and oscillates in time. Finally, we find that the above statistical features of the trajectories transcend the nature of noises, be it additive or multiplicative, and generalize to other self-propelled systems that are not necessarily actin based. PMID:27415296

  4. Statistics of actin-propelled trajectories in noisy environments

    Science.gov (United States)

    Wen, Fu-Lai; Chen, Hsuan-Yi; Leung, Kwan-tai

    2016-06-01

    Actin polymerization is ubiquitously utilized to power the locomotion of eukaryotic cells and pathogenic bacteria in living systems. Inevitably, actin polymerization and depolymerization proceed in a fluctuating environment that renders the locomotion stochastic. Previously, we have introduced a deterministic model that manages to reproduce actin-propelled trajectories in experiments, but not to address fluctuations around them. To remedy this, here we supplement the deterministic model with noise terms. It enables us to compute the effects of fluctuating actin density and forces on the trajectories. Specifically, the mean-squared displacement (MSD) of the trajectories is computed and found to show a super-ballistic scaling with an exponent 3 in the early stage, followed by a crossover to a normal, diffusive scaling of exponent 1 in the late stage. For open-end trajectories such as straights and S-shaped curves, the time of crossover matches the decay time of orientational order of the velocities along trajectories, suggesting that it is the spreading of velocities that leads to the crossover. We show that the super-ballistic scaling of MSD arises from the initial, linearly increasing correlation of velocities, before time translational symmetry is established. When the spreading of velocities reaches a steady state in the long-time limit, short-range correlation then yields a diffusive scaling in MSD. In contrast, close-loop trajectories like circles exhibit localized periodic motion, which inhibits spreading. The initial super-ballistic scaling of MSD arises from velocity correlation that both linearly increases and oscillates in time. Finally, we find that the above statistical features of the trajectories transcend the nature of noises, be it additive or multiplicative, and generalize to other self-propelled systems that are not necessarily actin based.

  5. Macromolecular crowding gives rise to microviscosity, anomalous diffusion and accelerated actin polymerization

    Science.gov (United States)

    Rashid, Rafi; Chee, Stella Min Ling; Raghunath, Michael; Wohland, Thorsten

    2015-05-01

    Macromolecular crowding (MMC) has been used in various in vitro experimental systems to mimic in vivo physiology. This is because the crowded cytoplasm of cells contains many different types of solutes dissolved in an aqueous medium. MMC in the extracellular microenvironment is involved in maintaining stem cells in their undifferentiated state (niche) as well as in aiding their differentiation after they have travelled to new locations outside the niche. MMC at physiologically relevant fractional volume occupancies (FVOs) significantly enhances the adipogenic differentiation of human bone marrow-derived mesenchymal stem cells during chemically induced adipogenesis. The mechanism by which MMC produces this enhancement is not entirely known. In the context of extracellular collagen deposition, we have recently reported the importance of optimizing the FVO while minimizing the bulk viscosity. Two opposing properties will determine the net rate of a biochemical reaction: the negative effect of bulk viscosity and the positive effect of the excluded volume, the latter being expressed by the FVO. In this study we have looked more closely at the effect of viscosity on reaction rates. We have used fluorimetry to measure the rate of actin polymerization and fluorescence correlation spectroscopy (FCS) to measure diffusion of various probes in solutions containing the crowder Ficoll at physiological concentrations. Similar to its effect on collagen, Ficoll enhanced the actin polymerization rate despite increasing the bulk viscosity. Our FCS measurements reveal a relatively minor component of anomalous diffusion. In addition, our measurements do suggest that microviscosity becomes relevant in a crowded environment. We ruled out bulk viscosity as a cause of the rate enhancement by performing the actin polymerization assay in glycerol. These opposite effects of Ficoll and glycerol led us to conclude that microviscosity becomes relevant at the length scale of the reacting

  6. Regulation of actin dynamics by WNT-5A: implications for human airway smooth muscle contraction

    Science.gov (United States)

    Koopmans, Tim; Kumawat, Kuldeep; Halayko, Andrew J; Gosens, Reinoud

    2016-01-01

    A defining feature of asthma is airway hyperresponsiveness (AHR), which underlies the exaggerated bronchoconstriction response of asthmatics. The role of the airway smooth muscle (ASM) in AHR has garnered increasing interest over the years, but how asthmatic ASM differs from healthy ASM is still an active topic of debate. WNT-5A is increasingly expressed in asthmatic ASM and has been linked with Th2-high asthma. Due to its link with calcium and cytoskeletal remodelling, we propose that WNT-5A may modulate ASM contractility. We demonstrated that WNT-5A can increase maximum isometric tension in bovine tracheal smooth muscle strips. In addition, we show that WNT-5A is preferentially expressed in contractile human airway myocytes compared to proliferative cells, suggesting an active role in maintaining contractility. Furthermore, WNT-5A treatment drives actin polymerisation, but has no effect on intracellular calcium flux. Next, we demonstrated that WNT-5A directly regulates TGF-β1-induced expression of α-SMA via ROCK-mediated actin polymerization. These findings suggest that WNT-5A modulates fundamental mechanisms that affect ASM contraction and thus may be of relevance for AHR in asthma. PMID:27468699

  7. Actin-binding proteins from Burkholderia mallei and Burkholderia thailandensis can functionally compensate for the actin-based motility defect of a Burkholderia pseudomallei bimA mutant

    OpenAIRE

    Stevens, J. M.; Ulrich, R L; Taylor, L A; Wood, M W; DeShazer, D; M.P. Stevens; Galyov, E. E.

    2005-01-01

    Recently we identified a bacterial factor (BimA) required for actin-based motility of Burkholderia pseudomallei. Here we report that Burkholderia mallei and Burkholderia thailandensis are capable of actin-based motility in J774.2 cells and that BimA homologs of these bacteria can restore the actin-based motility defect of a B. pseudomallei bimA mutant. While the BimA homologs differ in their amino-terminal sequence, they interact directly with actin in vitro and vary in their ability to bind ...

  8. Actin-Binding Proteins from Burkholderia mallei and Burkholderia thailandensis Can Functionally Compensate for the Actin-Based Motility Defect of a Burkholderia pseudomallei bimA Mutant

    OpenAIRE

    Stevens, Joanne M; Ulrich, Ricky L.; Taylor, Lowrie A.; Wood, Michael W.; DeShazer, David; Stevens, Mark P.; Galyov, Edouard E.

    2005-01-01

    Recently we identified a bacterial factor (BimA) required for actin-based motility of Burkholderia pseudomallei. Here we report that Burkholderia mallei and Burkholderia thailandensis are capable of actin-based motility in J774.2 cells and that BimA homologs of these bacteria can restore the actin-based motility defect of a B. pseudomallei bimA mutant. While the BimA homologs differ in their amino-terminal sequence, they interact directly with actin in vitro and vary in their ability to bind ...

  9. Actinic skin damage and mortality--the First National Health and Nutrition Examination Survey Epidemiologic Follow-up Study.

    Directory of Open Access Journals (Sweden)

    Wei He

    Full Text Available BACKGROUND: Exposure to sunlight may decrease the risk of several diseases through the synthesis of vitamin D, whereas solar radiation is the main cause of some skin and eye diseases. However, to the best of our knowledge, the association of sun-induced skin damage with mortality remains unknown. METHODOLOGY/PRINCIPAL FINDINGS: Subjects were 8472 white participants aged 25-74 years in the First National Health and Nutrition Examination Survey Epidemiologic Follow-up Study. Cardiovascular disease mortality, cancer mortality, and all-cause mortality were obtained by either a death certificate or a proxy interview, or both. Actinic skin damage was examined and recorded by the presence and severity (absent, minimal, moderate, or severe of overall actinic skin damage and its components (i.e., fine telangiectasia, solar elastosis, and actinic keratoses. Cox regression and Kaplan-Meier methods were applied to explore the associations. A total of 672 cancer deaths, 1500 cardiovascular disease deaths, and 2969 deaths from all causes were documented through the follow-up between 1971 and 1992. After controlling for potential confounding variables, severe overall actinic skin damage was associated with a 45% higher risk for all-cause mortality (95% CI: 1.22, 1.72; P<0.001, moderate overall skin damage with a 20% higher risk (95% CI: 1.08., 1.32; P<0.001, and minimal overall skin damage with no significant mortality difference, when compared to those with no skin damage. Similar results were obtained for all-cause mortality with fine telangiectasia, solar elastosis, and actinic keratoses. The results were similar for cancer and cardiovascular disease mortality. CONCLUSIONS: The present study gives an indication of an association of actinic skin damage with cardiovascular disease, cancer and all-cause mortality in white subjects. Given the lack of support in the scientific literature and potential unmeasured confounding factors, this finding should be

  10. Polymer models of chromosome (re)organization

    Science.gov (United States)

    Mirny, Leonid

    Chromosome Conformation Capture technique (Hi-C) provides comprehensive information about frequencies of spatial interactions between genomic loci. Inferring 3D organization of chromosomes from these data is a challenging biophysical problem. We develop a top-down approach to biophysical modeling of chromosomes. Starting with a minimal set of biologically motivated interactions we build ensembles of polymer conformations that can reproduce major features observed in Hi-C experiments. I will present our work on modeling organization of human metaphase and interphase chromosomes. Our works suggests that active processes of loop extrusion can be a universal mechanism responsible for formation of domains in interphase and chromosome compaction in metaphase.

  11. Dynamics of actin cables in polarized growth of the filamentous fungus Aspergillus nidulans

    Directory of Open Access Journals (Sweden)

    Anna eBergs

    2016-05-01

    Full Text Available Highly polarized growth of filamentous fungi requires a continuous supply of proteins and lipids to the hyphal tip. This transport is managed by vesicle trafficking via the actin and microtubule cytoskeletons and their associated motor proteins. Particularly, actin cables originating from the hyphal tip are essential for hyphal growth. Although specific marker proteins to visualize actin cables have been developed in filamentous fungi, the exact organization and dynamics of actin cables has remained elusive. Here we visualized actin cables using tropomyosin (TpmA and Lifeact fused to fluorescent proteins in Aspergillus nidulans and studied the dynamics and regulation. GFP tagged TpmA visualized dynamic actin cables formed from the hyphal tip with cycles of elongation and shrinkage. The elongation and shrinkage rates of actin cables were similar and approximately 0.6 μm/s. Comparison of actin markers revealed that high concentrations of Lifeact reduced actin dynamics. Simultaneous visualization of actin cables and microtubules suggests temporally and spatially coordinated polymerization and depolymerization between the two cytoskeletons. Our results provide new insights into the molecular mechanism of ordered polarized growth regulated by actin cables and microtubules.

  12. The conserved Tarp actin binding domain is important for chlamydial invasion.

    Directory of Open Access Journals (Sweden)

    Travis J Jewett

    Full Text Available The translocated actin recruiting phosphoprotein (Tarp is conserved among all pathogenic chlamydial species. Previous reports identified single C. trachomatis Tarp actin binding and proline rich domains required for Tarp mediated actin nucleation. A peptide antiserum specific for the Tarp actin binding domain was generated and inhibited actin polymerization in vitro and C. trachomatis entry in vivo, indicating an essential role for Tarp in chlamydial pathogenesis. Sequence analysis of Tarp orthologs from additional chlamydial species and C. trachomatis serovars indicated multiple putative actin binding sites. In order to determine whether the identified actin binding domains are functionally conserved, GST-Tarp fusions from multiple chlamydial species were examined for their ability to bind and nucleate actin. Chlamydial Tarps harbored variable numbers of actin binding sites and promoted actin nucleation as determined by in vitro polymerization assays. Our findings indicate that Tarp mediated actin binding and nucleation is a conserved feature among diverse chlamydial species and this function plays a critical role in bacterial invasion of host cells.

  13. Restricted morphological and behavioral abnormalities following ablation of β-actin in the brain.

    Directory of Open Access Journals (Sweden)

    Thomas R Cheever

    Full Text Available The local translation of β-actin is one mechanism proposed to regulate spatially-restricted actin polymerization crucial for nearly all aspects of neuronal development and function. However, the physiological significance of localized β-actin translation in neurons has not yet been demonstrated in vivo. To investigate the role of β-actin in the mammalian central nervous system (CNS, we characterized brain structure and function in a CNS-specific β-actin knock-out mouse (CNS-ActbKO. β-actin was rapidly ablated in the embryonic mouse brain, but total actin levels were maintained through upregulation of other actin isoforms during development. CNS-ActbKO mice exhibited partial perinatal lethality while survivors presented with surprisingly restricted histological abnormalities localized to the hippocampus and cerebellum. These tissue morphology defects correlated with profound hyperactivity as well as cognitive and maternal behavior impairments. Finally, we also identified localized defects in axonal crossing of the corpus callosum in CNS-ActbKO mice. These restricted defects occurred despite the fact that primary neurons lacking β-actin in culture were morphologically normal. Altogether, we identified novel roles for β-actin in promoting complex CNS tissue architecture while also demonstrating that distinct functions for the ubiquitously expressed β-actin are surprisingly restricted in vivo.

  14. Preliminarily Investigating the Polymorphism of Self-organized Actin Filament in Vitro by Atomic Force Microscope

    Institute of Scientific and Technical Information of China (English)

    Jun ZHANG; Yuan-Liang WANG; Xin-Yong Chen; Chuang-Long HE; Chao CHENG; Yang CAO

    2004-01-01

    With the atomic force microscope (AFM), we preliminarily investigated the large-scale structure of actin filaments formed in low concentration protein solution (5 μg/ml) via self-organization without the presence of any F-actin dynamic interfering factors (such as phalloidin) in vitro. It was found that the G-actin could be polymerized into ordered filamentous structures with different diameter from the slimmest filament of single F-actin to giant filament in tree-like branched aggregates. The observed polymerized actin filaments, to which our most intense attention was attracted, was discretely distributed and showed obvious polymorphism distinctly different from those in the presence of phalloidin or actin binding proteins (fimbrin, gelsolin, etc.) in previous experiments. Latter structures were mainly composed of single F-actin and/or multifilaments clearly consisting of several single F-actin. The experimental results clearly demonstrated that non-interference with the F-actin intrinsic dynamics in self-organizing could lead to the polymorphism of actin filamentous structures, and further analysis implied that the disturbance of normal F-actin dynamics by many factors could prevent the emergence of structural polymorphism, more often than not, give rise to formation of specific structures instead and different interference would bring about various particular structures under certain conditions.

  15. Re organ ization,Design an d Practice on Teaching Contents in Electromagnetism Curriculum%《电磁学》教学内容的重组设计与实施

    Institute of Scientific and Technical Information of China (English)

    张锡娟; 张月明; 殷英

    2015-01-01

    针对低年级大学生学习存在的问题,以《电磁学》课程为例,将教学内容重新组织与设计,把《电磁学》分为讲授、自学和探究3个模块。通过这3个模块的设计与实施,激发学生的学习兴趣,帮助学生习得概念,促进学生主动学习,培养学生学会管理时间和自我监控,提高学生查阅资料、分析问题、深入探究的能力。%For the problems about Lower Grade Students learning courses ,to“electromagnetism”course as an example , we re organized and redesigned the teaching contents ,divided the“electromagnetic”into three modules including teaching , self study and exploring .By designing and implementing these three modules ,We want to stimulate students′interests in study ,help them learn concepts and promote students′active learning ,train the students learn to manage time and self monitoring and finally these can improve students′capabilities of access to information ,analizing the problem ,in depth exploration .

  16. Isolation and identification of actin-binding proteins in Plasmodium falciparum by affinity chromatography

    Directory of Open Access Journals (Sweden)

    Claudia Forero

    2000-06-01

    Full Text Available The invasion of the erythrocyte by Plasmodium falciparum depends on the ability of the merozoite to move through the membrane invagination. This ability is probably mediated by actin dependent motors. Using affinity columns with G-actin and F-actin we isolated actin binding proteins from the parasite. By immunoblotting and immunoprecipitation with specific antibodies we identified the presence of tropomyosin, myosin, a-actinin, and two different actins in the eluate corresponding to F-actin binding proteins. In addition to these, a 240-260 kDa doublet, different in size from the erythrocyte spectrin, reacted with an antibody against human spectrin. All the above mentioned proteins were metabolically radiolabeled when the parasite was cultured with 35S-methionine. The presence of these proteins in P. falciparum is indicative of a complex cytoskeleton and supports the proposed role for an actin-myosin motor during invasion.

  17. Endocytosis-dependent coordination of multiple actin regulators is required for wound healing.

    Science.gov (United States)

    Matsubayashi, Yutaka; Coulson-Gilmer, Camilla; Millard, Tom H

    2015-08-01

    The ability to heal wounds efficiently is essential for life. After wounding of an epithelium, the cells bordering the wound form dynamic actin protrusions and/or a contractile actomyosin cable, and these actin structures drive wound closure. Despite their importance in wound healing, the molecular mechanisms that regulate the assembly of these actin structures at wound edges are not well understood. In this paper, using Drosophila melanogaster embryos, we demonstrate that Diaphanous, SCAR, and WASp play distinct but overlapping roles in regulating actin assembly during wound healing. Moreover, we show that endocytosis is essential for wound edge actin assembly and wound closure. We identify adherens junctions (AJs) as a key target of endocytosis during wound healing and propose that endocytic remodeling of AJs is required to form "signaling centers" along the wound edge that control actin assembly. We conclude that coordination of actin assembly, AJ remodeling, and membrane traffic is required for the construction of a motile leading edge during wound healing.

  18. Cytotoxic effects of incense particles in relation to oxidative stress, the cell cycle and F-actin assembly.

    Science.gov (United States)

    Chuang, Hsiao-Chi; Jones, Tim; Chen, Tzu-Tao; BéruBé, Kelly

    2013-07-18

    Epidemiological studies have suggested that combustion-derived smoke, such as that produced during incense burning, is a deleterious air pollutant. It is capable of initiating oxidative stress and mutation; however, the related apoptotic processes remain unclear. In order to elucidate the biological mechanisms of reactive oxygen species (ROS)-induced respiratory toxicology, alveolar epithelial A549 cells were exposed to incense particulate matter (PM), with and without antioxidant N-acetyl-l-cysteine (NAC). The cross-linking associations between oxidative capacity, cell cycle events, actin cytoskeletal dynamics and intracellular calcium signals were investigated. An incense PM suspension caused significant oxidative stress in A549 cells, as shown by inhibition of the cell cycle at G1 and G2/M check-points, and the induction of apoptosis at Sub-G1. At the same time, alterations in the F-actin filamentous assemblies were observed. The levels of intracellular Ca(2+) were increased after incense PM exposure. Antioxidant NAC treatment revealed that oxidative stress and F-actin remodelling was significantly mitigated. This suggests that ROS accumulation could alter cell cycle regulation and anomalous remodelling of the cortical cytoskeleton that allowed impaired cells to enter into apoptosis. This study has elucidated the integral patho-physiological interactions of incense PM and the potential mechanisms for the development of ROS-driven respiratory impairment.

  19. Formin-mediated actin polymerization cooperates with Mushroom body defect (Mud)-Dynein during Frizzled-Dishevelled spindle orientation.

    Science.gov (United States)

    Johnston, Christopher A; Manning, Laurina; Lu, Michelle S; Golub, Ognjen; Doe, Chris Q; Prehoda, Kenneth E

    2013-10-01

    To position the mitotic spindle, cytoskeletal components must be coordinated to generate cortical forces on astral microtubules. Although the dynein motor is common to many spindle orientation systems, 'accessory pathways' are often also required. In this work, we identified an accessory spindle orientation pathway in Drosophila that functions with Dynein during planar cell polarity, downstream of the Frizzled (Fz) effector Dishevelled (Dsh). Dsh contains a PDZ ligand and a Dynein-recruiting DEP domain that are both required for spindle orientation. The Dsh PDZ ligand recruits Canoe/Afadin and ultimately leads to Rho GTPase signaling mediated through RhoGEF2. The formin Diaphanous (Dia) functions as the Rho effector in this pathway, inducing F-actin enrichment at sites of cortical Dsh. Chimeric protein experiments show that the Dia-actin accessory pathway can be replaced by an independent kinesin (Khc73) accessory pathway for Dsh-mediated spindle orientation. Our results define two 'modular' spindle orientation pathways and show an essential role for actin regulation in Dsh-mediated spindle orientation.

  20. Feedback Interactions of Polymerized Actin with the Cell Membrane: Waves, Pulses, and Oscillations

    Science.gov (United States)

    Carlsson, Anders

    Polymerized filaments of the protein actin have crucial functions in cell migration, and in bending the cell membrane to drive endocytosis or the formation of protrusions. The nucleation and polymerization of actin filaments are controlled by upstream agents in the cell membrane, including nucleation-promoting factors (NPFs) that activate the Arp2/3 complex to form new branches on pre-existing filaments. But polymerized actin (F-actin) also feeds back on the assembly of NPFs. We explore the effects of the resulting feedback loop of F-actin and NPFs on two phenomena: actin pulses that drive endocytosis in yeast, and actin waves traveling along the membrane of several cell types. In our model of endocytosis in yeast, the actin network is grown explicitly in three dimensions, exerts a negative feedback interaction on localized patch of NPFs in the membrane, and bends the membrane by exerting a distribution of forces. This model explains observed actin and NPF pulse dynamics, and the effects of several interventions including i) NPF mutations, ii) inhibition of actin polymerization, and iii) deletion of a protein that allows F-actin to bend the cell membrane. The model predicts that mutation of the active region of an NPF will enhance the accumulation of that NPF, and we confirm this prediction by quantitative fluorescence microscopy. For actin waves, we treat a similar model, with NPFs distributed over a larger region of the cell membrane. This model naturally generates actin waves, and predicts a transition from wave behavior to spatially localized oscillations when NPFs are confined to a small region. We also predict a transition from waves to static polarization as the negative-feedback coupling between F-actin and the NPFs is reduced. Supported by NIGMS Grant R01 GM107667.

  1. Mapping of the Mouse Actin Capping Protein Beta Subunit Gene

    Directory of Open Access Journals (Sweden)

    Cooper John A

    2000-07-01

    Full Text Available Abstract Background Capping protein (CP, a heterodimer of α and β subunits, is found in all eukaryotes. CP binds to the barbed ends of actin filaments in vitro and controls actin assembly and cell motility in vivo. Vertebrates have three isoforms of CPβ produced by alternatively splicing from one gene; lower organisms have one gene and one isoform. Results We isolated genomic clones corresponding to the β subunit of mouse CP and identified its chromosomal location by interspecies backcross mapping. Conclusions The CPβ gene (Cappb1 mapped to Chromosome 4 between Cdc42 and D4Mit312. Three mouse mutations, snubnose, curly tail, and cribriform degeneration, map in the vicinity of the β gene.

  2. Actin Remodeling and Polymerization Forces Control Dendritic Spine Morphology

    CERN Document Server

    Miermans, Karsten; Storm, Cornelis; Hoogenraad, Casper

    2015-01-01

    Dendritic spines are small membranous structures that protrude from the neuronal dendrite. Each spine contains a synaptic contact site that may connect its parent dendrite to the axons of neighboring neurons. Dendritic spines are markedly distinct in shape and size, and certain types of stimulation prompt spines to evolve, in fairly predictable fashion, from thin nascent morphologies to the mushroom-like shapes associated with mature spines. This striking progression is coincident with the (re)configuration of the neuronal network during early development, learning and memory formation, and has been conjectured to be part of the machinery that encodes these processes at the scale of individual neuronal connections. It is well established that the structural plasticity of spines is strongly dependent upon the actin cytoskeleton inside the spine. A general framework that details the precise role of actin in directing the transitions between the various spine shapes is lacking. We address this issue, and present...

  3. Extra-nuclear signaling of progesterone receptor to breast cancer cell movement and invasion through the actin cytoskeleton.

    Directory of Open Access Journals (Sweden)

    Xiao-Dong Fu

    Full Text Available Progesterone plays a role in breast cancer development and progression but the effects on breast cancer cell movement or invasion have not been fully explored. In this study, we investigate the actions of natural progesterone and of the synthetic progestin medroxyprogesterone acetate (MPA on actin cytoskeleton remodeling and on breast cancer cell movement and invasion. In particular, we characterize the nongenomic signaling cascades implicated in these actions. T47-D breast cancer cells display enhanced horizontal migration and invasion of three-dimensional matrices in the presence of both progestins. Exposure to the hormones triggers a rapid remodeling of the actin cytoskeleton and the formation of membrane ruffles required for cell movement, which are dependent on the rapid phosphorylation of the actin-regulatory protein moesin. The extra-cellular small GTPase RhoA/Rho-associated kinase (ROCK-2 cascade plays central role in progesterone- and MPA-induced moesin activation, cell migration and invasion. In the presence of progesterone, progesterone receptor A (PRA interacts with the G protein G alpha(13, while MPA drives PR to interact with tyrosine kinase c-Src and to activate phosphatidylinositol-3 kinase, leading to the activation of RhoA/ROCK-2. In conclusion, our findings manifest that progesterone and MPA promote breast cancer cell movement via rapid actin cytoskeleton remodeling, which are mediated by moesin activation. These events are triggered by RhoA/ROCK-2 cascade through partially differing pathways by the two compounds. These results provide original mechanistic explanations for the effects of progestins on breast cancer progression and highlight potential targets to treat endocrine-sensitive breast cancers.

  4. Internal Motility in Stiffening Actin-Myosin Networks

    OpenAIRE

    Uhde, Joerg; Keller, Manfred; Sackmann, Erich; Parmeggiani, Andrea; Frey, Erwin

    2003-01-01

    We present a study on filamentous actin solutions containing heavy meromyosin subfragments of myosin II motor molecules. We focus on the viscoelastic phase behavior and internal dynamics of such networks during ATP depletion. Upon simultaneously using micro-rheology and fluorescence microscopy as complementary experimental tools, we find a sol-gel transition accompanied by a sudden onset of directed filament motion. We interpret the sol-gel transition in terms of myosin II enzymology, and sug...

  5. IFT88 influences chondrocyte actin organization and biomechanics

    OpenAIRE

    Z. Wang; Wann, A.K.T.; Thompson, C L; Hassen, A.; Wang, W; Knight, M.M.

    2016-01-01

    Summary Objectives Primary cilia are microtubule based organelles which control a variety of signalling pathways important in cartilage development, health and disease. This study examines the role of the intraflagellar transport (IFT) protein, IFT88, in regulating fundamental actin organisation and mechanics in articular chondrocytes. Methods The study used an established chondrocyte cell line with and without hypomorphic mutation of IFT88 (IFT88orpk). Confocal microscopy was used to quantif...

  6. Topical therapies for skin cancer and actinic keratosis.

    OpenAIRE

    Haque, T.; Rahman, K. M.; Thurston, D E; Hadgraft, J; Lane, M. E.

    2015-01-01

    The global incidence of skin cancer and actinic keratosis (AK) has increased dramatically in recent years. Although many tumours are treated with surgery or radiotherapy topical therapy has a place in the management of certain superficial skin neoplasms and AK. This review considers skin physiology, non-melanoma skin cancer (NMSC), the relationship between AK and skin cancer and drugs administered topically for these conditions. The dermal preparations for management of NMSC and AK are discus...

  7. Myosin lever arm directs collective motion on cellular actin network.

    Science.gov (United States)

    Hariadi, Rizal F; Cale, Mario; Sivaramakrishnan, Sivaraj

    2014-03-18

    The molecular motor myosin teams up to drive muscle contraction, membrane traffic, and cell division in biological cells. Myosin function in cells emerges from the interaction of multiple motors tethered to a scaffold, with surrounding actin filaments organized into 3D networks. Despite the importance of myosin function, the influence of intermotor interactions on collective motion remains poorly understood. In this study, we used precisely engineered myosin assemblies to examine emergence in collective myosin movement. We report that tethering multiple myosin VI motors, but not myosin V motors, modifies their movement trajectories on keratocyte actin networks. Single myosin V and VI dimers display similar skewed trajectories, albeit in opposite directions, when traversing the keratocyte actin network. In contrast, tethering myosin VI motors, but not myosin V motors, progressively straightens the trajectories with increasing myosin number. Trajectory shape of multimotor scaffolds positively correlates with the stiffness of the myosin lever arm. Swapping the flexible myosin VI lever arm for the relatively rigid myosin V lever increases trajectory skewness, and vice versa. A simplified model of coupled motor movement demonstrates that the differences in flexural rigidity of the two myosin lever arms is sufficient to account for the differences in observed behavior of groups of myosin V and VI motors. In accordance with this model trajectory, shapes for scaffolds containing both myosin V and VI are dominated by the myosin with a stiffer lever arm. Our findings suggest that structural features unique to each myosin type may confer selective advantages in cellular functions.

  8. Maintenance of asymmetric cellular localization of an auxin transport protein through interaction with the actin cytoskeleton

    Science.gov (United States)

    Muday, G. K.

    2000-01-01

    In shoots, polar auxin transport is basipetal (that is, from the shoot apex toward the base) and is driven by the basal localization of the auxin efflux carrier complex. The focus of this article is to summarize the experiments that have examined how the asymmetric distribution of this protein complex is controlled and the significance of this polar distribution. Experimental evidence suggests that asymmetries in the auxin efflux carrier may be established through localized secretion of Golgi vesicles, whereas an attachment of a subunit of the efflux carrier to the actin cytoskeleton may maintain this localization. In addition, the idea that this localization of the efflux carrier may control both the polarity of auxin movement and more globally regulate developmental polarity is explored. Finally, evidence indicating that the gravity vector controls auxin transport polarity is summarized and possible mechanisms for the environmentally induced changes in auxin transport polarity are discussed.

  9. Contractile units in disordered actomyosin bundles arise from F-actin buckling

    CERN Document Server

    Lenz, Martin; Gardel, Margaret L; Dinner, Aaron R

    2012-01-01

    Bundles of filaments and motors are central to contractility in cells. The classic example is striated muscle, where actomyosin contractility is mediated by highly organized sarcomeres which act as fundamental contractile units. However, many contractile bundles in vivo and in vitro lack sarcomeric organization. Here we propose a model for how contractility can arise in actomyosin bundles without sarcomeric organization and validate its predictions with experiments on a reconstituted system. In the model, internal stresses in frustrated arrangements of motors with diverse velocities cause filaments to buckle, leading to overall shortening. We describe the onset of buckling in the presence of stochastic actin-myosin detachment and predict that buckling-induced contraction occurs in an intermediate range of motor densities. We then calculate the size of the "contractile units" associated with this process. Consistent with these results, our reconstituted actomyosin bundles contract at relatively high motor dens...

  10. Successful use of a topical mixture with ozolipoile in the treatment of actinic ulcers

    Directory of Open Access Journals (Sweden)

    Inchingolo F

    2015-04-01

    Full Text Available Francesco Inchingolo,1 Angelo Tarullo,2 Raffaele Cagiano,2 Gianpiero Resta,3 Gianna Dipalma,3 Angelo Michele Inchingolo,3 Antonella Tarullo,3 Salvatore Scacco,4 Massimo Marrelli,5,6,* Luigi Corti,7,* Marco Tatullo8,* 1Department of Interdisciplinary Medicine, University of Bari “Aldo Moro”, Bari, 2Department of Biomedical Sciences and Human Oncology, University of Bari “Aldo Moro”, Bari, 3Private Practice, Dental Clinic, Bari, 4Department of Basic Medical Sciences, Neurosciences and Sensorial Organs, University of Bari “Aldo Moro”, Bari, 5Maxillofacial Unit, Calabrodental Clinic, Crotone, 6Healthcare Center, Marrelli Hospital, Crotone, 7UOC of Radiotherapy and Nuclear Medicine IOV – IRCCS, University of Padova, Padova, 8Biomedical Section, Tecnologica Research Institute, Crotone, Italy *These authors equally contributed to this research paper Purpose: Post-radiation skin damage is the result of alterations produced in the irradiated zone. There are different studies aimed at verifying the effectiveness of several kinds of molecules in the treatment of radiation-induced skin damage. The purpose of this study is to test a mixture with a formulation containing several natural active ingredients on actinic ulcers in patients receiving radiation therapies. Patients and methods: The authors recruited 13 patients, and randomly divided them into a test group (T1 and a control group (T2. The patients in both groups were undergoing radiotherapy. The T1 group was treated with a mixture called ozolipoile, whereas the T2 group was administered hyaluronic acid gel followed by non-ablative laser therapy. We evaluated the obtained results, the time of clinical healing, the reduction of pain, and each side effect, comparing all data between the T1 and T2 groups. Results: The average Visual Analog Scale results showed decrease in pain in both groups; however, while the T1 group showed a significant decrease in the values, the T2 group reported a

  11. LATS1 tumor suppressor is a novel actin-binding protein and negative regulator of actin polymerization

    Institute of Scientific and Technical Information of China (English)

    Stacy Visser-Grieve; Zhonghua Zhou; Yi-Min She; He Huang; Terry D Cyr; Tian Xu; Xiaolong Yang

    2011-01-01

    Dear Editor,The LATS tumor suppressor,conserved from Drosophila (dlats) to humans (LATS1,LATS2),plays a vital role in maintaining cellular homeostasis in humans since loss of either LATS1 or LATS2 leads to the development of numerous cancer types such as breast cancer and leukemia [1].Apart from its roles as a Ser/Thr kinase within the emerging Hippo pathway regulating cell proliferation and apoptosis,ultimately leading to the control of organ size and tumorigenesis [2],LATS is also implicated in a broad range of functions including regulation of genetic stability,transcription,and protein stability [1 ].Recently,tumor suppressors have also been shown to affect the later stages of tumorigenesis,including metastasis.Among this group of metastasis regulators are genes that can directly affect actin dynamics by binding to F-actin,such as the tumor suppressors p53 [3],NF2 [4] and APC [5].

  12. Molecular mechanical differences between isoforms of contractile actin in the presence of isoforms of smooth muscle tropomyosin.

    OpenAIRE

    Lennart Hilbert; Genevieve Bates; Roman, Horia N.; Jenna L Blumenthal; Zitouni, Nedjma B.; Apolinary Sobieszek; Mackey, Michael C.; Anne-Marie Lauzon

    2013-01-01

    The proteins involved in smooth muscle's molecular contractile mechanism - the anti-parallel motion of actin and myosin filaments driven by myosin heads interacting with actin - are found as different isoforms. While their expression levels are altered in disease states, their relevance to the mechanical interaction of myosin with actin is not sufficiently understood. Here, we analyzed in vitro actin filament propulsion by smooth muscle myosin for [Formula: see text]-actin ([Formula: see text...

  13. Ubiquitin ligase TRIM3 controls hippocampal plasticity and learning by regulating synaptic γ-actin levels.

    Science.gov (United States)

    Schreiber, Joerg; Végh, Marlene J; Dawitz, Julia; Kroon, Tim; Loos, Maarten; Labonté, Dorthe; Li, Ka Wan; Van Nierop, Pim; Van Diepen, Michiel T; De Zeeuw, Chris I; Kneussel, Matthias; Meredith, Rhiannon M; Smit, August B; Van Kesteren, Ronald E

    2015-11-01

    Synaptic plasticity requires remodeling of the actin cytoskeleton. Although two actin isoforms, β- and γ-actin, are expressed in dendritic spines, the specific contribution of γ-actin in the expression of synaptic plasticity is unknown. We show that synaptic γ-actin levels are regulated by the E3 ubiquitin ligase TRIM3. TRIM3 protein and Actg1 transcript are colocalized in messenger ribonucleoprotein granules responsible for the dendritic targeting of messenger RNAs. TRIM3 polyubiquitylates γ-actin, most likely cotranslationally at synaptic sites. Trim3(-/-) mice consequently have increased levels of γ-actin at hippocampal synapses, resulting in higher spine densities, increased long-term potentiation, and enhanced short-term contextual fear memory consolidation. Interestingly, hippocampal deletion of Actg1 caused an increase in long-term fear memory. Collectively, our findings suggest that temporal control of γ-actin levels by TRIM3 is required to regulate the timing of hippocampal plasticity. We propose a model in which TRIM3 regulates synaptic γ-actin turnover and actin filament stability and thus forms a transient inhibitory constraint on the expression of hippocampal synaptic plasticity. PMID:26527743

  14. Altered Cell Mechanics from the Inside: Dispersed Single Wall Carbon Nanotubes Integrate with and Restructure Actin

    Directory of Open Access Journals (Sweden)

    Mohammad F. Islam

    2012-05-01

    Full Text Available With a range of desirable mechanical and optical properties, single wall carbon nanotubes (SWCNTs are a promising material for nanobiotechnologies. SWCNTs also have potential as biomaterials for modulation of cellular structures. Previously, we showed that highly purified, dispersed SWCNTs grossly alter F-actin inside cells. F-actin plays critical roles in the maintenance of cell structure, force transduction, transport and cytokinesis. Thus, quantification of SWCNT-actin interactions ranging from molecular, sub-cellular and cellular levels with both structure and function is critical for developing SWCNT-based biotechnologies. Further, this interaction can be exploited, using SWCNTs as a unique actin-altering material. Here, we utilized molecular dynamics simulations to explore the interactions of SWCNTs with actin filaments. Fluorescence lifetime imaging microscopy confirmed that SWCNTs were located within ~5 nm of F-actin in cells but did not interact with G-actin. SWCNTs did not alter myosin II sub-cellular localization, and SWCNT treatment in cells led to significantly shorter actin filaments. Functionally, cells with internalized SWCNTs had greatly reduced cell traction force. Combined, these results demonstrate direct, specific SWCNT alteration of F-actin structures which can be exploited for SWCNT-based biotechnologies and utilized as a new method to probe fundamental actin-related cellular processes and biophysics.

  15. Actin and myosin regulate cytoplasm stiffness in plant cells: a study using optical tweezers.

    Science.gov (United States)

    van der Honing, Hannie S; de Ruijter, Norbert C A; Emons, Anne Mie C; Ketelaar, Tijs

    2010-01-01

    Here, we produced cytoplasmic protrusions with optical tweezers in mature BY-2 suspension cultured cells to study the parameters involved in the movement of actin filaments during changes in cytoplasmic organization and to determine whether stiffness is an actin-related property of plant cytoplasm. Optical tweezers were used to create cytoplasmic protrusions resembling cytoplasmic strands. Simultaneously, the behavior of the actin cytoskeleton was imaged. After actin filament depolymerization, less force was needed to create cytoplasmic protrusions. During treatment with the myosin ATPase inhibitor 2,3-butanedione monoxime, more trapping force was needed to create and maintain cytoplasmic protrusions. Thus, the presence of actin filaments and, even more so, the deactivation of a 2,3-butanedione monoxime-sensitive factor, probably myosin, stiffens the cytoplasm. During 2,3-butanedione monoxime treatment, none of the tweezer-formed protrusions contained filamentous actin, showing that a 2,3-butanedione monoxime-sensitive factor, probably myosin, is responsible for the movement of actin filaments, and implying that myosin serves as a static cross-linker of actin filaments when its motor function is inhibited. The presence of actin filaments does not delay the collapse of cytoplasmic protrusions after tweezer release. Myosin-based reorganization of the existing actin cytoskeleton could be the basis for new cytoplasmic strand formation, and thus the production of an organized cytoarchitecture.

  16. Characterization of engineered actin binding proteins that control filament assembly and structure.

    Directory of Open Access Journals (Sweden)

    Crista M Brawley

    Full Text Available BACKGROUND: Eukaryotic cells strictly regulate the structure and assembly of their actin filament networks in response to various stimuli. The actin binding proteins that control filament assembly are therefore attractive targets for those who wish to reorganize actin filaments and reengineer the cytoskeleton. Unfortunately, the naturally occurring actin binding proteins include only a limited set of pointed-end cappers, or proteins that will block polymerization from the slow-growing end of actin filaments. Of the few that are known, most are part of large multimeric complexes that are challenging to manipulate. METHODOLOGY/PRINCIPAL FINDINGS: We describe here the use of phage display mutagenesis to generate of a new class of binding protein that can be targeted to the pointed-end of actin. These proteins, called synthetic antigen binders (sABs, are based on an antibody-like scaffold where sequence diversity is introduced into the binding loops using a novel "reduced genetic code" phage display library. We describe effective strategies to select and screen for sABs that ensure the generated sABs bind to the pointed-end surface of actin exclusively. CONCLUSIONS/SIGNIFICANCE: From our set of pointed-end binders, we identify three sABs with particularly useful properties to systematically probe actin dynamics: one protein that caps the pointed end, a second that crosslinks actin filaments, and a third that severs actin filaments and promotes disassembly.

  17. Genome-wide RNAi screen for nuclear actin reveals a network of cofilin regulators.

    Science.gov (United States)

    Dopie, Joseph; Rajakylä, Eeva K; Joensuu, Merja S; Huet, Guillaume; Ferrantelli, Evelina; Xie, Tiao; Jäälinoja, Harri; Jokitalo, Eija; Vartiainen, Maria K

    2015-07-01

    Nuclear actin plays an important role in many processes that regulate gene expression. Cytoplasmic actin dynamics are tightly controlled by numerous actin-binding proteins, but regulation of nuclear actin has remained unclear. Here, we performed a genome-wide RNA interference (RNAi) screen in Drosophila cells to identify proteins that influence either nuclear polymerization or import of actin. We validate 19 factors as specific hits, and show that Chinmo (known as Bach2 in mammals), SNF4Aγ (Prkag1 in mammals) and Rab18 play a role in nuclear localization of actin in both fly and mammalian cells. We identify several new regulators of cofilin activity, and characterize modulators of both cofilin kinases and phosphatase. For example, Chinmo/Bach2, which regulates nuclear actin levels also in vivo, maintains active cofilin by repressing the expression of the kinase Cdi (Tesk in mammals). Finally, we show that Nup98 and lamin are candidates for regulating nuclear actin polymerization. Our screen therefore reveals new aspects of actin regulation and links nuclear actin to many cellular processes.

  18. Expression of Chlamydomonas actin-gfp fusion gene in to-bacco suspension cell and polymerization of the actin-gfp protein in vitro

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    The fusion gene of actin (cDNA of Chlamydo- monas reinhardtii) and green fluorescence protein (gfp) had been constructed into two expression vectors which could be expressed in E. coli and tobacco suspension cells BY2. The correct expression was observed in E. coli and BY2 with a fluorescence microscopy. The fusion protein, which took part in the membrane skeleton, was mainly located peripherally along the membrane, specially the fusion protein was dis-tributed around nucleus and cell plate, while the fusion pro-tein also forms F-actin in the cell. The fusion protein was purified from Bl21plus by ammonium sulfate fractionation, ion exchange chromatography and hydrophobic interaction chromatography. The purified production could polymerize into F-actin when the actin polymerizing buffer was added. It was demonstrated that the characteristics and function of actin in Chlamydomonas was similar with those of animals and higher plants.

  19. The Formin Diaphanous Regulates Myoblast Fusion through Actin Polymerization and Arp2/3 Regulation.

    Science.gov (United States)

    Deng, Su; Bothe, Ingo; Baylies, Mary K

    2015-08-01

    The formation of multinucleated muscle cells through cell-cell fusion is a conserved process from fruit flies to humans. Numerous studies have shown the importance of Arp2/3, its regulators, and branched actin for the formation of an actin structure, the F-actin focus, at the fusion site. This F-actin focus forms the core of an invasive podosome-like structure that is required for myoblast fusion. In this study, we find that the formin Diaphanous (Dia), which nucleates and facilitates the elongation of actin filaments, is essential for Drosophila myoblast fusion. Following cell recognition and adhesion, Dia is enriched at the myoblast fusion site, concomitant with, and having the same dynamics as, the F-actin focus. Through analysis of Dia loss-of-function conditions using mutant alleles but particularly a dominant negative Dia transgene, we demonstrate that reduction in Dia activity in myoblasts leads to a fusion block. Significantly, no actin focus is detected, and neither branched actin regulators, SCAR or WASp, accumulate at the fusion site when Dia levels are reduced. Expression of constitutively active Dia also causes a fusion block that is associated with an increase in highly dynamic filopodia, altered actin turnover rates and F-actin distribution, and mislocalization of SCAR and WASp at the fusion site. Together our data indicate that Dia plays two roles during invasive podosome formation at the fusion site: it dictates the level of linear F-actin polymerization, and it is required for appropriate branched actin polymerization via localization of SCAR and WASp. These studies provide new insight to the mechanisms of cell-cell fusion, the relationship between different regulators of actin polymerization, and invasive podosome formation that occurs in normal development and in disease.

  20. A peek into tropomyosin binding and unfolding on the actin filament.

    Directory of Open Access Journals (Sweden)

    Abhishek Singh

    Full Text Available BACKGROUND: Tropomyosin is a prototypical coiled coil along its length with subtle variations in structure that allow interactions with actin and other proteins. Actin binding globally stabilizes tropomyosin. Tropomyosin-actin interaction occurs periodically along the length of tropomyosin. However, it is not well understood how tropomyosin binds actin. PRINCIPAL FINDINGS: Tropomyosin's periodic binding sites make differential contributions to two components of actin binding, cooperativity and affinity, and can be classified as primary or secondary sites. We show through mutagenesis and analysis of recombinant striated muscle alpha-tropomyosins that primary actin binding sites have a destabilizing coiled-coil interface, typically alanine-rich, embedded within a non-interface recognition sequence. Introduction of an Ala cluster in place of the native, more stable interface in period 2 and/or period 3 sites (of seven increased the affinity or cooperativity of actin binding, analysed by cosedimentation and differential scanning calorimetry. Replacement of period 3 with period 5 sequence, an unstable region of known importance for cooperative actin binding, increased the cooperativity of binding. Introduction of the fluorescent probe, pyrene, near the mutation sites in periods 2 and 3 reported local instability, stabilization by actin binding, and local unfolding before or coincident with dissociation from actin (measured using light scattering, and chain dissociation (analyzed using circular dichroism. CONCLUSIONS: This, and previous work, suggests that regions of tropomyosin involved in binding actin have non-interface residues specific for interaction with actin and an unstable interface that is locally stabilized upon binding. The destabilized interface allows residues on the coiled-coil surface to obtain an optimal conformation for interaction with actin by increasing the number of local substates that the side chains can sample. We suggest

  1. The Formin Diaphanous Regulates Myoblast Fusion through Actin Polymerization and Arp2/3 Regulation.

    Directory of Open Access Journals (Sweden)

    Su Deng

    2015-08-01

    Full Text Available The formation of multinucleated muscle cells through cell-cell fusion is a conserved process from fruit flies to humans. Numerous studies have shown the importance of Arp2/3, its regulators, and branched actin for the formation of an actin structure, the F-actin focus, at the fusion site. This F-actin focus forms the core of an invasive podosome-like structure that is required for myoblast fusion. In this study, we find that the formin Diaphanous (Dia, which nucleates and facilitates the elongation of actin filaments, is essential for Drosophila myoblast fusion. Following cell recognition and adhesion, Dia is enriched at the myoblast fusion site, concomitant with, and having the same dynamics as, the F-actin focus. Through analysis of Dia loss-of-function conditions using mutant alleles but particularly a dominant negative Dia transgene, we demonstrate that reduction in Dia activity in myoblasts leads to a fusion block. Significantly, no actin focus is detected, and neither branched actin regulators, SCAR or WASp, accumulate at the fusion site when Dia levels are reduced. Expression of constitutively active Dia also causes a fusion block that is associated with an increase in highly dynamic filopodia, altered actin turnover rates and F-actin distribution, and mislocalization of SCAR and WASp at the fusion site. Together our data indicate that Dia plays two roles during invasive podosome formation at the fusion site: it dictates the level of linear F-actin polymerization, and it is required for appropriate branched actin polymerization via localization of SCAR and WASp. These studies provide new insight to the mechanisms of cell-cell fusion, the relationship between different regulators of actin polymerization, and invasive podosome formation that occurs in normal development and in disease.

  2. Regulation of T cell receptor signaling by the actin cytoskeleton and poroelastic cytoplasm

    Science.gov (United States)

    Beemiller, Peter; Krummel, Matthew F.

    2013-01-01

    Summary The actin cytoskeleton plays essential roles in modulating T-cell activation. Most models of T-cell receptor (TCR) triggering, signalosome assembl, y and immune synapse formation invoke actin-dependent mechanisms. As T cells are constitutively motile cells, TCR triggering and signaling occur against a cytoskeletal backdrop that is constantly remodeling. While the interplay between actin dynamics and TCR signaling have been the focus of research for many years, much of the work in T cells has considered actin largely for its ‘scaffolding’ function. We examine the roles of the actin cytoskeleton in TCR signaling and immune synapse formation with an emphasis on how poroelasticity, an ensemble feature of actin dynamics with the cytosol, relates to how T cells respond to stimulation. PMID:24117819

  3. Regulation of T-cell receptor signaling by the actin cytoskeleton and poroelastic cytoplasm.

    Science.gov (United States)

    Beemiller, Peter; Krummel, Matthew F

    2013-11-01

    The actin cytoskeleton plays essential roles in modulating T-cell activation. Most models of T-cell receptor (TCR) triggering signalosome assembly and immune synapse formation invoke actin-dependent mechanisms. As T cells are constitutively motile cells, TCR triggering and signaling occur against a cytoskeletal backdrop that is constantly remodeling. While the interplay between actin dynamics and TCR signaling have been the focus of research for many years, much of the work in T cells has considered actin largely for its 'scaffolding' function. We examine the roles of the actin cytoskeleton in TCR signaling and immune synapse formation with an emphasis on how poroelasticity, an ensemble feature of actin dynamics with the cytosol, relates to how T cells respond to stimulation. PMID:24117819

  4. The interaction between the adaptor protein APS and Enigma is involved in actin organisation

    DEFF Research Database (Denmark)

    Barres, Romain; Gonzalez, Teresa; Le Marchand-Brustel, Yannick;

    2005-01-01

    and APS were partially co-localised with F-actin in small ruffling structures. Insulin increased the complex formation between APS and Enigma and their co-localisation in large F-actin containing ruffles. While in NIH-3T3 and HeLa cells the co-expression of both Enigma and APS did not modify the actin...... cytoskeleton organisation, expression of Enigma alone led to the formation of F-actin clusters. Similar alteration in actin cytoskeleton organisation was observed in cells expressing both Enigma and APS with a mutation in the NPTY motif. These results identify Enigma as a novel APS-binding protein and suggest...... that the APS/Enigma complex plays a critical role in actin cytoskeleton organisation....

  5. Cloning and Characterization of an Abalone (Haliotis discus hannai) Actin Gene

    Institute of Scientific and Technical Information of China (English)

    MA Hongming; XU Wei; MAI Kangsen; LIUFU Zhiguo; CHEN Hong

    2004-01-01

    An actin encoding gene was cloned by using RT-PCR, 3' RACE and 5' RACE from abalone Haliotis discus hannai. The full length of the gene is 1532 base pairs, which contains a long 3' untranslated region of 307 base pairs and 79 base pairs of 5' untranslated sequence. The open reading frame encodes 376 amino acid residues. Sequence comparison with those of human and other mollusks showed high conservation among species at amino acid level. The identities was 96%, 97% and 96% respectively compared with Aplysia californica, Biomphalaria glabrata and Homo sapience β-actin. It is also indicated that this actin is more similar to the human cytoplasmic actin(β-actin)than to human muscle actin.

  6. Plant pathogenic bacteria target the actin microfilament network involved in the trafficking of disease defense components.

    Science.gov (United States)

    Jelenska, Joanna; Kang, Yongsung; Greenberg, Jean T

    2014-01-01

    Cells of infected organisms transport disease defense-related molecules along actin filaments to deliver them to their sites of action to combat the pathogen. To accommodate higher demand for intracellular traffic, plant F-actin density increases transiently during infection or treatment of Arabidopsis with pathogen-associated molecules. Many animal and plant pathogens interfere with actin polymerization and depolymerization to avoid immune responses. Pseudomonas syringae, a plant extracellular pathogen, injects HopW1 effector into host cells to disrupt the actin cytoskeleton and reduce vesicle movement in order to elude defense responses. In some Arabidopsis accessions, however, HopW1 is recognized and causes resistance via an actin-independent mechanism. HopW1 targets isoform 7 of vegetative actin (ACT7) that is regulated by phytohormones and environmental factors. We hypothesize that dynamic changes of ACT7 filaments are involved in plant immunity. PMID:25551177

  7. Direct Microtubule-Binding by Myosin-10 Orients Centrosomes toward Retraction Fibers and Subcortical Actin Clouds.

    Science.gov (United States)

    Kwon, Mijung; Bagonis, Maria; Danuser, Gaudenz; Pellman, David

    2015-08-10

    Positioning of centrosomes is vital for cell division and development. In metazoan cells, spindle positioning is controlled by a dynamic pool of subcortical actin that organizes in response to the position of retraction fibers. These actin "clouds" are proposed to generate pulling forces on centrosomes and mediate spindle orientation. However, the motors that pull astral microtubules toward these actin structures are not known. Here, we report that the unconventional myosin, Myo10, couples actin-dependent forces from retraction fibers and subcortical actin clouds to centrosomes. Myo10-mediated centrosome positioning requires its direct microtubule binding. Computational image analysis of large microtubule populations reveals a direct effect of Myo10 on microtubule dynamics and microtubule-cortex interactions. Myo10's role in centrosome positioning is distinct from, but overlaps with, that of dynein. Thus, Myo10 plays a key role in integrating the actin and microtubule cytoskeletons to position centrosomes and mitotic spindles. PMID:26235048

  8. Actin based processes that could determine the cytoplasmic architecture of plant cells.

    Science.gov (United States)

    van der Honing, Hannie S; Emons, Anne Mie C; Ketelaar, Tijs

    2007-05-01

    Actin polymerisation can generate forces that are necessary for cell movement, such as the propulsion of a class of bacteria, including Listeria, and the protrusion of migrating animal cells. Force generation by the actin cytoskeleton in plant cells has not been studied. One process in plant cells that is likely to depend on actin-based force generation is the organisation of the cytoplasm. We compare the function of actin binding proteins of three well-studied mammalian models that depend on actin-based force generation with the function of their homologues in plants. We predict the possible role of these proteins, and thus the role of actin-based force generation, in the production of cytoplasmic organisation in plant cells.

  9. Inhibiting actin depolymerization enhances osteoblast differentiation and bone formation in human stromal stem cells

    DEFF Research Database (Denmark)

    Chen, Li; Shi, Kaikai; Frary, Charles Edward;

    2015-01-01

    Remodeling of the actin cytoskeleton through actin dynamics is involved in a number of biological processes, but its role in human stromal (skeletal) stem cells (hMSCs) differentiation is poorly understood. In the present study, we demonstrated that stabilizing actin filaments by inhibiting gene...... expression of the two main actin depolymerizing factors (ADFs): Cofilin 1 (CFL1) and Destrin (DSTN) in hMSCs, enhanced cell viability and differentiation into osteoblastic cells (OB) in vitro, as well as heterotopic bone formation in vivo. Similarly, treating hMSC with Phalloidin, which is known to stabilize...... polymerized actin filaments, increased hMSCs viability and OB differentiation. Conversely, Cytocholasin D, an inhibitor of actin polymerization, reduced cell viability and inhibited OB differentiation of hMSC. At a molecular level, preventing Cofilin phosphorylation through inhibition of LIM domain kinase 1...

  10. Effect of cytochalasins on F-actin and morphology of Ehrlich ascites tumor cells

    DEFF Research Database (Denmark)

    Mills, J W; Falsig Pedersen, S; Walmod, P S;

    2000-01-01

    that, in intact cells, different cytochalasins can have varying effects on cell morphology and F-actin content and organization. To examine this problem in more detail, we analyzed the effects of cytochalasins on the cell morphology of and F-actin content and organization in Ehrlich ascites tumor (EAT......Cytochalasins have been used extensively to probe the role of F-actin in different aspects of cellular function. Most of the data obtained are interpreted on the basis of the well-established depolymerizing effects of cytochalasins on F-actin preparations in vitro. However, some evidence indicates......) cells. After a 3-min exposure to 0.5 microM cytochalasin D, B, or E, F-actin content was equally reduced in all cases and this correlated with a reduction in the amount of cortical F-actin associated with the EAT cell membrane. However, only with CE was cell morphology markedly altered...

  11. When fat is not bad: the regulation of actin dynamics by phospholipid signaling molecules

    Directory of Open Access Journals (Sweden)

    Roman ePleskot

    2014-01-01

    Full Text Available The actin cytoskeleton plays a key role in the plant morphogenesis and is involved in polar cell growth, movement of subcellular organelles, cell division, and plant defense. Organization of actin cytoskeleton undergoes dynamic remodeling in response to internal developmental cues and diverse environmental signals. This dynamic behavior is regulated by numerous actin-binding proteins that integrate various signaling pathways. Production of the signaling lipids phosphatidylinositol 4,5-bisphosphate and phosphatidic acid affects the activity and subcellular distribution of several actin-binding proteins, and typically correlates with increased actin polymerization. Here we review current knowledge of the inter-regulatory dynamics between signaling phospholipids and the actin cytoskeleton in plant cells.

  12. Regulation of T cell receptor signaling by the actin cytoskeleton and poroelastic cytoplasm

    OpenAIRE

    Beemiller, Peter; Krummel, Matthew F.

    2013-01-01

    The actin cytoskeleton plays essential roles in modulating T-cell activation. Most models of T-cell receptor (TCR) triggering, signalosome assembl, y and immune synapse formation invoke actin-dependent mechanisms. As T cells are constitutively motile cells, TCR triggering and signaling occur against a cytoskeletal backdrop that is constantly remodeling. While the interplay between actin dynamics and TCR signaling have been the focus of research for many years, much of the work in T cells has ...

  13. Actin Polymerization Controls the Organization of WASH Domains at the Surface of Endosomes

    OpenAIRE

    Emmanuel Derivery; Emmanuèle Helfer; Véronique Henriot; Alexis Gautreau

    2012-01-01

    Sorting of cargoes in endosomes occurs through their selective enrichment into sorting platforms, where transport intermediates are generated. The WASH complex, which directly binds to lipids, activates the Arp2/3 complex and hence actin polymerization onto such sorting platforms. Here, we analyzed the role of actin polymerization in the physiology of endosomal domains containing WASH using quantitative image analysis. Actin depolymerization is known to enlarge endosomes. Using a novel coloca...

  14. Hypogelsolinemia, a disorder of the extracellular actin scavenger system, in patients with multiple sclerosis

    OpenAIRE

    Janmey Paul A; Szmitkowski Maciej; Drozdowski Wiesław; Mroczko Barbara; Wen Qi; Ciccarelli Nicholas J; Kułakowska Alina; Bucki Robert

    2010-01-01

    Abstract Background Extracellular gelsolin (GSN) and GC-globulin/Vitamin D-binding protein (DBP) appear to play an important role in clearing the actin from extracellular fluids and in modulating cellular responses to anionic bioactive lipids. In this study we hypothesized that cellular actin release and/or increase in bioactive lipids associated with multiple sclerosis (MS) development will translate into alteration of the actin scavenger system protein concentrations in blood and cerebrospi...

  15. Opposing Roles for Actin in Cdc42p PolarizationD⃞

    OpenAIRE

    Irazoqui, Javier E.; Howell, Audrey S.; Theesfeld, Chandra L.; Lew, Daniel J.

    2005-01-01

    In animal and fungal cells, the monomeric GTPase Cdc42p is a key regulator of cell polarity that itself exhibits a polarized distribution in asymmetric cells. Previous work showed that in budding yeast, Cdc42p polarization is unaffected by depolymerization of the actin cytoskeleton (Ayscough et al., J. Cell Biol. 137, 399–416, 1997). Surprisingly, we now report that unlike complete actin depolymerization, partial actin depolymerization leads to the dispersal of Cdc42p from the polarization si...

  16. Cell elasticity is regulated by the tropomyosin isoform composition of the actin cytoskeleton.

    Science.gov (United States)

    Jalilian, Iman; Heu, Celine; Cheng, Hong; Freittag, Hannah; Desouza, Melissa; Stehn, Justine R; Bryce, Nicole S; Whan, Renee M; Hardeman, Edna C; Fath, Thomas; Schevzov, Galina; Gunning, Peter W

    2015-01-01

    The actin cytoskeleton is the primary polymer system within cells responsible for regulating cellular stiffness. While various actin binding proteins regulate the organization and dynamics of the actin cytoskeleton, the proteins responsible for regulating the mechanical properties of cells are still not fully understood. In the present study, we have addressed the significance of the actin associated protein, tropomyosin (Tpm), in influencing the mechanical properties of cells. Tpms belong to a multi-gene family that form a co-polymer with actin filaments and differentially regulate actin filament stability, function and organization. Tpm isoform expression is highly regulated and together with the ability to sort to specific intracellular sites, result in the generation of distinct Tpm isoform-containing actin filament populations. Nanomechanical measurements conducted with an Atomic Force Microscope using indentation in Peak Force Tapping in indentation/ramping mode, demonstrated that Tpm impacts on cell stiffness and the observed effect occurred in a Tpm isoform-specific manner. Quantitative analysis of the cellular filamentous actin (F-actin) pool conducted both biochemically and with the use of a linear detection algorithm to evaluate actin structures revealed that an altered F-actin pool does not absolutely predict changes in cell stiffness. Inhibition of non-muscle myosin II revealed that intracellular tension generated by myosin II is required for the observed increase in cell stiffness. Lastly, we show that the observed increase in cell stiffness is partially recapitulated in vivo as detected in epididymal fat pads isolated from a Tpm3.1 transgenic mouse line. Together these data are consistent with a role for Tpm in regulating cell stiffness via the generation of specific populations of Tpm isoform-containing actin filaments.

  17. Cell stress promotes the association of phosphorylated HspB1 with F-actin.

    Directory of Open Access Journals (Sweden)

    Joseph P Clarke

    Full Text Available Previous studies have suggested that the small heat shock protein, HspB1, has a direct influence on the dynamics of cytoskeletal elements, in particular, filamentous actin (F-actin polymerization. In this study we have assessed the influence of HspB1 phosphorylation on its interaction(s with F-actin. We first determined the distribution of endogenous non-phosphorylated HspB1, phosphorylated HspB1 and F-actin in neuroendocrine PC12 cells by immunocytochemistry and confocal microscopy. We then investigated a potential direct interaction between HspB1 with F-actin by precipitating F-actin directly with biotinylated phalloidin followed by Western analyses; the reverse immunoprecipitation of HspB1 was also carried out. The phosphorylation influence of HspB1 in this interaction was investigated by using pharmacologic inhibition of p38 MAPK. In control cells, HspB1 interacts with F-actin as a predominantly non-phosphorylated protein, but subsequent to stress there is a redistribution of HspB1 to the cytoskeletal fraction and a significantly increased association of pHspB1 with F-actin. Our data demonstrate HspB1 is found in a complex with F-actin both in phosphorylated and non-phosphorylated forms, with an increased association of pHspB1 with F-actin after heat stress. Overall, our study combines both cellular and biochemical approaches to show cellular localization and direct demonstration of an interaction between endogenous HspB1 and F-actin using methodolgy that specifically isolates F-actin.

  18. Temperature change does not affect force between single actin filaments and HMM from rabbit muscles.

    OpenAIRE

    Kawai, M.; Kawaguchi, K; M. Saito; Ishiwata, S

    2000-01-01

    The temperature dependence of sliding force, velocity, and unbinding force was studied on actin filaments when they were placed on heavy meromyosin (HMM) attached to a glass surface. A fluorescently labeled actin filament was attached to the gelsolin-coated surface of a 1-microm polystyrene bead. The bead was trapped by optical tweezers, and HMM-actin interaction was performed at 20-35 degrees C to examine whether force is altered by the temperature change. Our experiments demonstrate that sl...

  19. Production and characterization of polyclonal antibody against a synthetic peptide from β-actin protein

    OpenAIRE

    Nazila Amini; Mohadeseh Naghi Vishteh; Omid Zarei; Reza Hadavi; Negah Ahmadvand; Hodjattallah Rabbani; Mahmood Jeddi-Tehrani

    2014-01-01

    Objective(s):Antibodies against actin, as one of the most widely studied structural and multifunctional housekeeping proteins in eukaryotic cells, are used as internal loading controls in western blot analyses. The aim of this study was to produce polyclonal antibody against a synthetic peptide derived from N-terminal region of β-actin protein to be used as a protein loading control in western blot and other assay systems. Materials and Methods: A synthetic peptide derived from β-actin protei...

  20. Actin and nuclear myosin Ⅰ are associated with RNAP Ⅱ and function in gene transcription

    Institute of Scientific and Technical Information of China (English)

    ZHU XiaoJuan; HUANG BaiQu; WANG XingZhi; HAO Shui; ZENG XianLu

    2007-01-01

    The presence of actin in the nucleus as well as its functions in various nuclear processes has been made clear in the past few years. Actin is known to be a part of chromatin-remodeling complexes BAF,which are required for maximal ATPase activity of the Brg1 component of the BAF complex. Moreover,the essential roles of acfin in transcription mediated by RNA polymerases Ⅰ, Ⅱ and Ⅲ have been demonstrated recently. On the other hand, a myosin Ⅰ isoform, which contains a unique NH2-terminal extension for nucleus localization, has been specifically localized in nucleus. As is well known, myosin Ⅰis an actin-binding protein and plays an important role in various cellular activities. Though actin and nuclear myosin Ⅰ (NM Ⅰ) have been implicated to play distinct roles in gene expression, there has been no evidence for the actin-myosin interaction that might be involved in gene transcription mediated by RNA polymerase Ⅱ (RNAP Ⅱ). Here we show evidence that both actin and NM Ⅰ are associated with RNAP Ⅱ in nucleus by using co-localization and co-IP assays, and they may act together on gene transcription.The antibodies against β-actin or NM Ⅰ can block RNA synthesis in a eukaryotic in vitro transcription system with template DNA comprising the promoter and the coding region of human autocrine motility factor receptor (hAMFR) gene; the antibodies pre-adsorbed with purified actin and NM Ⅰ have no effect in transcriptional inhibition, indicating that the inhibition of transcription by anti-actin and anti-NM Ⅰ is specific. These results suggest a direct involvement of actin-myosin complexes in regulating transcription. It also implicates that actin and NM Ⅰ may co-exist in a same complex with RNAP Ⅱ and the interaction of RNAP Ⅱ with actin and NM Ⅰ functions in the RNAP Ⅱ-mediated transcription.

  1. Mammalian Fat1 cadherin regulates actin dynamics and cell–cell contact

    OpenAIRE

    Tanoue, Takuji; Takeichi, Masatoshi

    2004-01-01

    Fat cadherins form a distinct subfamily of the cadherin gene superfamily, and are featured by their unusually large extracellular domain. In this work, we investigated the function of a mammalian Fat cadherin. Fat1 was localized at filopodial tips, lamellipodial edges, and cell–cell boundaries, overlapping with dynamic actin structures. RNA interference–mediated knockdown of Fat1 resulted in disorganization of cell junction–associated F-actin and other actin fibers/cables, disturbance of cell...

  2. Hypotonicity and cell volume regulation in shark rectal gland: role of organic osmolytes and F-actin.

    Science.gov (United States)

    Ziyadeh, F N; Mills, J W; Kleinzeller, A

    1992-03-01

    Hypotonic stress (reduction of external tonicity from approximately 900 mosM and 295 mM NaCl to approximately 600 mosM and 135 mM NaCl) produced a relatively slow regulatory volume decrease (RVD) in dogfish shark (Squalus acanthias) rectal gland cells. During the 5-h experiment, cell K+ content remained unchanged; cell content of Na+ and Cl- dropped in the initial swelling phase by some 50% (reflecting the corresponding reduction in medium NaCl), and then remained unchanged during volume recovery phase. Also, cellular fluxes of 86Rb+ and urea were not affected by hypotonic stress. However, hypotonicity enhanced 10- to 20-fold the efflux of organic cell osmolytes taurine, betaine, and trimethyloxamine, and this accounted for the loss of osmotically obliged water during RVD. Enhancement of osmolyte efflux by hypotonic stress was abolished by readjusting the low-Na+ saline to isotonicity (approximately 900 mosM) with innocuous cations (choline+, Li+, or N-methylglucamine+). The results suggest that reduction of medium tonicity may be the determinant for the RVD response to hypotonic stress. The above properties of the observed RVD were also displayed when studying changes on cell F-actin at the basolateral cell face; hypotonic stress (medium with 135 mM NaCl) produced a rapid disappearance of fluorescence related to this cytoskeletal component, whereas no such changes were seen in low-Na+ salines made isotonic with choline or N-methylglucamine chloride nor in a saline made hyposmolar by omitting urea. Hence, hypotonicity is required to affect F-actin organization (depolymerization?). These changes of F-actin fluorescence are transient; they were completed within 5-10 min of hypotonic stress, and afterwards a gradual reconstitution of cell F-actin organization was seen. The above observations are consistent with the assumption that, in shark rectal gland cells, transient loss of cytoskeleton (F-actin) organization at the basolateral cell face, induced by hypotonicity

  3. Transportation of nanoscale cargoes by myosin propelled actin filaments.

    Directory of Open Access Journals (Sweden)

    Malin Persson

    Full Text Available Myosin II propelled actin filaments move ten times faster than kinesin driven microtubules and are thus attractive candidates as cargo-transporting shuttles in motor driven lab-on-a-chip devices. In addition, actomyosin-based transportation of nanoparticles is useful in various fundamental studies. However, it is poorly understood how actomyosin function is affected by different number of nanoscale cargoes, by cargo size, and by the mode of cargo-attachment to the actin filament. This is studied here using biotin/fluorophores, streptavidin, streptavidin-coated quantum dots, and liposomes as model cargoes attached to monomers along the actin filaments ("side-attached" or to the trailing filament end via the plus end capping protein CapZ. Long-distance transportation (>100 µm could be seen for all cargoes independently of attachment mode but the fraction of motile filaments decreased with increasing number of side-attached cargoes, a reduction that occurred within a range of 10-50 streptavidin molecules, 1-10 quantum dots or with just 1 liposome. However, as observed by monitoring these motile filaments with the attached cargo, the velocity was little affected. This also applied for end-attached cargoes where the attachment was mediated by CapZ. The results with side-attached cargoes argue against certain models for chemomechanical energy transduction in actomyosin and give important insights of relevance for effective exploitation of actomyosin-based cargo-transportation in molecular diagnostics and other nanotechnological applications. The attachment of quantum dots via CapZ, without appreciable modulation of actomyosin function, is useful in fundamental studies as exemplified here by tracking with nanometer accuracy.

  4. Actin, RhoA, and Rab11 Participation during Encystment in Entamoeba invadens

    Directory of Open Access Journals (Sweden)

    M. Herrera-Martínez

    2013-01-01

    Full Text Available In the genus Entamoeba, actin reorganization is necessary for cyst differentiation; however, its role is still unknown. The aim of this work was to investigate the role of actin and encystation-related proteins during Entamoeba invadens encystation. Studied proteins were actin, RhoA, a small GTPase involved through its effectors in the rearrangement of the actin cytoskeleton; Rab11, a protein involved in the transport of encystation vesicles; and enolase, as an encystment vesicles marker. Results showed a high level of polymerized actin accompanied by increased levels of RhoA-GTP during cell rounding and loss of vacuoles. Cytochalasin D, an actin polymerization inhibitor, and Y27632, an inhibitor of RhoA activity, reduced encystment in 80%. These inhibitors also blocked cell rounding, disposal of vacuoles, and the proper formation of the cysts wall. At later times, F-actin and Rab11 colocalized with enolase, suggesting that Rab11 could participate in the transport of the cyst wall components through the F-actin cytoskeleton. These results suggest that actin cytoskeleton rearrangement is playing a decisive role in determining cell morphology changes and helping with the transport of cell wall components to the cell surface during encystment of E. invadens.

  5. Actin, RhoA, and Rab11 participation during encystment in Entamoeba invadens.

    Science.gov (United States)

    Herrera-Martínez, M; Hernández-Ramírez, V I; Lagunes-Guillén, A E; Chávez-Munguía, B; Talamás-Rohana, P

    2013-01-01

    In the genus Entamoeba, actin reorganization is necessary for cyst differentiation; however, its role is still unknown. The aim of this work was to investigate the role of actin and encystation-related proteins during Entamoeba invadens encystation. Studied proteins were actin, RhoA, a small GTPase involved through its effectors in the rearrangement of the actin cytoskeleton; Rab11, a protein involved in the transport of encystation vesicles; and enolase, as an encystment vesicles marker. Results showed a high level of polymerized actin accompanied by increased levels of RhoA-GTP during cell rounding and loss of vacuoles. Cytochalasin D, an actin polymerization inhibitor, and Y27632, an inhibitor of RhoA activity, reduced encystment in 80%. These inhibitors also blocked cell rounding, disposal of vacuoles, and the proper formation of the cysts wall. At later times, F-actin and Rab11 colocalized with enolase, suggesting that Rab11 could participate in the transport of the cyst wall components through the F-actin cytoskeleton. These results suggest that actin cytoskeleton rearrangement is playing a decisive role in determining cell morphology changes and helping with the transport of cell wall components to the cell surface during encystment of E. invadens.

  6. Actin-myosin network is required for proper assembly of influenza virus particles

    International Nuclear Information System (INIS)

    Actin filaments are known to play a central role in cellular dynamics. After polymerization of actin, various actin-crosslinking proteins including non-muscle myosin II facilitate the formation of spatially organized actin filament networks. The actin-myosin network is highly expanded beneath plasma membrane. The genome of influenza virus (vRNA) replicates in the cell nucleus. Then, newly synthesized vRNAs are nuclear-exported to the cytoplasm as ribonucleoprotein complexes (vRNPs), followed by transport to the beneath plasma membrane where virus particles assemble. Here, we found that, by inhibiting actin-myosin network formation, the virus titer tends to be reduced and HA viral spike protein is aggregated on the plasma membrane. These results indicate that the actin-myosin network plays an important role in the virus formation. - Highlights: • Actin-myosin network is important for the influenza virus production. • HA forms aggregations at the plasma membrane in the presence of blebbistatin. • M1 is recruited to the budding site through the actin-myosin network

  7. Rapid non-equilibrium turnover fluidizes entangled F-actin solutions

    Science.gov (United States)

    McCall, Patrick M.; Kovar, David R.; Gardel, Margaret L.

    The actin cytoskeleton of living cells is a semiflexible polymer network which regulates cell division, motility, and morphogenesis by controlling cell shape. These complex shape-changing processes require both mechanical deformation and remodeling of the actin cytoskeleton. Molecular motors generate internal forces to drive deformation, while cytoskeletal remodeling is regulated by non-equilibrium polymer turnover. Although the mechanical properties of equilibrium actin filament (F-actin) networks are well-described by theories of semiflexible polymers, these theories do not incorporate the effects of non-equilibrium turnover. To address this experimentally, we developed a model system in which both the turnover rate and the length distribution of purified F-actin can be tuned independently at steady-state through the combined action of actin regulatory proteins. Specifically we tune the concentrations of cofilin, profilin, and formin to regulate F-actin severing, recycling, and nucleation, respectively. We find that the actin turnover rate can be tuned by cofilin up to 25-fold (31 +/- 2 subunits/sec/filament). Surprisingly, changes in turnover rate have no effect on the steady-state F-actin length distribution, which is instead set by formin concentration. Passive microrheology measurements show that increased turnover leads to striking fluidization in both entangled and crosslinked networks. Non-equilibrium turnover thus enables modulation of network mechanics, which impacts force transmission and material deformation.

  8. Cell-cycle regulation of formin-mediated actin cable assembly.

    Science.gov (United States)

    Miao, Yansong; Wong, Catherine C L; Mennella, Vito; Michelot, Alphée; Agard, David A; Holt, Liam J; Yates, John R; Drubin, David G

    2013-11-19

    Assembly of appropriately oriented actin cables nucleated by formin proteins is necessary for many biological processes in diverse eukaryotes. However, compared with knowledge of how nucleation of dendritic actin filament arrays by the actin-related protein-2/3 complex is regulated, the in vivo regulatory mechanisms for actin cable formation are less clear. To gain insights into mechanisms for regulating actin cable assembly, we reconstituted the assembly process in vitro by introducing microspheres functionalized with the C terminus of the budding yeast formin Bni1 into extracts prepared from yeast cells at different cell-cycle stages. EM studies showed that unbranched actin filament bundles were reconstituted successfully in the yeast extracts. Only extracts enriched in the mitotic cyclin Clb2 were competent for actin cable assembly, and cyclin-dependent kinase 1 activity was indispensible. Cyclin-dependent kinase 1 activity also was found to regulate cable assembly in vivo. Here we present evidence that formin cell-cycle regulation is conserved in vertebrates. The use of the cable-reconstitution system to test roles for the key actin-binding proteins tropomyosin, capping protein, and cofilin provided important insights into assembly regulation. Furthermore, using mass spectrometry, we identified components of the actin cables formed in yeast extracts, providing the basis for comprehensive understanding of cable assembly and regulation.

  9. Actin-myosin network is required for proper assembly of influenza virus particles

    Energy Technology Data Exchange (ETDEWEB)

    Kumakura, Michiko; Kawaguchi, Atsushi, E-mail: ats-kawaguchi@md.tsukuba.ac.jp; Nagata, Kyosuke, E-mail: knagata@md.tsukuba.ac.jp

    2015-02-15

    Actin filaments are known to play a central role in cellular dynamics. After polymerization of actin, various actin-crosslinking proteins including non-muscle myosin II facilitate the formation of spatially organized actin filament networks. The actin-myosin network is highly expanded beneath plasma membrane. The genome of influenza virus (vRNA) replicates in the cell nucleus. Then, newly synthesized vRNAs are nuclear-exported to the cytoplasm as ribonucleoprotein complexes (vRNPs), followed by transport to the beneath plasma membrane where virus particles assemble. Here, we found that, by inhibiting actin-myosin network formation, the virus titer tends to be reduced and HA viral spike protein is aggregated on the plasma membrane. These results indicate that the actin-myosin network plays an important role in the virus formation. - Highlights: • Actin-myosin network is important for the influenza virus production. • HA forms aggregations at the plasma membrane in the presence of blebbistatin. • M1 is recruited to the budding site through the actin-myosin network.

  10. Fertilization in Torenia fournieri: actin organization and nuclear behavior in the central cell and primary endosperm

    Institute of Scientific and Technical Information of China (English)

    YUAN; Ming(袁明); FU; Ying(傅缨); WANG; Feng(王凤); HUANG; Bingquan(黄炳权); Sze-Yong; Zee(徐是雄); Peter; K.Hepler

    2002-01-01

    Studies of the living embryo sacs of Torenia fournieri reveal that the actin cytoskeleton undergoes dramatic changes that correlate with nuclear migration within the central cell and the primary endosperm. Before pollination, actin filaments appear as short bundles randomly distributed in the cortex of the central cell. Two days after anthesis, they become organized into a distinct actin network. At this stage the secondary nucleus, which is located in the central region of the central cell, possesses an associated array of short actin filaments. Soon after pollination, the actin filaments become fragmented in the micropylar end and the secondary nucleus is located next to the egg apparatus. After fertilization, the primary endosperm nucleus moves away from the egg cell and actin filaments reorganize into a prominent network in the cytoplasm of the primary endosperm. Disruption of the actin cytoskeleton with latrunculin A and cytochalasin B indicates that actin is involved in the migration of the nucleus in the central cell. Our data also suggest that the dynamics of actin cytoskeleton may be responsible for the reorganization of the central cell and primary endosperm cytoplasm during fertilization.

  11. Interaction between microtubules and the Drosophila formin Cappuccino and its effect on actin assembly.

    Science.gov (United States)

    Roth-Johnson, Elizabeth A; Vizcarra, Christina L; Bois, Justin S; Quinlan, Margot E

    2014-02-14

    Formin family actin nucleators are potential coordinators of the actin and microtubule cytoskeletons, as they can both nucleate actin filaments and bind microtubules in vitro. To gain a more detailed mechanistic understanding of formin-microtubule interactions and formin-mediated actin-microtubule cross-talk, we studied microtubule binding by Cappuccino (Capu), a formin involved in regulating actin and microtubule organization during Drosophila oogenesis. We found that two distinct domains within Capu, FH2 and tail, work together to promote high-affinity microtubule binding. The tail domain appears to bind microtubules through nonspecific charge-based interactions. In contrast, distinct residues within the FH2 domain are important for microtubule binding. We also report the first visualization of a formin polymerizing actin filaments in the presence of microtubules. Interestingly, microtubules are potent inhibitors of the actin nucleation activity of Capu but appear to have little effect on Capu once it is bound to the barbed end of an elongating filament. Because Capu does not simultaneously bind microtubules and assemble actin filaments in vitro, its actin assembly and microtubule binding activities likely require spatial and/or temporal regulation within the Drosophila oocyte.

  12. TWISTED DWARF1 Mediates the Action of Auxin Transport Inhibitors on Actin Cytoskeleton Dynamics.

    Science.gov (United States)

    Zhu, Jinsheng; Bailly, Aurelien; Zwiewka, Marta; Sovero, Valpuri; Di Donato, Martin; Ge, Pei; Oehri, Jacqueline; Aryal, Bibek; Hao, Pengchao; Linnert, Miriam; Burgardt, Noelia Inés; Lücke, Christian; Weiwad, Matthias; Michel, Max; Weiergräber, Oliver H; Pollmann, Stephan; Azzarello, Elisa; Mancuso, Stefano; Ferro, Noel; Fukao, Yoichiro; Hoffmann, Céline; Wedlich-Söldner, Roland; Friml, Jiří; Thomas, Clément; Geisler, Markus

    2016-04-01

    Plant growth and architecture is regulated by the polar distribution of the hormone auxin. Polarity and flexibility of this process is provided by constant cycling of auxin transporter vesicles along actin filaments, coordinated by a positive auxin-actin feedback loop. Both polar auxin transport and vesicle cycling are inhibited by synthetic auxin transport inhibitors, such as 1-N-naphthylphthalamic acid (NPA), counteracting the effect of auxin; however, underlying targets and mechanisms are unclear. Using NMR, we map the NPA binding surface on the Arabidopsis thaliana ABCB chaperone TWISTED DWARF1 (TWD1). We identify ACTIN7 as a relevant, although likely indirect, TWD1 interactor, and show TWD1-dependent regulation of actin filament organization and dynamics and that TWD1 is required for NPA-mediated actin cytoskeleton remodeling. The TWD1-ACTIN7 axis controls plasma membrane presence of efflux transporters, and as a consequence act7 and twd1 share developmental and physiological phenotypes indicative of defects in auxin transport. These can be phenocopied by NPA treatment or by chemical actin (de)stabilization. We provide evidence that TWD1 determines downstream locations of auxin efflux transporters by adjusting actin filament debundling and dynamizing processes and mediating NPA action on the latter. This function appears to be evolutionary conserved since TWD1 expression in budding yeast alters actin polarization and cell polarity and provides NPA sensitivity. PMID:27053424

  13. Ca2+/Calmodulin-dependent Protein Kinase IV-mediated LIM Kinase Activation Is Critical for Calcium Signal-induced Neurite Outgrowth*

    OpenAIRE

    Takemura, Miyohiko; Mishima, Toshiaki; Wang, Yan; Kasahara, Jiro; Fukunaga, Kohji; Ohashi, Kazumasa; Mizuno, Kensaku

    2009-01-01

    Actin cytoskeletal remodeling is essential for neurite outgrowth. LIM kinase 1 (LIMK1) regulates actin cytoskeletal remodeling by phosphorylating and inactivating cofilin, an actin filament-disassembling factor. In this study, we investigated the role of LIMK1 in calcium signal-induced neurite outgrowth. The calcium ionophore ionomycin induced LIMK1 activation and cofilin phosphorylation in Neuro-2a neuroblastoma cells. Knockdown of LIMK1 or expression of a kinase-dead mutant of LIMK1 suppres...

  14. Intra-axonal myosin and actin in nerve regeneration.

    Science.gov (United States)

    McQuarrie, Irvine G; Lund, Linda M

    2009-10-01

    A focused review of sciatic nerve regeneration in the rat model, based on research conducted by the authors, is presented. We examine structural proteins carried distally in the axon by energy-requiring motor enzymes, using protein chemistry and molecular biology techniques in combination with immunohistochemistry. Relevant findings from other laboratories are cited and discussed. The general conclusion is that relatively large amounts of actin and tubulin are required to construct a regenerating axon and that these materials mainly originate in the parent axon. The motor enzymes that carry these proteins forward as macromolecules include kinesin and dynein but probably also include myosin. PMID:19927086

  15. Colocalization of synapsin and actin during synaptic vesicle recycling

    DEFF Research Database (Denmark)

    Bloom, Ona; Evergren, Emma; Tomilin, Nikolay;

    2003-01-01

    activity, however, synapsin was detected in the pool of vesicles proximal to the active zone. In addition, actin and synapsin were found colocalized in a dynamic filamentous cytomatrix at the sites of synaptic vesicle recycling, endocytic zones. Synapsin immunolabeling was not associated with clathrin......-coated intermediates but was found on vesicles that appeared to be recycling back to the cluster. Disruption of synapsin function by microinjection of antisynapsin antibodies resulted in a prominent reduction of the cytomatrix at endocytic zones of active synapses. Our data suggest that in addition to its known...

  16. Plasma Gelsolin and Circulating Actin Correlate with Hemodialysis Mortality

    OpenAIRE

    Lee, Po-Shun; Sampath, Kartik; Karumanchi, S. Ananth; Tamez, Hector; Bhan, Ishir; Isakova, Tamara; Gutierrez, Orlando M.; Wolf, Myles; Chang, Yuchiao; Stossel, Thomas P.; Thadhani, Ravi

    2009-01-01

    Plasma gelsolin (pGSN) binds actin and bioactive mediators to localize inflammation. Low pGSN correlates with adverse outcomes in acute injury, whereas administration of recombinant pGSN reduces mortality in experimental sepsis. We found that mean pGSN levels of 150 patients randomly selected from 10,044 starting chronic hemodialysis were 140 ± 42 mg/L, 30 to 50% lower than levels reported for healthy individuals. In a larger sample, we performed a case-control analysis to evaluate the relati...

  17. Internal Motility in Stiffening Actin-Myosin Networks

    CERN Document Server

    Uhde, J; Sackmann, E; Parmeggiani, A; Frey, E; Uhde, Joerg; Keller, Manfred; Sackmann, Erich; Parmeggiani, Andrea; Frey, Erwin

    2003-01-01

    We present a study on filamentous actin solutions containing heavy meromyosin subfragments of myosin II motor molecules. We focus on the viscoelastic phase behavior and internal dynamics of such networks during ATP depletion. Upon simultaneously using micro-rheology and fluorescence microscopy as complementary experimental tools, we find a sol-gel transition accompanied by a sudden onset of directed filament motion. We interpret the sol-gel transition in terms of myosin II enzymology, and suggest a "zipping" mechanism to explain the filament motion in the vicinity of the sol-gel transition.

  18. A Multimodular Tensegrity Model of an Actin Stress Fiber

    Science.gov (United States)

    Luo, Yaozhi; Xu, Xian; Lele, Tanmay; Kumar, Sanjay; Ingber, Donald E.

    2008-01-01

    Stress fibers are contractile bundles in the cytoskeleton that stabilize cell structure by exerting traction forces on extracellular matrix. Individual stress fibers are molecular bundles composed of parallel actin and myosin filaments linked by various actin-binding proteins, which are organized end-on-end in a sarcomere-like pattern within an elongated three-dimensional network. While measurements of single stress fibers in living cells show that they behave like tensed viscoelastic fibers, precisely how this mechanical behavior arises from this complex supramolecular arrangement of protein components remains unclear. Here we show that computationally modeling a stress fiber as a multi-modular tensegrity network can predict several key behaviors of stress fibers measured in living cells, including viscoelastic retraction, fiber splaying after severing, non-uniform contraction, and elliptical strain of a puncture wound within the fiber. The tensegrity model also can explain how they simultaneously experience passive tension and generate active contraction forces; in contrast, a tensed cable net model predicts some, but not all, of these properties. Thus, tensegrity models may provide a useful link between molecular and cellular scale mechanical behaviors, and represent a new handle on multi-scale modeling of living materials. PMID:18632107

  19. Chronic actinic dermatitis - A study of clinical features

    Directory of Open Access Journals (Sweden)

    Somani Vijay

    2005-01-01

    Full Text Available Background: Chronic actinic dermatitis (CAD, one of the immune mediated photo-dermatoses, comprises a spectrum of conditions including persistent light reactivity, photosensitive eczema and actinic reticuloid. Diagnostic criteria were laid down about 20 years back, but clinical features are the mainstay in diagnosis. In addition to extreme sensitivity to UVB, UVA and/or visible light, about three quarters of patients exhibit contact sensitivity to several allergens, which may contribute to the etiopathogenesis of CAD. This study was undertaken to examine the clinical features of CAD in India and to evaluate the relevance of patch testing and photo-aggravation testing in the diagnosis of CAD. Methods: The clinical data of nine patients with CAD were analyzed. Histopathology, patch testing and photo-aggravation testing were also performed. Results: All the patients were males. The average age of onset was 57 years. The first episode was usually noticed in the beginning of summer. Later the disease gradually tended to be perennial, without any seasonal variations. The areas affected were mainly the photo-exposed areas in all patients, and the back in three patients. Erythroderma was the presenting feature in two patients. The palms and soles were involved in five patients. Patch testing was positive in seven of nine patients. Conclusions: The diagnosis of CAD mainly depended upon the history and clinical features. The incidence of erythroderma and palmoplantar eczema was high in our series. Occupation seems to play a role in the etiopathogenesis of CAD.

  20. 5DFRXXL region of long myosin light chain kinase causes F-actin bundle formation

    Institute of Scientific and Technical Information of China (English)

    YANG Chunxiang; WEI Dongmei; CHEN Chen; YU Weiping; ZHU Minsheng

    2005-01-01

    Long myosin light chain kinase (L-MLCK) contains five DFRXXL motifs with ability to bind F-actin. Binding stoichiometry data indicated that each DFRXXL motif might bind each G-actin, but its biological significance remained unknown. We hypothesized that L-MLCK might act as an F-actin bundle peptides by its multiple binding sites of 5DFRXXL motifs to actin. In order to characterize F-actin-bundle formation properties of 5DFRXXL region of long myosin light chain kinase, we expressed and purified 5DFRXXL peptides tagged with HA in vitro. The properties of 5DFRXXL peptides binding to myofilaments or F-actin were analyzed by binding stoichiometries assays. The results indicated that 5DFRXXL peptides bound to myofilaments or F-actin with high affinity. KD values of 5DFRXXL binding to myofilaments and F-actin were 0.45 and 0.41 μmol/L, re- spectively. Cross-linking assay demonstrated that 5DFRXXL peptides could bundle F-actin efficiently. Typical F-actin bundles were observed morphologically through determina- tion of confocal and electron microscopy after adding 5DFRXXL peptides. After transfection of pEGFP-5DFRXXL plasmid into eukaryocyte, spike structure was observed around cell membrane edge. We guess that such structure formation may be attributable to F-actin over-bundle forma- tion caused by 5DFRXXL peptides. Therefore, we suppose that L-MLCK may be a new bundling protein and somehow play a certain role in organization of cell skeleton besides mediating cell contraction by it kinase activity.

  1. Stability of actin-lysozyme complexes formed in cystic fibrosis disease.

    Science.gov (United States)

    Mohammadinejad, Sarah; Ghamkhari, Behnoush; Abdolmaleki, Sarah

    2016-08-21

    Finding the conditions for destabilizing actin-lysozyme complexes is of biomedical importance in preventing infections in cystic fibrosis. In this manuscript, the effects of different charge-mutants of lysozyme and salt concentration on the stability of actin-lysozyme complexes are studied using Langevin dynamics simulation. A coarse-grained model of F-actin is used in which both its twist and bending rigidities are considered. We observe that the attraction between F-actins is stronger in the presence of wild-type lysozymes relative to the mutated lysozymes of lower charges. By calculating the potential of mean force between F-actins, we conclude that the stability of actin-lysozyme complexes is decreased by reducing the charge of lysozyme mutants. The distributions of different lysozyme charge-mutants show that wild-type (+9e) lysozymes are mostly accumulated in the center of triangles formed by three adjacent F-actins, while lysozyme mutants of charges +7e and +5e occupy the bridging regions between F-actins. Low-charge mutants of lysozyme (+3e) distribute uniformly around F-actins. A rough estimate of the electrostatic energy for these different distributions proves that the distribution in which lysozymes reside in the center of triangles leads to more stable complexes. Also our results in the presence of a salt suggest that at physiological salt concentration of airway, F-actin complexes are not formed by charge-reduced mutants of lysozyme. The findings are interesting because if we can design charge-reduced lysozyme mutants with considerable antibacterial activity, they are not sequestered inside F-actin aggregates and can play their role as antibacterial agents against airway infection. PMID:27436705

  2. Lgr4 and Lgr5 drive the formation of long actin-rich cytoneme-like membrane protrusions

    Science.gov (United States)

    Snyder, Joshua C.; Rochelle, Lauren K.; Marion, Sébastien; Lyerly, H. Kim; Barak, Larry S.; Caron, Marc G.

    2015-01-01

    ABSTRACT Embryonic development and adult tissue homeostasis require precise information exchange between cells and their microenvironment to coordinate cell behavior. A specialized class of ultra-long actin-rich filopodia, termed cytonemes, provides one mechanism for this spatiotemporal regulation of extracellular cues. We provide here a mechanism whereby the stem-cell marker Lgr5, and its family member Lgr4, promote the formation of cytonemes. Lgr4- and Lgr5-induced cytonemes exceed lengths of 80 µm, are generated through stabilization of nascent filopodia from an underlying lamellipodial-like network and functionally provide a pipeline for the transit of signaling effectors. As proof-of-principle, we demonstrate that Lgr5-induced cytonemes act as conduits for cell signaling by demonstrating that the actin motor and filopodial cargo carrier protein myosin X (Myo10) and the G-protein-coupled receptor (GPCR) signaling effector β-arrestin-2 (Arrb2) transit into cytonemes. This work delineates a biological function for Lgr4 and Lgr5 and provides the rationale to fully investigate Lgr4 and Lgr5 function and cytonemes in mammalian stem cell and cancer stem cell behavior. PMID:25653388

  3. Induction of actin disruption and downregulation of P-glycoprotein expression by solamargine in multidrug-resistant K562/A02 cells

    Institute of Scientific and Technical Information of China (English)

    LI Xia; ZHAO Ying; JI Mei; LIU Shan-shan; CUI Min; LOU Hong-xiang

    2011-01-01

    Background Solamargine (SM), a steroidal glycoalkaloid isolated from the Chinese herb Solarium incanum, has been shown to inhibit the growth of some cancer cell lines and induce significant apoptosis. However, the effects of SM on multidrug-resistant (MDR) cells and the molecular mechanisms involved are poorly understood. The purpose of this study was to evaluate the anti-MDR effects of SM and the associated mechanisms in MDR K562/A02 cells.Methods The cytotoxicity of SM was measured by 3-(4,5-dimethylthiazol)-2,5-diphenyltetrazolium bromide (MTT) assay. The 14',6-diamidino-2-phenylindole (DAPI) nuclear staining and flow cytometry were used to detect SM-induced apoptosis. The mRNA expression of P-glycoprotein (P-gp) was investigated by real-time PCR (RT-PCR). Western blotting was used to determine the expression of Bcl-2, Bax, and actin. The changes in the morphology of actin were examined with immunofluorescence staining.Results MTT results showed that SM effectively killed the MDR sublines K562/A02, KB/VCR, and H460/paclitaxel (Taxol), and their parental cell lines K562, KB, and H460 to an equivalent or more sensitive degree. Based on the results by flow cytometry and immunostaining, the pro-apoptotic effects of SM were observed in MDR K562/A02 cells. Furthermore, the RT-PCR results showed that SM induced the downregulation of MDR1 mRNA. In addition, the expression of P-gp and actin was decreased in the SM-treated cells, as measured by western blotting and immunostaining.Conclusions These results demonstrate that SM effectively triggers apoptosis in MDR tumor cells, which is associated with actin disruption and downregulation of MDR1 expression. This compound may merit further investigation as a potential therapeutic agent that bypasses the MDR mechanism for the treatment of MDR tumors.

  4. Actin based processes that could determine the cytoplasmic architecture of plant cells

    NARCIS (Netherlands)

    Honing, van der H.S.; Emons, A.M.C.; Ketelaar, M.J.

    2007-01-01

    Actin polymerisation can generate forces that are necessary for cell movement, such as the propulsion of a class of bacteria, including Listeria, and the protrusion of migrating animal cells. Force generation by the actin cytoskeleton in plant cells has not been studied. One process in plant cells t

  5. The Actin-Binding Protein α-Adducin Is Required for Maintaining Axon Diameter

    Directory of Open Access Journals (Sweden)

    Sérgio Carvalho Leite

    2016-04-01

    Full Text Available The actin-binding protein adducin was recently identified as a component of the neuronal subcortical cytoskeleton. Here, we analyzed mice lacking adducin to uncover the function of this protein in actin rings. α-adducin knockout mice presented progressive axon enlargement in the spinal cord and optic and sciatic nerves, followed by axon degeneration and loss. Using stimulated emission depletion super-resolution microscopy, we show that a periodic subcortical actin cytoskeleton is assembled in every neuron type inspected including retinal ganglion cells and dorsal root ganglia neurons. In neurons devoid of adducin, the actin ring diameter increased, although the inter-ring periodicity was maintained. In vitro, the actin ring diameter adjusted as axons grew, suggesting the lattice is dynamic. Our data support a model in which adducin activity is not essential for actin ring assembly and periodicity but is necessary to control the diameter of both actin rings and axons and actin filament growth within rings.

  6. Aspects of plant cell growth and the actin cytoskeleton: lessons from root hairs

    NARCIS (Netherlands)

    Ruijter, de N.C.A.

    1999-01-01

    The main topic the thesis addresses is the role of the actin cytoskeleton in the growth process of plant cells. Plant growth implies a combination of cell division and cell expansion. The cytoskeleton, which exists of microtubules and actin filaments, plays a major role in both processes. Before cel

  7. β-Actin protein expression differs in the submandibular glands of male and female mice.

    Science.gov (United States)

    Chen, Gang; Zou, Ye; Zhang, Xuan; Xu, Lingfei; Hu, Qiaoyun; Li, Ting; Yao, Chenjuan; Yu, Shali; Wang, Xiaoke; Wang, Chun

    2016-07-01

    β-actin, a cytoskeletal protein, is the most widely used housekeeping gene. Although housekeeping genes are expressed in all tissues, the β-actin gene is expressed in certain cell types because of differential binding of transcriptional factors to the regulatory elements of the gene. The expression and localization of β-actin protein in the submandibular glands (SMG) of mice were investigated in this study, using Western blot analysis and immunohistochemistry. In ICR and C57BL/6J mice, the levels of β-actin protein in the SMG of females are significantly higher than those in the SMG of males. β-actin protein is majorly distributed in acinar cells of SMG. There is no significant difference in the expression level of β-actin protein between females and castrated males. After castrated male ICR mice are treated with 10 mg/kg/day testosterone propionate (TP) for 3 weeks, the levels of β-actin protein in SMG decrease. The numbers of duct per unit area increase, whereas the numbers of acinus per unit area decrease after TP administration. These data suggest that β-actin protein is mainly distributed in acinar cells of SMG and results in a marked sexual dimorphism in mice. PMID:27079296

  8. Calcium influx through CRAC channels controls actin organization and dynamics at the immune synapse.

    Science.gov (United States)

    Hartzell, Catherine A; Jankowska, Katarzyna I; Burkhardt, Janis K; Lewis, Richard S

    2016-01-01

    T cell receptor (TCR) engagement opens Ca(2+) release-activated Ca(2+) (CRAC) channels and triggers formation of an immune synapse between T cells and antigen-presenting cells. At the synapse, actin reorganizes into a concentric lamellipod and lamella with retrograde actin flow that helps regulate the intensity and duration of TCR signaling. We find that Ca(2+) influx is required to drive actin organization and dynamics at the synapse. Calcium acts by promoting actin depolymerization and localizing actin polymerization and the actin nucleation promotion factor WAVE2 to the periphery of the lamellipod while suppressing polymerization elsewhere. Ca(2+)-dependent retrograde actin flow corrals ER tubule extensions and STIM1/Orai1 complexes to the synapse center, creating a self-organizing process for CRAC channel localization. Our results demonstrate a new role for Ca(2+) as a critical regulator of actin organization and dynamics at the synapse, and reveal potential feedback loops through which Ca(2+) influx may modulate TCR signaling. PMID:27440222

  9. A dynamin-actin interaction is required for vesicle scission during endocytosis in yeast.

    Science.gov (United States)

    Palmer, Sarah E; Smaczynska-de Rooij, Iwona I; Marklew, Christopher J; Allwood, Ellen G; Mishra, Ritu; Johnson, Simeon; Goldberg, Martin W; Ayscough, Kathryn R

    2015-03-30

    Actin is critical for endocytosis in yeast cells, and also in mammalian cells under tension. However, questions remain as to how force generated through actin polymerization is transmitted to the plasma membrane to drive invagination and scission. Here, we reveal that the yeast dynamin Vps1 binds and bundles filamentous actin. Mutational analysis of Vps1 in a helix of the stalk domain identifies a mutant RR457-458EE that binds actin more weakly. In vivo analysis of Vps1 function demonstrates that the mutation disrupts endocytosis but not other functions of Vps1 such as vacuolar trafficking or peroxisome fission. The mutant Vps1 is stably expressed in cells and co-localizes with the endocytic reporters Abp1 and the amphiphysin Rvs167. Detailed analysis of individual endocytic patch behavior indicates that the mutation causes aberrant movements in later stages of endocytosis, consistent with a scission defect. Ultrastructural analysis of yeast cells using electron microscopy reveals a significant increase in invagination depth, further supporting a role for the Vps1-actin interaction during scission. In vitro analysis of the mutant protein demonstrates that--like wild-type Vps1--it is able to form oligomeric rings, but, critically, it has lost its ability to bundle actin filaments into higher-order structures. A model is proposed in which actin filaments bind Vps1 during invagination, and this interaction is important to transduce the force of actin polymerization to the membrane to drive successful scission.

  10. Opposing Roles for Actin in Cdc42p PolarizationD⃞

    Science.gov (United States)

    Irazoqui, Javier E.; Howell, Audrey S.; Theesfeld, Chandra L.; Lew, Daniel J.

    2005-01-01

    In animal and fungal cells, the monomeric GTPase Cdc42p is a key regulator of cell polarity that itself exhibits a polarized distribution in asymmetric cells. Previous work showed that in budding yeast, Cdc42p polarization is unaffected by depolymerization of the actin cytoskeleton (Ayscough et al., J. Cell Biol. 137, 399–416, 1997). Surprisingly, we now report that unlike complete actin depolymerization, partial actin depolymerization leads to the dispersal of Cdc42p from the polarization site in unbudded cells. We provide evidence that dispersal is due to endocytosis associated with cortical actin patches and that actin cables are required to counteract the dispersal and maintain Cdc42p polarity. Thus, although Cdc42p is initially polarized in an actin-independent manner, maintaining that polarity may involve a reinforcing feedback between Cdc42p and polarized actin cables to counteract the dispersing effects of actin-dependent endocytosis. In addition, we report that once a bud has formed, polarized Cdc42p becomes more resistant to dispersal, revealing an unexpected difference between unbudded and budded cells in the organization of the polarization site. PMID:15616194

  11. Calcium influx through CRAC channels controls actin organization and dynamics at the immune synapse

    Science.gov (United States)

    Hartzell, Catherine A; Jankowska, Katarzyna I; Burkhardt, Janis K; Lewis, Richard S

    2016-01-01

    T cell receptor (TCR) engagement opens Ca2+ release-activated Ca2+ (CRAC) channels and triggers formation of an immune synapse between T cells and antigen-presenting cells. At the synapse, actin reorganizes into a concentric lamellipod and lamella with retrograde actin flow that helps regulate the intensity and duration of TCR signaling. We find that Ca2+ influx is required to drive actin organization and dynamics at the synapse. Calcium acts by promoting actin depolymerization and localizing actin polymerization and the actin nucleation promotion factor WAVE2 to the periphery of the lamellipod while suppressing polymerization elsewhere. Ca2+-dependent retrograde actin flow corrals ER tubule extensions and STIM1/Orai1 complexes to the synapse center, creating a self-organizing process for CRAC channel localization. Our results demonstrate a new role for Ca2+ as a critical regulator of actin organization and dynamics at the synapse, and reveal potential feedback loops through which Ca2+ influx may modulate TCR signaling. DOI: http://dx.doi.org/10.7554/eLife.14850.001 PMID:27440222

  12. Drosophila Fascin is a novel downstream target of prostaglandin signaling during actin remodeling.

    Science.gov (United States)

    Groen, Christopher M; Spracklen, Andrew J; Fagan, Tiffany N; Tootle, Tina L

    2012-12-01

    Although prostaglandins (PGs)-lipid signals produced downstream of cyclooxygenase (COX) enzymes-regulate actin cytoskeletal dynamics, their mechanisms of action are unknown. We previously established Drosophila oogenesis, in particular nurse cell dumping, as a new model to determine how PGs regulate actin remodeling. PGs, and thus the Drosophila COX-like enzyme Pxt, are required for both the parallel actin filament bundle formation and the cortical actin strengthening required for dumping. Here we provide the first link between Fascin (Drosophila Singed, Sn), an actin-bundling protein, and PGs. Loss of either pxt or fascin results in similar actin defects. Fascin interacts, both pharmacologically and genetically, with PGs, as reduced Fascin levels enhance the effects of COX inhibition and synergize with reduced Pxt levels to cause both parallel bundle and cortical actin defects. Conversely, overexpression of Fascin in the germline suppresses the effects of COX inhibition and genetic loss of Pxt. These data lead to the conclusion that PGs regulate Fascin to control actin remodeling. This novel interaction has implications beyond Drosophila, as both PGs and Fascin-1, in mammalian systems, contribute to cancer cell migration and invasion.

  13. EhCoactosin stabilizes actin filaments in the protist parasite Entamoeba histolytica.

    Directory of Open Access Journals (Sweden)

    Nitesh Kumar

    2014-09-01

    Full Text Available Entamoeba histolytica is a protist parasite that is the causative agent of amoebiasis, and is a highly motile organism. The motility is essential for its survival and pathogenesis, and a dynamic actin cytoskeleton is required for this process. EhCoactosin, an actin-binding protein of the ADF/cofilin family, participates in actin dynamics, and here we report our studies of this protein using both structural and functional approaches. The X-ray crystal structure of EhCoactosin resembles that of human coactosin-like protein, with major differences in the distribution of surface charges and the orientation of terminal regions. According to in vitro binding assays, full-length EhCoactosin binds both F- and G-actin. Instead of acting to depolymerize or severe F-actin, EhCoactosin directly stabilizes the polymer. When EhCoactosin was visualized in E. histolytica cells using either confocal imaging or total internal reflectance microscopy, it was found to colocalize with F-actin at phagocytic cups. Over-expression of this protein stabilized F-actin and inhibited the phagocytic process. EhCoactosin appears to be an unusual type of coactosin involved in E. histolytica actin dynamics.

  14. Changes in actin dynamics are involved in salicylic acid signaling pathway.

    Science.gov (United States)

    Matoušková, Jindřiška; Janda, Martin; Fišer, Radovan; Sašek, Vladimír; Kocourková, Daniela; Burketová, Lenka; Dušková, Jiřina; Martinec, Jan; Valentová, Olga

    2014-06-01

    Changes in actin cytoskeleton dynamics are one of the crucial players in many physiological as well as non-physiological processes in plant cells. Positioning of actin filament arrays is necessary for successful establishment of primary lines of defense toward pathogen attack, depolymerization leads very often to the enhanced susceptibility to the invading pathogen. On the other hand it was also shown that the disruption of actin cytoskeleton leads to the induction of defense response leading to the expression of PATHOGENESIS RELATED proteins (PR). In this study we show that pharmacological actin depolymerization leads to the specific induction of genes in salicylic acid pathway but not that involved in jasmonic acid signaling. Life imaging of leafs of Arabidopsis thaliana with GFP-tagged fimbrin (GFP-fABD2) treated with 1 mM salicylic acid revealed rapid disruption of actin filaments resembling the pattern viewed after treatment with 200 nM latrunculin B. The effect of salicylic acid on actin filament fragmentation was prevented by exogenous addition of phosphatidic acid, which binds to the capping protein and thus promotes actin polymerization. The quantitative evaluation of actin filament dynamics is also presented.

  15. Drosophila myosin-XX functions as an actin-binding protein to facilitate the interaction between Zyx102 and actin.

    Science.gov (United States)

    Cao, Yang; White, Howard D; Li, Xiang-Dong

    2014-01-21

    The class XX myosin is a member of the diverse myosin superfamily and exists in insects and several lower invertebrates. DmMyo20, the class XX myosin in Drosophila, is encoded by dachs, which functions as a crucial downstream component of the Fat signaling pathway, influencing growth, affinity, and gene expression during development. Sequence analysis shows that DmMyo20 contains a unique N-terminal extension, the motor domain, followed by one IQ motif, and a C-terminal tail. To investigate the biochemical properties of DmMyo20, we expressed several DmMyo20 truncated constructs containing the motor domain in the baculovirus/Sf9 system. We found that the motor domain of DmMyo20 had neither ATPase activity nor the ability to bind to ATP, suggesting that DmMyo20 does not function as a molecular motor. We found that the motor domain of DmMyo20 could specifically bind to actin filaments in an ATP-independent manner and enhance the interaction between actin filaments and Zyx102, a downstream component of DmMyo20 in the Fat signaling pathway. These results suggest that DmMyo20 functions as a scaffold protein, but not as a molecular motor, in a signaling pathway controlling cell differentiation.

  16. Engineering an artificial amoeba propelled by nanoparticle-triggered actin polymerization

    Energy Technology Data Exchange (ETDEWEB)

    Yi Jinsoo; Schmidt, Jacob; Chien Aichi; Montemagno, Carlo D [Department of Bioengineering, University of California Los Angeles, 420 Westwood Plaza, 7523 Boelter Hall, Los Angeles, CA 90095-1600 (United States)], E-mail: montemcd@ucmail.uc.edu

    2009-02-25

    We have engineered an amoeba system combining nanofabricated inorganic materials with biological components, capable of propelling itself via actin polymerization. The nanofabricated materials have a mechanism similar to the locomotion of the Listeria monocytogenes, food poisoning bacteria. The propulsive force generation utilizes nanoparticles made from nickel and gold functionalized with the Listeria monocytogenes transmembrane protein, ActA. These Listeria-mimic nanoparticles were in concert with actin, actin binding proteins, ATP (adenosine triphosphate) and encapsulated within a lipid vesicle. This system is an artificial cell, such as a vesicle, where artificial nanobacteria and actin polymerization machinery are used in driving force generators inside the cell. The assembled structure was observed to crawl on a glass surface analogously to an amoeba, with the speed of the movement dependent on the amount of actin monomers and ATP present.

  17. Engineering an artificial amoeba propelled by nanoparticle-triggered actin polymerization

    International Nuclear Information System (INIS)

    We have engineered an amoeba system combining nanofabricated inorganic materials with biological components, capable of propelling itself via actin polymerization. The nanofabricated materials have a mechanism similar to the locomotion of the Listeria monocytogenes, food poisoning bacteria. The propulsive force generation utilizes nanoparticles made from nickel and gold functionalized with the Listeria monocytogenes transmembrane protein, ActA. These Listeria-mimic nanoparticles were in concert with actin, actin binding proteins, ATP (adenosine triphosphate) and encapsulated within a lipid vesicle. This system is an artificial cell, such as a vesicle, where artificial nanobacteria and actin polymerization machinery are used in driving force generators inside the cell. The assembled structure was observed to crawl on a glass surface analogously to an amoeba, with the speed of the movement dependent on the amount of actin monomers and ATP present.

  18. Covalent immobilization of myosin for in-vitro motility of actin

    Indian Academy of Sciences (India)

    Ellis Bagga; Sunita Kumari; Rajesh Kumar; Rakesh Kumar; R P Bajpai; Lalit M Bharadwaj

    2005-11-01

    The present study reports the covalent immobilization of myosin on glass surface and in-vitro motility of actin-myosin biomolecular motor. Myosin was immobilized on poly-L-lysine coated glass using heterobifunctional cross linker EDC and characterized by AFM. The in-vitro motility of actin was carried out on the immobilized myosin. It was observed that velocity of actin over myosin increases with increasing actin concentration (0.4-1.0 mg/ml) and was found in the range of 0.40-3.25 m/s. The motility of actin-myosin motor on artificial surfaces is of immense importance for developing nanodevices for healthcare and engineering applications.

  19. Echinococcus granulosus: Cloning and Functional in Vitro Characterization of an Actin Filament Fragmenting Protein.

    Science.gov (United States)

    Cortez-Herrera, E; Yamamoto, R R; Rodrigues, J J; Farias, S E; Ferreira, H B; Zaha, A

    2001-04-01

    We report the isolation and characterization of an Echinococcus granulosus gene that codes for a protein with actin filament fragmenting and nucleating activities (EgAFFP). The genomic region corresponding to the EgAFFP gene presents a coding sequence of 1110 bp that is interrupted by eight introns. The EgAFFP deduced amino acid sequence is about 40% homologous to those of several members of the gelsolin family, such as Physarum polycephalum fragmin, Dictyostelium discoideum severin, and Lumbricus terrestris actin modulator. As do other proteins of the same family, EgAFFP presents three repeated domains, each one characterized by internal conserved amino acid motifs. Assays with fluorescence-labeled actin showed that the full-length recombinant EgAFFP effectively binds actin monomers in both a calcium-dependent and calcium-independent manner and also presents actin nucleating and severing activities.

  20. Rk1, a ginsenoside, is a new blocker of vascular leakage acting through actin structure remodeling.

    Science.gov (United States)

    Maeng, Yong-Sun; Maharjan, Sony; Kim, Jeong-Hun; Park, Jeong-Hill; Suk Yu, Young; Kim, Young-Myoung; Kwon, Young-Guen

    2013-01-01

    Endothelial barrier integrity is essential for vascular homeostasis and increased vascular permeability and has been implicated in many pathological processes, including diabetic retinopathy. Here, we investigated the effect of Rk1, a ginsenoside extracted from sun ginseng, on regulation of endothelial barrier function. In human retinal endothelial cells, Rk1 strongly inhibited permeability induced by VEGF, advanced glycation end-product, thrombin, or histamine. Furthermore, Rk1 significantly reduced the vessel leakiness of retina in a diabetic mouse model. This anti-permeability activity of Rk1 is correlated with enhanced stability and positioning of tight junction proteins at the boundary between cells. Signaling experiments revealed that Rk1 induces phosphorylation of myosin light chain and cortactin, which are critical regulators for the formation of the cortical actin ring structure and endothelial barrier. These findings raise the possibility that ginsenoside Rk1 could be exploited as a novel prototype compound for the prevention of human diseases that are characterized by vascular leakage. PMID:23894330

  1. Phospholipase C and myosin light chain kinase inhibition define a common step in actin regulation during cytokinesis

    Directory of Open Access Journals (Sweden)

    Fabian Lacramioara

    2007-05-01

    Full Text Available Abstract Background Phosphatidylinositol 4,5-bisphosphate (PIP2 is required for successful completion of cytokinesis. In addition, both PIP2 and phosphoinositide-specific phospholipase C (PLC have been localized to the cleavage furrow of dividing mammalian cells. PLC hydrolyzes PIP2 to yield diacylglycerol (DAG and inositol trisphosphate (IP3, which in turn induces calcium (Ca2+ release from the ER. Several studies suggest PIP2 must be hydrolyzed continuously for continued cleavage furrow ingression. The majority of these studies employ the N-substituted maleimide U73122 as an inhibitor of PLC. However, the specificity of U73122 is unclear, as its active group closely resembles the non-specific alkylating agent N-ethylmaleimide (NEM. In addition, the pathway by which PIP2 regulates cytokinesis remains to be elucidated. Results Here we compared the effects of U73122 and the structurally unrelated PLC inhibitor ET-18-OCH3 (edelfosine on cytokinesis in crane-fly and Drosophila spermatocytes. Our data show that the effects of U73122 are indeed via PLC because U73122 and ET-18-OCH3 produced similar effects on cell morphology and actin cytoskeleton organization that were distinct from those caused by NEM. Furthermore, treatment with the myosin light chain kinase (MLCK inhibitor ML-7 caused cleavage furrow regression and loss of both F-actin and phosphorylated myosin regulatory light chain from the contractile ring in a manner similar to treatment with U73122 and ET-18-OCH3. Conclusion We have used multiple inhibitors to examine the roles of PLC and MLCK, a predicted downstream target of PLC regulation, in cytokinesis. Our results are consistent with a model in which PIP2 hydrolysis acts via Ca2+ to activate myosin via MLCK and thereby control actin dynamics during constriction of the contractile ring.

  2. Compartmentalisation of cAMP-dependent signalling in blood platelets: The role of lipid rafts and actin polymerisation.

    Science.gov (United States)

    Raslan, Zaher; Naseem, Khalid M

    2015-01-01

    Prostacyclin (PGI2) inhibits blood platelets through the activation of membrane adenylyl cyclases (ACs) and cyclic adenosine 3',5'-monophosphate (cAMP)-mediated signalling. However, the molecular mechanism controlling cAMP signalling in blood platelet remains unclear, and in particular how individual isoforms of AC and protein kinase A (PKA) are coordinated to target distinct substrates in order to modulate platelet activation. In this study, we demonstrate that lipid rafts and the actin cytoskeleton may play a key role in regulating platelet responses to cAMP downstream of PGI2. Disruption of lipid rafts with methyl-beta-cyclodextrin (MβCD) increased platelet sensitivity to PGI2 and forskolin, a direct AC cyclase activator, resulting in greater inhibition of collagen-stimulated platelet aggregation. In contrast, platelet inhibition by the direct activator of PKA, 8-CPT-6-Phe-cAMP was unaffected by MβCD treatment. Consistent with the functional data, lipid raft disruption increased PGI2-stimulated cAMP formation and proximal PKA-mediated signalling events. Platelet inhibition, cAMP formation and phosphorylation of PKA substrates in response to PGI2 were also increased in the presence of cytochalasin D, indicating a role for actin cytoskeleton in signalling in response to PGI2. A potential role for lipid rafts in cAMP signalling is strengthened by our finding that a pool of ACV/VI and PKA was partitioned into lipid rafts. Our data demonstrate partial compartmentalisation of cAMP signalling machinery in platelets, where lipid rafts and the actin cytoskeleton regulate the inhibitory effects induced by PGI2. The increased platelet sensitivity to cAMP-elevating agents signalling upon raft and cytoskeleton disruption suggests that these compartments act to restrain basal cAMP signalling.

  3. Yersinia outer protein YopE affects the actin cytoskeleton in Dictyostelium discoideum through targeting of multiple Rho family GTPases

    LENUS (Irish Health Repository)

    Vlahou, Georgia

    2009-07-14

    Abstract Background All human pathogenic Yersinia species share a virulence-associated type III secretion system that translocates Yersinia effector proteins into host cells to counteract infection-induced signaling responses and prevent phagocytosis. Dictyostelium discoideum has been recently used to study the effects of bacterial virulence factors produced by internalized pathogens. In this study we explored the potential of Dictyostelium as model organism for analyzing the effects of ectopically expressed Yersinia outer proteins (Yops). Results The Yersinia pseudotuberculosis virulence factors YopE, YopH, YopM and YopJ were expressed de novo within Dictyostelium and their effects on growth in axenic medium and on bacterial lawns were analyzed. No severe effect was observed for YopH, YopJ and YopM, but expression of YopE, which is a GTPase activating protein for Rho GTPases, was found to be highly detrimental. GFP-tagged YopE expressing cells had less conspicuous cortical actin accumulation and decreased amounts of F-actin. The actin polymerization response upon cAMP stimulation was impaired, although chemotaxis was unaffected. YopE also caused reduced uptake of yeast particles. These alterations are probably due to impaired Rac1 activation. We also found that YopE predominantly associates with intracellular membranes including the Golgi apparatus and inhibits the function of moderately overexpressed RacH. Conclusion The phenotype elicited by YopE in Dictyostelium can be explained, at least in part, by inactivation of one or more Rho family GTPases. It further demonstrates that the social amoeba Dictyostelium discoideum can be used as an efficient and easy-to-handle model organism in order to analyze the function of a translocated GAP protein of a human pathogen.

  4. Myofibril growth during cardiac hypertrophy is regulated through dual phosphorylation and acetylation of the actin capping protein CapZ.

    Science.gov (United States)

    Lin, Ying-Hsi; Warren, Chad M; Li, Jieli; McKinsey, Timothy A; Russell, Brenda

    2016-08-01

    The mechanotransduction signaling pathways initiated in heart muscle by increased mechanical loading are known to lead to long-term transcriptional changes and hypertrophy, but the rapid events for adaptation at the sarcomeric level are not fully understood. The goal of this study was to test the hypothesis that actin filament assembly during cardiomyocyte growth is regulated by post-translational modifications (PTMs) of CapZβ1. In rapidly hypertrophying neonatal rat ventricular myocytes (NRVMs) stimulated by phenylephrine (PE), two-dimensional gel electrophoresis (2DGE) of CapZβ1 revealed a shift toward more negative charge. Consistent with this, mass spectrometry identified CapZβ1 phosphorylation on serine-204 and acetylation on lysine-199, two residues which are near the actin binding surface of CapZβ1. Ectopic expression of dominant negative PKCɛ (dnPKCɛ) in NRVMs blunted the PE-induced increase in CapZ dynamics, as evidenced by the kinetic constant (Kfrap) of fluorescence recovery after photobleaching (FRAP), and concomitantly reduced phosphorylation and acetylation of CapZβ1. Furthermore, inhibition of class I histone deacetylases (HDACs) increased lysine-199 acetylation on CapZβ1, which increased Kfrap of CapZ and stimulated actin dynamics. Finally, we show that PE treatment of NRVMs results in decreased binding of HDAC3 to myofibrils, suggesting a signal-dependent mechanism for the regulation of sarcomere-associated CapZβ1 acetylation. Taken together, this dual regulation through phosphorylation and acetylation of CapZβ1 provides a novel model for the regulation of myofibril growth during cardiac hypertrophy. PMID:27185186

  5. Coupling of the hydration water dynamics and the internal dynamics of actin detected by quasielastic neutron scattering

    International Nuclear Information System (INIS)

    Highlights: ► Quasielastic neutron scattering spectra of F-actin and G-actin were measured. ► Analysis of the samples in D2O and H2O provided the spectra of hydration water. ► The first layer hydration water around F-actin is less mobile than around G-actin. ► This difference in hydration water is in concert with the internal dynamics of actin. ► Water outside the first layer behaves bulk-like but influenced by the first layer. -- Abstract: In order to characterize dynamics of water molecules around F-actin and G-actin, quasielastic neutron scattering experiments were performed on powder samples of F-actin and G-actin, hydrated either with D2O or H2O, at hydration ratios of 0.4 and 1.0. By combined analysis of the quasielastic neutron scattering spectra, the parameter values characterizing the dynamics of the water molecules in the first hydration layer and those of the water molecules outside of the first layer were obtained. The translational diffusion coefficients (DT) of the hydration water in the first layer were found to be 1.2 × 10−5 cm2/s and 1.7 × 10−5 cm2/s for F-actin and G-actin, respectively, while that for bulk water was 2.8 × 10−5 cm2/s. The residence times were 6.6 ps and 5.0 ps for F-actin and G-actin, respectively, while that for bulk water was 0.62 ps. These differences between F-actin and G-actin, indicating that the hydration water around G-actin is more mobile than that around F-actin, are in concert with the results of the internal dynamics of F-actin and G-actin, showing that G-actin fluctuates more rapidly than F-actin. This implies that the dynamics of the hydration water is coupled to the internal dynamics of the actin molecules. The DT values of the water molecules outside of the first hydration layer were found to be similar to that of bulk water though the residence times are strongly affected by the first hydration layer. This supports the recent observation on intracellular water that shows bulk-like behavior

  6. c-Jun N-terminal kinase phosphorylation of MARCKSL1 determines actin stability and migration in neurons and in cancer cells.

    Science.gov (United States)

    Björkblom, Benny; Padzik, Artur; Mohammad, Hasan; Westerlund, Nina; Komulainen, Emilia; Hollos, Patrik; Parviainen, Lotta; Papageorgiou, Anastassios C; Iljin, Kristiina; Kallioniemi, Olli; Kallajoki, Markku; Courtney, Michael J; Mågård, Mats; James, Peter; Coffey, Eleanor T

    2012-09-01

    Cell migration is a fundamental biological function, critical during development and regeneration, whereas deregulated migration underlies neurological birth defects and cancer metastasis. MARCKS-like protein 1 (MARCKSL1) is widely expressed in nervous tissue, where, like Jun N-terminal protein kinase (JNK), it is required for neural tube formation, though the mechanism is unknown. Here we show that MARCKSL1 is directly phosphorylated by JNK on C-terminal residues (S120, T148, and T183). This phosphorylation enables MARCKSL1 to bundle and stabilize F-actin, increase filopodium numbers and dynamics, and retard migration in neurons. Conversely, when MARCKSL1 phosphorylation is inhibited, actin mobility increases and filopodium formation is compromised whereas lamellipodium formation is enhanced, as is cell migration. We find that MARCKSL1 mRNA is upregulated in a broad range of cancer types and that MARCKSL1 protein is strongly induced in primary prostate carcinomas. Gene knockdown in prostate cancer cells or in neurons reveals a critical role for MARCKSL1 in migration that is dependent on the phosphorylation state; phosphomimetic MARCKSL1 (MARCKSL1(S120D,T148D,T183D)) inhibits whereas dephospho-MARCKSL1(S120A,T148A,T183A) induces migration. In summary, these data show that JNK phosphorylation of MARCKSL1 regulates actin homeostasis, filopodium and lamellipodium formation, and neuronal migration under physiological conditions and that, when ectopically expressed in prostate cancer cells, MARCKSL1 again determines cell movement.

  7. Possible roles of actin and myosin during anaphase chromosome movements in locust spermatocytes.

    Science.gov (United States)

    Fabian, Lacramioara; Forer, Arthur

    2007-01-01

    We tested whether the mechanisms of chromosome movement during anaphase in locust (Locusta migratoria L.) spermatocytes might be similar to those described for crane-fly spermatocytes. Actin and myosin have been implicated in anaphase chromosome movements in crane-fly spermatocytes, as indicated by the effects of inhibitors and by the localisations of actin and myosin in spindles. In this study, we tested whether locust spermatocyte spindles also utilise actin and myosin, and whether actin is involved in microtubule flux. Living locust spermatocytes were treated with inhibitors of actin (latrunculin B and cytochalasin D), myosin (BDM), or myosin phosphorylation (Y-27632 and ML-7). We added drugs (individually) during anaphase. Actin inhibitors alter anaphase: chromosomes either completely stop moving, slow, or sometimes accelerate. The myosin inhibitor, BDM, also alters anaphase: in most cases, the chromosomes drastically slow or stop. ML-7, an inhibitor of MLCK, causes chromosomes to stop, slow, or sometimes accelerate, similar to actin inhibitors. Y-27632, an inhibitor of Rho-kinase, drastically slows or stops anaphase chromosome movements. The effects of the drugs on anaphase movement are reversible: most of the half-bivalents resumed movement at normal speed after these drugs were washed out. Actin and myosin were present in the spindles in locations consistent with their possible involvement in force production. Microtubule flux along kinetochore fibres is an actin-dependent process, since LatB completely removes or drastically reduces the gap in microtubule acetylation at the kinetochore. These results suggest that actin and myosin are involved in anaphase chromosome movements in locust spermatocytes. PMID:17922265

  8. Simultaneous recordings of force and sliding movement between a myosin-coated glass microneedle and actin cables in vitro.

    OpenAIRE

    Chaen, S; Oiwa, K; Shimmen, T; Iwamoto, H; Sugi, H

    1989-01-01

    To elucidate the molecular mechanism of muscle contraction resulting from the ATP-dependent actin-myosin interaction, we constructed an assay system with which both the force and the movement produced by the actin-myosin interaction in vitro can be simultaneously recorded and analyzed. The assay system consisted of the giant internodal cells of an alga, Nitellopsis obtusa, which contain well-organized arrays of actin filaments (actin cables) running along the cell long axis, and a glass micro...

  9. The Role of Actin-Capping Protein and Src signalling in tissue growth and apoptosis during Drosophila wing development

    OpenAIRE

    Jezowska, Barbara Zofia

    2012-01-01

    Dissertation presented to obtain the Ph.D degree in Developmental Biology The actin cytoskeleton controls numerous cellular processes, including cell morphology and polarity, endocytosis, intracellular trafficking, contractility and cell division. Actin filament growth, stability and disassembly are controlled by a plethora of actin-binding proteins. Among them Capping Protein is a highly conserved αβ heterodimer, which binds the barbed ends of actin filaments, inhibiting addit...

  10. Vital role for the Plasmodium actin capping protein (CP) beta-subunit in motility of malaria sporozoites

    OpenAIRE

    Ganter, Markus; Schüler, Herwig; Matuschewski, Kai

    2009-01-01

    Successful malaria transmission from the mosquito vector to the mammalian host depends crucially on active sporozoite motility. Sporozoite locomotion and host cell invasion are driven by the parasite's own actin/myosin motor. A unique feature of this motor machinery is the presence of very short subpellicular actin filaments. Therefore, F-actin stabilizing proteins likely play a central role in parasite locomotion. Here, we investigated the role of the Plasmodium berghei actin capping protein...

  11. Sensitivity of an actinic reticuloid cell strain to near-ultraviolet radiation and its modification by Trolox-C, a vitamin E analogue

    Energy Technology Data Exchange (ETDEWEB)

    Kralli, A.; Moss, S.H.

    1987-06-01

    Near-UV radiation (365 nm)-induced lethality, as measured by colony-forming ability, showed an actinic reticuloid cell strain to be sensitive relative to normal human fibroblasts, when irradiated at 25/sup 0/C. This effect was not seen after far-UV (254 nm) irradiation. Trolox-C, a water-soluble analogue of vitamin E, incorporated in the pre-irradiation growth medium or in the post-irradiation plating medium, protected the actinic reticuloid cells to the extent that they were as resistant as normal cells. Plating medium containing Trolox-C did not provide differential protection against inactivation of the two cell strains by wavelengths in the far-UV region. The protection provided by Trolox-C, an analogue of the natural antioxidant vitamin E, suggests some free radical involvement in the aetiology of the disease.

  12. The sensitivity of an actinic reticuloid cell strain to near-ultraviolet radiation and its modification by trolox-C, a vitamin E analogue.

    Science.gov (United States)

    Kralli, A; Moss, S H

    1987-06-01

    Near-UV radiation (365 nm)-induced lethality, as measured by colony-forming ability, showed an actinic reticuloid cell strain to be sensitive relative to normal human fibroblasts, when irradiated at 25 degrees C. This effect was not seen after far-UV (254 nm) irradiation. Trolox-C, a water-soluble analogue of vitamin E, incorporated in the pre-irradiation growth medium or in the post-irradiation plating medium, protected the actinic reticuloid cells to the extent that they were as resistant as normal cells. Plating medium containing Trolox-C did not provide differential protection against inactivation of the two cell strains by wavelengths in the far-UV region. The protection provided by Trolox-C, an analogue of the natural antioxidant vitamin E, suggests some free radical involvement in the aetiology of the disease.

  13. Actin filaments growing against a barrier with fluctuating shape

    CERN Document Server

    Sadhu, Raj Kumar

    2016-01-01

    We study force generation by a set of parallel actin filaments growing against a non-rigid obstacle, in presence of an external load. The filaments polymerize by either moving the whole obstacle, with a large energy cost, or by causing local distortion in its shape which costs much less energy. The non-rigid obstacle also has local thermal fluctuations due to which its shape can change with time and we describe this using fluctuations in the height profile of a one dimensional interface with Kardar-Parisi-Zhang dynamics. We find the shape fluctuations of the barrier strongly affects the force generation mechanism. The qualitative nature of the force-velocity curve is crucially determined by the relative time-scale of filament and barrier dynamics. The height profile of the barrier also shows interesting variation with the external load. Our analytical calculation within mean-field theory shows reasonable agreement with our simulation results.

  14. Desmosome dynamics in migrating epithelial cells requires the actin cytoskeleton

    Science.gov (United States)

    Roberts, Brett J.; Pashaj, Anjeza; Johnson, Keith R.; Wahl, James K.

    2011-01-01

    Re-modeling of epithelial tissues requires that the cells in the tissue rearrange their adhesive contacts in order to allow cells to migrate relative to neighboring cells. Desmosomes are prominent adhesive structures found in a variety of epithelial tissues that are believed to inhibit cell migration and invasion. Mechanisms regulating desmosome assembly and stability in migrating cells are largely unknown. In this study we established a cell culture model to examine the fate of desmosomal components during scratch wound migration. Desmosomes are rapidly assembled between epithelial cells at the lateral edges of migrating cells and structures are transported in a retrograde fashion while the structures become larger and mature. Desmosome assembly and dynamics in this system are dependent on the actin cytoskeleton prior to being associated with the keratin intermediate filament cytoskeleton. These studies extend our understanding of desmosome assembly and provide a system to examine desmosome assembly and dynamics during epithelial cell migration. PMID:21945137

  15. Actin filaments on myosin beds: The velocity distribution

    Science.gov (United States)

    Bourdieu, L.; Magnasco, M. O.; Winkelmann, D. A.; Libchaber, A.

    1995-12-01

    In vitro studies of actin filaments sliding on a myosin-coated surface are analyzed, filament by filament, at a sampling rate of 30 per second. For each filament, the mean arc length coordinate is computed and histograms of instantaneous velocities, along the arc length, are established. Two types of motion are observed, depending on the experimental conditions. The first one is characterized by a homogeneous flow, with well defined velocities. In this regime, specific defects are a constitutive part of the flow. It is observed at high temperature, at high myosin coverage, and with a particular mode of attachment of myosin to the surface. The second regime shows no clear velocity selection, but a broadband distribution. It is characterized by high friction and is observed at low temperature or low myosin density. (c) 1995 The American Physical Society

  16. Actin filaments growing against a barrier with fluctuating shape

    Science.gov (United States)

    Sadhu, Raj Kumar; Chatterjee, Sakuntala

    2016-06-01

    We study force generation by a set of parallel actin filaments growing against a nonrigid obstacle, in the presence of an external load. The filaments polymerize by either moving the whole obstacle, with a large energy cost, or by causing local distortion in its shape which costs much less energy. The nonrigid obstacle also has local thermal fluctuations due to which its shape can change with time and we describe this using fluctuations in the height profile of a one-dimensional interface with Kardar-Parisi-Zhang dynamics. We find the shape fluctuations of the barrier strongly affect the force generation mechanism. The qualitative nature of the force-velocity curve is crucially determined by the relative time scale of filament and barrier dynamics. The height profile of the barrier also shows interesting variation with the external load. Our analytical calculations within mean-field theory show reasonable agreement with our simulation results.

  17. Prokaryotic DNA segregation by an actin-like filament

    DEFF Research Database (Denmark)

    Møller-Jensen, Jakob; Bugge Jensen, Rasmus; Löwe, Jan;

    2002-01-01

    The mechanisms responsible for prokaryotic DNA segregation are largely unknown. The partitioning locus (par) encoded by the Escherichia coli plasmid R1 actively segregates its replicon to daughter cells. We show here that the ParM ATPase encoded by par forms dynamic actin-like filaments...... was ATP dependent, and depolymerization of ParM filaments required nucleotide hydrolysis. Our in vivo and in vitro results indicate that ParM polymerization generates the force required for directional movement of plasmids to opposite cell poles and that the ParR-parC complex functions as a nucleation...... point for ParM polymerization. Hence, we provide evidence for a simple prokaryotic analogue of the eukaryotic mitotic spindle apparatus....

  18. Percolation mechanism drives actin gels to the critically connected state

    Science.gov (United States)

    Lee, Chiu Fan; Pruessner, Gunnar

    2016-05-01

    Cell motility and tissue morphogenesis depend crucially on the dynamic remodeling of actomyosin networks. An actomyosin network consists of an actin polymer network connected by cross-linker proteins and motor protein myosins that generate internal stresses on the network. A recent discovery shows that for a range of experimental parameters, actomyosin networks contract to clusters with a power-law size distribution [J. Alvarado, Nat. Phys. 9, 591 (2013), 10.1038/nphys2715]. Here, we argue that actomyosin networks can exhibit a robust critical signature without fine-tuning because the dynamics of the system can be mapped onto a modified version of percolation with trapping (PT), which is known to show critical behavior belonging to the static percolation universality class without the need for fine-tuning of a control parameter. We further employ our PT model to generate experimentally testable predictions.

  19. Calcium and actin in the saga of awakening oocytes

    Energy Technology Data Exchange (ETDEWEB)

    Santella, Luigia, E-mail: santella@szn.it; Limatola, Nunzia; Chun, Jong T.

    2015-04-24

    The interaction of the spermatozoon with the egg at fertilization remains one of the most fascinating mysteries of life. Much of our scientific knowledge on fertilization comes from studies on sea urchin and starfish, which provide plenty of gametes. Large and transparent, these eggs have served as excellent model systems for studying egg activation and embryo development in seawater, a plain natural medium. Starfish oocytes allow the study of the cortical, cytoplasmic and nuclear changes during the meiotic maturation process, which can also be triggered in vitro by hormonal stimulation. These morphological and biochemical changes ensure successful fertilization of the eggs at the first metaphase. On the other hand, sea urchin eggs are fertilized after the completion of meiosis, and are particularly suitable for the study of sperm–egg interaction, early events of egg activation, and embryonic development, as a large number of mature eggs can be fertilized synchronously. Starfish and sea urchin eggs undergo abrupt changes in the cytoskeleton and ion fluxes in response to the fertilizing spermatozoon. The plasma membrane and cortex of an egg thus represent “excitable media” that quickly respond to the stimulus with the Ca{sup 2+} swings and structural changes. In this article, we review some of the key findings on the rapid dynamic rearrangements of the actin cytoskeleton in the oocyte/egg cortex upon hormonal or sperm stimulation and their roles in the modulation of the Ca{sup 2+} signals and in the control of monospermic fertilization. - Highlights: • Besides microtubules, microfilaments may anchor the nucleus to oocyte surface. • The cortical Ca{sup 2+} flash and wave at fertilization mirror electrical membrane change. • Artificial egg activation lacks microvilli extension in the perivitelline space. • Calcium is necessary but not sufficient for cortical granules exocytosis. • Actin cytoskeleton modulates Ca{sup 2+} release at oocyte maturation

  20. Mechanical Properties of Re-constituted Actin Networks at an Oil/Water Interface Determined by Microrheology

    NARCIS (Netherlands)

    Ershov, D.S.; Cohen Stuart, M.A.; Gucht, van der J.

    2012-01-01

    There have been various attempts to investigate the mechanical properties of the actin cortex in cells, but the factors that control them remain poorly understood. To make progress, we develop a reconstituted model of the actin cortex that mimics its structure. We attach actin filaments to lipids li

  1. 25 Years of Tension over Actin Binding to the Cadherin Cell Adhesion Complex: The Devil is in the Details.

    Science.gov (United States)

    Nelson, W James; Weis, William I

    2016-07-01

    Over the past 25 years, there has been a conceptual (re)evolution in understanding how the cadherin cell adhesion complex, which contains F-actin-binding proteins, binds to the actin cytoskeleton. There is now good synergy between structural, biochemical, and cell biological results that the cadherin-catenin complex binds to F-actin under force. PMID:27166091

  2. Comparative genome analysis of cortactin and HSI : the significance of the F-actin binding repeat domain

    NARCIS (Netherlands)

    van Rossum, AGSH; Schuuring-Scholtes, E; Seggelen, VV; Kluin, PM; Schuuring, E

    2005-01-01

    Background: In human carcinomas, overexpression of cortactin correlates with poor prognosis. Cortactin is an F-actin-binding protein involved in cytoskeletal rearrangements and cell migration by promoting actin-related protein (Arp)2/3 mediated actin polymerization. It shares a high amino acid seque

  3. Interactions between the Yeast SM22 Homologue Scp1 and Actin Demonstrate the Importance of Actin Bundling in Endocytosis*S⃞

    Science.gov (United States)

    Gheorghe, Dana M.; Aghamohammadzadeh, Soheil; Rooij, Iwona I. Smaczynska-de; Allwood, Ellen G.; Winder, Steve J.; Ayscough, Kathryn R.

    2008-01-01

    The yeast SM22 homologue Scp1 has previously been shown to act as an actin-bundling protein in vitro. In cells, Scp1 localizes to the cortical actin patches that form as part of the invagination process during endocytosis, and its function overlaps with that of the well characterized yeast fimbrin homologue Sac6p. In this work we have used live cell imaging to demonstrate the importance of key residues in the Scp1 actin interface. We have defined two actin binding domains within Scp1 that allow the protein to both bind and bundle actin without the need for dimerization. Green fluorescent protein-tagged mutants of Scp1 also indicate that actin localization does not require the putative phosphorylation site Ser-185 to be functional. Deletion of SCP1 has few discernable effects on cell growth and morphology. However, we reveal that scp1 deletion is compensated for by up-regulation of Sac6. Furthermore, Scp1 levels are increased in the absence of sac6. The presence of compensatory pathways to up-regulate Sac6 or Scp1 levels in the absence of the other suggest that maintenance of sufficient bundling activity is critical within the cell. Analysis of cortical patch assembly and movement during endocytosis reveals a previously undetected role for Scp1 in movement of patches away from the plasma membrane. Additionally, we observe a dramatic increase in patch lifetime in a strain lacking both sac6 and scp1, demonstrating the central role played by actin-bundling proteins in the endocytic process. PMID:18400761

  4. Ena/VASP Enabled is a highly processive actin polymerase tailored to self-assemble parallel-bundled F-actin networks with Fascin.

    Science.gov (United States)

    Winkelman, Jonathan D; Bilancia, Colleen G; Peifer, Mark; Kovar, David R

    2014-03-18

    Filopodia are exploratory finger-like projections composed of multiple long, straight, parallel-bundled actin filaments that protrude from the leading edge of migrating cells. Drosophila melanogaster Enabled (Ena) is a member of the Ena/vasodilator-stimulated phosphoprotein protein family, which facilitates the assembly of filopodial actin filaments that are bundled by Fascin. However, the mechanism by which Ena and Fascin promote the assembly of uniformly thick F-actin bundles that are capable of producing coordinated protrusive forces without buckling is not well understood. We used multicolor evanescent wave fluorescence microscopy imaging to follow individual Ena molecules on both single and Fascin-bundled F-actin in vitro. Individual Ena tetramers increase the elongation rate approximately two- to threefold and inhibit capping protein by remaining processively associated with the barbed end for an average of ∼10 s in solution, for ∼60 s when immobilized on a surface, and for ∼110 s when multiple Ena tetramers are clustered on a surface. Ena also can gather and simultaneously elongate multiple barbed ends. Collectively, these properties could facilitate the recruitment of Fascin and initiate filopodia formation. Remarkably, we found that Ena's actin-assembly properties are tunable on Fascin-bundled filaments, facilitating the formation of filopodia-like F-actin networks without tapered barbed ends. Ena-associated trailing barbed ends in Fascin-bundled actin filaments have approximately twofold more frequent and approximately fivefold longer processive runs, allowing them to catch up with leading barbed ends efficiently. Therefore, Fascin and Ena cooperate to extend and maintain robust filopodia of uniform thickness with aligned barbed ends by a unique mechanistic cycle.

  5. Asiaticoside Inhibits TNF-α-Induced Endothelial Hyperpermeability of Human Aortic Endothelial Cells.

    Science.gov (United States)

    Fong, Lai Yen; Ng, Chin Theng; Zakaria, Zainul Amiruddin; Baharuldin, Mohamad Taufik Hidayat; Arifah, Abdul Kadir; Hakim, Muhammad Nazrul; Zuraini, Ahmad

    2015-10-01

    The increase in endothelial permeability often promotes edema formation in various pathological conditions. Tumor necrosis factor-alpha (TNF-α), a pro-atherogenic cytokine, impairs endothelial barrier function and causes endothelial dysfunction in early stage of atherosclerosis. Asiaticoside, one of the triterpenoids derived from Centella asiatica, is known to possess antiinflammatory activity. In order to examine the role of asiaticoside in preserving the endothelial barrier, we assessed its effects on endothelial hyperpermeability and disruption of actin filaments evoked by TNF-α in human aortic endothelial cells (HAEC). TNF-α caused an increase in endothelial permeability to fluorescein isothiocyanate (FITC)-dextran. Asiaticoside pretreatment significantly suppressed TNF-α-induced increased permeability. Asiaticoside also prevented TNF-α-induced actin redistribution by suppressing stress fiber formation. However, the increased F to G actin ratio stimulated by TNF-α was not changed by asiaticoside. Cytochalasin D, an actin depolymerizing agent, was used to correlate the anti-hyperpermeability effect of asiaticoside with actin cytoskeleton. Surprisingly, asiaticoside failed to prevent cytochalasin D-induced increased permeability. These results suggest that asiaticoside protects against the disruption of endothelial barrier and actin rearrangement triggered by TNF-α without a significant change in total actin pool. However, asiaticoside seems to work by other mechanisms to maintain the integrity of endothelial barrier rather than stabilizing the F-actin organization.

  6. Hierarchical Cross-linked F-actin Networks: Understanding Structure and Assembly

    Science.gov (United States)

    Hirst, Linda; Nguyen, Lam

    2009-11-01

    The protein, F-actin provides us with an interesting system in which to investigate the assembly properties of semi-flexible filaments in the presence of cross-linkers. Recently it was observed that F-actin, in the presence of the cross-linker alpha-actinin at high molar ratios will generate a novel hierarchical network of filament bundles. We investigate this system using coarse-grained molecular dynamics (MD) simulation, confocal microscopy and x-ray scattering. We have studied the F-actin/alpha-actinin system in detail with different actin conc. (C) and alpha-actinin/actin molar ratios (gamma). Confocal microscopy and analysis shows that the assembled systems fall into one of 3 phases depending on C and gamma: (1) loosely connected network of F-actin and bundles, (2) loosely connected network of dense domains and (3) uniform network of bundles. This can be explained and replicated using MD simulation. We have also examined different types of cross-linkers to represent the proteins, fascin and filamin. Results show that phase formation is related to the flexibility in binding between F-actin and cross-linkers. This degree of freedom, possible with longer cross-linkers allows the formation of branch points and thus bundle networks.

  7. LeftyA decreases Actin Polymerization and Stiffness in Human Endometrial Cancer Cells

    Science.gov (United States)

    Salker, Madhuri S.; Schierbaum, Nicolas; Alowayed, Nour; Singh, Yogesh; Mack, Andreas F.; Stournaras, Christos; Schäffer, Tilman E.; Lang, Florian

    2016-01-01

    LeftyA, a cytokine regulating stemness and embryonic differentiation, down-regulates cell proliferation and migration. Cell proliferation and motility require actin reorganization, which is under control of ras-related C3 botulinum toxin substrate 1 (Rac1) and p21 protein-activated kinase 1 (PAK1). The present study explored whether LeftyA modifies actin cytoskeleton, shape and stiffness of Ishikawa cells, a well differentiated endometrial carcinoma cell line. The effect of LeftyA on globular over filamentous actin ratio was determined utilizing Western blotting and flow cytometry. Rac1 and PAK1 transcript levels were measured by qRT-PCR as well as active Rac1 and PAK1 by immunoblotting. Cell stiffness (quantified by the elastic modulus), cell surface area and cell volume were studied by atomic force microscopy (AFM). As a result, 2 hours treatment with LeftyA (25 ng/ml) significantly decreased Rac1 and PAK1 transcript levels and activity, depolymerized actin, and decreased cell stiffness, surface area and volume. The effect of LeftyA on actin polymerization was mimicked by pharmacological inhibition of Rac1 and PAK1. In the presence of the Rac1 or PAK1 inhibitor LeftyA did not lead to significant further actin depolymerization. In conclusion, LeftyA leads to disruption of Rac1 and Pak1 activity with subsequent actin depolymerization, cell softening and cell shrinkage. PMID:27404958

  8. Actin Cytoskeleton Manipulation by Effector Proteins Secreted by Diarrheagenic Escherichia coli Pathotypes

    Directory of Open Access Journals (Sweden)

    Fernando Navarro-Garcia

    2013-01-01

    Full Text Available The actin cytoskeleton is a dynamic structure necessary for cell and tissue organization, including the maintenance of epithelial barriers. Disruption of the epithelial barrier coincides with alterations of the actin cytoskeleton in several disease states. These disruptions primarily affect the paracellular space, which is normally regulated by tight junctions. Thereby, the actin cytoskeleton is a common and recurring target of bacterial virulence factors. In order to manipulate the actin cytoskeleton, bacteria secrete and inject toxins and effectors to hijack the host cell machinery, which interferes with host-cell pathways and with a number of actin binding proteins. An interesting model to study actin manipulation by bacterial effectors is Escherichia coli since due to its genome plasticity it has acquired diverse genetic mobile elements, which allow having different E. coli varieties in one bacterial species. These E. coli pathotypes, including intracellular and extracellular bacteria, interact with epithelial cells, and their interactions depend on a specific combination of virulence factors. In this paper we focus on E. coli effectors that mimic host cell proteins to manipulate the actin cytoskeleton. The study of bacterial effector-cytoskeleton interaction will contribute not only to the comprehension of the molecular causes of infectious diseases but also to increase our knowledge of cell biology.

  9. WAVE binds Ena/VASP for enhanced Arp2/3 complex-based actin assembly.

    Science.gov (United States)

    Havrylenko, Svitlana; Noguera, Philippe; Abou-Ghali, Majdouline; Manzi, John; Faqir, Fahima; Lamora, Audrey; Guérin, Christophe; Blanchoin, Laurent; Plastino, Julie

    2015-01-01

    The WAVE complex is the main activator of the Arp2/3 complex for actin filament nucleation and assembly in the lamellipodia of moving cells. Other important players in lamellipodial protrusion are Ena/VASP proteins, which enhance actin filament elongation. Here we examine the molecular coordination between the nucleating activity of the Arp2/3 complex and the elongating activity of Ena/VASP proteins for the formation of actin networks. Using an in vitro bead motility assay, we show that WAVE directly binds VASP, resulting in an increase in Arp2/3 complex-based actin assembly. We show that this interaction is important in vivo as well, for the formation of lamellipodia during the ventral enclosure event of Caenorhabditis elegans embryogenesis. Ena/VASP's ability to bind F-actin and profilin-complexed G-actin are important for its effect, whereas Ena/VASP tetramerization is not necessary. Our data are consistent with the idea that binding of Ena/VASP to WAVE potentiates Arp2/3 complex activity and lamellipodial actin assembly.

  10. Structural Transition of Actin Filament in a Cell-Sized Water Droplet with a Phospholipid Membrane

    CERN Document Server

    Hase, M

    2005-01-01

    Actin filament, F-actin, is a semiflexible polymer with a negative charge, and is one of the main constituents on cell membranes. To clarify the effect of cross-talk between a phospholipid membrane and actin filaments in cells, we conducted microscopic observations on the structural changes in actin filaments in a cell-sized (several tens of micrometers in diameter) water droplet coated with a phospholipid membrane such as phosphatidylserine (PS; negatively-charged head group) or phosphatidylethanolamine (PE; neutral head group) as a simple model of a living cell membrane. With PS, actin filaments are distributed uniformly in the water phase without adsorption onto the membrane surface between 2 and 6 mM Mg2+, while between 6 and 12 mM Mg2+, actin filaments are adsorbed onto the inner membrane surface. With PE, actin filaments are uniformly adsorbed onto the inner membrane surface between 2 and 12 mM Mg2+. With both PS and PE membranes, at Mg2+ concentrations higher than 12 mM, thick bundles are formed in the...

  11. The interplay between neuronal activity and actin dynamics mimic the setting of an LTD synaptic tag.

    Science.gov (United States)

    Szabó, Eszter C; Manguinhas, Rita; Fonseca, Rosalina

    2016-09-21

    Persistent forms of plasticity, such as long-term depression (LTD), are dependent on the interplay between activity-dependent synaptic tags and the capture of plasticity-related proteins. We propose that the synaptic tag represents a structural alteration that turns synapses permissive to change. We found that modulation of actin dynamics has different roles in the induction and maintenance of LTD. Inhibition of either actin depolymerisation or polymerization blocks LTD induction whereas only the inhibition of actin depolymerisation blocks LTD maintenance. Interestingly, we found that actin depolymerisation and CaMKII activation are involved in LTD synaptic-tagging and capture. Moreover, inhibition of actin polymerisation mimics the setting of a synaptic tag, in an activity-dependent manner, allowing the expression of LTD in non-stimulated synapses. Suspending synaptic activation also restricts the time window of synaptic capture, which can be restored by inhibiting actin polymerization. Our results support our hypothesis that modulation of the actin cytoskeleton provides an input-specific signal for synaptic protein capture.

  12. Interaction of actin with plasminogen/plasmin system: mechanisms and physiological role

    Directory of Open Access Journals (Sweden)

    Tykhomyrov A. A.

    2012-12-01

    Full Text Available In the present review, we have summarized and analyzed the literature data concerning cooperation between multifunctional proteins, the components of plasminogen/plasmin system and actin. The mechanisms underlying intermolecular interactions and the role of plasminogen kringle domains in protein-protein recognition are reviewed. A particular attention is paid to extracellular actin that serves as a surface protein of plasma membrane in various cells. A putative role of surface actin as the universal «non-hemostatic» center of plasminogen activation is discussed. The exposition of cytoskeletal actin on the outer surface of cellular membrane is thought to be a phenomenon, which is involved in both normal cell functioning and development of pathologies. In particular, the mechanism of plasminogen fragmentation on the surface of cancer cells mediated by actin, which results in generation of endogenous suppressors of tumor growth and metastazing (angiostatins, is described. It has been acknowledged that the plasminogen/plasmin system in concert with surface actin regulates releasing biologically active substances, e. g. catecholamines. The comprehensive assessment of plasminogen/plasmin system and surface actin exposition is proposed to be a criterion of functional status of cells and can be used as a diagnostic parameter at various pathologies.

  13. The Actin Filament-Binding Protein Coronin Regulates Motility in Plasmodium Sporozoites

    Science.gov (United States)

    Bane, Kartik S.; Singer, Mirko; Reinig, Miriam; Klug, Dennis; Heiss, Kirsten; Baum, Jake; Mueller, Ann-Kristin; Frischknecht, Friedrich

    2016-01-01

    Parasites causing malaria need to migrate in order to penetrate tissue barriers and enter host cells. Here we show that the actin filament-binding protein coronin regulates gliding motility in Plasmodium berghei sporozoites, the highly motile forms of a rodent malaria-causing parasite transmitted by mosquitoes. Parasites lacking coronin show motility defects that impair colonization of the mosquito salivary glands but not migration in the skin, yet result in decreased transmission efficiency. In non-motile sporozoites low calcium concentrations mediate actin-independent coronin localization to the periphery. Engagement of extracellular ligands triggers an intracellular calcium release followed by the actin-dependent relocalization of coronin to the rear and initiation of motility. Mutational analysis and imaging suggest that coronin organizes actin filaments for productive motility. Using coronin-mCherry as a marker for the presence of actin filaments we found that protein kinase A contributes to actin filament disassembly. We finally speculate that calcium and cAMP-mediated signaling regulate a switch from rapid parasite motility to host cell invasion by differentially influencing actin dynamics. PMID:27409081

  14. Cortical instability drives periodic supracellular actin pattern formation in epithelial tubes.

    Science.gov (United States)

    Hannezo, Edouard; Dong, Bo; Recho, Pierre; Joanny, Jean-François; Hayashi, Shigeo

    2015-07-14

    An essential question of morphogenesis is how patterns arise without preexisting positional information, as inspired by Turing. In the past few years, cytoskeletal flows in the cell cortex have been identified as a key mechanism of molecular patterning at the subcellular level. Theoretical and in vitro studies have suggested that biological polymers such as actomyosin gels have the property to self-organize, but the applicability of this concept in an in vivo setting remains unclear. Here, we report that the regular spacing pattern of supracellular actin rings in the Drosophila tracheal tubule is governed by a self-organizing principle. We propose a simple biophysical model where pattern formation arises from the interplay of myosin contractility and actin turnover. We validate the hypotheses of the model using photobleaching experiments and report that the formation of actin rings is contractility dependent. Moreover, genetic and pharmacological perturbations of the physical properties of the actomyosin gel modify the spacing of the pattern, as the model predicted. In addition, our model posited a role of cortical friction in stabilizing the spacing pattern of actin rings. Consistently, genetic depletion of apical extracellular matrix caused strikingly dynamic movements of actin rings, mirroring our model prediction of a transition from steady to chaotic actin patterns at low cortical friction. Our results therefore demonstrate quantitatively that a hydrodynamical instability of the actin cortex can trigger regular pattern formation and drive morphogenesis in an in vivo setting.

  15. AIP1 acts with cofilin to control actin dynamics during epithelial morphogenesis.

    Science.gov (United States)

    Chu, Dandan; Pan, Hanshuang; Wan, Ping; Wu, Jing; Luo, Jun; Zhu, Hong; Chen, Jiong

    2012-10-01

    During epithelial morphogenesis, cells not only maintain tight adhesion for epithelial integrity but also allow dynamic intercellular movement to take place within cell sheets. How these seemingly opposing processes are coordinated is not well understood. Here, we report that the actin disassembly factors AIP1 and cofilin are required for remodeling of adherens junctions (AJs) during ommatidial precluster formation in Drosophila eye epithelium, a highly stereotyped cell rearrangement process which we describe in detail in our live imaging study. AIP1 is enriched together with F-actin in the apical region of preclusters, whereas cofilin displays a diffuse and uniform localization pattern. Cofilin overexpression completely rescues AJ remodeling defects caused by AIP1 loss of function, and cofilin physically interacts with AIP1. Pharmacological reduction of actin turnover results in similar AJ remodeling defects and decreased turnover of E-cadherin, which also results from AIP1 deficiency, whereas an F-actin-destabilizing drug affects AJ maintenance and epithelial integrity. Together with other data on actin polymerization, our results suggest that AIP1 enhances cofilin-mediated actin disassembly in the apical region of precluster cells to promote remodeling of AJs and thus intercellular movement, but also that robust actin polymerization promotes AJ general adhesion and integrity during the remodeling process.

  16. The Hippo pathway polarizes the actin cytoskeleton during collective migration of Drosophila border cells.

    Science.gov (United States)

    Lucas, Eliana P; Khanal, Ichha; Gaspar, Pedro; Fletcher, Georgina C; Polesello, Cedric; Tapon, Nicolas; Thompson, Barry J

    2013-06-10

    Collective migration of Drosophila border cells depends on a dynamic actin cytoskeleton that is highly polarized such that it concentrates around the outer rim of the migrating cluster of cells. How the actin cytoskeleton becomes polarized in these cells to enable collective movement remains unknown. Here we show that the Hippo signaling pathway links determinants of cell polarity to polarization of the actin cytoskeleton in border cells. Upstream Hippo pathway components localize to contacts between border cells inside the cluster and signal through the Hippo and Warts kinases to polarize actin and promote border cell migration. Phosphorylation of the transcriptional coactivator Yorkie (Yki)/YAP by Warts does not mediate the function of this pathway in promoting border cell migration, but rather provides negative feedback to limit the speed of migration. Instead, Warts phosphorylates and inhibits the actin regulator Ena to activate F-actin Capping protein activity on inner membranes and thereby restricts F-actin polymerization mainly to the outer rim of the migrating cluster.

  17. Cloning and sequence analysis of β-actin gene from Aedes albopictus (Diptera: Culicidae)

    Institute of Scientific and Technical Information of China (English)

    Weijie Wang; Xiaobang Hu; Donghui Zhang; Jianhua Jiao; Yan Sun; Lei Ma; Changliang Zhu

    2007-01-01

    Objective: To obtain the complete β-actin gene from Aedes albopictus. Methods: Total RNA was extracted from C6/36 cells. Degenerate primers were designed based on the β-actin sequences of An. gambiae, Ae. aegypti, Cx. pipiens pallens and D.melanogaster. By RT-PCR, the product was amplified, purified, cloned into the pGT vector and sequenced. The β-actin sequence was aligned and phylogenetically analyzed by the BLAST program and the CLUSTAL W program. Results: A sequence of 1132 bp including an open reading frame of 1131 bp was obtained (GenBank DQ657949). The deduced protein had 376 amino acids.Aligned to SWISS-PROT, it exhibited a high level of identity with β-actins from Anopheles, Drosophila and Culex at the amino acid sequence level. Phylogenetic analysis indicated that Ae. albopictus β-actin was much more homologous with invertebrate β-actin than with vertebrate β-actin. Conclusion: The gene may be used as the internal control in the experiments of Ae. albopictus.

  18. Identification of a novel actin-dependent signal transducing module allows for the targeted degradation of GLI1.

    Science.gov (United States)

    Schneider, Philipp; Bayo-Fina, Juan Miguel; Singh, Rajeev; Kumar Dhanyamraju, Pavan; Holz, Philipp; Baier, Aninja; Fendrich, Volker; Ramaswamy, Annette; Baumeister, Stefan; Martinez, Elisabeth D; Lauth, Matthias

    2015-01-01

    The Down syndrome-associated DYRK1A kinase has been reported as a stimulator of the developmentally important Hedgehog (Hh) pathway, but cells from Down syndrome patients paradoxically display reduced Hh signalling activity. Here we find that DYRK1A stimulates GLI transcription factor activity through phosphorylation of general nuclear localization clusters. In contrast, in vivo and in vitro experiments reveal that DYRK1A kinase can also function as an inhibitor of endogenous Hh signalling by negatively regulating ABLIM proteins, the actin cytoskeleton and the transcriptional co-activator MKL1 (MAL). As a final effector of the DYRK1A-ABLIM-actin-MKL1 sequence, we identify the MKL1 interactor Jumonji domain demethylase 1A (JMJD1A) as a novel Hh pathway component stabilizing the GLI1 protein in a demethylase-independent manner. Furthermore, a Jumonji-specific small-molecule antagonist represents a novel and powerful inhibitor of Hh signal transduction by inducing GLI1 protein degradation in vitro and in vivo. PMID:26310823

  19. The Nf-actin gene is an important factor for food-cup formation and cytotoxicity of pathogenic Naegleria fowleri.

    Science.gov (United States)

    Sohn, Hae-Jin; Kim, Jong-Hyun; Shin, Myeong-Heon; Song, Kyoung-Ju; Shin, Ho-Joon

    2010-03-01

    Naegleria fowleri destroys target cells by trogocytosis, a phagocytosis mechanism, and a process of piecemeal ingestion of target cells by food-cups. Phagocytosis is an actin-dependent process that involves polymerization of monomeric G-actin into filamentous F-actin. However, despite the numerous studies concerning phagocytosis, its role in the N. fowleri food-cup formation related with trogocytosis has been poorly reported. In this study, we cloned and characterized an Nf-actin gene to elucidate the role of Nf-actin gene in N. fowleri pathogenesis. The Nf-actin gene is composed of 1,128-bp and produced a 54.1-kDa recombinant protein (Nf-actin). The sequence identity was 82% with nonpathogenic Naegleria gruberi but has no sequence identity with other mammals or human actin gene. Anti-Nf-actin polyclonal antibody was produced in BALB/c mice immunized with recombinant Nf-actin. The Nf-actin was localized on the cytoplasm, pseudopodia, and especially, food-cup structure (amoebastome) in N. fowleri trophozoites using immunofluorescence assay. When N. fowleri co-cultured with Chinese hamster ovary cells, Nf-actin was observed to localize around on phagocytic food-cups. We also observed that N. fowleri treated with cytochalasin D as actin polymerization inhibitor or transfected with antisense oligomer of Nf-actin gene had shown the reduced ability of food-cup formation and in vitro cytotoxicity. Finally, it suggests that Nf-actin plays an important role in phagocytic activity of pathogenic N. fowleri.

  20. Comparative genome analysis reveals a conserved family of actin-like proteins in apicomplexan parasites

    Directory of Open Access Journals (Sweden)

    Sibley L David

    2005-12-01

    Full Text Available Abstract Background The phylum Apicomplexa is an early-branching eukaryotic lineage that contains a number of important human and animal pathogens. Their complex life cycles and unique cytoskeletal features distinguish them from other model eukaryotes. Apicomplexans rely on actin-based motility for cell invasion, yet the regulation of this system remains largely unknown. Consequently, we focused our efforts on identifying actin-related proteins in the recently completed genomes of Toxoplasma gondii, Plasmodium spp., Cryptosporidium spp., and Theileria spp. Results Comparative genomic and phylogenetic studies of apicomplexan genomes reveals that most contain only a single conventional actin and yet they each have 8–10 additional actin-related proteins. Among these are a highly conserved Arp1 protein (likely part of a conserved dynactin complex, and Arp4 and Arp6 homologues (subunits of the chromatin-remodeling machinery. In contrast, apicomplexans lack canonical Arp2 or Arp3 proteins, suggesting they lost the Arp2/3 actin polymerization complex on their evolutionary path towards intracellular parasitism. Seven of these actin-like proteins (ALPs are novel to apicomplexans. They show no phylogenetic associations to the known Arp groups and likely serve functions specific to this important group of intracellular parasites. Conclusion The large diversity of actin-like proteins in apicomplexans suggests that the actin protein family has diverged to fulfill various roles in the unique biology of intracellular parasites. Conserved Arps likely participate in vesicular transport and gene expression, while apicomplexan-specific ALPs may control unique biological traits such as actin-based gliding motility.

  1. Retinoids and glucocorticoids have opposite effects on actin cytoskeleton rearrangement in hippocampal HT22 cells.

    Science.gov (United States)

    Hélène, Roumes; Julie, Brossaud; Aloïs, Lemelletier; Marie-Pierre, Moisan; Véronique, Pallet; Anabelle, Redonnet; Jean-Benoît, Corcuff

    2016-02-01

    A chronic excess of glucocorticoids elicits deleterious effects in the hippocampus. Conversely, retinoic acid plays a major role in aging brain plasticity. As synaptic plasticity depends on mechanisms related to cell morphology, we investigated the involvement of retinoic acid and glucocorticoids in the remodelling of the HT22 neurons actin cytoskeleton. Cells exhibited a significantly more elongated shape with retinoic acid and a rounder shape with dexamethasone; retinoic acid reversed the effects of dexamethasone. Actin expression and abundance were unchanged by retinoic acid or dexamethasone but F-actin organization was dramatically modified. Indeed, retinoic acid and dexamethasone increased (70 ± 7% and 176 ± 5%) cortical actin while retinoic acid suppressed the effect of dexamethasone (90 ± 6%). Retinoic acid decreased (-22 ± 9%) and dexamethasone increased (134 ± 16%) actin stress fibres. Retinoic acid also suppressed the effect of dexamethasone (-21 ± 7%). Spectrin is a key protein in the actin network remodelling. Its abundance was decreased by retinoic acid and increased by dexamethasone (-21 ± 11% and 52 ± 10%). However, retinoic acid did not modify the effect of dexamethasone (48 ± 7%). Calpain activity on spectrin was increased by retinoic acid and decreased by dexamethasone (26 ± 14% and -57 ± 5%); retinoic acid mildly but significantly modified the effect of dexamethasone (-44 ± 7%). The calpain inhibitor calpeptin suppressed the effects of retinoic acid and dexamethasone on cell shape and actin stress fibres remodelling but did not modify the effects on cortical actin. Retinoic acid and dexamethasone have a dramatic but mainly opposite effect on actin cytoskeleton remodelling. These effects originate, at least partly, from calpain activity. PMID:26748244

  2. Retinoids and glucocorticoids have opposite effects on actin cytoskeleton rearrangement in hippocampal HT22 cells.

    Science.gov (United States)

    Hélène, Roumes; Julie, Brossaud; Aloïs, Lemelletier; Marie-Pierre, Moisan; Véronique, Pallet; Anabelle, Redonnet; Jean-Benoît, Corcuff

    2016-02-01

    A chronic excess of glucocorticoids elicits deleterious effects in the hippocampus. Conversely, retinoic acid plays a major role in aging brain plasticity. As synaptic plasticity depends on mechanisms related to cell morphology, we investigated the involvement of retinoic acid and glucocorticoids in the remodelling of the HT22 neurons actin cytoskeleton. Cells exhibited a significantly more elongated shape with retinoic acid and a rounder shape with dexamethasone; retinoic acid reversed the effects of dexamethasone. Actin expression and abundance were unchanged by retinoic acid or dexamethasone but F-actin organization was dramatically modified. Indeed, retinoic acid and dexamethasone increased (70 ± 7% and 176 ± 5%) cortical actin while retinoic acid suppressed the effect of dexamethasone (90 ± 6%). Retinoic acid decreased (-22 ± 9%) and dexamethasone increased (134 ± 16%) actin stress fibres. Retinoic acid also suppressed the effect of dexamethasone (-21 ± 7%). Spectrin is a key protein in the actin network remodelling. Its abundance was decreased by retinoic acid and increased by dexamethasone (-21 ± 11% and 52 ± 10%). However, retinoic acid did not modify the effect of dexamethasone (48 ± 7%). Calpain activity on spectrin was increased by retinoic acid and decreased by dexamethasone (26 ± 14% and -57 ± 5%); retinoic acid mildly but significantly modified the effect of dexamethasone (-44 ± 7%). The calpain inhibitor calpeptin suppressed the effects of retinoic acid and dexamethasone on cell shape and actin stress fibres remodelling but did not modify the effects on cortical actin. Retinoic acid and dexamethasone have a dramatic but mainly opposite effect on actin cytoskeleton remodelling. These effects originate, at least partly, from calpain activity.

  3. A feedback loop between dynamin and actin recruitment during clathrin-mediated endocytosis.

    Directory of Open Access Journals (Sweden)

    Marcus J Taylor

    Full Text Available Clathrin-mediated endocytosis proceeds by a sequential series of reactions catalyzed by discrete sets of protein machinery. The final reaction in clathrin-mediated endocytosis is membrane scission, which is mediated by the large guanosine triophosphate hydrolase (GTPase dynamin and which may involve the actin-dependent recruitment of N-terminal containing BIN/Amphiphysin/RVS domain containing (N-BAR proteins. Optical microscopy has revealed a detailed picture of when and where particular protein types are recruited in the ∼20-30 s preceding scission. Nevertheless, the regulatory mechanisms and functions that underpin protein recruitment are not well understood. Here we used an optical assay to investigate the coordination and interdependencies between the recruitment of dynamin, the actin cytoskeleton, and N-BAR proteins to individual clathrin-mediated endocytic scission events. These measurements revealed that a feedback loop exists between dynamin and actin at sites of membrane scission. The kinetics of dynamin, actin, and N-BAR protein recruitment were modulated by dynamin GTPase activity. Conversely, acute ablation of actin dynamics using latrunculin-B led to a ∼50% decrease in the incidence of scission, an ∼50% decrease in the amplitude of dynamin recruitment, and abolished actin and N-BAR recruitment to scission events. Collectively these data suggest that dynamin, actin, and N-BAR proteins work cooperatively to efficiently catalyze membrane scission. Dynamin controls its own recruitment to scission events by modulating the kinetics of actin and N-BAR recruitment to sites of scission. Conversely actin serves as a dynamic scaffold that concentrates dynamin and N-BAR proteins at sites of scission.

  4. The Drosophila planar polarity gene multiple wing hairs directly regulates the actin cytoskeleton.

    Science.gov (United States)

    Lu, Qiuheng; Schafer, Dorothy A; Adler, Paul N

    2015-07-15

    The evolutionarily conserved frizzled/starry night (fz/stan) pathway regulates planar cell polarity (PCP) in vertebrates and invertebrates. This pathway has been extensively studied in the Drosophila wing, where it is manifested by an array of distally pointing cuticular hairs. Using in vivo imaging we found that, early in hair growth, cells have multiple actin bundles and hairs that subsequently fuse into a single growing hair. The downstream PCP gene multiple wing hairs (mwh) plays a key role in this process and acts to antagonize the actin cytoskeleton. In mwh mutants hair initiation is not limited to a small region at the distal edge of pupal wing cells as in wild type, resulting in multiple hairs with aberrant polarity. Extra actin bundles/hairs are formed and do not completely fuse, in contrast to wild type. As development proceeded additional hairs continued to form, further increasing hair number. We identified a fragment of Mwh with in vivo rescue activity and that bound and bundled F-actin filaments and inhibited actin polymerization in in vitro actin assays. The loss of these activities can explain the mwh mutant phenotype. Our data suggest a model whereby, prior to hair initiation, proximally localized Mwh inhibits actin polymerization resulting in polarized activation of the cytoskeleton and hair formation on the distal side of wing cells. During hair growth Mwh is found in growing hairs, where we suggest it functions to promote the fusion of actin bundles and inhibit the formation of additional actin bundles that could lead to extra hairs.

  5. Prediction and dissection of widely-varying association rate constants of actin-binding proteins.

    Directory of Open Access Journals (Sweden)

    Xiaodong Pang

    Full Text Available Actin is an abundant protein that constitutes a main component of the eukaryotic cytoskeleton. Its polymerization and depolymerization are regulated by a variety of actin-binding proteins. Their functions range from nucleation of actin polymerization to sequestering G-actin in 1∶1 complexes. The kinetics of forming these complexes, with rate constants varying at least three orders of magnitude, is critical to the distinct regulatory functions. Previously we have developed a transient-complex theory for computing protein association mechanisms and association rate constants. The transient complex refers to an intermediate in which the two associating proteins have near-native separation and relative orientation but have yet to form short-range specific interactions of the native complex. The association rate constant is predicted as k(a = k(a0 e(-ΔG(el*/k(BT, where k(a0 is the basal rate constant for reaching the transient complex by free diffusion, and the Boltzmann factor captures the bias of long-range electrostatic interactions. Here we applied the transient-complex theory to study the association kinetics of seven actin-binding proteins with G-actin. These proteins exhibit three classes of association mechanisms, due to their different molecular shapes and flexibility. The 1000-fold k(a variations among them can mostly be attributed to disparate electrostatic contributions. The basal rate constants also showed variations, resulting from the different shapes and sizes of the interfaces formed by the seven actin-binding proteins with G-actin. This study demonstrates the various ways that actin-binding proteins use physical properties to tune their association mechanisms and rate constants to suit distinct regulatory functions.

  6. Insulin induces PKC-dependent proliferation of mesenteric vascular smooth muscle cells from hypertensive patients

    Institute of Scientific and Technical Information of China (English)

    Xukai WANG; Yan WANG; Chenming YANG; Ying WAN; Xianwen JI

    2006-01-01

    Background and objectives Proliferation of human vascular smooth muscle cells (VSMCs) induced by hyperinsulinemia is a very common clinical pathology. Extensive research has focused on PKC (Protein kinase C)-MAPK (mitogen-activated protein kinase)intracellular signal transduction and the phenotypic modulation accompanied by reorganization of intracellular F-actins in VSMCs.Methods DNA synthesis, signaling of ERK1/2 MAPKs, and changes in α-smooth muscle (SM) actin and F-actin were studied in hypertensive and normotensive human arterial VSMCs exposed to insulin and PMA with and without the PKC inhibitor, GF109203X.Results Differences among cell types in MAPK signaling, α-SM actin, and F-actin isoforms in VSMCs harvested from the arteries of patients with essential hypertension (EH) and normotension (NT) were identified in response to insulin treatment. Proliferation and activation of MAPK were more pronounced in EH VSMCs than in NEH VSMCs. Insulin exposure decreased expression of α-SM actin and was accompanied by rearrangement of intracellular F-actins in VSMCs, especially in the EH group. These effects were reversed by treatment with the PKC inhibitor. Conclusions Human mesenteric VSMCs of EH and NT patients differed in proliferation, MAPK signaling, and degree of changes in α-SM actin and F-actin isoforms immediately following insulin exposure in vitro.

  7. Hierarchical self-assembly of actin in micro-confinements using microfluidics

    OpenAIRE

    Deshpande, Siddharth; Pfohl, Thomas

    2012-01-01

    We present a straightforward microfluidics system to achieve step-by-step reaction sequences in a diffusion-controlled manner in quasi two-dimensional micro-confinements. We demonstrate the hierarchical self-organization of actin (actin monomers—entangled networks of filaments—networks of bundles) in a reversible fashion by tuning the Mg2+ ion concentration in the system. We show that actin can form networks of bundles in the presence of Mg2+ without any cross-linking proteins. The properties...

  8. Stabilization of F-actin prevents cAMP-elicited Cl- secretion in T84 cells.

    OpenAIRE

    Shapiro, M.; Matthews, J.; Hecht, G; Delp, C; Madara, J. L.

    1991-01-01

    T84 cells, a human intestinal epithelial cell line, serve as a model of electrogenic Cl- secretion. We find that cAMP-elicited Cl- secretion in T84 cells is accompanied by a marked redistribution of F-actin in the basolateral portion of the cell. To prevent this F-actin redistribution and thereby assess its importance to Cl- secretion, we have defined simple conditions under which this model epithelium can be loaded with nitrobenzoxadiazole (NBD)-phallicidin. This reagent binds F-actin with h...

  9. Modeling of the motion of the actin filament on the myosin motility assays

    Science.gov (United States)

    Young, Yuan; Shelley, Mike

    2007-11-01

    In motility assays, cytoskeletal actin filaments (actin filaments) glide over a surface coated with motor proteins, and the different modes of motion provide a simple measure of the force exerted by the motor proteins (Bourdieu, 1995). Motivated by these experiments, we consider the actin filament as a slender, elastic filament immersed in Stokesian flow, driven by a tangential forcing that mimics the force by the motor proteins. We find qualitative agreement on several points between our analysis and simulations and experimental observations. Furthermore, we study the correlation between filament transport and the characteristics of motion with the spatial pattern of motor protein density.

  10. Differential thymosin β10 expression levels and actin filament organization in tumor cell lines with different metastatic potential

    Institute of Scientific and Technical Information of China (English)

    刘从容; 马春树; 宁钧宇; 由江峰; 廖松林; 郑杰

    2004-01-01

    Background To investigate the differential expression levels of thymosin β10 (Tβ1O) and the corresponding changes of actin filament organization in human tumor cell lines with different metastatic potential.Methods Four groups of nine human tumor cell lines with different metastatic potential were analyzed for the amount of Tβ10 mRNAs by Northern blot and for their peptide expression levels by immunohistochemistry. The filamentous actin (F-actin)was observed by staining of TRITC-phalloidin to detect changes in actin organization. Results In comparison with non-/weakly metastatic counterparts, TβIO was upregulated in highly metastatic human lung cancer, malignant melanoma and breast cancer cell lines. Staining of TRITC-phalloidin revealed less actin bundles, a fuzzy network of shorter filaments and some F-actin aggregates in the highly metastatic tumor cells. Meanwhile, the actin filaments were robust and orderly arranged in the non-/weakly metastatic cancer cell lines.Conclusion Tβ10 levels correlate positively with the metastatic capacity in human tumors currently examined. The increasing metastatic potential of tumor cells is accompanied by a loss of F-actin,poorly arranged actin skeleton organizations and presence of F-actin aggregates. There is a consistent correlation between the elevated TβIO expression and the disrupted actin skeleton.

  11. Characterization of ring-like F-actin structure as a mechanical partner for spindle positioning in mitosis.

    Directory of Open Access Journals (Sweden)

    Huan Lu

    Full Text Available Proper spindle positioning and orientation are essential for accurate mitosis which requires dynamic interactions between microtubule and actin filament (F-actin. Although mounting evidence demonstrates the role of F-actin in cortical cytoskeleton dynamics, it remains elusive as to the structure and function of F-actin-based networks in spindle geometry. Here we showed a ring-like F-actin structure surrounding the mitotic spindle which forms since metaphase and maintains in MG132-arrested metaphase HeLa cells. This cytoplasmic F-actin structure is relatively isotropic and less dynamic. Our computational modeling of spindle position process suggests a possible mechanism by which the ring-like F-actin structure can regulate astral microtubule dynamics and thus mitotic spindle orientation. We further demonstrated that inhibiting Plk1, Mps1 or Myosin, and disruption of microtubules or F-actin polymerization perturbs the formation of the ring-like F-actin structure and alters spindle position and symmetric division. These findings reveal a previously unrecognized but important link between mitotic spindle and ring-like F-actin network in accurate mitosis and enables the development of a method to theoretically illustrate the relationship between mitotic spindle and cytoplasmic F-actin.

  12. Lifeact-mEGFP reveals a dynamic apical F-actin network in tip growing plant cells.

    Directory of Open Access Journals (Sweden)

    Luis Vidali

    Full Text Available BACKGROUND: Actin is essential for tip growth in plants. However, imaging actin in live plant cells has heretofore presented challenges. In previous studies, fluorescent probes derived from actin-binding proteins often alter growth, cause actin bundling and fail to resolve actin microfilaments. METHODOLOGY/PRINCIPAL FINDINGS: In this report we use Lifeact-mEGFP, an actin probe that does not affect the dynamics of actin, to visualize actin in the moss Physcomitrella patens and pollen tubes from Lilium formosanum and Nicotiana tobaccum. Lifeact-mEGFP robustly labels actin microfilaments, particularly in the apex, in both moss protonemata and pollen tubes. Lifeact-mEGFP also labels filamentous actin structures in other moss cell types, including cells of the gametophore. CONCLUSIONS/SIGNIFICANCE: Lifeact-mEGFP, when expressed at optimal levels does not alter moss protonemal or pollen tube growth. We suggest that Lifeact-mEGFP represents an exciting new versatile probe for further studies of actin's role in tip growing plant cells.

  13. Actinic Mask Inspection at the ALS Initial Design Review

    Energy Technology Data Exchange (ETDEWEB)

    Barty, A; Chapman, H; Sweeney, D; Levesque, R; Bokor, J; Gullikson, E; Jong, S; Liu, Y; Yi, M; Denbeaux, G; Goldberg, K; Naulleau, P; Denham, P; Rekawa, S; Baston, P; Tackaberry, R; Barale, P

    2003-03-05

    This report is the first milestone report for the actinic mask blank inspection project conducted at the VNL, which forms sub-section 3 of the Q1 2003 mask blank technology transfer program at the VNL. Specifically this report addresses deliverable 3.1.1--design review and preliminary tool design. The goal of this project is to design an actinic mask inspection tool capable of operating in two modes: high-speed scanning for the detection of multilayer defects (inspection mode), and a high-resolution aerial image mode in which the image emulates the imaging illumination conditions of a stepper system (aerial image or AIM mode). The purpose and objective of these two modes is as follows: (1) Defect inspection mode--This imaging mode is designed to scan large areas of the mask for defects EUV multilayer coatings. The goal is to detect the presence of multilayer defects on a mask blank and to store the co-ordinates for subsequent review in AIM mode, thus it is not essential that the illumination and imaging conditions match that of a production stepper. Potential uses for this imaging mode include: (a) Correlating the results obtained using actinic inspection with results obtained using other non-EUV defect inspection systems to verify that the non-EUV scanning systems are detecting all critical defects; (b) Gaining sufficient information to associate defects with particular processes, such as various stages of the multilayer deposition or different modes of operation of the deposition tool; and (c) Assessing the density and EUV impact of surface and multilayer anomalies. Because of the low defect density achieved using current multilayer coating technology it is necessary to be able to efficiently scan large areas of the mask in order to obtain sufficient statistics for use in cross-correlation experiments. Speed of operation as well as sensitivity is therefore key to operation in defect inspection mode. (2) Aerial Image Microscope (AIM) mode--In AIM mode the tool is

  14. ZEB1 drives prometastatic actin cytoskeletal remodeling by downregulating miR-34a expression

    Science.gov (United States)

    Ahn, Young-Ho; Gibbons, Don L.; Chakravarti, Deepavali; Creighton, Chad J.; Rizvi, Zain H.; Adams, Henry P.; Pertsemlidis, Alexander; Gregory, Philip A.; Wright, Josephine A.; Goodall, Gregory J.; Flores, Elsa R.; Kurie, Jonathan M.

    2012-01-01

    Metastatic cancer is extremely difficult to treat, and the presence of metastases greatly reduces a cancer patient’s likelihood of long-term survival. The ZEB1 transcriptional repressor promotes metastasis through downregulation of microRNAs (miRs) that are strong inducers of epithelial differentiation and inhibitors of stem cell factors. Given that each miR can target multiple genes with diverse functions, we posited that the prometastatic network controlled by ZEB1 extends beyond these processes. We tested this hypothesis using a mouse model of human lung adenocarcinoma metastasis driven by ZEB1, human lung carcinoma cells, and human breast carcinoma cells. Transcriptional profiling studies revealed that ZEB1 controls the expression of numerous oncogenic and tumor-suppressive miRs, including miR-34a. Ectopic expression of miR-34a decreased tumor cell invasion and metastasis, inhibited the formation of promigratory cytoskeletal structures, suppressed activation of the RHO GTPase family, and regulated a gene expression signature enriched in cytoskeletal functions and predictive of outcome in human lung adenocarcinomas. We identified several miR-34a target genes, including Arhgap1, which encodes a RHO GTPase activating protein that was required for tumor cell invasion. These findings demonstrate that ZEB1 drives prometastatic actin cytoskeletal remodeling by downregulating miR-34a expression and provide a compelling rationale to develop miR-34a as a therapeutic agent in lung cancer patients. PMID:22850877

  15. IDENTIFICATION OF NOVEL FIBROBLAST GROWTH FACTOR RECEPTOR 3 GENE MUTATIONS IN ACTINIC CHEILITIS

    Science.gov (United States)

    Chou, Annie; Dekker, Nusi; Jordan, Richard C.K.

    2009-01-01

    Objective Activating mutations in the fibroblast growth factor receptor 3 (FGFR3) gene are responsible for several craniosynostosis and chondrodysplasia syndromes as well as some human cancers including bladder and cervical carcinoma. Despite a high frequency in some benign skin disorders, FGFR3 mutations have not been reported in cutaneous malignancies. Actinic cheilitis (AC) is a sun-induced premalignancy affecting the lower lip that frequently progresses to squamous cell carcinoma (SCC). The objective of this study was to determine if FGFR3 gene mutations are present in AC and SCC of the lip. Study Design DNA was extracted and purified from micro-dissected, formalin-fixed, paraffin-embedded tissue sections of 20 cases of AC and SCC arising in AC. Exons 7, 15, and 17 were PCR amplified and direct sequenced. Results Four novel somatic mutations in the FGFR3 gene were identified: exon 7 mutation 742C→T (amino acid change R248C), exon 15 mutations 1850A→G (D617G) and 1888G→A (V630M), and exon 17 mutation 2056G→A (E686K). Grade of dysplasia did not correlate with presence of mutations. Conclusion The frequency of FGFR3 receptor mutations suggests a functional role for the FGFR3 receptor in the development of epithelial di