WorldWideScience

Sample records for affect actin cytoskeletal

  1. 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...

  2. 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.

  3. 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.

  4. A Legionella Effector Disrupts Host Cytoskeletal Structure by Cleaving Actin

    Science.gov (United States)

    Liu, Yao; Zhu, Wenhan; Tan, Yunhao; Nakayasu, Ernesto S.; Staiger, Christopher J.

    2017-01-01

    Legionella pneumophila, the etiological agent of Legionnaires’ disease, replicates intracellularly in protozoan and human hosts. Successful colonization and replication of this pathogen in host cells requires the Dot/Icm type IVB secretion system, which translocates approximately 300 effector proteins into the host cell to modulate various cellular processes. In this study, we identified RavK as a Dot/Icm substrate that targets the host cytoskeleton and reduces actin filament abundance in mammalian cells upon ectopic expression. RavK harbors an H95EXXH99 motif associated with diverse metalloproteases, which is essential for the inhibition of yeast growth and for the induction of cell rounding in HEK293T cells. We demonstrate that the actin protein itself is the cellular target of RavK and that this effector cleaves actin at a site between residues Thr351 and Phe352. Importantly, RavK-mediated actin cleavage also occurs during L. pneumophila infection. Cleavage by RavK abolishes the ability of actin to form polymers. Furthermore, an F352A mutation renders actin resistant to RavK-mediated cleavage; expression of the mutant in mammalian cells suppresses the cell rounding phenotype caused by RavK, further establishing that actin is the physiological substrate of RavK. Thus, L. pneumophila exploits components of the host cytoskeleton by multiple effectors with distinct mechanisms, highlighting the importance of modulating cellular processes governed by the actin cytoskeleton in the intracellular life cycle of this pathogen. PMID:28129393

  5. Formins: Actin nucleators that regulate cytoskeletal dynamics during spermatogenesis.

    Science.gov (United States)

    Li, Nan; Mruk, Dolores D; Tang, Elizabeth I; Wong, Chris Kc; Lee, Will M; Silvestrini, Bruno; Cheng, C Yan

    2015-01-01

    Formins are a growing class of actin nucleation proteins that promote the polymerization of actin microfilaments, forming long stretches of actin microfilaments to confer actin filament bundling in mammalian cells. As such, microfilament bundles can be formed in specific cellular domains, in particular in motile mammalian cells, such as filopodia. Since ectoplasmic specialization (ES), a testis-specific adherens junction (AJ), at the Sertoli cell-cell and Sertoli-spermatid interface is constituted by arrays of actin microfilament bundles, it is likely that formins are playing a significant physiological role on the homeostasis of ES during the epithelial cycle of spermatogenesis. In this Commentary, we provide a timely discussion on formin 1 which was recently shown to be a crucial regulator of actin microfilaments at the ES in the rat testis (Li N et al. Endocrinology, 2015, in press; DOI: 10.1210/en.2015-1161, PMID:25901598). We also highlight research that is needed to unravel the functional significance of formins in spermatogenesis.

  6. Aggregatibacter actinomycetemcomitans lipopolysaccharide affects human gingival fibroblast cytoskeletal organization.

    Science.gov (United States)

    Gutiérrez-Venegas, Gloria; Contreras-Marmolejo, Luis Arturo; Román-Alvárez, Patricia; Barajas-Torres, Carolina

    2008-04-01

    The cytoskeleton is a dynamic structure that plays a key role in maintaining cell morphology and function. This study investigates the effect of bacterial wall lipopolysaccharide (LPS), a strong inflammatory agent, on the dynamics and organization of actin, tubulin, vimentin, and vinculin proteins in human gingival fibroblasts (HGF). A time-dependent study showed a noticeable change in actin architecture after 1.5 h of incubation with LPS (1 microg/ml) with the formation of orthogonal fibers and further accumulation of actin filament at the cell periphery by 24 h. When 0.01-10 microg/ml of LPS was added to human gingival fibroblast cultures, cells acquired a round, flat shape and gradually developed cytoplasmic ruffling. Lipopolysaccharides extracted from Aggregatibacter actinomycetemcomitans periodontopathogenic bacteria promoted alterations in F-actin stress fibres of human gingival cells. Normally, human gingival cells have F-actin fibres that are organized in linear distribution throughout the cells, extending along the cell's length. LPS-treated cells exhibited changes in cytoskeletal protein organization, and F-actin was reorganized by the formation of bundles underneath and parallel to the cell membrane. We also found the reorganization of the vimentin network into vimentin bundling after 1.5 h of treatment. HGF cells exhibited diffuse and granular gamma-tubulin stain. There was no change in LPS-treated HGF. However, vinculin plaques distributed in the cell body diminished after LPS treatment. We conclude that the dynamic and structured organization of cytoskeletal filaments and actin assembly in human gingival fibroblasts is altered by LPS treatment and is accompanied by a decrease in F-actin pools.

  7. Beta-actin deficiency with oxidative posttranslational modifications in Rett syndrome erythrocytes: insights into an altered cytoskeletal organization.

    Directory of Open Access Journals (Sweden)

    Alessio Cortelazzo

    Full Text Available Beta-actin, a critical player in cellular functions ranging from cell motility and the maintenance of cell shape to transcription regulation, was evaluated in the erythrocyte membranes from patients with typical Rett syndrome (RTT and methyl CpG binding protein 2 (MECP2 gene mutations. RTT, affecting almost exclusively females with an average frequency of 1∶10,000 female live births, is considered the second commonest cause of severe cognitive impairment in the female gender. Evaluation of beta-actin was carried out in a comparative cohort study on red blood cells (RBCs, drawn from healthy control subjects and RTT patients using mass spectrometry-based quantitative analysis. We observed a decreased expression of the beta-actin isoforms (relative fold changes for spots 1, 2 and 3: -1.82±0.15, -2.15±0.06, and -2.59±0.48, respectively in pathological RBCs. The results were validated by western blotting and immunofluorescence microscopy. In addition, beta-actin from RTT patients also showed a dramatic increase in oxidative posttranslational modifications (PTMs as the result of its binding with the lipid peroxidation product 4-hydroxy-2-nonenal (4-HNE. Our findings demonstrate, for the first time, a beta-actin down-regulation and oxidative PTMs for RBCs of RTT patients, thus indicating an altered cytoskeletal organization.

  8. 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

  9. The skeleton in the closet: actin cytoskeletal remodeling in β-cell function.

    Science.gov (United States)

    Arous, Caroline; Halban, Philippe A

    2015-10-01

    Over the last few decades, biomedical research has considered not only the function of single cells but also the importance of the physical environment within a whole tissue, including cell-cell and cell-extracellular matrix interactions. Cytoskeleton organization and focal adhesions are crucial sensors for cells that enable them to rapidly communicate with the physical extracellular environment in response to extracellular stimuli, ensuring proper function and adaptation. The involvement of the microtubular-microfilamentous cytoskeleton in secretion mechanisms was proposed almost 50 years ago, since when the evolution of ever more sensitive and sophisticated methods in microscopy and in cell and molecular biology have led us to become aware of the importance of cytoskeleton remodeling for cell shape regulation and its crucial link with signaling pathways leading to β-cell function. Emerging evidence suggests that dysfunction of cytoskeletal components or extracellular matrix modification influences a number of disorders through potential actin cytoskeleton disruption that could be involved in the initiation of multiple cellular functions. Perturbation of β-cell actin cytoskeleton remodeling could arise secondarily to islet inflammation and fibrosis, possibly accounting in part for impaired β-cell function in type 2 diabetes. This review focuses on the role of actin remodeling in insulin secretion mechanisms and its close relationship with focal adhesions and myosin II.

  10. Nano-ZnO leads to tubulin macrotube assembly and actin bundling, triggering cytoskeletal catastrophe and cell necrosis

    Science.gov (United States)

    García-Hevia, Lorena; Valiente, Rafael; Martín-Rodríguez, Rosa; Renero-Lecuna, Carlos; González, Jesús; Rodríguez-Fernández, Lidia; Aguado, Fernando; Villegas, Juan C.; Fanarraga, Mónica L.

    2016-05-01

    Zinc is a crucial element in biology that plays chief catalytic, structural and protein regulatory roles. Excess cytoplasmic zinc is toxic to cells so there are cell-entry and intracellular buffering mechanisms that control intracellular zinc availability. Tubulin and actin are two zinc-scavenging proteins that are essential components of the cellular cytoskeleton implicated in cell division, migration and cellular architecture maintenance. Here we demonstrate how exposure to different ZnO nanostructures, namely ZnO commercial nanoparticles and custom-made ZnO nanowires, produce acute cytotoxic effects in human keratinocytes (HaCat) and epithelial cells (HeLa) triggering a dose-dependent cell retraction and collapse. We show how engulfed ZnO nanoparticles dissolve intracellularly, triggering actin filament bundling and structural changes in microtubules, transforming these highly dynamic 25 nm diameter polymers into rigid macrotubes of tubulin, severely affecting cell proliferation and survival. Our results demonstrate that nano-ZnO causes acute cytoskeletal collapse that triggers necrosis, followed by a late reactive oxygen species (ROS)-dependent apoptotic process.Zinc is a crucial element in biology that plays chief catalytic, structural and protein regulatory roles. Excess cytoplasmic zinc is toxic to cells so there are cell-entry and intracellular buffering mechanisms that control intracellular zinc availability. Tubulin and actin are two zinc-scavenging proteins that are essential components of the cellular cytoskeleton implicated in cell division, migration and cellular architecture maintenance. Here we demonstrate how exposure to different ZnO nanostructures, namely ZnO commercial nanoparticles and custom-made ZnO nanowires, produce acute cytotoxic effects in human keratinocytes (HaCat) and epithelial cells (HeLa) triggering a dose-dependent cell retraction and collapse. We show how engulfed ZnO nanoparticles dissolve intracellularly, triggering actin

  11. Identification and immunolocalization of actin cytoskeletal components in light- and dark-adapted octopus retinas.

    Science.gov (United States)

    De Velasco, B; Martinez, J M; Ochoa, G H; Miller, A M; Clark, Y M; Matsumoto, B; Robles, L J

    1999-06-01

    speculate that these proteins and actin remain associated with an avillar membrane that connects opposing sets of rhabdomeres in light-adapted retinas. Association of these cytoskeletal proteins with the avillar membrane would constitute a pool of proteins that could be recruited for rapid microvillus formation from the previously avillar region.

  12. 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.

  13. Cytoskeletal remodeling in differentiated vascular smooth muscle is actin isoform dependent and stimulus dependent.

    Science.gov (United States)

    Kim, Hak Rim; Gallant, Cynthia; Leavis, Paul C; Gunst, Susan J; Morgan, Kathleen G

    2008-09-01

    Dynamic remodeling of the actin cytoskeleton plays an essential role in the migration and proliferation of vascular smooth muscle cells. It has been suggested that actin remodeling may also play an important functional role in nonmigrating, nonproliferating differentiated vascular smooth muscle (dVSM). In the present study, we show that contractile agonists increase the net polymerization of actin in dVSM, as measured by the differential ultracentrifugation of vascular smooth muscle tissue and the costaining of single freshly dissociated cells with fluorescent probes specific for globular and filamentous actin. Furthermore, induced alterations of the actin polymerization state, as well as actin decoy peptides, inhibit contractility in a stimulus-dependent manner. Latrunculin pretreatment or actin decoy peptides significantly inhibit contractility induced by a phorbol ester or an alpha-agonist, but these procedures have no effect on contractions induced by KCl. Aorta dVSM expresses alpha-smooth muscle actin, beta-actin, nonmuscle gamma-actin, and smooth muscle gamma-actin. The incorporation of isoform-specific cell-permeant synthetic actin decoy peptides, as well as isoform-specific probing of cell fractions and two-dimensional gels, demonstrates that actin remodeling during alpha-agonist contractions involves the remodeling of primarily gamma-actin and, to a lesser extent, beta-actin. Taken together, these results show that net isoform- and agonist-dependent increases in actin polymerization regulate vascular contractility.

  14. 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.

  15. Phosphorylation of the cytoskeletal protein CAP1 controls its association with cofilin and actin.

    Science.gov (United States)

    Zhou, Guo-Lei; Zhang, Haitao; Wu, Huhehasi; Ghai, Pooja; Field, Jeffrey

    2014-12-01

    Cell signaling can control the dynamic balance between filamentous and monomeric actin by modulating actin regulatory proteins. One family of actin regulating proteins that controls actin dynamics comprises cyclase-associated proteins 1 and 2 (CAP1 and 2, respectively). However, cell signals that regulate CAPs remained unknown. We mapped phosphorylation sites on mouse CAP1 and found S307 and S309 to be regulatory sites. We further identified glycogen synthase kinase 3 as a kinase phosphorylating S309. The phosphomimetic mutant S307D/S309D lost binding to its partner cofilin and, when expressed in cells, caused accumulation of actin stress fibers similar to that in cells with reduced CAP expression. In contrast, the non-phosphorylatable S307A/S309A mutant showed drastically increased cofilin binding and reduced binding to actin. These results suggest that the phosphorylation serves to facilitate release of cofilin for a subsequent cycle of actin filament severing. Moreover, our results suggest that S307 and S309 function in tandem; neither the alterations in binding cofilin and/or actin, nor the defects in rescuing the phenotype of the enlarged cell size in CAP1 knockdown cells was observed in point mutants of either S307 or S309. In summary, we identify a novel regulatory mechanism of CAP1 through phosphorylation.

  16. Actin Cytoskeletal Organization in Drosophila Germline Ring Canals Depends on Kelch Function in a Cullin-RING E3 Ligase.

    Science.gov (United States)

    Hudson, Andrew M; Mannix, Katelynn M; Cooley, Lynn

    2015-11-01

    The Drosophila Kelch protein is required to organize the ovarian ring canal cytoskeleton. Kelch binds and cross-links F-actin in vitro, and it also functions with Cullin 3 (Cul3) as a component of a ubiquitin E3 ligase. How these two activities contribute to cytoskeletal remodeling in vivo is not known. We used targeted mutagenesis to investigate the mechanism of Kelch function. We tested a model in which Cul3-dependent degradation of Kelch is required for its function, but we found no evidence to support this hypothesis. However, we found that mutant Kelch deficient in its ability to interact with Cul3 failed to rescue the kelch cytoskeletal defects, suggesting that ubiquitin ligase activity is the principal activity required in vivo. We also determined that the proteasome is required with Kelch to promote the ordered growth of the ring canal cytoskeleton. These results indicate that Kelch organizes the cytoskeleton in vivo by targeting a protein substrate for degradation by the proteasome.

  17. Estrogen effects on actin cytoskeletal and endocytic proteins associated with tubulobulbar complex disruption in rat testes.

    Science.gov (United States)

    Upadhyay, Rahul D; Kumar, Anita V; Sonawane, Shobha; Gaonkar, Reshma; Balasinor, Nafisa H

    2013-10-01

    Tubulobulbar complexes (TBCs), evaginations of mature spermatids, penetrate into the surrounding Sertoli cell cytoplasm of testis seminiferous epithelium during rat spermatogenesis. These structures prepare mature spermatids for their release into the seminiferous tubular lumen via a process called spermiation. Based on their functions of transient attachment and endocytosis, many actin-regulatory and endocytic proteins are associated with TBCs. Previously, exogenous 17β-estradiol administration to adult male rats showed spermiation failure that was attributed to TBC disruption. To determine the molecular basis of estrogen-induced TBC disruption, we examined the expressions and localizations of actin-regulatory proteins, endocytic proteins, Rho-GTPases, and phosphorylation in TBCs during sperm release. Results demonstrated absence of neural Wiscott Aldrich syndrome protein, cortactin, adaptor-related protein complex 2 sigma-1 subunit, dynamin 2, cell division control protein 42, and phosphocortactin in the concavity of spermatid head where TBCs are present without change in their protein expression levels. Absence of these proteins could have led to collapse of the TBC structure which is involved in its formation and function.

  18. Cytoskeletal actin networks in motile cells are critically self-organized systems synchronized by mechanical interactions.

    Science.gov (United States)

    Cardamone, Luca; Laio, Alessandro; Torre, Vincent; Shahapure, Rajesh; DeSimone, Antonio

    2011-08-23

    Growing networks of actin fibers are able to organize into compact, stiff two-dimensional structures inside lamellipodia of crawling cells. We put forward the hypothesis that the growing actin network is a critically self-organized system, in which long-range mechanical stresses arising from the interaction with the plasma membrane provide the selective pressure leading to organization. We show that a simple model based only on this principle reproduces the stochastic nature of lamellipodia protrusion (growth periods alternating with fast retractions) and several of the features observed in experiments: a growth velocity initially insensitive to the external force; the capability of the network to organize its orientation; a load-history-dependent growth velocity. Our model predicts that the spectrum of the time series of the height of a growing lamellipodium decays with the inverse of the frequency. This behavior is a well-known signature of self-organized criticality and is confirmed by unique optical tweezer measurements performed in vivo on neuronal growth cones.

  19. Biogenesis of actin-like bacterial cytoskeletal filaments destined for positioning prokaryotic magnetic organelles.

    Science.gov (United States)

    Pradel, Nathalie; Santini, Claire-Lise; Bernadac, Alain; Fukumori, Yoshihiro; Wu, Long-Fei

    2006-11-14

    Magnetosomes comprise a magnetic nanocrystal surrounded by a lipid bilayer membrane. These unique prokaryotic organelles align inside magnetotactic bacterial cells and serve as an intracellular compass allowing the bacteria to navigate along the geomagnetic field in aquatic environments. Cryoelectron tomography of Magnetospirillum strains has revealed that the magnetosome chain is surrounded by a network of filaments that may be composed of MamK given that the filaments are absent in the mamK mutant cells. The process of the MamK filament assembly is unknown. Here we prove the authenticity of the MamK filaments and show that MamK exhibits linear distribution inside Magnetospirillum sp. cells even in the area without magnetosomes. The mamK gene alone is sufficient to direct the synthesis of straight filaments in Escherichia coli, and one extremity of the MamK filaments is located at the cellular pole. By using dual fluorescent labeling of MamK, we found that MamK nucleates at multiple sites and assembles into mosaic filaments. Time-lapse experiments reveal that the assembly of the MamK filaments is a highly dynamic and kinetically asymmetrical process. MamK bundles might initiate the formation of a new filament or associate to one preexistent filament. Our results demonstrate the mechanism of biogenesis of prokaryotic cytoskeletal filaments that are structurally and functionally distinct from the known MreB and ParM filaments. In addition to positioning magnetosomes, other hypothetical functions of the MamK filaments in magnetotaxis might include anchoring magnetosomes and being involved in magnetic reception.

  20. Loss of actin cytoskeletal function and EDS1 activity, in combination, severely compromises non-host resistance in Arabidopsis against wheat powdery mildew.

    Science.gov (United States)

    Yun, Byung-Wook; Atkinson, Helen A; Gaborit, Charlotte; Greenland, Andy; Read, Nick D; Pallas, Jacqueline A; Loake, Gary J

    2003-06-01

    Plant immunity against the majority of the microbial pathogens is conveyed by a phenomenon known as non-host resistance (NHR). This defence mechanism affords durable protection to plant species against given species of phytopathogens. We investigated the genetic basis of NHR in Arabidopsis against the wheat powdery mildew fungus Blumeria graminis f. sp. tritici (Bgt). Both primary and appressorial germ tubes were produced from individual Bgt conidia on the surface of the Arabidopsis leaves. Attempted infection occasionally resulted in successful penetration, which led to the development of an abnormal unilateral haustorium. Inoculation of a series of Arabidopsis defence-related mutants with Bgt resulted in the attenuation of reactive oxygen intermediate (ROI) production and salicylic acid (SA)-dependent defence gene expression in eds1, pad4 and nahG plants, which are known to be defective in some aspects of host resistance. Furthermore, Bgt often developed bilateral haustoria in the mutant Arabidopsis lines that closely resembled those formed in wheat. A similar decrease in NHR was observed following treatment of the wild-type Arabidopsis plants with cytochalasin E, an inhibitor of actin microfilament polymerisation. In eds1 mutants, inhibition of actin polymerisation severely compromised NHR in Arabidopsis against Bgt. This permitted completion of the Bgt infection cycle on these plants. Therefore, actin cytoskeletal function and EDS1 activity, in combination, are major contributors to NHR in Arabidopsis against wheat powdery mildew.

  1. Cytoskeletal heart-enriched actin-associated protein (CHAP) is expressed in striated and smooth muscle cells in chick and mouse during embryonic and adult stages.

    Science.gov (United States)

    van Eldik, Willemijn; Beqqali, Abdelaziz; Monshouwer-Kloots, Jantine; Mummery, Christine; Passier, Robert

    2011-01-01

    We recently identified a new Z-disc protein, CHAP (Cytoskeletal Heart-enriched Actin-associated Protein), which is expressed in striated muscle and plays an important role during embryonic muscle development in mouse and zebrafish. Here, we confirm and further extend these findings by (i) the identification and characterization of the CHAP orthologue in chick and (ii) providing a detailed analysis of CHAP expression in mouse during embryonic and adult stages. Chick CHAP contains a PDZ domain and a nuclear localization signal, resembling the human and mouse CHAPa. CHAP is expressed in the developing heart and somites, as well as muscle precursors of the limb buds in mouse and chick embryos. CHAP expression in heart and skeletal muscle is maintained in adult mice, both in slow and fast muscle fibers. Moreover, besides expression in striated muscle, we demonstrate that CHAP is expressed in smooth muscle cells of aorta, carotid and coronary arteries in adult mice, but not during embryonic development.

  2. 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...

  3. LL-37 induces polymerization and bundling of actin and affects actin structure.

    Directory of Open Access Journals (Sweden)

    Asaf Sol

    Full Text Available Actin exists as a monomer (G-actin which can be polymerized to filaments F-actin that under the influence of actin-binding proteins and polycations bundle and contribute to the formation of the cytoskeleton. Bundled actin from lysed cells increases the viscosity of sputum in lungs of cystic fibrosis patients. The human host defense peptide LL-37 was previously shown to induce actin bundling and was thus hypothesized to contribute to the pathogenicity of this disease. In this work, interactions between actin and the cationic LL-37 were studied by optical, proteolytic and surface plasmon resonance methods and compared to those obtained with scrambled LL-37 and with the cationic protein lysozyme. We show that LL-37 binds strongly to CaATP-G-actin while scrambled LL-37 does not. While LL-37, at superstoichiometric LL-37/actin concentrations polymerizes MgATP-G-actin, at lower non-polymerizing concentrations LL-37 inhibits actin polymerization by MgCl(2 or NaCl. LL-37 bundles Mg-F-actin filaments both at low and physiological ionic strength when in equimolar or higher concentrations than those of actin. The LL-37 induced bundles are significantly less sensitive to increase in ionic strength than those induced by scrambled LL-37 and lysozyme. LL-37 in concentrations lower than those needed for actin polymerization or bundling, accelerates cleavage of both monomer and polymer actin by subtilisin. Our results indicate that the LL-37-actin interaction is partially electrostatic and partially hydrophobic and that a specific actin binding sequence in the peptide is responsible for the hydrophobic interaction. LL-37-induced bundles, which may contribute to the accumulation of sputum in cystic fibrosis, are dissociated very efficiently by DNase-1 and also by cofilin.

  4. 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.

  5. 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.

  6. Cytoskeletal dynamics

    DEFF Research Database (Denmark)

    Bendix, Pól Martin

    2015-01-01

    I worked with reconstitutted contractile acto-myosin systems containing mainly actin, actin cross-linkers and myosin motors. Contractility and rheology of such systems was studied using confocal microscopy and rheology....

  7. The reversible increase in tight junction permeability induced by capsaicin is mediated via cofilin-actin cytoskeletal dynamics and decreased level of occludin.

    Directory of Open Access Journals (Sweden)

    Tomoko Shiobara

    Full Text Available Previous results demonstrated that capsaicin induces the reversible tight junctions (TJ opening via cofilin activation. The present study investigated the mechanisms underlying the reversible TJ opening and compared the effect to the irreversible opening induced by actin inhibitors. Capsaicin treatment induced the F-actin alteration unique to capsaicin compared to actin-interacting agents such as latrunculin A, which opens TJ irreversibly. Along with TJ opening, capsaicin decreased the level of F-actin at bicellular junctions but increased it at tricellular junctions accompanied with its concentration on the apical side of the lateral membrane. No change in TJ protein localization was observed upon exposure to capsaicin, but the amount of occludin was decreased significantly. In addition, cosedimentation analyses suggested a decrease in the interactions forming TJ, thereby weakening TJ tightness. Introduction of cofilin, LIMK and occludin into the cell monolayers confirmed their contribution to the transepithelial electrical resistance decrease. Finally, exposure of monolayers to capsaicin augmented the paracellular passage of both charged and uncharged compounds, as well as of insulin, indicating that capsaicin can be employed to modulate epithelial permeability. Our results demonstrate that capsaicin induces TJ opening through a unique mechanism, and suggest that it is a new type of paracellular permeability enhancer.

  8. Purification of Tetrahymena cytoskeletal proteins.

    Science.gov (United States)

    Honts, Jerry E

    2012-01-01

    Like all eukaryotic cells, Tetrahymena thermophila contains a rich array of cytoskeletal proteins, some familiar and some novel. A detailed analysis of the structure, function, and interactions of these proteins requires procedures for purifying the individual protein components. Procedures for the purification of actin and tubulin from Tetrahymena are reviewed, followed by a description of a procedure that yields proteins from the epiplasmic layer and associated structures, including the tetrins. Finally, the challenges and opportunities for future advances are assessed.

  9. 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.

  10. Selective ablation of the androgen receptor in mouse sertoli cells affects sertoli cell maturation, barrier formation and cytoskeletal development.

    Directory of Open Access Journals (Sweden)

    Ariane Willems

    Full Text Available The observation that mice with a selective ablation of the androgen receptor (AR in Sertoli cells (SC (SCARKO mice display a complete block in meiosis supports the contention that SC play a pivotal role in the control of germ cell development by androgens. To delineate the physiological and molecular mechanism responsible for this control, we compared tubular development in pubertal SCARKO mice and littermate controls. Particular attention was paid to differences in SC maturation, SC barrier formation and cytoskeletal organization and to the molecular mediators potentially involved. Functional analysis of SC barrier development by hypertonic perfusion and lanthanum permeation techniques and immunohistochemical analysis of junction formation showed that SCARKO mice still attempt to produce a barrier separating basal and adluminal compartment but that barrier formation is delayed and defective. Defective barrier formation was accompanied by disturbances in SC nuclear maturation (immature shape, absence of prominent, tripartite nucleoli and SC polarization (aberrant positioning of SC nuclei and cytoskeletal elements such as vimentin. Quantitative RT-PCR was used to study the transcript levels of genes potentially related to the described phenomena between day 8 and 35. Differences in the expression of SC genes known to play a role in junction formation could be shown from day 8 for Cldn11, from day 15 for Cldn3 and Espn, from day 20 for Cdh2 and Jam3 and from day 35 for ZO-1. Marked differences were also noted in the transcript levels of several genes that are also related to cell adhesion and cytoskeletal dynamics but that have not yet been studied in SC (Actn3, Ank3, Anxa9, Scin, Emb, Mpzl2. It is concluded that absence of a functional AR in SC impedes the remodeling of testicular tubules expected at the onset of spermatogenesis and interferes with the creation of the specific environment needed for germ cell development.

  11. Simulated Microgravity Induced Cytoskeletal Rearrangements are Modulated by Protooncogenes

    Science.gov (United States)

    Melhado, C. D.; Sanford, G. L.; Bosah, F.; Harris-Hooker, S.

    1998-01-01

    Microgravity is the environment living systems encounter during space flight and gravitational unloading is the effect of this environment on living systems. The cell, being a multiphasic chemical system, is a useful starting point to study the potential impact of gravity unloading on physiological function. In the absence of gravity, sedimentation of organelles including chromosomes, mitochondria, nuclei, the Golgi apparatus, vacuoles, and the endoplasmic reticulum may be affected. Most of these organelles, however, are somewhat held in place by cytoskeleton. Hansen and Igber suggest that intermediate filaments act to stabilize the nuleus against rotational movement, and integrate cell and nuclear structure. The tensegrity theory supports the idea that mechanical or physical forces alters the cytoskeletal structures of a cell resulting in the changes in cell: matrix interactions and receptor-signaling coupling. This type of stress to the cytoskeleton may be largely responsible regulating cell shape, growth, movement and metabolism. Mouse MC3T3 El cells under microgravity exhibited significant cytoskeletal changes and alterations in cell growth. The alterations in cytoskeleton architecture may be due to changes in the expression of actin related proteins or integrins. Philopott and coworkers reported on changes in the distribution of microtubule and cytoskeleton elements in the cells of heart tissue from space flight rats and those centrifuged at 1.7g. Other researchers have showed that microgravity reduced EGF-induced c-fos and c-jun expression compared to 1 g controls. Since c-fos and c-jun are known regulators of cell growth, it is likely that altered signal transduction involving protooncogenes may play a crucial role in the reduced growth and alterations in cytoskeletal arrangements found during space flight. It is clear that a microgravity environment induces a number of changes in cell shape, cell surface molecules, gene expression, and cytoskeletal

  12. Effects of oridonin on cytoskeletal protein F-actin in human pancreatic carcinoma cells%冬凌草甲素对胰腺癌细胞骨架蛋白F-actin的影响

    Institute of Scientific and Technical Information of China (English)

    刘军楼; 沈洪; 徐力; 杨继兵; 于希忠; 孙志岭

    2015-01-01

    Background and purpose:Traditional Chinese medicine with notable effect and little adverse reaction is increasingly concerned about the medical profession because of its great potential and advantage in treating pancreatic carcinoma. In this experiment, we studied the effects of oridonin on apoptosis and cytoskeletal protein F-actin in human pancreatic carcinoma SW1990 cells. Methods:SW1990 cells in culture medium were treated with different concentrations of oridonin. The inhibitory rate of the cells was measured by MTT assay. Morphology of cell apoptosis was observed by DAPI stain and cell apoptotic rate was detected by lfow cytometry (FCM). The morphological changes of F-actin were observed by laser confocal microscopy. Results:The growth of human pancreatic carcinoma SW1990 cells was signiifcantly inhibited by oridonin. Apoptosis morphological changes including condensation of chromatin and nuclear fragmentation were observed clearly by DAPI stain. The early apoptotic rate of SW1990 cells treated with 25, 50μmol/L oridonin was signiifcantly higher than that of the control group (3.78±0.46, 9.51±0.63 vs 0.73±0.06, P<0.05), and the late apoptotic rate and cell necrosis rate were also signiifcantly higher than that of the control group (14.40±1.78, 20.53±2.54 vs 4.16±0.31, P<0.05). F-actin was showed from polymerization to depolymerization after oridonin treatment. Conclusion:Oridonin can obviously inhibit the proliferation and induce apoptosis of SW1990 cells. The mechanisms may involve the depolymerization of F-actin after treatment with oridonin.%背景与目的:中医药治疗肿瘤不良反应低且疗效显著,在防治胰腺癌方面有较大的潜力与优势,日益受到国内、外医学界的关注。本研究观察中草药冬凌草的有效成分冬凌草甲素对人胰腺癌SW1990凋亡及细胞骨架蛋白F-actin的影响。方法:以不同浓度的冬凌草甲素作用于体外培养的SW1990细胞,采用MTT法检测细胞生长

  13. Bidirectional Interplay between Vimentin Intermediate Filaments and Contractile Actin Stress Fibers

    Directory of Open Access Journals (Sweden)

    Yaming Jiu

    2015-06-01

    Full Text Available The actin cytoskeleton and cytoplasmic intermediate filaments contribute to cell migration and morphogenesis, but the interplay between these two central cytoskeletal elements has remained elusive. Here, we find that specific actin stress fiber structures, transverse arcs, interact with vimentin intermediate filaments and promote their retrograde flow. Consequently, myosin-II-containing arcs are important for perinuclear localization of the vimentin network in cells. The vimentin network reciprocally restricts retrograde movement of arcs and hence controls the width of flat lamellum at the leading edge of the cell. Depletion of plectin recapitulates the vimentin organization phenotype of arc-deficient cells without affecting the integrity of vimentin filaments or stress fibers, demonstrating that this cytoskeletal cross-linker is required for productive interactions between vimentin and arcs. Collectively, our results reveal that plectin-mediated interplay between contractile actomyosin arcs and vimentin intermediate filaments controls the localization and dynamics of these two cytoskeletal systems and is consequently important for cell morphogenesis.

  14. 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...

  15. Collective dynamics of active cytoskeletal networks.

    Directory of Open Access Journals (Sweden)

    Simone Köhler

    Full Text Available Self organization mechanisms are essential for the cytoskeleton to adapt to the requirements of living cells. They rely on the intricate interplay of cytoskeletal filaments, crosslinking proteins and molecular motors. Here we present an in vitro minimal model system consisting of actin filaments, fascin and myosin-II filaments exhibiting pulsatile collective dynamics and superdiffusive transport properties. Both phenomena rely on the complex competition of crosslinking molecules and motor filaments in the network. They are only observed if the relative strength of the binding of myosin-II filaments to the actin network allows exerting high enough forces to unbind actin/fascin crosslinks. This is shown by varying the binding strength of the acto-myosin bond and by combining the experiments with phenomenological simulations based on simple interaction rules.

  16. Importance of a Lys113-Glu195 intermonomer ionic bond in F-actin stabilization and regulation by yeast formins Bni1p and Bnr1p.

    Science.gov (United States)

    Wen, Kuo-Kuang; McKane, Melissa; Rubenstein, Peter A

    2013-06-28

    Proper actin cytoskeletal function requires actin's ability to generate a stable filament and requires that this reaction be regulated by actin-binding proteins via allosteric effects on the actin. A proposed ionic interaction in the actin filament interior between Lys(113) of one monomer and Glu(195) of a monomer in the apposing strand potentially fosters cross-strand stabilization and allosteric communication between the filament interior and exterior. We interrupted the potential interaction by creating either K113E or E195K actin. By combining the two, we also reversed the interaction with a K113E/E195K (E/K) mutant. In all cases, we isolated viable cells expressing only the mutant actin. Either single mutant cell displays significantly decreased growth in YPD medium. This deficit is rescued in the double mutant. All three mutants display abnormal phalloidin cytoskeletal staining. K113E actin exhibits a critical concentration of polymerization 4 times higher than WT actin, nucleates more poorly, and forms shorter filaments. Restoration of the ionic bond, E/K, eliminates most of these problems. E195K actin behaves much more like WT actin, indicating accommodation of the neighboring lysines. Both Bni1 and Bnr1 formin FH1-FH2 fragment accelerate polymerization of WT, E/K, and to a lesser extent E195K actin. Bni1p FH1-FH2 dramatically inhibits K113E actin polymerization, consistent with barbed end capping. However, Bnr1p FH1-FH2 restores K113E actin polymerization, forming single filaments. In summary, the proposed ionic interaction plays an important role in filament stabilization and in the propagation of allosteric changes affecting formin regulation in an isoform-specific fashion.

  17. Piracy of decay-accelerating factor (CD55) signal transduction by the diffusely adhering strain Escherichia coli C1845 promotes cytoskeletal F-actin rearrangements in cultured human intestinal INT407 cells.

    Science.gov (United States)

    Peiffer, I; Servin, A L; Bernet-Camard, M F

    1998-09-01

    Diffusely adhering Escherichia coli (DAEC) C1845 (clinical isolate) harboring the fimbrial adhesin F1845 can infect cultured human differentiated intestinal epithelial cells; this process is followed by the disassembly of the actin network in the apical domain. The aim of this study was to examine the mechanism by which DAEC C1845 promotes F-actin rearrangements. For this purpose, we used a human embryonic intestinal cell line (INT407) expressing the membrane-associated glycosylphosphatidylinositol (GPI) protein-anchored decay-accelerating factor (DAF), the receptor of the F1845 adhesin. We show here that infection of INT407 cells by DAEC C1845 can provoke dramatic F-actin rearrangements without cell entry. Clustering of phosphotyrosines was observed, revealing that the DAEC C1845-DAF interaction involves the recruitment of signal transduction molecules. A pharmacological approach with a subset of inhibitors of signal transduction molecules was used to identify the cascade of signal transduction molecules that are coupled to the DAF, that are activated upon infection, and that promote the F-actin rearrangements. DAEC C1845-induced F-actin rearrangements can be blocked dose dependently by protein tyrosine kinase, phospholipase Cgamma, phosphatidylinositol 3-kinase, protein kinase C, and Ca2+ inhibitors. F-actin rearrangements and blocking by inhibitors were observed after infection of the cells with two E. coli recombinants carrying the plasmids containing the fimbrial adhesin F1845 or the fimbrial hemagglutinin Dr, belonging to the same family of adhesins. These findings show that the DAEC Dr family of pathogens promotes alterations in the intestinal cell cytoskeleton by piracy of the DAF-GPI signal cascade without bacterial cell entry.

  18. Biotechnological aspects of cytoskeletal regulation in plants.

    Science.gov (United States)

    Komis, George; Luptovciak, Ivan; Doskocilova, Anna; Samaj, Jozef

    2015-11-01

    The cytoskeleton is a protein-based intracellular superstructure that evolved early after the appearance of bacterial prokaryotes. Eventually cytoskeletal proteins and their macromolecular assemblies were established in eukaryotes and assumed critical roles in cell movements, intracellular organization, cell division and cell differentiation. In biomedicine the small-molecules targeting cytoskeletal elements are in the frontline of anticancer research with plant-derived cytoskeletal drugs such as Vinca alkaloids and toxoids, being routinely used in the clinical practice. Moreover, plants are also major material, food and energy resources for human activities ranging from agriculture, textile industry, carpentry, energy production and new material development to name some few. Most of these inheritable traits are associated with cell wall synthesis and chemical modification during primary and secondary plant growth and inevitably are associated with the dynamics, organization and interactions of the plant cytoskeleton. Taking into account the vast intracellular spread of microtubules and actin microfilaments the cytoskeleton collectively assumed central roles in plant growth and development, in determining the physical stance of plants against the forces of nature and becoming a battleground between pathogenic invaders and the defense mechanisms of plant cells. This review aims to address the role of the plant cytoskeleton in manageable features of plants including cellulose biosynthesis with implications in wood and fiber properties, in biofuel production and the contribution of plant cytoskeletal elements in plant defense responses against pathogens or detrimental environmental conditions. Ultimately the present work surveys the potential of cytoskeletal proteins as platforms of plant genetic engineering, nominating certain cytoskeletal proteins as vectors of favorable traits in crops and other economically important plants.

  19. Chorein Sensitive Arrangement of Cytoskeletal Architecture

    Directory of Open Access Journals (Sweden)

    Sabina Honisch

    2015-08-01

    Full Text Available Background/Aims: Chorein is a protein expressed in various cell types. Loss of function mutations of the chorein encoding gene VPS13A lead to chorea-acanthocytosis, an autosomal recessive genetic disease characterized by movement disorder and behavioral abnormalities. Recent observations revealed that chorein is a powerful regulator of actin cytoskeleton in erythrocytes, platelets, K562 and endothelial HUVEC cells. Methods: In the present study we have used Western blotting to study actin polymerization dynamics, laser scanning microscopy to evaluate in detail the role of chorein in microfilaments, microtubules and intermediate filaments cytoskeleton architecture and RT-PCR to assess gene transcription of the cytoskeletal proteins. Results: We report here powerful depolymerization of actin microfilaments both, in erythrocytes and fibroblasts isolated from chorea-acanthocytosis patients. Along those lines, morphological analysis of fibroblasts from chorea-acanthocytosis patients showed disarranged microtubular network, when compared to fibroblasts from healthy donors. Similarly, the intermediate filament networks of desmin and cytokeratins showed significantly disordered organization with clearly diminished staining in patient's fibroblasts. In line with this, RT-PCR analysis revealed significant downregulation of desmin and cytokeratin gene transcripts. Conclusion: Our results provide for the first time evidence that defective chorein is accompanied by significant structural disorganization of all cytoskeletal structures in human fibroblasts from chorea-acanthocytosis patients.

  20. Intracellular cytoskeletal elements and cytoskeletons in bacteria.

    Science.gov (United States)

    Madkour, Mohamed H F; Mayer, Frank

    2007-01-01

    Within a short period of time after the discovery of bacterial cytoskletons, major progress had been made in areas such as general spatial layout of cytoskeletons, their involvement in a variety of cellfunctions (shape control, cell division, chromosome segregation, cell motility). This progress was achieved by application of advanced investigation techniques. Homologs of eukaryotic actin, tubulin, and intermediate filaments were found in bacteria; cytoskeletal proteins not closely or not at all related to any of these major cytoskeletal proteins were discovered in a number of bacteria such as Mycoplasmas, Spiroplasmas, Spirochetes, Treponema, Caulobacter. A structural role for bacterial elongation factor Tu was indicated. On the basis of this new thinking, new approaches in biotechnology and new drugs are on the way.

  1. Cooperation of the BTB-Zinc finger protein, Abrupt, with cytoskeletal regulators in Drosophila epithelial tumorigenesis

    Directory of Open Access Journals (Sweden)

    Nezaket Turkel

    2015-08-01

    Full Text Available The deregulation of cell polarity or cytoskeletal regulators is a common occurrence in human epithelial cancers. Moreover, there is accumulating evidence in human epithelial cancer that BTB-ZF genes, such as Bcl6 and ZBTB7A, are oncogenic. From our previous studies in the vinegar fly, Drosophila melanogaster, we have identified a cooperative interaction between a mutation in the apico-basal cell polarity regulator Scribble (Scrib and overexpression of the BTB-ZF protein Abrupt (Ab. Herein, we show that co-expression of ab with actin cytoskeletal regulators, RhoGEF2 or Src64B, in the developing eye-antennal epithelial tissue results in the formation of overgrown amorphous tumours, whereas ab and DRac1 co-expression leads to non-cell autonomous overgrowth. Together with ab, these genes affect the expression of differentiation genes, resulting in tumours locked in a progenitor cell fate. Finally, we show that the expression of two mammalian genes related to ab, Bcl6 and ZBTB7A, which are oncogenes in mammalian epithelial cancers, significantly correlate with the upregulation of cytoskeletal genes or downregulation of apico-basal cell polarity neoplastic tumour suppressor genes in colorectal, lung and other human epithelial cancers. Altogether, this analysis has revealed that upregulation of cytoskeletal regulators cooperate with Abrupt in Drosophila epithelial tumorigenesis, and that high expression of human BTB-ZF genes, Bcl6 and ZBTB7A, shows significant correlations with cytoskeletal and cell polarity gene expression in specific epithelial tumour types. This highlights the need for further investigation of the cooperation between these genes in mammalian systems.

  2. Oestradiol and progesterone differentially alter cytoskeletal protein expression and flame cell morphology in Taenia crassiceps.

    Science.gov (United States)

    Ambrosio, Javier R; Ostoa-Saloma, Pedro; Palacios-Arreola, M Isabel; Ruíz-Rosado, Azucena; Sánchez-Orellana, Pedro L; Reynoso-Ducoing, Olivia; Nava-Castro, Karen E; Martínez-Velázquez, Nancy; Escobedo, Galileo; Ibarra-Coronado, Elizabeth G; Valverde-Islas, Laura; Morales-Montor, Jorge

    2014-09-01

    We examined the effects of oestradiol (E2) and progesterone (P4) on cytoskeletal protein expression in the helminth Taenia crassiceps - specifically actin, tubulin and myosin. These proteins assemble into flame cells, which constitute the parasite excretory system. Total protein extracts were obtained from E2- and P4-treated T. crassiceps cysticerci and untreated controls, and analysed by one- and two-dimensional protein electrophoresis, flow cytometry, immunofluorescence and videomicroscopy. Exposure of T. crassiceps cysticerci to E2 and P4 induced differential protein expression patterns compared with untreated controls. Changes in actin, tubulin and myosin expression were confirmed by flow cytometry of parasite cells and immunofluorescence. In addition, parasite morphology was altered in response to E2 and P4 versus controls. Flame cells were primarily affected at the level of the ciliary tuft, in association with the changes in actin, tubulin and myosin. We conclude that oestradiol and progesterone act directly on T. crassiceps cysticerci, altering actin, tubulin and myosin expression and thus affecting the assembly and function of flame cells. Our results increase our understanding of several aspects of the molecular crosstalk between host and parasite, which might be useful in designing anthelmintic drugs that exclusively impair parasitic proteins which mediate cell signaling and pathogenic reproduction and establishment.

  3. Rho/Rho-dependent kinase affects locomotion and actin-myosin II activity of Amoeba proteus.

    Science.gov (United States)

    Kłopocka, W; Redowicz, M J

    2004-10-01

    The highly motile free-living unicellular organism Amoeba proteus has been widely used as a model to study cell motility. However, the molecular mechanisms underlying its unique locomotion are still scarcely known. Recently, we have shown that blocking the amoebae's endogenous Rac- and Rho-like proteins led to distinct and irreversible changes in the appearance of these large migrating cells as well as to a significant inhibition of their locomotion. In order to elucidate the mechanism of the Rho pathway, we tested the effects of blocking the endogenous Rho-dependent kinase (ROCK) by anti-ROCK antibodies and Y-27632, (+)-(R)-trans-4-(1-aminoethyl)-N-(4-pyridyl)cyclohexanecarboxamide dihydrochloride, a specific inhibitor of ROCK, on migrating amoebae and the effect of the Rho and ROCK inhibition on the actin-activated Mg-ATPase of the cytosolic fraction of the amoebae. Amoebae microinjected with anti-ROCK inhibitors remained contracted and strongly attached to the glass surface and exhibited an atypical locomotion. Despite protruding many pseudopodia that were advancing in various directions, the amoebae could not effectively move. Immunofluorescence studies showed that ROCK-like protein was dispersed throughout the cytoplasm and was also found in the regions of actin-myosin II interaction during both isotonic and isometric contraction. The Mg-ATPase activity was about two- to threefold enhanced, indicating that blocking the Rho/Rho-dependent kinase activated myosin. It is possible then that in contrast to the vertebrate cells, the inactivation of Rho/Rho-dependent kinase in amoebae leads to the activation of myosin II and to the observed hypercontracted cells which cannot exert effective locomotion.

  4. Patterning and lifetime of plasma membrane-localized cellulose synthase is dependent on actin organization in Arabidopsis interphase cells.

    Science.gov (United States)

    Sampathkumar, Arun; Gutierrez, Ryan; McFarlane, Heather E; Bringmann, Martin; Lindeboom, Jelmer; Emons, Anne-Mie; Samuels, Lacey; Ketelaar, Tijs; Ehrhardt, David W; Persson, Staffan

    2013-06-01

    The actin and microtubule cytoskeletons regulate cell shape across phyla, from bacteria to metazoans. In organisms with cell walls, the wall acts as a primary constraint of shape, and generation of specific cell shape depends on cytoskeletal organization for wall deposition and/or cell expansion. In higher plants, cortical microtubules help to organize cell wall construction by positioning the delivery of cellulose synthase (CesA) complexes and guiding their trajectories to orient newly synthesized cellulose microfibrils. The actin cytoskeleton is required for normal distribution of CesAs to the plasma membrane, but more specific roles for actin in cell wall assembly and organization remain largely elusive. We show that the actin cytoskeleton functions to regulate the CesA delivery rate to, and lifetime of CesAs at, the plasma membrane, which affects cellulose production. Furthermore, quantitative image analyses revealed that actin organization affects CesA tracking behavior at the plasma membrane and that small CesA compartments were associated with the actin cytoskeleton. By contrast, localized insertion of CesAs adjacent to cortical microtubules was not affected by the actin organization. Hence, both actin and microtubule cytoskeletons play important roles in regulating CesA trafficking, cellulose deposition, and organization of cell wall biogenesis.

  5. Fractal dimension as a measure of altered actin cytoskeleton in MC3T3-E1 cells under simulated microgravity using 3-D/2-D clinostats.

    Science.gov (United States)

    Qian, A R; Li, D; Han, J; Gao, X; Di, S M; Zhang, W; Hu, L F; Shang, Peng

    2012-05-01

    Osteoblasts, the bone-forming cells, respond to various mechanical forces, such as stretch and fluid shear force in essentially similar ways. The cytoskeleton, as the load-bearing architecture of the cell, is sensitive to altered inertial forces. Disruption of the cytoskeleton will result in alteration of cellular structure and function. However, it is difficult to quantitatively illustrate cytoskeletal rearrangement because of the complexity of cytoskeletal structure. Usually, the morphological changes in actin organization caused by external stimulus are basically descriptive. In this study, fractal dimensions (D) analysis was used to quantify the morphological changes in the actin cytoskeleton of osteoblast-like cells (MC3T3-E1) under simulated microgravity using 3-D/2-D clinostats. The ImageJ software was used to count the fractal dimension of actin cytoskeleton by box-counting methods. Real-time PCR and immunofluroscent assays were used to further confirm the results obtained by fractal dimension analysis. The results showed significant decreases in D value of actin cytoskeleton, β-actin mRNA expression, and the mean fluorescence intensity of F-actin in osteoblast-like cells after 24 or 48 h of incubation under 3-D/2-D clinorotation condition compared with control. The findings indicate that 3-D/2-D clinorotation affects both actin cytoskeleton architecture and mRNA expression, and fractal may be a promising approach for quantitative analysis of the changes in cytoskeleton in different environments.

  6. The role of cyclase-associated protein in regulating actin filament dynamics – more than a monomer-sequestration factor

    OpenAIRE

    Ono, Shoichiro

    2013-01-01

    Dynamic reorganization of the actin cytoskeleton is fundamental to a number of cell biological events. A variety of actin-regulatory proteins modulate polymerization and depolymerization of actin and contribute to actin cytoskeletal reorganization. Cyclase-associated protein (CAP) is a conserved actin-monomer-binding protein that has been studied for over 20 years. Early studies have shown that CAP sequesters actin monomers; recent studies, however, have revealed more active roles of CAP in a...

  7. Bacterial actin and tubulin homologs in cell growth and division.

    Science.gov (United States)

    Busiek, Kimberly K; Margolin, William

    2015-03-16

    In contrast to the elaborate cytoskeletal machines harbored by eukaryotic cells, such as mitotic spindles, cytoskeletal structures detectable by typical negative stain electron microscopy are generally absent from bacterial cells. As a result, for decades it was thought that bacteria lacked cytoskeletal machines. Revolutions in genomics and fluorescence microscopy have confirmed the existence not only of smaller-scale cytoskeletal structures in bacteria, but also of widespread functional homologs of eukaryotic cytoskeletal proteins. The presence of actin, tubulin, and intermediate filament homologs in these relatively simple cells suggests that primitive cytoskeletons first arose in bacteria. In bacteria such as Escherichia coli, homologs of tubulin and actin directly interact with each other and are crucial for coordinating cell growth and division. The function and direct interactions between these proteins will be the focus of this review.

  8. Patterning and Lifetime of Plasma Membrane-Localized Cellulose Synthase Is Dependent on Actin Organization in Arabidopsis Interphase Cells1[W

    Science.gov (United States)

    Sampathkumar, Arun; Gutierrez, Ryan; McFarlane, Heather E.; Bringmann, Martin; Lindeboom, Jelmer; Emons, Anne-Mie; Samuels, Lacey; Ketelaar, Tijs; Ehrhardt, David W.; Persson, Staffan

    2013-01-01

    The actin and microtubule cytoskeletons regulate cell shape across phyla, from bacteria to metazoans. In organisms with cell walls, the wall acts as a primary constraint of shape, and generation of specific cell shape depends on cytoskeletal organization for wall deposition and/or cell expansion. In higher plants, cortical microtubules help to organize cell wall construction by positioning the delivery of cellulose synthase (CesA) complexes and guiding their trajectories to orient newly synthesized cellulose microfibrils. The actin cytoskeleton is required for normal distribution of CesAs to the plasma membrane, but more specific roles for actin in cell wall assembly and organization remain largely elusive. We show that the actin cytoskeleton functions to regulate the CesA delivery rate to, and lifetime of CesAs at, the plasma membrane, which affects cellulose production. Furthermore, quantitative image analyses revealed that actin organization affects CesA tracking behavior at the plasma membrane and that small CesA compartments were associated with the actin cytoskeleton. By contrast, localized insertion of CesAs adjacent to cortical microtubules was not affected by the actin organization. Hence, both actin and microtubule cytoskeletons play important roles in regulating CesA trafficking, cellulose deposition, and organization of cell wall biogenesis. PMID:23606596

  9. 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.

  10. 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.

  11. The DCR protein TTC3 affects differentiation and Golgi compactness in neurons through specific actin-regulating pathways.

    Directory of Open Access Journals (Sweden)

    Gaia Elena Berto

    Full Text Available In neuronal cells, actin remodeling plays a well known role in neurite extension but is also deeply involved in the organization of intracellular structures, such as the Golgi apparatus. However, it is still not very clear which mechanisms may regulate actin dynamics at the different sites. In this report we show that high levels of the TTC3 protein, encoded by one of the genes of the Down Syndrome Critical Region (DCR, prevent neurite extension and disrupt Golgi compactness in differentiating primary neurons. These effects largely depend on the capability of TTC3 to promote actin polymerization through signaling pathways involving RhoA, ROCK, CIT-N and PIIa. However, the functional relationships between these molecules differ significantly if considering the TTC3 activity on neurite extension or on Golgi organization. Finally, our results reveal an unexpected stage-dependent requirement for F-actin in Golgi organization at different stages of neuronal differentiation.

  12. 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.

  13. Redox regulation of actin by thioredoxin-1 is mediated by the interaction of the proteins via cysteine 62.

    Science.gov (United States)

    Wang, Xiaogang; Ling, Shukuan; Zhao, Dingsheng; Sun, Qiao; Li, Qi; Wu, Feng; Nie, Jielin; Qu, Lina; Wang, Bo; Shen, Xun; Bai, Yanqiang; Li, Yingxian; Li, Yinghui

    2010-09-01

    Actin is a highly conserved protein in eukaryotic cells, and has been identified as one of the main redox targets by redox proteomics under oxidative stress. However, little is known about the mechanisms of regulation of the redox state of actin. In this study, we investigated how thioredoxin-1 (Trx1) affected the redox state of actin and its polymerization under oxidative stress in SH-SY5Y cells. Trx1 decreased the levels of reactive oxygen species (ROS) in the cells, and cysteine residues at positions 32, 35, and 69 of the Trx1 protein were active sites for redox regulation. Actin could be kept in a reduced state by Trx1 under H(2)O(2) stimulation. A physical interaction was found to exist between actin and Trx1. Cysteine 62 in Trx1 was the key site that interacted with actin, and it was required to maintain cellular viability and anti-apoptotic function. Taken together, these results suggested that Trx1 could protect cells from apoptosis under oxidative stress not only by increasing the total antioxidant capability and decreasing the ROS levels, but also by stabilizing the actin cytoskeletal system, which cooperatively contributed to the enhancement of cell viability and worked against apoptosis.

  14. Modulators of cytoskeletal reorganization in CA1 hippocampal neurons show increased expression in patients at mid-stage Alzheimer's disease.

    Directory of Open Access Journals (Sweden)

    Patricia F Kao

    Full Text Available During the progression of Alzheimer's disease (AD, hippocampal neurons undergo cytoskeletal reorganization, resulting in degenerative as well as regenerative changes. As neurofibrillary tangles form and dystrophic neurites appear, sprouting neuronal processes with growth cones emerge. Actin and tubulin are indispensable for normal neurite development and regenerative responses to injury and neurodegenerative stimuli. We have previously shown that actin capping protein beta2 subunit, Capzb2, binds tubulin and, in the presence of tau, affects microtubule polymerization necessary for neurite outgrowth and normal growth cone morphology. Accordingly, Capzb2 silencing in hippocampal neurons resulted in short, dystrophic neurites, seen in neurodegenerative diseases including AD. Here we demonstrate the statistically significant increase in the Capzb2 expression in the postmortem hippocampi in persons at mid-stage, Braak and Braak stage (BB III-IV, non-familial AD in comparison to controls. The dynamics of Capzb2 expression in progressive AD stages cannot be attributed to reactive astrocytosis. Moreover, the increased expression of Capzb2 mRNA in CA1 pyramidal neurons in AD BB III-IV is accompanied by an increased mRNA expression of brain derived neurotrophic factor (BDNF receptor tyrosine kinase B (TrkB, mediator of synaptic plasticity in hippocampal neurons. Thus, the up-regulation of Capzb2 and TrkB may reflect cytoskeletal reorganization and/or regenerative response occurring in hippocampal CA1 neurons at a specific stage of AD progression.

  15. Comparative genome analysis of cortactin and HS1: the significance of the F-actin binding repeat domain

    Directory of Open Access Journals (Sweden)

    Seggelen Vera

    2005-02-01

    Full Text Available Abstract 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 (Arp2/3 mediated actin polymerization. It shares a high amino acid sequence and structural similarity to hematopoietic lineage cell-specific protein 1 (HS1 although their functions differ considerable. In this manuscript we describe the genomic organization of these two genes in a variety of species by a combination of cloning and database searches. Based on our analysis, we predict the genesis of the actin-binding repeat domain during evolution. Results Cortactin homologues exist in sponges, worms, shrimps, insects, urochordates, fishes, amphibians, birds and mammalians, whereas HS1 exists in vertebrates only, suggesting that both genes have been derived from an ancestor cortactin gene by duplication. In agreement with this, comparative genome analysis revealed very similar exon-intron structures and sequence homologies, especially over the regions that encode the characteristic highly conserved F-actin-binding repeat domain. Cortactin splice variants affecting this F-actin-binding domain were identified not only in mammalians, but also in amphibians, fishes and birds. In mammalians, cortactin is ubiquitously expressed except in hematopoietic cells, whereas HS1 is mainly expressed in hematopoietic cells. In accordance with their distinct tissue specificity, the putative promoter region of cortactin is different from HS1. Conclusions Comparative analysis of the genomic organization and amino acid sequences of cortactin and HS1 provides inside into their origin and evolution. Our analysis shows that both genes originated from a gene duplication event and subsequently HS1 lost two repeats, whereas cortactin gained one repeat. Our analysis genetically underscores the significance of the F-actin binding domain in

  16. Depolymerization of cortical actin inhibits UT-A1 urea transporter endocytosis but promotes forskolin-stimulated membrane trafficking.

    Science.gov (United States)

    Xu, Gang; Su, Hua; Carter, Conner B; Fröhlich, Otto; Chen, Guangping

    2012-04-01

    The cytoskeleton participates in many aspects of transporter protein regulation. In this study, by using yeast two-hybrid screening, we identified the cytoskeletal protein actin as a binding partner with the UT-A1 urea transporter. This suggests that actin plays a role in regulating UT-A1 activity. Actin specifically binds to the carboxyl terminus of UT-A1. A serial mutation study shows that actin binding to UT-A1's carboxyl terminus was abolished when serine 918 was mutated to alanine. In polarized UT-A1-MDCK cells, cortical filamentous (F) actin colocalizes with UT-A1 at the apical membrane and the subapical cytoplasm. In the cell surface, both actin and UT-A1 are distributed in the lipid raft microdomains. Disruption of the F-actin cytoskeleton by latrunculin B resulted in UT-A1 accumulation in the cell membrane as measured by biotinylation. This effect was mainly due to inhibition of UT-A1 endocytosis in both clathrin and caveolin-mediated endocytic pathways. In contrast, actin depolymerization facilitated forskolin-stimulated UT-A1 trafficking to the cell surface. Functionally, depolymerization of actin by latrunculin B significantly increased UT-A1 urea transport activity in an oocyte expression system. Our study shows that cortical F-actin not only serves as a structural protein, but directly interacts with UT-A1 and plays an important role in controlling UT-A1 cell surface expression by affecting both endocytosis and trafficking, therefore regulating UT-A1 bioactivity.

  17. Emergence of large-scale cell morphology and movement from local actin filament growth dynamics.

    Directory of Open Access Journals (Sweden)

    Catherine I Lacayo

    2007-09-01

    Full Text Available Variations in cell migration and morphology are consequences of changes in underlying cytoskeletal organization and dynamics. We investigated how these large-scale cellular events emerge as direct consequences of small-scale cytoskeletal molecular activities. Because the properties of the actin cytoskeleton can be modulated by actin-remodeling proteins, we quantitatively examined how one such family of proteins, enabled/vasodilator-stimulated phosphoprotein (Ena/VASP, affects the migration and morphology of epithelial fish keratocytes. Keratocytes generally migrate persistently while exhibiting a characteristic smooth-edged "canoe" shape, but may also exhibit less regular morphologies and less persistent movement. When we observed that the smooth-edged canoe keratocyte morphology correlated with enrichment of Ena/VASP at the leading edge, we mislocalized and overexpressed Ena/VASP proteins and found that this led to changes in the morphology and movement persistence of cells within a population. Thus, local changes in actin filament dynamics due to Ena/VASP activity directly caused changes in cell morphology, which is coupled to the motile behavior of keratocytes. We also characterized the range of natural cell-to-cell variation within a population by using measurable morphological and behavioral features--cell shape, leading-edge shape, filamentous actin (F-actin distribution, cell speed, and directional persistence--that we have found to correlate with each other to describe a spectrum of coordinated phenotypes based on Ena/VASP enrichment at the leading edge. This spectrum stretched from smooth-edged, canoe-shaped keratocytes--which had VASP highly enriched at their leading edges and migrated fast with straight trajectories--to more irregular, rounder cells migrating slower with less directional persistence and low levels of VASP at their leading edges. We developed a mathematical model that accounts for these coordinated cell-shape and

  18. A cytoskeletal localizing domain in the cyclase-associated protein, CAP/Srv2p, regulates access to a distant SH3-binding site.

    Science.gov (United States)

    Yu, J; Wang, C; Palmieri, S J; Haarer, B K; Field, J

    1999-07-09

    In the yeast, Saccharomyces cerevisiae, adenylyl cyclase consists of a 200-kDa catalytic subunit (CYR1) and a 70-kDa subunit (CAP/SRV2). CAP/Srv2p assists the small G protein Ras to activate adenylyl cyclase. CAP also regulates the cytoskeleton through an actin sequestering activity and is directed to cortical actin patches by a proline-rich SH3-binding site (P2). In this report we analyze the role of the actin cytoskeleton in Ras/cAMP signaling. Two alleles of CAP, L16P(Srv2) and R19T (SupC), first isolated in genetic screens for mutants that attenuate cAMP levels, reduced adenylyl cyclase binding, and cortical actin patch localization. A third mutation, L27F, also failed to localize but showed no loss of either cAMP signaling or adenylyl cyclase binding. However, all three N-terminal mutations reduced CAP-CAP multimer formation and SH3 domain binding, although the SH3-binding site is about 350 amino acids away. Finally, disruption of the actin cytoskeleton with latrunculin-A did not affect the cAMP phenotypes of the hyperactive Ras2(Val19) allele. These data identify a novel region of CAP that controls access to the SH3-binding site and demonstrate that cytoskeletal localization of CAP or an intact cytoskeleton per se is not necessary for cAMP signaling.

  19. CAS-1, a C. elegans cyclase-associated protein, is required for sarcomeric actin assembly in striated muscle

    OpenAIRE

    Nomura, Kazumi; Ono, Kanako; Ono, Shoichiro

    2012-01-01

    Assembly of contractile apparatuses in striated muscle requires precisely regulated reorganization of the actin cytoskeletal proteins into sarcomeric organization. Regulation of actin filament dynamics is one of the essential processes of myofibril assembly, but the mechanism of actin regulation in striated muscle is not clearly understood. Actin depolymerizing factor (ADF)/cofilin is a key enhancer of actin filament dynamics in striated muscle in both vertebrates and nematodes. Here, we repo...

  20. 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...

  1. Increased actin polymerization and stabilization interferes with neuronal function and survival in the AMPKγ mutant Loechrig.

    Directory of Open Access Journals (Sweden)

    Mandy Cook

    Full Text Available loechrig (loe mutant flies are characterized by progressive neuronal degeneration, behavioral deficits, and early death. The mutation is due to a P-element insertion in the gene for the γ-subunit of the trimeric AMP-activated protein kinase (AMPK complex, whereby the insertion affects only one of several alternative transcripts encoding a unique neuronal isoform. AMPK is a cellular energy sensor that regulates a plethora of signaling pathways, including cholesterol and isoprenoid synthesis via its downstream target hydroxy-methylglutaryl (HMG-CoA reductase. We recently showed that loe interferes with isoprenoid synthesis and increases the prenylation and thereby activation of RhoA. During development, RhoA plays an important role in neuronal outgrowth by activating a signaling cascade that regulates actin dynamics. Here we show that the effect of loe/AMPKγ on RhoA prenylation leads to a hyperactivation of this signaling pathway, causing increased phosphorylation of the actin depolymerizating factor cofilin and accumulation of filamentous actin. Furthermore, our results show that the resulting cytoskeletal changes in loe interfere with neuronal growth and disrupt axonal integrity. Surprisingly, these phenotypes were enhanced by expressing the Slingshot (SSH phosphatase, which during development promotes actin depolymerization by dephosphorylating cofilin. However, our studies suggest that in the adult SSH promotes actin polymerization, supporting in vitro studies using human SSH1 that suggested that SSH can also stabilize and bundle filamentous actin. Together with the observed increase in SSH levels in the loe mutant, our experiments suggest that in mature neurons SSH may function as a stabilization factor for filamentous actin instead of promoting actin depolymerization.

  2. Cytoskeletal Requirements for Hepatitis C Virus (HCV) RNA Synthesis in the HCV Replicon Cell Culture System

    OpenAIRE

    Bost, Anne G.; Venable, Daryl; Liu, Lifei; Heinz, Beverly A.

    2003-01-01

    Hepatitis C virus (HCV) induces microtubule aggregates in infected hepatocytes. To determine if cytoskeletal elements are important for HCV RNA synthesis, we examined the effect of cytoskeleton inhibitors on HCV replicon transcription in Huh7 cells. The data demonstrate that HCV replication complex-mediated RNA synthesis requires microtubule and actin polymerization.

  3. Cytoskeletal requirements for hepatitis C virus (HCV) RNA synthesis in the HCV replicon cell culture system.

    Science.gov (United States)

    Bost, Anne G; Venable, Daryl; Liu, Lifei; Heinz, Beverly A

    2003-04-01

    Hepatitis C virus (HCV) induces microtubule aggregates in infected hepatocytes. To determine if cytoskeletal elements are important for HCV RNA synthesis, we examined the effect of cytoskeleton inhibitors on HCV replicon transcription in Huh7 cells. The data demonstrate that HCV replication complex-mediated RNA synthesis requires microtubule and actin polymerization.

  4. Altered cytoskeletal structures in transformed cells exhibiting obviously metastatic capabilities

    Institute of Scientific and Technical Information of China (English)

    LINZHONGXIANG; WUBINGQUAN; 等

    1990-01-01

    Cytoskeletal changes in transformed cells (LM-51) eshibiting obviously metastatic capabilities were investigated by utilization of double-fluorescent labelling through combinations of:(1) tubulin indirect immunofluorescence plus Rhodamine-phalloidin staining of F-actins;(2) indirect immunofluorescent staining with α-actinin polyclonal-and vinculin monoclonal antibodies.The LM-51 cells which showed metastatic index of >50% were derived from lung metastasis in nude mice after subcutaneous inoculation of human highly metastatic tumor DNA transfected NIH3T3 cell transformants.The parent NIH3T3 cells exhibited well-organized microtubules,prominent stress fibers and adhesion plaques while their transformants showed remarkable cytoskeletal alterations:(1)reduced microtubules but increased MTOC fluorescence;(2)disrupted stress fibers and fewer adhesion plaques with their protein components redistributed in the cytoplasm;(3)Factin-and α-actinin/vinculin aggregates appeared in the cytoplasm.These aggregates were dot-like,varied in size(0.1-0.4μm) and number,located near the ventral surface of the cells.TPA-induced actin/vinculin bodies were studied too.Indications that actin and α-actinin/vinculin redistribution might be important alterations involved in the expression of metastatic capabilities of LM-51 transformed cells were discussed.

  5. Actin filament dynamics are dominated by rapid growth and severing activity in the Arabidopsis cortical array

    OpenAIRE

    Staiger, Christopher J.; Sheahan, Michael B.; Khurana, Parul; Wang,Xia; McCurdy, David W.; Blanchoin, Laurent

    2009-01-01

    Metazoan cells harness the power of actin dynamics to create cytoskeletal arrays that stimulate protrusions and drive intracellular organelle movements. In plant cells, the actin cytoskeleton is understood to participate in cell elongation; however, a detailed description and molecular mechanism(s) underpinning filament nucleation, growth, and turnover are lacking. Here, we use variable-angle epifluorescence microscopy (VAEM) to examine the organization and dynamics of the cortical cytoskelet...

  6. Antibodies covalently immobilized on actin filaments for fast myosin driven analyte transport.

    Directory of Open Access Journals (Sweden)

    Saroj Kumar

    Full Text Available Biosensors would benefit from further miniaturization, increased detection rate and independence from external pumps and other bulky equipment. Whereas transportation systems built around molecular motors and cytoskeletal filaments hold significant promise in the latter regard, recent proof-of-principle devices based on the microtubule-kinesin motor system have not matched the speed of existing methods. An attractive solution to overcome this limitation would be the use of myosin driven propulsion of actin filaments which offers motility one order of magnitude faster than the kinesin-microtubule system. Here, we realized a necessary requirement for the use of the actomyosin system in biosensing devices, namely covalent attachment of antibodies to actin filaments using heterobifunctional cross-linkers. We also demonstrated consistent and rapid myosin II driven transport where velocity and the fraction of motile actin filaments was negligibly affected by the presence of antibody-antigen complexes at rather high density (>20 µm(-1. The results, however, also demonstrated that it was challenging to consistently achieve high density of functional antibodies along the actin filament, and optimization of the covalent coupling procedure to increase labeling density should be a major focus for future work. Despite the remaining challenges, the reported advances are important steps towards considerably faster nanoseparation than shown for previous molecular motor based devices, and enhanced miniaturization because of high bending flexibility of actin filaments.

  7. 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

  8. c-myc mRNA in cytoskeletal-bound polysomes in fibroblasts.

    Science.gov (United States)

    Hesketh, J E; Campbell, G P; Whitelaw, P F

    1991-03-01

    3T3 fibroblasts were treated sequentially with 25 mM-KCl/0.05% Nonidet P40, 130 mM-KCl/0.05% Nonidet P40 and finally with 1% Nonidet P40/1% deoxycholate in order to release free, cytoskeletal-bound and membrane-bound polysomes respectively. The membrane-bound fraction was enriched in the mRNA for the membrane protein beta 2-microglobulin, whereas the cytoskeletal-bound polysomes were enriched in c-myc mRNA. Actin mRNA was present in both free and cytoskeletal-bound polysomes. The results suggest that cytoskeletal-bound polysomes are involved in the translation of specific mRNA species.

  9. 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.

  10. 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

  11. Coiled coils and SAH domains in cytoskeletal molecular motors.

    Science.gov (United States)

    Peckham, Michelle

    2011-10-01

    Cytoskeletal motors include myosins, kinesins and dyneins. Myosins move along tracks of actin filaments, whereas kinesins and dyneins move along microtubules. Many of these motors are involved in trafficking cargo in cells. However, myosins are mostly monomeric, whereas kinesins are mostly dimeric, owing to the presence of a coiled coil. Some myosins (myosins 6, 7 and 10) contain an SAH (single α-helical) domain, which was originally thought to be a coiled coil. These myosins are now known to be monomers, not dimers. The differences between SAH domains and coiled coils are described and the potential roles of SAH domains in molecular motors are discussed.

  12. Classic 18.5- and 21.5-kDa myelin basic protein isoforms associate with cytoskeletal and SH3-domain proteins in the immortalized N19-oligodendroglial cell line stimulated by phorbol ester and IGF-1.

    Science.gov (United States)

    Smith, Graham S T; Homchaudhuri, Lopamudra; Boggs, Joan M; Harauz, George

    2012-06-01

    The 18.5-kDa classic myelin basic protein (MBP) is an intrinsically disordered protein arising from the Golli (Genes of Oligodendrocyte Lineage) gene complex and is responsible for compaction of the myelin sheath in the central nervous system. This MBP splice isoform also has a plethora of post-translational modifications including phosphorylation, deimination, methylation, and deamidation, that reduce its overall net charge and alter its protein and lipid associations within oligodendrocytes (OLGs). It was originally thought that MBP was simply a structural component of myelin; however, additional investigations have demonstrated that MBP is multi-functional, having numerous protein-protein interactions with Ca²⁺-calmodulin, actin, tubulin, and proteins with SH3-domains, and it can tether these proteins to a lipid membrane in vitro. Here, we have examined cytoskeletal interactions of classic 18.5-kDa MBP, in vivo, using early developmental N19-OLGs transfected with fluorescently-tagged MBP, actin, tubulin, and zonula occludens 1 (ZO-1). We show that MBP redistributes to distinct 'membrane-ruffled' regions of the plasma membrane where it co-localizes with actin and tubulin, and with the SH3-domain-containing proteins cortactin and ZO-1, when stimulated with PMA, a potent activator of the protein kinase C pathway. Moreover, using phospho-specific antibody staining, we show an increase in phosphorylated Thr98 MBP (human sequence numbering) in membrane-ruffled OLGs. Previously, Thr98 phosphorylation of MBP has been shown to affect its conformation, interactions with other proteins, and tethering of other proteins to the membrane in vitro. Here, MBP and actin were also co-localized in new focal adhesion contacts induced by IGF-1 stimulation in cells grown on laminin-2. This study supports a role for classic MBP isoforms in cytoskeletal and other protein-protein interactions during membrane and cytoskeletal remodeling in OLGs.

  13. Persistent nuclear actin filaments inhibit transcription by RNA polymerase II.

    Science.gov (United States)

    Serebryannyy, Leonid A; Parilla, Megan; Annibale, Paolo; Cruz, Christina M; Laster, Kyle; Gratton, Enrico; Kudryashov, Dmitri; Kosak, Steven T; Gottardi, Cara J; de Lanerolle, Primal

    2016-09-15

    Actin is abundant in the nucleus and it is clear that nuclear actin has important functions. However, mystery surrounds the absence of classical actin filaments in the nucleus. To address this question, we investigated how polymerizing nuclear actin into persistent nuclear actin filaments affected transcription by RNA polymerase II. Nuclear filaments impaired nuclear actin dynamics by polymerizing and sequestering nuclear actin. Polymerizing actin into stable nuclear filaments disrupted the interaction of actin with RNA polymerase II and correlated with impaired RNA polymerase II localization, dynamics, gene recruitment, and reduced global transcription and cell proliferation. Polymerizing and crosslinking nuclear actin in vitro similarly disrupted the actin-RNA-polymerase-II interaction and inhibited transcription. These data rationalize the general absence of stable actin filaments in mammalian somatic nuclei. They also suggest a dynamic pool of nuclear actin is required for the proper localization and activity of RNA polymerase II.

  14. An actin cytoskeleton with evolutionarily conserved functions in the absence of canonical actin-binding proteins.

    Science.gov (United States)

    Paredez, Alexander R; Assaf, Zoe June; Sept, David; Timofejeva, Ljudmilla; Dawson, Scott C; Wang, Chung-Ju Rachel; Cande, W Z

    2011-04-12

    Giardia intestinalis, a human intestinal parasite and member of what is perhaps the earliest-diverging eukaryotic lineage, contains the most divergent eukaryotic actin identified to date and is the first eukaryote known to lack all canonical actin-binding proteins (ABPs). We sought to investigate the properties and functions of the actin cytoskeleton in Giardia to determine whether Giardia actin (giActin) has reduced or conserved roles in core cellular processes. In vitro polymerization of giActin produced filaments, indicating that this divergent actin is a true filament-forming actin. We generated an anti-giActin antibody to localize giActin throughout the cell cycle. GiActin localized to the cortex, nuclei, internal axonemes, and formed C-shaped filaments along the anterior of the cell and a flagella-bundling helix. These structures were regulated with the cell cycle and in encysting cells giActin was recruited to the Golgi-like cyst wall processing vesicles. Knockdown of giActin demonstrated that giActin functions in cell morphogenesis, membrane trafficking, and cytokinesis. Additionally, Giardia contains a single G protein, giRac, which affects the Giardia actin cytoskeleton independently of known target ABPs. These results imply that there exist ancestral and perhaps conserved roles for actin in core cellular processes that are independent of canonical ABPs. Of medical significance, the divergent giActin cytoskeleton is essential and commonly used actin-disrupting drugs do not depolymerize giActin structures. Therefore, the giActin cytoskeleton is a promising drug target for treating giardiasis, as we predict drugs that interfere with the Giardia actin cytoskeleton will not affect the mammalian host.

  15. Cryptococcus neoformans Is Internalized by Receptor-Mediated or ‘Triggered’ Phagocytosis, Dependent on Actin Recruitment

    Science.gov (United States)

    Guerra, Caroline Rezende; Seabra, Sergio Henrique; de Souza, Wanderley; Rozental, Sonia

    2014-01-01

    Cryptococcosis by the encapsulated yeast Cryptococcus neoformans affects mostly immunocompromised individuals and is a frequent neurological complication in AIDS patients. Recent studies support the idea that intracellular survival of Cryptococcus yeast cells is important for the pathogenesis of cryptococcosis. However, the initial steps of Cryptococcus internalization by host cells remain poorly understood. Here, we investigate the mechanism of Cryptococcus neoformans phagocytosis by peritoneal macrophages using confocal and electron microscopy techniques, as well as flow cytometry quantification, evaluating the importance of fungal capsule production and of host cell cytoskeletal elements for fungal phagocytosis. Electron microscopy analyses revealed that capsular and acapsular strains of C. neoformans are internalized by macrophages via both ‘zipper’ (receptor-mediated) and ‘trigger’ (membrane ruffle-dependent) phagocytosis mechanisms. Actin filaments surrounded phagosomes of capsular and acapsular yeasts, and the actin depolymerizing drugs cytochalasin D and latrunculin B inhibited yeast internalization and actin recruitment to the phagosome area. In contrast, nocodazole and paclitaxel, inhibitors of microtubule dynamics decreased internalization but did not prevent actin recruitment to the site of phagocytosis. Our results show that different uptake mechanisms, dependent on both actin and tubulin dynamics occur during yeast internalization by macrophages, and that capsule production does not affect the mode of Cryptococcus uptake by host cells. PMID:24586631

  16. 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.

  17. Nucleus-associated actin in Amoeba proteus.

    Science.gov (United States)

    Berdieva, Mariia; Bogolyubov, Dmitry; Podlipaeva, Yuliya; Goodkov, Andrew

    2016-10-01

    The presence, spatial distribution and forms of intranuclear and nucleus-associated cytoplasmic actin were studied in Amoeba proteus with immunocytochemical approaches. Labeling with different anti-actin antibodies and staining with TRITC-phalloidin and fluorescent deoxyribonuclease I were used. We showed that actin is abundant within the nucleus as well as in the cytoplasm of A. proteus cells. According to DNase I experiments, the predominant form of intranuclear actin is G-actin which is associated with chromatin strands. Besides, unpolymerized actin was shown to participate in organization of a prominent actin layer adjacent to the outer surface of nuclear envelope. No significant amount of F-actin was found in the nucleus. At the same time, the amoeba nucleus is enclosed in a basket-like structure formed by circumnuclear actin filaments and bundles connected with global cytoplasmic actin cytoskeleton. A supposed architectural function of actin filaments was studied by treatment with actin-depolymerizing agent latrunculin A. It disassembled the circumnuclear actin system, but did not affect the intranuclear chromatin structure. The results obtained for amoeba cells support the modern concept that actin is involved in fundamental nuclear processes that have evolved in the cells of multicellular organisms.

  18. Oocyte and embryonic cytoskeletal defects caused by mutations in the Drosophila swallow gene.

    Science.gov (United States)

    Meng, Jing; Stephenson, Edwin C

    2002-06-01

    The maternal effect gene swallow ( swa) of Drosophila is required for bicoid and htsN4 mRNA localization during oogenesis. Swallow is also required for additional, poorly understood, functions in early embryogenesis. We have examined the cytoskeleton in swa mutant oocytes and embryos by immunocytochemistry and confocal microscopy. Mid- and late-stage swaoocytes have defective cytoplasmic actin networks. Stage-10 oocytes have solid actin clumps and hollow actin spheres in the subcortical layer, and late-stage oocytes have uniformly distributed hollow actin spheres in the subcortical layer and in deeper cytoplasm. Swa preblastoderm embryos have uneven and irregularly distributed actin at the cortex, and defective subcortical actin networks that contain hollow and solid spheres. In swa syncytial blastoderm embryos, the abnormal actin cytoskeleton is associated with defects in nuclear distribution, migration and cleavage. Actin cytoskeletal defects correlate with spindle defects, suggesting that the abnormal organization of the actin cytoskeleton allows interaction of mitotic spindles, which induces defective nuclear divisions and loss of nuclei from the surface of the embryo.

  19. Cytosolic Proteins From Tobacco Pollen Tubes That Crosslink Microtubules and Actin Filaments In Vitro Are Metabolic Enzymes

    NARCIS (Netherlands)

    Romagnoli, Silvia; Faleri, Claudia; Bini, Luca; Baskin, Tobias I.; Cresti, Mauro

    2010-01-01

    In plant cells, many processes require cooperative action of both microtubules and actin filaments, but proteins mediating interactions between these cytoskeletal members are mostly undiscovered. Here, we attempt to identify such proteins by affinity purification. Cytosol from Nicotiana tabacum (tob

  20. Interplay between cytoskeletal polymerization and the chondrogenic phenotype in chondrocytes passaged in monolayer culture.

    Science.gov (United States)

    Parreno, Justin; Nabavi Niaki, Mortah; Andrejevic, Katarina; Jiang, Amy; Wu, Po-Han; Kandel, Rita A

    2017-02-01

    Tubulin and actin exist as monomeric units that polymerize to form either microtubules or filamentous actin. As the polymerization status (monomeric/polymeric ratio) of tubulin and/or actin have been shown to be important in regulating gene expression and phenotype in non-chondrocyte cells, the objective of this study was to examine the role of cytoskeletal polymerization on the chondrocyte phenotype. We hypothesized that actin and/or tubulin polymerization status modulates the chondrocyte phenotype during monolayer culture as well as in 3D culture during redifferentiation. To test this hypothesis, articular chondrocytes were grown and passaged in 2D monolayer culture. Cell phenotype was investigated by assessing cell morphology (area and circularity), actin/tubulin content, organization and polymerization status, as well as by determination of proliferation, fibroblast and cartilage matrix gene expression with passage number. Bovine chondrocytes became larger, more elongated, and had significantly (P  0.05) modulated, actin polymerization was increased in bovine P2 cells. Actin depolymerization, but not tubulin depolymerization, promoted the chondrocyte phenotype by inducing cell rounding, increasing aggrecan and reducing COL1 expression. Knockdown of actin depolymerization factor, cofilin, in these cells induced further P2 cell actin polymerization and increased COL1 gene expression. To confirm that actin status regulated COL1 gene expression in human P2 chondrocytes, human P2 chondrocytes were exposed to cytochalasin D. Cytochalasin D decreased COL1 gene expression in human passaged chondrocytes. Furthermore, culture of bovine P2 chondrocytes in 3D culture on porous bone substitute resulted in actin depolymerization, which correlated with decreased expression of COL1 and proliferation molecules. In 3D cultures, aggrecan gene expression was increased by cytochalasin D treatment and COL1 was further decreased. These results reveal that actin polymerization

  1. G-actin regulates rapid induction of actin nucleation by mDia1 to restore cellular actin polymers.

    Science.gov (United States)

    Higashida, Chiharu; Suetsugu, Shiro; Tsuji, Takahiro; Monypenny, James; Narumiya, Shuh; Watanabe, Naoki

    2008-10-15

    mDia1 belongs to the formin family of proteins that share FH1 and FH2 domains. Although formins play a critical role in the formation of many actin-based cellular structures, the physiological regulation of formin-mediated actin assembly within the cell is still unknown. Here we show that cells possess an acute actin polymer restoration mechanism involving mDia1. By using single-molecule live-cell imaging, we found that several treatments including low-dose G-actin-sequestering drugs and unpolymerizable actin mutants activate mDia1 to initiate fast directional movement. The FH2 region, the core domain for actin nucleation, is sufficient to respond to latrunculin B (LatB) to increase its actin nucleation frequency. Simulation analysis revealed an unexpected paradoxical effect of LatB that leads to a several fold increase in free G-actin along with an increase in total G-actin. These results indicate that in cells, the actin nucleation frequency of mDia1 is enhanced not only by Rho, but also strongly through increased catalytic efficiency of the FH2 domain. Consistently, frequent actin nucleation by mDia1 was found around sites of vigorous actin disassembly. Another major actin nucleator, the Arp2/3 complex, was not affected by the G-actin increase induced by LatB. Taken together, we propose that transient accumulation of G-actin works as a cue to promote mDia1-catalyzed actin nucleation to execute rapid reassembly of actin filaments.

  2. Identification and characterization of a set of conserved and new regulators of cytoskeletal organization, cell morphology and migration

    Directory of Open Access Journals (Sweden)

    Suryavanshi Narendra

    2011-08-01

    Full Text Available Abstract Background Cell migration is essential during development and in human disease progression including cancer. Most cell migration studies concentrate on known or predicted components of migration pathways. Results Here we use data from a genome-wide RNAi morphology screen in Drosophila melanogaster cells together with bioinformatics to identify 26 new regulators of morphology and cytoskeletal organization in human cells. These include genes previously implicated in a wide range of functions, from mental retardation, Down syndrome and Huntington's disease to RNA and DNA-binding genes. We classify these genes into seven groups according to phenotype and identify those that affect cell migration. We further characterize a subset of seven genes, FAM40A, FAM40B, ARC, FMNL3, FNBP3/FBP11, LIMD1 and ZRANB1, each of which has a different effect on cell shape, actin filament distribution and cell migration. Interestingly, in several instances closely related isoforms with a single Drosophila homologue have distinct phenotypes. For example, FAM40B depletion induces cell elongation and tail retraction defects, whereas FAM40A depletion reduces cell spreading. Conclusions Our results identify multiple regulators of cell migration and cytoskeletal signalling that are highly conserved between Drosophila and humans, and show that closely related paralogues can have very different functions in these processes.

  3. Cytoskeletal abnormalities in relation with meiotic competence and ageing in porcine and bovine oocytes during in vitro maturation.

    Science.gov (United States)

    Somfai, T; Kikuchi, K; Kaneda, M; Akagi, S; Watanabe, S; Mizutani, E; Haraguchi, S; Dang-Nguyen, T Q; Inaba, Y; Geshi, M; Nagai, T

    2011-10-01

    We investigated the frequencies of cytoskeletal anomalies in metaphase-II (M-II) and incompetent [arrested at an immature metaphase (IM) stage] porcine and bovine oocytes during in vitro maturation (IVM) in relation with ageing by immunostaining and confocal microscopy. In porcine oocytes, meiotic arrest at the IM stage was associated with abnormalities of cortical actin but not with abnormal spindles. Prolongation of IVM culture to 52 h did not affect microfilament and spindle abnormalities, but reduced the microfilament-rich area overlaying the spindle. Meiotic arrest of bovine oocytes at the IM stage was associated with degenerations of microfilaments, and the frequencies of abnormal spindles were also higher than those of M-II oocytes. Ageing of bovine oocytes (IVM for 30 h) did not affect cortical microfilaments but increased the frequency of spindle alterations in both M-II and IM bovine oocytes. These results suggest that, in both species, altered ability of oocytes to polymerize F-actin might be a possible reason for the failure of polar body extrusion during IVM. Also, there seem to be differences between the two species in the sensitivity of oocytes to suffer ageing-related spindle damages.

  4. Impact of actin filament stabilization on adult hippocampal and olfactory bulb neurogenesis.

    Science.gov (United States)

    Kronenberg, Golo; Gertz, Karen; Baldinger, Tina; Kirste, Imke; Eckart, Sarah; Yildirim, Ferah; Ji, Shengbo; Heuser, Isabella; Schröck, Helmut; Hörtnagl, Heide; Sohr, Reinhard; Djoufack, Pierre Chryso; Jüttner, René; Glass, Rainer; Przesdzing, Ingo; Kumar, Jitender; Freyer, Dorette; Hellweg, Rainer; Kettenmann, Helmut; Fink, Klaus Benno; Endres, Matthias

    2010-03-03

    Rearrangement of the actin cytoskeleton is essential for dynamic cellular processes. Decreased actin turnover and rigidity of cytoskeletal structures have been associated with aging and cell death. Gelsolin is a Ca(2+)-activated actin-severing protein that is widely expressed throughout the adult mammalian brain. Here, we used gelsolin-deficient (Gsn(-/-)) mice as a model system for actin filament stabilization. In Gsn(-/-) mice, emigration of newly generated cells from the subventricular zone into the olfactory bulb was slowed. In vitro, gelsolin deficiency did not affect proliferation or neuronal differentiation of adult neural progenitors cells (NPCs) but resulted in retarded migration. Surprisingly, hippocampal neurogenesis was robustly induced by gelsolin deficiency. The ability of NPCs to intrinsically sense excitatory activity and thereby implement coupling between network activity and neurogenesis has recently been established. Depolarization-induced [Ca(2+)](i) increases and exocytotic neurotransmitter release were enhanced in Gsn(-/-) synaptosomes. Importantly, treatment of Gsn(-/-) synaptosomes with mycotoxin cytochalasin D, which, like gelsolin, produces actin disassembly, decreased enhanced Ca(2+) influx and subsequent exocytotic norepinephrine release to wild-type levels. Similarly, depolarization-induced glutamate release from Gsn(-/-) brain slices was increased. Furthermore, increased hippocampal neurogenesis in Gsn(-/-) mice was associated with a special microenvironment characterized by enhanced density of perfused vessels, increased regional cerebral blood flow, and increased endothelial nitric oxide synthase (NOS-III) expression in hippocampus. Together, reduced filamentous actin turnover in presynaptic terminals causes increased Ca(2+) influx and, subsequently, elevated exocytotic neurotransmitter release acting on neural progenitors. Increased neurogenesis in Gsn(-/-) hippocampus is associated with a special vascular niche for neurogenesis.

  5. Distribution of cytoskeletal proteins, integrins, leukocyte adhesion molecules and extracellular matrix proteins in plastic-embedded human and rat kidneys

    NARCIS (Netherlands)

    van Goor, H; Coers, W; van der Horst, MLC; Suurmeijer, AJH

    2001-01-01

    OBJECTIVE: To study the distribution of cytoskeletal proteins (actin, alpha -actinin, vinculin, beta -tubulin, keratin, vimentin, desmin), adhesion molecules for cell-matrix interations (very later antigens [VLA1-6], beta1, beta2 [CD18], vitronectin receptor [alphav beta3], CD 11b), leukocyte adhesi

  6. 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.

  7. On the significance of microtubule flexural behavior in cytoskeletal mechanics.

    Directory of Open Access Journals (Sweden)

    Mehrdad Mehrbod

    Full Text Available Quantitative description of cell mechanics has challenged biological scientists for the past two decades. Various structural models have been attempted to analyze the structure of the cytoskeleton. One important aspect that has been largely ignored in all these modeling approaches is related to the flexural and buckling behavior of microtubular filaments. The objective of this paper is to explore the influence of this flexural and buckling behavior in cytoskeletal mechanics.In vitro the microtubules are observed to buckle in the first mode, reminiscent of a free, simply-supported beam. In vivo images of microtubules, however, indicate that the buckling mostly occurs in higher modes. This buckling mode switch takes place mostly because of the lateral support of microtubules via their connections to actin and intermediate filaments. These lateral loads are exerted throughout the microtubule length and yield a considerable bending behavior that, unless properly accounted for, would produce erroneous results in the modeling and analysis of the cytoskeletal mechanics.One of the promising attempts towards mechanical modeling of the cytoskeleton is the tensegrity model, which simplifies the complex network of cytoskeletal filaments into a combination merely of tension-bearing actin filaments and compression-bearing microtubules. Interestingly, this discrete model can qualitatively explain many experimental observations in cell mechanics. However, evidence suggests that the simplicity of this model may undermine the accuracy of its predictions, given the model's underlying assumption that "every single member bears solely either tensile or compressive behavior," i.e. neglecting the flexural behavior of the microtubule filaments. We invoke an anisotropic continuum model for microtubules and compare the bending energy stored in a single microtubule with its axial strain energy at the verge of buckling. Our results suggest that the bending energy can

  8. Actinic cheilitis: A review

    Directory of Open Access Journals (Sweden)

    Elangovan Somasundaram

    2015-01-01

    Full Text Available Actinic cheilitis (AC is a chronic inflammatory disorder of the lips that is caused by prolonged exposure to sunlight in susceptible individuals. It affects the vermilion region of the lower lip almost exclusively. UV-B rays with a wavelength of 290-320 nm are held responsible for the sunlight-induced damage. The exact mechanism of the development of AC is unclear. It is considered to be potentially malignant.

  9. 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.

  10. 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.

  11. FIBROBLAST CYTOSKELETAL REMODELING CONTRIBUTES TO CONNECTIVE TISSUE TENSION

    Science.gov (United States)

    Langevin, Helene M.; Bouffard, Nicole A.; Fox, James R.; Palmer, Bradley M.; Wu, Junru; Iatridis, James C.; Barnes, William D.; Badger, Gary J.; Howe, Alan K.

    2011-01-01

    The viscoelastic behavior of connective tissue is generally attributed to the material properties of the extracellular matrix rather than cellular activity. We have previously shown that fibroblasts within areolar connective tissue exhibit dynamic cytoskeletal remodeling within minutes in response to tissue stretch ex vivo and in vivo. Here, we tested the hypothesis that fibroblasts, through this cytoskeletal remodeling, actively contribute to the viscoelastic behavior of the whole tissue. We measured significantly increased tissue tension when cellular function was broadly inhibited by sodium azide and when cytoskeletal dynamics were compromised by disrupting microtubules (with colchicine) or actomyosin contractility (via Rho kinase inhibition). These treatments led to a decrease in cell body cross-sectional area and cell field perimeter (obtained by joining the end of all of a fibroblast’s processes). Suppressing lamellipodia formation by inhibiting Rac-1 decreased cell body cross-sectional area but did not affect cell field perimeter or tissue tension. Thus, by changing shape, fibroblasts can dynamically modulate the viscoelastic behavior of areolar connective tissue through Rho-dependent cytoskeletal mechanisms. These results have broad implications for our understanding of the dynamic interplay of forces between fibroblasts and their surrounding matrix, as well as for the neural, vascular and immune cell populations residing within connective tissue. PMID:20945345

  12. Fibroblast cytoskeletal remodeling contributes to connective tissue tension.

    Science.gov (United States)

    Langevin, Helene M; Bouffard, Nicole A; Fox, James R; Palmer, Bradley M; Wu, Junru; Iatridis, James C; Barnes, William D; Badger, Gary J; Howe, Alan K

    2011-05-01

    The visco-elastic behavior of connective tissue is generally attributed to the material properties of the extracellular matrix rather than cellular activity. We have previously shown that fibroblasts within areolar connective tissue exhibit dynamic cytoskeletal remodeling within minutes in response to tissue stretch ex vivo and in vivo. Here, we tested the hypothesis that fibroblasts, through this cytoskeletal remodeling, actively contribute to the visco-elastic behavior of the whole tissue. We measured significantly increased tissue tension when cellular function was broadly inhibited by sodium azide and when cytoskeletal dynamics were compromised by disrupting microtubules (with colchicine) or actomyosin contractility (via Rho kinase inhibition). These treatments led to a decrease in cell body cross-sectional area and cell field perimeter (obtained by joining the end of all of a fibroblast's processes). Suppressing lamellipodia formation by inhibiting Rac-1 decreased cell body cross-sectional area but did not affect cell field perimeter or tissue tension. Thus, by changing shape, fibroblasts can dynamically modulate the visco-elastic behavior of areolar connective tissue through Rho-dependent cytoskeletal mechanisms. These results have broad implications for our understanding of the dynamic interplay of forces between fibroblasts and their surrounding matrix, as well as for the neural, vascular, and immune cell populations residing within connective tissue.

  13. Nuclear Actin in Development and Transcriptional Reprogramming.

    Science.gov (United States)

    Misu, Shinji; Takebayashi, Marina; Miyamoto, Kei

    2017-01-01

    Actin is a highly abundant protein in eukaryotic cells and dynamically changes its polymerized states with the help of actin-binding proteins. Its critical function as a constituent of cytoskeleton has been well-documented. Growing evidence demonstrates that actin is also present in nuclei, referred to as nuclear actin, and is involved in a number of nuclear processes, including transcriptional regulation and chromatin remodeling. The contribution of nuclear actin to transcriptional regulation can be explained by its direct interaction with transcription machineries and chromatin remodeling factors and by controlling the activities of transcription factors. In both cases, polymerized states of nuclear actin affect the transcriptional outcome. Nuclear actin also plays an important role in activating strongly silenced genes in somatic cells for transcriptional reprogramming. When these nuclear functions of actin are considered, it is plausible to speculate that nuclear actin is also implicated in embryonic development, in which numerous genes need to be activated in a well-coordinated manner. In this review, we especially focus on nuclear actin's roles in transcriptional activation, reprogramming and development, including stem cell differentiation and we discuss how nuclear actin can be an important player in development and cell differentiation.

  14. [Photodynamic therapy for actinic cheilitis].

    Science.gov (United States)

    Castaño, E; Comunión, A; Arias, D; Miñano, R; Romero, A; Borbujo, J

    2009-12-01

    Actinic cheilitis is a subtype of actinic keratosis that mainly affects the lower lip and has a higher risk of malignant transformation. Its location on the labial mucosa influences the therapeutic approach. Vermilionectomy requires local or general anesthetic and is associated with a risk of an unsightly scar, and the treatment with 5-fluorouracil or imiquimod lasts for several weeks and the inflammatory reaction can be very intense. A number of authors have used photodynamic therapy as an alternative to the usual treatments. We present 3 patients with histologically confirmed actinic cheilitis treated using photodynamic therapy with methyl aminolevulinic acid as the photosensitizer and red light at 630 nm. The clinical response was good, with no recurrences after 3 to 6 months of follow-up. Our experience supports the use of photodynamic therapy as a good alternative for the treatment of actinic cheilitis.

  15. Nonlinear electrodynamics in cytoskeletal protein lattices

    Energy Technology Data Exchange (ETDEWEB)

    Hameroff, S.R.; Smith, S.A.; Watt, R.C.

    1983-01-01

    Cytoskeletal lattice proteins including microtubules are particularly involved in dynamic regulation of intracellular movements and activities. This paper considers possibilities and implications of biological information processing due to coupling of Davydov solitons, Frohlich coherent oscillations and other nonlinear electrodynamic phenomena to conformational states of the grid-like polymer subunits of cytoskeletal microtubules. 39 references.

  16. Rho GTPase-formin pairs in cytoskeletal remodelling.

    Science.gov (United States)

    Eisenmann, Kathryn M; Peng, Jun; Wallar, Bradley J; Alberts, Arthur S

    2005-01-01

    Diaphanous-related formins (Drfs) are members of a conserved formin family of actin-nucleating proteins and are thought to act as Rho GTPase effectors in signal transduction pathways that govern gene expression, cytoskeletal remodelling and cell division. In vitro evidence suggests that the three mammalian Drf proteins--mDia1, mDia2 and mDia3-have distinct GTPase-binding specificities. However, much of our current understanding of GTPase-Drf partnerships in mammalian cell signalling is based on expression studies using Drfs missing their unique GTPase-binding domains. We have employed fluorescence resonance energy transfer (FRET) and gene targeting approaches to identify the function of different GTPase-formin pairs in cell signalling. These studies have allowed us to uncover new roles for Drf proteins in cytoskeletal remodelling and novel regulatory mechanisms whereby GTPases influence formin function. Our genetic experiments strongly suggest that Drfs cooperate with other GTPase effector proteins, including the gene product of the Wiskott-Aldrich syndrome gene, WASP, during the regulation of cell proliferation. Further, the Drf gene knockout experiments indicate that this family of formins has a role in cancer pathophysiology.

  17. Reorganization of cytoskeletal proteins of mouse oocytes mediated by integrins

    Institute of Scientific and Technical Information of China (English)

    YUE Limin; ZHANG Lei; HE Yaping; ZHANG Jinhu; ZHENG Jie; HE Yanfang; ZHENG Yu; ZHANG Jie; ZHANG Li

    2004-01-01

    To study whether integrins on cell membrane ligate with intracellular cytoskeletal proteins and mediate their reorganization in egg activation, female mice were used for superovulation. The zona-free oocytes were incubated separately with specific ligand of integrins,an active RGD peptide, in vitro for certain period of time. RGE peptide and mouse capacitated sperm were used as controls. Freshly ovulated oocytes and those treated with different factors were immunostained with FITC-labeled anti-actin antibody, then detected with confocal microscope. The results demonstrated that freshly ovulated mouse oocytes, oocytes incubated for 2 h in vitro and those treated with control RGE peptide for 15 min showed hardly visible fluorescene or only thin fluorescence in plasma membrane region. Oocytes coincubated with sperms for 15 min and those treated with active RGD peptide for 10 min, 30 min and 2 hours respectively had strong and thick fluorescence in the plasma membrane and cortical region of oocytes, and some of them showed asymmetrically fluorescent distribution. It is proved that integrins on membrane are ligated directly with cytoskeletal protein. Integrins binding with their ligands regulate reorganization of cytoskelal protein, which may be involved in transmembrane signaling in egg activation.

  18. Decavanadate interactions with actin: inhibition of G-actin polymerization and stabilization of decameric vanadate.

    Science.gov (United States)

    Ramos, Susana; Manuel, Miguel; Tiago, Teresa; Duarte, Rui; Martins, Jorge; Gutiérrez-Merino, Carlos; Moura, José J G; Aureliano, Manuel

    2006-11-01

    Decameric vanadate species (V10) inhibit the rate and the extent of G-actin polymerization with an IC50 of 68+/-22 microM and 17+/-2 microM, respectively, whilst they induce F-actin depolymerization at a lower extent. On contrary, no effect on actin polymerization and depolymerization was detected for 2mM concentration of "metavanadate" solution that contains ortho and metavanadate species, as observed by combining kinetic with (51)V NMR spectroscopy studies. Although at 25 degrees C, decameric vanadate (10 microM) is unstable in the assay medium, and decomposes following a first-order kinetic, in the presence of G-actin (up to 8 microM), the half-life increases 5-fold (from 5 to 27 h). However, the addition of ATP (0.2mM) in the medium not only prevents the inhibition of G-actin polymerization by V10 but it also decreases the half-life of decomposition of decameric vanadate species from 27 to 10h. Decameric vanadate is also stabilized by the sarcoplasmic reticulum vesicles, which raise the half-life time from 5 to 18h whereas no effects were observed in the presence of phosphatidylcholine liposomes, myosin or G-actin alone. It is proposed that the "decavanadate" interaction with G-actin, favored by the G-actin polymerization, stabilizes decameric vanadate species and induces inhibition of G-actin polymerization. Decameric vanadate stabilization by cytoskeletal and transmembrane proteins can account, at least in part, for decavanadate toxicity reported in the evaluation of vanadium (V) effects in biological systems.

  19. Lamin A/C and emerin regulate MKL1/SRF activity by modulating actin dynamics

    Science.gov (United States)

    Ho, Chin Yee; Jaalouk, Diana E.; Vartiainen, Maria K.; Lammerding, Jan

    2013-01-01

    Laminopathies, caused by mutations in the LMNA gene encoding the nuclear envelope proteins lamins A and C, represent a diverse group of diseases that include Emery-Dreifuss Muscular Dystrophy (EDMD), dilated cardiomyopathy (DCM), limb-girdle muscular dystrophy, and Hutchison-Gilford progeria syndrome (HGPS).1 The majority of LMNA mutations affect skeletal and cardiac muscle by mechanisms that remain incompletely understood. Loss of structural function and disturbed interaction of mutant lamins with (tissue-specific) transcription factors have been proposed to explain the tissue-specific phenotypes.1 We report here that lamin A/C-deficient (Lmna−/−) and Lmna N195K mutant cells have impaired nuclear translocation and downstream signaling of the mechanosensitive transcription factor megakaryoblastic leukaemia 1 (MKL1), a myocardin family member that is pivotal in cardiac development and function.2 Disturbed nucleo-cytoplasmic shuttling of MKL1 was caused by altered actin dynamics in Lmna−/− and N195K mutant cells. Ectopic expression of the nuclear envelope protein emerin, which is mislocalized in Lmna mutant cells and also linked to EDMD and DCM, restored MKL1 nuclear translocation and rescued actin dynamics in mutant cells. These findings present a novel mechanism that could provide insight into the disease etiology for the cardiac phenotype in many laminopathies, whereby lamins A/C and emerin regulate gene expression through modulation of nuclear and cytoskeletal actin polymerization. PMID:23644458

  20. Lamin A/C and emerin regulate MKL1-SRF activity by modulating actin dynamics.

    Science.gov (United States)

    Ho, Chin Yee; Jaalouk, Diana E; Vartiainen, Maria K; Lammerding, Jan

    2013-05-23

    Laminopathies, caused by mutations in the LMNA gene encoding the nuclear envelope proteins lamins A and C, represent a diverse group of diseases that include Emery-Dreifuss muscular dystrophy (EDMD), dilated cardiomyopathy (DCM), limb-girdle muscular dystrophy, and Hutchison-Gilford progeria syndrome. Most LMNA mutations affect skeletal and cardiac muscle by mechanisms that remain incompletely understood. Loss of structural function and altered interaction of mutant lamins with (tissue-specific) transcription factors have been proposed to explain the tissue-specific phenotypes. Here we report in mice that lamin-A/C-deficient (Lmna(-/-)) and Lmna(N195K/N195K) mutant cells have impaired nuclear translocation and downstream signalling of the mechanosensitive transcription factor megakaryoblastic leukaemia 1 (MKL1), a myocardin family member that is pivotal in cardiac development and function. Altered nucleo-cytoplasmic shuttling of MKL1 was caused by altered actin dynamics in Lmna(-/-) and Lmna(N195K/N195K) mutant cells. Ectopic expression of the nuclear envelope protein emerin, which is mislocalized in Lmna mutant cells and also linked to EDMD and DCM, restored MKL1 nuclear translocation and rescued actin dynamics in mutant cells. These findings present a novel mechanism that could provide insight into the disease aetiology for the cardiac phenotype in many laminopathies, whereby lamin A/C and emerin regulate gene expression through modulation of nuclear and cytoskeletal actin polymerization.

  1. Immunohistochemical observation of actin filaments in epithelial cells encircling the taste pore cavity of rat fungiform papillae

    OpenAIRE

    Y Shiba; Uchida, T.; K Wakida; Komiyama, S; Ohishi, Y.

    2009-01-01

    Epithelial cells are connected to each other around taste pores in rat fungiform papillae. Cytoskeletal components are responsible for the maintenance of intracellular adhesion, and we investigated the identification and localization of actin filaments around taste pores. On the basis of observations made by immunohistochemical transmission electron microscopy comparing with confocal laser scanning microscopy using actin-lectin double staining, actin filaments were found to be localized, enci...

  2. Fluorescence staining of the actin cytoskeleton in living cells with 7-nitrobenz-2-oxa-1,3-diazole-phallacidin.

    OpenAIRE

    Barak, L S; Yocum, R R; Nothnagel, E A; Webb, W W

    1980-01-01

    An active fluorescent derivative of the actin-binding mushroom toxin phallacidin has been synthesized. Convenient methods were developed to stain actin cytoskeletal structures in living and fixed cultured animal cells and actively streaming algal cells. Actin binding specificity was demonstrated by competitive binding experiments and comparative staining of well-known structures. Large populations of living animal cells in culture were readily stained by using a relatively mild lysolecithin p...

  3. A Model for Stress Fiber Realignment Caused by Cytoskeletal Fluidization During Cyclic Stretching.

    Science.gov (United States)

    Pirentis, Athanassios P; Peruski, Elizabeth; Iordan, Andreea L; Stamenović, Dimitrije

    2011-03-01

    Uniaxial cyclic substrate stretching results in a concerted change of cytoskeletal organization such that stress fibers (SFs) realign away from the direction of stretching. Recent experiments revealed that brief transient stretch promptly ablates cellular contractile stress by means of cytoskeletal fluidization, followed by a slow stress recovery by means of resolidification. This, in turn, suggests that fluidization, resolidification and SF realignment may be linked together during stretching. We propose a mathematical model that simulates the effects of fluidization and resolidification on cytoskeletal contractile stress in order to investigate how these phenomena affect cytoskeletal realignment in response to pure uniaxial stretching of the substrate. The model comprises of individual elastic SFs anchored at the endpoints to an elastic substrate. Employing the global stability convention, the model predicts that in response to repeated stretch-unstretch cycles, SFs tend to realign in the direction perpendicular to stretching, consistent with data from the literature. The model is used to develop a computational scheme for predicting changes in cell orientation and polarity during stretching and how they relate to the underlying alterations in the cytoskeletal organization. We conclude that depletion of cytoskeletal contractile stress by means of fluidization and subsequent stress recovery by means of resolidification may play a key role in reorganization of cytoskeletal SFs in response to uniaxial stretching of the substrate.

  4. 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.

  5. 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...

  6. Implications of oxidovanadium(IV) binding to actin.

    Science.gov (United States)

    Ramos, Susana; Almeida, Rui M; Moura, José J G; Aureliano, Manuel

    2011-06-01

    Oxidovanadium(IV), a cationic species (VO(2+)) of vanadium(IV), binds to several proteins, including actin. Upon titration with oxidovanadium(IV), approximately 100% quenching of the intrinsic fluorescence of monomeric actin purified from rabbit skeletal muscle (G-actin) was observed, with a V(50) of 131 μM, whereas for the polymerized form of actin (F-actin) 75% of quenching was obtained and a V(50) value of 320 μM. Stern-Volmer plots were used to estimate an oxidovanadium(IV)-actin dissociation constant, with K(d) of 8.2 μM and 64.1 μM VOSO(4), for G-actin and F-actin, respectively. These studies reveal the presence of a high affinity binding site for oxidovanadium(IV) in actin, producing local conformational changes near the tryptophans most accessible to water in the three-dimensional structure of actin. The actin conformational changes, also confirmed by (1)H NMR, are accompanied by changes in G-actin hydrophobic surface, but not in F-actin. The (1)H NMR spectra of G-actin treated with oxidovanadium(IV) clearly indicates changes in the resonances ascribed to methyl group and aliphatic regions as well as to aromatics and peptide-bond amide region. In parallel, it was verified that oxidovanadium(IV) prevents the G-actin polymerization into F-actin. In the 0-200 μM range, VOSO(4) inhibits 40% of the extent of polymerization with an IC(50) of 15.1 μM, whereas 500 μM VOSO(4) totally suppresses actin polymerization. The data strongly suggest that oxidovanadium(IV) binds to actin at specific binding sites preventing actin polymerization. By affecting actin structure and function, oxidovanadium(IV) might be responsible for many cellular effects described for vanadium.

  7. The nucleotide receptor P2X7 mediates actin reorganization and membrane blebbing in RAW 264.7 macrophages via p38 MAP kinase and Rho.

    Science.gov (United States)

    Pfeiffer, Zachary A; Aga, Mini; Prabhu, Usha; Watters, Jyoti J; Hall, David J; Bertics, Paul J

    2004-06-01

    Extracellular nucleotides regulate macrophage function via P2X nucleotide receptors that form ligand-gated ion channels. In particular, P2X7 activation is characterized by pore formation, membrane blebbing, and cytokine release. P2X7 is also linked to mitogen-activated protein kinases (MAPK) and Rho-dependent pathways, which are known to affect cytoskeletal structure in other systems. As cytoskeletal function is critical for macrophage behavior, we have tested the importance of these pathways in actin filament reorganization during P2X7 stimulation in RAW 264.7 macrophages. We observed that the P2X7 agonists adenosine 5'-triphosphate (ATP) and 3'-O-(4-benzoylbenzoyl) ATP (BzATP) stimulated actin reorganization and concomitant membrane blebbing within 5 min. Disruption of actin filaments with cytochalasin D attenuated membrane blebbing but not P2X7-dependent pore formation or extracellular-regulated kinase (ERK)1/ERK2 and p38 activation, suggesting that these latter processes do not require intact actin filaments. However, we provide evidence that p38 MAPK and Rho activation but not ERK1/ERK2 activation is important for P2X7-mediated actin reorganization and membrane blebbing. First, activation of p38 and Rho was detected within 5 min of BzATP treatment, which is coincident with membrane blebbing. Second, the p38 inhibitors SB202190 and SB203580 reduced nucleotide-induced blebbing and actin reorganization, whereas the MAPK kinase-1/2 inhibitor U0126, which blocks ERK1/ERK2 activation, had no discernable effect. Third, the Rho-selective inhibitor C3 exoenzyme and the Rho effector kinase, Rho-associated coiled-coil kinase, inhibitor Y-27632, markedly attenuated BzATP-stimulated actin reorganization and membrane blebbing. These data support a model wherein p38- and Rho-dependent pathways are critical for P2X7-dependent actin reorganization and membrane blebbing, thereby facilitating P2X7 involvement in macrophage inflammatory responses.

  8. Multiscale modeling and mechanics of filamentous actin cytoskeleton.

    Science.gov (United States)

    Yamaoka, Hidetaka; Matsushita, Shinji; Shimada, Yoshitaka; Adachi, Taiji

    2012-03-01

    The adaptive structure and functional changes of the actin cytoskeleton are induced by its mechanical behavior at various temporal and spatial scales. In particular, the mechanical behaviors at different scales play important roles in the mechanical functions of various cells, and these multiscale phenomena require clarification. To establish a milestone toward achieving multiscale modeling and simulation, this paper reviews mathematical analyses and simulation methods applied to the mechanics of the filamentous actin cytoskeleton. The actin cytoskeleton demonstrates characteristic behaviors at every temporal and spatial scale, and mathematical models and simulation methods can be applied to each level of actin cytoskeletal structure ranging from the molecular to the network level. This paper considers studies on mathematical models and simulation methods based on the molecular dynamics, coarse-graining, and continuum dynamics approaches. Every temporal and spatial scale of actin cytoskeletal structure is considered, and it is expected that discrete and continuum dynamics ranging from functional expression at the molecular level to macroscopic functional expression at the whole cell level will be developed and applied to multiscale modeling and simulation.

  9. Drosophila Imp iCLIP identifies an RNA assemblage coordinating F-actin formation

    DEFF Research Database (Denmark)

    Hansen, Heidi Theil; Rasmussen, Simon Horskjær; Adolph, Sidsel Kramshøj;

    2015-01-01

    CLIP) technologies in Drosophila cells to identify transcripts associated with cytoplasmic ribonucleoproteins (RNPs) containing the RNA-binding protein Imp. RESULTS: We find extensive binding of Imp to 3'UTRs of transcripts that are involved in F-actin formation. A common denominator of the RNA-protein interface....... This demonstrates a physiological significance of the defined RNA regulon. CONCLUSIONS: Our data imply that Drosophila Imp RNPs may function as cytoplasmic mRNA assemblages that encode proteins which participate in actin cytoskeletal remodeling. Thus, they may facilitate co-ordinated protein expression in sub...... is the presence of multiple motifs with a central UA-rich element flanked by CA-rich elements. Experiments in single cells and intact flies reveal compromised actin cytoskeletal dynamics associated with low Imp levels. The former shows reduced F-actin formation and the latter exhibits abnormal neuronal patterning...

  10. 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.

  11. Cytoskeletal protein translation and expression in the rat brain are stressor-dependent and region-specific.

    Directory of Open Access Journals (Sweden)

    Petra Sántha

    Full Text Available Stress is an integral component of life that can sometimes cause a critical overload, depending on the qualitative and quantitative natures of the stressors. The involvement of actin, the predominant component of dendritic integrity, is a plausible candidate factor in stress-induced neuronal cytoskeletal changes. The major aim of this study was to compare the effects of three different stress conditions on the transcription and translation of actin-related cytoskeletal genes in the rat brain. Male Wistar rats were exposed to one or other of the frequently used models of physical stress, i.e. electric foot shock stress (EFSS, forced swimming stress (FSS, or psychosocial stress (PSS for periods of 3, 7, 14, or 21 days. The relative mRNA and protein expressions of β-actin, cofilin and mitogen-activated protein kinase 1 (MAPK-1 were determined by qRT- PCR and western blotting from hippocampus and frontal cortex samples. Stressor-specific alterations in both β-actin and cofilin expression levels were seen after stress. These alterations were most pronounced in response to EFSS, and exhibited a U-shaped time course. FSS led to a significant β-actin mRNA expression elevation in the hippocampus and the frontal cortex after 3 and 7 days, respectively, without any subsequent change. PSS did not cause any change in β-actin or cofilin mRNA or protein expression in the examined brain regions. EFSS, FSS and PSS had no effect on the expression of MAPK-1 mRNA at any tested time point. These findings indicate a very delicate, stress type-dependent regulation of neuronal cytoskeletal components in the rat hippocampus and frontal cortex.

  12. Disease-associated mutations in the actin-binding domain of filamin B cause cytoplasmic focal accumulations correlating with disease severity

    DEFF Research Database (Denmark)

    Daniel, Philip B; Morgan, Tim; Alanay, Yasemin;

    2012-01-01

    repeats surrounding the flexible hinge 1 region of the FLNB rod domain. Despite being positioned in domains that bind actin, it is unknown if these mutations perturb cytoskeletal structure. Expression of several full-length FLNB constructs containing ABD mutations resulted in the appearance of actin...

  13. Cytoskeletal dynamics of the teleostean fin ray during fin epimorphic regeneration.

    Science.gov (United States)

    Santos-Ruiz, Leonor; Santamaría, Jesús Alberto; Becerra, José

    2005-04-01

    Teleost fishes can regenerate their fins by epimorphic regeneration, a process that involves the transition of the formerly quiescent tissues of the stump to an active, growing state. This involves dynamic modifications of cell phenotype and behavior that must rely on alterations of the cytoskeleton. We have studied the spatial and temporal distribution of three main components of the cytoskeleton (actin, keratin and vimentin) in the regenerating fin, in order to establish putative relationships between cell cytoskeleton and cell behavior. According to our results, the massive rearrangement undergone by the epidermis right after injury, which takes place by cell migration, correlates with a transient down-regulation of keratin and a strong up-regulation of actin in the epidermal cells. During the subsequent epidermal growth, based on cell proliferation, keratin normal pattern is recovered while actin is down-regulated, although not to normal (quiescent) levels. The epidermal basal layer in contact with the blastema displays a particular cytoskeletal profile, different to that of the rest of the epidermal cells, which reflects its special features. In the connective tissue compartment, somatic cells do not contain vimentin, but keratin, as intermediate filament. Proliferative and migrative activation of these cells after injury correlates with actin up-regulation. Although this initial activation does not involve keratin down-regulation, blastemal cells were later observed to lack keratin, suggesting that such cytoskeletal modification might be needed for connective tissue cells to dedifferentiate and form the blastema. Cell differentiation in the newly formed, regenerated ray is accompanied by actin down-regulation and keratin up-regulation.

  14. CAS-1, a C. elegans cyclase-associated protein, is required for sarcomeric actin assembly in striated muscle.

    Science.gov (United States)

    Nomura, Kazumi; Ono, Kanako; Ono, Shoichiro

    2012-09-01

    Assembly of contractile apparatuses in striated muscle requires precisely regulated reorganization of the actin cytoskeletal proteins into sarcomeric organization. Regulation of actin filament dynamics is one of the essential processes of myofibril assembly, but the mechanism of actin regulation in striated muscle is not clearly understood. Actin depolymerizing factor (ADF)/cofilin is a key enhancer of actin filament dynamics in striated muscle in both vertebrates and nematodes. Here, we report that CAS-1, a cyclase-associated protein in Caenorhabditis elegans, promotes ADF/cofilin-dependent actin filament turnover in vitro and is required for sarcomeric actin organization in striated muscle. CAS-1 is predominantly expressed in striated muscle from embryos to adults. In vitro, CAS-1 binds to actin monomers and enhances exchange of actin-bound ATP/ADP even in the presence of UNC-60B, a muscle-specific ADF/cofilin that inhibits the nucleotide exchange. As a result, CAS-1 and UNC-60B cooperatively enhance actin filament turnover. The two proteins also cooperate to shorten actin filaments. A cas-1 mutation is homozygous lethal with defects in sarcomeric actin organization. cas-1-mutant embryos and worms have aggregates of actin in muscle cells, and UNC-60B is mislocalized to the aggregates. These results provide genetic and biochemical evidence that cyclase-associated protein is a critical regulator of sarcomeric actin organization in striated muscle.

  15. 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.

  16. The role of cyclase-associated protein in regulating actin filament dynamics - more than a monomer-sequestration factor.

    Science.gov (United States)

    Ono, Shoichiro

    2013-08-01

    Dynamic reorganization of the actin cytoskeleton is fundamental to a number of cell biological events. A variety of actin-regulatory proteins modulate polymerization and depolymerization of actin and contribute to actin cytoskeletal reorganization. Cyclase-associated protein (CAP) is a conserved actin-monomer-binding protein that has been studied for over 20 years. Early studies have shown that CAP sequesters actin monomers; recent studies, however, have revealed more active roles of CAP in actin filament dynamics. CAP enhances the recharging of actin monomers with ATP antagonistically to ADF/cofilin, and also promotes the severing of actin filaments in cooperation with ADF/cofilin. Self-oligomerization and binding to other proteins regulate activities and localization of CAP. CAP has crucial roles in cell signaling, development, vesicle trafficking, cell migration and muscle sarcomere assembly. This Commentary discusses the recent advances in our understanding of the functions of CAP and its implications as an important regulator of actin cytoskeletal dynamics, which are involved in various cellular activities.

  17. The role of CHAP in muscle development, heart disease and actin signaling

    NARCIS (Netherlands)

    Eldik, Willemijn Lisette van

    2013-01-01

    In this thesis we investigated a novel Z-disc protein, cytoskeletal heart-enriched actin-associated protein (CHAP). Two isoforms of CHAP exist, encoded by one gene. The longer isoform CHAPa is predominately expressed in adult tissues, whereas CHAPb is expressed during cardiac and skeletal developmen

  18. Identification of obscure yet conserved actin-associated proteins in Giardia lamblia.

    Science.gov (United States)

    Paredez, Alexander R; Nayeri, Arash; Xu, Jennifer W; Krtková, Jana; Cande, W Zacheus

    2014-06-01

    Consistent with its proposed status as an early branching eukaryote, Giardia has the most divergent actin of any eukaryote and lacks core actin regulators. Although conserved actin-binding proteins are missing from Giardia, its actin is utilized similarly to that of other eukaryotes and functions in core cellular processes such as cellular organization, endocytosis, and cytokinesis. We set out to identify actin-binding proteins in Giardia using affinity purification coupled with mass spectroscopy (multidimensional protein identification technology [MudPIT]) and have identified >80 putative actin-binding proteins. Several of these have homology to conserved proteins known to complex with actin for functions in the nucleus and flagella. We validated localization and interaction for seven of these proteins, including 14-3-3, a known cytoskeletal regulator with a controversial relationship to actin. Our results indicate that although Giardia lacks canonical actin-binding proteins, there is a conserved set of actin-interacting proteins that are evolutionarily indispensable and perhaps represent some of the earliest functions of the actin cytoskeleton.

  19. Cell shape, cytoskeletal mechanics, and cell cycle control in angiogenesis

    Science.gov (United States)

    Ingber, D. E.; Prusty, D.; Sun, Z.; Betensky, H.; Wang, N.

    1995-01-01

    Capillary endothelial cells can be switched between growth and differentiation by altering cell-extracellular matrix interactions and thereby, modulating cell shape. Studies were carried out to determine when cell shape exerts its growth-regulatory influence during cell cycle progression and to explore the role of cytoskeletal structure and mechanics in this control mechanism. When G0-synchronized cells were cultured in basic fibroblast growth factor (FGF)-containing defined medium on dishes coated with increasing densities of fibronectin or a synthetic integrin ligand (RGD-containing peptide), cell spreading, nuclear extension, and DNA synthesis all increased in parallel. To determine the minimum time cells must be adherent and spread on extracellular matrix (ECM) to gain entry into S phase, cells were removed with trypsin or induced to retract using cytochalasin D at different times after plating. Both approaches revealed that cells must remain extended for approximately 12-15 h and hence, most of G1, in order to enter S phase. After this restriction point was passed, normally 'anchorage-dependent' endothelial cells turned on DNA synthesis even when round and in suspension. The importance of actin-containing microfilaments in shape-dependent growth control was confirmed by culturing cells in the presence of cytochalasin D (25-1000 ng ml-1): dose-dependent inhibition of cell spreading, nuclear extension, and DNA synthesis resulted. In contrast, induction of microtubule disassembly using nocodazole had little effect on cell or nuclear spreading and only partially inhibited DNA synthesis. Interestingly, combination of nocodazole with a suboptimal dose of cytochalasin D (100 ng ml-1) resulted in potent inhibition of both spreading and growth, suggesting that microtubules are redundant structural elements which can provide critical load-bearing functions when microfilaments are partially compromised. Similar synergism between nocodazole and cytochalasin D was observed

  20. A versatile framework for simulating the dynamic mechanical structure of cytoskeletal networks

    CERN Document Server

    Freedman, Simon L; Hocky, Glen M; Dinner, Aaron R

    2016-01-01

    Computer simulations can aid in our understanding of how collective materials properties emerge from interactions between simple constituents. Here, we introduce a coarse- grained model of networks of actin filaments, myosin motors, and crosslinking proteins that enables simulation at biologically relevant time and length scales. We demonstrate that the model, with a consistent parameterization, qualitatively and quantitatively captures a suite of trends observed experimentally, including the statistics of filament fluctuations, mechanical responses to shear, motor motilities, and network rearrangements. The model can thus serve as a platform for interpretation and design of cytoskeletal materials experiments, as well as for further development of simulations incorporating active elements.

  1. Immunohistochemical observation of actin filaments in epithelial cells encircling the taste pore cavity of rat fungiform papillae

    Directory of Open Access Journals (Sweden)

    Y Shiba

    2009-12-01

    Full Text Available Epithelial cells are connected to each other around taste pores in rat fungiform papillae. Cytoskeletal components are responsible for the maintenance of intracellular adhesion, and we investigated the identification and localization of actin filaments around taste pores. On the basis of observations made by immunohistochemical transmission electron microscopy comparing with confocal laser scanning microscopy using actin-lectin double staining, actin filaments were found to be localized, encircling the squeezed taste pore cavity, in epithelial cells a few micrometers below the papilla surface. In addition, these observations suggest that the organization of actin filaments around taste pores might be involved in the constriction of taste pores.

  2. p-Chloromercuribenzoate-induced dissociation of cytoskeletal proteins in red blood cells of rats.

    Science.gov (United States)

    Kunimoto, M; Shibata, K; Miura, T

    1987-12-11

    Effects of p-chloromercuribenzoate (PCMB) on the cytoskeletal organization of rat red blood cells were studied. Upon incubation with 50 microM PCMB in 10 mM Tris-HCl (pH 7.4) at 37 degrees C for 30 min, 80% of actin and 45% of spectrin were released from the ghosts, resulting in the fragmentation of ghost membranes. Addition of 2 mM Mg2+ or 0.1 M KCl, or lowering incubation temperature to 0 degree C substantially inhibited the solubilization of the cytoskeletal proteins and the fragmentation of ghost membranes, which enable to examine the effects of PCMB on the interaction between transmembrane proteins and the peripheral cytoskeletal network. Decreased recoveries of transmembrane proteins, such as band 3 and glycophorin, in Triton shell fraction were observed in the ghosts incubated with PCMB either in the presence of Mg2+ or at 0 degree C. PCMB also inhibited the in vitro association of purified spectrin with spectrin-depleted inside-out vesicles through interaction with proteins in the vesicle, such as bands 2.1 and 3. In the PCMB-treated ghosts, intramembrane particles were highly aggregated, which further supports the PCMB-induced dissociation of the transmembrane proteins from the cytoskeletal network. The decreased recovery of glycophorin in the Triton shell fraction also observed in intact red blood cells upon incubation with PCMB. These results suggest that the main action of PCMB on red cell membranes under physiological condition, at higher ionic strength and in the presence of Mg2+, is to dissociate transmembrane proteins from the peripheral cytoskeletal network, which may modify functions of these proteins.

  3. Mechanics and dynamics of reconstituted cytoskeletal systems.

    Science.gov (United States)

    Jensen, Mikkel H; Morris, Eliza J; Weitz, David A

    2015-11-01

    The intracellular cytoskeleton is an active dynamic network of filaments and associated binding proteins that control key cellular properties, such as cell shape and mechanics. Due to the inherent complexity of the cell, reconstituted model systems have been successfully employed to gain an understanding of the fundamental physics governing cytoskeletal processes. Here, we review recent advances and key aspects of these reconstituted systems. We focus on the importance of assembly kinetics and dynamic arrest in determining network mechanics, and highlight novel emergent behavior occurring through interactions between cytoskeletal components in more complex networks incorporating multiple biopolymers and molecular motors.

  4. Actin dynamics shape microglia effector functions.

    Science.gov (United States)

    Uhlemann, Ria; Gertz, Karen; Boehmerle, Wolfgang; Schwarz, Tobias; Nolte, Christiane; Freyer, Dorette; Kettenmann, Helmut; Endres, Matthias; Kronenberg, Golo

    2016-06-01

    Impaired actin filament dynamics have been associated with cellular senescence. Microglia, the resident immune cells of the brain, are emerging as a central pathophysiological player in neurodegeneration. Microglia activation, which ranges on a continuum between classical and alternative, may be of critical importance to brain disease. Using genetic and pharmacological manipulations, we studied the effects of alterations in actin dynamics on microglia effector functions. Disruption of actin dynamics did not affect transcription of genes involved in the LPS-triggered classical inflammatory response. By contrast, in consequence of impaired nuclear translocation of phospho-STAT6, genes involved in IL-4 induced alternative activation were strongly downregulated. Functionally, impaired actin dynamics resulted in reduced NO secretion and reduced release of TNFalpha and IL-6 from LPS-stimulated microglia and of IGF-1 from IL-4 stimulated microglia. However, pathological stabilization of the actin cytoskeleton increased LPS-induced release of IL-1beta and IL-18, which belong to an unconventional secretory pathway. Reduced NO release was associated with decreased cytoplasmic iNOS protein expression and decreased intracellular arginine uptake. Furthermore, disruption of actin dynamics resulted in reduced microglia migration, proliferation and phagocytosis. Finally, baseline and ATP-induced [Ca(2+)]int levels were significantly increased in microglia lacking gelsolin, a key actin-severing protein. Together, the dynamic state of the actin cytoskeleton profoundly and distinctly affects microglia behaviours. Disruption of actin dynamics attenuates M2 polarization by inhibiting transcription of alternative activation genes. In classical activation, the role of actin remodelling is complex, does not relate to gene transcription and shows a major divergence between cytokines following conventional and unconventional secretion.

  5. Location of and post-mortem changes in some cytoskeletal proteins in pork and cod muscle

    DEFF Research Database (Denmark)

    Morrison, E.H.; Bremner, Allan; Purslow, P.P.

    2000-01-01

    The cytoskeletal proteins actin, nebulin, spectrin, desmin, vinculin and talin were labelled immunohistochemically in sections of muscle from commercially available pigs and cod (Gadus morhua) taken pre-rigor and from samples stored for several days. Actin, nebulin and spectrin gave similar...... labelling in fish. Labelling for talin in pork muscle was intense at the sarcolemma but was not present in samples stored for 4 days. In contrast, the label for talin was concentrated at the myotendinous junction of the cod muscle throughout the storage period. These are the first reports of the detection...... and location of spectrin and vinculin in fish muscle and of the location of talin. The results are discussed in terms of muscle structure, function and post-mortem tenderisation. (C) 2000 Society of Chemical Industry....

  6. Actin restructuring during Salmonella typhimurium infection investigated by confocal and super-resolution microscopy

    Science.gov (United States)

    Han, Jason J.; Kunde, Yuliya A.; Hong-Geller, Elizabeth; Werner, James H.

    2014-01-01

    We have used super-resolution optical microscopy and confocal microscopy to visualize the cytoskeletal restructuring of HeLa cells that accompanies and enables Salmonella typhimurium internalization. Herein, we report the use of confocal microscopy to verify and explore infection conditions that would be compatible with super-resolution optical microscopy, using Alexa-488 labeled phalloidin to stain the actin cytoskeletal network. While it is well known that actin restructuring and cytoskeletal rearrangements often accompany and assist in bacterial infection, most studies have employed conventional diffraction-limited fluorescence microscopy to explore these changes. Here we show that the superior spatial resolution provided by single-molecule localization methods (such as direct stochastic optical reconstruction microscopy) enables more precise visualization of the nanoscale changes in the actin cytoskeleton that accompany bacterial infection. In particular, we found that a thin (100-nm) ring of actin often surrounds an invading bacteria 10 to 20 min postinfection, with this ring being transitory in nature. We estimate that a few hundred monofilaments of actin surround the S. typhimurium in this heretofore unreported bacterial internalization intermediate.

  7. Actin restructuring during Salmonella typhimurium infection investigated by confocal and super-resolution microscopy.

    Science.gov (United States)

    Han, Jason J; Kunde, Yuliya A; Hong-Geller, Elizabeth; Werner, James H

    2014-01-01

    We have used super-resolution optical microscopy and confocal microscopy to visualize the cytoskeletal restructuring of HeLa cells that accompanies and enables Salmonella typhimurium internalization. Herein, we report the use of confocal microscopy to verify and explore infection conditions that would be compatible with super-resolution optical microscopy, using Alexa-488 labeled phalloidin to stain the actin cytoskeletal network. While it is well known that actin restructuring and cytoskeletal rearrangements often accompany and assist in bacterial infection, most studies have employed conventional diffraction-limited fluorescence microscopy to explore these changes. Here we show that the superior spatial resolution provided by single-molecule localization methods (such as direct stochastic optical reconstruction microscopy) enables more precise visualization of the nanoscale changes in the actin cytoskeleton that accompany bacterial infection. In particular, we found that a thin (100-nm) ring of actin often surrounds an invading bacteria 10 to 20 min postinfection, with this ring being transitory in nature. We estimate that a few hundred monofilaments of actin surround the S. typhimurium in this heretofore unreported bacterial internalization intermediate.

  8. Vascular Endothelial-Cadherin Regulates Cytoskeletal Tension, Cell Spreading, and Focal Adhesions by Stimulating RhoAD⃞

    Science.gov (United States)

    Nelson, Celeste M.; Pirone, Dana M.; Tan, John L.; Chen, Christopher S.

    2004-01-01

    Changes in vascular endothelial (VE)-cadherin–mediated cell-cell adhesion and integrin-mediated cell-matrix adhesion coordinate to affect the physical and mechanical rearrangements of the endothelium, although the mechanisms for such cross talk remain undefined. Herein, we describe the regulation of focal adhesion formation and cytoskeletal tension by intercellular VE-cadherin engagement, and the molecular mechanism by which this occurs. Increasing the density of endothelial cells to increase cell-cell contact decreased focal adhesions by decreasing cell spreading. This contact inhibition of cell spreading was blocked by disrupting VE-cadherin engagement with an adenovirus encoding dominant negative VE-cadherin. When changes in cell spreading were prevented by culturing cells on a micropatterned substrate, VE-cadherin–mediated cell-cell contact paradoxically increased focal adhesion formation. We show that VE-cadherin engagement mediates each of these effects by inducing both a transient and sustained activation of RhoA. Both the increase and decrease in cell-matrix adhesion were blocked by disrupting intracellular tension and signaling through the Rho-ROCK pathway. In all, these findings demonstrate that VE-cadherin signals through RhoA and the actin cytoskeleton to cross talk with cell-matrix adhesion and thereby define a novel pathway by which cell-cell contact alters the global mechanical and functional state of cells. PMID:15075376

  9. Real-time dynamics of emerging actin networks in cell-mimicking compartments.

    Directory of Open Access Journals (Sweden)

    Siddharth Deshpande

    Full Text Available Understanding the cytoskeletal functionality and its relation to other cellular components and properties is a prominent question in biophysics. The dynamics of actin cytoskeleton and its polymorphic nature are indispensable for the proper functioning of living cells. Actin bundles are involved in cell motility, environmental exploration, intracellular transport and mechanical stability. Though the viscoelastic properties of actin-based structures have been extensively probed, the underlying microstructure dynamics, especially their disassembly, is not fully understood. In this article, we explore the rich dynamics and emergent properties exhibited by actin bundles within flow-free confinements using a microfluidic set-up and epifluorescence microscopy. After forming entangled actin filaments within cell-sized quasi two-dimensional confinements, we induce their bundling using three different fundamental mechanisms: counterion condensation, depletion interactions and specific protein-protein interactions. Intriguingly, long actin filaments form emerging networks of actin bundles via percolation leading to remarkable properties such as stress generation and spindle-like intermediate structures. Simultaneous sharing of filaments in different links of the network is an important parameter, as short filaments do not form networks but segregated clusters of bundles instead. We encounter a hierarchical process of bundling and its subsequent disassembly. Additionally, our study suggests that such percolated networks are likely to exist within living cells in a dynamic fashion. These observations render a perspective about differential cytoskeletal responses towards numerous stimuli.

  10. The kinesin-like proteins, KAC1/2, regulate actin dynamics underlying chloroplast light-avoidance in Physcomitrella patens

    Institute of Scientific and Technical Information of China (English)

    Zhiyuan Shen; Yen-Chen Liu; Jeffrey P Bibeau; Kyle P Lemoi; Erkan Tzel; Luis Vidali

    2015-01-01

    In plants, light determines chloroplast position;these organelles show avoidance and accumulation re-sponses in high and low fluence-rate light, respectively. Chloroplast motility in response to light is driven by cytoskeletal elements. The actin cytoskeleton mediates chloroplast photorelocation responses in Arabidopsis thali-ana. In contrast, in the moss Physcomitrella patens, both, actin filaments and microtubules can transport chloroplasts. Because of the surprising evidence that two kinesin-like proteins (called KACs) are important for actin-dependent chloroplast photorelocation in vascular plants, we wanted to determine the cytoskeletal system responsible for the function of these proteins in moss. We performed gene-specific silencing using RNA interference in P. patens. We confirmed existing reports using gene knockouts, that PpKAC1 and PpKAC2 are required for chloroplast dispersion under uniform white light conditions, and that the two proteins are functionally equivalent. To address the specific cytoskeletal elements responsible for motility, this loss-of-function approach was combined with cytoskeleton-targeted drug studies. We found that, in P. patens, these KACs mediate the chloroplast light-avoidance response in an actin filament-dependent, rather than a microtubule-dependent manner. Using correlation-decay analysis of cytoskeletal dynamics, we found that PpKAC stabilizes cortical actin filaments, but has no effect on microtubule dynamics.

  11. Rab11 and Actin Cytoskeleton Participate in Giardia lamblia Encystation, Guiding the Specific Vesicles to the Cyst Wall

    Science.gov (United States)

    Castillo-Romero, Araceli; Leon-Avila, Gloria; Wang, Ching C.; Perez Rangel, Armando; Camacho Nuez, Minerva; Garcia Tovar, Carlos; Ayala-Sumuano, Jorge Tonatiuh; Luna-Arias, Juan Pedro; Hernandez, Jose Manuel

    2010-01-01

    Background Giardia passes through two stages during its life cycle, the trophozoite and the cyst. Cyst formation involves the synthesis of cyst wall proteins (CWPs) and the transport of CWPs into encystation-specific vesicles (ESVs). Active vesicular trafficking is essential for encystation, but the molecular machinery driving vesicular trafficking remains unknown. The Rab proteins are involved in the targeting of vesicles to several intracellular compartments through their association with cytoskeletal motor proteins. Methodology and Principal Findings In this study, we found a relationship between Rab11 and the actin cytoskeleton in CWP1 transport. Confocal microscopy showed Rab11 was distributed throughout the entire trophozoite, while in cysts it was translocated to the periphery of the cell, where it colocalized with ESVs and microfilaments. Encystation was also accompanied by changes in rab11 mRNA expression. To evaluate the role of microfilaments in encystation, the cells were treated with latrunculin A. Scanning electron microscopy showed this treatment resulted in morphological damages to encysted parasites. The intensity of fluorescence-labeled Rab11 and CWP1 in ESVs and cyst walls was reduced, and rab11 and cwp1 mRNA levels were down-regulated. Furthermore, knocking down Rab11 with a hammerhead ribozyme resulted in an up to 80% down-regulation of rab11 mRNA. Although this knockdown did not appear lethal for trophozoites and did not affect cwp1 expression during the encystation, confocal images showed CWP1 was redistributed throughout the cytosol. Conclusions and Significance Our results indicate that Rab11 participates in the early and late encystation stages by regulating CWP1 localization and the actin-mediated transport of ESVs towards the periphery. In addition, alterations in the dynamics of actin affected rab11 and cwp1 expression. Our results provide new information about the molecules involved in Giardia encystation and suggest that Rab11 and

  12. Rab11 and actin cytoskeleton participate in Giardia lamblia encystation, guiding the specific vesicles to the cyst wall.

    Directory of Open Access Journals (Sweden)

    Araceli Castillo-Romero

    Full Text Available BACKGROUND: Giardia passes through two stages during its life cycle, the trophozoite and the cyst. Cyst formation involves the synthesis of cyst wall proteins (CWPs and the transport of CWPs into encystation-specific vesicles (ESVs. Active vesicular trafficking is essential for encystation, but the molecular machinery driving vesicular trafficking remains unknown. The Rab proteins are involved in the targeting of vesicles to several intracellular compartments through their association with cytoskeletal motor proteins. METHODOLOGY AND PRINCIPAL FINDINGS: In this study, we found a relationship between Rab11 and the actin cytoskeleton in CWP1 transport. Confocal microscopy showed Rab11 was distributed throughout the entire trophozoite, while in cysts it was translocated to the periphery of the cell, where it colocalized with ESVs and microfilaments. Encystation was also accompanied by changes in rab11 mRNA expression. To evaluate the role of microfilaments in encystation, the cells were treated with latrunculin A. Scanning electron microscopy showed this treatment resulted in morphological damages to encysted parasites. The intensity of fluorescence-labeled Rab11 and CWP1 in ESVs and cyst walls was reduced, and rab11 and cwp1 mRNA levels were down-regulated. Furthermore, knocking down Rab11 with a hammerhead ribozyme resulted in an up to 80% down-regulation of rab11 mRNA. Although this knockdown did not appear lethal for trophozoites and did not affect cwp1 expression during the encystation, confocal images showed CWP1 was redistributed throughout the cytosol. CONCLUSIONS AND SIGNIFICANCE: Our results indicate that Rab11 participates in the early and late encystation stages by regulating CWP1 localization and the actin-mediated transport of ESVs towards the periphery. In addition, alterations in the dynamics of actin affected rab11 and cwp1 expression. Our results provide new information about the molecules involved in Giardia encystation and

  13. The actin cytoskeleton may control the polar distribution of an auxin transport protein

    Science.gov (United States)

    Muday, G. K.; Hu, S.; Brady, S. R.; Davies, E. (Principal Investigator)

    2000-01-01

    The gravitropic bending of plants has long been linked to the changes in the transport of the plant hormone auxin. To understand the mechanism by which gravity alters auxin movement, it is critical to know how polar auxin transport is initially established. In shoots, polar auxin transport is basipetal (i.e., from the shoot apex toward the base). It is driven by the basal localization of the auxin efflux carrier complex. One mechanism for localizing this efflux carrier complex to the basal membrane may be through attachment to the actin cytoskeleton. The efflux carrier protein complex is believed to consist of several polypeptides, including a regulatory subunit that binds auxin transport inhibitors, such as naphthylphthalamic acid (NPA). Several lines of experimentation have been used to determine if the NPA binding protein interacts with actin filaments. The NPA binding protein has been shown to partition with the actin cytoskeleton during detergent extraction. Agents that specifically alter the polymerization state of the actin cytoskeleton change the amount of NPA binding protein and actin recovered in these cytoskeletal pellets. Actin-affinity columns were prepared with polymers of actin purified from zucchini hypocotyl tissue. NPA binding activity was eluted in a single peak from the actin filament column. Cytochalasin D, which fragments the actin cytoskeleton, was shown to reduce polar auxin transport in zucchini hypocotyls. The interaction of the NPA binding protein with the actin cytoskeleton may localize it in one plane of the plasma membrane, and thereby control the polarity of auxin transport.

  14. Actin Rings of Power.

    Science.gov (United States)

    Schwayer, Cornelia; Sikora, Mateusz; Slováková, Jana; Kardos, Roland; Heisenberg, Carl-Philipp

    2016-06-20

    Circular or ring-like actin structures play important roles in various developmental and physiological processes. Commonly, these rings are composed of actin filaments and myosin motors (actomyosin) that, upon activation, trigger ring constriction. Actomyosin ring constriction, in turn, has been implicated in key cellular processes ranging from cytokinesis to wound closure. Non-constricting actin ring-like structures also form at cell-cell contacts, where they exert a stabilizing function. Here, we review recent studies on the formation and function of actin ring-like structures in various morphogenetic processes, shedding light on how those different rings have been adapted to fulfill their specific roles.

  15. A Steric Antagonism of Actin Polymerization by a Salmonella Virulence Protein

    Energy Technology Data Exchange (ETDEWEB)

    Margarit,S.; Davidson, W.; Frego, L.; Stebbins, F.

    2006-01-01

    Salmonella spp. require the ADP-ribosyltransferase activity of the SpvB protein for intracellular growth and systemic virulence. SpvB covalently modifies actin, causing cytoskeletal disruption and apoptosis. We report here the crystal structure of the catalytic domain of SpvB, and we show by mass spectrometric analysis that SpvB modifies actin at Arg177, inhibiting its ATPase activity. We also describe two crystal structures of SpvB-modified, polymerization-deficient actin. These structures reveal that ADP-ribosylation does not lead to dramatic conformational changes in actin, suggesting a model in which this large family of toxins inhibits actin polymerization primarily through steric disruption of intrafilament contacts.

  16. Multiscale View of Cytoskeletal Mechanoregulation of Cell and Tissue Polarity.

    Science.gov (United States)

    Luxenburg, Chen; Geiger, Benjamin

    2017-01-01

    The ability of cells to generate, maintain, and repair tissues with complex architecture, in which distinct cells function as coherent units, relies on polarity cues. Polarity can be described as an asymmetry along a defined axis, manifested at the molecular, structural, and functional levels. Several types of cell and tissue polarities were described in the literature, including front-back, apical-basal, anterior-posterior, and left-right polarity. Extensive research provided insights into the specific regulators of each polarization process, as well as into generic elements that affect all types of polarities. The actin cytoskeleton and the associated adhesion structures are major regulators of most, if not all, known forms of polarity. Actin filaments exhibit intrinsic polarity and their ability to bind many proteins including the mechanosensitive adhesion and motor proteins, such as myosins, play key roles in cell polarization. The actin cytoskeleton can generate mechanical forces and together with the associated adhesions, probe the mechanical, structural, and chemical properties of the environment, and transmit signals that impact numerous biological processes, including cell polarity. In this article we highlight novel mechanisms whereby the mechanical forces and actin-adhesion complexes regulate cell and tissue polarity in a variety of natural and experimental systems.

  17. Role of the PI3K regulatory subunit in the control of actin organization and cell migration

    OpenAIRE

    Jiménez, Concepción; Armas Portela, Rosario; Mellado, Mario; Rodríguez Frade, José M.; Collard, John; Serrano, Antonio; Martínez-Alonso, Carlos; Ávila, Jesús; Carrera, Ana C.

    2000-01-01

    Cell migration represents an important cellular response that utilizes cytoskeletal reorganization as its driving force. Here, we describe a new signaling cascade linking PDGF receptor stimulation to actin rearrangements and cell migration. We demonstrate that PDGF activates Cdc42 and its downstream effector N-WASP to mediate filopodia formation, actin stress fiber disassembly, and a reduction in focal adhesion complexes. Induction of the Cdc42 pathway is independent of phosphoinositide 3-kin...

  18. Cytoskeletal dynamics and cell signaling during planar polarity establishment in the Drosophila embryonic denticle.

    Science.gov (United States)

    Price, Meredith H; Roberts, David M; McCartney, Brooke M; Jezuit, Erin; Peifer, Mark

    2006-02-01

    Many epithelial cells are polarized along the plane of the epithelium, a property termed planar cell polarity. The Drosophila wing and eye imaginal discs are the premier models of this process. Many proteins required for polarity establishment and its translation into cytoskeletal polarity were identified from studies of those tissues. More recently, several vertebrate tissues have been shown to exhibit planar cell polarity. Striking similarities and differences have been observed when different tissues exhibiting planar cell polarity are compared. Here we describe a new tissue exhibiting planar cell polarity - the denticles, hair-like projections of the Drosophila embryonic epidermis. We describe in real time the changes in the actin cytoskeleton that underlie denticle development, and compare this with the localization of microtubules, revealing new aspects of cytoskeletal dynamics that may have more general applicability. We present an initial characterization of the localization of several actin regulators during denticle development. We find that several core planar cell polarity proteins are asymmetrically localized during the process. Finally, we define roles for the canonical Wingless and Hedgehog pathways and for core planar cell polarity proteins in denticle polarity.

  19. Analysis of the expression of cytoskeletal proteins of Taenia crassiceps ORF strain cysticerci (Cestoda).

    Science.gov (United States)

    Reynoso-Ducoing, Olivia; Valverde-Islas, Laura; Paredes-Salomon, Cristina; Pérez-Reyes, América; Landa, Abraham; Robert, Lilia; Mendoza, Guillermo; Ambrosio, Javier R

    2014-05-01

    The Taenia crassiceps ORF strain is used to generate a murine model of cysticercosis, which is used for diagnosis, evaluation of drugs, and vaccination. This particular strain only exists as cysticerci, is easily maintained under in vivo and in vitro conditions, and offers an excellent model for studying the cytoskeletons of cestodes. In this study, several experimental approaches were used to determine the tissue expression of its cytoskeletal proteins. The techniques used were microscopy (video, confocal, and transmission electron), one-dimensional (1D) and two-dimensional (2D) electrophoresis, immunochemistry, and mass spectrometry. The tissue expression of actin, tubulin, and paramyosin was assessed using microscopy, and their protein isoforms were determined with 1D and 2D electrophoresis and immunochemistry. Nineteen spots were excised from a proteomic gel and identified by liquid chromatography-tandem mass spectrometry and immunochemistry. The proteins identified were classic cytoskeletal proteins, metabolic enzymes, and proteins with diverse biological functions, but mainly involved in detoxification activities. Research suggests that most noncytoskeletal proteins interact with actin or tubulin, and the results of the present study suggest that the proteins identified may be involved in supporting the dynamics and plasticity of the cytoskeleton of T. crassiceps cysticerci. These results contribute to our knowledge of the cellular biology and physiology of cestodes.

  20. The phosphorylation status and cytoskeletal remodeling of striatal astrocytes treated with quinolinic acid

    Energy Technology Data Exchange (ETDEWEB)

    Pierozan, Paula; Ferreira, Fernanda; Ortiz de Lima, Bárbara; Gonçalves Fernandes, Carolina [Departamento de Bioquímica, Instituto de Ciências Básicas da Saúde, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS 90035-003 (Brazil); Totarelli Monteforte, Priscila; Castro Medaglia, Natalia de; Bincoletto, Claudia; Soubhi Smaili, Soraya [Departamento de Farmacologia, Universidade Federal de São Paulo (UNIFESP/EPM), São Paulo, SP (Brazil); Pessoa-Pureur, Regina, E-mail: rpureur@ufrgs.br [Departamento de Bioquímica, Instituto de Ciências Básicas da Saúde, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS 90035-003 (Brazil)

    2014-04-01

    Quinolinic acid (QUIN) is a glutamate agonist which markedly enhances the vulnerability of neural cells to excitotoxicity. QUIN is produced from the amino acid tryptophan through the kynurenine pathway (KP). Dysregulation of this pathway is associated with neurodegenerative conditions. In this study we treated striatal astrocytes in culture with QUIN and assayed the endogenous phosphorylating system associated with glial fibrillary acidic protein (GFAP) and vimentin as well as cytoskeletal remodeling. After 24 h incubation with 100 µM QUIN, cells were exposed to {sup 32}P-orthophosphate and/or protein kinase A (PKA), protein kinase dependent of Ca{sup 2+}/calmodulin II (PKCaMII) or protein kinase C (PKC) inhibitors, H89 (20 μM), KN93 (10 μM) and staurosporin (10 nM), respectively. Results showed that hyperphosphorylation was abrogated by PKA and PKC inhibitors but not by the PKCaMII inhibitor. The specific antagonists to ionotropic NMDA and non-NMDA (50 µM DL-AP5 and CNQX, respectively) glutamate receptors as well as to metabotropic glutamate receptor (mGLUR; 50 µM MCPG), mGLUR1 (100 µM MPEP) and mGLUR5 (10 µM 4C3HPG) prevented the hyperphosphorylation provoked by QUIN. Also, intra and extracellular Ca{sup 2+} quelators (1 mM EGTA; 10 µM BAPTA-AM, respectively) prevented QUIN-mediated effect, while Ca{sup 2+} influx through voltage-dependent Ca{sup 2+} channel type L (L-VDCC) (blocker: 10 µM verapamil) is not implicated in this effect. Morphological analysis showed dramatically altered actin cytoskeleton with concomitant change of morphology to fusiform and/or flattened cells with retracted cytoplasm and disruption of the GFAP meshwork, supporting misregulation of actin cytoskeleton. Both hyperphosphorylation and cytoskeletal remodeling were reversed 24 h after QUIN removal. Astrocytes are highly plastic cells and the vulnerability of astrocyte cytoskeleton may have important implications for understanding the neurotoxicity of QUIN in neurodegenerative

  1. Real space flight travel is associated with ultrastructural changes, cytoskeletal disruption and premature senescence of HUVEC.

    Science.gov (United States)

    Kapitonova, M Y; Muid, S; Froemming, G R A; Yusoff, W N W; Othman, S; Ali, A M; Nawawi, H M

    2012-12-01

    Microgravity, hypergravity, vibration, ionizing radiation and temperature fluctuations are major factors of outer space flight affecting human organs and tissues. There are several reports on the effect of space flight on different human cell types of mesenchymal origin while information regarding changes to vascular endothelial cells is scarce. Ultrastructural and cytophysiological features of macrovascular endothelial cells in outer space flight and their persistence during subsequent culturing were demonstrated in the present investigation. At the end of the space flight, endothelial cells displayed profound changes indicating cytoskeletal lesions and increased cell membrane permeability. Readapted cells of subsequent passages exhibited persisting cytoskeletal changes, decreased metabolism and cell growth indicating cellular senescence.

  2. Vitreous-induced cytoskeletal rearrangements via the Rac1 GTPase-dependent signaling pathway in human retinal pigment epithelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Huang, Xionggao [State Key Ophthalmic Laboratory, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou (China); Department of Ophthalmology, Hainan Medical College, Haikou (China); Wei, Yantao; Ma, Haizhi [State Key Ophthalmic Laboratory, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou (China); Zhang, Shaochong, E-mail: zhshaochong@163.com [State Key Ophthalmic Laboratory, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou (China)

    2012-03-09

    Highlights: Black-Right-Pointing-Pointer Vitreous induces morphological changes and cytoskeletal rearrangements in RPE cells. Black-Right-Pointing-Pointer Rac1 is activated in vitreous-transformed RPE cells. Black-Right-Pointing-Pointer Rac inhibition prevents morphological changes in vitreous-transformed RPE cells. Black-Right-Pointing-Pointer Rac inhibition suppresses cytoskeletal rearrangements in vitreous-transformed RPE cells. Black-Right-Pointing-Pointer The vitreous-induced effects are mediated by a Rac1 GTPase/LIMK1/cofilin pathway. -- Abstract: Proliferative vitreoretinopathy (PVR) is mainly caused by retinal pigment epithelial (RPE) cell migration, invasion, proliferation and transformation into fibroblast-like cells that produce the extracellular matrix (ECM). The vitreous humor is known to play an important role in PVR. An epithelial-to-mesenchymal transdifferentiation (EMT) of human RPE cells induced by 25% vitreous treatment has been linked to stimulation of the mesenchymal phenotype, migration and invasion. Here, we characterized the effects of the vitreous on the cell morphology and cytoskeleton in human RPE cells. The signaling pathway that mediates these effects was investigated. Serum-starved RPE cells were incubated with 25% vitreous, and the morphological changes were examined by phase-contrast microscopy. Filamentous actin (F-actin) was examined by immunofluorescence and confocal microscopy. Protein phosphorylation of AKT, ERK1/2, Smad2/3, LIM kinase (LIMK) 1 and cofilin was analyzed by Western blot analysis. Vitreous treatment induced cytoskeletal rearrangements, activated Rac1 and enhanced the phosphorylation of AKT, ERK1/2 and Smad2/3. When the cells were treated with a Rac activation-specific inhibitor, the cytoskeletal rearrangements were prevented, and the phosphorylation of Smad2/3 was blocked. Vitreous treatment also enhanced the phosphorylation of LIMK1 and cofilin and the Rac inhibitor blocked this effect. We propose that vitreous

  3. Cytoskeletal network morphology regulates intracellular transport dynamics

    CERN Document Server

    Ando, David; Huang, Kerwyn Casey; Gopinathan, Ajay

    2016-01-01

    Intracellular transport is essential for maintaining proper cellular function in most eukaryotic cells, with perturbations in active transport resulting in several types of disease. Efficient delivery of critical cargos to specific locations is accomplished through a combination of passive diffusion and active transport by molecular motors that ballistically move along a network of cytoskeletal filaments. Although motor-based transport is known to be necessary to overcome cytoplasmic crowding and the limited range of diffusion within reasonable time scales, the topological features of the cytoskeletal network that regulate transport efficiency and robustness have not been established. Using a continuum diffusion model, we observed that the time required for cellular transport was minimized when the network was localized near the nucleus. In simulations that explicitly incorporated network spatial architectures, total filament mass was the primary driver of network transit times. However, filament traps that r...

  4. 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.

  5. 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.

  6. Actin as a potential target for decavanadate.

    Science.gov (United States)

    Ramos, Susana; Moura, José J G; Aureliano, Manuel

    2010-12-01

    ATP prevents G-actin cysteine oxidation and vanadyl formation specifically induced by decavanadate, suggesting that the oxometalate-protein interaction is affected by the nucleotide. The ATP exchange rate is increased by 2-fold due to the presence of decavanadate when compared with control actin (3.1×10(-3) s(-1)), and an apparent dissociation constant (k(dapp)) of 227.4±25.7 μM and 112.3±8.7 μM was obtained in absence or presence of 20 μM V(10), respectively. Moreover, concentrations as low as 50 μM of decameric vanadate species (V(10)) increases the relative G-actin intrinsic fluorescence intensity by approximately 80% whereas for a 10-fold concentration of monomeric vanadate (V(1)) no effects were observed. Upon decavanadate titration, it was observed a linear increase in G-actin hydrophobic surface (2.6-fold), while no changes were detected for V(1) (0-200 μM). Taken together, three major ideas arise: i) ATP prevents decavanadate-induced G-actin cysteine oxidation and vanadate reduction; ii) decavanadate promotes actin conformational changes resulting on its inactivation, iii) decavanadate has an effect on actin ATP binding site. Once it is demonstrated that actin is a new potential target for decavanadate, being the ATP binding site a suitable site for decavanadate binding, it is proposed that some of the biological effects of vanadate can be, at least in part, explained by decavanadate interactions with actin.

  7. Molecular architecture of synaptic actin cytoskeleton in hippocampal neurons reveals a mechanism of dendritic spine morphogenesis.

    Science.gov (United States)

    Korobova, Farida; Svitkina, Tatyana

    2010-01-01

    Excitatory synapses in the brain play key roles in learning and memory. The formation and functions of postsynaptic mushroom-shaped structures, dendritic spines, and possibly of presynaptic terminals, rely on actin cytoskeleton remodeling. However, the cytoskeletal architecture of synapses remains unknown hindering the understanding of synapse morphogenesis. Using platinum replica electron microscopy, we characterized the cytoskeletal organization and molecular composition of dendritic spines, their precursors, dendritic filopodia, and presynaptic boutons. A branched actin filament network containing Arp2/3 complex and capping protein was a dominant feature of spine heads and presynaptic boutons. Surprisingly, the spine necks and bases, as well as dendritic filopodia, also contained a network, rather than a bundle, of branched and linear actin filaments that was immunopositive for Arp2/3 complex, capping protein, and myosin II, but not fascin. Thus, a tight actin filament bundle is not necessary for structural support of elongated filopodia-like protrusions. Dynamically, dendritic filopodia emerged from densities in the dendritic shaft, which by electron microscopy contained branched actin network associated with dendritic microtubules. We propose that dendritic spine morphogenesis begins from an actin patch elongating into a dendritic filopodium, which tip subsequently expands via Arp2/3 complex-dependent nucleation and which length is modulated by myosin II-dependent contractility.

  8. Cyclase-associated protein (CAP) acts directly on F-actin to accelerate cofilin-mediated actin severing across the range of physiological pH.

    Science.gov (United States)

    Normoyle, Kieran P M; Brieher, William M

    2012-10-12

    Fast actin depolymerization is necessary for cells to rapidly reorganize actin filament networks. Utilizing a Listeria fluorescent actin comet tail assay to monitor actin disassembly rates, we observed that although a mixture of actin disassembly factors (cofilin, coronin, and actin-interacting protein 1 is sufficient to disassemble actin comet tails in the presence of physiological G-actin concentrations this mixture was insufficient to disassemble actin comet tails in the presence of physiological F-actin concentrations. Using biochemical complementation, we purified cyclase-associated protein (CAP) from thymus extracts as a factor that protects against the inhibition of excess F-actin. CAP has been shown to participate in actin dynamics but has been thought to act by liberating cofilin from ADP·G-actin monomers to restore cofilin activity. However, we found that CAP augments cofilin-mediated disassembly by accelerating the rate of cofilin-mediated severing. We also demonstrated that CAP acts directly on F-actin and severs actin filaments at acidic, but not neutral, pH. At the neutral pH characteristic of cytosol in most mammalian cells, we demonstrated that neither CAP nor cofilin are capable of severing actin filaments. However, the combination of CAP and cofilin rapidly severed actin at all pH values across the physiological range. Therefore, our results reveal a new function for CAP in accelerating cofilin-mediated actin filament severing and provide a mechanism through which cells can maintain high actin turnover rates without having to alkalinize cytosol, which would affect many biochemical reactions beyond actin depolymerization.

  9. Run-and-pause dynamics of cytoskeletal motor proteins

    Science.gov (United States)

    Hafner, Anne E.; Santen, Ludger; Rieger, Heiko; Shaebani, M. Reza

    2016-11-01

    Cytoskeletal motor proteins are involved in major intracellular transport processes which are vital for maintaining appropriate cellular function. When attached to cytoskeletal filaments, the motor exhibits distinct states of motility: active motion along the filaments, and pause phase in which it remains stationary for a finite time interval. The transition probabilities between motion and pause phases are asymmetric in general, and considerably affected by changes in environmental conditions which influences the efficiency of cargo delivery to specific targets. By considering the motion of individual non-interacting molecular motors on a single filament as well as a dynamic filamentous network, we present an analytical model for the dynamics of self-propelled particles which undergo frequent pause phases. The interplay between motor processivity, structural properties of filamentous network, and transition probabilities between the two states of motility drastically changes the dynamics: multiple transitions between different types of anomalous diffusive dynamics occur and the crossover time to the asymptotic diffusive or ballistic motion varies by several orders of magnitude. We map out the phase diagrams in the space of transition probabilities, and address the role of initial conditions of motion on the resulting dynamics.

  10. 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.

  11. Mechanics of composite cytoskeletal and extracellular networks

    Science.gov (United States)

    Das, Moumita

    2014-03-01

    Living cells sense and respond to mechanical forces in their surroundings. This mechanical response is mainly due to the cell cytoskeleton, and its interaction with the extracellular matrix (ECM). The cell cytoskeleton is a composite polymeric scaffold made of many different types of protein filaments and crosslinking proteins. Two major filament systems in the cytoskeleton are actin filaments (F-actin) and microtubules (MTs). Actin filaments are semiflexible, while the much stiffer MTs behave as rigid rods. I shall discuss theories that help understand how the direct coupling to the surrounding F-actin matrix allows intracellular MTs to bear large compressive forces and controls the range of force transmission along the MTs, and how the MTs not only enhance the stiffness of the cell cytoskeleton, but can also dramatically endow an initially nearly incompressible F-actin matrix with enhanced compressibility relative to its shear compliance. A second source of compositeness in the cytoskeleton is the presences of different types of crosslinkers that can interact cooperatively leading to enhanced mechanical rigidity and tunable response. Like the cytoskeleton, the ECM is also a polymeric composite. It is primarily composed of a mesh of fibrous proteins, mainly stiff collagen filaments, and a comparatively flexible gel of proteoglycans and hyaluronan. I shall discuss a model that shows how the interplay between the collagen network and the background elastic gel leads to a mechanically robust ECM.

  12. Patterning and lifetime of plasma membrane-localized cellulose synthase is dependent on actin organization in Arabidopsis interphase cells

    NARCIS (Netherlands)

    Sampathkumar, A.; Gutierrez, R.; McFarlane, H.E.; Bringmann, M.; Lindeboom, J.J.; Emons, A.M.C.; Samuels, L.; Ketelaar, T.; Ehrhardt, D.W.; Persson, S.

    2013-01-01

    The actin and microtubule cytoskeletons regulate cell shape across phyla, from bacteria to metazoans. In organisms with cell walls, the wall acts as a primary constraint of shape, and generation of specific cell shape depends on cytoskeletal organization for wall deposition and/or cell expansion. In

  13. Drosophila comes of age as a model system for understanding the function of cytoskeletal proteins in cells, tissues, and organisms.

    Science.gov (United States)

    Rodal, Avital A; Del Signore, Steven J; Martin, Adam C

    2015-05-01

    For the last 100 years, Drosophila melanogaster has been a powerhouse genetic system for understanding mechanisms of inheritance, development, and behavior in animals. In recent years, advances in imaging and genetic tools have led to Drosophila becoming one of the most effective systems for unlocking the subcellular functions of proteins (and particularly cytoskeletal proteins) in complex developmental settings. In this review, written for non-Drosophila experts, we will discuss critical technical advances that have enabled these cell biological insights, highlighting three examples of cytoskeletal discoveries that have arisen as a result: (1) regulation of Arp2/3 complex in myoblast fusion, (2) cooperation of the actin filament nucleators Spire and Cappuccino in establishment of oocyte polarity, and (3) coordination of supracellular myosin cables. These specific examples illustrate the unique power of Drosophila both to uncover new cytoskeletal structures and functions, and to place these discoveries in a broader in vivo context, providing insights that would have been impossible in a cell culture model or in vitro. Many of the cellular structures identified in Drosophila have clear counterparts in mammalian cells and tissues, and therefore elucidating cytoskeletal functions in Drosophila will be broadly applicable to other organisms.

  14. Actinic lichen nitidus

    Directory of Open Access Journals (Sweden)

    Loretta Davis

    2010-01-01

    Full Text Available We present the case of a 29-year-old black female with an initial clinical and histopathologic diagnosis of actinic lichen nitidus. Three years later, she presented with scattered hyperpigmented macules with oval pink/viol­aceous plaques bilaterally on her forearms and on her neck, clinically consistent with actinic lichen planus. She was treated with topical steroids at each visit, with subsequent resolution of her lesions. In this report, we discuss the spectrum of actinic lichenoid dermatoses and of disease that presents even in the same patient.

  15. 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.

  16. Small-molecule intramimics of formin autoinhibition: a new strategy to target the cytoskeletal remodeling machinery in cancer cells.

    Science.gov (United States)

    Lash, L Leanne; Wallar, Bradley J; Turner, Julie D; Vroegop, Steven M; Kilkuskie, Robert E; Kitchen-Goosen, Susan M; Xu, H Eric; Alberts, Arthur S

    2013-11-15

    Although the cancer cell cytoskeleton is a clinically validated target, few new strategies have emerged for selectively targeting cell division by modulating the cytoskeletal structure, particularly ways that could avoid the cardiotoxic and neurotoxic effects of current agents such as taxanes. We address this gap by describing a novel class of small-molecule agonists of the mammalian Diaphanous (mDia)-related formins, which act downstream of Rho GTPases to assemble actin filaments, and their organization with microfilaments to establish and maintain cell polarity during migration and asymmetric division. GTP-bound Rho activates mDia family members by disrupting the interaction between the DID and DAD autoregulatory domains, which releases the FH2 domain to modulate actin and microtubule dynamics. In screening for DID-DAD disruptors that activate mDia, we identified two molecules called intramimics (IMM-01 and -02) that were sufficient to trigger actin assembly and microtubule stabilization, serum response factor-mediated gene expression, cell-cycle arrest, and apoptosis. In vivo analysis of IMM-01 and -02 established their ability to slow tumor growth in a mouse xenograft model of colon cancer. Taken together, our work establishes the use of intramimics and mDia-related formins as a new general strategy for therapeutic targeting of the cytoskeletal remodeling machinery of cancer cells.

  17. The role of actin networks in cellular mechanosensing

    Science.gov (United States)

    Azatov, Mikheil

    behavior as in cancer metastasis. In addition to stiffness, the local geometry or topography of the surface has been shown to modulate the movement, morphology, and cytoskeletal organization of cells. However, the effect of topography on fluctuations of intracellular structures, which arise from motor driven activity on a viscoelastic actin network are not known. I have used nanofabricated substrates with parallel ridges to show that the cell shape, the actin cytoskeleton and focal adhesions all align along the direction of the ridges, exhibiting a biphasic dependence on the spacing between ridges. I further demonstrated that palladin bands along actin stress fibers undergo a complex diffusive motion with velocities aligned along the direction of ridges. These results provide insight into the mechanisms of cellular mechanosensing of the environment, suggesting a complex interplay between the actin cytoskeleton and cellular adhesions in coordinating cellular response to surface topography. Overall, this work has advanced our understanding of mechanisms that govern cellular responses to their physical environment.

  18. 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.

  19. Run-and-tumble dynamics of cytoskeletal motor proteins

    CERN Document Server

    Hafner, Anne E; Rieger, Heiko; Shaebani, M Reza

    2016-01-01

    Cytoskeletal motor proteins are involved in major intracellular transport processes which are vital for maintaining appropriate cellular function. The motor exhibits distinct states of motility: active motion along filaments, and effectively stationary phase in which it detaches from the filaments and performs passive diffusion in the vicinity of the detachment point due to cytoplasmic crowding. The transition rates between motion and pause phases are asymmetric in general, and considerably affected by changes in environmental conditions which influences the efficiency of cargo delivery to specific targets. By considering the motion of molecular motor on a single filament as well as a dynamic filamentous network, we present an analytical model for the dynamics of self-propelled particles which undergo frequent pause phases. The interplay between motor processivity, structural properties of filamentous network, and transition rates between the two states of motility drastically changes the dynamics: multiple t...

  20. Activation of 5-HT7 receptor stimulates neurite elongation through mTOR, Cdc42 and actin filaments dynamics.

    Science.gov (United States)

    Speranza, Luisa; Giuliano, Teresa; Volpicelli, Floriana; De Stefano, M Egle; Lombardi, Loredana; Chambery, Angela; Lacivita, Enza; Leopoldo, Marcello; Bellenchi, Gian C; di Porzio, Umberto; Crispino, Marianna; Perrone-Capano, Carla

    2015-01-01

    Recent studies have indicated that the serotonin receptor subtype 7 (5-HT7R) plays a crucial role in shaping neuronal morphology during embryonic and early postnatal life. Here we show that pharmacological stimulation of 5-HT7R using a highly selective agonist, LP-211, enhances neurite outgrowth in neuronal primary cultures from the cortex, hippocampus and striatal complex of embryonic mouse brain, through multiple signal transduction pathways. All these signaling systems, involving mTOR, the Rho GTPase Cdc42, Cdk5, and ERK, are known to converge on the reorganization of cytoskeletal proteins that subserve neurite outgrowth. Indeed, our data indicate that neurite elongation stimulated by 5-HT7R is modulated by drugs affecting actin polymerization. In addition, we show, by 2D Western blot analyses, that treatment of neuronal cultures with LP-211 alters the expression profile of cofilin, an actin binding protein involved in microfilaments dynamics. Furthermore, by using microfluidic chambers that physically separate axons from the soma and dendrites, we demonstrate that agonist-dependent activation of 5-HT7R stimulates axonal elongation. Our results identify for the first time several signal transduction pathways, activated by stimulation of 5-HT7R, that converge to promote cytoskeleton reorganization and consequent modulation of axonal elongation. Therefore, the activation of 5-HT7R might represent one of the key elements regulating CNS connectivity and plasticity during development.

  1. Activation of 5-HT7 receptor stimulates neurite elongation through mTOR, Cdc42 and actin filaments dynamics

    Directory of Open Access Journals (Sweden)

    Luisa eSperanza

    2015-03-01

    Full Text Available Recent studies have indicated that the serotonin receptor subtype 7 (5-HT7R plays a crucial role in shaping neuronal morphology during embryonic and early postnatal life. Here we show that pharmacological stimulation of 5-HT7R using a highly selective agonist, LP-211, enhances neurite outgrowth in neuronal primary cultures from the cortex, hippocampus and striatal complex of embryonic mouse brain, through multiple signal transduction pathways. All these signaling systems, involving mTOR, the Rho GTPase Cdc42, Cdk5 and ERK, are known to converge on the reorganization of cytoskeletal proteins that subserve neurite outgrowth. Indeed, our data indicate that neurite elongation stimulated by 5-HT7R is modulated by drugs affecting actin polymerization.In addition, we show, by 2D western blot analyses, that treatment of neuronal cultures with LP-211 alters the expression profile of cofilin, an actin binding protein involved in microfilaments dynamics. Furthermore, by using microfluidic chambers that physically separate axons from the soma and dendrites, we demonstrate that agonist-dependent activation of 5-HT7R stimulates axonal elongation. Our results identify for the first time several signal transduction pathways, activated by stimulation of 5-HT7R, that converge to promote cytoskeleton reorganization and consequent modulation of axonal elongation. Therefore, the activation of 5-HT7R might represent one of the key elements regulating CNS connectivity and plasticity during development.

  2. Erythrocyte shape abnormalities, membrane oxidative damage, and β-actin alterations: an unrecognized triad in classical autism.

    Science.gov (United States)

    Ciccoli, Lucia; De Felice, Claudio; Paccagnini, Eugenio; Leoncini, Silvia; Pecorelli, Alessandra; Signorini, Cinzia; Belmonte, Giuseppe; Guerranti, Roberto; Cortelazzo, Alessio; Gentile, Mariangela; Zollo, Gloria; Durand, Thierry; Valacchi, Giuseppe; Rossi, Marcello; Hayek, Joussef

    2013-01-01

    Autism spectrum disorders (ASDs) are a complex group of neurodevelopment disorders steadily rising in frequency and treatment refractory, where the search for biological markers is of paramount importance. Although red blood cells (RBCs) membrane lipidomics and rheological variables have been reported to be altered, with some suggestions indicating an increased lipid peroxidation in the erythrocyte membrane, to date no information exists on how the oxidative membrane damage may affect cytoskeletal membrane proteins and, ultimately, RBCs shape in autism. Here, we investigated RBC morphology by scanning electron microscopy in patients with classical autism, that is, the predominant ASDs phenotype (age range: 6-26 years), nonautistic neurodevelopmental disorders (i.e., "positive controls"), and healthy controls (i.e., "negative controls"). A high percentage of altered RBCs shapes, predominantly elliptocytes, was observed in autistic patients, but not in both control groups. The RBCs altered morphology in autistic subjects was related to increased erythrocyte membrane F2-isoprostanes and 4-hydroxynonenal protein adducts. In addition, an oxidative damage of the erythrocyte membrane β-actin protein was evidenced. Therefore, the combination of erythrocyte shape abnormalities, erythrocyte membrane oxidative damage, and β-actin alterations constitutes a previously unrecognized triad in classical autism and provides new biological markers in the diagnostic workup of ASDs.

  3. Erythrocyte Shape Abnormalities, Membrane Oxidative Damage, and β-Actin Alterations: An Unrecognized Triad in Classical Autism

    Directory of Open Access Journals (Sweden)

    Lucia Ciccoli

    2013-01-01

    Full Text Available Autism spectrum disorders (ASDs are a complex group of neurodevelopment disorders steadily rising in frequency and treatment refractory, where the search for biological markers is of paramount importance. Although red blood cells (RBCs membrane lipidomics and rheological variables have been reported to be altered, with some suggestions indicating an increased lipid peroxidation in the erythrocyte membrane, to date no information exists on how the oxidative membrane damage may affect cytoskeletal membrane proteins and, ultimately, RBCs shape in autism. Here, we investigated RBC morphology by scanning electron microscopy in patients with classical autism, that is, the predominant ASDs phenotype (age range: 6–26 years, nonautistic neurodevelopmental disorders (i.e., “positive controls”, and healthy controls (i.e., “negative controls”. A high percentage of altered RBCs shapes, predominantly elliptocytes, was observed in autistic patients, but not in both control groups. The RBCs altered morphology in autistic subjects was related to increased erythrocyte membrane F2-isoprostanes and 4-hydroxynonenal protein adducts. In addition, an oxidative damage of the erythrocyte membrane β-actin protein was evidenced. Therefore, the combination of erythrocyte shape abnormalities, erythrocyte membrane oxidative damage, and β-actin alterations constitutes a previously unrecognized triad in classical autism and provides new biological markers in the diagnostic workup of ASDs.

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

    Science.gov (United States)

    Estes, Kathleen A; Szumowski, Suzannah C; Troemel, Emily R

    2011-09-01

    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.

  5. 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.

  6. The role of actin turnover in retrograde actin network flow in neuronal growth cones.

    Directory of Open Access Journals (Sweden)

    David Van Goor

    Full Text Available The balance of actin filament polymerization and depolymerization maintains a steady state network treadmill in neuronal growth cones essential for motility and guidance. Here we have investigated the connection between depolymerization and treadmilling dynamics. We show that polymerization-competent barbed ends are concentrated at the leading edge and depolymerization is distributed throughout the peripheral domain. We found a high-to-low G-actin gradient between peripheral and central domains. Inhibiting turnover with jasplakinolide collapsed this gradient and lowered leading edge barbed end density. Ultrastructural analysis showed dramatic reduction of leading edge actin filament density and filament accumulation in central regions. Live cell imaging revealed that the leading edge retracted even as retrograde actin flow rate decreased exponentially. Inhibition of myosin II activity before jasplakinolide treatment lowered baseline retrograde flow rates and prevented leading edge retraction. Myosin II activity preferentially affected filopodial bundle disassembly distinct from the global effects of jasplakinolide on network turnover. We propose that growth cone retraction following turnover inhibition resulted from the persistence of myosin II contractility even as leading edge assembly rates decreased. The buildup of actin filaments in central regions combined with monomer depletion and reduced polymerization from barbed ends suggests a mechanism for the observed exponential decay in actin retrograde flow. Our results show that growth cone motility is critically dependent on continuous disassembly of the peripheral actin network.

  7. Effects of latrunculin B on the actin cytoskeleton and hyphal growth in Phytophthora infestans.

    Science.gov (United States)

    Ketelaar, Tijs; Meijer, Harold J G; Spiekerman, Marjolein; Weide, Rob; Govers, Francine

    2012-12-01

    The actin cytoskeleton is conserved in all eukaryotes, but its functions vary among different organisms. In oomycetes, the function of the actin cytoskeleton has received relatively little attention. We have performed a bioinformatics study and show that oomycete actin genes fall within a distinct clade that is divergent from plant, fungal and vertebrate actin genes. To obtain a better understanding of the functions of the actin cytoskeleton in hyphal growth of oomycetes, we studied the actin organization in Phytophthora infestans hyphae and the consequences of treatment with the actin depolymerising drug latrunculin B (latB). This revealed that latB treatment causes a concentration dependent inhibition of colony expansion and aberrant hyphal growth. The most obvious aberrations observed upon treatment with 0.1 μM latB were increased hyphal branching and irregular tube diameters whereas at higher concentrations latB (0.5 and 1 μM) tips of expanding hyphae changed into balloon-like shapes. This aberrant growth correlated with changes in the organization of the actin cytoskeleton. In untreated hyphae, staining with fluorescently tagged phalloidin revealed two populations of actin filaments: long, axially oriented actin filament cables and cortical actin filament plaques. Two hyphal subtypes were recognized, one containing only plaques and the other containing both cables and plaques. In the latter, some hyphae had an apical zone without actin filament plaques. Upon latB treatment, the proportion of hyphae without actin filament cables increased and there were more hyphae with a short apical zone without actin filament plaques. In general, actin filament plaques were more resilient against actin depolymerisation than actin filament cables. Besides disturbing hyphal growth and actin organization, actin depolymerisation also affected the positioning of nuclei. In the presence of latB, the distance between nuclei and the hyphal tip decreased, suggesting that the actin

  8. Photodynamic therapy for the treatment of actinic cheilitis.

    Science.gov (United States)

    Kodama, Makiko; Watanabe, Daisuke; Akita, Yoichi; Tamada, Yasuhiko; Matsumoto, Yoshinari

    2007-10-01

    Although actinic cheilitis is a common disease, it should be treated carefully because it can undergo malignant transformation. We report a case of actinic cheilitis treated with photodynamic therapy (PDT) using 5-aminolevulinic acid (ALA), with satisfactory outcome in both clinical and pathological aspects. Actinic cheilitis is a pathologic condition affecting mainly the lower lip caused by long-term exposure of the lips to the UV radiation in sunlight. Analogous to actinic keratosis of the skin, actinic cheilitis is considered as a precancerous lesion and it may develop into squamous cell carcinoma. We report a case of actinic cheilitis treated with PDT using ALA, with satisfactory outcome in both clinical and pathological aspects.

  9. Purification of recombinant human and Drosophila septin hexamers for TIRF assays of actin-septin filament assembly.

    Science.gov (United States)

    Mavrakis, M; Tsai, F-C; Koenderink, G H

    2016-01-01

    Septins are guanine nucleotide-binding proteins that are conserved from fungi to humans. Septins assemble into heterooligomeric complexes and higher-order structures with key roles in various cellular functions including cell migration and division. The mechanisms by which septins assemble and interact with other cytoskeletal elements like actin remain elusive. A powerful approach to address this question is by cell-free reconstitution of purified cytoskeletal proteins combined with fluorescence microscopy. Here, we describe procedures for the purification of recombinant Drosophila and human septin hexamers from Escherichia coli and reconstitution of actin-septin coassembly. These procedures can be used to compare assembly of Drosophila and human septins and their coassembly with the actin cytoskeleton by total internal reflection fluorescence microscopy.

  10. Electrostatics control actin filament nucleation and elongation kinetics.

    Science.gov (United States)

    Crevenna, Alvaro H; Naredi-Rainer, Nikolaus; Schönichen, André; Dzubiella, Joachim; Barber, Diane L; Lamb, Don C; Wedlich-Söldner, Roland

    2013-04-26

    The actin cytoskeleton is a central mediator of cellular morphogenesis, and rapid actin reorganization drives essential processes such as cell migration and cell division. Whereas several actin-binding proteins are known to be regulated by changes in intracellular pH, detailed information regarding the effect of pH on the actin dynamics itself is still lacking. Here, we combine bulk assays, total internal reflection fluorescence microscopy, fluorescence fluctuation spectroscopy techniques, and theory to comprehensively characterize the effect of pH on actin polymerization. We show that both nucleation and elongation are strongly enhanced at acidic pH, with a maximum close to the pI of actin. Monomer association rates are similarly affected by pH at both ends, although dissociation rates are differentially affected. This indicates that electrostatics control the diffusional encounter but not the dissociation rate, which is critical for the establishment of actin filament asymmetry. A generic model of protein-protein interaction, including electrostatics, explains the observed pH sensitivity as a consequence of charge repulsion. The observed pH effect on actin in vitro agrees with measurements of Listeria propulsion in pH-controlled cells. pH regulation should therefore be considered as a modulator of actin dynamics in a cellular environment.

  11. 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.

  12. Drosophila cyfip regulates synaptic development and endocytosis by suppressing filamentous actin assembly.

    Science.gov (United States)

    Zhao, Lu; Wang, Dan; Wang, Qifu; Rodal, Avital A; Zhang, Yong Q

    2013-04-01

    The formation of synapses and the proper construction of neural circuits depend on signaling pathways that regulate cytoskeletal structure and dynamics. After the mutual recognition of a growing axon and its target, multiple signaling pathways are activated that regulate cytoskeletal dynamics to determine the morphology and strength of the connection. By analyzing Drosophila mutations in the cytoplasmic FMRP interacting protein Cyfip, we demonstrate that this component of the WAVE complex inhibits the assembly of filamentous actin (F-actin) and thereby regulates key aspects of synaptogenesis. Cyfip regulates the distribution of F-actin filaments in presynaptic neuromuscular junction (NMJ) terminals. At cyfip mutant NMJs, F-actin assembly was accelerated, resulting in shorter NMJs, more numerous satellite boutons, and reduced quantal content. Increased synaptic vesicle size and failure to maintain excitatory junctional potential amplitudes under high-frequency stimulation in cyfip mutants indicated an endocytic defect. cyfip mutants exhibited upregulated bone morphogenetic protein (BMP) signaling, a major growth-promoting pathway known to be attenuated by endocytosis at the Drosophila NMJ. We propose that Cyfip regulates synapse development and endocytosis by inhibiting actin assembly.

  13. F-actin-like filaments formed by plasmid segregation protein ParM

    DEFF Research Database (Denmark)

    van den Ent, Fusinita; Møller-Jensen, Jakob; Amos, Linda A.;

    2002-01-01

    It was the general belief that DNA partitioning in prokaryotes is independent of a cytoskeletal structure, which in eukaryotic cells is indispensable for DNA segregation. Recently, however, immunofluorescence microscopy revealed highly dynamic, filamentous structures along the longitudinal axis...... of Escherichia coli formed by ParM, a plasmid-encoded protein required for accurate segregation of low-copy-number plasmid R1. We show here that ParM polymerizes into double helical protofilaments with a longitudinal repeat similar to filamentous actin (F-actin) and MreB filaments that maintain the cell shape...

  14. ZEB1 drives prometastatic actin cytoskeletal remodeling by downregulating miR-34a expression

    OpenAIRE

    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 proc...

  15. Differential remodeling of actin cytoskeleton architecture by profilin isoforms leads to distinct effects on cell migration and invasion.

    Science.gov (United States)

    Mouneimne, Ghassan; Hansen, Scott D; Selfors, Laura M; Petrak, Lara; Hickey, Michele M; Gallegos, Lisa L; Simpson, Kaylene J; Lim, James; Gertler, Frank B; Hartwig, John H; Mullins, R Dyche; Brugge, Joan S

    2012-11-13

    Dynamic actin cytoskeletal reorganization is integral to cell motility. Profilins are well-characterized regulators of actin polymerization; however, functional differences among coexpressed profilin isoforms are not well defined. Here, we demonstrate that profilin-1 and profilin-2 differentially regulate membrane protrusion, motility, and invasion; these processes are promoted by profilin-1 and suppressed by profilin-2. Compared to profilin-1, profilin-2 preferentially drives actin polymerization by the Ena/VASP protein, EVL. Profilin-2 and EVL suppress protrusive activity and cell motility by an actomyosin contractility-dependent mechanism. Importantly, EVL or profilin-2 downregulation enhances invasion in vitro and in vivo. In human breast cancer, lower EVL expression correlates with high invasiveness and poor patient outcome. We propose that profilin-2/EVL-mediated actin polymerization enhances actin bundling and suppresses breast cancer cell invasion.

  16. Three-dimensional Organization of Layered Apical Cytoskeletal Networks Associated with Mouse Airway Tissue Development

    Science.gov (United States)

    Tateishi, Kazuhiro; Nishida, Tomoki; Inoue, Kanako; Tsukita, Sachiko

    2017-03-01

    The cytoskeleton is an essential cellular component that enables various sophisticated functions of epithelial cells by forming specialized subcellular compartments. However, the functional and structural roles of cytoskeletons in subcellular compartmentalization are still not fully understood. Here we identified a novel network structure consisting of actin filaments, intermediate filaments, and microtubules directly beneath the apical membrane in mouse airway multiciliated cells and in cultured epithelial cells. Three-dimensional imaging by ultra-high voltage electron microscopy and immunofluorescence revealed that the morphological features of each network depended on the cell type and were spatiotemporally integrated in association with tissue development. Detailed analyses using Odf2 mutant mice, which lack ciliary basal feet and apical microtubules, suggested a novel contribution of the intermediate filaments to coordinated ciliary beating. These findings provide a new perspective for viewing epithelial cell differentiation and tissue morphogenesis through the structure and function of apical cytoskeletal networks.

  17. Cytoskeletal Linker Protein Dystonin Is Not Critical to Terminal Oligodendrocyte Differentiation or CNS Myelination.

    Directory of Open Access Journals (Sweden)

    Samantha F Kornfeld

    Full Text Available Oligodendrocyte differentiation and central nervous system myelination require massive reorganization of the oligodendrocyte cytoskeleton. Loss of specific actin- and tubulin-organizing factors can lead to impaired morphological and/or molecular differentiation of oligodendrocytes, resulting in a subsequent loss of myelination. Dystonin is a cytoskeletal linker protein with both actin- and tubulin-binding domains. Loss of function of this protein results in a sensory neuropathy called Hereditary Sensory Autonomic Neuropathy VI in humans and dystonia musculorum in mice. This disease presents with severe ataxia, dystonic muscle and is ultimately fatal early in life. While loss of the neuronal isoforms of dystonin primarily leads to sensory neuron degeneration, it has also been shown that peripheral myelination is compromised due to intrinsic Schwann cell differentiation abnormalities. The role of this cytoskeletal linker in oligodendrocytes, however, remains unclear. We sought to determine the effects of the loss of neuronal dystonin on oligodendrocyte differentiation and central myelination. To address this, primary oligodendrocytes were isolated from a severe model of dystonia musculorum, Dstdt-27J, and assessed for morphological and molecular differentiation capacity. No defects could be discerned in the differentiation of Dstdt-27J oligodendrocytes relative to oligodendrocytes from wild-type littermates. Survival was also compared between Dstdt-27J and wild-type oligodendrocytes, revealing no significant difference. Using a recently developed migration assay, we further analysed the ability of primary oligodendrocyte progenitor cell motility, and found that Dstdt-27J oligodendrocyte progenitor cells were able to migrate normally. Finally, in vivo analysis of oligodendrocyte myelination was done in phenotype-stage optic nerve, cerebral cortex and spinal cord. The density of myelinated axons and g-ratios of Dstdt-27J optic nerves was normal, as

  18. Directed actin assembly and motility.

    Science.gov (United States)

    Boujemaa-Paterski, Rajaa; Galland, Rémi; Suarez, Cristian; Guérin, Christophe; Théry, Manuel; Blanchoin, Laurent

    2014-01-01

    The actin cytoskeleton is a key component of the cellular architecture. However, understanding actin organization and dynamics in vivo is a complex challenge. Reconstitution of actin structures in vitro, in simplified media, allows one to pinpoint the cellular biochemical components and their molecular interactions underlying the architecture and dynamics of the actin network. Previously, little was known about the extent to which geometrical constraints influence the dynamic ultrastructure of these networks. Therefore, in order to study the balance between biochemical and geometrical control of complex actin organization, we used the innovative methodologies of UV and laser patterning to design a wide repertoire of nucleation geometries from which we assembled branched actin networks. Using these methods, we were able to reconstitute complex actin network organizations, closely related to cellular architecture, to precisely direct and control their 3D connections. This methodology mimics the actin networks encountered in cells and can serve in the fabrication of innovative bioinspired systems.

  19. In vivo rescue of alveolar macrophages from SP-A knockout mice with exogenous SP-A nearly restores a wild type intracellular proteome; actin involvement

    Directory of Open Access Journals (Sweden)

    Floros Joanna

    2011-10-01

    Full Text Available Abstract Background Mice lacking surfactant protein-A (SP-A-/-; knockout; KO exhibit increased vulnerability to infection and injury. Although many bronchoalveolar lavage (BAL protein differences between KO and wild-type (WT are rapidly reversed in KO after infection, their clinical course is still compromised. We studied the impact of SP-A on the alveolar macrophage (AM proteome under basal conditions. Male SP-A KO mice were SP-A-treated (5 micrograms/mouse and sacrificed in 6 or 18 hr. The AM proteomes of KO, SP-A-treated KO, and WT mice were studied by 2D-DIGE coupled with MALDI-ToF/ToF and AM actin distribution was examined by phalloidon staining. Results We observed: a significant differences from KO in WT or exogenous SP-A-treated in 45 of 76 identified proteins (both increases and decreases. These included actin-related/cytoskeletal proteins (involved in motility, phagocytosis, endocytosis, proteins of intracellular signaling, cell differentiation/regulation, regulation of inflammation, protease/chaperone function, and proteins related to Nrf2-mediated oxidative stress response pathway; b SP-A-induced changes causing the AM proteome of the KO to resemble that of WT; and c that SP-A treatment altered cell size and F-actin distribution. Conclusions These differences are likely to enhance AM function. The observations show for the first time that acute in vivo SP-A treatment of KO mice, under basal or unstimulated conditions, affects the expression of multiple AM proteins, alters F-actin distribution, and can restore much of the WT phenotype. We postulate that the SP-A-mediated expression profile of the AM places it in a state of "readiness" to successfully conduct its innate immune functions and ensure lung health.

  20. 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.

  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.

  2. 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.

  3. Expression patterns of ubiquitin, heat shock protein 70, alpha-actin and beta-actin over the molt cycle in the abdominal muscle of marine shrimp Litopenaeus vannamei.

    Science.gov (United States)

    Cesar, Jose Renato O; Yang, Jinzeng

    2007-05-01

    Crustacean muscle growth is discontinuous due to molt cycle. To characterize molt-related gene expression patterns, we studied the mRNA levels of molecular chaperone-ubiquitin and heat shock protein 70 (Hsp 70) in comparison with muscle protein alpha-actin and beta-actin in marine shrimp Litopenaeus vannamei. Total RNA from abdominal muscle was isolated from 3-month-old animals in six different molt stages. The mRNA levels of target genes were detected by reverse-transcriptase-multiplex PCR and expressed as the ratio to elongation factor-1alpha. Ubiquitin mRNA levels were relatively steady over all stages of the molt cycle. Hsp70 levels were not detectable in early postmolt and late premolt stages, but showed a progressive increase from late postmolt to intermolt stages. Expression levels of alpha-actin gene were lower during postmolt, reached a plateau in intermolt and remained relatively high in premolt stage. Levels of beta-actin increased progressively from postmolt to intermolt, reaching a maximum value in premolt. Therefore, the mRNAs encoding for ubiquitin and Hsp 70 in abdominal muscle did not increase significantly in premolt stages, which is typically associated with claw muscle degradation. Muscle structural alpha-actin and cytoskeletal beta-actin were increased during intermolt and premolt stages, suggesting high muscle growth during these stages in the abdominal muscle of the L. vannamei.

  4. An actin monomer binding activity localizes to the carboxyl-terminal half of the Saccharomyces cerevisiae cyclase-associated protein.

    Science.gov (United States)

    Freeman, N L; Chen, Z; Horenstein, J; Weber, A; Field, J

    1995-03-10

    The Saccharomyces cerevisiae adenylyl cyclase complex contains at least two subunits, a 200-kDa catalytic subunit and a 70-kDa cyclase-associated protein, CAP (also called Srv2p). Genetic studies suggested two roles for CAP, one as a positive regulator of cAMP levels in yeast and a second role as a cytoskeletal regulator. We present evidence showing that CAP sequesters monomeric actin (Kd in the range of 0.5-5 microM), decreasing actin incorporation into actin filaments. Anti-CAP monoclonal antibodies co-immunoprecipitate a protein with a molecular size of about 46 kDa. When CAP was purified from yeast using an anti-CAP monoclonal antibody column, the 46-kDa protein co-purified with a stoichiometry of about 1:1 with CAP. Western blots identified the 46-kDa protein as yeast actin. CAP also bound to muscle actin in vitro in immunoprecipitation assays and falling ball viscometry assays. Experiments with pyrene-labeled actin demonstrated that CAP sequesters actin monomers. The actin monomer binding activity is localized to the carboxyl-terminal half of CAP. Together, these data suggest that yeast CAP regulates the yeast cytoskeleton by sequestering actin monomers.

  5. 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, R. A.

    2016-01-01

    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......MSC by regulating cytoskeleton organization. Targeting TAGLN is a plausible approach to enrich for committed hMSC cells needed for regenerative medicine application....

  6. Purinoreceptor P2X7 Regulation of Ca(2+) Mobilization and Cytoskeletal Rearrangement Is Required for Corneal Reepithelialization after Injury.

    Science.gov (United States)

    Minns, Martin S; Teicher, Gregory; Rich, Celeste B; Trinkaus-Randall, Vickery

    2016-02-01

    The process of wound healing involves a complex network of signaling pathways working to promote rapid cell migration and wound closure. Activation of purinergic receptors by secreted nucleotides plays a major role in calcium mobilization and the subsequent calcium-dependent signaling that is essential for proper healing. The role of the purinergic receptor P2X7 in wound healing is still relatively unknown. We demonstrate that P2X7 expression increases at the leading edge of corneal epithelium after injury in an organ culture model, and that this change occurs despite an overall decrease in P2X7 expression throughout the epithelium. Inhibition of P2X7 prevents this change in localization after injury and impairs wound healing. In cell culture, P2X7 inhibition attenuates the amplitude and duration of injury-induced calcium mobilization in cells at the leading edge. Immunofluorescence analysis of scratch-wounded cells reveals that P2X7 inhibition results in an overall decrease in the number of focal adhesions along with a concentration of focal adhesions at the wound margin. Live cell imaging of green fluorescent protein-labeled actin and talin shows that P2X7 inhibition alters actin cytoskeletal rearrangements and focal adhesion dynamics after injury. Together, these data demonstrate that P2X7 plays a critical role in mediating calcium signaling and coordinating cytoskeletal rearrangement at the leading edge, both of which processes are early signaling events necessary for proper epithelial wound healing.

  7. Purinoreceptor P2X7 Regulation of Ca2+ Mobilization and Cytoskeletal Rearrangement Is Required for Corneal Reepithelialization after Injury

    Science.gov (United States)

    Minns, Martin S.; Teicher, Gregory; Rich, Celeste B.; Trinkaus-Randall, Vickery

    2017-01-01

    The process of wound healing involves a complex network of signaling pathways working to promote rapid cell migration and wound closure. Activation of purinergic receptors by secreted nucleotides plays a major role in calcium mobilization and the subsequent calcium-dependent signaling that is essential for proper healing. The role of the purinergic receptor P2X7 in wound healing is still relatively unknown. We demonstrate that P2X7 expression increases at the leading edge of corneal epithelium after injury in an organ culture model, and that this change occurs despite an overall decrease in P2X7 expression throughout the epithelium. Inhibition of P2X7 prevents this change in localization after injury and impairs wound healing. In cell culture, P2X7 inhibition attenuates the amplitude and duration of injury-induced calcium mobilization in cells at the leading edge. Immunofluorescence analysis of scratch-wounded cells reveals that P2X7 inhibition results in an overall decrease in the number of focal adhesions along with a concentration of focal adhesions at the wound margin. Live cell imaging of green fluorescent protein–labeled actin and talin shows that P2X7 inhibition alters actin cytoskeletal rearrangements and focal adhesion dynamics after injury. Together, these data demonstrate that P2X7 plays a critical role in mediating calcium signaling and coordinating cytoskeletal rearrangement at the leading edge, both of which processes are early signaling events necessary for proper epithelial wound healing. PMID:26683661

  8. beta-Dystroglycan modulates the interplay between actin and microtubules in human-adhered platelets.

    Science.gov (United States)

    Cerecedo, Doris; Cisneros, Bulmaro; Suárez-Sánchez, Rocío; Hernández-González, Enrique; Galván, Iván

    2008-05-01

    To maintain the continuity of an injured blood vessel, platelets change shape, secrete granule contents, adhere, aggregate, and retract in a haemostatic plug. Ordered arrays of microtubules, microfilaments, and associated proteins are responsible for these platelet responses. In full-spread platelets, microfilament bundles in association with other cytoskeleton proteins are anchored in focal contacts. Recent studies in migrating cells suggest that co-ordination and direct physical interaction of microtubules and actin network modulate adhesion development. In platelets, we have proposed a feasible association between these two cytoskeletal systems, as well as the participation of the dystrophin-associated protein complex, as part of the focal adhesion complex. The present study analysed the participation of microtubules and actin during the platelet adhesion process. Confocal microscopy, fluorescence resonance transfer energy and immunoprecipitation assays were used to provide evidence of a cross-talk between these two cytoskeletal systems. Interestingly, beta-dystroglycan was found to act as an interplay protein between actin and microtubules and an additional communication between these two cytoskeleton networks was maintained through proteins of focal adhesion complex. Altogether our data are indicative of a dynamic co-participation of actin filaments and microtubules in modulating focal contacts to achieve platelet function.

  9. Spectrin-dependent and -independent association of F-actin with the erythrocyte membrane.

    Science.gov (United States)

    Cohen, C M; Foley, S F

    1980-08-01

    Binding of F-actin to spectrin-actin-depleted erythrocyte membrane inside-out vesicles was measured using [3H]F-actin. F-actin binding to vesicles at 25 degrees C was stimulated 5-10 fold by addition of spectrin dimers or tetramers to vesicles. Spectrin tetramer was twice as effective as dimer in stimulating actin binding, but neither tetramer nor dimer stimulated binding at 4 degrees C. The addition of purified erythrocyte membrane protein band 4.1 to spectrin-reconstituted vesicles doubled their actin-binding capacity. Trypsinization of unreconstituted vesicles that contain ghosts, decreased their F-actin-binding capacity by 70%. Whereas little or none of the residual spectrin was affected by trypsinization, band 4.1 was significantly degraded. Our results show that spectrin can anchor actin filaments to the cytoplasmic surface of erythrocyte membranes and suggest that band 4.1 may be importantly involved in the association.

  10. The actin-interacting protein AIP1 is essential for actin organization and plant development

    NARCIS (Netherlands)

    Ketelaar, T.; Anthony, R.G.; Voigt, B.; Menzel, D.; Hussey, P.J.

    2004-01-01

    Cell division, growth, and cytoplasmic organization require a dynamic actin cytoskeleton. The filamentous actin (F-actin) network is regulated by actin binding proteins that modulate actin dynamics. These actin binding proteins often have cooperative interactions [1 and 2]. In particular, actin inte

  11. Reinforcement versus fluidization in cytoskeletal mechanoresponsiveness.

    Directory of Open Access Journals (Sweden)

    Ramaswamy Krishnan

    Full Text Available Every adherent eukaryotic cell exerts appreciable traction forces upon its substrate. Moreover, every resident cell within the heart, great vessels, bladder, gut or lung routinely experiences large periodic stretches. As an acute response to such stretches the cytoskeleton can stiffen, increase traction forces and reinforce, as reported by some, or can soften and fluidize, as reported more recently by our laboratory, but in any given circumstance it remains unknown which response might prevail or why. Using a novel nanotechnology, we show here that in loading conditions expected in most physiological circumstances the localized reinforcement response fails to scale up to the level of homogeneous cell stretch; fluidization trumps reinforcement. Whereas the reinforcement response is known to be mediated by upstream mechanosensing and downstream signaling, results presented here show the fluidization response to be altogether novel: it is a direct physical effect of mechanical force acting upon a structural lattice that is soft and fragile. Cytoskeletal softness and fragility, we argue, is consistent with early evolutionary adaptations of the eukaryotic cell to material properties of a soft inert microenvironment.

  12. Reinforcement versus fluidization in cytoskeletal mechanoresponsiveness.

    Science.gov (United States)

    Krishnan, Ramaswamy; Park, Chan Young; Lin, Yu-Chun; Mead, Jere; Jaspers, Richard T; Trepat, Xavier; Lenormand, Guillaume; Tambe, Dhananjay; Smolensky, Alexander V; Knoll, Andrew H; Butler, James P; Fredberg, Jeffrey J

    2009-01-01

    Every adherent eukaryotic cell exerts appreciable traction forces upon its substrate. Moreover, every resident cell within the heart, great vessels, bladder, gut or lung routinely experiences large periodic stretches. As an acute response to such stretches the cytoskeleton can stiffen, increase traction forces and reinforce, as reported by some, or can soften and fluidize, as reported more recently by our laboratory, but in any given circumstance it remains unknown which response might prevail or why. Using a novel nanotechnology, we show here that in loading conditions expected in most physiological circumstances the localized reinforcement response fails to scale up to the level of homogeneous cell stretch; fluidization trumps reinforcement. Whereas the reinforcement response is known to be mediated by upstream mechanosensing and downstream signaling, results presented here show the fluidization response to be altogether novel: it is a direct physical effect of mechanical force acting upon a structural lattice that is soft and fragile. Cytoskeletal softness and fragility, we argue, is consistent with early evolutionary adaptations of the eukaryotic cell to material properties of a soft inert microenvironment.

  13. Recent advances into vanadyl, vanadate and decavanadate interactions with actin.

    Science.gov (United States)

    Ramos, S; Moura, J J G; Aureliano, M

    2012-01-01

    Although the number of papers about "vanadium" has doubled in the last decade, the studies about "vanadium and actin" are scarce. In the present review, the effects of vanadyl, vanadate and decavanadate on actin structure and function are compared. Decavanadate (51)V NMR signals, at -516 ppm, broadened and decreased in intensity upon actin titration, whereas no effects were observed for vanadate monomers, at -560 ppm. Decavanadate is the only species inducing actin cysteine oxidation and vanadyl formation, both processes being prevented by the natural ligand of the protein, ATP. Vanadyl titration with monomeric actin (G-actin), analysed by EPR spectroscopy, reveals a 1:1 binding stoichiometry and a K(d) of 7.5 μM(-1). Both decavanadate and vanadyl inhibited G-actin polymerization into actin filaments (F-actin), with a IC(50) of 68 and 300 μM, respectively, as analysed by light scattering assays, whereas no effects were detected for vanadate up to 2 mM. However, only vanadyl (up to 200 μM) induces 100% of G-actin intrinsic fluorescence quenching, whereas decavanadate shows an opposite effect, which suggests the presence of vanadyl high affinity actin binding sites. Decavanadate increases (2.6-fold) the actin hydrophobic surface, evaluated using the ANSA probe, whereas vanadyl decreases it (15%). Both vanadium species increased the ε-ATP exchange rate (k = 6.5 × 10(-3) s(-1) and 4.47 × 10(-3) s(-1) for decavanadate and vanadyl, respectively). Finally, (1)H NMR spectra of G-actin treated with 0.1 mM decavanadate clearly indicate that major alterations occur in protein structure, which are much less visible in the presence of ATP, confirming the preventive effect of the nucleotide on the decavanadate interaction with the protein. Putting it all together, it is suggested that actin, which is involved in many cellular processes, might be a potential target not only for decavanadate but above all for vanadyl. By affecting actin structure and function, vanadium can

  14. Actin capping protein alpha maintains vestigial-expressing cells within the Drosophila wing disc epithelium.

    Science.gov (United States)

    Janody, Florence; Treisman, Jessica E

    2006-09-01

    Tissue patterning must be translated into morphogenesis through cell shape changes mediated by remodeling of the actin cytoskeleton. We have found that Capping protein alpha (Cpa) and Capping protein beta (Cpb), which prevent extension of the barbed ends of actin filaments, are specifically required in the wing blade primordium of the Drosophila wing disc. cpa or cpb mutant cells in this region, but not in the remainder of the wing disc, are extruded from the epithelium and undergo apoptosis. Excessive actin filament polymerization is not sufficient to explain this phenotype, as loss of Cofilin or Cyclase-associated protein does not cause cell extrusion or death. Misexpression of Vestigial, the transcription factor that specifies the wing blade, both increases cpa transcription and makes cells dependent on cpa for their maintenance in the epithelium. Our results suggest that Vestigial specifies the cytoskeletal changes that lead to morphogenesis of the adult wing.

  15. Live Cell Imaging Reveals Structural Associations between the Actin and Microtubule Cytoskeleton in Arabidopsis [W] [OA

    Science.gov (United States)

    Sampathkumar, Arun; Lindeboom, Jelmer J.; Debolt, Seth; Gutierrez, Ryan; Ehrhardt, David W.; Ketelaar, Tijs; Persson, Staffan

    2011-01-01

    In eukaryotic cells, the actin and microtubule (MT) cytoskeletal networks are dynamic structures that organize intracellular processes and facilitate their rapid reorganization. In plant cells, actin filaments (AFs) and MTs are essential for cell growth and morphogenesis. However, dynamic interactions between these two essential components in live cells have not been explored. Here, we use spinning-disc confocal microscopy to dissect interaction and cooperation between cortical AFs and MTs in Arabidopsis thaliana, utilizing fluorescent reporter constructs for both components. Quantitative analyses revealed altered AF dynamics associated with the positions and orientations of cortical MTs. Reorganization and reassembly of the AF array was dependent on the MTs following drug-induced depolymerization, whereby short AFs initially appeared colocalized with MTs, and displayed motility along MTs. We also observed that light-induced reorganization of MTs occurred in concert with changes in AF behavior. Our results indicate dynamic interaction between the cortical actin and MT cytoskeletons in interphase plant cells. PMID:21693695

  16. MicroRNAs miR-143 and miR-145 modulate cytoskeletal dynamics and responsiveness of smooth muscle cells to injury

    Science.gov (United States)

    Xin, Mei; Small, Eric M.; Sutherland, Lillian B.; Qi, Xiaoxia; McAnally, John; Plato, Craig F.; Richardson, James A.; Bassel-Duby, Rhonda; Olson, Eric N.

    2009-01-01

    Vascular injury triggers dedifferentiation and cytoskeletal remodeling of smooth muscle cells (SMCs), culminating in vessel occlusion. Serum response factor (SRF) and its coactivator, myocardin, play a central role in the control of smooth muscle phenotypes by regulating the expression of cytoskeletal genes. We show that SRF and myocardin regulate a cardiovascular-specific microRNA (miRNA) cluster encoding miR-143 and miR-145. To assess the functions of these miRNAs in vivo, we systematically deleted them singly and in combination in mice. Mice lacking both miR-143 and miR-145 are viable and do not display overt abnormalities in smooth muscle differentiation, although they show a significant reduction in blood pressure due to reduced vascular tone. Remarkably, however, neointima formation in response to vascular injury is profoundly impeded in mice lacking these miRNAs, due to disarray of actin stress fibers and diminished migratory activity of SMCs. These abnormalities reflect the regulation of a cadre of modulators of SRF activity and actin dynamics by miR-143 and miR-145. Thus, miR-143 and miR-145 act as integral components of the regulatory network whereby SRF controls cytoskeletal remodeling and phenotypic switching of SMCs during vascular disease. PMID:19720868

  17. Random walks of cytoskeletal motors in open and closed compartments

    NARCIS (Netherlands)

    Lipowsky, R.; Klumpp, S.

    2001-01-01

    Random walks of molecular motors, which bind to and unbind from cytoskeletal filaments, are studied theoretically. The bound and unbound motors undergo directed and nondirected motion, respectively. Motors in open compartments exhibit anomalous drift velocities. Motors in closed compartments generat

  18. 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.

  19. 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.

  20. Deranged smooth muscle α-actin as a biomarker of intestinal pseudo-obstruction: a controlled multinational case series

    Science.gov (United States)

    Knowles, C H; Silk, D B A; Darzi, A; Veress, B; Feakins, R; Raimundo, A H; Crompton, T; Browning, E C; Lindberg, G; Martin, J E

    2004-01-01

    Background and aims: Chronic idiopathic intestinal pseudo-obstruction (CIIP) is a severe motility disorder associated with significant morbidity. Several histopathological (neuropathic and myopathic) phenotypes have been described but only a single adult with jejunal smooth (circular) muscle α-actin deficiency. We present a prospective multinational case series investigating smooth muscle α-actin deficiency as a biomarker of this disease. Methods: A total of 115 fully clinically and physiologically (including prolonged (24 hour) ambulatory jejunal manometry) characterised CIIP patients from three European centres were studied. Immunohistochemical localisation of actins and other cytoskeletal proteins were performed on laparoscopic full thickness jejunal biopsies and compared with adult controls. Distribution of α-actin was also characterised in other gut regions and in the developing human alimentary tract. Results: Twenty eight of 115 (24%) CIIP patient biopsies had absent (n = 22) or partial (n = 6) jejunal smooth muscle α-actin immunostaining in the circular muscle layer. In contrast, smooth muscle α-actin staining was preserved in the longitudinal muscle and in adult jejunal controls (n = 20). Comparative study of other adult alimentary tract regions and fetal small intestine, suggested significant spatial and temporal variations in smooth muscle α-actin expression. Conclusions: The ability to modulate α-smooth muscle actin expression, evident in development, is maintained in adult life and may be influenced by disease, rendering it a valuable biomarker even in the absence of other structural abnormalities. PMID:15479676

  1. Skeletal muscle-specific ablation of gamma(cyto-actin does not exacerbate the mdx phenotype.

    Directory of Open Access Journals (Sweden)

    Kurt W Prins

    Full Text Available We previously documented a ten-fold increase in gamma(cyto-actin expression in dystrophin-deficient skeletal muscle and hypothesized that increased gamma(cyto-actin expression may participate in an adaptive cytoskeletal remodeling response. To explore whether increased gamma(cyto-actin fortifies the cortical cytoskeleton in dystrophic skeletal muscle, we generated double knockout mice lacking both dystrophin and gamma(cyto-actin specifically in skeletal muscle (ms-DKO. Surprisingly, dystrophin-deficient mdx and ms-DKO mice presented with comparable levels of myofiber necrosis, membrane instability, and deficits in muscle function. The lack of an exacerbated phenotype in ms-DKO mice suggests gamma(cyto-actin and dystrophin function in a common pathway. Finally, because both mdx and ms-DKO skeletal muscle showed similar levels of utrophin expression and presented with identical dystrophies, we conclude utrophin can partially compensate for the loss of dystrophin independent of a gamma(cyto-actin-utrophin interaction.

  2. Microtubule anchoring by cortical actin bundles prevents streaming of the oocyte cytoplasm.

    Science.gov (United States)

    Wang, Ying; Riechmann, Veit

    2008-01-01

    The localisation of the determinants of the body axis during Drosophila oogenesis is dependent on the microtubule (MT) cytoskeleton. Mutations in the actin binding proteins Profilin, Cappuccino (Capu) and Spire result in premature streaming of the cytoplasm and a reorganisation of the oocyte MT network. As a consequence, the localisation of axis determinants is abolished in these mutants. It is unclear how actin regulates the organisation of the MTs, or what the spatial relationship between these two cytoskeletal elements is. Here, we report a careful analysis of the oocyte cytoskeleton. We identify thick actin bundles at the oocyte cortex, in which the minus ends of the MTs are embedded. Disruption of these bundles results in cortical release of the MT minus ends, and premature onset of cytoplasmic streaming. Thus, our data indicate that the actin bundles anchor the MTs minus ends at the oocyte cortex, and thereby prevent streaming of the cytoplasm. We further show that actin bundle formation requires Profilin but not Capu and Spire. Thus, our results support a model in which Profilin acts in actin bundle nucleation, while Capu and Spire link the bundles to MTs. Finally, our data indicate how cytoplasmic streaming contributes to the reorganisation of the MT cytoskeleton. We show that the release of the MT minus ends from the cortex occurs independently of streaming, while the formation of MT bundles is streaming dependent.

  3. 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.

  4. 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

  5. Early changes in Huntington's disease patient brains involve alterations in cytoskeletal and synaptic elements.

    Science.gov (United States)

    DiProspero, Nicholas A; Chen, Er-Yun; Charles, Vinod; Plomann, Markus; Kordower, Jeffrey H; Tagle, Danilo A

    2004-09-01

    Huntington's disease (HD) is caused by a polyglutamine repeat expansion in the N-terminus of the huntingtin protein. Huntingtin is normally present in the cytoplasm where it may interact with structural and synaptic elements. The mechanism of HD pathogenesis remains unknown but studies indicate a toxic gain-of-function possibly through aberrant protein interactions. To investigate whether early degenerative changes in HD involve alterations of cytoskeletal and vesicular components, we examined early cellular changes in the frontal cortex of HD presymptomatic (PS), early pathological grade (grade 1) and late-stage (grade 3 and 4) patients as compared to age-matched controls. Morphologic analysis using silver impregnation revealed a progressive decrease in neuronal fiber density and organization in pyramidal cell layers beginning in presymptomatic HD cases. Immunocytochemical analyses for the cytoskeletal markers alpha -tubulin, microtubule-associated protein 2, and phosphorylated neurofilament demonstrated a concomitant loss of staining in early grade cases. Immunoblotting for synaptic proteins revealed a reduction in complexin 2, which was marked in some grade 1 HD cases and significantly reduced in all late stage cases. Interestingly, we demonstrate that two synaptic proteins, dynamin and PACSIN 1, which were unchanged by immunoblotting, showed a striking loss by immunocytochemistry beginning in early stage HD tissue suggesting abnormal distribution of these proteins. We propose that mutant huntingtin affects proteins involved in synaptic function and cytoskeletal integrity before symptoms develop which may influence early disease onset and/or progression.

  6. Magnetic manipulation of actin orientation, polymerization, and gliding on myosin using superparamagnetic iron oxide particles.

    Science.gov (United States)

    Chen, Yun; Guzik, Stephanie; Sumner, James P; Moreland, John; Koretsky, Alan P

    2011-02-11

    The actin cytoskeleton controls cell shape, motility, as well as intracellular molecular trafficking. The ability to remotely manipulate actin is therefore highly desirable as a tool to probe and manipulate biological processes at the molecular level. We demonstrate actin manipulation by labeling actin filaments with superparamagnetic iron oxide particles (IOPs) and applying a uniform magnetic field to affect actin orientation, polymerization and gliding on myosin. We show for the first time magnetic manipulation of magnetizable actin filaments at the molecular level while gliding on a bed of myosin molecules and during polymerization. A model for the magnetic alignment and guiding mechanism is proposed based on the torque from the induced molecular anisotropy due to interactions between neighboring IOPs distributed along magnetically labeled actin molecules.

  7. Ring closure in actin polymers

    Science.gov (United States)

    Sinha, Supurna; Chattopadhyay, Sebanti

    2017-03-01

    We present an analysis for the ring closure probability of semiflexible polymers within the pure bend Worm Like Chain (WLC) model. The ring closure probability predicted from our analysis can be tested against fluorescent actin cyclization experiments. We also discuss the effect of ring closure on bend angle fluctuations in actin polymers.

  8. The impact of tropomyosins on actin filament assembly is isoform specific.

    Science.gov (United States)

    Janco, Miro; Bonello, Teresa T; Byun, Alex; Coster, Adelle C F; Lebhar, Helene; Dedova, Irina; Gunning, Peter W; Böcking, Till

    2016-07-01

    Tropomyosin (Tpm) is an α helical coiled-coil dimer that forms a co-polymer along the actin filament. Tpm is involved in the regulation of actin's interaction with binding proteins as well as stabilization of the actin filament and its assembly kinetics. Recent studies show that multiple Tpm isoforms also define the functional properties of distinct actin filament populations within a cell. Subtle structural variations within well conserved Tpm isoforms are the key to their functional specificity. Therefore, we purified and characterized a comprehensive set of 8 Tpm isoforms (Tpm1.1, Tpm1.12, Tpm1.6, Tpm1.7, Tpm1.8, Tpm2.1, Tpm3.1, and Tpm4.2), using well-established actin co-sedimentation and pyrene fluorescence polymerization assays. We observed that the apparent affinity (Kd(app)) to filamentous actin varied in all Tpm isoforms between ∼0.1-5 μM with similar values for both, skeletal and cytoskeletal actin filaments. The data did not indicate any correlation between affinity and size of Tpm molecules, however high molecular weight (HMW) isoforms Tpm1.1, Tpm1.6, Tpm1.7 and Tpm2.1, showed ∼3-fold higher cooperativity compared to low molecular weight (LMW) isoforms Tpm1.12, Tpm1.8, Tpm3.1, and Tpm4.2. The rate of actin filament elongation in the presence of Tpm2.1 increased, while all other isoforms decreased the elongation rate by 27-85 %. Our study shows that the biochemical properties of Tpm isoforms are finely tuned and depend on sequence variations in alternatively spliced regions of Tpm molecules.

  9. VAMP-7 links granule exocytosis to actin reorganization during platelet activation.

    Science.gov (United States)

    Koseoglu, Secil; Peters, Christian G; Fitch-Tewfik, Jennifer L; Aisiku, Omozuanvbo; Danglot, Lydia; Galli, Thierry; Flaumenhaft, Robert

    2015-07-30

    Platelet activation results in profound morphologic changes accompanied by release of granule contents. Recent evidence indicates that fusion of granules with the plasma membrane during activation provides auxiliary membrane to cover growing actin structures. Yet little is known about how membrane fusion is coupled with actin reorganization. Vesicle-associated membrane protein (VAMP)-7 is found on platelet vesicles and possesses an N-terminal longin domain capable of linking exocytosis to cytoskeletal remodeling. We have evaluated platelets from VAMP-7(-/-) mice to determine whether this VAMP isoform contributes to granule release and platelet spreading. VAMP-7(-/-) platelets demonstrated a partial defect in dense granule exocytosis and impaired aggregation. α Granule exocytosis from VAMP-7(-/-) platelets was diminished both in vitro and in vivo during thrombus formation. Consistent with a role of VAMP-7 in cytoskeletal remodeling, spreading on matrices was decreased in VAMP-7(-/-) platelets compared to wild-type controls. Immunoprecipitation of VAMP-7 revealed an association with VPS9-domain ankyrin repeat protein (VARP), an adaptor protein that interacts with both membrane-bound and cytoskeleton proteins and with Arp2/3. VAMP-7, VARP, and Arp2/3 localized to the platelet periphery during spreading. These studies demonstrate that VAMP-7 participates in both platelet granule secretion and spreading and suggest a mechanism whereby VAMP-7 links granule exocytosis with actin reorganization.

  10. 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)

  11. Estradiol influences the mechanical properties of human fetal osteoblasts through cytoskeletal changes

    Energy Technology Data Exchange (ETDEWEB)

    Muthukumaran, Padmalosini [Department of Bioengineering, National University of Singapore (Singapore); Lim, Chwee Teck [Department of Bioengineering, National University of Singapore (Singapore); Department of Mechanical Engineering, National University of Singapore (Singapore); Mechanobiology Institute, National University of Singapore (Singapore); Singapore-MIT Alliance for Research and Technology (SMART), National University of Singapore (Singapore); Lee, Taeyong, E-mail: bielt@nus.edu.sg [Department of Bioengineering, National University of Singapore (Singapore)

    2012-07-06

    Highlights: Black-Right-Pointing-Pointer Estradiol induced stiffness changes of osteoblasts were quantified using AFM. Black-Right-Pointing-Pointer Estradiol causes significant decrease in the stiffness of osteoblasts. Black-Right-Pointing-Pointer Decreased stiffness was caused by decreased density of f-actin network. Black-Right-Pointing-Pointer Stiffness changes were not associated with mineralized matrix of osteoblasts. Black-Right-Pointing-Pointer Estradiol increases inherent alkaline phosphatase activity of osteoblasts. -- Abstract: Estrogen is known to have a direct effect on bone forming osteoblasts and bone resorbing osteoclasts. The cellular and molecular effects of estrogen on osteoblasts and osteoblasts-like cells have been extensively studied. However, the effect of estrogen on the mechanical property of osteoblasts has not been studied yet. It is important since mechanical property of the mechanosensory osteoblasts could be pivotal to its functionality in bone remodeling. This is the first study aimed to assess the direct effect of estradiol on the apparent elastic modulus (E{sup Asterisk-Operator }) and corresponding cytoskeletal changes of human fetal osteoblasts (hFOB 1.19). The cells were cultured in either medium alone or medium supplemented with {beta}-estradiol and then subjected to Atomic Force Microscopy indentation (AFM) to determine E{sup Asterisk-Operator }. The underlying changes in cytoskeleton were studied by staining the cells with TRITC-Phalloidin. Following estradiol treatment, the cells were also tested for proliferation, alkaline phosphatase activity and mineralization. With estradiol treatment, E{sup Asterisk-Operator} of osteoblasts significantly decreased by 43-46%. The confocal images showed that the changes in f-actin network observed in estradiol treated cells can give rise to the changes in the stiffness of the cells. Estradiol also increases the inherent alkaline phosphatase activity of the cells. Estradiol induced stiffness

  12. Actinic cheilitis with a familial pattern: An unusual case

    Directory of Open Access Journals (Sweden)

    Surekha Murthi

    2014-01-01

    Full Text Available Actinic cheilitis is a chronic inflammatory disorder of the lip, affecting the lower lip mainly, caused by cumulative long-term effects of ultraviolet (UV radiation in sunlight. It is a premalignant condition with a malignancy potential of 6-10%. It is reported that it almost exclusively occurs in fair-skinned people and those who work outdoors. However, it has recently been reported that actinic cheilitis is not exclusive to fair-skinned people. It is most common in middle-aged or older male patients. Diagnosis of actinic cheilitis is mainly based on demographical, clinical, and histopathological findings. Factors such as socioeconomic status, smoking, dietary habits, and genetic predisposition are also associated with lip cancer. Here, we present a case of actinic cheilitis in two siblings and in their mother, showing a familial pattern. An incisional biopsy of the lower lip in the mother showed severe dysplastic changes indicating transformation to squamous cell carcinoma.

  13. Loss of γ-cytoplasmic actin triggers myofibroblast transition of human epithelial cells.

    Science.gov (United States)

    Lechuga, Susana; Baranwal, Somesh; Li, Chao; Naydenov, Nayden G; Kuemmerle, John F; Dugina, Vera; Chaponnier, Christine; Ivanov, Andrei I

    2014-10-15

    Transdifferentiation of epithelial cells into mesenchymal cells and myofibroblasts plays an important role in tumor progression and tissue fibrosis. Such epithelial plasticity is accompanied by dramatic reorganizations of the actin cytoskeleton, although mechanisms underlying cytoskeletal effects on epithelial transdifferentiation remain poorly understood. In the present study, we observed that selective siRNA-mediated knockdown of γ-cytoplasmic actin (γ-CYA), but not β-cytoplasmic actin, induced epithelial-to-myofibroblast transition (EMyT) of different epithelial cells. The EMyT manifested by increased expression of α-smooth muscle actin and other contractile proteins, along with inhibition of genes responsible for cell proliferation. Induction of EMyT in γ-CYA-depleted cells depended on activation of serum response factor and its cofactors, myocardial-related transcriptional factors A and B. Loss of γ-CYA stimulated formin-mediated actin polymerization and activation of Rho GTPase, which appear to be essential for EMyT induction. Our findings demonstrate a previously unanticipated, unique role of γ-CYA in regulating epithelial phenotype and suppression of EMyT that may be essential for cell differentiation and tissue fibrosis.

  14. Syndapin promotes pseudocleavage furrow formation by actin organization in the syncytial Drosophila embryo.

    Science.gov (United States)

    Sherlekar, Aparna; Rikhy, Richa

    2016-07-01

    Coordinated membrane and cytoskeletal remodeling activities are required for membrane extension in processes such as cytokinesis and syncytial nuclear division cycles in Drosophila Pseudocleavage furrow membranes in the syncytial Drosophila blastoderm embryo show rapid extension and retraction regulated by actin-remodeling proteins. The F-BAR domain protein Syndapin (Synd) is involved in membrane tubulation, endocytosis, and, uniquely, in F-actin stability. Here we report a role for Synd in actin-regulated pseudocleavage furrow formation. Synd localized to these furrows, and its loss resulted in short, disorganized furrows. Synd presence was important for the recruitment of the septin Peanut and distribution of Diaphanous and F-actin at furrows. Synd and Peanut were both absent in furrow-initiation mutants of RhoGEF2 and Diaphanous and in furrow-progression mutants of Anillin. Synd overexpression in rhogef2 mutants reversed its furrow-extension phenotypes, Peanut and Diaphanous recruitment, and F-actin organization. We conclude that Synd plays an important role in pseudocleavage furrow extension, and this role is also likely to be crucial in cleavage furrow formation during cell division.

  15. Kaempferol inhibits Entamoeba histolytica growth by altering cytoskeletal functions.

    Science.gov (United States)

    Bolaños, Verónica; Díaz-Martínez, Alfredo; Soto, Jacqueline; Marchat, Laurence A; Sanchez-Monroy, Virginia; Ramírez-Moreno, Esther

    2015-11-01

    The flavonoid kaempferol obtained from Helianthemum glomeratum, an endemic Mexican medicinal herb used to treat gastrointestinal disorders, has been shown to inhibit growth of Entamoeba histolytica trophozoites in vitro; however, the mechanisms associated with this activity have not been documented. Several works reported that kaempferol affects cytoskeleton in mammalian cells. In order to gain insights into the action mechanisms involved in the anti-amoebic effect of kaempferol, here we evaluated the effect of this compound on the pathogenic events driven by the cytoskeleton during E. histolytica infection. We also carried out a two dimensional gel-based proteomic analysis to evidence modulated proteins that could explain the phenotypical changes observed in trophozoites. Our results showed that kaempferol produces a dose-dependent effect on trophozoites growth and viability with optimal concentration being 27.7 μM. Kaempferol also decreased adhesion, it increased migration and phagocytic activity, but it did not affect erythrocyte binding nor cytolytic capacity of E. histolytica. Congruently, proteomic analysis revealed that the cytoskeleton proteins actin, myosin II heavy chain and cortexillin II were up-regulated in response to kaempferol treatment. In conclusion, kaempferol anti-amoebic effects were associated with deregulation of proteins related with cytoskeleton, which altered invasion mechanisms.

  16. Fisetin antagonizes cell fusion, cytoskeletal organization and bone resorption in RANKL-differentiated murine macrophages.

    Science.gov (United States)

    Kim, Yun-Ho; Kim, Jung-Lye; Lee, Eun-Jung; Park, Sin-Hye; Han, Seon-Young; Kang, Soon Ah; Kang, Young-Hee

    2014-03-01

    Osteoclastogenesis is comprised of several stage s including progenitor survival, differentiation to mononuclear preosteoclasts, cell fusion to multinuclear mature osteoclasts, and activation to osteoclasts with bone resorbing activity. Botanical antioxidants are now being increasingly investigated for their health-promoting effects on bone. This study investigated that fisetin, a flavonol found naturally in many fruits and vegetables, suppressed osteoclastogenesis by disturbing receptor activator of nuclear factor (NF)-κB ligand (RANKL)-mediated signaling pathway and demoting osteoclastogenic protein induction. Nontoxic fisetin at ≤10 μM inhibited the induction of RANK, tumor necrosis factor receptor associated factor 6 (TRAF6) and the activation of NF-κB in RANKL-stimulated RAW 264.7 macrophages. In RANKL-differentiated osteoclasts cell fusion protein of E-cadherin was induced, which was dampened by fisetin. The formation of tartrate-resistance acid phosphatase-positive multinucleated osteoclasts was suppressed by adding fisetin to RANKL-exposed macrophages. It was also found that fisetin reduced actin ring formation and gelsolin induction of osteclasts enhanced by RANKL through disturbing c-Src-proline-rich tyrosine kinase 2 signaling. Fisetin deterred preosteoclasts from the cell-cell fusion and the organization of the cytoskeleton to seal the resorbing area and to secret protons for bone resorption. Consistently, the 5 day-treatment of fisetin diminished RANKL-induced cellular expression of carbonic anhydrase II and integrin β3 concurrently with a reduction of osteoclast bone-resorbing activity. Therefore, fisetin was a natural therapeutic agent retarding osteoclast fusion and cytoskeletal organization such as actin rings and ruffled boarder, which is a property of mature osteoclasts and is required for osteoclasts to resorb bone.

  17. 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.

  18. Actin cytoskeleton: putting a CAP on actin polymerization.

    Science.gov (United States)

    Stevenson, V A; Theurkauf, W E

    2000-10-05

    Two recent studies have identified a Drosophila homolog of cyclase-associated protein (CAP) as a developmentally important negative regulator of actin polymerization that may also directly mediate signal transduction.

  19. Involvement of actin rearrangements within the amygdala and the dorsal hippocampus in aversive memories of drug withdrawal in acute morphine-dependent rats.

    Science.gov (United States)

    Hou, Yuan-Yuan; Lu, Bin; Li, Mu; Liu, Yao; Chen, Jie; Chi, Zhi-Qiang; Liu, Jing-Gen

    2009-09-30

    Aversive memories of drug withdrawal can generate a motivational state leading to compulsive drug taking. Changes in synaptic plasticity may be involved in the formation of aversive memories. Dynamic rearrangement of the cytoskeletal actin, a major structural component of the dendritic spine, regulates synaptic plasticity. Here, the potential involvement of actin rearrangements in the induction of aversive memories of morphine withdrawal was examined. We found that lesions of the amygdala or dorsal hippocampus (DH) but not nucleus accumbens (NAc) impaired conditioned place aversion (CPA) of acute morphine-dependent rats. Accordingly, conditioned morphine withdrawal induced actin rearrangements in the amygdala and the DH but not in the NAc. In addition, we found that conditioned morphine withdrawal also increased activity-regulated cytoskeletal-associated protein (Arc) expression in the amygdala but not in the DH, although actin rearrangements were observed in both areas. We further found that inhibition of actin rearrangements by intra-amygdala or intra-DH injections of latrunculin A, an inhibitor of actin polymerization, significantly attenuated CPA. Furthermore, we found that manipulation of amygdala beta-adrenoceptor activity by its antagonist propranolol and agonist clenbuterol differentially altered actin rearrangements in the DH. Therefore, our findings reveal that actin rearrangements in the amygdala and the DH are required for the acquisition and consolidation of the aversive memories of drug withdrawal and that the beta-noradrenergic system within the amygdala modulates aversive memory consolidation by regulating actin rearrangements but not Arc protein expression in the DH, which is distinct from its role in modulation of inhibitory avoidance memory.

  20. The actin-microtubule cross-linking activity of Drosophila Short stop is regulated by intramolecular inhibition.

    Science.gov (United States)

    Applewhite, Derek A; Grode, Kyle D; Duncan, Mara C; Rogers, Stephen L

    2013-09-01

    Actin and microtubule dynamics must be precisely coordinated during cell migration, mitosis, and morphogenesis--much of this coordination is mediated by proteins that physically bridge the two cytoskeletal networks. We have investigated the regulation of the Drosophila actin-microtubule cross-linker Short stop (Shot), a member of the spectraplakin family. Our data suggest that Shot's cytoskeletal cross-linking activity is regulated by an intramolecular inhibitory mechanism. In its inactive conformation, Shot adopts a "closed" conformation through interactions between its NH(2)-terminal actin-binding domain and COOH-terminal EF-hand-GAS2 domain. This inactive conformation is targeted to the growing microtubule plus end by EB1. On activation, Shot binds along the microtubule through its COOH-terminal GAS2 domain and binds to actin with its NH(2)-terminal tandem CH domains. We propose that this mechanism allows Shot to rapidly cross-link dynamic microtubules in response to localized activating signals at the cell cortex.

  1. Allosteric regulation by cooperative conformational changes of actin filaments drives mutually exclusive binding with cofilin and myosin.

    Science.gov (United States)

    Ngo, Kien Xuan; Umeki, Nobuhisa; Kijima, Saku T; Kodera, Noriyuki; Ueno, Hiroaki; Furutani-Umezu, Nozomi; Nakajima, Jun; Noguchi, Taro Q P; Nagasaki, Akira; Tokuraku, Kiyotaka; Uyeda, Taro Q P

    2016-10-20

    Heavy meromyosin (HMM) of myosin II and cofilin each binds to actin filaments cooperatively and forms clusters along the filaments, but it is unknown whether the two cooperative bindings are correlated and what physiological roles they have. Fluorescence microscopy demonstrated that HMM-GFP and cofilin-mCherry each bound cooperatively to different parts of actin filaments when they were added simultaneously in 0.2 μM ATP, indicating that the two cooperative bindings are mutually exclusive. In 0.1 mM ATP, the motor domain of myosin (S1) strongly inhibited the formation of cofilin clusters along actin filaments. Under this condition, most actin protomers were unoccupied by S1 at any given moment, suggesting that transiently bound S1 alters the structure of actin filaments cooperatively and/or persistently to inhibit cofilin binding. Consistently, cosedimentation experiments using copolymers of actin and actin-S1 fusion protein demonstrated that the fusion protein affects the neighboring actin protomers, reducing their affinity for cofilin. In reciprocal experiments, cofilin-actin fusion protein reduced the affinity of neighboring actin protomers for S1. Thus, allosteric regulation by cooperative conformational changes of actin filaments contributes to mutually exclusive cooperative binding of myosin II and cofilin to actin filaments, and presumably to the differential localization of both proteins in cells.

  2. Functional Analysis of Actin-Binding Proteins in the Central Nervous System of Drosophila.

    Science.gov (United States)

    He, Qi; Roblodowski, Christopher

    2016-01-01

    Using Drosophila actin-binding protein Dunc-115 as model system, this chapter describes a MARCM (mosaic analysis with a repressible cell marker)-based method for analyzing cytoskeletal components for their functions in the nervous system. Following a concise description about the principle, a step-by-step protocol is provided for generating the needed stocks and for histological analysis. Additional details and explanations have been given in the accompanying notes. Together, this should form a practical and sufficient recipe for performing at the single-cell-level loss-of-function and gain-of-function analyses of proteins associated with the cytoskeleton.

  3. Polarized actin structural dynamics in response to cyclic uniaxial stretch

    Science.gov (United States)

    Huang, Lawrence; Helmke, Brian P.

    2014-01-01

    Endothelial cell (EC) alignment to directional flow or stretch supports anti-inflammatory functions, but mechanisms controlling polarized structural adaptation in response to physical cues remain unclear. This study aimed to determine whether factors associated with early actin edge ruffling implicated in cell polarization are prerequisite for stress fiber (SF) reorientation in response to cyclic uniaxial stretch. Time-lapse analysis of EGFP-actin in confluent ECs showed that onset of either cyclic uniaxial or equibiaxial stretch caused a non-directional increase in edge ruffling. Edge activity was concentrated in a direction perpendicular to the stretch axis after 60 min, consistent with the direction of SF alignment. Rho-kinase inhibition caused reorientation of both stretch-induced edge ruffling and SF alignment parallel to the stretch axis. Arp2/3 inhibition attenuated stretch-induced cell elongation and disrupted polarized edge dynamics and microtubule organizing center reorientation, but it had no effect on the extent of SF reorientation. Disrupting localization of p21-activated kinase (PAK) did not prevent stretch-induced SF reorientation, suggesting that this Rac effector is not critical in regulating stretch-induced cytoskeletal remodeling. Overall, these results suggest that directional edge ruffling is not a primary mechanism that guides SF reorientation in response to stretch; the two events are coincident but not causal. PMID:25821527

  4. 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.

  5. 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.

  6. Mimicking the mechanical properties of the cell cortex by the self-assembly of an actin cortex in vesicles

    Science.gov (United States)

    Luo, Tianzhi; Srivastava, Vasudha; Ren, Yixin; Robinson, Douglas N.

    2014-04-01

    The composite of the actin cytoskeleton and plasma membrane plays important roles in many biological events. Here, we employed the emulsion method to synthesize artificial cells with biomimetic actin cortex in vesicles and characterized their mechanical properties. We demonstrated that the emulsion method provides the flexibility to adjust the lipid composition and protein concentrations in artificial cells to achieve the desired size distribution, internal microstructure, and mechanical properties. Moreover, comparison of the cortical elasticity measured for reconstituted artificial cells to that of real cells, including those manipulated using genetic depletion and pharmacological inhibition, strongly supports that actin cytoskeletal proteins are dominant over lipid molecules in cortical mechanics. Our study indicates that the assembly of biological systems in artificial cells with purified cellular components provides a powerful way to answer biological questions.

  7. The Strip-Hippo Pathway Regulates Synaptic Terminal Formation by Modulating Actin Organization at the Drosophila Neuromuscular Synapses

    Directory of Open Access Journals (Sweden)

    Chisako Sakuma

    2016-08-01

    Full Text Available Synapse formation requires the precise coordination of axon elongation, cytoskeletal stability, and diverse modes of cell signaling. The underlying mechanisms of this interplay, however, remain unclear. Here, we demonstrate that Strip, a component of the striatin-interacting phosphatase and kinase (STRIPAK complex that regulates these processes, is required to ensure the proper development of synaptic boutons at the Drosophila neuromuscular junction. In doing so, Strip negatively regulates the activity of the Hippo (Hpo pathway, an evolutionarily conserved regulator of organ size whose role in synapse formation is currently unappreciated. Strip functions genetically with Enabled, an actin assembly/elongation factor and the presumptive downstream target of Hpo signaling, to modulate local actin organization at synaptic termini. This regulation occurs independently of the transcriptional co-activator Yorkie, the canonical downstream target of the Hpo pathway. Our study identifies a previously unanticipated role of the Strip-Hippo pathway in synaptic development, linking cell signaling to actin organization.

  8. 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.

  9. 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.

  10. 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.

  11. The neurogenic basic helix–loop–helix transcription factor NeuroD6 concomitantly increases mitochondrial mass and regulates cytoskeletal organization in the early stages of neuronal differentiation

    Directory of Open Access Journals (Sweden)

    Kristin Kathleen Baxter

    2009-09-01

    Full Text Available Mitochondria play a central role during neurogenesis by providing energy in the form of ATP for cytoskeletal remodelling, outgrowth of neuronal processes, growth cone activity and synaptic activity. However, the fundamental question of how differentiating neurons control mitochondrial biogenesis remains vastly unexplored. Since our previous studies have shown that the neurogenic bHLH (basic helix–loop–helix transcription factor NeuroD6 is sufficient to induce differentiation of the neuronal progenitor-like PC12 cells and that it triggers expression of mitochondrial-related genes, we investigated whether NeuroD6 could modulate the mitochondrial biomass using our PC12-ND6 cellular paradigm. Using a combination of flow cytometry, confocal microscopy and mitochondrial fractionation, we demonstrate that NeuroD6 stimulates maximal mitochondrial mass at the lamellipodia stage, thus preceding axonal growth. NeuroD6 triggers remodelling of the actin and microtubule networks in conjunction with increased expression of the motor protein KIF5B, thus promoting mitochondrial movement in developing neurites with accumulation in growth cones. Maintenance of the NeuroD6-induced mitochondrial mass requires an intact cytoskeletal network, as its disruption severely reduces mitochondrial mass. The present study provides the first evidence that NeuroD6 plays an integrative role in co-ordinating increase in mitochondrial mass with cytoskeletal remodelling, suggestive of a role of this transcription factor as a co-regulator of neuronal differentiation and energy metabolism.

  12. 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.

  13. 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

  14. [Cytoskeletal reorganization in hepatocytes of the regenerating mouse liver].

    Science.gov (United States)

    Gleĭberman, A S; Troianovskiĭ, S M; Bannikov, G A

    1984-12-01

    The intracellular pattern of prekeratin and actin filaments has been studied on sections of mouse livers regenerating after CCl4 injury. Monoclonal antibodies against one of liver prekeratins and monospecific polyclonal actin antibodies were used in the indirect immunofluorescent test. The presence of alpha-fetoprotein and bile canaliculi antigen was also monitored during regeneration. In control livers, prekeratin and actin filaments formed thick bundles adjacent to plasma membranes. The cytoplasmic prekeratin network was unmarked. In contrast to the latter, the bright well developed intracytoplasmic prekeratin network and intracytoplasmic actin fibers were identified in the perinecrotic hepatocytes by the 3d-4th day of regeneration. This rearrangement of the cytoskeleton coincided in time with the appearance of alpha-fetoprotein and the loss of the bile canaliculi antigen in the perinecrotic hepatocytes.

  15. The dynamics of filament assembly define cytoskeletal network morphology

    Science.gov (United States)

    Foffano, Giulia; Levernier, Nicolas; Lenz, Martin

    2016-12-01

    The actin cytoskeleton is a key component in the machinery of eukaryotic cells, and it self-assembles out of equilibrium into a wide variety of biologically crucial structures. Although the molecular mechanisms involved are well characterized, the physical principles governing the spatial arrangement of actin filaments are not understood. Here we propose that the dynamics of actin network assembly from growing filaments results from a competition between diffusion, bundling and steric hindrance, and is responsible for the range of observed morphologies. Our model and simulations thus predict an abrupt dynamical transition between homogeneous and strongly bundled networks as a function of the actin polymerization rate. This suggests that cells may effect dramatic changes to their internal architecture through minute modifications of their nonequilibrium dynamics. Our results are consistent with available experimental data.

  16. 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.

  17. Chorein, the protein responsible for chorea-acanthocytosis, interacts with β-adducin and β-actin.

    Science.gov (United States)

    Shiokawa, Nari; Nakamura, Masayuki; Sameshima, Mieko; Deguchi, Akiko; Hayashi, Takehiro; Sasaki, Natsuki; Sano, Akira

    2013-11-08

    Chorea-acanthocytosis (ChAc) is an autosomal, recessive hereditary disease characterized by striatal neurodegeneration and acanthocytosis, and caused by loss of function mutations in the vacuolar protein sorting 13 homolog A (VPS13A) gene. VPS13A encodes chorein whose physiological function at the molecular level is poorly understood. In this study, we show that chorein interacts with β-adducin and β-actin. We first compare protein expression in human erythrocyte membranes using proteomic analysis. Protein levels of β-adducin isoform 1 and β-actin are markedly decreased in erythrocyte membranes from a ChAc patient. Subsequent co-immunoprecipitation (co-IP) and reverse co-IP assays using extracts from chorein-overexpressing human embryonic kidney 293 (HEK293) cells, shows that β-adducin (isoforms 1 and 2) and β-actin interact with chorein. Immunocytochemical analysis using chorein-overexpressing HEK293 cells demonstrates co-localization of chorein with β-adducin and β-actin. In addition, immunoreactivity of β-adducin isoform 1 is significantly decreased in the striatum of gene-targeted ChAc-model mice. Adducin and actin are membrane cytoskeletal proteins, involved in synaptic function. Expression of β-adducin is restricted to the brain and hematopoietic tissues, corresponding to the main pathological lesions of ChAc, and thereby implicating β-adducin and β-actin in ChAc pathogenesis.

  18. 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.

  19. Cytoskeletal and membrane dynamics during higher plant cytokinesis.

    Science.gov (United States)

    McMichael, Colleen M; Bednarek, Sebastian Y

    2013-03-01

    Following mitosis, cytoplasm, organelles and genetic material are partitioned into daughter cells through the process of cytokinesis. In somatic cells of higher plants, two cytoskeletal arrays, the preprophase band and the phragmoplast, facilitate the positioning and de novo assembly of the plant-specific cytokinetic organelle, the cell plate, which develops across the division plane and fuses with the parental plasma membrane to yield distinct new cells. The coordination of cytoskeletal and membrane dynamics required to initiate, assemble and shape the cell plate as it grows toward the mother cell cortex is dependent upon a large array of proteins, including molecular motors, membrane tethering, fusion and restructuring factors and biosynthetic, structural and regulatory elements. This review focuses on the temporal and molecular requirements of cytokinesis in somatic cells of higher plants gleaned from recent studies using cell biology, genetics, pharmacology and biochemistry.

  20. Intracellular transport driven by cytoskeletal motors: General mechanisms and defects

    CERN Document Server

    Appert-Rolland, Cecile; Santen, Ludger

    2015-01-01

    Cells are strongly out-of-equilibrium systems driven by continuous energy supply. They carry out many vital functions requiring active transport of various ingredients and organelles, some being small, others being large. The cytoskeleton, composed of three types of filaments, determines the shape of the cell and plays a role in cell motion. It also serves as a road network for the so-called cytoskeletal motors. These molecules can attach to a cytoskeletal filament, perform directed motion, possibly carrying along some cargo, and then detach. It is a central issue to understand how intracellular transport driven by molecular motors is regulated, in particular because its breakdown is one of the signatures of some neuronal diseases like the Alzheimer. We give a survey of the current knowledge on microtubule based intracellular transport. We first review some biological facts obtained from experiments, and present some modeling attempts based on cellular automata. We start with background knowledge on the origi...

  1. IQGAP proteins are integral components of cytoskeletal regulation.

    Science.gov (United States)

    Briggs, Michael W; Sacks, David B

    2003-06-01

    IQGAP1 is a scaffolding protein that binds to a diverse array of signalling and structural molecules. By interacting with its target proteins, human IQGAP1 participates in multiple cellular functions, including Ca(2+)/calmodulin signalling, cytoskeletal architecture, CDC42 and Rac signalling, E-cadherin-mediated cell-cell adhesion and beta-catenin-mediated transcription. Yeast IQGAP homologues are important regulators of cellular morphogenesis because they are required for budding and cytokinesis. Here we discuss the structure and function of IQGAP1 as a member of the family of IQGAP proteins and summarize the current knowledge about IQGAP1 and IQGAP2. Collectively, these data reveal that IQGAP1 is a fundamental regulator of cytoskeletal function.

  2. The role of formin tails in actin nucleation, processive elongation, and filament bundling.

    Science.gov (United States)

    Vizcarra, Christina L; Bor, Batbileg; Quinlan, Margot E

    2014-10-31

    Formins are multidomain proteins that assemble actin in a wide variety of biological processes. They both nucleate and remain processively associated with growing filaments, in some cases accelerating filament growth. The well conserved formin homology 1 and 2 domains were originally thought to be solely responsible for these activities. Recently a role in nucleation was identified for the Diaphanous autoinhibitory domain (DAD), which is C-terminal to the formin homology 2 domain. The C-terminal tail of the Drosophila formin Cappuccino (Capu) is conserved among FMN formins but distinct from other formins. It does not have a DAD domain. Nevertheless, we find that Capu-tail plays a role in filament nucleation similar to that described for mDia1 and other formins. Building on this, replacement of Capu-tail with DADs from other formins tunes nucleation activity. Capu-tail has low-affinity interactions with both actin monomers and filaments. Removal of the tail reduces actin filament binding and bundling. Furthermore, when the tail is removed, we find that processivity is compromised. Despite decreased processivity, the elongation rate of filaments is unchanged. Again, replacement of Capu-tail with DADs from other formins tunes the processive association with the barbed end, indicating that this is a general role for formin tails. Our data show a role for the Capu-tail domain in assembling the actin cytoskeleton, largely mediated by electrostatic interactions. Because of its multifunctionality, the formin tail is a candidate for regulation by other proteins during cytoskeletal rearrangements.

  3. IQGAP proteins are integral components of cytoskeletal regulation

    OpenAIRE

    2003-01-01

    IQGAP1 is a scaffolding protein that binds to a diverse array of signalling and structural molecules. By interacting with its target proteins, human IQGAP1 participates in multiple cellular functions, including Ca2+/calmodulin signalling, cytoskeletal architecture, CDC42 and Rac signalling, E-cadherin-mediated cell–cell adhesion and β-catenin-mediated transcription. Yeast IQGAP homologues are important regulators of cellular morphogenesis because they are required for budding and cytokinesis....

  4. 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.

  5. 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.

  6. Hijacking host cell highways: manipulation of the host actin cytoskeleton by obligate intracellular bacterial pathogens

    Directory of Open Access Journals (Sweden)

    Punsiri M Colonne

    2016-09-01

    Full Text Available Intracellular bacterial pathogens replicate within eukaryotic cells and display unique adaptations that support key infection events including invasion, replication, immune evasion, and dissemination. From invasion to dissemination, all stages of the intracellular bacterial life cycle share the same three-dimensional cytosolic space containing the host cytoskeleton. For successful infection and replication, many pathogens hijack the cytoskeleton using effector proteins introduced into the host cytosol by specialized secretion systems. A subset of effectors contains eukaryotic-like motifs that mimic host proteins to exploit signaling and modify specific cytoskeletal components such as actin and microtubules. Cytoskeletal rearrangement promotes numerous events that are beneficial to the pathogen, including internalization of bacteria, subversion of cell intrinsic immunity, structural support for bacteria-containing vacuoles, altered vesicular trafficking, actin-dependent bacterial movement, and pathogen dissemination. This review highlights a diverse group of obligate intracellular bacterial pathogens that manipulate the host cytoskeleton to thrive within eukaryotic cells and discusses underlying molecular mechanisms that promote these dynamic host-pathogen interactions.

  7. 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.

  8. 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.

  9. Actin-based propulsion of functionalized hard versus fluid spherical objects

    Science.gov (United States)

    Delatour, Vincent; Shekhar, Shashank; Reymann, Anne-Cécile; Didry, Dominique; Diêp Lê, Kim Hô; Romet-Lemonne, Guillaume; Helfer, Emmanuèle; Carlier, Marie-France

    2008-02-01

    The directed polymerization of a branched actin network against a functionalized surface drives cell protrusions and organelle propulsion in living cells. Solid microspheres or giant unilamellar vesicles, functionalized with neural Wiskott-Aldrich syndrome protein (N-WASP), initiate the formation of a branched actin array using actin-related protein 2/3 (Arp2/3) complex, when placed in a motility assay reconstituted with pure proteins. These systems are useful biomimetic models of actin-based propulsion that allow to address how the interplay between the physical properties of the functionalized surface and the dynamics of the actin cytoskeleton determines motile behavior. Both solid beads and deformable vesicles display either continuous or saltatory propulsive motions, which are analyzed comparatively; we show that the deformability of liposomes and the mobility of N-WASP at the lipid surface affect the dynamic and structural parameters of the actin meshwork. Our results indicate that beads and vesicles use different mechanisms to translate insertional polymerization of actin at their surface into directed movement: stress relaxation within the actin gel prevents the accumulation of filaments at the front of moving beads, while segregation of nucleators reduces actin polymerization at the front of moving vesicles.

  10. Three-dimensional structure of actin filaments and of an actin gel made with actin-binding protein.

    Science.gov (United States)

    Niederman, R; Amrein, P C; Hartwig, J

    1983-05-01

    Purified muscle actin and mixtures of actin and actin-binding protein were examined in the transmission electron microscope after fixation, critical point drying, and rotary shadowing. The three-dimensional structure of the protein assemblies was analyzed by a computer-assisted graphic analysis applicable to generalized filament networks. This analysis yielded information concerning the frequency of filament intersections, the filament length between these intersections, the angle at which filaments branch at these intersections, and the concentration of filaments within a defined volume. Purified actin at a concentration of 1 mg/ml assembled into a uniform mass of long filaments which overlap at random angles between 0 degrees and 90 degrees. Actin in the presence of macrophage actin-binding protein assembled into short, straight filaments, organized in a perpendicular branching network. The distance between branch points was inversely related to the molar ratio of actin-binding protein to actin. This distance was what would be predicted if actin filaments grew at right angles off of nucleation sites on the two ends of actin-binding protein dimers, and then annealed. The results suggest that actin in combination with actin-binding protein self-assembles to form a three-dimensional network resembling the peripheral cytoskeleton of motile cells.

  11. Hierarchical self-organization of cytoskeletal active networks

    CERN Document Server

    Gordon, Daniel; Keasar, Chen; Farago, Oded

    2012-01-01

    The structural reorganization of the actin cytoskeleton is facilitated through the action of motor proteins that crosslink the actin filaments and transport them relative to each other. Here, we present a combined experimental-computational study that probes the dynamic evolution of mixtures of actin filaments and clusters of myosin motors. While on small spatial and temporal scales the system behaves in a very noisy manner, on larger scales it evolves into several well distinct patterns such as bundles, asters, and networks. These patterns are characterized by junctions with high connectivity, whose formation is possible due to the organization of the motors in "oligoclusters" (intermediate-size aggregates). The simulations reveal that the self-organization process proceeds through a series of hierarchical steps, starting from local microscopic moves and ranging up to the macroscopic large scales where the steady-state structures are formed. Our results shed light into the mechanisms involved in processes li...

  12. Morphological Transformation and Force Generation of Active Cytoskeletal Networks

    Science.gov (United States)

    Maruri, Daniel; Kamm, Roger D.

    2017-01-01

    Cells assemble numerous types of actomyosin bundles that generate contractile forces for biological processes, such as cytokinesis and cell migration. One example of contractile bundles is a transverse arc that forms via actomyosin-driven condensation of actin filaments in the lamellipodia of migrating cells and exerts significant forces on the surrounding environments. Structural reorganization of a network into a bundle facilitated by actomyosin contractility is a physiologically relevant and biophysically interesting process. Nevertheless, it remains elusive how actin filaments are reoriented, buckled, and bundled as well as undergo tension buildup during the structural reorganization. In this study, using an agent-based computational model, we demonstrated how the interplay between the density of myosin motors and cross-linking proteins and the rigidity, initial orientation, and turnover of actin filaments regulates the morphological transformation of a cross-linked actomyosin network into a bundle and the buildup of tension occurring during the transformation. PMID:28114384

  13. Actinic cheilitis in dental practice.

    Science.gov (United States)

    Savage, N W; McKay, C; Faulkner, C

    2010-06-01

    Actinic cheilitis is a potentially premalignant condition involving predominantly the vermilion of the lower lip. The aim of the current paper was to review the clinical presentation of actinic cheilitis and demonstrate the development of management plans using a series of cases. These are designed to provide immediate treatment where required but also to address the medium and long-term requirements of the patient. The authors suggest that the clinical examination of lips and the assessment of actinic cheilitis and other lip pathology become a regular part of the routine soft tissue examination undertaken as a part of the periodic examination of dental patients. Early recognition of actinic cheilitis can allow the development of strategies for individual patients that prevent progression. These are based on past sun exposure, future lifestyle changes and the daily use of emollient sunscreens, broad-brimmed hats and avoidance of sun exposure during the middle of the day. This is a service that is not undertaken as a matter of routine in general medical practice as patients are not seen with the regularity of dental patients and generally not under the ideal examination conditions available in the dental surgery.

  14. The Gas2 family protein Pigs is a microtubule +TIP that affects cytoskeleton organisation.

    Science.gov (United States)

    Girdler, Gemma C; Applewhite, Derek A; Perry, Wick M G; Rogers, Stephen L; Röper, Katja

    2016-01-01

    Coordination between different cytoskeletal systems is crucial for many cell biological functions, including cell migration and mitosis, and also plays an important role during tissue morphogenesis. Proteins of the class of cytoskeletal crosslinkers, or cytolinkers, have the ability to interact with more than one cytoskeletal system at a time and are prime candidates to mediate any coordination. One such class comprises the Gas2-like proteins, combining a conserved calponin-homology-type actin-binding domain and a Gas2 domain predicted to bind microtubules (MTs). This domain combination is also found in spectraplakins, huge cytolinkers that play important roles in many tissues in both invertebrates and vertebrates. Here, we dissect the ability of the single Drosophila Gas2-like protein Pigs to interact with both actin and MT cytoskeletons, both in vitro and in vivo, and illustrate complex regulatory interactions that determine the localisation of Pigs to and its effects on the cytoskeleton.

  15. 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.

  16. 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.

  17. Cyclase-associated protein 1 (CAP1) promotes cofilin-induced actin dynamics in mammalian nonmuscle cells.

    Science.gov (United States)

    Bertling, Enni; Hotulainen, Pirta; Mattila, Pieta K; Matilainen, Tanja; Salminen, Marjo; Lappalainen, Pekka

    2004-05-01

    Cyclase-associated proteins (CAPs) are highly conserved actin monomer binding proteins present in all eukaryotes. However, the mechanism by which CAPs contribute to actin dynamics has been elusive. In mammals, the situation is further complicated by the presence of two CAP isoforms whose differences have not been characterized. Here, we show that CAP1 is widely expressed in mouse nonmuscle cells, whereas CAP2 is the predominant isoform in developing striated muscles. In cultured NIH3T3 and B16F1 cells, CAP1 is a highly abundant protein that colocalizes with cofilin-1 to dynamic regions of the cortical actin cytoskeleton. Analysis of CAP1 knockdown cells demonstrated that this protein promotes rapid actin filament depolymerization and is important for cell morphology, migration, and endocytosis. Interestingly, depletion of CAP1 leads to an accumulation of cofilin-1 into abnormal cytoplasmic aggregates and to similar cytoskeletal defects to those seen in cofilin-1 knockdown cells, demonstrating that CAP1 is required for proper subcellular localization and function of ADF/cofilin. Together, these data provide the first direct in vivo evidence that CAP promotes rapid actin dynamics in conjunction with ADF/cofilin and is required for several central cellular processes in mammals.

  18. An actin cytoskeleton with evolutionarily conserved functions in the absence of canonical actin-binding proteins

    OpenAIRE

    Paredez, Alexander R.; Assaf, Zoe June; Sept, David; Timofejeva, Ljudmilla; Dawson, Scott C.; Wang, Chung-Ju Rachel; Cande, W. Z.

    2011-01-01

    Giardia intestinalis, a human intestinal parasite and member of what is perhaps the earliest-diverging eukaryotic lineage, contains the most divergent eukaryotic actin identified to date and is the first eukaryote known to lack all canonical actin-binding proteins (ABPs). We sought to investigate the properties and functions of the actin cytoskeleton in Giardia to determine whether Giardia actin (giActin) has reduced or conserved roles in core cellular processes. In vitro polymerization of gi...

  19. Cytoskeletal changes induced by allosteric modulators of calcium-sensing receptor in esophageal epithelial cells.

    Science.gov (United States)

    Abdulnour-Nakhoul, Solange; Brown, Karen L; Rabon, Edd C; Al-Tawil, Youhanna; Islam, Mohammed T; Schmieg, John J; Nakhoul, Nazih L

    2015-11-01

    The calcium-sensing receptor (CaSR), a G-protein-coupled receptor, plays a role in glandular and fluid secretion in the gastrointestinal tract, and regulates differentiation and proliferation of epithelial cells. We examined the expression of CaSR in normal and pathological conditions of human esophagus and investigated the effect of a CaSR agonist, cinacalcet (CCT), and antagonist, calhex (CHX), on cell growth and cell-cell junctional proteins in primary cultures of porcine stratified squamous esophageal epithelium. We used immunohistochemistry and Western analysis to monitor expression of CaSR and cell-cell adhesion molecules, and MTT assay to monitor cell proliferation in cultured esophageal cells. CCT treatment significantly reduced proliferation, changed the cell shape from polygonal to spindle-like, and caused redistribution of E-cadherin and β-catenin from the cell membrane to the cytoplasm. Furthermore, it reduced expression of β-catenin by 35% (P < 0.02) and increased expression of a proteolysis cleavage fragment of E-cadherin, Ecad/CFT2, by 2.3 folds (P < 0.01). On the other hand, CHX treatment enhanced cell proliferation by 27% (P < 0.01), increased the expression of p120-catenin by 24% (P < 0.04), and of Rho, a GTPase involved in cytoskeleton remodeling, by 18% (P < 0.03). In conclusion, CaSR is expressed in normal esophagus as well as in Barrett's, esophageal adenocarcinoma, squamous cell carcinoma, and eosinophilic esophagitis. Long-term activation of CaSR with CCT disrupted the cadherin-catenin complex, induced cytoskeletal remodeling, actin fiber formation, and redistribution of CaSR to the nuclear area. These changes indicate a significant and complex role of CaSR in epithelial remodeling and barrier function of esophageal cells.

  20. Altering the cellular mechanical force balance results in integrated changes in cell, cytoskeletal and nuclear shape

    Science.gov (United States)

    Sims, J. R.; Karp, S.; Ingber, D. E.

    1992-01-01

    Studies were carried out with capillary endothelial cells cultured on fibronectin (FN)-coated dishes in order to analyze the mechanism of cell and nuclear shape control by extracellular matrix (ECM). To examine the role of the cytoskeleton in shape determination independent of changes in transmembrane osmotic pressure, membranes of adherent cells were permeabilized with saponin (25 micrograms/ml) using a buffer that maintains the functional integrity of contractile microfilaments. Real-time videomicroscopic studies revealed that addition of 250 microM ATP resulted in time-dependent retraction and rounding of permeabilized cells and nuclei in a manner similar to that observed in intact living cells following detachment using trypsin-EDTA. Computerized image analysis confirmed that permeabilized cells remained essentially rigid in the absence of ATP and that retraction was stimulated in a dose-dependent manner as the concentration of ATP was raised from 10 to 250 microM. Maximal rounding occurred by 30 min with projected cell and nuclear areas being reduced by 69 and 41%, respectively. ATP-induced rounding was also accompanied by a redistribution of microfilaments resulting in formation of a dense net of F-actin surrounding retracted nuclei. Importantly, ATP-stimulated changes in cell, cytoskeletal, and nuclear form were prevented in permeabilized cells using a synthetic myosin peptide (IRICRKG) that has been previously shown to inhibit actomyosin filament sliding in muscle. In contrast, both the rate and extent of cell and nuclear rounding were increased in permeabilized cells exposed to ATP when the soluble FN peptide, GRGDSP, was used to dislodge immobilized FN from cell surface integrin receptors.(ABSTRACT TRUNCATED AT 250 WORDS).

  1. Cytoskeletal mechanics in pressure-overload cardiac hypertrophy

    Science.gov (United States)

    Tagawa, H.; Wang, N.; Narishige, T.; Ingber, D. E.; Zile, M. R.; Cooper, G. 4th

    1997-01-01

    We have shown that the cellular contractile dysfunction characteristic of pressure-overload cardiac hypertrophy results not from an abnormality intrinsic to the myofilament portion of the cardiocyte cytoskeleton but rather from an increased density of the microtubule component of the extramyofilament portion of the cardiocyte cytoskeleton. To determine how, in physical terms, this increased microtubule density mechanically overloads the contractile apparatus at the cellular level, we measured cytoskeletal stiffness and apparent viscosity in isolated cardiocytes via magnetic twisting cytometry, a technique by which magnetically induced force is applied directly to the cytoskeleton through integrin-coupled ferromagnetic beads coated with Arg-Gly-Asp (RGD) peptide. Measurements were made in two groups of cardiocytes from cats with right ventricular (RV) hypertrophy induced by pulmonary artery banding: (1) those from the pressure-overloaded RV and (2) those from the normally loaded same-animal control left ventricle (LV). Cytoskeletal stiffness increased almost twofold, from 8.53 +/- 0.77 dyne/cm2 in the normally loaded LV cardiocytes to 16.46 +/- 1.32 dyne/cm2 in the hypertrophied RV cardiocytes. Cytoskeletal apparent viscosity increased almost fourfold, from 20.97 +/- 1.92 poise in the normally loaded LV cardiocytes to 87.85 +/- 6.95 poise in the hypertrophied RV cardiocytes. In addition to these baseline data showing differing stiffness and, especially, apparent viscosity in the two groups of cardiocytes, microtubule depolymerization by colchicine was found to return both the stiffness and the apparent viscosity of the pressure overload-hypertrophied RV cells fully to normal. Conversely, microtubule hyperpolymerization by taxol increased the stiffness and apparent viscosity values of normally loaded LV cardiocytes to the abnormal values given above for pressure-hypertrophied RV cardiocytes. Thus, increased microtubule density constitutes primarily a viscous load on

  2. Integrin α5β1-fimbriae binding and actin rearrangement are essential for Porphyromonas gingivalis invasion of osteoblasts and subsequent activation of the JNK pathway

    Directory of Open Access Journals (Sweden)

    Zhang Wenjian

    2013-01-01

    Full Text Available Abstract Background Chronic periodontitis is an infectious disease of the periodontium, which includes the gingival epithelium, periodontal ligament and alveolar bone. The signature clinical feature of periodontitis is resorption of alveolar bone and subsequent tooth loss. The Gram-negative oral anaerobe, Porphyromonas gingivalis, is strongly associated with periodontitis, and it has been shown previously that P. gingivalis is capable of invading osteoblasts in a dose- and time-dependent manner resulting in inhibition of osteoblast differentiation and mineralization in vitro. It is not yet clear which receptors and cytoskeletal components mediate the invasive process, nor how the signaling pathways and viability of osteoblasts are affected by bacterial internalization. This study aimed to investigate these issues using an in vitro model system involving the inoculation of P. gingivalis ATCC 33277 into primary osteoblast cultures. Results It was found that binding between P. gingivalis fimbriae and integrin α5β1 on osteoblasts, and subsequent peripheral condensation of actin, are essential for entry of P. gingivalis into osteoblasts. The JNK pathway was activated in invaded osteoblasts, and apoptosis was induced by repeated infections. Conclusions These observations indicate that P. gingivalis manipulates osteoblast function to promote its initial intracellular persistence by prolonging the host cell life span prior to its intercellular dissemination via host cell lysis. The identification of molecules critical to the interaction between P. gingivalis and osteoblasts will facilitate the development of new therapeutic strategies for the prevention of periodontal bone loss.

  3. Use of the Aspergillus oryzae actin gene promoter in a novel reporter system for exploring antifungal compounds and their target genes.

    Science.gov (United States)

    Marui, Junichiro; Yoshimi, Akira; Hagiwara, Daisuke; Fujii-Watanabe, Yoshimi; Oda, Ken; Koike, Hideaki; Tamano, Koichi; Ishii, Tomoko; Sano, Motoaki; Machida, Masayuki; Abe, Keietsu

    2010-08-01

    Demand for novel antifungal drugs for medical and agricultural uses has been increasing because of the diversity of pathogenic fungi and the emergence of drug-resistant strains. Genomic resources for various living species, including pathogenic fungi, can be utilized to develop novel and effective antifungal compounds. We used Aspergillus oryzae as a model to construct a reporter system for exploring novel antifungal compounds and their target genes. The comprehensive gene expression analysis showed that the actin-encoding actB gene was transcriptionally highly induced by benomyl treatment. We therefore used the actB gene to construct a novel reporter system for monitoring responses to cytoskeletal stress in A. oryzae by introducing the actB promoter::EGFP fusion gene. Distinct fluorescence was observed in the reporter strain with minimum background noise in response to not only benomyl but also compounds inhibiting lipid metabolism that is closely related to cell membrane integrity. The fluorescent responses indicated that the reporter strain can be used to screen for lead compounds affecting fungal microtubule and cell membrane integrity, both of which are attractive antifungal targets. Furthermore, the reporter strain was shown to be technically applicable for identifying novel target genes of antifungal drugs triggering perturbation of fungal microtubules or membrane integrity.

  4. Remodeling of the fibroblast cytoskeletal architecture during the replication cycle of Ectromelia virus: A morphological in vitro study in a murine cell line.

    Science.gov (United States)

    Szulc-Dabrowska, Lidia; Gregorczyk, Karolina P; Struzik, Justyna; Boratynska-Jasinska, Anna; Szczepanowska, Joanna; Wyzewski, Zbigniew; Toka, Felix N; Gierynska, Malgorzata; Ostrowska, Agnieszka; Niemialtowski, Marek G

    2016-08-01

    Ectromelia virus (ECTV, the causative agent of mousepox), which represents the same genus as variola virus (VARV, the agent responsible for smallpox in humans), has served for years as a model virus for studying mechanisms of poxvirus-induced disease. Despite increasing knowledge on the interaction between ECTV and its natural host-the mouse-surprisingly, still little is known about the cell biology of ECTV infection. Because pathogen interaction with the cytoskeleton is still a growing area of research in the virus-host cell interplay, the aim of the present study was to evaluate the consequences of ECTV infection on the cytoskeleton in a murine fibroblast cell line. The viral effect on the cytoskeleton was reflected by changes in migration of the cells and rearrangement of the architecture of tubulin, vimentin, and actin filaments. The virus-induced cytoskeletal rearrangements observed in these studies contributed to the efficient cell-to-cell spread of infection, which is an important feature of ECTV virulence. Additionally, during later stages of infection L929 cells produced two main types of actin-based cellular protrusions: short (actin tails and "dendrites") and long (cytoplasmic corridors). Due to diversity of filopodial extensions induced by the virus, we suggest that ECTV represents a valuable new model for studying processes and pathways that regulate the formation of cytoskeleton-based cellular structures. © 2016 Wiley Periodicals, Inc.

  5. Extending the molecular clutch beyond actin-based cell motility

    Science.gov (United States)

    Havrylenko, Svitlana; Mezanges, Xavier; Batchelder, Ellen; Plastino, Julie

    2014-10-01

    Many cell movements occur via polymerization of the actin cytoskeleton beneath the plasma membrane at the front of the cell, forming a protrusion called a lamellipodium, while myosin contraction squeezes forward the back of the cell. In what is known as the ‘molecular clutch’ description of cell motility, forward movement results from the engagement of the acto-myosin motor with cell-matrix adhesions, thus transmitting force to the substrate and producing movement. However during cell translocation, clutch engagement is not perfect, and as a result, the cytoskeleton slips with respect to the substrate, undergoing backward (retrograde) flow in the direction of the cell body. Retrograde flow is therefore inversely proportional to cell speed and depends on adhesion and acto-myosin dynamics. Here we asked whether the molecular clutch was a general mechanism by measuring motility and retrograde flow for the Caenorhabditis elegans sperm cell in different adhesive conditions. These cells move by adhering to the substrate and emitting a dynamic lamellipodium, but the sperm cell does not contain an acto-myosin cytoskeleton. Instead the lamellipodium is formed by the assembly of major sperm protein, which has no biochemical or structural similarity to actin. We find that these cells display the same molecular clutch characteristics as acto-myosin containing cells. We further show that retrograde flow is produced both by cytoskeletal assembly and contractility in these cells. Overall this study shows that the molecular clutch hypothesis of how polymerization is transduced into motility via adhesions is a general description of cell movement regardless of the composition of the cytoskeleton.

  6. Magnolol inhibits migration of vascular smooth muscle cells via cytoskeletal remodeling pathway to attenuate neointima formation

    Energy Technology Data Exchange (ETDEWEB)

    Karki, Rajendra [Division of Pharmacology and Toxicology, School of Pharmacy, University of Missouri-Kansas City (United States); Department of Oriental Medicine Resources, Mokpo National University (Korea, Republic of); Kim, Seong-Bin [Jeollanamdo Development Institute for Korean Traditional Medicine, Jangheung gun, Jeollanamdo (Korea, Republic of); Kim, Dong-Wook, E-mail: dbkim@mokpo.ac.kr [Department of Oriental Medicine Resources, Mokpo National University (Korea, Republic of)

    2013-12-10

    Background: Increased proliferation and migration of vascular smooth muscle cells (VSMCs) contribute importantly to the formation of both atherosclerotic and restenotic lesions. The objective of this study was to investigate the effect of magnolol on VSMC migration. Methods: The proteolytic activity of matrix metalloproteinases (MMPs) in tumor necrosis factor alpha (TNF-α) stimulated VSMCs was performed by gelatin zymography. VSMC migration was assessed by wound healing and Boyden chamber methods. Collagen induced VSMC adhesion was determined by spectrofluorimeter and stress fibers formation was evaluated by fluorescence microscope. The expression of signaling molecules involved in stress fibers formation was determined by western blot. The phosphorylation of myosin light chain (MLC20) was determined by urea-glycerol polyacrylamide gel electrophoresis. Immunohistochemistry was performed to determine the expression of β1-integrin and collagen type I in the injured carotid arteries of rats on day 35 after vascular injury. Results: VSMC migration was strongly inhibited by magnolol without affecting MMPs expression. Also, magnolol inhibited β1-integrin expression, FAK phosphorylation and RhoA and Cdc42 activation to inhibit the collagen induced stress fibers formation. Moreover, magnolol inhibited the phosphorylation of MLC20. Our in vivo results showed that magnolol inhibited β1-integrin expression, collagen type I deposition and FAK phosphorylation in injured carotid arteries without affecting MMP-2 activity. Conclusions: Magnolol inhibited VSMC migration via inhibition of cytoskeletal remodeling pathway to attenuate neointima formation. General significance: This study provides a rationale for further evaluation of magnolol for the management of atherosclerosis and restenosis. - Highlights: • Magnolol strongly inhibited migration of VSMCs. • Magnolol inhibited stress fibers formation. • MLC20 phosphorylation was also inhibited by magnolol. • Anti

  7. 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

  8. 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.

  9. Substrate, focal adhesions, and actin filaments: a mechanical unit with a weak spot for mechanosensitive proteins

    Science.gov (United States)

    Kirchenbüchler, David; Born, Simone; Kirchgeßner, Norbert; Houben, Sebastian; Hoffmann, Bernd; Merkel, Rudolf

    2010-05-01

    Mechanosensing is a vital prerequisite for dynamic remodeling of focal adhesions and cytoskeletal structures upon substrate deformation. For example, tissue formation, directed cell orientation or cell differentiation are regulated by such mechanosensing processes. Focal adhesions and the actin cytoskeleton are believed to be involved in these processes, but where mechanosensing molecules are located and how elastic substrate, focal adhesions and the cytoskeleton couple with each other upon substrate deformation still remains obscure. To approach these questions we have developed a sensitive method to apply defined spatially decaying deformation fields to cells cultivated on ultrasoft elastic substrates and to accurately quantify the resulting displacements of the actin cytoskeleton, focal adhesions, as well as the substrate. Displacement fields were recorded in live cell microscopy by tracking either signals from fluorescent proteins or marker particles in the substrate. As model cell type we used myofibroblasts. These cells are characterized by highly stable adhesion and force generating structures but are still able to detect mechanical signals with high sensitivity. We found a rigid connection between substrate and focal adhesions. Furthermore, stress fibers were found to be barely extendable almost over their whole lengths. Plastic deformation took place only at the very ends of actin filaments close to focal adhesions. As a result, this area became elongated without extension of existing actin filaments by polymerization. Both ends of the stress fibers were mechanically coupled with detectable plastic deformations on either site. Interestingly, traction force dependent substrate deformation fields remained mostly unaffected even when stress fiber elongations were released. These data argue for a location of mechanosensing proteins at the ends of actin stress fibers and describe, except for these domains, the whole system to be relatively rigid for tensile

  10. Cytoskeletal Reorganization Drives Mesenchymal Condensation and Regulates Downstream Molecular Signaling.

    Directory of Open Access Journals (Sweden)

    Poulomi Ray

    Full Text Available Skeletal condensation occurs when specified mesenchyme cells self-organize over several days to form a distinctive cartilage template. Here, we determine how and when specified mesenchyme cells integrate mechanical and molecular information from their environment, forming cartilage condensations in the pharyngeal arches of chick embryos. By disrupting cytoskeletal reorganization, we demonstrate that dynamic cell shape changes drive condensation and modulate the response of the condensing cells to Fibroblast Growth Factor (FGF, Bone Morphogenetic Protein (BMP and Transforming Growth Factor beta (TGF-β signaling pathways. Rho Kinase (ROCK-driven actomyosin contractions and Myosin II-generated differential cell cortex tension regulate these cell shape changes. Disruption of the condensation process inhibits the differentiation of the mesenchyme cells into chondrocytes, demonstrating that condensation regulates the fate of the mesenchyme cells. We also find that dorsal and ventral condensations undergo distinct cell shape changes. BMP signaling is instructive for dorsal condensation-specific cell shape changes. Moreover, condensations exhibit ventral characteristics in the absence of BMP signaling, suggesting that in the pharyngeal arches ventral morphology is the ground pattern. Overall, this study characterizes the interplay between cytoskeletal dynamics and molecular signaling in a self-organizing system during tissue morphogenesis.

  11. Fourier and fractal analysis of cytoskeletal morphology altered by xenobiotics

    Science.gov (United States)

    Crosta, Giovanni F.; Urani, Chiara; Fumarola, Laura

    2003-06-01

    The cytoskeletal microtubules (MTs) of rat hepatocytes treated by Benomyl (a fungicide) were imaged by means of immunofluorescent staining and optical microscopy. Images of untreated, or control (C), and of treated (T) cells were processed both by fractal and Fourier analysis. The C-MTs had contour fractal dimensions higher (>= 1.4) than those of T-MTs (enhancement," which corresponds to the application of a (pseudo)differential operator to the image. Enhanced spectra were interpolated by a polynomial, q, of degree 39, from which morphological descriptors were extracted. Descriptors from Fourier analysis made image classification possible. Principal components analysis was applied to the descriptors. In the plane of the first two components, {z1,z2}, the minimum spanning tree was drawn. Images of T-MTs formed a single cluster, whereas images of C-MTs formed two clusters, C1 and C2. The component z1 correlated positively with the local maxima and minima of q, which reflected differences between T and C in power spectral density in the 1 to 2000 cycles/mm spatial frequency band. The difference between C1 and C2 was ascribed to anisotropy of the MT bundles. The implemented image classifier is capable of telling differences in cytoskeletal organization. As a consequence the method can become a tool for testing cytotoxicity and for extracting quantitative information about intracellular alterations of various origin.

  12. THRUMIN1 is a light-regulated actin-bundling protein involved in chloroplast motility.

    Science.gov (United States)

    Whippo, Craig W; Khurana, Parul; Davis, Phillip A; DeBlasio, Stacy L; DeSloover, Daniel; Staiger, Christopher J; Hangarter, Roger P

    2011-01-11

    Chloroplast movement in response to changing light conditions optimizes photosynthetic light absorption. This repositioning is stimulated by blue light perceived via the phototropin photoreceptors and is transduced to the actin cytoskeleton. Some actin-based motility systems use filament reorganizations rather than myosin-based translocations. Recent research favors the hypothesis that chloroplast movement is driven by actin reorganization at the plasma membrane, but no proteins affecting chloroplast movements have been shown to associate with both the plasma membrane and actin filaments in vivo. Here we identified THRUMIN1 as a critical link between phototropin photoreceptor activity at the plasma membrane and actin-dependent chloroplast movements. THRUMIN1 bundles filamentous actin in vitro, and it localizes to the plasma membrane and displays light- and phototropin-dependent localization to microfilaments in vivo. These results suggest that phototropin-induced actin bundling via THRUMIN1 is important for chloroplast movement. A mammalian homolog of THRUMIN1, GRXCR1, has been implicated in auditory responses and hair cell stereocilla development as a regulator of actin architecture. Studies of THRUMIN1 will help elucidate the function of this family of eukaryotic proteins.

  13. Keeping it all together: auxin-actin crosstalk in plant development.

    Science.gov (United States)

    Zhu, Jinsheng; Geisler, Markus

    2015-08-01

    Polar auxin transport and the action of the actin cytoskeleton are tightly interconnected, which is documented by the finding that auxin transporters reach their final destination by active movement of secretory vesicles along F-actin tracks. Moreover, auxin transporter polarity and flexibility is thought to depend on transporter cycling that requires endocytosis and exocytosis of vesicles. In this context, we have reviewed the current literature on an involvement of the actin cytoskeleton in polar auxin transport and identify known similarities and differences in its structure, function and dynamics in comparison to non-plant organisms. By describing how auxin modulates actin expression and actin organization and how actin and its stability affects auxin-transporter endocytosis and recycling, we discuss the current knowledge on regulatory auxin-actin feedback loops. We focus on known effects of auxin and of auxin transport inhibitors on the stability and organization of actin and examine the functionality of auxin and/or auxin transport inhibitor-binding proteins with respect to their suitability to integrate auxin/auxin transport inhibitor action. Finally, we indicate current difficulties in the interpretation of organ, time and concentration-dependent auxin/auxin transport inhibitor treatments and formulate simple future experimental guidelines.

  14. The actin multigene family of Paramecium tetraurelia

    Directory of Open Access Journals (Sweden)

    Wagner Erika

    2007-03-01

    Full Text Available Abstract Background A Paramecium tetraurelia pilot genome project, the subsequent sequencing of a Megabase chromosome as well as the Paramecium genome project aimed at gaining insight into the genome of Paramecium. These cells display a most elaborate membrane trafficking system, with distinct, predictable pathways in which actin could participate. Previously we had localized actin in Paramecium; however, none of the efforts so far could proof the occurrence of actin in the cleavage furrow of a dividing cell, despite the fact that actin is unequivocally involved in cell division. This gave a first hint that Paramecium may possess actin isoforms with unusual characteristics. The genome project gave us the chance to search the whole Paramecium genome, and, thus, to identify and characterize probably all actin isoforms in Paramecium. Results The ciliated protozoan, P. tetraurelia, contains an actin multigene family with at least 30 members encoding actin, actin-related and actin-like proteins. They group into twelve subfamilies; a large subfamily with 10 genes, seven pairs and one trio with > 82% amino acid identity, as well as three single genes. The different subfamilies are very distinct from each other. In comparison to actins in other organisms, P. tetraurelia actins are highly divergent, with identities topping 80% and falling to 30%. We analyzed their structure on nucleotide level regarding the number and position of introns. On amino acid level, we scanned the sequences for the presence of actin consensus regions, for amino acids of the intermonomer interface in filaments, for residues contributing to ATP binding, and for known binding sites for myosin and actin-specific drugs. Several of those characteristics are lacking in several subfamilies. The divergence of P. tetraurelia actins and actin-related proteins between different P. tetraurelia subfamilies as well as with sequences of other organisms is well represented in a phylogenetic

  15. 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

  16. 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.

  17. The design of MACs (minimal actin cortices).

    Science.gov (United States)

    Vogel, Sven K; Heinemann, Fabian; Chwastek, Grzegorz; Schwille, Petra

    2013-11-01

    The actin cell cortex in eukaryotic cells is a key player in controlling and maintaining the shape of cells, and in driving major shape changes such as in cytokinesis. It is thereby constantly being remodeled. Cell shape changes require forces acting on membranes that are generated by the interplay of membrane coupled actin filaments and assemblies of myosin motors. Little is known about how their interaction regulates actin cell cortex remodeling and cell shape changes. Because of the vital importance of actin, myosin motors and the cell membrane, selective in vivo experiments and manipulations are often difficult to perform or not feasible. Thus, the intelligent design of minimal in vitro systems for actin-myosin-membrane interactions could pave a way for investigating actin cell cortex mechanics in a detailed and quantitative manner. Here, we present and discuss the design of several bottom-up in vitro systems accomplishing the coupling of actin filaments to artificial membranes, where key parameters such as actin densities and membrane properties can be varied in a controlled manner. Insights gained from these in vitro systems may help to uncover fundamental principles of how exactly actin-myosin-membrane interactions govern actin cortex remodeling and membrane properties for cell shape changes.

  18. 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.

  19. Drosophila Rho-associated kinase (Drok) links Frizzled-mediated planar cell polarity signaling to the actin cytoskeleton.

    Science.gov (United States)

    Winter, C G; Wang, B; Ballew, A; Royou, A; Karess, R; Axelrod, J D; Luo, L

    2001-04-06

    Frizzled (Fz) and Dishevelled (Dsh) are components of an evolutionarily conserved signaling pathway that regulates planar cell polarity. How this signaling pathway directs asymmetric cytoskeletal reorganization and polarized cell morphology remains unknown. Here, we show that Drosophila Rho-associated kinase (Drok) works downstream of Fz/Dsh to mediate a branch of the planar polarity pathway involved in ommatidial rotation in the eye and in restricting actin bundle formation to a single site in developing wing cells. The primary output of Drok signaling is regulating the phosphorylation of nonmuscle myosin regulatory light chain, and hence the activity of myosin II. Drosophila myosin VIIA, the homolog of the human Usher Syndrome 1B gene, also functions in conjunction with this newly defined portion of the Fz/Dsh signaling pathway to regulate the actin cytoskeleton.

  20. Expression of type I collagen and tenascin C is regulated by actin polymerization through MRTF in dedifferentiated chondrocytes.

    Science.gov (United States)

    Parreno, Justin; Raju, Sneha; Niaki, Mortah Nabavi; Andrejevic, Katarina; Jiang, Amy; Delve, Elizabeth; Kandel, Rita

    2014-10-16

    This study examined actin regulation of fibroblast matrix genes in dedifferentiated chondrocytes. We demonstrated that dedifferentiated chondrocytes exhibit increased actin polymerization, nuclear localization of myocardin related transcription factor (MRTF), increased type I collagen (col1) and tenascin C (Tnc) gene expression, and decreased Sox9 gene expression. Induction of actin depolymerization by latrunculin treatment or cell rounding, reduced MRTF nuclear localization, repressed col1 and Tnc expression, and increased Sox9 gene expression in dedifferentiated chondrocytes. Treatment of passaged chondrocytes with MRTF inhibitor repressed col1 and Tnc expression, but did not affect Sox9 expression. Our results show that actin polymerization regulates fibroblast matrix gene expression through MRTF in passaged chondrocytes.

  1. 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.

  2. 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.

  3. 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.

  4. Insight into the assembly properties and functional organisation of the magnetotactic bacterial actin-like homolog, MamK.

    Directory of Open Access Journals (Sweden)

    Sanjiv Sonkaria

    Full Text Available Magnetotactic bacteria (MTB synthesize magnetosomes, which are intracellular vesicles comprising a magnetic particle. A series of magnetosomes arrange themselves in chains to form a magnetic dipole that enables the cell to orient itself along the Earth's magnetic field. MamK, an actin-like homolog of MreB has been identified as a central component in this organisation. Gene deletion, fluorescence microscopy and in vitro studies have yielded mechanistic differences in the filament assembly of MamK with other bacterial cytoskeletal proteins within the cell. With little or no information on the structural and behavioural characteristics of MamK outside the cell, the mamK gene from Magnetospirillium gryphiswaldense was cloned and expressed to better understand the differences in the cytoskeletal properties with its bacterial homologues MreB and acitin. Despite the low sequence identity shared between MamK and MreB (22% and actin (18%, the behaviour of MamK monitored by light scattering broadly mirrored that of its bacterial cousin MreB primarily in terms of its pH, salt, divalent metal-ion and temperature dependency. The broad size variability of MamK filaments revealed by light scattering studies was supported by transmission electron microscopy (TEM imaging. Filament morphology however, indicated that MamK conformed to linearly orientated filaments that appeared to be distinctly dissimilar compared to MreB suggesting functional differences between these homologues. The presence of a nucleotide binding domain common to actin-like proteins was demonstrated by its ability to function both as an ATPase and GTPase. Circular dichroism and structural homology modelling showed that MamK adopts a protein fold that is consistent with the 'classical' actin family architecture but with notable structural differences within the smaller domains, the active site region and the overall surface electrostatic potential.

  5. R-(+)-perillyl alcohol-induced cell cycle changes, altered actin cytoskeleton, and decreased ras and p34(cdc2) expression in colonic adenocarcinoma SW480 cells.

    Science.gov (United States)

    Cerda, S R; Wilkinson, J; Thorgeirsdottir, S; Broitman, S A

    1999-01-01

    Monoterpenes as S-(-)-perillyl alcohol (PA) have been shown to inhibit the isoprenylation of such growth regulatory proteins as ras. In this study, we investigated the effects of the R-(+) enantiomer of PA on cell cycle, signaling, and cytoskeletal control in the colonic adenocarcinoma cell line SW480, which carries a K-ras mutation. Cell cycle analysis by flow cytometry of SW480 cells treated with 1 mM PA for 24 hours demonstrated an increase in the number of cells in G0/G1 with a decrease in S phase, compared with untreated control cells. These cell cycle changes correlated with an inhibition of protein isoprenylation from (14)C-mevalonate and decreased expression of the cell cycle regulatory kinase p34(cdc2). Additionally, PA-treated cells acquired a flattened morphology with a condensation of cytoskeletal actin spikes to the periphery. This was in contrast to treatment with 15 microM mevinolin (MVN), a direct mevalonate synthesis inhibitor, which imparted to SW480 cells a more rounded and spindly morphology, associated with the depolymerization of actin microfilaments. Together, these data suggest that fluctuations in mevalonate and isoprenoid pools may involve different morphologic phenomenon. Because ras mediated signaling is related to the organization of the actin cytoskeleton, we investigated the effects of PA on the isoprenylation of ras. Although MVN treatment inhibited ras farnesylation, PA treatment decreased the expression of total ras protein. In summary, R-(+)-PA-induced cell signaling events correlated with alterations in the organization of cytoskeletal actin and decreased protein expression of growth regulatory proteins, such as ras and cdc2 kinase. These effects may contribute to the growth inhibitory activity of R-(+)-PA.

  6. The cytoskeletal protein Zyxin inhibits Shh signaling during the CNS patterning in Xenopus laevis through interaction with the transcription factor Gli1.

    Science.gov (United States)

    Martynova, Natalia Y; Ermolina, Ludmila V; Ermakova, Galina V; Eroshkin, Fedor M; Gyoeva, Fatima K; Baturina, Natalia S; Zaraisky, Andrey G

    2013-08-01

    Zyxin is a cytoskeletal protein that controls cell movements by regulating actin filaments assembly, but it can also modulate gene expression owing to its interactions with the proteins involved in signaling cascades. Therefore, identification of proteins that interact with Zyxin in embryonic cells is a promising way to unravel mechanisms responsible for coupling of two major components of embryogenesis: morphogenetic movements and cell differentiation. Now we show that in Xenopus laevis embryos Zyxin can bind to and suppress activity of the primary effector of Sonic hedgehog (Shh) signaling cascade, the transcription factor Gli1. By using loss- and gain-of-function approaches, we demonstrate that Zyxin is essential for reduction of Shh signaling within the dorsal part of the neural tube of X. laevis embryo. Thus, our finding discloses a novel function of Zyxin in fine tuning of the central neural system patterning which is based on the ventral-to-dorsal gradient of Shh signaling.

  7. A conserved proline-rich region of the Saccharomyces cerevisiae cyclase-associated protein binds SH3 domains and modulates cytoskeletal localization.

    Science.gov (United States)

    Freeman, N L; Lila, T; Mintzer, K A; Chen, Z; Pahk, A J; Ren, R; Drubin, D G; Field, J

    1996-02-01

    Saccharomyces cerevisiae cyclase-associated protein (CAP or Srv2p) is multifunctional. The N-terminal third of CAP binds to adenylyl cyclase and has been implicated in adenylyl cyclase activation in vivo. The widely conserved C-terminal domain of CAP binds to monomeric actin and serves an important cytoskeletal regulatory function in vivo. In addition, all CAP homologs contain a centrally located proline-rich region which has no previously identified function. Recently, SH3 (Src homology 3) domains were shown to bind to proline-rich regions of proteins. Here we report that the proline-rich region of CAP is recognized by the SH3 domains of several proteins, including the yeast actin-associated protein Abp1p. Immunolocalization experiments demonstrate that CAP colocalizes with cortical actin-containing structures in vivo and that a region of CAP containing the SH3 domain binding site is required for this localization. We also demonstrate that the SH3 domain of yeast Abp1p and that of the yeast RAS protein guanine nucleotide exchange factor Cdc25p complex with adenylyl cyclase in vitro. Interestingly, the binding of the Cdc25p SH3 domain is not mediated by CAP and therefore may involve direct binding to adenylyl cyclase or to an unidentified protein which complexes with adenylyl cyclase. We also found that CAP homologous from Schizosaccharomyces pombe and humans bind SH3 domains. The human protein binds most strongly to the SH3 domain from the abl proto-oncogene. These observations identify CAP as an SH3 domain-binding protein and suggest that CAP mediates interactions between SH3 domain proteins and monomeric actin.

  8. 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.

  9. Functional characterization of skeletal F-actin labeled on the NH2-terminal segment of residues 1-28.

    Science.gov (United States)

    Bertrand, R; Chaussepied, P; Audemard, E; Kassab, R

    1989-05-15

    Rabbit skeletal alpha-actin was covalently labeled in the filamentous state by the fluorescent nucleophile, N-(5-sulfo-1-naphthyl)ethylenediamine (EDANS) in the presence of the carboxyl group activator 1-(3-dimethyl-aminopropyl)-3-ethylcarbodiimide (EDC). The coupling reaction was continued until the incorporation of nearly 1 mol EDANS/mol actin. After limited proteolytic digestion of the labeled protein and chromatographic identification of the EDANS-peptides, about 80% of the attached fluorophore was found on the actin segment of residues 1-28, most probably within the N-terminal acidic region of residues 1-7. A minor labeling site was located on the segment that consists of residues 40-113. No label was incorporated into the COOH-terminal moiety consisting of residues 113-375. The isolated EDANS-G-actin undergoes polymerization in the presence of salts but at a rate significantly greater than unlabeled actin. The EDANS-F-actin could be complexed to skeletal chymotryptic myosin subfragment 1 (S-1) and to tropomyosin. The complex formed between EDANS-F-actin and S-1 could not be further crosslinked by EDC but the two proteins were readily joined by glutaraldehyde as observed for native actin-S-1, suggesting that the EDANS-substituted carboxyl site is also involved in the EDC crosslinking of native actin to S-1. Moreover, the EDANS labeling of F-actin resulted in a 20-fold increase in the Km of the actin-activated Mg2+.ATPase of S-1. Thus, this labeling, while it did not much affect the rigor actin-S-1 interaction, changes the actin binding to the S-1-nucleotide complexes significantly. The selective introduction of a variety of spectral probes, like EDANS, or other classes of fluorophores, on the N-terminal region of actin, through the reported carbodiimide coupling reaction, would provide several different derivatives valuable for assessing the functional role of the negatively charged N-terminus of actin during its interaction with myosin and other actin

  10. 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.

  11. Towards Experimental Tests of Quantum Effects in Cytoskeletal Proteins

    CERN Document Server

    Mershin, A; Miller, J H; Nawarathna, D; Skoulakis, E M C; Mavromatos, Nikolaos E; Kolomenskij, A A; Schüssler, H A; Luduena, R F; Nanopoulos, Dimitri V; Mershin, Andreas; Sanabria, Hugo; Miller, John H.; Nawarathna, Dharmakeerthna; Skoulakis, Efthimios M.C.; Mavromatos, Nikolaos E.; Kolomenskii, Alexadre A.; Schuessler, Hans A.; Luduena, Richard F.; Nanopoulos, Dimitri V.

    2005-01-01

    It has become increasingly evident that fabrication of novel biomaterials through molecular self-assembly is going to play a significant role in material science and possibly the information technology of the future. Tubulin, microtubules (MTs) and the cytoskeleton are dynamic, self-assembling systems and we asked whether their structure and function contain the clues on how to fabricate biomolecular information processing devices. Here we review our neurobiological studies of transgenic Drosophila that strongly suggest the microtubular cytoskeleton is near the 'front lines' of intracellular information manipulation and storage. We also establish that spectroscopic techniques such as refractometry, surface plasmon resonance sensing and dielectric spectroscopy, coupled with molecular dynamic simulations and (quantum) electrodynamic analytical theory are useful tools in the study of the electrodynamic and possible quantum effects in cytoskeletal proteins. Implicit in our driving question is the possibility that...

  12. Cytoskeletal re-arrangement in TGF-β1-induced alveolar epithelial-mesenchymal transition studied by atomic force microscopy and high-content analysis.

    Science.gov (United States)

    Buckley, Stephen T; Medina, Carlos; Davies, Anthony M; Ehrhardt, Carsten

    2012-04-01

    Epithelial-mesenchymal transition (EMT) is closely implicated in the pathogenesis of idiopathic pulmonary fibrosis. Associated with this phenotypic transition is the acquisition of an elongated cell morphology and establishment of stress fibers. The extent to which these EMT-associated changes influence cellular mechanics is unclear. We assessed the biomechanical properties of alveolar epithelial cells (A549) following exposure to TGF-β1. Using atomic force microscopy, changes in cell stiffness and surface membrane features were determined. Stimulation with TGF-β1 gave rise to a significant increase in stiffness, which was augmented by a collagen I matrix. Additionally, TGF-β1-treated cells exhibited a rougher surface profile with notable protrusions. Simultaneous quantitative examination of the morphological attributes of stimulated cells using an image-based high-content analysis system revealed dramatic alterations in cell shape, F-actin content and distribution. Together, these investigations point to a strong correlation between the cytoskeletal-associated cellular architecture and the mechanical dynamics of alveolar epithelial cells undergoing EMT. From the Clinical Editor: Epithelial-mesenchymal transition is implicated in the pathogenesis of pulmonary fibrosis. Using atomic force microscopy, the authors demonstrate a strong correlation between the cytoskeletal-associated cellular architecture and the mechanical dynamics of alveolar epithelial cells undergoing mesenchymal transition.

  13. 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.

  14. Dynamic Actin Gene Family Evolution in Primates

    Directory of Open Access Journals (Sweden)

    Liucun Zhu

    2013-01-01

    Full Text Available Actin is one of the most highly conserved proteins and plays crucial roles in many vital cellular functions. In most eukaryotes, it is encoded by a multigene family. Although the actin gene family has been studied a lot, few investigators focus on the comparison of actin gene family in relative species. Here, the purpose of our study is to systematically investigate characteristics and evolutionary pattern of actin gene family in primates. We identified 233 actin genes in human, chimpanzee, gorilla, orangutan, gibbon, rhesus monkey, and marmoset genomes. Phylogenetic analysis showed that actin genes in the seven species could be divided into two major types of clades: orthologous group versus complex group. Codon usages and gene expression patterns of actin gene copies were highly consistent among the groups because of basic functions needed by the organisms, but much diverged within species due to functional diversification. Besides, many great potential pseudogenes were found with incomplete open reading frames due to frameshifts or early stop codons. These results implied that actin gene family in primates went through “birth and death” model of evolution process. Under this model, actin genes experienced strong negative selection and increased the functional complexity by reproducing themselves.

  15. 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].

  16. Fyn Mediates High Glucose-Induced Actin Cytoskeleton Reorganization of Podocytes via Promoting ROCK Activation In Vitro

    Directory of Open Access Journals (Sweden)

    Zhimei Lv

    2016-01-01

    Full Text Available Fyn, a member of the Src family of tyrosine kinases, is a key regulator in cytoskeletal remodeling in a variety of cell types. Recent studies have demonstrated that Fyn is responsible for nephrin tyrosine phosphorylation, which will result in polymerization of actin filaments and podocyte damage. Thus detailed involvement of Fyn in podocytes is to be elucidated. In this study, we investigated the potential role of Fyn/ROCK signaling and its interactions with paxillin. Our results presented that high glucose led to filamentous actin (F-actin rearrangement in podocytes, accompanied by paxillin phosphorylation and increased cell motility, during which Fyn and ROCK were markedly activated. Gene knockdown of Fyn by siRNA showed a reversal effect on high glucose-induced podocyte damage and ROCK activation; however, inhibition of ROCK had no significant effects on Fyn phosphorylation. These observations demonstrate that in vitro Fyn mediates high glucose-induced actin cytoskeleton remodeling of podocytes via promoting ROCK activation and paxillin phosphorylation.

  17. Actin-associated protein palladin is required for migration behavior and differentiation potential of C2C12 myoblast cells

    Energy Technology Data Exchange (ETDEWEB)

    Nguyen, Ngoc Uyen Nhi; Liang, Vincent Roderick; Wang, Hao-Ven, E-mail: hvwang@mail.ncku.edu.tw

    2014-09-26

    Highlights: • Palladin is involved in myogenesis in vitro. • Palladin knockdown by siRNA increases myoblast proliferation, viability and differentiation. • Palladin knockdown decreases C2C12 myoblast migration ability. - Abstract: The actin-associated protein palladin has been shown to be involved in differentiation processes in non-muscle tissues. However, but its function in skeletal muscle has rarely been studied. Palladin plays important roles in the regulation of diverse actin-related signaling in a number of cell types. Since intact actin-cytoskeletal remodeling is necessary for myogenesis, in the present study, we pursue to investigate the role of actin-associated palladin in skeletal muscle differentiation. Palladin in C2C12 myoblasts is knocked-down using specific small interfering RNA (siRNA). The results show that down-regulation of palladin decreased migratory activity of mouse skeletal muscle C2C12 myoblasts. Furthermore, the depletion of palladin enhances C2C12 vitality and proliferation. Of note, the loss of palladin promotes C2C12 to express the myosin heavy chain, suggesting that palladin has a role in the modulation of C2C12 differentiation. It is thus proposed that palladin is required for normal C2C12 myogenesis in vitro.

  18. The pericellular environment regulates cytoskeletal development and the differentiation of mesenchymal stem cells and determines their response to hydrostatic pressure

    Directory of Open Access Journals (Sweden)

    AJ Steward

    2013-02-01

    Full Text Available The objective of this study was to examine the interplay between matrix stiffness and hydrostatic pressure (HP in regulating chondrogenesis of mesenchymal stem cells (MSCs and to further elucidate the mechanotransductive roles of integrins and the cytoskeleton. MSCs were seeded into 1 %, 2 % or 4 % agarose hydrogels and exposed to cyclic hydrostatic pressure. In a permissive media, the stiffer hydrogels supported an osteogenic phenotype, with little evidence of chondrogenesis observed regardless of the matrix stiffness. In a chondrogenic media, the stiffer gels suppressed cartilage matrix production and gene expression, with the addition of RGDS (an integrin blocker found to return matrix synthesis to similar levels as in the softer gels. Vinculin, actin and vimentin organisation all adapted within stiffer hydrogels, with the addition of RGDS again preventing these changes. While the stiffer gels inhibited chondrogenesis, they enhanced mechanotransduction of HP. RGDS suppressed the mechanotransduction of HP, suggesting a role for integrin binding as a regulator of both matrix stiffness and HP. Intermediate filaments also appear to play a role in the mechanotransduction of HP, as only vimentin organisation adapted in response to this mechanical stimulus. To conclude, the results of this study demonstrate that matrix density and/or stiffness modulates the development of the pericellular matrix and consequently integrin binding and cytoskeletal structure. The study further suggests that physiological cues such as HP enhance chondrogenesis of MSCs as the pericellular environment matures and the cytoskeleton adapts, and points to a novel role for vimentin in the transduction of HP.

  19. 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...

  20. 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.

  1. 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.

  2. The formin DAD domain plays dual roles in autoinhibition and actin nucleation.

    Science.gov (United States)

    Gould, Christopher J; Maiti, Sankar; Michelot, Alphée; Graziano, Brian R; Blanchoin, Laurent; Goode, Bruce L

    2011-03-08

    Formins are a large family of actin assembly-promoting proteins with many important biological roles. However, it has remained unclear how formins nucleate actin polymerization. All other nucleators are known to recruit actin monomers as a central part of their mechanisms. However, the actin-nucleating FH2 domain of formins lacks appreciable affinity for monomeric actin. Here, we found that yeast and mammalian formins bind actin monomers but that this activity requires their C-terminal DAD domains. Furthermore, we observed that the DAD works in concert with the FH2 to enhance nucleation without affecting the rate of filament elongation. We dissected this mechanism in mDia1, mapped nucleation activity to conserved residues in the DAD, and demonstrated that DAD roles in nucleation and autoinhibition are separable. Furthermore, DAD enhancement of nucleation was independent of contributions from the FH1 domain to nucleation. Together, our data show that (1) the DAD has dual functions in autoinhibition and nucleation; (2) the FH1, FH2, and DAD form a tripartite nucleation machine; and (3) formins nucleate by recruiting actin monomers and therefore are more similar to other nucleators than previously thought.

  3. Multicolour Multilevel STED nanoscopy of Actin/Spectrin Organization at Synapses.

    Science.gov (United States)

    Sidenstein, Sven C; D'Este, Elisa; Böhm, Marvin J; Danzl, Johann G; Belov, Vladimir N; Hell, Stefan W

    2016-05-25

    Superresolution fluorescence microscopy of multiple fluorophores still requires development. Here we present simultaneous three-colour stimulated emission depletion (STED) nanoscopy relying on a single STED beam at 620 nm. Toggling the STED beam between two or more power levels ("multilevelSTED") optimizes resolution and contrast in all colour channels, which are intrinsically co-aligned and well separated. Three-colour recording is demonstrated by imaging the nanoscale cytoskeletal organization in cultured hippocampal neurons. The down to ~35 nm resolution identified periodic actin/betaII spectrin lattices along dendrites and spines; however, at presynaptic and postsynaptic sites, these patterns were found to be absent. Both our multicolour scheme and the 620 nm STED line should be attractive for routine STED microscopy applications.

  4. Multicolour Multilevel STED nanoscopy of Actin/Spectrin Organization at Synapses

    Science.gov (United States)

    Sidenstein, Sven C.; D'Este, Elisa; Böhm, Marvin J.; Danzl, Johann G.; Belov, Vladimir N.; Hell, Stefan W.

    2016-05-01

    Superresolution fluorescence microscopy of multiple fluorophores still requires development. Here we present simultaneous three-colour stimulated emission depletion (STED) nanoscopy relying on a single STED beam at 620 nm. Toggling the STED beam between two or more power levels (“multilevelSTED”) optimizes resolution and contrast in all colour channels, which are intrinsically co-aligned and well separated. Three-colour recording is demonstrated by imaging the nanoscale cytoskeletal organization in cultured hippocampal neurons. The down to ~35 nm resolution identified periodic actin/betaII spectrin lattices along dendrites and spines; however, at presynaptic and postsynaptic sites, these patterns were found to be absent. Both our multicolour scheme and the 620 nm STED line should be attractive for routine STED microscopy applications.

  5. The maternal-to-zygotic transition targets actin to promote robustness during morphogenesis.

    Science.gov (United States)

    Zheng, Liuliu; Sepúlveda, Leonardo A; Lua, Rhonald C; Lichtarge, Olivier; Golding, Ido; Sokac, Anna Marie

    2013-11-01

    Robustness is a property built into biological systems to ensure stereotypical outcomes despite fluctuating inputs from gene dosage, biochemical noise, and the environment. During development, robustness safeguards embryos against structural and functional defects. Yet, our understanding of how robustness is achieved in embryos is limited. While much attention has been paid to the role of gene and signaling networks in promoting robust cell fate determination, little has been done to rigorously assay how mechanical processes like morphogenesis are designed to buffer against variable conditions. Here we show that the cell shape changes that drive morphogenesis can be made robust by mechanisms targeting the actin cytoskeleton. We identified two novel members of the Vinculin/α-Catenin Superfamily that work together to promote robustness during Drosophila cellularization, the dramatic tissue-building event that generates the primary epithelium of the embryo. We find that zygotically-expressed Serendipity-α (Sry-α) and maternally-loaded Spitting Image (Spt) share a redundant, actin-regulating activity during cellularization. Spt alone is sufficient for cellularization at an optimal temperature, but both Spt plus Sry-α are required at high temperature and when actin assembly is compromised by genetic perturbation. Our results offer a clear example of how the maternal and zygotic genomes interact to promote the robustness of early developmental events. Specifically, the Spt and Sry-α collaboration is informative when it comes to genes that show both a maternal and zygotic requirement during a given morphogenetic process. For the cellularization of Drosophilids, Sry-α and its expression profile may represent a genetic adaptive trait with the sole purpose of making this extreme event more reliable. Since all morphogenesis depends on cytoskeletal remodeling, both in embryos and adults, we suggest that robustness-promoting mechanisms aimed at actin could be effective at

  6. The maternal-to-zygotic transition targets actin to promote robustness during morphogenesis.

    Directory of Open Access Journals (Sweden)

    Liuliu Zheng

    2013-11-01

    Full Text Available Robustness is a property built into biological systems to ensure stereotypical outcomes despite fluctuating inputs from gene dosage, biochemical noise, and the environment. During development, robustness safeguards embryos against structural and functional defects. Yet, our understanding of how robustness is achieved in embryos is limited. While much attention has been paid to the role of gene and signaling networks in promoting robust cell fate determination, little has been done to rigorously assay how mechanical processes like morphogenesis are designed to buffer against variable conditions. Here we show that the cell shape changes that drive morphogenesis can be made robust by mechanisms targeting the actin cytoskeleton. We identified two novel members of the Vinculin/α-Catenin Superfamily that work together to promote robustness during Drosophila cellularization, the dramatic tissue-building event that generates the primary epithelium of the embryo. We find that zygotically-expressed Serendipity-α (Sry-α and maternally-loaded Spitting Image (Spt share a redundant, actin-regulating activity during cellularization. Spt alone is sufficient for cellularization at an optimal temperature, but both Spt plus Sry-α are required at high temperature and when actin assembly is compromised by genetic perturbation. Our results offer a clear example of how the maternal and zygotic genomes interact to promote the robustness of early developmental events. Specifically, the Spt and Sry-α collaboration is informative when it comes to genes that show both a maternal and zygotic requirement during a given morphogenetic process. For the cellularization of Drosophilids, Sry-α and its expression profile may represent a genetic adaptive trait with the sole purpose of making this extreme event more reliable. Since all morphogenesis depends on cytoskeletal remodeling, both in embryos and adults, we suggest that robustness-promoting mechanisms aimed at actin

  7. The Role of Formin Tails in Actin Nucleation, Processive Elongation, and Filament Bundling*

    Science.gov (United States)

    Vizcarra, Christina L.; Bor, Batbileg; Quinlan, Margot E.

    2014-01-01

    Formins are multidomain proteins that assemble actin in a wide variety of biological processes. They both nucleate and remain processively associated with growing filaments, in some cases accelerating filament growth. The well conserved formin homology 1 and 2 domains were originally thought to be solely responsible for these activities. Recently a role in nucleation was identified for the Diaphanous autoinhibitory domain (DAD), which is C-terminal to the formin homology 2 domain. The C-terminal tail of the Drosophila formin Cappuccino (Capu) is conserved among FMN formins but distinct from other formins. It does not have a DAD domain. Nevertheless, we find that Capu-tail plays a role in filament nucleation similar to that described for mDia1 and other formins. Building on this, replacement of Capu-tail with DADs from other formins tunes nucleation activity. Capu-tail has low-affinity interactions with both actin monomers and filaments. Removal of the tail reduces actin filament binding and bundling. Furthermore, when the tail is removed, we find that processivity is compromised. Despite decreased processivity, the elongation rate of filaments is unchanged. Again, replacement of Capu-tail with DADs from other formins tunes the processive association with the barbed end, indicating that this is a general role for formin tails. Our data show a role for the Capu-tail domain in assembling the actin cytoskeleton, largely mediated by electrostatic interactions. Because of its multifunctionality, the formin tail is a candidate for regulation by other proteins during cytoskeletal rearrangements. PMID:25246531

  8. F- and G-actin homeostasis regulates mechanosensitive actin nucleation by formins.

    Science.gov (United States)

    Higashida, Chiharu; Kiuchi, Tai; Akiba, Yushi; Mizuno, Hiroaki; Maruoka, Masahiro; Narumiya, Shuh; Mizuno, Kensaku; Watanabe, Naoki

    2013-04-01

    Physical force evokes rearrangement of the actin cytoskeleton. Signalling pathways such as tyrosine kinases, stretch-activated Ca(2+) channels and Rho GTPases are involved in force sensing. However, how signals are transduced to actin assembly remains obscure. Here we show mechanosensitive actin polymerization by formins (formin homology proteins). Cells overexpressing mDia1 increased the amount of F-actin on release of cell tension. Fluorescence single-molecule speckle microscopy revealed rapid induction of processive actin assembly by mDia1 on cell cortex deformation. mDia1 lacking the Rho-binding domain and other formins exhibited mechanosensitive actin nucleation, suggesting Rho-independent activation. Mechanosensitive actin nucleation by mDia1 required neither Ca(2+) nor kinase signalling. Overexpressing LIM kinase abrogated the induction of processive mDia1. Furthermore, s-FDAPplus (sequential fluorescence decay after photoactivation) analysis revealed a rapid actin monomer increase on cell cortex deformation. Our direct visualization of the molecular behaviour reveals a mechanosensitive actin filament regeneration mechanism in which G-actin released by actin remodelling plays a pivotal role.

  9. Cytoskeletal regulation dominates temperature-sensitive proteomic changes of hibernation in forebrain of 13-lined ground squirrels.

    Directory of Open Access Journals (Sweden)

    Allyson G Hindle

    Full Text Available 13-lined ground squirrels, Ictidomys tridecemlineatus, are obligate hibernators that transition annually between summer homeothermy and winter heterothermy - wherein they exploit episodic torpor bouts. Despite cerebral ischemia during torpor and rapid reperfusion during arousal, hibernator brains resist damage and the animals emerge neurologically intact each spring. We hypothesized that protein changes in the brain underlie winter neuroprotection. To identify candidate proteins, we applied a sensitive 2D gel electrophoresis method to quantify protein differences among forebrain extracts prepared from ground squirrels in two summer, four winter and fall transition states. Proteins that differed among groups were identified using LC-MS/MS. Only 84 protein spots varied significantly among the defined states of hibernation. Protein changes in the forebrain proteome fell largely into two reciprocal patterns with a strong body temperature dependence. The importance of body temperature was tested in animals from the fall; these fall animals use torpor sporadically with body temperatures mirroring ambient temperatures between 4 and 21°C as they navigate the transition between summer homeothermy and winter heterothermy. Unlike cold-torpid fall ground squirrels, warm-torpid individuals strongly resembled the homeotherms, indicating that the changes observed in torpid hibernators are defined by body temperature, not torpor per se. Metabolic enzymes were largely unchanged despite varied metabolic activity across annual and torpor-arousal cycles. Instead, the majority of the observed changes were cytoskeletal proteins and their regulators. While cytoskeletal structural proteins tended to differ seasonally, i.e., between summer homeothermy and winter heterothermy, their regulatory proteins were more strongly affected by body temperature. Changes in the abundance of various isoforms of the microtubule assembly and disassembly regulatory proteins

  10. Rac1 at the crossroad of actin dynamics and neuroinflammation in Amyotrophic Lateral Sclerosis

    Directory of Open Access Journals (Sweden)

    Nadia eD'Ambrosi

    2014-09-01

    Full Text Available Rac1 is a major player of the Rho family of small GTPases that controls multiple cell signaling pathways, such as the organization of cytoskeleton (including adhesion and motility, cell proliferation, apoptosis and activation of immune cells. In the nervous system, in particular, Rac1 GTPase plays a key regulatory function of both actin and microtubule cytoskeletal dynamics and thus it is central to axonal growth and stability, as well as dendrite and spine structural plasticity. Rac1 is also a crucial regulator of NADPH-dependent membrane oxidase (NOX, a prominent source of ROS, thus having a central role in the inflammatory response and neurotoxicity mediated by microglia cells in the nervous system. As such, alterations in Rac1 activity might well be involved in the processes that give rise to Amyotrophic Lateral Sclerosis (ALS, a complex syndrome where cytoskeletal disturbances in motor neurons and redox alterations in the inflammatory compartment play pivotal and synergic roles in the final disease outcomes. Here we will discuss the genetic and mechanistic evidence indicating the relevance of Rac1 dysregulation in the pathogenesis of ALS.

  11. Association of dopamine D(3) receptors with actin-binding protein 280 (ABP-280).

    Science.gov (United States)

    Li, Ming; Li, Chuanyu; Weingarten, Paul; Bunzow, James R; Grandy, David K; Zhou, Qun Yong

    2002-03-01

    Proteins that bind to G protein-coupled receptors have been identified as regulators of receptor localization and signaling. In our previous studies, a cytoskeletal protein, actin-binding protein 280 (ABP-280), was found to associate with the third cytoplasmic loop of dopamine D(2) receptors. In this study, we demonstrate that ABP-280 also interacts with dopamine D(3) receptors, but not with D(4) receptors. Similar to the dopamine D(2) receptor, the D(3)/ABP-280 association is of signaling importance. In human melanoma M2 cells lacking ABP-280, D(3) receptors were unable to inhibit forskolin-stimulated cyclic AMP (cAMP) production significantly. D(4) receptors, however, exhibited a similar degree of inhibition of forskolin-stimulated cAMP production in ABP-280-deficient M2 cells and ABP-280-replent M2 subclones (A7 cells). Further experiments revealed that the D(3)/ABP-280 interaction was critically dependent upon a 36 amino acid carboxyl domain of the D(3) receptor third loop, which is conserved in the D(2) receptor but not in the D(4) receptor. Our results demonstrate a subtype-specific regulation of dopamine D(2)-family receptor signaling by the cytoskeletal protein ABP-280.

  12. 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.

  13. 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...

  14. 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.

  15. Cytoskeletal organization of bee ovarian follicles during oogenesis.

    Science.gov (United States)

    Patrício, Karina; da Cruz-Landim, Carminda; Machado-Santelli, Gláucia Maria

    2011-01-01

    The germ cells in the germarium of the bee meroistic polytrophic ovarian cysts remain interconnected by cytoplasmic bridges as a result of incomplete cell division. These intercellular bridges form a distribution pathway for the substances that initially determine which of the cystocytes will become oocyte and later conduct the products synthesized by the nurse cells to the oocyte. In the present work, the presence and distribution of cytoskeleton components, actin and tubulin were studied in ovaries of queens of Apis mellifera and Scaptotrigona postica, two eusocial species, using antibody against α- and β-tubulin and FITC-phalloidin, aiming to shed light on the role of these cytoskeleton elements in oogenesis. The immunofluorescent preparations were analyzed by laser scanning confocal microscopy. F-actin was detected in the intercellular bridges of both species. The tubulin distribution in cell cytoplasm of A. mellifera and S. postica also displayed similar pattern. The role of these elements in the oogenetic events responsible for both cell support and motility is discussed.

  16. Cytoskeletal proteins participate in conserved viral strategies across kingdoms of life.

    Science.gov (United States)

    Erb, Marcella L; Pogliano, Joe

    2013-12-01

    The discovery of tubulin-like cytoskeletal proteins carried on the genomes of bacteriophages that are actively used for phage propagation during both the lytic and lysogenic cycle have revealed that there at least two ways that viruses can utilize a cytoskeleton; co-opt the host cytoskeleton or bring their own homologues. Either strategy underscores the deep evolutionary relationship between viruses and cytoskeletal proteins and points to a conservation of viral strategies that crosses the kingdoms of life. Here we review some of the most recent discoveries about tubulin cytoskeletal elements encoded by phages and compare them to some of the strategies utilized by the gammaherpesvirues of mammalian cells.

  17. Dendritic cell podosome dynamics does not depend on the F-actin regulator SWAP-70.

    Directory of Open Access Journals (Sweden)

    Anne Götz

    Full Text Available In addition to classical adhesion structures like filopodia or focal adhesions, dendritic cells similar to macrophages and osteoclasts assemble highly dynamic F-actin structures called podosomes. They are involved in cellular processes such as extracellular matrix degradation, bone resorption by osteoclasts, and trans-cellular diapedesis of lymphocytes. Besides adhesion and migration, podosomes enable dendritic cells to degrade connective tissue by matrix metalloproteinases. SWAP-70 interacts with RhoGTPases and F-actin and regulates migration of dendritic cells. SWAP-70 deficient osteoclasts are impaired in F-actin-ring formation and bone resorption. In the present study, we demonstrate that SWAP-70 is not required for podosome formation and F-actin turnover in dendritic cells. Furthermore, we found that toll-like receptor 4 ligand induced podosome disassembly and podosome-mediated matrix degradation is not affected by SWAP-70 in dendritic cells. Thus, podosome formation and function in dendritic cells is independent of SWAP-70.

  18. A LIM Domain Protein from Tobacco Involved in Actin-Bundling and Histone Gene Transcription

    Institute of Scientific and Technical Information of China (English)

    Danièle Moes; Sabrina Gatti; Céline Hoffmann; Monika Dieterle; Flora Moreau; Katrin Neumann; Marc Schumacher

    2013-01-01

    The two LIM domain-containing proteins from plants (LIMs) typically exhibit a dual cytoplasmic-nuclear distribution,suggesting that,in addition to their previously described roles in actin cytoskeleton organization,they participate in nuclear processes.Using a south-western blot-based screen aimed at identifying factors that bind to plant histone gene promoters,we isolated a positive clone containing the tobacco LIM protein WLIM2 (NtWLIM2) cDNA.Using both green fluorescent protein (GFP) fusion-and immunology-based strategies,we provide clear evidence that NtWLIM2 localizes to the actin cytoskeleton,the nucleus,and the nucleolus.Interestingly,the disruption of the actin cytoskeleton by latrunculin B significantly increases NtWLIM2 nuclear fraction,pinpointing a possible novel cytoskeletal-nuclear crosstalk.Biochemical and electron microscopy experiments reveal the ability of NtWLIM2 to directly bind to actin filaments and to crosslink the latter into thick actin bundles.Electrophoretic mobility shift assays show that NtWLIM2 specifically binds to the conserved octameric cis-elements (Oct) of the Arabidopsis histone H4A748 gene promoter and that this binding largely relies on both LIM domains.Importantly,reporter-based experiments conducted in Arabidopsis and tobacco protoplasts confirm the ability of NtWLIM2 to bind to and activate the H4A748 gene promoter in live cells.Expression studies indicate the constitutive presence of NtWLIM2 mRNA and NtWLIM2 protein during tobacco BY-2 cell proliferation and cell cycle progression,suggesting a role of NtWLIM2 in the activation of basal histone gene expression.Interestingly,both live cell and in vitro data support NtWLIM2 di/oligomerization.We propose that NtWLIM2 functions as an actin-stabilizing protein,which,upon cytoskeleton remodeling,shuttles to the nucleus in order to modify gene expression.

  19. A model of cytoskeletal reorientation in response to substrate stretching

    Directory of Open Access Journals (Sweden)

    Lazopoulos K.A.

    2008-01-01

    Full Text Available Living adherent cells change their orientation in response to substrate stretching such that their cytoskeletal components reorganize in a new direction. To study this phenomenon, we model the cytoskeleton as a planar system of elastic cables and struts both pinned at their endpoints to a flat flexible substrate. Tensed (pre-strained cables represent acting stress fibers, whereas compression-bearing struts represent microtubules. We assume that in response to uniaxial substrate stretching the model reorients and deforms into a new configuration that minimizes its total potential energy. Using the Maxwell's global stability criterion, we find global minima configurations during static extension and compression of the substrate. Based on these results, we predict reorientation during cyclic stretching of the substrate. We find that in response to cyclic stretching cells either reorient transversely to the direction of stretching, or exhibit multiple configurations symmetrically distributed relative to the direction of stretching. These predictions are consistent with experimental data on living cells from the literature.

  20. 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.

  1. GROWTH AND MORPHOLOGY OF POLYMER-ACTIN COMPLEXES

    Institute of Scientific and Technical Information of China (English)

    Hyuck Joon Kwon; Kazuhiro Shikinaka; Akira Kakugo; Hidemitsu Furukawa; Yoshihito Osada; Jian Ping Gong

    2007-01-01

    F-actins are semi-flexible polyelectrolytes and can be assembled into large polymer-actin complex with polymorphism through electrostatic interaction with polycations. This study investigates the structural phase behavior and the growth of polymer-actin complexes in terms of its longitudinal and lateral sizes. Our results show that formation of polymer-actin complexes is cooperative, and morphology and growth of polymer-actin complexes depend on polycation species and concentrations of polycation and salt in a constant actin concentration. We found that the longitudinal growth and lateral growth of polymer-actin complexes are dominated by different factors. This induces the structural polymorphism of polymer-actin complexes. Major factors to influence the polymorphism of polymer-actin complexes in polyelectrolyte system have been discussed. Our results indicate that the semi-flexible polyelectrolyte nature of F-actins is important for controlling the morphology and growth of actin architectures in cell.

  2. Brain-Specific Cytoskeletal Damage Markers in Cerebrospinal Fluid: Is There a Common Pattern between Amyotrophic Lateral Sclerosis and Primary Progressive Multiple Sclerosis?

    Directory of Open Access Journals (Sweden)

    Ahmed Abdelhak

    2015-07-01

    Full Text Available Many neurodegenerative disorders share a common pathophysiological pathway involving axonal degeneration despite different etiological triggers. Analysis of cytoskeletal markers such as neurofilaments, protein tau and tubulin in cerebrospinal fluid (CSF may be a useful approach to detect the process of axonal damage and its severity during disease course. In this article, we review the published literature regarding brain-specific CSF markers for cytoskeletal damage in primary progressive multiple sclerosis and amyotrophic lateral sclerosis in order to evaluate their utility as a biomarker for disease progression in conjunction with imaging and histological markers which might also be useful in other neurodegenerative diseases associated with affection of the upper motor neurons. A long-term benefit of such an approach could be facilitating early diagnostic and prognostic tools and assessment of treatment efficacy of disease modifying drugs.

  3. Brain-Specific Cytoskeletal Damage Markers in Cerebrospinal Fluid: Is There a Common Pattern between Amyotrophic Lateral Sclerosis and Primary Progressive Multiple Sclerosis?

    Science.gov (United States)

    Abdelhak, Ahmed; Junker, Andreas; Brettschneider, Johannes; Kassubek, Jan; Ludolph, Albert C; Otto, Markus; Tumani, Hayrettin

    2015-07-31

    Many neurodegenerative disorders share a common pathophysiological pathway involving axonal degeneration despite different etiological triggers. Analysis of cytoskeletal markers such as neurofilaments, protein tau and tubulin in cerebrospinal fluid (CSF) may be a useful approach to detect the process of axonal damage and its severity during disease course. In this article, we review the published literature regarding brain-specific CSF markers for cytoskeletal damage in primary progressive multiple sclerosis and amyotrophic lateral sclerosis in order to evaluate their utility as a biomarker for disease progression in conjunction with imaging and histological markers which might also be useful in other neurodegenerative diseases associated with affection of the upper motor neurons. A long-term benefit of such an approach could be facilitating early diagnostic and prognostic tools and assessment of treatment efficacy of disease modifying drugs.

  4. 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).

  5. 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.

  6. The Infection of Cucumber (Cucumis sativus L.) Roots by Meloidogyne incognita Alters the Expression of Actin-Depolymerizing Factor (ADF) Genes, Particularly in Association with Giant Cell Formation

    Science.gov (United States)

    Liu, Bin; Liu, Xingwang; Liu, Ying; Xue, Shudan; Cai, Yanling; Yang, Sen; Dong, Mingming; Zhang, Yaqi; Liu, Huiling; Zhao, Binyu; Qi, Changhong; Zhu, Ning; Ren, Huazhong

    2016-01-01

    Cucumber (Cucumis sativus L.) is threatened by substantial yield losses due to the south root-knot nematode (Meloidogyne incognita). However, understanding of the molecular mechanisms underlying the process of nematode infection is still limited. In this study, we found that M. incognita infection affected the structure of cells in cucumber roots and treatment of the cytoskeleton inhibitor (cytochalasin D) reduced root-knot nematode (RKN) parasitism. It is known that Actin-Depolymerizing Factor (ADF) affects cell structure, as well as the organization of the cytoskeleton. To address the hypothesis that nematode-induced abnormal cell structures and cytoskeletal rearrangements might be mediated by the ADF genes, we identified and characterized eight cucumber ADF (CsADF) genes. Phylogenetic analysis showed that the cucumber ADF gene family is grouped into four ancient subclasses. Expression analysis revealed that CsADF1, CsADF2-1, CsADF2-2, CsADF2-3 (Subclass I), and CsADF6 (Subclass III) have higher transcript levels than CsADF7-1, CsADF7-2 (Subclass II genes), and CsADF5 (Subclass IV) in roots. Members of subclass I genes (CsADF1, CsADF2-1, CsADF2-2, and CsADF2-3), with the exception of CsADF2-1, exhibited a induction of expression in roots 14 days after their inoculation (DAI) with nematodes. However, the expression of subclass II genes (CsADF7-1 and CsADF7-2) showed no significant change after inoculation. The transcript levels of CsADF6 (Subclass III) showed a specific induction at 21 DAI, while CsADF5 (Subclass IV) was weakly expressed in roots, but was strongly up-regulated as early as 7 DAI. In addition, treatment of roots with cytochalasin D caused an approximately 2-fold down-regulation of the CsADF genes in the treated plants. These results suggest that CsADF gene mediated actin dynamics are associated with structural changes in roots as a consequence of M. incognita infection. PMID:27695469

  7. The Infection of Cucumber (Cucumis sativus L. Roots by Meloidogyne incognita Alters the Expression of Actin-Depolymerizing Factor (ADF Genes, Particularly in Association with Giant Cell Formation

    Directory of Open Access Journals (Sweden)

    Bin Liu

    2016-09-01

    Full Text Available Cucumber (Cucumis sativus L. is threatened by substantial yield losses due to the south root-knot nematode (Meloidogyne incognita. However, understanding of the molecular mechanisms underlying the process of nematode infection is still limited. In this study, we found that M. incognita infection affected the structure of cells in cucumber roots and treatment of the cytoskeleton inhibitor (cytochalasin D reduced root-knot nematode (RKN parasitism. It is known that Actin-Depolymerizing Factor (ADF affects cell structure, as well as the organization of the cytoskeleton. To address the hypothesis that nematode-induced abnormal cell structures and cytoskeletal rearrangements might be mediated by the ADF genes, we identified and characterized eight cucumber ADF (CsADF genes. Phylogenetic analysis showed that the cucumber ADF gene family is grouped into four ancient subclasses. Expression analysis revealed that CsADF1, CsADF2-1, CsADF2-2, CsADF2-3 (Subclass I and CsADF6 (Subclass III have higher transcript levels than CsADF7-1, CsADF7-2 (Subclass II genes and CsADF5 (Subclass IV in roots. Members of subclass I genes (CsADF1, CsADF2-1, CsADF2-2 and CsADF2-3, with the exception of CsADF2-1, exhibited a induction of expression in roots 14 days after their inoculation (DAI with nematodes. However, the expression of subclass II genes (CsADF7-1 and CsADF7-2 showed no significant change after inoculation. The transcript levels of CsADF6 (Subclass III showed a specific induction at 21 DAI, while CsADF5 (Subclass IV was weakly expressed in roots, but was strongly up-regulated as early as 7 DAI. In addition, treatment of roots with cytochalasin D caused an approximately two-fold down-regulation of the CsADF genes in the treated plants. These results suggest that CsADF gene mediated actin dynamics are associated with structural changes in roots as a consequence of M. incognita infection.

  8. 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.

  9. Balancing spatially regulated β-actin translation and dynamin-mediated endocytosis is required to assemble functional epithelial monolayers.

    Science.gov (United States)

    Cruz, Lissette A; Vedula, Pavan; Gutierrez, Natasha; Shah, Neel; Rodriguez, Steven; Ayee, Brian; Davis, Justin; Rodriguez, Alexis J

    2015-12-01

    Regulating adherens junction complex assembly/disassembly is critical to maintaining epithelial homeostasis in healthy epithelial tissues. Consequently, adherens junction structure and function is often perturbed in clinically advanced tumors of epithelial origin. Some of the most studied factors driving adherens junction complex perturbation in epithelial cancers are transcriptional and epigenetic down-regulation of E-cadherin expression. However, numerous reports demonstrate that post-translational regulatory mechanisms such as endocytosis also regulate early phases of epithelial-mesenchymal transition and metastatic progression. In already assembled healthy epithelia, E-cadherin endocytosis recycles cadherin-catenin complexes to regulate the number of mature adherens junctions found at cell-cell contact sites. However, following de novo epithelial cell-cell contact, endocytosis negatively regulates adherens junction assembly by removing E-cadherin from the cell surface. By contrast, following de novo epithelial cell-cell contact, spatially localized β-actin translation drives cytoskeletal remodeling and consequently E-cadherin clustering at cell-cell contact sites and therefore positively regulates adherens junction assembly. In this report we demonstrate that dynamin-mediated endocytosis and β-actin translation-dependent cadherin-catenin complex anchoring oppose each other following epithelial cell-cell contact. Consequently, the final extent of adherens junction assembly depends on which of these processes is dominant following epithelial cell-cell contact. We expressed β-actin transcripts impaired in their ability to properly localize monomer synthesis (Δ3'UTR) in MDCK cells to perturb actin filament remodeling and anchoring, and demonstrate the resulting defect in adherens junction structure and function is rescued by inhibiting dynamin mediated endocytosis. Therefore, we demonstrate balancing spatially regulated β-actin translation and dynamin

  10. Reduced myelin basic protein and actin-related gene expression in visual cortex in schizophrenia.

    Science.gov (United States)

    Matthews, Paul R; Eastwood, Sharon L; Harrison, Paul J

    2012-01-01

    Most brain gene expression studies of schizophrenia have been conducted in the frontal cortex or hippocampus. The extent to which alterations occur in other cortical regions is not well established. We investigated primary visual cortex (Brodmann area 17) from the Stanley Neuropathology Consortium collection of tissue from 60 subjects with schizophrenia, bipolar disorder, major depression, or controls. We first carried out a preliminary array screen of pooled RNA, and then used RT-PCR to quantify five mRNAs which the array identified as differentially expressed in schizophrenia (myelin basic protein [MBP], myelin-oligodendrocyte glycoprotein [MOG], β-actin [ACTB], thymosin β-10 [TB10], and superior cervical ganglion-10 [SCG10]). Reduced mRNA levels were confirmed by RT-PCR for MBP, ACTB and TB10. The MBP reduction was limited to transcripts containing exon 2. ACTB and TB10 mRNAs were also decreased in bipolar disorder. None of the transcripts were altered in subjects with major depression. Reduced MBP mRNA in schizophrenia replicates findings in other brain regions and is consistent with oligodendrocyte involvement in the disorder. The decreases in expression of ACTB, and the actin-binding protein gene TB10, suggest changes in cytoskeletal organisation. The findings confirm that the primary visual cortex shows molecular alterations in schizophrenia and extend the evidence for a widespread, rather than focal, cortical pathophysiology.

  11. Demonstration of actin filament stress fibers in microvascular endothelial cells in situ.

    Science.gov (United States)

    Nehls, V; Drenckhahn, D

    1991-07-01

    We have developed a method for immunostaining the microvascular tree of rat mesenteric windows in situ. The procedure consists of three steps, i.e., mild fixation with formaldehyde, controlled proteolytic digestion of the mesothelial layer, and permeabilization with acetone. Discrimination between different microvascular segments was possible by double-fluorescent staining with antibodies to the smooth muscle isoform of alpha-actin and to nonmuscle myosin from platelets. Antibodies to nonmuscle myosin labeled numerous longitudinally oriented cables in endothelial cells of all microvascular segments (arterioles, metarterioles, pre-, mid-, and postcapillaries, small venules). Occasionally, the myosin-containing cables displayed the interrupted sarcomere-like staining pattern that is diagnostic for stress fibers. In contrast, staining of actin filaments with phalloidin-rhodamin resulted in a noninterrupted, continuous fluorescence of the stress fibers. A possible functional role of microvascular endothelial stress fibers is to serve as a tensile cytoskeletal scaffold that stabilizes the tubular, three-dimensional geometry of microvessels and, in addition, to help the endothelium resist the shear forces created by blood flow and by collision with red and white blood cells.

  12. Reduced myelin basic protein and actin-related gene expression in visual cortex in schizophrenia.

    Directory of Open Access Journals (Sweden)

    Paul R Matthews

    Full Text Available Most brain gene expression studies of schizophrenia have been conducted in the frontal cortex or hippocampus. The extent to which alterations occur in other cortical regions is not well established. We investigated primary visual cortex (Brodmann area 17 from the Stanley Neuropathology Consortium collection of tissue from 60 subjects with schizophrenia, bipolar disorder, major depression, or controls. We first carried out a preliminary array screen of pooled RNA, and then used RT-PCR to quantify five mRNAs which the array identified as differentially expressed in schizophrenia (myelin basic protein [MBP], myelin-oligodendrocyte glycoprotein [MOG], β-actin [ACTB], thymosin β-10 [TB10], and superior cervical ganglion-10 [SCG10]. Reduced mRNA levels were confirmed by RT-PCR for MBP, ACTB and TB10. The MBP reduction was limited to transcripts containing exon 2. ACTB and TB10 mRNAs were also decreased in bipolar disorder. None of the transcripts were altered in subjects with major depression. Reduced MBP mRNA in schizophrenia replicates findings in other brain regions and is consistent with oligodendrocyte involvement in the disorder. The decreases in expression of ACTB, and the actin-binding protein gene TB10, suggest changes in cytoskeletal organisation. The findings confirm that the primary visual cortex shows molecular alterations in schizophrenia and extend the evidence for a widespread, rather than focal, cortical pathophysiology.

  13. HGF Modulates Actin Cytoskeleton Remodeling and Contraction in Testicular Myoid Cells

    Directory of Open Access Journals (Sweden)

    Angela Catizone

    2015-01-01

    Full Text Available The presence of the HGF/Met system in the testicular myoid cells was first discovered by our group. However, the physiological role of this pathway remains poorly understood. We previously reported that HGF increases uPA secretion and TGF-β activation in cultured tubular fragments and that HGF is maximally expressed at Stages VII–VIII of the seminiferous epithelium cycle, when myoid cell contraction occurs. It is well known that the HGF/Met pathway is involved in cytoskeletal remodeling; moreover, the interaction of uPA with its receptor, uPAR, as well as the activation of TGF-β have been reported to be related to the actin cytoskeleton contractility of smooth muscle cells. Herein, we report that HGF induces actin cytoskeleton remodeling in vitro in isolated myoid cells and myoid cell contraction in cultured seminiferous tubules. To better understand these phenomena, we evaluated: (1 the regulation of the uPA machinery in isolated myoid cells after HGF administration; and (2 the effect of uPA or Met inhibition on HGF-treated tubular fragments. Because uPA activates latent TGF-β, the secretion of this factor was also evaluated. We found that both uPA and TGF-β activation increase after HGF administration. In testicular tubular fragments, HGF-induced TGF-β activation and myoid cell contraction are abrogated by uPA or Met inhibitor administration.

  14. Low ozone concentrations stimulate cytoskeletal organization, mitochondrial activity and nuclear transcription

    Directory of Open Access Journals (Sweden)

    M. Costanzo

    2015-04-01

    Full Text Available Ozone therapy is a modestly invasive procedure based on the regeneration capabilities of low ozone concentrations and used in medicine as an alternative/adjuvant treatment for different diseases. However, the cellular mechanisms accounting for the positive effects of mild ozonization are still largely unexplored. To this aim, in the present study the effects of low ozone concentrations (1 to 20 µg O3/mL O2 on structural and functional cell features have been investigated in vitro by using morphological, morphometrical, cytochemical and immunocytochemical techniques at bright field, fluorescence and transmission electron microscopy. Cells exposed to pure O2 or air served as controls. The results demonstrated that the effects of ozoneadministration are dependent on gas concentration, and the cytoskeletal organization, mitochondrial activity and nuclear transcription may be differently affected. This suggests that, to ensure effective and permanent metabolic cell activation, ozone treatments should take into account the cytological and cytokinetic features of the different tissues. 

  15. Mechanics model for actin-based motility.

    Science.gov (United States)

    Lin, Yuan

    2009-02-01

    We present here a mechanics model for the force generation by actin polymerization. The possible adhesions between the actin filaments and the load surface, as well as the nucleation and capping of filament tips, are included in this model on top of the well-known elastic Brownian ratchet formulation. A closed form solution is provided from which the force-velocity relationship, summarizing the mechanics of polymerization, can be drawn. Model predictions on the velocity of moving beads driven by actin polymerization are consistent with experiment observations. This model also seems capable of explaining the enhanced actin-based motility of Listeria monocytogenes and beads by the presence of Vasodilator-stimulated phosphoprotein, as observed in recent experiments.

  16. Evidence for physical and functional interactions among two Saccharomyces cerevisiae SH3 domain proteins, an adenylyl cyclase-associated protein and the actin cytoskeleton.

    Science.gov (United States)

    Lila, T; Drubin, D G

    1997-02-01

    In a variety of organisms, a number of proteins associated with the cortical actin cytoskeleton contain SH3 domains, suggesting that these domains may provide the physical basis for functional interactions among structural and regulatory proteins in the actin cytoskeleton. We present evidence that SH3 domains mediate at least two independent functions of the Saccharomyces cerevisiae actin-binding protein Abp1p in vivo. Abp1p contains a single SH3 domain that has recently been shown to bind in vitro to the adenylyl cyclase-associated protein Srv2p. Immunofluorescence analysis of Srv2p subcellular localization in strains carrying mutations in either ABP1 or SRV2 reveals that the Abp1p SH3 domain mediates the normal association of Srv2p with the cortical actin cytoskeleton. We also show that a site in Abp1p itself is specifically bound by the SH3 domain of the actin-associated protein Rvs167p. Genetic analysis provides evidence that Abp1p and Rvs167p have functions that are closely interrelated. Abp1 null mutations, like rvs167 mutations, result in defects in sporulation and reduced viability under certain suboptimal growth conditions. In addition, mutations in ABP1 and RVS167 yield similar profiles of genetic "synthetic lethal" interactions when combined with mutations in genes encoding other cytoskeletal components. Mutations which specifically disrupt the SH3 domain-mediated interaction between Abp1p and Srv2p, however, show none of the shared phenotypes of abp1 and rvs167 mutations. We conclude that the Abp1p SH3 domain mediates the association of Srv2p with the cortical actin cytoskeleton, and that Abp1p performs a distinct function that is likely to involve binding by the Rvs167p SH3 domain. Overall, work presented here illustrates how SH3 domains can integrate the activities of multiple actin cytoskeleton proteins in response to varying environmental conditions.

  17. 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.

  18. A potential yeast actin allosteric conduit dependent on hydrophobic core residues val-76 and trp-79.

    Science.gov (United States)

    Wen, Kuo-Kuang; McKane, Melissa; Stokasimov, Ema; Fields, Jonathon; Rubenstein, Peter A

    2010-07-02

    Intramolecular allosteric interactions responsible for actin conformational regulation are largely unknown. Previous work demonstrated that replacing yeast actin Val-76 with muscle actin Ile caused decreased nucleotide exchange. Residue 76 abuts Trp-79 in a six-residue linear array beginning with Lys-118 on the surface and ending with His-73 in the nucleotide cleft. To test if altering the degree of packing of these two residues would affect actin dynamics, we constructed V76I, W79F, and W79Y single mutants as well as the Ile-76/Phe-79 and Ile-76/Tyr-79 double mutants. Tyr or Phe should decrease crowding and increase protein flexibility. Subsequent introduction of Ile should restore packing and dampen changes. All mutants showed decreased growth in liquid medium. W79Y alone was severely osmosensitive and exhibited vacuole abnormalities. Both properties were rescued by Ile-76. Phe-79 or Tyr decreased the thermostability of actin and increased its nucleotide exchange rate. These effects, generally greater for Tyr than for Phe, were reversed by introduction of Ile-76. HD exchange showed that the mutations caused propagated conformational changes to all four subdomains. Based on results from phosphate release and light-scattering assays, single mutations affected polymerization in the order of Ile, Phe, and Tyr from least to most. Introduction of Ile-76 partially rescued the polymerization defects caused by either Tyr-79 or Phe-79. Thus, alterations in crowding of the 76-79 residue pair can strongly affect actin conformation and behavior, and these results support the theory that the amino acid array in which they are located may play a central role in actin regulation.

  19. Cross-linking study on skeletal muscle actin: properties of suberimidate-treated actin.

    Science.gov (United States)

    Ohara, O; Takahashi, S; Ooi, T; Fujiyoshi, Y

    1982-06-01

    Cross-linking experiments were performed on muscle skeletal actin, using imidoesters of various chain lengths. Chemical analyses on all products except one (derived from succinimidate) show evidence of the presence of intramolecular cross-links in the molecule. The detailed properties of suberimidate-treated actin (SA) are as follows: SA contains nearly 1 mol of intramolecular cross-link per mol of actin and less than 15% of intermolecularly cross-linked products. Even at a low salt concentration, SA is polymeric, exchanges slowly its bound nucleotide with free nucleotides in solution, and shows an F-actin-type CD spectrum. Electron micrographs of SA reveal that SA exists actually as fibrous polymers in solutions of low ionic strength, although the fibers seem to be less rigid than those at high salt concentration. The F-form of SA at a high salt concentration is indistinguishable from intact F-actin. SA can bind heavy meromyosin and activate the ATPase of heavy meromyosin as observed for intact F-actin. Tropomyosin binds SA only at a high salt concentration. These results show that SA possesses the properties of F-actin even in media of low salt concentration, which are favorable for depolymerization of F-actin. Thus, we may infer that the conformation of SA is frozen in the F-state of actin by the introduction of intramolecular cross-links in the protein.

  20. Molecular characterization of Toxoplasma gondii formin 3, an actin nucleator dispensable for tachyzoite growth and motility.

    Science.gov (United States)

    Daher, Wassim; Klages, Natacha; Carlier, Marie-France; Soldati-Favre, Dominique

    2012-03-01

    Toxoplasma gondii belongs to the phylum Apicomplexa, a group of obligate intracellular parasites that rely on gliding motility to enter host cells. Drugs interfering with the actin cytoskeleton block parasite motility, host cell invasion, and egress from infected cells. Myosin A, profilin, formin 1, formin 2, and actin-depolymerizing factor have all been implicated in parasite motility, yet little is known regarding the importance of actin polymerization and other myosins for the remaining steps of the parasite lytic cycle. Here we establish that T. gondii formin 3 (TgFRM3), a newly described formin homology 2 domain (FH2)-containing protein, binds to Toxoplasma actin and nucleates rabbit actin assembly in vitro. TgFRM3 expressed as a transgene exhibits a patchy localization at several distinct structures within the parasite. Disruption of the TgFRM3 gene by double homologous recombination in a ku80-ko strain reveals no vital function for tachyzoite propagation in vitro, which is consistent with its weak level of expression in this life stage. Conditional stabilization of truncated forms of TgFRM3 suggests that different regions of the molecule contribute to distinct localizations. Moreover, expression of TgFRM3 lacking the C-terminal domain severely affects parasite growth and replication. This work provides a first insight into how this specialized formin, restricted to the group of coccidia, completes its actin-nucleating activity.

  1. Simiate is an Actin binding protein involved in filopodia dynamics and arborisation of neurons.

    Directory of Open Access Journals (Sweden)

    Kristin eDerlig

    2014-04-01

    Full Text Available The Actin cytoskeleton constitutes the functional base for a multitude of cellular processes extending from motility and migration to cell mechanics and morphogenesis. The latter is particularly important to neuronal cells since the accurate functioning of the brain crucially depends on the correct arborisation of neurons, a process that requires the formation of several dozens to hundreds of dendritic branches. Recently, a model was proposed where different transcription factors are detailed to distinct facets and phases of dendritogenesis and exert their function by acting on the Actin cytoskeleton, however, the proteins involved as well as the underlying molecular mechanisms are largely unknown.Here, we demonstrate that Simiate, a protein previously indicated to activate transcription, directly associates with both, G- and F-Actin and in doing so, affects Actin polymerisation and Actin turnover in living cells. Imaging studies illustrate that Simiate particularly influences filopodia dynamics and specifically increases the branching of proximal, but not distal dendrites of developing neurons. The data suggests that Simiate functions as a direct molecular link between transcription regulation on one side, and dendritogenesis on the other, wherein Simiate serves to coordinate the development of proximal and distal dendrites by acting on the Actin cytoskeleton of filopodia and on transcription regulation, hence supporting the novel model.

  2. Dynamic buckling of actin within filopodia

    DEFF Research Database (Denmark)

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

    2015-01-01

    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...... a filopodium and holding it while measuring the cellular response, we also monitor and analyze the waiting times for the first buckle observed in the fluorescently labeled actin shaft....

  3. 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.

  4. [When and why treat actinic keratoses?].

    Science.gov (United States)

    Wulf, Hans Christian

    2014-02-03

    Actinic keratoses (AK) are small, inflamed, hyperkeratotic, sunprovoked lesions which may progress to squamous cell carcinoma (SCC). There are two main reasons for treating AK: one is as prophylaxis against SCC, the other is because of cosmetic discomfort, with clothes getting caught in the hyperkeratotic AK. Visible AK and neighbouring invisible AK should be treated. As AK are provoked by UV radiation, protection against UV is essential. This paper comments on a Cochrane review: "Interventions for actinic keratosis" and treatments avaliable in Denmark.

  5. Influence of simulated microgravity on the activation of the small GTPase Rho involved in cytoskeletal formation – molecular cloning and sequencing of bovine leukemia-associated guanine nucleotide exchange factor

    Directory of Open Access Journals (Sweden)

    Seki Masaya

    2006-06-01

    Full Text Available Abstract Background The irregular formation of cytoskeletal fibers in spaceflown experimental cells has been observed, but the disorganization process of fibers is still poorly understood. It is well known that the activation of the small GTPase Rho leads to actin stress fibers assembly. This study was performed to evaluate the effect of simulated microgravity on the activation of Rho that is involved in actin fiber remodeling in cells. Results Clinorotation influences actin fiber remodeling and its related signaling pathways that involve the small GTPase Rho. Actin stress fiber remodeling was significantly inhibited to a greater extent in cells cultured under clinorotation than in static cultured cells. From the gene and protein expression analyses, we found that the expression level of leukemia-associated Rho guanine nucleotide exchange factor (LARG, which activates Rho, was downregulated under clinorotation. Moreover, we identified the full-length LARG cDNA. The amount of GTP-bound RhoA, that is, the active form of RhoA, decreased under this condition. Conclusion The activation of the small GTPase Rho was influenced by simulated microgravity generated by a three-dimensional (3D clinostat. Furthermore, the full-length cDNA of bovine LARG, a member of the Rho guanine nucleotide exchange factor (GEF family, was identified, and its gene expression was observed to be downregulated under clinorotation. This downregulation subsequently resulted in the repression of RhoA activation. These results indicated that the disorganization of the actin fibers was caused by the inhibition of Rho activation by 3D clinorotation.

  6. 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.

  7. 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

  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. 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.

  10. 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.

  11. Structure of a Longitudinal Actin Dimer Assembled by Tandem W Domains: Implications for Actin Filament Nucleation

    Energy Technology Data Exchange (ETDEWEB)

    Rebowski, Grzegorz; Namgoong, Suk; Boczkowska, Malgorzata; Leavis, Paul C.; Navaza, Jorge; Dominguez, Roberto (IBS); (BBRI); (UPENN-MED)

    2013-11-20

    Actin filament nucleators initiate polymerization in cells in a regulated manner. A common architecture among these molecules consists of tandem WASP homology 2 domains (W domains) that recruit three to four actin subunits to form a polymerization nucleus. We describe a low-resolution crystal structure of an actin dimer assembled by tandem W domains, where the first W domain is cross-linked to Cys374 of the actin subunit bound to it, whereas the last W domain is followed by the C-terminal pointed end-capping helix of thymosin {beta}4. While the arrangement of actin subunits in the dimer resembles that of a long-pitch helix of the actin filament, important differences are observed. These differences result from steric hindrance of the W domain with intersubunit contacts in the actin filament. We also determined the structure of the first W domain of Vibrio parahaemolyticus VopL cross-linked to actin Cys374 and show it to be nearly identical with non-cross-linked W-Actin structures. This result validates the use of cross-linking as a tool for the study of actin nucleation complexes, whose natural tendency to polymerize interferes with most structural methods. Combined with a biochemical analysis of nucleation, the structures may explain why nucleators based on tandem W domains with short inter-W linkers have relatively weak activity, cannot stay bound to filaments after nucleation, and are unlikely to influence filament elongation. The findings may also explain why nucleation-promoting factors of the Arp2/3 complex, which are related to tandem-W-domain nucleators, are ejected from branch junctions after nucleation. We finally show that the simple addition of the C-terminal pointed end-capping helix of thymosin {beta}4 to tandem W domains can change their activity from actin filament nucleation to monomer sequestration.

  12. Structure of a longitudinal actin dimer assembled by tandem w domains: implications for actin filament nucleation.

    Science.gov (United States)

    Rebowski, Grzegorz; Namgoong, Suk; Boczkowska, Malgorzata; Leavis, Paul C; Navaza, Jorge; Dominguez, Roberto

    2010-10-15

    Actin filament nucleators initiate polymerization in cells in a regulated manner. A common architecture among these molecules consists of tandem WASP homology 2 domains (W domains) that recruit three to four actin subunits to form a polymerization nucleus. We describe a low-resolution crystal structure of an actin dimer assembled by tandem W domains, where the first W domain is cross-linked to Cys374 of the actin subunit bound to it, whereas the last W domain is followed by the C-terminal pointed end-capping helix of thymosin β4. While the arrangement of actin subunits in the dimer resembles that of a long-pitch helix of the actin filament, important differences are observed. These differences result from steric hindrance of the W domain with intersubunit contacts in the actin filament. We also determined the structure of the first W domain of Vibrio parahaemolyticus VopL cross-linked to actin Cys374 and show it to be nearly identical with non-cross-linked W-Actin structures. This result validates the use of cross-linking as a tool for the study of actin nucleation complexes, whose natural tendency to polymerize interferes with most structural methods. Combined with a biochemical analysis of nucleation, the structures may explain why nucleators based on tandem W domains with short inter-W linkers have relatively weak activity, cannot stay bound to filaments after nucleation, and are unlikely to influence filament elongation. The findings may also explain why nucleation-promoting factors of the Arp2/3 complex, which are related to tandem-W-domain nucleators, are ejected from branch junctions after nucleation. We finally show that the simple addition of the C-terminal pointed end-capping helix of thymosin β4 to tandem W domains can change their activity from actin filament nucleation to monomer sequestration.

  13. 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.

  14. Microtubule-Actin Cross-Linking Factor 1: Domains, Interaction Partners, and Tissue-Specific Functions.

    Science.gov (United States)

    Goryunov, Dmitry; Liem, Ronald K H

    2016-01-01

    The cytoskeleton of most eukaryotic cells is composed of three principal filamentous components: actin filaments, microtubules (MTs), and intermediate filaments. It is a highly dynamic system that plays crucial roles in a wide range of cellular processes, including migration, adhesion, cytokinesis, morphogenesis, intracellular traffic and signaling, and structural flexibility. Among the large number of cytoskeleton-associated proteins characterized to date, microtubule-actin cross-linking factor 1 (MACF1) is arguably the most versatile integrator and modulator of cytoskeleton-related processes. MACF1 belongs to the plakin family of proteins, and within it, to the spectraplakin subfamily. These proteins are characterized by the ability to bridge MT and actin cytoskeletal networks in a dynamic fashion, which underlies their involvement in the regulation of cell migration, axonal extension, and vesicular traffic. Studying MACF1 functions has provided insights not only into the regulation of the cytoskeleton but also into molecular mechanisms of both normal cellular physiology and cellular pathology. Multiple MACF1 isoforms exist, composed of a large variety of alternatively spliced domains. Each of these domains mediates a specific set of interactions and functions. These functions are manifested in tissue and cell-specific phenotypes observed in conditional MACF1 knockout mice. The conditional models described to date reveal critical roles of MACF1 in mammalian skin, nervous system, heart muscle, and intestinal epithelia. Complete elimination of MACF1 is early embryonic lethal, indicating an essential role for MACF1 in early development. Further studies of MACF1 domains and their interactions will likely reveal multiple new roles of this protein in various tissues.

  15. 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.

  16. 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.

  17. 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

  18. Effect of different methods of cryopreservation on the cytoskeletal integrity of dromedary camel (Camelus dromedarius) embryos.

    Science.gov (United States)

    Skidmore, J A; Schoevers, E; Stout, T A E

    2009-07-01

    This study examined the effect of different methods of cryopreservation on the cytoskeletal integrity of camel embryos. A total of 32 embryos were recovered on Days 6 and 7 after ovulation and measured before being frozen using either a conventional slow-cooling technique (n=12: six Day 6 and six Day 7 embryos) or vitrification (n=12: four Day 6 and eight Day 7). The remaining 8 'control' embryos (four Day 6 and four Day 7) were not cryopreserved but instead incubated in holding medium for 30 min. After thawing, warming or incubation, the embryos were stained with 4,6-diamino-2-phenylindole dihydrochloride (DAPI) to identify dead cells. Subsequently, the embryos were fixed in 4% paraformaldehyde, permeabilized and labelled with Alexa Fluor 488-Phalloidin to enable assessment of cytoskeleton integrity. Vitrified-warmed embryos contained a significantly higher percentage of dead cells than either conventionally frozen embryos or controls (P or =0.07). Whereas embryo size did not affect the number of dead cells in conventionally frozen embryos, vitrified-warmed embryos >300 microm in diameter had a significantly higher percentage of dead cells than embryos < or =300 microm (P=0.01). Cytoskeleton integrity was also affected by both freezing method and embryo diameter. All 8 control embryos had a Grade I cytoskeleton, compared with only 2/24 (8.3%) frozen or vitrified embryos. Of the 8 slow-frozen or vitrified embryos with a Grade III cytoskeleton post-thaw, 7 had been vitrified and 6 were larger (Day 7) embryos. These results indicate that while both slow-freezing and vitrification of camel embryos lead to cytoskeleton disruption and cell death, embryo quality is better preserved by slow-freezing.

  19. 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.

  20. 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.

  1. The unusual dynamics of parasite actin result from isodesmic polymerization.

    Science.gov (United States)

    Skillman, Kristen M; Ma, Christopher I; Fremont, Daved H; Diraviyam, Karthikeyan; Cooper, John A; Sept, David; Sibley, L David

    2013-01-01

    Previous reports have indicated that parasite actins are short and inherently unstable, despite being required for motility. Here we re-examine the polymerization properties of actin in Toxoplasma gondii, unexpectedly finding that it exhibits isodesmic polymerization in contrast to the conventional nucleation-elongation process of all previously studied actins from both eukaryotes and bacteria. Polymerization kinetics of actin in T. gondii lacks both a lag phase and critical concentration, normally characteristic of actins. Unique among actins, the kinetics of assembly can be fit with a single set of rate constants for all subunit interactions, without need for separate nucleation and elongation rates. This isodesmic model accurately predicts the assembly, disassembly and the size distribution of actin filaments in T. gondii in vitro, providing a mechanistic explanation for actin dynamics in vivo. Our findings expand the repertoire of mechanisms by which actin polymerization is governed and offer clues about the evolution of self-assembling, stabilized protein polymers.

  2. 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.

  3. 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.

  4. 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.

  5. Plant villins:Versatile actin regulatory proteins

    Institute of Scientific and Technical Information of China (English)

    Shanjin Huang; Xiaolu Qu; Ruihui Zhang

    2015-01-01

    Regulation of actin dynamics is a central theme in cel biology that is important for different aspects of cel physiology. Vil in, a member of the vil in/gelsolin/fragmin superfamily of proteins, is an important regulator of actin. Vil ins contain six gelsolin homology domains (G1–G6) and an extra headpiece domain. In contrast to their mammalian counterparts, plant vil ins are expressed widely, implying that plant vil ins play a more general role in regulating actin dynamics. Some plant vil ins have a defined role in modifying actin dynamics in the pol en tube;most of their in vivo activities remain to be ascertained. Recently, our understanding of the functions and mechanisms of action for plant vil ins has progressed rapidly, primarily due to the advent of Arabidopsis thaliana genetic approaches and imaging capabilities that can visualize actin dynamics at the single filament level in vitro and in living plant cel s. In this review, we focus on discussing the biochemical activities and modes of regulation of plant vil ins. Here, we present current understand-ing of the functions of plant vil ins. Final y, we highlight some of the key unanswered questions regarding the functions and regulation of plant vil ins for future research.

  6. The cytoskeletal inhibitors latrunculin A and blebbistatin exert antitumorigenic properties in human hepatocellular carcinoma cells by interfering with intracellular HuR trafficking

    Energy Technology Data Exchange (ETDEWEB)

    Doller, Anke; Badawi, Amel [Pharmazentrum Frankfurt/ZAFES, Klinikum der Goethe-Universität Frankfurt, Frankfurt/Main (Germany); Schmid, Tobias; Brauß, Thilo [Institut für Biochemie I (Pathobiochemie), Klinikum der Goethe-Universität Frankfurt, Frankfurt/Main (Germany); Pleli, Thomas [Medizinische Klinik 1, Schwerpunkt Gastroenterologie und Hepatologie, Klinikum der Goethe-Universität Frankfurt, Frankfurt/Main (Germany); Meyer zu Heringdorf, Dagmar [Pharmazentrum Frankfurt/ZAFES, Klinikum der Goethe-Universität Frankfurt, Frankfurt/Main (Germany); Piiper, Albrecht [Medizinische Klinik 1, Schwerpunkt Gastroenterologie und Hepatologie, Klinikum der Goethe-Universität Frankfurt, Frankfurt/Main (Germany); Pfeilschifter, Josef [Pharmazentrum Frankfurt/ZAFES, Klinikum der Goethe-Universität Frankfurt, Frankfurt/Main (Germany); Eberhardt, Wolfgang, E-mail: w.eberhardt@em.uni-frankfurt.de [Pharmazentrum Frankfurt/ZAFES, Klinikum der Goethe-Universität Frankfurt, Frankfurt/Main (Germany)

    2015-01-01

    The impact of the RNA-binding protein HuR for the post-transcriptional deregulation of tumor-relevant genes is well established. Despite of elevations in HuR expression levels, an increase in cytoplasmic HuR abundance in many cases correlates with a high grade of malignancy. Here, we demonstrated that administration of the actin-depolymerizing macrolide latrunculin A, or blebbistatin, an inhibitor of myosin II ATPase activity, caused a dose- and time-dependent reduction in the high cytoplasmic HuR content of HepG2 and Huh7 hepatocellular carcinoma (HCC) cells. Subcellular fractionation revealed that in addition, both inhibitors strongly attenuated cytoskeletal and membrane-bound HuR abundance and conversely increased the HuR amount in nuclear cell fractions. Concomitant with changes in intracellular HuR localization, both cytoskeletal inhibitors markedly decreased the half-lives of cyclooxygenase-2 (COX-2), cyclin A and cyclin D{sub 1} encoding mRNAs resulting in a significant reduction in their expression levels in HepG2 cells. Importantly, a similar reduction in the expression of these HuR targets was achieved by a RNA interference (RNAi)-mediated knockdown of either HuR or nonmuscle myoin IIA. Using polysomal fractionation, we further demonstrate that the decrease in cytoplasmic HuR by latrunculin A or blebbistatin is accompanied by a marked change in the allocation of HuR and its mRNA cargo from polysomes to ribonucleoprotein (RNP) particles. Functionally, the basal migration and prostaglandin E{sub 2} synthesis are similarly impaired in inhibitor-treated and stable HuR-knockdown HepG2 cells. Our data demonstrate that interfering with the actomyosin-dependent HuR trafficking may comprise a valid therapeutic option for antagonizing pathologic posttranscriptional gene expression by HuR and furthermore emphasize the potential benefit of HuR inhibitory strategies for treatment of HCC. - Highlights: • We tested the effects of latrunculin A and blebbistatin on

  7. The NAV2 homolog Sickie regulates F-actin-mediated axonal growth in Drosophila mushroom body neurons via the non-canonical Rac-Cofilin pathway.

    Science.gov (United States)

    Abe, Takashi; Yamazaki, Daisuke; Murakami, Satoshi; Hiroi, Makoto; Nitta, Yohei; Maeyama, Yuko; Tabata, Tetsuya

    2014-12-01

    The Rac-Cofilin pathway is essential for cytoskeletal remodeling to control axonal development. Rac signals through the canonical Rac-Pak-LIMK pathway to suppress Cofilin-dependent axonal growth and through a Pak-independent non-canonical pathway to promote outgrowth. Whether this non-canonical pathway converges to promote Cofilin-dependent F-actin reorganization in axonal growth remains elusive. We demonstrate that Sickie, a homolog of the human microtubule-associated protein neuron navigator 2, cell-autonomously regulates axonal growth of Drosophila mushroom body (MB) neurons via the non-canonical pathway. Sickie was prominently expressed in the newborn F-actin-rich axons of MB neurons. A sickie mutant exhibited axonal growth defects, and its phenotypes were rescued by exogenous expression of Sickie. We observed phenotypic similarities and genetic interactions among sickie and Rac-Cofilin signaling components. Using the MARCM technique, distinct F-actin and phospho-Cofilin patterns were detected in developing axons mutant for sickie and Rac-Cofilin signaling regulators. The upregulation of Cofilin function alleviated the axonal defect of the sickie mutant. Epistasis analyses revealed that Sickie suppresses the LIMK overexpression phenotype and is required for Pak-independent Rac1 and Slingshot phosphatase to counteract LIMK. We propose that Sickie regulates F-actin-mediated axonal growth via the non-canonical Rac-Cofilin pathway in a Slingshot-dependent manner.

  8. The Ovary of Tubifex tubifex (Clitellata, Naididae, Tubificinae Is Composed of One, Huge Germ-Line Cyst that Is Enriched with Cytoskeletal Components.

    Directory of Open Access Journals (Sweden)

    Anna Z Urbisz

    Full Text Available Recent studies on the ovary organization and oogenesis in Tubificinae have revealed that their ovaries are small polarized structures that are composed of germ cells in subsequent stages of oogenesis that are associated with somatic cells. In syncytial cysts, as a rule, each germ cell is connected to the central cytoplasmic mass, the cytophore, via only one stable intercellular bridge (ring canal. In this paper we present detailed data about the composition of germ-line cysts in Tubifex tubifex with special emphasis on the occurrence and distribution of the cytoskeletal elements. Using fixed material and live cell imaging techniques, we found that the entire ovary of T. tubifex is composed of only one, huge multicellular germ-line cyst, which may contain up to 2,600 cells. Its architecture is broadly similar to the cysts that are found in other clitellate annelids, i.e. a common, anuclear cytoplasmic mass in the center of the cyst and germ cells that are connected to it via intercellular bridges. The cytophore in the T. tubifex cyst extends along the long axis of the ovary in the form of elongated and branched cytoplasmic strands. Rhodamine-coupled phalloidin staining revealed that the prominent strands of actin filaments occur inside the cytophore. Similar to the cytophore, F-actin strands are branched and they are especially well developed in the middle and outermost parts of the ovary. Microfilaments are also present in the ring canals that connect the germ cells with the cytophore in the narrow end of the ovary. Using TubulinTracker, we found that the microtubules form a prominent network of loosely and evenly distributed tubules inside the cytophore as well as in every germ cell. The well-developed cytoskeletal elements in T. tubifex ovary seem to ensure the integrity of such a huge germ-line cyst of complex (germ cells-ring canals-cytophore organization. A comparison between the cysts that are described here and other well-known female

  9. The Ovary of Tubifex tubifex (Clitellata, Naididae, Tubificinae) Is Composed of One, Huge Germ-Line Cyst that Is Enriched with Cytoskeletal Components.

    Science.gov (United States)

    Urbisz, Anna Z; Chajec, Łukasz; Świątek, Piotr

    2015-01-01

    Recent studies on the ovary organization and oogenesis in Tubificinae have revealed that their ovaries are small polarized structures that are composed of germ cells in subsequent stages of oogenesis that are associated with somatic cells. In syncytial cysts, as a rule, each germ cell is connected to the central cytoplasmic mass, the cytophore, via only one stable intercellular bridge (ring canal). In this paper we present detailed data about the composition of germ-line cysts in Tubifex tubifex with special emphasis on the occurrence and distribution of the cytoskeletal elements. Using fixed material and live cell imaging techniques, we found that the entire ovary of T. tubifex is composed of only one, huge multicellular germ-line cyst, which may contain up to 2,600 cells. Its architecture is broadly similar to the cysts that are found in other clitellate annelids, i.e. a common, anuclear cytoplasmic mass in the center of the cyst and germ cells that are connected to it via intercellular bridges. The cytophore in the T. tubifex cyst extends along the long axis of the ovary in the form of elongated and branched cytoplasmic strands. Rhodamine-coupled phalloidin staining revealed that the prominent strands of actin filaments occur inside the cytophore. Similar to the cytophore, F-actin strands are branched and they are especially well developed in the middle and outermost parts of the ovary. Microfilaments are also present in the ring canals that connect the germ cells with the cytophore in the narrow end of the ovary. Using TubulinTracker, we found that the microtubules form a prominent network of loosely and evenly distributed tubules inside the cytophore as well as in every germ cell. The well-developed cytoskeletal elements in T. tubifex ovary seem to ensure the integrity of such a huge germ-line cyst of complex (germ cells-ring canals-cytophore) organization. A comparison between the cysts that are described here and other well-known female germ-line cysts is

  10. p38MAPK, Rho/ROCK and PKC pathways are involved in influenza-induced cytoskeletal rearrangement and hyperpermeability in PMVEC via phosphorylating ERM.

    Science.gov (United States)

    Zhang, Chenyue; Wu, Ying; Xuan, Zinan; Zhang, Shujing; Wang, Xudan; Hao, Yu; Wu, Jun; Zhang, Shu

    2014-11-04

    Severe influenza infections are featured by acute lung injury, a syndrome of pulmonary microvascular leak. A growing number of evidences have shown that the pulmonary microvascular endothelial cells (PMVEC) are critical target of influenza virus, promoting microvascular leak. It is reported that there are multiple mechanisms by which influenza virus could elicit increased pulmonary endothelial permeability, in both direct and indirect manners. Ezrin/radixin/moesin family proteins, the linkers between plasma membrane and actin cytoskeleton, have been reported to be involved in cell adhesion, motility and may modulate endothelial permeability. Studies have also shown that ERM is phosphorylated in response to various stimuli via p38MAPK, Rho/ROCK or PKC pathways. However, it is unclear that whether influenza infection could induce ERM phosphorylation and its relocalization. In the present study, we have found that there are cytoskeletal reorganization and permeability increases in the course of influenza virus infection, accompanied by upregulated levels of p-ERM. p-ERM's aggregation along the periphery of PMVEC upon influenza virus infection was detected via confocal microscopy. Furthermore, we sought to determine the role of p38MAPK, Rho/ROCK and PKC pathways in ERM phosphorylation as well as their involvement in influenza virus-induced endothelial malfunction. The activation of p38MAPK, Rho/ROCK and PKC pathways upon influenza virus stimulation were observed, as evidenced by the evaluation of phosphorylated p38 (p-p38), phosphorylated MKK (p-MKK) in p38MAPK pathway, ROCK1 in Rho/ROCK pathway and phosphorylated PKC (p-PKC) in PKC pathway. We also showed that virus-induced ERM phosphorylation was reduced by using p38MAPK inhibitor, SB203580 (20 μM), Rho/ROCK inhibitor, Y27632 (20 μM), PKC inhibitor, LY317615 (10 μM). Additionally, influenza virus-induced F-actin reorganization and hyperpermeability were attenuated by pretreatment with SB203580, Y27632 and LY317615

  11. Treatment Options for Actinic Keratosis

    Science.gov (United States)

    ... Skin Cancer Skin Cancer Screening Research Skin Cancer Treatment (PDQ®)–Patient Version General Information About Skin Cancer ... Certain factors affect prognosis (chance of recovery) and treatment options. The prognosis (chance of recovery ) depends mostly ...

  12. Cytoskeletal arrangement and its intercellular connection in wheat young leaf cells

    Institute of Scientific and Technical Information of China (English)

    SEIXIANGYUN; LINGCHENGJIAN

    1993-01-01

    By using the techniques of partial digestion of cell wall and selective extraction,we examined the cytoskeleton of wheat yong leaf cells under scanning electron microscope(SEM).A 3-dimensional cytoskeletal system,showing some new features,was observed.The cortical network located beneath the cross wall was an anastomosing organization.The association of nucleus with the cell wall by some skeletal filaments was also found.It is notice able that there were cytoskeletal filaments,which passed through cell wall and connected together with cytoskeletal arrays of adjacent cells,Thus,it is possible that an integral skeletal network existed within the yong leaf tissue of wheat.

  13. Cytosolic SYT/SS18 isoforms are actin-associated proteins that function in matrix-specific adhesion.

    Directory of Open Access Journals (Sweden)

    Jaehong Kim

    Full Text Available SYT (SYnovial sarcoma Translocated gene or SS18 is widely produced as two isoforms, SYT/L and SYT/S, that are thought to function in the nucleus as transcriptional coactivators. Using isoform-specific antibodies, we detected a sizable pool of SYT isoforms in the cytosol where the proteins were organized into filamentous arrays. Actin and actin-associated proteins co-immunoprecipitated with SYT isoforms, which also co-sedimented and co-localized with the actin cytoskeleton in cultured cells and tissues. The association of SYT with actin bundles was extensive yet stopped short of the distal ends at focal adhesions. Disruption of the actin cytoskeleton also led to a breakdown of the filamentous organization of SYT isoforms in the cytosol. RNAi ablation of SYT/L alone or both isoforms markedly impaired formation of stress fibers and focal adhesions but did not affect formation of cortical actin bundles. Furthermore, ablation of SYT led to markedly impaired adhesion and spreading on fibronectin and laminin-111 but not on collagen types I or IV. These findings indicate that cytoplasmic SYT isoforms interact with actin filaments and function in the ability cells to bind and react to specific extracellular matrices.

  14. Cell cycle regulation and cytoskeletal remodelling are critical processes in the nutritional programming of embryonic development.

    Directory of Open Access Journals (Sweden)

    Angelina Swali

    Full Text Available Many mechanisms purport to explain how nutritional signals during early development are manifested as disease in the adult offspring. While these describe processes leading from nutritional insult to development of the actual pathology, the initial underlying cause of the programming effect remains elusive. To establish the primary drivers of programming, this study aimed to capture embryonic gene and protein changes in the whole embryo at the time of nutritional insult rather than downstream phenotypic effects. By using a cross-over design of two well established models of maternal protein and iron restriction we aimed to identify putative common "gatekeepers" which may drive nutritional programming.Both protein and iron deficiency in utero reduced the nephron complement in adult male Wistar and Rowett Hooded Lister rats (P<0.05. This occurred in the absence of damage to the glomerular ultrastructure. Microarray, proteomic and pathway analyses identified diet-specific and strain-specific gatekeeper genes, proteins and processes which shared a common association with the regulation of the cell cycle, especially the G1/S and G2/M checkpoints, and cytoskeletal remodelling. A cell cycle-specific PCR array confirmed the down-regulation of cyclins with protein restriction and the up-regulation of apoptotic genes with iron deficiency.The timing and experimental design of this study have been carefully controlled to isolate the common molecular mechanisms which may initiate the sequelae of events involved in nutritional programming of embryonic development. We propose that despite differences in the individual genes and proteins affected in each strain and with each diet, the general response to nutrient deficiency in utero is perturbation of the cell cycle, at the level of interaction with the cytoskeleton and the mitotic checkpoints, thereby diminishing control over the integrity of DNA which is allowed to replicate. These findings offer novel

  15. CHUP1 mediates actin-based light-induced chloroplast avoidance movement in the moss Physcomitrella patens.

    Science.gov (United States)

    Usami, Hiroka; Maeda, Takuma; Fujii, Yusuke; Oikawa, Kazusato; Takahashi, Fumio; Kagawa, Takatoshi; Wada, Masamitsu; Kasahara, Masahiro

    2012-12-01

    Chloroplasts change their intracellular distribution in response to light intensity. CHUP1 (CHLOROPLAST UNUSUAL POSITIONING1) is indispensable for this response in Arabidopsis thaliana. However, involvement of CHUP1 in light-induced chloroplast movement is unknown in other plants. In this study, CHUP1 orthologues were isolated from a moss, Physcomitrella patens, and a fern, Adiantum capillus-veneris, by cDNA library screening and PCR cloning based on the P. patens genome sequence. Functional motifs found in CHUP1 of A. thaliana were conserved among the CHUP1 orthologues. In addition to the putative functional regions, the C-terminal regions (approximately 250 amino acids), which are unique in CHUP1s, were highly conserved. Green fluorescent protein (GFP) fusions of P. patens CHUP1s (PpCHUP1A, PpCHUP1B and PpCHUP1C) were transiently expressed in protoplast cells. All GFP fusions were localized on the chloroplasts. Light-induced chloroplast avoidance movement of chup1 disruptants of P. patens was examined in the presence of cytoskeletal inhibitors because of the utilization of both microtubules and actin filaments for the movement in P. patens. When actin filaments were disrupted by cytochalasin B, the wild type (WT) and all chup1 disruptants showed chloroplast avoidance movement. However, when microtubules were disrupted by Oryzalin, chloroplasts in ∆chup1A and ∆chup1A/B rarely moved and stayed in the strong light-irradiated area. On the other hand, WT, ∆chup1B and ∆chup1C showed chloroplast avoidance movement. These results suggest that PpCHUP1A predominantly mediates the actin-based light-induced chloroplast avoidance movement. This study reveals that CHUP1 functions on the chloroplasts and is involved in the actin-based light-induced chloroplast avoidance movement in P. patens.

  16. De novo actin polymerization is required for model Hirano body formation in Dictyostelium

    Directory of Open Access Journals (Sweden)

    Yun Dong

    2016-06-01

    Full Text Available Hirano bodies are eosinophilic, actin-rich inclusions found in autopsied brains in numerous neurodegenerative diseases. The mechanism of Hirano body formation is unknown. Mass spectrometry analysis was performed to identify proteins from partially purified model Hirano bodies from Dictyostelium. This analysis identified proteins primarily belonging to ribosomes, proteasomes, mitochondria and cytoskeleton. Profilin, Arp/2/3 and WASH identified by mass spectrometry were found to colocalise with model Hirano bodies. Due to their roles in actin regulation, we selected these proteins for further investigation. Inhibition of the Arp2/3 complex by CK666 prevented formation of model Hirano bodies. Since Arp2/3 activation occurs via the WASH or WAVE complex, we next investigated how these proteins affect Hirano body formation. Whereas model Hirano bodies could form in WASH-deficient cells, they failed to form in cells lacking HSPC300, a member of the WAVE complex. We identified other proteins required for Hirano body formation that include profilin and VASP, an actin nucleation factor. In the case of VASP, both its G- and F-actin binding domains were required for model Hirano body formation. Collectively, our results indicate that de novo actin polymerization is required to form model Hirano bodies.

  17. Focal adhesion kinase is required for actin polymerization and remodeling of the cytoskeleton during sperm capacitation

    Science.gov (United States)

    Roa-Espitia, Ana L.; Hernández-Rendón, Eva R.; Baltiérrez-Hoyos, Rafael; Muñoz-Gotera, Rafaela J.; Cote-Vélez, Antonieta; Jiménez, Irma; González-Márquez, Humberto

    2016-01-01

    ABSTRACT Several focal adhesion proteins are known to cooperate with integrins to link the extracellular matrix to the actin cytoskeleton; as a result, many intracellular signaling pathways are activated and several focal adhesion complexes are formed. However, how these proteins function in mammalian spermatozoa remains unknown. We confirm the presence of focal adhesion proteins in guinea pig spermatozoa, and we explore their role during capacitation and the acrosome reaction, and their relationship with the actin cytoskeleton. Our results suggest the presence of a focal adhesion complex formed by β1-integrin, focal adhesion kinase (FAK), paxillin, vinculin, talin, and α-actinin in the acrosomal region. Inhibition of FAK during capacitation affected the protein tyrosine phosphorylation associated with capacitation that occurs within the first few minutes of capacitation, which caused the acrosome reaction to become increasingly Ca2+ dependent and inhibited the polymerization of actin. The integration of vinculin and talin into the complex, and the activation of FAK and paxillin during capacitation, suggests that the complex assembles at this time. We identify that vinculin and α-actinin increase their interaction with F-actin while it remodels during capacitation, and that during capacitation focal adhesion complexes are structured. FAK contributes to acrosome integrity, likely by regulating the polymerization and the remodeling of the actin cytoskeleton. PMID:27402964

  18. Actin cytoskeleton contributes to the elastic modulus of embryonic tendon during early development.

    Science.gov (United States)

    Schiele, Nathan R; von Flotow, Friedrich; Tochka, Zachary L; Hockaday, Laura A; Marturano, Joseph E; Thibodeau, Jeffrey J; Kuo, Catherine K

    2015-06-01

    Tendon injuries are common and heal poorly. Strategies to regenerate or replace injured tendons are challenged by an incomplete understanding of normal tendon development. Our previous study showed that embryonic tendon elastic modulus increases as a function of developmental stage. Inhibition of enzymatic collagen crosslink formation abrogated increases in tendon elastic modulus at late developmental stages, but did not affect increases in elastic modulus of early stage embryonic tendons. Here, we aimed to identify potential contributors to the mechanical properties of these early stage embryonic tendons. We characterized tendon progenitor cells in early stage embryonic tendons, and the influence of actin cytoskeleton disruption on tissue elastic modulus. Cells were closely packed in embryonic tendons, and did not change in density during early development. We observed an organized network of actin filaments that seemed contiguous between adjacent cells. The actin filaments exhibited a crimp pattern with a period and amplitude that matched the crimp of collagen fibers at each developmental stage. Chemical disruption of the actin cytoskeleton decreased tendon tissue elastic modulus, measured by atomic force microscopy. Our results demonstrate that early developmental stage embryonic tendons possess a well organized actin cytoskeleton network that contributes significantly to tendon tissue mechanical properties.

  19. Actin-dependence of the chloroplast cold positioning response in the liverwort Marchantia polymorpha L.

    Directory of Open Access Journals (Sweden)

    Shun Kimura

    2016-09-01

    Full Text Available The subcellular positioning of chloroplasts can be changed by alterations in the environment such as light and temperature. For example, in leaf mesophyll cells, chloroplasts localize along anticlinal cell walls under high-intensity light, and along periclinal cell walls under low-intensity light. These types of positioning responses are involved in photosynthetic optimization. In light-mediated chloroplast positioning responses, chloroplasts move to the appropriate positions in an actin-dependent manner, although some exceptions also depend on microtubule. Even under low-intensity light, at low temperature (e.g., 5°C, chloroplasts localize along anticlinal cell walls; this phenomenon is termed chloroplast cold positioning. In this study, we analyzed whether chloroplast cold positioning is dependent on actin filaments and/or microtubules in the liverwort Marchantia polymorpha L. When liverwort cells were treated with drugs for the de-polymerization of actin filaments, chloroplast cold positioning was completely inhibited. In contrast, chloroplast cold positioning was not affected by treatment with a drug for the de-polymerization of microtubules. These observations indicate the actin-dependence of chloroplast cold positioning in M. polymorpha. Actin filaments during the chloroplast cold positioning response were visualized by using fluorescent probes based on fluorescent proteins in living liverwort cells, and thus, their behavior during the chloroplast cold positioning response was documented.

  20. Shielding of the Geomagnetic Field Alters Actin Assembly and Inhibits Cell Motility in Human Neuroblastoma Cells.

    Science.gov (United States)

    Mo, Wei-Chuan; Zhang, Zi-Jian; Wang, Dong-Liang; Liu, Ying; Bartlett, Perry F; He, Rong-Qiao

    2016-03-31

    Accumulating evidence has shown that absence of the geomagnetic field (GMF), the so-called hypomagnetic field (HMF) environment, alters the biological functions in seemingly non-magnetosensitive cells and organisms, which indicates that the GMF could be sensed by non-iron-rich and non-photo-sensing cells. The underlying mechanisms of the HMF effects on those cells are closely related to their GMF sensation but remain poorly understood so far. Previously, we found that the HMF represses expressions of genes associated with cell migration and cytoskeleton assembly in human neuroblastoma cells (SH-SY5Y cell line). Here, we measured the HMF-induced changes on cell morphology, adhesion, motility and actin cytoskeleton in SH-SY5Y cells. The HMF inhibited cell adhesion and migration accompanied with a reduction in cellular F-actin amount. Moreover, following exposure to the HMF, the number of cell processes was reduced and cells were smaller in size and more round in shape. Furthermore, disordered kinetics of actin assembly in vitro were observed during exposure to the HMF, as evidenced by the presence of granule and meshed products. These results indicate that elimination of the GMF affects assembly of the motility-related actin cytoskeleton, and suggest that F-actin is a target of HMF exposure and probably a mediator of GMF sensation.

  1. Mutations in smooth muscle alpha-actin (ACTA2) lead to thoracic aortic aneurysms and dissections.

    Science.gov (United States)

    Guo, Dong-Chuan; Pannu, Hariyadarshi; Tran-Fadulu, Van; Papke, Christina L; Yu, Robert K; Avidan, Nili; Bourgeois, Scott; Estrera, Anthony L; Safi, Hazim J; Sparks, Elizabeth; Amor, David; Ades, Lesley; McConnell, Vivienne; Willoughby, Colin E; Abuelo, Dianne; Willing, Marcia; Lewis, Richard A; Kim, Dong H; Scherer, Steve; Tung, Poyee P; Ahn, Chul; Buja, L Maximilian; Raman, C S; Shete, Sanjay S; Milewicz, Dianna M

    2007-12-01

    The major function of vascular smooth muscle cells (SMCs) is contraction to regulate blood pressure and flow. SMC contractile force requires cyclic interactions between SMC alpha-actin (encoded by ACTA2) and the beta-myosin heavy chain (encoded by MYH11). Here we show that missense mutations in ACTA2 are responsible for 14% of inherited ascending thoracic aortic aneurysms and dissections (TAAD). Structural analyses and immunofluorescence of actin filaments in SMCs derived from individuals heterozygous for ACTA2 mutations illustrate that these mutations interfere with actin filament assembly and are predicted to decrease SMC contraction. Aortic tissues from affected individuals showed aortic medial degeneration, focal areas of medial SMC hyperplasia and disarray, and stenotic arteries in the vasa vasorum due to medial SMC proliferation. These data, along with the previously reported MYH11 mutations causing familial TAAD, indicate the importance of SMC contraction in maintaining the structural integrity of the ascending aorta.

  2. 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.

  3. SYP73 Anchors the ER to the Actin Cytoskeleton for Maintenance of ER Integrity and Streaming in Arabidopsis.

    Science.gov (United States)

    Cao, Pengfei; Renna, Luciana; Stefano, Giovanni; Brandizzi, Federica

    2016-12-05

    The endoplasmic reticulum (ER) is an essential organelle that spreads throughout the cytoplasm as one interconnected network of narrow tubules and dilated cisternae that enclose a single lumen. The ER network undergoes extensive remodeling, which critically depends on membrane-cytoskeleton interactions [1]. In plants, the ER is also highly mobile, and its streaming contributes significantly to the movement of other organelles [2, 3]. The remodeling and motility of the plant ER rely mainly on actin [4] and to a minor extent on microtubules [5]. Although a three-way interaction between the ER, cytosolic myosin-XI, and F-actin mediates the plant ER streaming [6], the mechanisms underlying stable interaction of the ER membrane with actin are unknown. Early electron microscopy studies suggested a direct attachment of the plant ER with actin filaments [7, 8], but it is plausible that yet-unknown proteins facilitate anchoring of the ER membrane with the cytoskeleton. We demonstrate here that SYP73, a member of the plant Syp7 subgroup of SNARE proteins [9] containing actin-binding domains, is a novel ER membrane-associated actin-binding protein. We show that overexpression of SYP73 causes a striking rearrangement of the ER over actin and that, similar to mutations of myosin-XI [4, 10, 11], loss of SYP73 reduces ER streaming and affects overall ER network morphology and plant growth. We propose a model for plant ER remodeling whereby the dynamic rearrangement and streaming of the ER network depend on the propelling action of myosin-XI over actin coupled with a SYP73-mediated bridging, which dynamically anchors the ER membrane with actin filaments.

  4. 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.

  5. 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.

  6. Freely suspended actin cortex models on arrays of microfabricated pillars

    NARCIS (Netherlands)

    Roos, Wouter H.; Roth, Alexander; Konle, Johannes; Presting, Hartmut; Sackmann, Erich; Spatz, Joachim P.

    2003-01-01

    Actin networking across pillar-tops: Actin filaments have been self-assembled onto microscopic silicon pillars, forming quasi-two-dimensional networks (see graphic) and creating novel possibilities for mimicking functions of the cellular actin cortex on solid-state devices.

  7. Actin cytoskeleton demonstration in Trichomonas vaginalis and in other trichomonads.

    Science.gov (United States)

    Brugerolle, G; Bricheux, G; Coffe, G

    1996-01-01

    The flagellate form of Trichomonas vaginalis (T v) transforms to amoeboid cells upon adherence to converslips. They grow and their nuclei divide without undergoing cytokinesis, yielding giant cells and a monolayer of T v F-actin was demonstrated in Trichomonas vaginalis by fluorescence microscopy using phalloidin and an anti-actin mAb which labelled the cytoplasm of both the flagellate and amoeboid forms. Comparative electrophoresis and immunoblotting established that the actin band has the same 42 kDa as muscle actin, but 2-D electrophoresis resolved the actin band into four spots; the two major spots observed were superimposable with major muscle actin isoforms. Electron microscopy demonstrated an ectoplasmic microfibrillar layer along the adhesion zone of amoeboid T v adhering to coverslips. Immunogold staining, using anti-actin monoclonal antibodies demonstrated that this layer was mainly composed of actin microfilaments. A comparative immunoblotting study comprising seven trichomonad species showed that all trichomonads studied expressed actin. The mAb Sigma A-4700 specific for an epitope on the actin C-terminal sequence labelled only actin of Trichomonas vaginalis, Tetratrichomonas gallinarum. Trichomitus batrachorum and Hypotrichomonas acosta, but not the actin of Tritrichomonas foetus, Tritrichomonas augusta and Monocercomonas sp. This discrimination between a 'trichomonas branch' and a 'tritrichomonas branch' is congruent with inferred sequence phylogeny from SSu rRNA and with classical phylogeny of trichomonads.

  8. 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.

  9. 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.

  10. Guidance of subcellular tubulogenesis by actin under the control of a synaptotagmin-like protein and Moesin.

    Science.gov (United States)

    JayaNandanan, N; Mathew, Renjith; Leptin, Maria

    2014-01-01

    Apical membranes in many polarized epithelial cells show specialized morphological adaptations that fulfil distinct physiological functions. The air-transporting tubules of Drosophila tracheal terminal cells represent an extreme case of membrane specialization. Here we show that Bitesize (Btsz), a synaptotagmin-like protein family member, is needed for luminal membrane morphogenesis. Unlike in multicellular tubes and other epithelia, where it influences apical integrity by affecting adherens junctions, Btsz here acts at a distance from junctions. Localized at the luminal membrane through its tandem C2 domain, it recruits activated Moesin. Both proteins are needed for the integrity of the actin cytoskeleton at the luminal membrane, but not for other pools of F-actin in the cell, nor do actin-dependent processes at the outer membrane, such as filopodial activity or membrane growth depend on Btsz. Btsz and Moesin guide luminal membrane morphogenesis through organizing actin and allowing the incorporation of membrane containing the apical determinant Crumbs.

  11. Unconventional actins and actin-binding proteins in human protozoan parasites.

    Science.gov (United States)

    Gupta, C M; Thiyagarajan, S; Sahasrabuddhe, A A

    2015-06-01

    Actin and its regulatory proteins play a key role in several essential cellular processes such as cell movement, intracellular trafficking and cytokinesis in most eukaryotes. While these proteins are highly conserved in higher eukaryotes, a number of unicellular eukaryotic organisms contain divergent forms of these proteins which have highly unusual biochemical and structural properties. Here, we review the biochemical and structural properties of these unconventional actins and their core binding proteins which are present in commonly occurring human protozoan parasites.

  12. 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...

  13. CYP1A-immunopositive proteins in bivalves identified as cytoskeletal and major vault proteins

    DEFF Research Database (Denmark)

    Grøsvik, Bjørn Einar; Jonsson, Henrik; Rodríguez-Ortega, Manuel J;

    2006-01-01

    To identify possible CYP1A-immunopositive proteins in bivalves, we used anti-fish CYP1A antibodies combined with one- and two-dimensional gel electrophoresis and mass spectrometry, and found that two of the main CYP1A-immunopositive proteins in digestive gland of Mytilus edulis, were cytoskeletal...

  14. Morphometric analysis of Mauthner axon cytoskeletal components in adult and subadult fish.

    Science.gov (United States)

    Alfei, L; Medolago-Albani, L; Zezze, G M; Stefanelli, A

    1992-01-01

    A previous cytoskeletal analysis on trout MA during developmental stages demonstrated, during the subadult stages, neurofilaments (NF) as main components as expressed by the high values of neurofilament to microtubules (MT) ratio which was found to be of the order of 300:1. Since the MA cytoskeletal composition is not known in the adult fish, the MA cytoskeletal composition has been compared to other axons of much smaller diameter of the fasciculus longitudinalis medialis (flm) among which the MA run in the ventral spinal cord. The following parameters were measured on conventional electron microscopy in MA and flm axons cross sections micrographs by means of a computer linked graphic tablet (Apple II): axonal caliber, number of microtubules (MT), microtubular (MT/microns2) and neurofilament (NF/microns2) densities. The analysis of these parameters demonstrated that neurofilaments are the main architectural components in the adult and subadult fish MA and flm axons. However, MA cytoskeletal composition differs from the other flm axons because of its particular very high ratio of neurofilaments to microtubules. The inverse relationship of axonal caliber to microtubular density, previously found in the trout during developmental stages and suggested also for many other vertebrate species, was further confirmed for flm axons which, with calibers 10 times smaller than MA, exhibit a microtubular density 10 times larger.

  15. Computational connectionism within neurons: A model of cytoskeletal automata subserving neural networks

    Science.gov (United States)

    Rasmussen, Steen; Karampurwala, Hasnain; Vaidyanath, Rajesh; Jensen, Klaus S.; Hameroff, Stuart

    1990-06-01

    “Neural network” models of brain function assume neurons and their synaptic connections to be the fundamental units of information processing, somewhat like switches within computers. However, neurons and synapses are extremely complex and resemble entire computers rather than switches. The interiors of the neurons (and other eucaryotic cells) are now known to contain highly ordered parallel networks of filamentous protein polymers collectively termed the cytoskeleton. Originally assumed to provide merely structural “bone-like” support, cytoskeletal structures such as microtubules are now recognized to organize cell interiors dynamically. The cytoskeleton is the internal communication network for the eucaryotic cell, both by means of simple transport and by means of coordinating extremely complicated events like cell division, growth and differentiation. The cytoskeleton may therefore be viewed as the cell's “nervous system”. Consequently the neuronal cytoskeleton may be involved in molecular level information processing which subserves higher, collective neuronal functions ultimately relating to cognition. Numerous models of information processing within the cytoskeleton (in particular, microtubules) have been proposed. We have utilized cellular automata as a means to model and demonstrate the potential for information processing in cytoskeletal microtubules. In this paper, we extend previous work and simulate associative learning in a cytoskeletal network as well as assembly and disassembly of microtubules. We also discuss possible relevance and implications of cytoskeletal information processing to cognition.

  16. Localization of actin in Moloney murine leukemia virus by immunoelectron microscopy.

    Science.gov (United States)

    Nermut, M V; Wallengren, K; Pager, J

    1999-07-20

    Immunoelectron microscopy was used to detect actin in wild-type (wt) Moloney murine leukemia virus (MoMuLV) and in virus-like particles (VLP) produced by recombinant Semliki Forest virus expressing only the MoMuLV gag polyprotein. Gold immunolabeling revealed the presence of actin on the surface of delipidized VLP and delipidized wt virus particles. Statistical evaluation of the number of colloidal gold particles per VLP revealed a large range of values and a prevalence of VLP with small numbers of gold particles. Labeling for actin was lost after prolonged treatment of VLP with 1% Nonidet-P40, high-pH buffer, or gelsolin. Gold immunolabeling with antibodies to gag proteins p15 (MA) and p12 and p30 (CA) was abundant and was not affected by treatment of VLP or wt virus with 1% Nonidet or gelsolin. VLP treated with a mixture of detergent and aldehyde fixatives showed more uniform and consistent labeling for actin than without fixatives. Negative staining or heavy metal shadowing revealed a globular surface of delipidized VLP. Stereomicrographs of gold-immunolabeled VLP showed that p15gag and p12gag were associated with the globular projections. Delipidized VLP were also well labeled with antibody to p30gag, which indicated that the gag shell permitted access of antibodies to p30gag and was therefore not a closely packed structure. Labeling for actin-binding proteins moesin and ezrin was negative in both the wt virus and the VLP. The absence of Gaussian distribution of actin in the sample of VLP suggests that actin is not a structural protein and its presence in MuLV virus particles may be fortuitous. This, however, does not rule out any possible role of actin in transport, assembly, budding, or release of virus particles, events which take place in the cytoplasm or at the plasma membrane. The site of actin in VLP is discussed in relation to the present knowledge of the molecular organization of the MuLV gag shell.

  17. 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.

  18. Quantification of Filamentous Actin (F-actin) Puncta in Rat Cortical Neurons.

    Science.gov (United States)

    Li, Hailong; Aksenova, Marina; Bertrand, Sarah J; Mactutus, Charles F; Booze, Rosemarie

    2016-02-10

    Filamentous actin protein (F-actin) plays a major role in spinogenesis, synaptic plasticity, and synaptic stability. Changes in dendritic F-actin rich structures suggest alterations in synaptic integrity and connectivity. Here we provide a detailed protocol for culturing primary rat cortical neurons, Phalloidin staining for F-actin puncta, and subsequent quantification techniques. First, the frontal cortex of E18 rat embryos are dissociated into low-density cell culture, then the neurons grown in vitro for at least 12-14 days. Following experimental treatment, the cortical neurons are stained with AlexaFluor 488 Phalloidin (to label the dendritic F-actin puncta) and microtubule-associated protein 2 (MAP2; to validate the neuronal cells and dendritic integrity). Finally, specialized software is used to analyze and quantify randomly selected neuronal dendrites. F-actin rich structures are identified on second order dendritic branches (length range 25-75 µm) with continuous MAP2 immunofluorescence. The protocol presented here will be a useful method for investigating changes in dendritic synapse structures subsequent to experimental treatments.

  19. Myosins 1 and 6, myosin light chain kinase, actin and microtubules cooperate during antibody-mediated internalisation and trafficking of membrane-expressed viral antigens in feline infectious peritonitis virus infected monocytes.

    Science.gov (United States)

    Dewerchin, Hannah L; Desmarets, Lowiese M; Noppe, Ytse; Nauwynck, Hans J

    2014-02-12

    Monocytes infected with feline infectious peritonitis virus, a coronavirus, express viral proteins in their plasma membranes. Upon binding of antibodies, these proteins are quickly internalised through a new clathrin- and caveolae-independent internalisation pathway. By doing so, the infected monocytes can escape antibody-dependent cell lysis. In the present study, we investigated which kinases and cytoskeletal proteins are of importance during internalisation and subsequent intracellular transport. The experiments showed that myosin light chain kinase (MLCK) and myosin 1 are crucial for the initiation of the internalisation. With co-localisation stainings, it was found that MLCK and myosin 1 co-localise with antigens even before internalisation started. Myosin 6 co-localised with the internalising complexes during passage through the cortical actin, were it might play a role in moving or disintegrating actin filaments, to overcome the actin barrier. One minute after internalisation started, vesicles had passed the cortical actin, co-localised with microtubules and association with myosin 6 was lost. The vesicles were further transported over the microtubules and accumulated at the microtubule organising centre after 10 to 30 min. Intracellular trafficking over microtubules was mediated by MLCK, myosin 1 and a small actin tail. Since inhibiting MLCK with ML-7 was so efficient in blocking the internalisation pathway, this target can be used for the development of a new treatment for FIPV.

  20. Aegeline from Aegle marmelos stimulates glucose transport via Akt and Rac1 signaling, and contributes to a cytoskeletal rearrangement through PI3K/Rac1.

    Science.gov (United States)

    Gautam, Sudeep; Ishrat, Nayab; Singh, Rohit; Narender, Tadigoppula; Srivastava, Arvind K

    2015-09-01

    Aegeline is an alkaloidal-amide, isolated from the leaves of Aegle marmelos and have shown antihyperglycemic as well as antidyslipidemic activities in the validated animal models of type 2 diabetes mellitus. Here we delineate, aegeline enhanced GLUT4 translocation mediated 2-deoxy-glucose uptake in both time and concentration-dependent manner. 2-deoxy-glucose uptake was completely stymied by the transport inhibitors (wortmannin and genistein) in C2C12 myotubes. Pharmacological inhibition of Akt (also known as protein kinase B) and Ras-related C3 botulinum toxin substrate 1 (Rac1) suggest that both Akt and Rac1 operate aegeline-stimulated glucose transport via distinct parallel pathways. Moreover, aegeline activates p21 protein-activated kinase 1 (PAK1) and cofilin (an actin polymerization regulator). Rac1 inhibitor (Rac1 inhib II) and PAK1 inhibitor (IPA-3) completely blocked aegeline-induced phosphorylation of cofilin and p21 protein-activated kinase 1 (PAK1). In summary, these findings suggest that aegeline stimulates the glucose transport through Akt and Rac1 dependent distinct parallel pathways and have cytoskeletal roles via stimulation of the PI3-kinase-Rac1-PAK1-cofilin pathway in the skeletal muscle cells. Therefore, multiple targets of aegeline in the improvement of insulin sensitivity of the skeletal muscle cells may be suggested.

  1. Polycystin-1 Induces Cell Migration by Regulating Phosphatidylinositol 3-kinase-dependent Cytoskeletal Rearrangements and GSK3β-dependent Cell–Cell Mechanical Adhesion

    Science.gov (United States)

    Boca, Manila; D'Amato, Lisa; Distefano, Gianfranco; Polishchuk, Roman S.; Germino, Gregory G.

    2007-01-01

    Polycystin-1 (PC-1) is a large plasma-membrane receptor encoded by the PKD1 gene mutated in autosomal dominant polycystic kidney disease (ADPKD). Although the disease is thought to be recessive on a molecular level, the precise mechanism of cystogenesis is unclear, although cytoarchitecture defects seem to be the most likely initiating events. Here we show that PC-1 regulates the actin cytoskeleton in renal epithelial cells (MDCK) and induces cell scattering and cell migration. All of these effects require phosphatidylinositol 3-kinase (PI3-K) activity. Consistent with these observations Pkd1−/− mouse embryonic fibroblasts (MEFs) have reduced capabilities to migrate compared with controls. PC-1 overexpressing MDCK cells are able to polarize normally with proper adherens and tight junctions formation, but show quick reabsorption of ZO-1, E-cadherin, and β-catenin upon wounding of a monolayer and a transient epithelial-to-mesenchymal transition (EMT) that favors a rapid closure of the wound and repolarization. Finally, we show that PC-1 is able to control the turnover of cytoskeletal-associated β-catenin through activation of GSK3β. Expression of a nondegradable form of β-catenin in PC-1 MDCK cells restores strong cell–cell mechanical adhesion. We propose that PC-1 might be a central regulator of epithelial plasticity and its loss results in impaired normal epithelial homeostasis. PMID:17671167

  2. Glycoprotein Ib-IX-V Complex Transmits Cytoskeletal Forces That Enhance Platelet Adhesion.

    Science.gov (United States)

    Feghhi, Shirin; Munday, Adam D; Tooley, Wes W; Rajsekar, Shreya; Fura, Adriane M; Kulman, John D; López, Jose A; Sniadecki, Nathan J

    2016-08-09

    Platelets bind to exposed vascular matrix at a wound site through a highly specialized surface receptor, glycoprotein (GP) Ib-IX-V complex, which recognizes von Willebrand factor (VWF) in the matrix. GPIb-IX-V is a catch bond for it becomes more stable as force is applied to it. After attaching to the wound site, platelets generate cytoskeletal forces to compact and reinforce the hemostatic plug. Here, we evaluated the role of the GPIb-IX-V complex in the transmission of cytoskeletal forces. We used arrays of flexible, silicone nanoposts to measure the contractility of individual platelets on VWF. We found that a significant proportion of cytoskeletal forces were transmitted to VWF through GPIb-IX-V, an unexpected finding given the widely held notion that platelet forces are transmitted exclusively through its integrins. In particular, we found that the interaction between GPIbα and the A1 domain of VWF mediates this force transmission. We also demonstrate that the binding interaction between GPIbα and filamin A is involved in force transmission. Furthermore, our studies suggest that cytoskeletal forces acting through GPIbα are involved in maintaining platelet adhesion when external forces are absent. Thus, the GPIb-IX-V/VWF bond is able to transmit force, and uses this force to strengthen the bond through a catch-bond mechanism. This finding expands our understanding of how platelets attach to sites of vascular injury, describing a new, to the best of our knowledge, mechanism in which the catch bonds of GPIb-IX-V/VWF can be supported by internal forces produced by cytoskeletal tension.

  3. Preservation of tissue microstructure and functionality during freezing by modulation of cytoskeletal structure.

    Science.gov (United States)

    Park, Seungman; Seawright, Angela; Park, Sinwook; Craig Dutton, J; Grinnell, Frederick; Han, Bumsoo

    2015-05-01

    Cryopreservation is one of the key enabling technologies for tissue engineering and regenerative medicine, which can provide reliable long-term storage of engineered tissues (ETs) without losing their functionality. However, it is still extremely difficult to design and develop cryopreservation protocols guaranteeing the post-thaw tissue functionality. One of the major challenges in cryopreservation is associated with the difficulty of identifying effective and less toxic cryoprotective agents (CPAs) to guarantee the post-thaw tissue functionality. In this study, thus, a hypothesis was tested that the modulation of the cytoskeletal structure of cells embedded in the extracellular matrix (ECM) can mitigate the freezing-induced changes of the functionality and can reduce the amount of CPA necessary to preserve the functionality of ETs during cryopreservation. In order to test this hypothesis, we prepared dermal equivalents by seeding fibroblasts in type I collagen matrices resulting in three different cytoskeletal structures. These ETs were exposed to various freeze/thaw (F/T) conditions with and without CPAs. The freezing-induced cell-fluid-matrix interactions and subsequent functional properties of the ETs were assessed. The results showed that the cytoskeletal structure and the use of CPA were strongly correlated to the preservation of the post-thaw functional properties. As the cytoskeletal structure became stronger via stress fiber formation, the ET's functionality was preserved better. It also reduced the necessary CPA concentration to preserve the post-thaw functionality. However, if the extent of the freezing-induced cell-fluid-matrix interaction was too excessive, the cytoskeletal structure was completely destroyed and the beneficial effects became minimal.

  4. 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 wit...... with severe actinic damage showed more impairment in HRQoL than those with mild disease. Correlations between instruments suggest that they are complementary as they measure different aspects of HRQoL and are used for different purposes....... with different severity levels of AK treated in dermatology specialist care using generic and disease-specific HRQoL instruments and to analyze their relationship. METHODS: AK patients who visited dermatological clinics in Denmark were included in an observational, cross-sectional, study in a multi...... instruments was analyzed with the Spearman correlation test. RESULTS: A total of 312 patients were included in the analyses. Patients reported impairment in the disease specific HRQoL instrument AKQoL (mean AKQoL 6.7, DLQI 2, EQ-5D-5 L 0.88, and EQ-VAS 79). HRQoL was least affected in patients with mild...

  5. 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.

  6. Transient state model of actin-based motility

    Institute of Scientific and Technical Information of China (English)

    2011-01-01

    We developed a transient model for actin-based motility.Diffusion of actin monomers was included in the formulation and its influence on the speed of actin-driven cargos was examined in detail.Our results clearly demonstrated how actin polymerization accelerates cargos that are initially stationary,as well as how steady-state is eventually reached.We also found that,due to polymerization and diffusion,actin monomer concentration near the load surface can be significantly lower than that in the rest of th...

  7. The 5’cap of Tobacco Mosaic Virus (TMV) is required for virion attachment to the actin/ER network during early infection

    DEFF Research Database (Denmark)

    Christensen, Nynne Meyn; Tilsner, Jens; Bell, Karen;

    to the motile cortical actin/ER network within minutes of injection. Granule movement on actin/ER was arrested by actin inhibitors indicating actindependent RNA movement. The 5’ methylguanosine TMV cap was shown to be required for vRNA anchoring to the ER. TMV vRNA lacking the 5’cap failed to form granules...

  8. Examination of actin and microtubule dependent APC localisations in living mammalian cells

    Directory of Open Access Journals (Sweden)

    Adams Matthew

    2006-01-01

    Full Text Available Abstract Background The trafficking of the adenomatous polyposis coli (APC tumour suppressor protein in mammalian cells is a perennially controversial topic. Immunostaining evidence for an actin-associated APC localisation at intercellular junctions has been previously presented, though live imaging of mammalian junctional APC has not been documented. Results Using live imaging of transfected COS-7 cells we observed intercellular junction-associated pools of GFP-APC in addition to previously documented microtubule-associated GFP-APC and a variety of minor localisations. Although both microtubule and junction-associated populations could co-exist within individual cells, they differed in their subcellular location, dynamic behaviour and sensitivity to cytoskeletal poisons. GFP-APC deletion mutant analysis indicated that a protein truncated immediately after the APC armadillo repeat domain retained the ability to localise to adhesive membranes in transfected cells. Supporting this, we also observed junctional APC immunostaining in cultures of human colorectal cancer cell line that express truncated forms of APC. Conclusion Our data indicate that APC can be found in two spatially separate populations at the cell periphery and these populations can co-exist in the same cell. The first localisation is highly dynamic and associated with microtubules near free edges and in cell vertices, while the second is comparatively static and is closely associated with actin at sites of cell-cell contact. Our imaging confirms that human GFP-APC possesses many of the localisations and behaviours previously seen by live imaging of Xenopus GFP-APC. However, we report the novel finding that GFP-APC puncta can remain associated with the ends of shrinking microtubules. Deletion analysis indicated that the N-terminal region of the APC protein mediated its junctional localisation, consistent with our observation that truncated APC proteins in colon cancer cell lines are

  9. Bundling Actin Filaments From Membranes: Some Novel Players

    Directory of Open Access Journals (Sweden)

    Clément eThomas

    2012-08-01

    Full Text Available Progress in live-cell imaging of the cytoskeleton has significantly extended our knowledge about the organization and dynamics of actin filaments near the plasma membrane of plant cells. Noticeably, two populations of filamentous structures can be distinguished. On the one hand, fine actin filaments which exhibit an extremely dynamic behavior basically characterized by fast polymerization and prolific severing events, a process referred to as actin stochastic dynamics. On the other hand, thick actin bundles which are composed of several filaments and which are comparatively more stable although they constantly remodel as well. There is evidence that the actin cytoskeleton plays critical roles in trafficking and signaling at both the cell cortex and organelle periphery but the exact contribution of actin bundles remains unclear. A common view is that actin bundles provide the long-distance tracks used by myosin motors to deliver their cargo to growing regions and accordingly play a particularly important role in cell polarization. However, several studies support that actin bundles are more than simple passive highways and display multiple and dynamic roles in the regulation of many processes, such as cell elongation, polar auxin transport, stomatal and chloroplast movement, and defense against pathogens. The list of identified plant actin-bundling proteins is ever expanding, supporting that plant cells shape structurally and functionally different actin bundles. Here I review the most recently characterized actin-bundling proteins, with a particular focus on those potentially relevant to membrane trafficking and/or signaling.

  10. Distributed actin turnover in the lamellipodium and FRAP kinetics.

    Science.gov (United States)

    Smith, Matthew B; Kiuchi, Tai; Watanabe, Naoki; Vavylonis, Dimitrios

    2013-01-08

    Studies of actin dynamics at the leading edge of motile cells with single-molecule speckle (SiMS) microscopy have shown a broad distribution of EGFP-actin speckle lifetimes and indicated actin polymerization and depolymerization over an extended region. Other experiments using FRAP with the same EGFP-actin as a probe have suggested, by contrast, that polymerization occurs exclusively at the leading edge. We performed FRAP experiments on XTC cells to compare SiMS to FRAP on the same cell type. We used speckle statistics obtained by SiMS to model the steady-state distribution and kinetics of actin in the lamellipodium. We demonstrate that a model with a single diffuse actin species is in good agreement with FRAP experiments. A model including two species of diffuse actin provides an even better agreement. The second species consists of slowly diffusing oligomers that associate to the F-actin network throughout the lamellipodium or break up into monomers after a characteristic time. Our work motivates studies to test the presence and composition of slowly diffusing actin species that may contribute to local remodeling of the actin network and increase the amount of soluble actin.

  11. Crystal structure and structure-based mutagenesis of actin-specific ADP-ribosylating toxin CPILE-a as novel enterotoxin

    Science.gov (United States)

    Toniti, Waraphan; Yoshida, Toru; Tsurumura, Toshiharu; Irikura, Daisuke; Monma, Chie; Kamata, Yoichi

    2017-01-01

    Unusual outbreaks of food poisoning in Japan were reported in which Clostridium perfringens was strongly suspected to be the cause based on epidemiological information and fingerprinting of isolates. The isolated strains lack the typical C. perfringens enterotoxin (CPE) but secrete a new enterotoxin consisting of two components: C. perfringens iota-like enterotoxin-a (CPILE-a), which acts as an enzymatic ADP-ribosyltransferase, and CPILE-b, a membrane binding component. Here we present the crystal structures of apo-CPILE-a, NAD+-CPILE-a and NADH-CPILE-a. Though CPILE-a structure has high similarity with known iota toxin-a (Ia) with NAD+, it possesses two extra-long protruding loops from G262-S269 and E402-K408 that are distinct from Ia. Based on the Ia–actin complex structure, we focused on actin-binding interface regions (I-V) including two protruding loops (PT) and examined how mutations in these regions affect the ADP-ribosylation activity of CPILE-a. Though some site-directed mutagenesis studies have already been conducted on the actin binding site of Ia, in the present study, mutagenesis studies were conducted against both α- and β/γ-actin in CPILE-a and Ia. Interestingly, CPILE-a ADP-ribosylates both α- and β/γ-actin, but its sensitivity towards β/γ-actin is 36% compared with α-actin. Our results contrast to that only C2-I ADP-ribosylates β/γ-actin. We also showed that PT-I and two convex-concave interactions in CPILE-a are important for actin binding. The current study is the first detailed analysis of site-directed mutagenesis in the actin binding region of Ia and CPILE-a against both α- and β/γ-actin. PMID:28199340

  12. Tropomyosin diffusion over actin subunits facilitates thin filament assembly

    Directory of Open Access Journals (Sweden)

    Stefan Fischer

    2016-01-01

    Full Text Available Coiled-coil tropomyosin binds to consecutive actin-subunits along actin-containing thin filaments. Tropomyosin molecules then polymerize head-to-tail to form cables that wrap helically around the filaments. Little is known about the assembly process that leads to continuous, gap-free tropomyosin cable formation. We propose that tropomyosin molecules diffuse over the actin-filament surface to connect head-to-tail to partners. This possibility is likely because (1 tropomyosin hovers loosely over the actin-filament, thus binding weakly to F-actin and (2 low energy-barriers provide tropomyosin freedom for 1D axial translation on F-actin. We consider that these unique features of the actin-tropomyosin interaction are the basis of tropomyosin cable formation.

  13. Tropomyosin diffusion over actin subunits facilitates thin filament assembly

    Science.gov (United States)

    Fischer, Stefan; Rynkiewicz, Michael J.; Moore, Jeffrey R.; Lehman, William

    2016-01-01

    Coiled-coil tropomyosin binds to consecutive actin-subunits along actin-containing thin filaments. Tropomyosin molecules then polymerize head-to-tail to form cables that wrap helically around the filaments. Little is known about the assembly process that leads to continuous, gap-free tropomyosin cable formation. We propose that tropomyosin molecules diffuse over the actin-filament surface to connect head-to-tail to partners. This possibility is likely because (1) tropomyosin hovers loosely over the actin-filament, thus binding weakly to F-actin and (2) low energy-barriers provide tropomyosin freedom for 1D axial translation on F-actin. We consider that these unique features of the actin-tropomyosin interaction are the basis of tropomyosin cable formation. PMID:26798831

  14. Design and evaluation of Actichip, a thematic microarray for the study of the actin cytoskeleton

    Directory of Open Access Journals (Sweden)

    Chalmel Frédéric

    2007-08-01

    Full Text Available Abstract Background The actin cytoskeleton plays a crucial role in supporting and regulating numerous cellular processes. Mutations or alterations in the expression levels affecting the actin cytoskeleton system or related regulatory mechanisms are often associated with complex diseases such as cancer. Understanding how qualitative or quantitative changes in expression of the set of actin cytoskeleton genes are integrated to control actin dynamics and organisation is currently a challenge and should provide insights in identifying potential targets for drug discovery. Here we report the development of a dedicated microarray, the Actichip, containing 60-mer oligonucleotide probes for 327 genes selected for transcriptome analysis of the human actin cytoskeleton. Results Genomic data and sequence analysis features were retrieved from GenBank and stored in an integrative database called Actinome. From these data, probes were designed using a home-made program (CADO4MI allowing sequence refinement and improved probe specificity by combining the complementary information recovered from the UniGene and RefSeq databases. Actichip performance was analysed by hybridisation with RNAs extracted from epithelial MCF-7 cells and human skeletal muscle. Using thoroughly standardised procedures, we obtained microarray images with excellent quality resulting in high data reproducibility. Actichip displayed a large dynamic range extending over three logs with a limit of sensitivity between one and ten copies of transcript per cell. The array allowed accurate detection of small changes in gene expression and reliable classification of samples based on the expression profiles of tissue-specific genes. When compared to two other oligonucleotide microarray platforms, Actichip showed similar sensitivity and concordant expression ratios. Moreover, Actichip was able to discriminate the highly similar actin isoforms whereas the two other platforms did not. Conclusion Our

  15. Transfer of a redox-signal through the cytosol by redox-dependent microcompartmentation of glycolytic enzymes at mitochondria and actin cytoskeleton

    Directory of Open Access Journals (Sweden)

    Joanna eWojtera-Kwiczor

    2013-01-01

    Full Text Available The cytosolic glyceraldehyde-3-phosphate dehydrogenase (GAPDH, EC 1.2.1.12, GapC plays an important role in glycolysis by providing the cell with ATP and NADH. Interestingly, despite its glycolytic function in the cytosol, GAPDH was reported to possess additional non-glycolytic activities, correlating with its nuclear or cytoskeletal localization in animal cells. In transiently transformed mesophyll protoplasts from Arabidopsis. thaliana colocalization and interaction of the glycolytic enzymes with the mitochondria and with the actin cytoskeleton was visualized by confocal laser scanning microscopy (cLSM using fluorescent protein fusions and by bimolecular fluorescence complementation (BiFC, respectively. Yeast two-hybrid screens, dot-blot overlay assays, and co-sedimentation assays were used to identify potential protein-protein interactions between two cytosolic GAPDH isoforms (GapC1, At3g04120; GapC2, At1g13440 from A. thaliana with the neighbouring glycolytic enzyme, fructose 1,6-bisphosphate aldolase (FBA6, At2g36460, the mitochondrial porin (VDAC3; At5g15090, and actin in vitro. From these experiments, a mitochondrial association is suggested for both glycolytic enzymes, GAPDH and aldolase, which appear to bind to the outer mitochondrial membrane, in a redox-dependent manner. In addition, both glycolytic enzymes were found to bind to F-actin in cosedimentation assays, and lead to bundling of purified rabbit actin, as visualized by cLSM. Actin binding and bundling occurred reversibly under oxidizing conditions. We speculate that such dynamic formation of microcompartments is part of a redox-dependent retrograde signal transduction network for adaptation upon oxidative stress.

  16. Photodamage induced by Zinc(II)-phthalocyanine to microtubules, actin, alpha-actinin and keratin of HeLa cells.

    Science.gov (United States)

    Juarranz, A; Espada, J; Stockert, J C; Villanueva, A; Polo, S; Domínguez, V; Cañete, M

    2001-03-01

    We have studied the photosensitizing effects of zinc(II)-phthalocyanine (ZnPc) on the cytoskeleton of HeLa cells using sublethal (10(-7) M, followed by 1 or 3 min of red light to induce 20%, LD20, or 60%, LD60, cell death, respectively) or lethal (5 x 10(-6) M and 15 min of irradiation, LD100) experimental conditions. The immunofluorescent analysis of the cytoskeleton showed a variable photodamage to microtubules (MT), actin microfilaments (AF) and intermediate filaments of keratin (KF), as well as on alpha-actinin, which was dependent on treatment conditions. Both sublethal treatments induced deep alterations on interphase and mitotic MT. The mitotic index increased with time with the maximum at 18 h (12%) or 24 h (14%) after LD20 or LD60, respectively. The alterations on AF and alpha-actinin were much more severe than those observed on KF at any evaluated time. With the exception of the KF, which remained partially organized, the MT and AF network was severely damaged by the lethal treatment. Western blot analysis for alpha-tubulin, G-actin and alpha-actinin from soluble and insoluble fractions confirmed the results observed by immunofluorescence, thus indicating that these cytoskeletal components are involved in cell damage and death by ZnPc photosensitization.

  17. Drosophila sosie functions with βH-Spectrin and actin organizers in cell migration, epithelial morphogenesis and cortical stability

    Directory of Open Access Journals (Sweden)

    Olivier Urwyler

    2012-08-01

    Morphogenesis in multicellular organisms requires the careful coordination of cytoskeletal elements, dynamic regulation of cell adhesion and extensive cell migration. sosie (sie is a novel gene required in various morphogenesis processes in Drosophila oogenesis. Lack of sie interferes with normal egg chamber packaging, maintenance of epithelial integrity and control of follicle cell migration, indicating that sie is involved in controlling epithelial integrity and cell migration. For these functions sie is required both in the germ line and in the soma. Consistent with this, Sosie localizes to plasma membranes in the germ line and in the somatic follicle cells and is predicted to present an EGF-like domain on the extracellular side. Two positively charged residues, C-terminal to the predicted transmembrane domain (on the cytoplasmic side, are required for normal plasma membrane localization of Sosie. Because sie also contributes to normal cortical localization of βH-Spectrin, it appears that cortical βH-Spectrin mediates some of the functions of sosie. sie also interacts with the genes coding for the actin organizers Filamin and Profilin and, in the absence of sie function, F-actin is less well organized and nurse cells frequently fuse.

  18. PI(3,5)P2 controls endosomal branched actin dynamics by regulating cortactin-actin interactions.

    Science.gov (United States)

    Hong, Nan Hyung; Qi, Aidong; Weaver, Alissa M

    2015-08-31

    Branched actin critically contributes to membrane trafficking by regulating membrane curvature, dynamics, fission, and transport. However, how actin dynamics are controlled at membranes is poorly understood. Here, we identify the branched actin regulator cortactin as a direct binding partner of phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2) and demonstrate that their interaction promotes turnover of late endosomal actin. In vitro biochemical studies indicated that cortactin binds PI(3,5)P2 via its actin filament-binding region. Furthermore, PI(3,5)P2 competed with actin filaments for binding to cortactin, thereby antagonizing cortactin activity. These findings suggest that PI(3,5)P2 formation on endosomes may remove cortactin from endosome-associated branched actin. Indeed, inhibition of PI(3,5)P2 production led to cortactin accumulation and actin stabilization on Rab7(+) endosomes. Conversely, inhibition of Arp2/3 complex activity greatly reduced cortactin localization to late endosomes. Knockdown of cortactin reversed PI(3,5)P2-inhibitor-induced actin accumulation and stabilization on endosomes. These data suggest a model in which PI(3,5)P2 binding removes cortactin from late endosomal branched actin networks and thereby promotes net actin turnover.

  19. RBC-NOS-dependent S-nitrosylation of cytoskeletal proteins improves RBC deformability.

    Directory of Open Access Journals (Sweden)

    Marijke Grau

    Full Text Available BACKGROUND: Red blood cells (RBC possess a nitric oxide synthase (RBC-NOS whose activation depends on the PI3-kinase/Akt kinase pathway. RBC-NOS-produced NO exhibits important biological functions like maintaining RBC deformability. Until now, the cellular target structure for NO, to exert its influence on RBC deformability, remains unknown. In the present study we analyzed the modification of RBC-NOS activity by pharmacological treatments, the resulting influence on RBC deformability and provide first evidence for possible target proteins of RBC-NOS-produced NO in the RBC cytoskeletal scaffold. METHODS/FINDINGS: Blood from fifteen male subjects was incubated with the NOS substrate L-arginine to directly stimulate enzyme activity. Direct inhibition of enzyme activity was induced by L-N5-(1-Iminoethyl-ornithin (L-NIO. Indirect stimulation and inhibition of RBC-NOS were achieved by applying insulin and wortmannin, respectively, substances known to affect PI3-kinase/Akt kinase pathway. The NO donor sodium nitroprusside (SNP and the NO scavenger 2-(4-carboxyphenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO were additionally applied as NO positive and negative controls, respectively. Immunohistochemical staining was used to determine phosphorylation and thus activation of RBC-NOS. As a marker for NO synthesis nitrite was measured in plasma and RBCs using chemiluminescence detection. S-nitrosylation of erythrocyte proteins was determined by biotin switch assay and modified proteins were identified using LC-MS. RBC deformability was determined by ektacytometry. The data reveal that activated RBC-NOS leads to increased NO production, S-nitrosylation of RBC proteins and RBC deformability, whereas RBC-NOS inhibition resulted in contrary effects. CONCLUSION/SIGNIFICANCE: This study first-time provides strong evidence that RBC-NOS-produced NO modifies RBC deformability through direct S-nitrosylation of cytoskeleton proteins, most likely α- and

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

    Directory of Open Access Journals (Sweden)

    Juan Du

    Full Text Available 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.

  1. 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.

  2. F-actin cytoskeleton and the fate of organelles in chromaffin cells.

    Science.gov (United States)

    Villanueva, José; Gimenez-Molina, Yolanda; Viniegra, Salvador; Gutiérrez, Luis M

    2016-06-01

    In addition to playing a fundamental structural role, the F-actin cytoskeleton in neuroendocrine chromaffin cells has a prominent influence on governing the molecular mechanism and regulating the secretory process. Performing such roles, the F-actin network might be essential to first transport, and later locate the cellular organelles participating in the secretory cycle. Chromaffin granules are transported from the internal cytosolic regions to the cell periphery along microtubular and F-actin structures. Once in the cortical region, they are embedded in the F-actin network where these vesicles experience restrictions in motility. Similarly, mitochondria transport is affected by both microtubule and F-actin inhibitors and suffers increasing motion restrictions when they are located in the cortical region. Therefore, the F-actin cortex is a key factor in defining the existence of two populations of cortical and perinuclear granules and mitochondria which could be distinguished by their different location and mobility. Interestingly, other important organelles for controlling intracellular calcium levels, such as the endoplasmic reticulum network, present clear differences in distribution and much lower mobility than chromaffin vesicles and mitochondria. Nevertheless, both mitochondria and the endoplasmic reticulum appear to distribute in the proximity of secretory sites to fulfill a pivotal role, forming triads with calcium channels ensuring the fine tuning of the secretory response. This review presents the contributions that provide the basis for our current view regarding the influence that F-actin has on the distribution of organelles participating in the release of catecholamines in chromaffin cells, and summarizes this knowledge in simple models. In chromaffin cells, organelles such as granules and mitochondria distribute forming cortical and perinuclear populations whereas others like the ER present homogenous distributions. In the present review we discuss

  3. 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.

  4. 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.

  5. Omega-3 fatty acids modulate Weibel-Palade body degranulation and actin cytoskeleton rearrangement in PMA-stimulated human umbilical vein endothelial cells.

    Science.gov (United States)

    Bürgin-Maunder, Corinna S; Brooks, Peter R; Russell, Fraser D

    2013-11-08

    Long chain omega-3 polyunsaturated fatty acids (LC n-3 PUFAs) produce cardiovascular benefits by improving endothelial function. Endothelial cells store von Willebrand factor (vWF) in cytoplasmic Weibel-Palade bodies (WPBs). We examined whether LC n-3 PUFAs regulate WPB degranulation using cultured human umbilical vein endothelial cells (HUVECs). HUVECs were incubated with or without 75 or 120 µM docosahexaenoic acid or eicosapentaenoic acid for 5 days at 37 °C. WPB degranulation was stimulated using phorbol 12-myristate 13-acetate (PMA), and this was assessed by immunocytochemical staining for vWF. Actin reorganization was determined using phalloidin-TRITC staining. We found that PMA stimulated WPB degranulation, and that this was significantly reduced by prior incubation of cells with LC n-3 PUFAs. In these cells, WPBs had rounded rather than rod-shaped morphology and localized to the perinuclear region, suggesting interference with cytoskeletal remodeling that is necessary for complete WPB degranulation. In line with this, actin rearrangement was altered in cells containing perinuclear WPBs, where cells exhibited a thickened actin rim in the absence of prominent cytoplasmic stress fibers. These findings indicate that LC n-3 PUFAs provide some protection against WBP degranulation, and may contribute to an improved understanding of the anti-thrombotic effects previously attributed to LC n-3 PUFAs.

  6. Omega-3 Fatty Acids Modulate Weibel-Palade Body Degranulation and Actin Cytoskeleton Rearrangement in PMA-Stimulated Human Umbilical Vein Endothelial Cells

    Directory of Open Access Journals (Sweden)

    Corinna S. Bürgin-Maunder

    2013-11-01

    Full Text Available Long chain omega-3 polyunsaturated fatty acids (LC n-3 PUFAs produce cardiovascular benefits by improving endothelial function. Endothelial cells store von Willebrand factor (vWF in cytoplasmic Weibel-Palade bodies (WPBs. We examined whether LC n-3 PUFAs regulate WPB degranulation using cultured human umbilical vein endothelial cells (HUVECs. HUVECs were incubated with or without 75 or 120 µM docosahexaenoic acid or eicosapentaenoic acid for 5 days at 37 °C. WPB degranulation was stimulated using phorbol 12-myristate 13-acetate (PMA, and this was assessed by immunocytochemical staining for vWF. Actin reorganization was determined using phalloidin-TRITC staining. We found that PMA stimulated WPB degranulation, and that this was significantly reduced by prior incubation of cells with LC n-3 PUFAs. In these cells, WPBs had rounded rather than rod-shaped morphology and localized to the perinuclear region, suggesting interference with cytoskeletal remodeling that is necessary for complete WPB degranulation. In line with this, actin rearrangement was altered in cells containing perinuclear WPBs, where cells exhibited a thickened actin rim in the absence of prominent cytoplasmic stress fibers. These findings indicate that LC n-3 PUFAs provide some protection against WBP degranulation, and may contribute to an improved understanding of the anti-thrombotic effects previously attributed to LC n-3 PUFAs.

  7. Dynamics of Actin Stress Fibers and Focal Adhesions during Slow Migration in Swiss 3T3 Fibroblasts: Intracellular Mechanism of Cell Turning

    Directory of Open Access Journals (Sweden)

    Michiko Sugawara

    2016-01-01

    Full Text Available To understand the mechanism regulating the spontaneous change in polarity that leads to cell turning, we quantitatively analyzed the dynamics of focal adhesions (FAs coupling with the self-assembling actin cytoskeletal structure in Swiss 3T3 fibroblasts. Fluorescent images were acquired from cells expressing GFP-actin and RFP-zyxin by laser confocal microscopy. On the basis of the maximum area, duration, and relocation distance of FAs extracted from the RFP-zyxin images, the cells could be divided into 3 regions: the front region, intermediate lateral region, and rear region. In the intermediate lateral region, FAs appeared close to the leading edge and were stabilized gradually as its area increased. Simultaneously, bundled actin stress fibers (SFs were observed vertically from the positions of these FAs, and they connected to the other SFs parallel to the leading edge. Finally, these connecting SFs fused to form a single SF with matured FAs at both ends. This change in SF organization with cell retraction in the first cycle of migration followed by a newly formed protrusion in the next cycle is assumed to lead to cell turning in migrating Swiss 3T3 fibroblasts.

  8. Probing cytoskeletal structures by coupling optical superresolution and AFM techniques for a correlative approach.

    Science.gov (United States)

    Chacko, Jenu Varghese; Zanacchi, Francesca Cella; Diaspro, Alberto

    2013-11-01

    In this article, we describe and show the application of some of the most advanced fluorescence superresolution techniques, STED AFM and STORM AFM microscopy towards imaging of cytoskeletal structures, such as microtubule filaments. Mechanical and structural properties can play a relevant role in the investigation of cytoskeletal structures of interest, such as microtubules, that provide support to the cell structure. In fact, the mechanical properties, such as the local stiffness and the elasticity, can be investigated by AFM force spectroscopy with tens of nanometers resolution. Force curves can be analyzed in order to obtain the local elasticity (and the Young's modulus calculation by fitting the force curves from every pixel of interest), and the combination with STED/STORM microscopy integrates the measurement with high specificity and yields superresolution structural information. This hybrid modality of superresolution-AFM working is a clear example of correlative multimodal microscopy.

  9. Proteomic profiling in Drosophila reveals potential Dube3a regulation of the actin cytoskeleton and neuronal homeostasis.

    Directory of Open Access Journals (Sweden)

    Laura Jensen

    Full Text Available The molecular defects associated with Angelman syndrome (AS and 15q duplication autism are directly correlated to expression levels of the E3 ubiquitin ligase protein UBE3A. Here we used Drosophila melanogaster to screen for the targets of this ubiquitin ligase under conditions of both decreased (as in AS or increased (as in dup(15 levels of the fly Dube3a or human UBE3A proteins. Using liquid phase isoelectric focusing of proteins from whole fly head extracts we identified a total of 50 proteins that show changes in protein, and in some cases transcriptional levels, when Dube3a fluctuates. We analyzed head extracts from cytoplasmic, nuclear and membrane fractions for Dube3a regulated proteins. Our results indicate that Dube3a is involved in the regulation of cellular functions related to ATP synthesis/metabolism, actin cytoskeletal integrity, both catabolism and carbohydrate metabolism as well as nervous system development and function. Sixty-two percent of the proteins were >50% identical to homologous human proteins and 8 have previously be shown to be ubiquitinated in the fly nervous system. Eight proteins may be regulated by Dube3a at the transcript level through the transcriptional co-activation function of Dube3a. We investigated one autism-associated protein, ATPα, and found that it can be ubiquitinated in a Dube3a dependent manner. We also found that Dube3a mutants have significantly less filamentous actin than wild type larvae consistent with the identification of actin targets regulated by Dube3a. The identification of UBE3A targets is the first step in unraveling the molecular etiology of AS and duplication 15q autism.

  10. 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

  11. Tyrosine kinase activity, cytoskeletal organization, and motility in human vascular endothelial cells.

    OpenAIRE

    1994-01-01

    Tyrosine phosphorylation of cytoskeletal proteins occurs during integrin-mediated cell adhesion to extracellular matrix proteins. We have investigated the role of tyrosine phosphorylation in the migration and initial spreading of human umbilical vein endothelial cells (HUVEC). Elevated phosphotyrosine concentrations were noted in the focal adhesions of HUVEC migrating into wounds. Anti-phosphotyrosine Western blots of extracts of wounded HUVEC monolayers demonstrated increased phosphorylation...

  12. Organization of cytoskeletal elements and organelles preceding growth cone emergence from an identified neuron in situ

    OpenAIRE

    1989-01-01

    The purpose of this study was to investigate the arrangement of cytoskeletal elements and organelles in an identified neuron in situ at the site of emergence of its growth cone just before and concurrent with the onset of axonogenesis. The Ti1 pioneer neurons are the first pair of afferent neurons to differentiate in embryonic grasshopper limbs. They arise at the distal tip of the limb bud epithelium, the daughter cells of a single precursor cell, the Pioneer Mother Cell (PMC). Using immunohi...

  13. The cytoskeletal protein Ndel1 regulates dynamin 2 GTPase activity.

    Directory of Open Access Journals (Sweden)

    Mathieu Chansard

    Full Text Available Cytoskeleton dynamics, membranes trafficking and positioning are essential for the proper functioning of any mammalian cell. The identification of the molecules and mechanisms that allow these cellular processes to interface is vital for understanding cell behaviors. Ndel1, the mammalian homolog of the Aspergillus nidulans NudE, organizes the cytoskeleton and regulates molecular motors, thereby impacting on the positioning of membranes. Hypothetically, Ndel1 can act in concert with enzymes controlling membrane trafficking (vesicle-mediated transport per se, but this idea has never been investigated. We now report that a pool of Ndel1 associates directly with Dynamin 2 (Dyn2, a large cytosolic GTPase involved in the trafficking of the AMPA receptor subunit GluR1. In vitro, Ndel1 enhances Dyn2 GTPase activity in its unassembled and assembled forms, without promoting oligomerization of the enzyme. In cells, gain and loss of function of Ndel1 recapitulate the effects of overexpression of Dyn2 and Dyn2 dominant negative with reduced GTPase activity on the intracellular localization of GluR1, respectively, without affecting the stability of microtubules. Together, these results indicate that Ndel1 regulates Dyn2 GTPase activity and impacts GluR1-containing membranes distribution in a manner reminiscent of Dyn2.

  14. Cytoskeletal Configuration Modulates Mechanically Induced Changes in Mesenchymal Stem Cell Osteogenesis, Morphology, and Stiffness

    Science.gov (United States)

    Pongkitwitoon, Suphannee; Uzer, Gunes; Rubin, Janet; Judex, Stefan

    2016-10-01

    Mesenchymal stem cells (MSC) responding to mechanical cues generated by physical activity is critical for skeletal development and remodeling. Here, we utilized low intensity vibrations (LIV) as a physiologically relevant mechanical signal and hypothesized that the confined cytoskeletal configuration imposed by 2D culture will enable human bone marrow MSCs (hBMSC) to respond more robustly when LIV is applied in-plane (horizontal-LIV) rather than out-of-plane (vertical-LIV). All LIV signals enhanced hBMSC proliferation, osteogenic differentiation, and upregulated genes associated with cytoskeletal structure. The cellular response was more pronounced at higher frequencies (100 Hz vs 30 Hz) and when applied in the horizontal plane. Horizontal but not vertical LIV realigned the cell cytoskeleton, culminating in increased cell stiffness. Our results show that applying very small oscillatory motions within the primary cell attachment plane, rather than perpendicular to it, amplifies the cell’s response to LIV, ostensibly facilitating a more effective transfer of intracellular forces. Transcriptional and structural changes in particular with horizontal LIV, together with the strong frequency dependency of the signal, emphasize the importance of intracellular cytoskeletal configuration in sensing and responding to high-frequency mechanical signals at low intensities.

  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. 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

  17. Human cytomegalovirus induces alteration of (-actin mRNA and microfilaments in human embryo fibroblast cells

    Institute of Scientific and Technical Information of China (English)

    林茂芳; 魏国庆; 黄河; 蔡真

    2004-01-01

    Objective: To investigate the infection of human embryo fibroblast cell line HF cells by CMV as well as the effects of CMV on β-actin mRNA and microfilaments. Methods: HF cells shape was observed after the infection of CMV. RT-PCR assay was used to detect the mRNA expression of CMV immediate early (IE) gene, β-actin and GAPDH genes of HF cells infected by CMV. CMV particles and cell microfilaments were detected with electron microscope. Results: Shape of HF cell changed after the infection by CMV. HF cells infected by CMV could express IE mRNA and the expression of β-actin mRNA decreased in a time- and titer-dependent manner compared with the uninfected HF cells whose expression of GAPDH mRNA did not change much. CMV particles were found with electron microscope in the cells. Microfilaments were ruptured and shortened after the infection of CMV. Conclusion: CMV can not only infect human embryo fibroblast cells line HF cells and replicate in the cells, but can also affect the expression of β-actin mRNA and the microfilaments.

  18. Modulation of cargo release from dense core granules by size and actin network.

    Science.gov (United States)

    Felmy, Felix

    2007-08-01

    During regulated fusion of secretory granules with the plasma membrane, a fusion pore first opens and then dilates. The dilating pore allows cargo proteins from the dense core to be released into the extracellular space. Using real-time evanescent field fluorescence microscopy of live PC12 cells, it was determined how rapidly proteins of different sizes escape from single granules after fusion. Tissue plasminogen activator (tPA)-Venus is released 40-fold slower than the three times smaller neuropeptide Y [NPY-monomeric GFP (mGFP)]. An NPY bearing two mGFPs in tandem [NPY-(mGFP)(2)] as an intermediate-sized fusion probe is released most slowly. Although, the time-course of release varies substantially for a given probe. Coexpression of beta-actin, actin-related protein 3 or mAbp1 slowed the release of the two larger cargo molecules but did not affect release of NPY-mGFP or of the granule-membrane-bound probe Vamp-pHluorin. Additionally, high concentrations of cytochalasin D slowed release of the tPA-Venus. Together these results suggest that fusion pore dilation is not the only determinate of release time-course and that actin rearrangements similar to those mediating actin-mediated motility influences the time-course of release without directly interfering with the granule membrane to cell membrane connection.

  19. 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.

  20. 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...

  1. WH2 domain: a small, versatile adapter for actin monomers.

    Science.gov (United States)

    Paunola, Eija; Mattila, Pieta K; Lappalainen, Pekka

    2002-02-20

    The actin cytoskeleton plays a central role in many cell biological processes. The structure and dynamics of the actin cytoskeleton are regulated by numerous actin-binding proteins that usually contain one of the few known actin-binding motifs. WH2 domain (WASP homology domain-2) is a approximately 35 residue actin monomer-binding motif, that is found in many different regulators of the actin cytoskeleton, including the beta-thymosins, ciboulot, WASP (Wiskott Aldrich syndrome protein), verprolin/WIP (WASP-interacting protein), Srv2/CAP (adenylyl cyclase-associated protein) and several uncharacterized proteins. The most highly conserved residues in the WH2 domain are important in beta-thymosin's interactions with actin monomers, suggesting that all WH2 domains may interact with actin monomers through similar interfaces. Our sequence database searches did not reveal any WH2 domain-containing proteins in plants. However, we found three classes of these proteins: WASP, Srv2/CAP and verprolin/WIP in yeast and animals. This suggests that the WH2 domain is an ancient actin monomer-binding motif that existed before the divergence of fungal and animal lineages.

  2. 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.

  3. Interaction of calponin with actin and its functional implications.

    Science.gov (United States)

    Kołakowski, J; Makuch, R; Stepkowski, D; Dabrowska, R

    1995-01-01

    Titration of F-actin with calponin causes the formation of two types of complexes. One, at saturation, contains a lower ratio of calponin to actin (0.5:1) and is insoluble at physiological ionic strength. The another is soluble, with a higher ratio of calponin to actin (1:1). Electron microscopy revealed that the former complex consists of paracrystalline bundles of actin filaments, whereas the latter consists of separate filaments. Ca(2+)-calmodulin causes dissociation of bundles with simultaneous increase in the number of separate calponin-containing filaments. Further increase in the calmodulin concentration results in full release of calponin from actin filaments. In motility assays, calponin, when added together with ATP to actin filaments complexed with immobilized myosin, evoked a decrease in both the number and velocity of moving actin filaments. Addition of calponin to actin filaments before their binding to myosin resulted in a formation of actin filament bundles which were dissociated by ATP. Images Figure 2 PMID:7864810

  4. 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.

  5. 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

  6. 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...... in conjunction with rotation enables the cell to explore a much larger 3-dimensional space and allows for more complex, and possibly stronger, interactions with the external environment.(2) Here we focus on how bending of the filopodial actin dynamically correlates with pulling on an optically trapped...

  7. Human CAP1 is a key factor in the recycling of cofilin and actin for rapid actin turnover.

    Science.gov (United States)

    Moriyama, Kenji; Yahara, Ichiro

    2002-04-15

    Cofilin-ADF (actin-depolymerizing factor) is an essential driver of actin-based motility. We discovered two proteins, p65 and p55, that are components of the actin-cofilin complex in a human HEK293 cell extract and identified p55 as CAP1/ASP56, a human homologue of yeast CAP/SRV2 (cyclase-associated protein). CAP is a bifunctional protein with an N-terminal domain that binds to Ras-responsive adenylyl cyclase and a C-terminal domain that inhibits actin polymerization. Surprisingly, we found that the N-terminal domain of CAP1, but not the C-terminal domain, is responsible for the interaction with the actin-cofilin complex. The N-terminal domain of CAP1 was also found to accelerate the depolymerization of F-actin at the pointed end, which was further enhanced in the presence of cofilin and/or the C-terminal domain of CAP1. Moreover, CAP1 and its C-terminal domain were observed to facilitate filament elongation at the barbed end and to stimulate ADP-ATP exchange on G-actin, a process that regenerates easily polymerizable G-actin. Although cofilin inhibited the nucleotide exchange on G-actin even in the presence of the C-terminal domain of CAP1, its N-terminal domain relieved this inhibition. Thus, CAP1 plays a key role in speeding up the turnover of actin filaments by effectively recycling cofilin and actin and through its effect on both ends of actin filament.

  8. F-actin-based extensions of the head cyst cell adhere to the maturing spermatids to maintain them in a tight bundle and prevent their premature release in Drosophila testis

    Directory of Open Access Journals (Sweden)

    Ray Krishanu

    2009-05-01

    Full Text Available Abstract Background In Drosophila, all the 64 clonally derived spermatocytes differentiate in syncytium inside two somatic-origin cyst cells. They elongate to form slender spermatids, which are individualized and then released into the seminal vesicle. During individualization, differentiating spermatids are organized in a tight bundle inside the cyst, which is expected to play an important role in sperm selection. However, actual significance of this process and its underlying mechanism are unclear. Results We show that dynamic F-actin-based processes extend from the head cyst cell at the start of individualization, filling the interstitial space at the rostral ends of the maturing spermatid bundle. In addition to actin, these structures contained lamin, beta-catenin, dynamin, myosin VI and several other filopodial components. Further, pharmacological and genetic analyses showed that cytoskeletal stability and dynamin function are essential for their maintenance. Disruption of these F-actin based processes was associated with spermatid bundle disassembly and premature sperm release inside the testis. Conclusion Altogether, our data suggests that the head cyst cell adheres to the maturing spermatid heads through F-actin-based extensions, thus maintaining them in a tight bundle. This is likely to regulate mature sperm release into the seminal vesicle. Overall, this process bears resemblance to mammalian spermiation.

  9. MRP-1/CD9 gene transduction regulates the actin cytoskeleton through the downregulation of WAVE2.

    Science.gov (United States)

    Huang, C-L; Ueno, M; Liu, D; Masuya, D; Nakano, J; Yokomise, H; Nakagawa, T; Miyake, M

    2006-10-19

    Motility-related protein-1 (MRP-1/CD9) is involved in cell motility. We studied the change in the actin cytoskeleton, and the expression of actin-related protein (Arp) 2 and Arp3 and the Wiskott-Aldrich syndrome protein (WASP) family according to MRP-1/CD9 gene transduction into HT1080 cells. The frequency of cells with lamellipodia was significantly lower in MRP-1/CD9-transfected HT1080 cells than in control HT1080 cells (PMRP-1/CD9 gene transduction affected the subcellular localization of Arp2 and Arp3 proteins. Furthermore, MRP-1/CD9 gene transduction induced a downregulation of WAVE2 expression (PMRP-1/CD9 monoclonal antibody inhibited downregulation of WAVE2 in MRP-1/CD9-transfected HT1080 cells (PMRP-1/CD9 gene transduction. Furthermore, downregulation of WAVE2 by transfection of WAVE2-specific small interfering RNA (siRNA) mimicked the morphological effects of MRP-1/CD9 gene transduction and suppressed cell motility. However, transfection of each siRNA for Wnt1, Wnt2b1 or Wnt5a did not affect WAVE2 expression. Transfection of WAVE2-specific siRNA also did not affect expressions of these Wnts. These results indicate that MRP-1/CD9 regulates the actin cytoskeleton by downregulating of the WAVE2, through the Wnt-independent signal pathway.

  10. Self-assembly of Artificial Actin Filaments

    Science.gov (United States)

    Grosenick, Christopher; Cheng, Shengfeng

    Actin Filaments are long, double-helical biopolymers that make up the cytoskeleton along with microtubules and intermediate filaments. In order to further understand the self-assembly process of these biopolymers, a model to recreate actin filament geometry was developed. A monomer in the shape of a bent rod with vertical and lateral binding sites was designed to assemble into single or double helices. With Molecular Dynamics simulations, a variety of phases were observed to form by varying the strength of the binding sites. Ignoring lateral binding sites, we have found a narrow range of binding strengths that lead to long single helices via various growth pathways. When lateral binding strength is introduced, double helices begin to form. These double helices self-assemble into substantially more stable structures than their single helix counterparts. We have found double helices to form long filaments at about half the vertical binding strength of single helices. Surprisingly, we have found that triple helices occasionally form, indicating the importance of structural regulation in the self-assembly of biopolymers.

  11. 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.

  12. ICAM-2 expression mediates a membrane-actin link, confers a nonmetastatic phenotype and reflects favorable tumor stage or histology in neuroblastoma.

    Directory of Open Access Journals (Sweden)

    Karina Jin Yoon

    Full Text Available The actin cytoskeleton is a primary determinant of tumor cell motility and metastatic potential. Motility and metastasis are thought to be regulated, in large part, by the interaction of membrane proteins with cytoplasmic linker proteins and of these linker proteins, in turn, with actin. However, complete membrane-to-actin linkages have been difficult to identify. We used co-immunoprecipitation and competitive peptide assays to show that intercellular adhesion molecule-2 (ICAM-2/alpha-actinin/actin may comprise such a linkage in neuroblastoma cells. ICAM-2 expression limited the motility of these cells and redistributed actin fibers in vitro, and suppressed development of disseminated tumors in an in vivo model of metastatic neuroblastoma. Consistent with these observations, immunohistochemical analysis demonstrated ICAM-2 expression in primary neuroblastoma tumors exhibiting features that are associated with limited metastatic disease and more favorable clinical outcome. In neuroblastoma cell lines, ICAM-2 expression did not affect AKT activation, tumorigenic potential or chemosensitivity, as has been reported for some types of transfected cells. The observed ICAM-2-mediated suppression of metastatic phenotype is a novel function for this protein, and the interaction of ICAM-2/alpha-actinin/actin represents the first complete membrane-linker protein-actin linkage to impact tumor cell motility in vitro and metastatic potential in an in vivo model. Current work focuses on identifying specific protein domains critical to the regulation of neuroblastoma cell motility and metastasis and on determining if these domains represent exploitable therapeutic targets.

  13. The influence of the spatial resolution of topographic input data on the accuracy of 3-D UV actinic flux and irradiance calculations

    Directory of Open Access Journals (Sweden)

    P. Weihs

    2011-10-01

    Full Text Available The aim of this study was to investigate the influence of the spatial resolution of a digital elevation map (DEM on the three-dimensional (3-D radiative transfer performance for both spectral ultraviolet (UV irradiance and actinic flux at 305 nm. Model simulations were performed for clear sky conditions for three case studies: the first and second one using three sites in the Innsbruck area and the third one using three sites at the Sonnblick Observatory and surrounding area. It was found that DEM resolution may change the altitude at some locations by up to 500 m, resulting in changes in the sky obscured by the horizon of up to 15%. The geographical distribution of UV irradiance and actinic flux shows that with larger pixel size, uncertainties in UV irradiance and actinic flux determination of up to 100% are possible. These large changes in incident irradiance and actinic flux with changing pixel size are strongly connected to shading effects. The effect of DEM pixel size on irradiance and actinic flux was studied at the six locations, and it was found that significant increases in irradiance and actinic flux with increasing DEM pixel size occurred at one valley location at high solar zenith angles in the Innsbruck area as well as for one steep valley location in the Sonnblick area. This increase in irradiance and actinic flux with increasing DEM resolution is most likely to be connected to shading effects affecting the reflections from the surroundings.

  14. 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...

  15. 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.

  16. 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.

  17. 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.

  18. Interaction of actin and the chloroplast protein import apparatus.

    Science.gov (United States)

    Jouhet, Juliette; Gray, John C

    2009-07-10

    Actin filaments are major components of the cytoskeleton and play numerous essential roles, including chloroplast positioning and plastid stromule movement, in plant cells. Actin is present in pea chloroplast envelope membrane preparations and is localized at the surface of the chloroplasts, as shown by agglutination of intact isolated chloroplasts by antibodies to actin. To identify chloroplast envelope proteins involved in actin binding, we have carried out actin co-immunoprecipitation and co-sedimentation experiments on detergent-solubilized pea chloroplast envelope membranes. Proteins co-immunoprecipitated with actin were identified by mass spectrometry and by Western blotting and included the Toc159, Toc75, Toc34, and Tic110 components of the TOC-TIC protein import apparatus. A direct interaction of actin with Escherichia coli-expressed Toc159, but not Toc33, was shown by co-sedimentation experiments, suggesting that Toc159 is the component of the TOC complex that interacts with actin on the cytosolic side of the outer envelope membrane. The physiological significance of this interaction is unknown, but it may play a role in the import of nuclear-encoded photosynthesis proteins.

  19. Yeast studies reveal moonlighting functions of the ancient actin cytoskeleton

    Science.gov (United States)

    Sattlegger, Evelyn; Chernova, Tatiana A.; Gogoi, Neeku M.; Pillai, Indu V.; Chernoff, Yury O.; Munn, Alan L.

    2014-01-01

    Classic functions of the actin cytoskeleton include control of cell size and shape and the internal organisation of cells. These functions are manifest in cellular processes of fundamental importance throughout biology such as the generation of cell polarity, cell migration, cell adhesion and cell division. However, studies in the unicellular model eukaryote Saccharomyces cerevisiae (Baker's yeast) are giving insights into other functions in which the actin cytoskeleton plays a critical role. These include endocytosis, control of protein translation and determination of protein 3-dimensional shape (especially conversion of normal cellular proteins into prions). Here we present a concise overview of these new "moonlighting" roles for the actin cytoskeleton and how some of these roles might lie at the heart of important molecular switches. This is an exciting time for researchers interested in the actin cytoskeleton. We show here how studies of actin are leading us into many new and exciting realms at the interface of genetics, biochemistry and cell biology. While many of the pioneering studies have been conducted using yeast, the conservation of the actin cytoskeleton and its component proteins throughout eukaryotes suggests that these new roles for the actin cytoskeleton may not be restricted to yeast cells but rather may reflect new roles for the actin cytoskeleton of all eukaryotes. PMID:25138357

  20. 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

  1. 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.

  2. Photodynamic therapy: treatment of choice for actinic cheilitis?

    Science.gov (United States)

    Rossi, R; Assad, G Bani; Buggiani, G; Lotti, T

    2008-01-01

    The major therapeutic approaches (5-fluorouracil, imiquimod, vermilionectomy, and CO(2) Laser ablation) for actinic cheilitis are aimed at avoiding and preventing a malignant transformation into invasive squamous cell carcinoma via destruction/removal of the damaged epithelium. Recently, photodynamic therapy (PDT) has been introduced as a therapeutic modality for epithelial skin tumors, with good efficacy/safety profile and good cosmetic results. Regarding actinic cheilitis, PDT could be considered a new therapeutic option? The target of our study was to evaluate the efficacy and tolerability of PDT in actinic cheilitis, using a methyl-ester of aminolevulinic acid (MAL) as topical photosensitizing agent and controlled the effects of the therapy for a 30-month follow-up period. MAL-PDT seems to be the ideal treatment for actinic cheilitis and other actinic keratosis, especially on exposed parts such as the face, joining tolerability and clinical efficacy with an excellent cosmetic outcome.

  3. Dynamics of actin evolution in dinoflagellates.

    Science.gov (United States)

    Kim, Sunju; Bachvaroff, Tsvetan R; Handy, Sara M; Delwiche, Charles F

    2011-04-01

    Dinoflagellates have unique nuclei and intriguing genome characteristics with very high DNA content making complete genome sequencing difficult. In dinoflagellates, many genes are found in multicopy gene families, but the processes involved in the establishment and maintenance of these gene families are poorly understood. Understanding the dynamics of gene family evolution in dinoflagellates requires comparisons at different evolutionary scales. Studies of closely related species provide fine-scale information relative to species divergence, whereas comparisons of more distantly related species provides broad context. We selected the actin gene family as a highly expressed conserved gene previously studied in dinoflagellates. Of the 142 sequences determined in this study, 103 were from the two closely related species, Dinophysis acuminata and D. caudata, including full length and partial cDNA sequences as well as partial genomic amplicons. For these two Dinophysis species, at least three types of sequences could be identified. Most copies (79%) were relatively similar and in nucleotide trees, the sequences formed two bushy clades corresponding to the two species. In comparisons within species, only eight to ten nucleotide differences were found between these copies. The two remaining types formed clades containing sequences from both species. One type included the most similar sequences in between-species comparisons with as few as 12 nucleotide differences between species. The second type included the most divergent sequences in comparisons between and within species with up to 93 nucleotide differences between sequences. In all the sequences, most variation occurred in synonymous sites or the 5' UnTranslated Region (UTR), although there was still limited amino acid variation between most sequences. Several potential pseudogenes were found (approximately 10% of all sequences depending on species) with incomplete open reading frames due to frameshifts or early stop

  4. A pseudo-spin model for the wall of cytoskeletal microtubule

    Institute of Scientific and Technical Information of China (English)

    Chen Ying; Qiu Xi-Jun; Li Ru-Xin

    2005-01-01

    A pseudo-spin model is intended to describe the physical dynamics of unbound electrons in the wall of cytoskeletal microtubule (MT). Due to the inherent symmetry of the structure and the electric properties in the MT, one may treat it as a one-dimensional ferroelectric system, and describe the nonlinear dynamics of dimer electric dipoles in one protofilament of the MT by virtue of the double-well potential. Consequently, the physical problem has been mapped onto the pseudo-spin system, and the mean-field approximation has been taken to get some physical results.