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Sample records for actin cytoskeletal structure

  1. Structure, chromosome location, and expression of the human. gamma. -actin gene: Differential evolution, location, and expression of the cytoskeletal BETA- and. gamma. -actin genes

    Erba, H.P.; Eddy, R.; Shows, T.; Kedes, L.; Gunning, P.

    1988-04-01

    The accumulation of the cytoskeletal ..beta..-and ..gamma..-actin mRNAs was determined in a variety of mouse tissues and organs. The ..beta..-iosform is always expressed in excess of the ..gamma..-isoform. However, the molar ratio of ..beta..- to ..gamma..-actin mRNA varies from 1.7 in kidney and testis to 12 in sarcomeric muscle to 114 in liver. The authors conclude that, whereas the cytoskeletal ..beta..- and ..gamma..-actins are truly coexpressed, their mRNA levels are subject to differential regulation between different cell types. The human ..gamma..-actin gene has been cloned and sequenced, and its chromosome location has been determined. The gene is located on human chromosome 17, unlike ..beta..-actin which is on chromosome 7. Thus, if these genes are also unlinked in the mouse, the coexpression of the ..beta..- and ..gamma..-actin genes in rodent tissues cannot be determined by gene linkage. Comparison of the human ..beta..- and ..gamma..-actin genes reveals that noncoding sequences in the 5'-flanking region and in intron III have been conserved since the duplication that gave rise to these two genes. In contrast, there are sequences in intron III and the 3'-untranslated region which are not present in the ..beta..-actin gene but are conserved between the human ..gamma..-actin and the Xenopus borealis type 1 actin genes. Such conserved noncoding sequences may contribute to the coexpression of ..beta..- and ..gamma..-actin or to the unique regulation and function of the ..gamma..-actin gene. Finally, the authors demonstrate that the human ..gamma..-actin gene is expressed after introduction into mouse L cells and C2 myoblasts and that, upon fusion of C2 cells to form myotubes, the human ..gamma..-actin gene is appropriately regulated.

  2. Actin dynamics and the elasticity of cytoskeletal networks

    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.

  3. [Cytoskeletal actin and its associated proteins. Some examples in Protista].

    Guillén, N; Carlier, M F; Brugerolle, G; Tardieux, I; Ausseil, J

    1998-06-01

    Many processes, cell motility being an example, require cells to remodel the actin cytoskeleton in response to both intracellular and extracellular signals. Reorganization of the actin cytoskeleton involves the rapid disassembly and reassembly of actin filaments, a phenomenon regulated by the action of particular actin-binding proteins. In recent years, an interest in studying actin regulation in unicellular organisms has arisen. Parasitic protozoan are among these organisms and studies of the cytoskeleton functions of these protozoan are relevant related to either cell biology or pathogenicity. To discuss recent data in this field, a symposium concerning "Actin and actin-binding proteins in protists" was held on May 8-11 in Paris, France, during the XXXV meeting of the French Society of Protistology. As a brief summary of the symposium we report here findings concerning the in vitro actin dynamic assembly, as well as the characterization of several actin-binding proteins from the parasitic protozoan Entamoeba histolytica, Trichomonas vaginalis and Plasmodium knowlesi. In addition, localization of actin in non-pathogen protists such as Prorocentrum micans and Crypthecodinium cohnii is also presented. The data show that some actin-binding proteins facilitate organization of filaments into higher order structures as pseudopods, while others have regulatory functions, indicating very particular roles for actin-binding proteins. One of the proteins discussed during the symposium, the actin depolymerizing factor ADF, was shown to enhance the treadmilling rate of actin filaments. In vitro, ADF binds to the ADP-bound forms of G-actin and F-actin, thereby participating in and changing the rate of actin assembly. Biochemical approaches allowed the identification of a protein complex formed by HSP/C70-cap32-34 which might also be involved in depolymerization of F-actin in P. knowlesi. Molecular and cellular approaches were used to identify proteins such as ABP-120 and myosin

  4. 2',3'-Cyclic nucleotide 3'-phosphodiesterase binds to actin-based cytoskeletal elements in an isoprenylation-independent manner.

    De Angelis, D A; Braun, P E

    1996-09-01

    2',3'-Cyclic nucleotide 3'-phosphodiesterase (CNP) is an isoprenylated protein enriched in myelin and oligodendrocytes but also present in several other tissues at low levels. CNP binds avidly to membranes and in addition possesses several characteristics of cytoskeletal proteins. The role of isoprenylation in the association of CNP with the cytoskeleton was analyzed by ectopic expression in L cells of epitope-tagged CNP1 and a non-isoprenylated mutant CNP1. Using nonionic detergent extraction, drug-mediated cytoskeletal disruption, and coimmunoprecipitation with an anti-actin antibody, we show that CNP1 is associated with actin-based cytoskeletal elements independently of its isoprenylation status. A control protein, p21c-H-ras, which is also modified by isoprenylation at its carboxyl-terminus, does not bind to cytoskeletal structures as judged by the same criteria. We present a model that accounts for the association of CNP1 with membranes and the cytoskeleton. PMID:8752099

  5. Cadmium-induced glutathionylation of actin occurs through a ROS-independent mechanism: Implications for cytoskeletal integrity

    Cadmium disrupts the actin cytoskeleton in rat mesangial cells, and we have previously shown that this involves a complex interplay involving activation of kinase signaling, protein translocation, and disruption of focal adhesions. Here we investigate the role that glutathionylation of actin plays in Cd2+-associated cytoskeletal reorganization. Low concentrations of Cd2+ (0.5–2 μM) caused an increase in actin glutathionylation by 6 h, whereas at higher concentrations glutathionylation remained at basal levels. Although oxidation with diamide increased glutathionylation, reactive oxygen species (ROS) were not involved in the Cd2+-dependent effect, as only Cd2+ concentrations above 2 μM were sufficient to increase ROS. However, low [Cd2+] increased total glutathione levels without affecting the ratio of reduced/oxidized glutathione, and inhibition of glutathione synthesis suppressed actin glutathionylation. Cadmium increased the activity of the enzyme glutaredoxin, which influences the equilibrium between glutathionylated and deglutathionylated proteins and thus may influence levels of glutathionylated actin. Together these observations show that cadmium-dependent effects on actin glutathionylation are affected by glutathione metabolism and not by direct effects of ROS on thiol chemistry. In vitro polymerization assays with glutathionylated actin show a decreased rate of polymerization. In contrast, immunofluorescence of cytoskeletal structure in intact cells suggests that increases in actin glutathionylation accompanying increased glutathione levels occurring under low Cd2+ exposure are protective in vivo, with cytoskeletal disruption ensuing only when higher Cd2+ concentrations increase ROS levels and prevent an increase in actin–glutathione conjugates. - Highlights: • Cadmium disrupts the actin cytoskeleton in mesangial cells. • Cadmium induces glutathionylation of actin at low concentrations. • Glutathionylation requires glutathione synthesis but is

  6. Cadmium-induced glutathionylation of actin occurs through a ROS-independent mechanism: Implications for cytoskeletal integrity

    Choong, Grace; Liu, Ying; Xiao, Weiqun; Templeton, Douglas M., E-mail: doug.templeton@utoronto.ca

    2013-10-15

    Cadmium disrupts the actin cytoskeleton in rat mesangial cells, and we have previously shown that this involves a complex interplay involving activation of kinase signaling, protein translocation, and disruption of focal adhesions. Here we investigate the role that glutathionylation of actin plays in Cd{sup 2+}-associated cytoskeletal reorganization. Low concentrations of Cd{sup 2+} (0.5–2 μM) caused an increase in actin glutathionylation by 6 h, whereas at higher concentrations glutathionylation remained at basal levels. Although oxidation with diamide increased glutathionylation, reactive oxygen species (ROS) were not involved in the Cd{sup 2+}-dependent effect, as only Cd{sup 2+} concentrations above 2 μM were sufficient to increase ROS. However, low [Cd{sup 2+}] increased total glutathione levels without affecting the ratio of reduced/oxidized glutathione, and inhibition of glutathione synthesis suppressed actin glutathionylation. Cadmium increased the activity of the enzyme glutaredoxin, which influences the equilibrium between glutathionylated and deglutathionylated proteins and thus may influence levels of glutathionylated actin. Together these observations show that cadmium-dependent effects on actin glutathionylation are affected by glutathione metabolism and not by direct effects of ROS on thiol chemistry. In vitro polymerization assays with glutathionylated actin show a decreased rate of polymerization. In contrast, immunofluorescence of cytoskeletal structure in intact cells suggests that increases in actin glutathionylation accompanying increased glutathione levels occurring under low Cd{sup 2+} exposure are protective in vivo, with cytoskeletal disruption ensuing only when higher Cd{sup 2+} concentrations increase ROS levels and prevent an increase in actin–glutathione conjugates. - Highlights: • Cadmium disrupts the actin cytoskeleton in mesangial cells. • Cadmium induces glutathionylation of actin at low concentrations.

  7. Dithiocarbamate propineb induces acetylcholine release through cytoskeletal actin depolymerization in PC12 cells.

    Viviani, Barbara; Bartesaghi, Stefano; Binaglia, Marco; Corsini, Emanuela; Boraso, Mariaserena; Grazi, Enrico; Galli, Corrado L; Marinovich, Marina

    2008-11-10

    Neurological complications as well as movement disorders are relevant symptoms in animals and humans chronically exposed to dithiocarbamates. Using rat pheochromocytoma cells differentiated by NGF (PC12), we investigated whether propineb affects acetylcholine (Ach) release and the molecular mechanisms involved. Propineb (0.001-100 nM) dose-dependently increased Ach release from PC12. Thus, 0.001-1 nM propineb-induced Ach release, reaching a maximal effect ( approximately 50%) at 0.1-1 nM. Higher concentrations of propineb (10-100 nM) caused a progressive disappearance of the effect. Chelation of extra- and intracellular Ca(2+) did not affect Ach release by propineb, which was prevented by the actin stabilizer jasplakinolide (500 nM). Accordingly, actin depolymerization was observed after exposure of differentiated PC12 to 0.1-1 nM propineb, a loss of effect was evident at higher concentrations (100 nM), and the effect was Ca(2+)-independent. Disulfiram, a related dithiocarbamate not coordinated with Zn(2+), also depolymerized actin, suggesting the involvement of the organic structure of dithiocarbamates rather than the leakage of Zn(2+). Nevertheless, propineb did not depolymerize actin in a cell-free system. These data suggest that dithiocarbamates, through the activation of intracellular cascade(s), impair cytoskeletal actin. This effect may contribute to affect synaptic vesicles processing resulting in an impaired cholinergic transmission. PMID:18822360

  8. A POROELASTIC MODEL FOR CELL CRAWLING INCLUDING MECHANICAL COUPLING BETWEEN CYTOSKELETAL CONTRACTION AND ACTIN POLYMERIZATION.

    Taber, L A; Shi, Y; Yang, L; Bayly, P V

    2011-01-01

    Much is known about the biophysical mechanisms involved in cell crawling, but how these processes are coordinated to produce directed motion is not well understood. Here, we propose a new hypothesis whereby local cytoskeletal contraction generates fluid flow through the lamellipodium, with the pressure at the front of the cell facilitating actin polymerization which pushes the leading edge forward. The contraction, in turn, is regulated by stress in the cytoskeleton. To test this hypothesis, finite element models for a crawling cell are presented. These models are based on nonlinear poroelasticity theory, modified to include the effects of active contraction and growth, which are regulated by mechanical feedback laws. Results from the models agree reasonably well with published experimental data for cell speed, actin flow, and cytoskeletal deformation in migrating fish epidermal keratocytes. The models also suggest that oscillations can occur for certain ranges of parameter values. PMID:21765817

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

    Katie Porter

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

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

    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. Xenopus cytoskeletal actin and human c-fos gene promoters share a conserved protein-binding site.

    Mohun, T; Garrett, N; Treisman, R

    1987-03-01

    Xenopus laevis cytoskeletal actin gene promoters contain a 20-bp sequence homologous to the serum response element (SRE) required for transient human c-fos gene transcription in response to serum factors. Both sequences bind the same factor in HeLa cell extracts, as shown by binding competition, DNase I and dimethylsulphate (DMS) protection and DMS interference assays. A similar protein is present in Xenopus laevis oocytes. Sequences containing the SRE homology are essential for constitutive activity of the actin promoter in both Xenopus and mouse cells, and a synthetic SRE functions as a promoter element in these cells. In mouse cells, transcription of both transfected Xenopus actin and actin/c-fos fusion genes is activated following serum stimulation. These data suggest that the SRE and its cognate protein form part of a regulatory pathway that has been highly conserved during evolution. PMID:3582369

  12. The Neurofibromatosis Type 2 Gene Product, merlin, Reverses the F-Actin Cytoskeletal Defects in Primary Human Schwannoma Cells

    Bashour, Anne-Marie; Meng, J.-J.; Ip, Wallace; MacCollin, Mia; Ratner, Nancy

    2002-01-01

    Schwannoma tumors, which occur sporadically and in patients with neurofibromatosis, account for 8% of intracranial tumors and can only be treated by surgical removal. Most schwannomas have biallelic mutations in the NF2 tumor suppressor gene. We previously showed that schwannoma-derived Schwann cells exhibit membrane ruffling and aberrant cell spreading when plated onto laminin, indicative of fundamental F-actin cytoskeletal defects. Here we expand these observations to a large group of spora...

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

    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.

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

    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. Altered cytoskeletal structures in transformed cells exhibiting obviously metastatic capabilities

    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.

  16. Arginine ADP-ribosylation mechanism based on structural snapshots of iota-toxin and actin complex

    Tsurumura, Toshiharu; Tsumori, Yayoi; Qiu, Hao; Oda, Masataka; Sakurai, Jun; Nagahama, Masahiro; Tsuge, Hideaki

    2012-01-01

    Clostridium perfringens iota-toxin (Ia) mono-ADP ribosylates Arg177 of actin, leading to cytoskeletal disorganization and cell death. To fully understand the reaction mechanism of arginine-specific mono-ADP ribosyl transferase, the structure of the toxin-substrate protein complex must be characterized. Recently, we solved the crystal structure of Ia in complex with actin and the nonhydrolyzable NAD+ analog βTAD (thiazole-4-carboxamide adenine dinucleotide); however, the structures of the NAD+...

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

    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. PMID:21825142

  18. The scaffolding protein IQGAP1 co-localizes with actin at the cytoplasmic face of the nuclear envelope: implications for cytoskeletal regulation

    Johnson, Michael A.; Henderson, Beric R.

    2012-01-01

    IQGAP1 is an important cytoskeletal regulator, known to act at the plasma membrane to bundle and cap actin filaments, and to tether the cortical actin meshwork to microtubules via plus-end binding proteins. Here we describe the novel subcellular localization of IQGAP1 at the cytoplasmic face of the nuclear envelope, where it co-located with F-actin. The IQGAP1 and F-actin staining overlapped that of microtubules at the nuclear envelope, revealing a pattern strikingly similar to that observed ...

  19. A multi-structural single cell model of force-induced interactions of cytoskeletal components.

    Barreto, Sara; Clausen, Casper H; Perrault, Cecile M; Fletcher, Daniel A; Lacroix, Damien

    2013-08-01

    Several computational models based on experimental techniques and theories have been proposed to describe cytoskeleton (CSK) mechanics. Tensegrity is a prominent model for force generation, but it cannot predict mechanics of individual CSK components, nor explain the discrepancies from the different single cell stimulating techniques studies combined with cytoskeleton-disruptors. A new numerical concept that defines a multi-structural 3D finite element (FE) model of a single-adherent cell is proposed to investigate the biophysical and biochemical differences of the mechanical role of each cytoskeleton component under loading. The model includes prestressed actin bundles and microtubule within cytoplasm and nucleus surrounded by the actin cortex. We performed numerical simulations of atomic force microscopy (AFM) experiments by subjecting the cell model to compressive loads. The numerical role of the CSK components was corroborated with AFM force measurements on U2OS-osteosarcoma cells and NIH-3T3 fibroblasts exposed to different cytoskeleton-disrupting drugs. Computational simulation showed that actin cortex and microtubules are the major components targeted in resisting compression. This is a new numerical tool that explains the specific role of the cortex and overcomes the difficulty of isolating this component from other networks in vitro. This illustrates that a combination of cytoskeletal structures with their own properties is necessary for a complete description of cellular mechanics. PMID:23702149

  20. Mapping the cytoskeletal prestress

    Park, Chan Young; Tambe, Dhananjay; Alencar, Adriano M.; Trepat, Xavier; Zhou, En Hua; Millet, Emil; Butler, James P.; Fredberg, Jeffrey J.

    2010-01-01

    Cell mechanical properties on a whole cell basis have been widely studied, whereas local intracellular variations have been less well characterized and are poorly understood. To fill this gap, here we provide detailed intracellular maps of regional cytoskeleton (CSK) stiffness, loss tangent, and rate of structural rearrangements, as well as their relationships to the underlying regional F-actin density and the local cytoskeletal prestress. In the human airway smooth muscle cell, we used micro...

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

    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.

  2. Reorganization of the actin cytoskeleton via transcriptional regulation of cytoskeletal/focal adhesion genes by myocardin-related transcription factors (MRTFs/MAL/MKLs)

    RhoA is a crucial regulator of stress fiber and focal adhesion formation through the activation of actin nucleation and polymerization. It also regulates the nuclear translocation of myocardin-related transcription factor-A and -B (MRTF-A/B, MAL or MKL 1/2), which are co-activators of serum response factor (SRF). In dominant-negative MRTF-A (DN-MRTF-A)-expressing NIH 3T3 cell lines, the expressions of several cytoskeletal/focal adhesion genes were down-regulated, and the formation of stress fiber and focal adhesion was severely diminished. MRTF-A/B-knockdown cells also exhibited such cytoskeletal defects. In reporter assays, both RhoA and MRTF-A enhanced promoter activities of these genes in a CArG-box-dependent manner, and DN-MRTF-A inhibited the RhoA-mediated activation of these promoters. In dominant-negative RhoA (RhoA-N19)-expressing NIH 3T3 cell lines, the nuclear translocation of MRTF-A/B was predominantly prevented, resulting in the reduced expression of cytoskeletal/focal adhesion proteins. Further, constitutive-active MRTF-A/B increased the expression of endogenous cytoskeletal/focal adhesion proteins, and thereby rescued the defective phenotype of stress fibers and focal adhesions in RhoA-N19 expressing cells. These results indicate that MRTF-A/B act as pivotal mediators of stress fiber and focal adhesion formation via the transcriptional regulation of a subset of cytoskeletal/focal adhesion genes

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

    Xiaorui Chen

    2013-06-01

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

  4. Cytoskeletal Mechanics

    Mofrad, Mohammad R. K.; Kamm, Roger D.

    2011-08-01

    1. Introduction and the biological basis for cell mechanics Mohammad R. K. Mofrad and Roger Kamm; 2. Experimental measurements of intracellular mechanics Paul Janmey and Christoph Schmidt; 3. The cytoskeleton as a soft glassy material Jeffrey Fredberg and Ben Fabry; 4. Continuum elastic or viscoelastic models for the cell Mohammad R. K. Mofrad, Helene Karcher and Roger Kamm; 5. Multiphasic models of cell mechanics Farshid Guuilak, Mansoor A. Haider, Lori A. Setton, Tod A. Laursen and Frank P. T. Baaijens; 6. Models of cytoskeletal mechanics based on tensegrity Dimitrije Stamenovic; 7. Cells, gels and mechanics Gerald H. Pollack; 8. Polymer-based models of cytoskeletal networks F. C. MacKintosh; 9. Cell dynamics and the actin cytoskeleton James L. McGrath and C. Forbes Dewey, Jr; 10. Active cellular motion: continuum theories and models Marc Herant and Micah Dembo; 11. Summary Mohammad R. K. Mofrad and Roger Kamm.

  5. Artificial cytoskeletal structures within enzymatically active bio-inorganic protocells.

    Kumar, Ravinash Krishna; Li, Mei; Olof, Sam N; Patil, Avinash J; Mann, Stephen

    2013-02-11

    The fabrication of enzymatically active, semi-permeable bio-inorganic protocells capable of self-assembling a cytoskeletal-like interior and undergoing small-molecule dephosphorylation reactions is described. Reversible disassembly of an amino acid-derived supramolecular hydrogel within the internalized reaction space is used to tune the enzymatic activity of the nanoparticle-bounded inorganic compartments. PMID:23027575

  6. Osteoclasts and monocytes have similar cytoskeletal structures and adhesion property in vitro.

    Zallone, A Z; Teti, A; Primavera, M V; Naldini, L; Marchisio, P. C.

    1983-01-01

    The distribution of some cytoskeletal structures (microtubules, microfilaments, intermediate filaments) has been studied by indirect immunofluorescence microscopy and affinity purified antibodies in osteoclasts isolated from medullary bone of laying hens and in hen blood monocytes cultured in vitro. Both cell types show similar patterns of distribution of cytoskeletal structures and this further supports the concept that these cells are closely related. Osteoclasts and monocytes are also simi...

  7. Structure of the F-actin-tropomyosin complex.

    von der Ecken, Julian; Müller, Mirco; Lehman, William; Manstein, Dietmar J; Penczek, Pawel A; Raunser, Stefan

    2015-03-01

    Filamentous actin (F-actin) is the major protein of muscle thin filaments, and actin microfilaments are the main component of the eukaryotic cytoskeleton. Mutations in different actin isoforms lead to early-onset autosomal dominant non-syndromic hearing loss, familial thoracic aortic aneurysms and dissections, and multiple variations of myopathies. In striated muscle fibres, the binding of myosin motors to actin filaments is mainly regulated by tropomyosin and troponin. Tropomyosin also binds to F-actin in smooth muscle and in non-muscle cells and stabilizes and regulates the filaments there in the absence of troponin. Although crystal structures for monomeric actin (G-actin) are available, a high-resolution structure of F-actin is still missing, hampering our understanding of how disease-causing mutations affect the function of thin muscle filaments and microfilaments. Here we report the three-dimensional structure of F-actin at a resolution of 3.7 Å in complex with tropomyosin at a resolution of 6.5 Å, determined by electron cryomicroscopy. The structure reveals that the D-loop is ordered and acts as a central region for hydrophobic and electrostatic interactions that stabilize the F-actin filament. We clearly identify map density corresponding to ADP and Mg(2+) and explain the possible effect of prominent disease-causing mutants. A comparison of F-actin with G-actin reveals the conformational changes during filament formation and identifies the D-loop as their key mediator. We also confirm that negatively charged tropomyosin interacts with a positively charged groove on F-actin. Comparison of the position of tropomyosin in F-actin-tropomyosin with its position in our previously determined F-actin-tropomyosin-myosin structure reveals a myosin-induced transition of tropomyosin. Our results allow us to understand the role of individual mutations in the genesis of actin- and tropomyosin-related diseases and will serve as a strong foundation for the targeted

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

    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.

  9. Bacterial Subversion of Host Actin Dynamics at the Plasma Membrane

    Carabeo, Rey

    2011-01-01

    Invasion of non-phagocytic cells by a number of bacterial pathogens involves the subversion of the actin cytoskeletal remodelling machinery to produce actin-rich cell surface projections designed to engulf the bacteria. The signalling that occurs to induce these actin-rich structures has considerable overlap amongst a diverse group of bacteria. The molecular organisation within these structures act in concert to internalise the invading pathogen. This dynamic process could be subdivided into ...

  10. Diffusion in Cytoplasm: Effects of Excluded Volume Due to Internal Membranes and Cytoskeletal Structures

    Novak, Igor L.; Kraikivski, Pavel; Slepchenko, Boris M.

    2009-01-01

    The intricate geometry of cytoskeletal networks and internal membranes causes the space available for diffusion in cytoplasm to be convoluted, thereby affecting macromolecule diffusivity. We present a first systematic computational study of this effect by approximating intracellular structures as mixtures of random overlapping obstacles of various shapes. Effective diffusion coefficients are computed using a fast homogenization technique. It is found that a simple two-parameter power law prov...

  11. Addition of electrophilic lipids to actin alters filament structure

    Pathophysiological processes associated with oxidative stress lead to the generation of reactive lipid species. Among them, lipids bearing unsaturated aldehyde or ketone moieties can form covalent adducts with cysteine residues and modulate protein function. Through proteomic techniques we have identified actin as a target for the addition of biotinylated analogs of the cyclopentenone prostaglandins 15-deoxy-Δ12,14-PGJ2 (15d-PGJ2) and PGA1 in NIH-3T3 fibroblasts. This modification could take place in vitro and mapped to the protein C-terminal end. Other electrophilic lipids, like the isoprostane 8-iso-PGA1 and 4-hydroxy-2-nonenal, also bound to actin. The C-terminal region of actin is important for monomer-monomer interactions and polymerization. Electron microscopy showed that actin treated with 15d-PGJ2 or 4-hydroxy-2-nonenal formed filaments which were less abundant and displayed shorter length and altered structure. Streptavidin-gold staining allowed mapping of biotinylated 15d-PGJ2 at sites of filament disruption. These results shed light on the structural implications of actin modification by lipid electrophiles

  12. Novel actin-like filament structure from Clostridium tetani.

    Popp, David; Narita, Akihiro; Lee, Lin Jie; Ghoshdastider, Umesh; Xue, Bo; Srinivasan, Ramanujam; Balasubramanian, Mohan K; Tanaka, Toshitsugu; Robinson, Robert C

    2012-06-15

    Eukaryotic F-actin is constructed from two protofilaments that gently wind around each other to form a helical polymer. Several bacterial actin-like proteins (Alps) are also known to form F-actin-like helical arrangements from two protofilaments, yet with varied helical geometries. Here, we report a unique filament architecture of Alp12 from Clostridium tetani that is constructed from four protofilaments. Through fitting of an Alp12 monomer homology model into the electron microscopy data, the filament was determined to be constructed from two antiparallel strands, each composed of two parallel protofilaments. These four protofilaments form an open helical cylinder separated by a wide cleft. The molecular interactions within single protofilaments are similar to F-actin, yet interactions between protofilaments differ from those in F-actin. The filament structure and assembly and disassembly kinetics suggest Alp12 to be a dynamically unstable force-generating motor involved in segregating the pE88 plasmid, which encodes the lethal tetanus toxin, and thus a potential target for drug design. Alp12 can be repeatedly cycled between states of polymerization and dissociation, making it a novel candidate for incorporation into fuel-propelled nanobiopolymer machines. PMID:22514279

  13. Mechanosensitive kinetic preference of actin-binding protein to actin filament

    Inoue, Yasuhiro; Adachi, Taiji

    2016-04-01

    The kinetic preference of actin-binding proteins to actin filaments is altered by external forces on the filament. Such an altered kinetic preference is largely responsible for remodeling the actin cytoskeletal structure in response to intracellular forces. During remodeling, actin-binding proteins and actin filaments interact under isothermal conditions, because the cells are homeostatic. In such a temperature homeostatic state, we can rigorously and thermodynamically link the chemical potential of actin-binding proteins to stresses on the actin filaments. From this relationship, we can construct a physical model that explains the force-dependent kinetic preference of actin-binding proteins to actin filaments. To confirm the model, we have analyzed the mechanosensitive alternation of the kinetic preference of Arp2/3 and cofilin to actin filaments. We show that this model captures the qualitative responses of these actin-binding proteins to the forces, as observed experimentally. Moreover, our theoretical results demonstrate that, depending on the structural parameters of the binding region, actin-binding proteins can show different kinetic responses even to the same mechanical signal tension, in which the double-helix nature of the actin filament also plays a critical role in a stretch-twist coupling of the filament.

  14. The role of the cofilin-actin rod stress response in neurodegenerative diseases uncovers potential new drug targets

    Munsie, Lise N.; Truant, Ray

    2012-01-01

    The cofilin-actin rod stress response is an actin cytoskeletal dynamic arrest that occurs in cells under a variety of stress conditions. Upon stress, the rapidly activated cofilin saturates actin filaments causing them to bundle into rod structures in either the nucleus or cytoplasm, halting actin polymerization and thus freeing ATP. Importantly, these rods dissociate quickly following relief of the transient stress. The rods form inappropriately in neurons involved in the progression of Alzh...

  15. Chorein Sensitive Arrangement of Cytoskeletal Architecture

    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.

  16. Cytoskeletal dynamics

    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....

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

    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...... 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....

  18. Structure of a Filament-Like Actin Trimer Bound to the Bacterial Effector VopL

    Zahm, Jacob A.; Padrick, Shae B.; Chen, Zhucheng; Pak, Chi W.; Yunus, Ali A.; Henry, Lisa; Tomchick, Diana R.; Chen, Zhe; Rosen, Michael K.

    2013-01-01

    Bacterial pathogens use secreted effector proteins to subvert host-cell defenses. VopL is an effector protein from Vibrio parahaemolyticus that nucleates actin filaments. VopL consists of a VopL C-terminal Domain (VCD) and a tandem array of three WASP homology 2 (WH2) motifs. Here we report the crystal structure of the VCD dimer bound to actin. The VCD binds three actin monomers in a spatial arrangement close to that in the canonical actin filament. In this configuration each actin can readil...

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

    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...

  20. Cell Forces and Cytoskeletal Order Parameters

    Discher, Dennis

    2012-02-01

    Nematic, Smectic and Isotropic Order parameters have found wide-spread use in characterizing all manner of soft matter systems, but have not yet been applied to characterize and understand the structures within living cells, particularly cytoskeletal structures. Several examples will be used to illustrate the utility of such analyses, ranging from experiments on stem cells attached to or in various elastic matrices to embryonic heart tissue and simulations of membrane cytoskeletons under all manner of stressing. Recently developed theory will be shown to apply in general with account of cell contractility, matrix elasticity and dimensionality as well as cell shape and a newly defined ``cytoskeletal polarizability.'' The latter property of cells is likely different between different cell types due to different amounts of key cytoskeletal components with some types of stem cells being more polarizable than others. Evidence of coupling to the nucleus as a viscoelastic inclusion will also be presented. [4pt] References: (1) P. Dalhaimer, D.E. Discher, T. Lubensky. Crosslinked actin networks exhibit liquid crystal elastomer behavior, including soft-mode elasticity. Nature Physics 3: 354-360 (2007). (2) A. Zemel, F.Rehfeldt, A.E.X. Brown, D.E. Discher, and S.A. Safran. Optimal matrix rigidity in the self-polarization of stem cells. Nature Physics 6: 468 - 473 (2010).

  1. Form, Fabric, and Function of a Flagellum-Associated Cytoskeletal Structure.

    Morriswood, Brooke

    2015-01-01

    Trypanosoma brucei is a uniflagellated protist and the causative agent of African trypanosomiasis, a neglected tropical disease. The single flagellum of T. brucei is essential to a number of cellular processes such as motility, and has been a longstanding focus of scientific enquiry. A number of cytoskeletal structures are associated with the flagellum in T. brucei, and one such structure-a multiprotein complex containing the repeat motif protein TbMORN1-is the focus of this review. The TbMORN1-containing complex, which was discovered less than ten years ago, is essential for the viability of the mammalian-infective form of T. brucei. The complex has an unusual asymmetric morphology, and is coiled around the flagellum to form a hook shape. Proteomic analysis using the proximity-dependent biotin identification (BioID) technique has elucidated a number of its components. Recent work has uncovered a role for TbMORN1 in facilitating protein entry into the cell, thus providing a link between the cytoskeleton and the endomembrane system. This review summarises the extant data on the complex, highlights the outstanding questions for future enquiry, and provides speculation as to its possible role in a size-exclusion mechanism for regulating protein entry. The review additionally clarifies the nomenclature associated with this topic, and proposes the adoption of the term "hook complex" to replace the former name "bilobe" to describe the complex. PMID:26540076

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

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

    2016-01-16

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

  3. Form, Fabric, and Function of a Flagellum-Associated Cytoskeletal Structure

    Brooke Morriswood

    2015-11-01

    Full Text Available Trypanosoma brucei is a uniflagellated protist and the causative agent of African trypanosomiasis, a neglected tropical disease. The single flagellum of T. brucei is essential to a number of cellular processes such as motility, and has been a longstanding focus of scientific enquiry. A number of cytoskeletal structures are associated with the flagellum in T. brucei, and one such structure—a multiprotein complex containing the repeat motif protein TbMORN1—is the focus of this review. The TbMORN1-containing complex, which was discovered less than ten years ago, is essential for the viability of the mammalian-infective form of T. brucei. The complex has an unusual asymmetric morphology, and is coiled around the flagellum to form a hook shape. Proteomic analysis using the proximity-dependent biotin identification (BioID technique has elucidated a number of its components. Recent work has uncovered a role for TbMORN1 in facilitating protein entry into the cell, thus providing a link between the cytoskeleton and the endomembrane system. This review summarises the extant data on the complex, highlights the outstanding questions for future enquiry, and provides speculation as to its possible role in a size-exclusion mechanism for regulating protein entry. The review additionally clarifies the nomenclature associated with this topic, and proposes the adoption of the term “hook complex” to replace the former name “bilobe” to describe the complex.

  4. Measurements and models of cytoskeletal rheology

    Kamm, Roger

    2006-11-01

    Much attention has recently focused on understanding the rheology of living cells and reconstituted actin gels using a variety of experimental methods (e.g., single- and multi-particle tracking, magnetic twisting cytometry, AFM indentation) and several different models or descriptors (e.g., biopolymer models, tensegrity, cellular solids, power-law rheology), but the debate continues regarding the fundamental basis for the experimental observations. Our recent studies examine the time-dependent behavior of neutrophils as they deform to enter a narrow channel with capillary-scale dimensions. A sudden drop in the shear modulus is observed, followed by recovery to pre-deformation values in < 1 minute. These rheological changes coincide with a reduction in f-actin content and a transient increase in calcium ion concentration [Ca^++], and the change in storage modulus can be prevented by calcium chelation, suggesting that these observations are causally linked. Cells lacking the ability to increase [Ca^++] also become activated more rapidly following deformation, and the time to activation is independent of intracellular strain rates, contrary to experiments lacking the chelating agent. To better understand these processes and the nature of cytoskeletal rheology in general, we have developed a Brownian dynamics model for cytoskeletal self-assembly and subsequent rheological measurement by single particle tracking. Cross-linking proteins are included possessing a range of properties that lead to a variety of cytoskeletal structures from a fine, homogeneous mesh to a structure containing large stress fibers of varying thickness. These results are described in a multi-dimensional phase space that takes into account the geometry, dimensions and stiffness of the cross-linkers.

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

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

    2016-06-01

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

  6. Probing GFP-actin diffusion in living cells using fluorescence correlation spectroscopy

    The cytoskeleton of eukaryotic cells is continuously remodeled by polymerization and depolymerization of actin. Consequently, the relative content of polymerized filamentous actin (F-actin) and monomeric globular actin (G-actin) is subject to temporal and spatial fluctuations. Since fluorescence correlation spectroscopy (FCS) can measure the diffusion of fluorescently labeled actin it seems likely that FCS allows us to determine the dynamics and hence indirectly the structural properties of the cytoskeleton components with high spatial resolution. To this end we investigate the FCS signal of GFP-actin in living Dictyostelium discoideum cells and explore the inherent spatial and temporal signatures of the actin cytoskeleton. Using the free green fluorescent protein (GFP) as a reference, we find that actin diffusion inside cells is dominated by G-actin and slower than diffusion in diluted cell extract. The FCS signal in the dense cortical F-actin network near the cell membrane is probed using the cytoskeleton protein LIM and is found to be slower than cytosolic G-actin diffusion. Furthermore, we show that polymerization of the cytoskeleton induced by Jasplakinolide leads to a substantial decrease of G-actin diffusion. Pronounced fluctuations in the distribution of the FCS correlation curves can be induced by latrunculin, which is known to induce actin waves. Our work suggests that the FCS signal of GFP-actin in combination with scanning or spatial correlation techniques yield valuable information about the local dynamics and concomitant cytoskeletal properties

  7. Characterization of engineered actin binding proteins that control filament assembly and structure.

    Crista M Brawley

    Full Text Available BACKGROUND: Eukaryotic cells strictly regulate the structure and assembly of their actin filament networks in response to various stimuli. The actin binding proteins that control filament assembly are therefore attractive targets for those who wish to reorganize actin filaments and reengineer the cytoskeleton. Unfortunately, the naturally occurring actin binding proteins include only a limited set of pointed-end cappers, or proteins that will block polymerization from the slow-growing end of actin filaments. Of the few that are known, most are part of large multimeric complexes that are challenging to manipulate. METHODOLOGY/PRINCIPAL FINDINGS: We describe here the use of phage display mutagenesis to generate of a new class of binding protein that can be targeted to the pointed-end of actin. These proteins, called synthetic antigen binders (sABs, are based on an antibody-like scaffold where sequence diversity is introduced into the binding loops using a novel "reduced genetic code" phage display library. We describe effective strategies to select and screen for sABs that ensure the generated sABs bind to the pointed-end surface of actin exclusively. CONCLUSIONS/SIGNIFICANCE: From our set of pointed-end binders, we identify three sABs with particularly useful properties to systematically probe actin dynamics: one protein that caps the pointed end, a second that crosslinks actin filaments, and a third that severs actin filaments and promotes disassembly.

  8. The effect of mouse twinfilin-1 on the structure and dynamics of monomeric actin.

    Takács-Kollár, Veronika; Nyitrai, Miklós; Hild, Gábor

    2016-07-01

    The effect of twinfilin-1 on the structure and dynamics of monomeric actin was investigated with fluorescence spectroscopy and differential scanning calorimetry experiments. Fluorescence anisotropy measurements proved that G-actin and twinfilin-1 could form a complex. Due to the formation of the complexes the dissociation of the nucleotide slowed down from the nucleotide-binding pocket of actin. Fluorescence quenching experiments showed that the accessibility of the actin bound ε-ATP decreased in the presence of twinfilin-1. Temperature dependent fluorescence resonance energy transfer and differential scanning calorimetry experiments revealed that the protein matrix of actin becomes more rigid and more heat resistant in the presence of twinfilin-1. The results suggest that the nucleotide binding cleft shifted into a more closed and stable conformational state of actin in the presence of twinfilin-1. PMID:27079635

  9. Structure, chromosome location, and expression of the human smooth muscle (enteric type). gamma. -actin gene: Evolution of six human actin genes

    Miwa, Takeshi; Manabe, Yoshihisa; Kamada, Shinji; Kakunaga, Takeo (Osaka Univ. (Japan)); Kurokawa, Kiyoshi; Ueyama, Hisao (Shiga Univ. of Medical Science, Seta (Japan)); Kanda, Naotoshi (Tokyo Women' s Medical Coll. (Japan)); Bruns, G. (Children' s Hospital, Boston, MA (United States))

    1991-06-01

    Recombinant phages that carry the human smooth muscle (enteric type) {gamma}-actin gene were isolated from human genomic DNA libraries. The amino acid sequence deduced from the nucleotide sequence matches those of cDNAs but differs from the protein sequence previously reported at one amino acid position, codon 359. The gene containing one 5{prime} untranslated exon and eight coding exons extends for 27 kb on human chromosome 2. The intron between codons 84 and 85 (site 3) is unique to the two smooth muscle actin genes. From characterized molecular structures of the six human actin isoform genes, the authors propose a hypothesis of evolutionary pathway of the actin gene family. A presumed ancestral actin isoform gene had introns at least sites, 1, 2, and 4 through 8. Cytoplasmic actin genes may have directly evolved from it through loss of introns at sites 5 and 6. However, through duplication of the ancestral actin gene with substitutions of many amino acids, a prototype of muscle actin genes had been created. Subsequently, striated muscle actin and smooth muscle actin genes may have evolved from this prototype by loss of an intron at site 4 and acquisition of a new intron at site 3, respectively.

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

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

    2002-01-01

    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...... compared with F-actin, despite the similar arrangement of the subunits within the filaments. Thus, there is now evidence for cytoskeletal structures, formed by actin-like filaments that are involved in plasmid partitioning in E.coli. Udgivelsesdato: Dec 16...

  11. Morphological change and crystal structure of skeletal muscle actin

    Actin from skeletal muscle was crystallized in fluorescent dye/acetone solutions. Three different polymorphic forms of the crystals were observed by polarization microscope and video systems. Ultrastructural observation and electron diffraction analysis of the crystals have been made using a 1 MeV electron microscope. The specimens were unstained or negatively stained with uranyl acetate. The diffraction spots of the crystals faded within twenty seconds. Minimum dose system and low temperature techniques were effective in taking highly resolved images and diffraction patterns of the crystals. Actin crystals diffracted well to 2 A resolution. The rod form of actin crystals is orthorhombic and the cell dimensions are 61 Ax41 Ax33 A. The unit cell contains one actin monomer. (orig.)

  12. Autothixotropy of Water and its Possible Importance for the Cytoskeletal Structures

    Vybíral, Bohumil

    2011-12-01

    an organization of water in cytoskeletal structures are outlined.

  13. Formation of actin networks in microfluidic concentration gradients

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

    2016-05-01

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

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

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

    2015-08-15

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

  15. Cell elasticity with altered cytoskeletal architectures across multiple cell types.

    Grady, Martha E; Composto, Russell J; Eckmann, David M

    2016-08-01

    The cytoskeleton is primarily responsible for providing structural support, localization and transport of organelles, and intracellular trafficking. The structural support is supplied by actin filaments, microtubules, and intermediate filaments, which contribute to overall cell elasticity to varying degrees. We evaluate cell elasticity in five different cell types with drug-induced cytoskeletal derangements to probe how actin filaments and microtubules contribute to cell elasticity and whether it is conserved across cell type. Specifically, we measure elastic stiffness in primary chondrocytes, fibroblasts, endothelial cells (HUVEC), hepatocellular carcinoma cells (HUH-7), and fibrosarcoma cells (HT 1080) subjected to two cytoskeletal destabilizers: cytochalasin D and nocodazole, which disrupt actin and microtubule polymerization, respectively. Elastic stiffness is measured by atomic force microscopy (AFM) and the disruption of the cytoskeleton is confirmed using fluorescence microscopy. The two cancer cell lines showed significantly reduced elastic moduli values (~0.5kPa) when compared to the three healthy cell lines (~2kPa). Non-cancer cells whose actin filaments were disrupted using cytochalasin D showed a decrease of 60-80% in moduli values compared to untreated cells of the same origin, whereas the nocodazole-treated cells showed no change in elasticity. Overall, we demonstrate actin filaments contribute more to elastic stiffness than microtubules but this result is cell type dependent. Cancer cells behaved differently, exhibiting increased stiffness as well as stiffness variability when subjected to nocodazole. We show that disruption of microtubule dynamics affects cancer cell elasticity, suggesting therapeutic drugs targeting microtubules be monitored for significant elastic changes. PMID:26874250

  16. Disassembly of actin structures by nanosecond pulsed electric field is a downstream effect of cell swelling

    Pakhomov, Andrei G.; Xiao, Shu; Pakhomova, Olga N.; Semenov, Iurii; Kuipers, Marjorie A.; Ibey, Bennett L.

    2014-01-01

    Disruption of the actin cytoskeleton structures was reported as one of the characteristic effects of nanosecond-duration pulsed electric field (nsPEF) in both mammalian and plant cells. We utilized CHO cells that expressed the monomeric fluorescent protein (mApple) tagged to actin to test if nsPEF modifies the cell actin directly or as a consequence of cell membrane permeabilization. A train of four 600-ns pulses at 19.2 kV/cm (2 Hz) caused immediate cell membrane poration manifested by YO-PR...

  17. Structural Transition of Actin Filament in a Cell-Sized Water Droplet with a Phospholipid Membrane

    Hase, M

    2005-01-01

    Actin filament, F-actin, is a semiflexible polymer with a negative charge, and is one of the main constituents on cell membranes. To clarify the effect of cross-talk between a phospholipid membrane and actin filaments in cells, we conducted microscopic observations on the structural changes in actin filaments in a cell-sized (several tens of micrometers in diameter) water droplet coated with a phospholipid membrane such as phosphatidylserine (PS; negatively-charged head group) or phosphatidylethanolamine (PE; neutral head group) as a simple model of a living cell membrane. With PS, actin filaments are distributed uniformly in the water phase without adsorption onto the membrane surface between 2 and 6 mM Mg2+, while between 6 and 12 mM Mg2+, actin filaments are adsorbed onto the inner membrane surface. With PE, actin filaments are uniformly adsorbed onto the inner membrane surface between 2 and 12 mM Mg2+. With both PS and PE membranes, at Mg2+ concentrations higher than 12 mM, thick bundles are formed in the...

  18. Characterization of ring-like F-actin structure as a mechanical partner for spindle positioning in mitosis.

    Huan Lu

    Full Text Available Proper spindle positioning and orientation are essential for accurate mitosis which requires dynamic interactions between microtubule and actin filament (F-actin. Although mounting evidence demonstrates the role of F-actin in cortical cytoskeleton dynamics, it remains elusive as to the structure and function of F-actin-based networks in spindle geometry. Here we showed a ring-like F-actin structure surrounding the mitotic spindle which forms since metaphase and maintains in MG132-arrested metaphase HeLa cells. This cytoplasmic F-actin structure is relatively isotropic and less dynamic. Our computational modeling of spindle position process suggests a possible mechanism by which the ring-like F-actin structure can regulate astral microtubule dynamics and thus mitotic spindle orientation. We further demonstrated that inhibiting Plk1, Mps1 or Myosin, and disruption of microtubules or F-actin polymerization perturbs the formation of the ring-like F-actin structure and alters spindle position and symmetric division. These findings reveal a previously unrecognized but important link between mitotic spindle and ring-like F-actin network in accurate mitosis and enables the development of a method to theoretically illustrate the relationship between mitotic spindle and cytoplasmic F-actin.

  19. Native globular actin has a thermodynamically unstable quasi-stationary structure with elements of intrinsic disorder.

    Kuznetsova, Irina M; Povarova, Olga I; Uversky, Vladimir N; Turoverov, Konstantin K

    2016-02-01

    The native form of globular actin, G-actin, is formed in vivo as a result of complex post-translational folding processes that require ATP energy expenditure and are assisted by the 70 kDa heat shock protein, prefoldin and chaperonin containing TCP-1. G-actin is stabilized by the binding of one ATP molecule and one Ca(2+) ion (or Mg(2+) in vivo). Chemical denaturants, heating or Ca(2+) removal transform native actin (N) into 'inactivated actin' (I), a compact oligomer comprising 14-16 subunits. Viscogenic and crowding agents slow this process but do not stop it. The lack of calcium in the solution accelerates the spontaneous N → I transition. Thus, native G-actin has a kinetically stable (as a result of the high free energy barrier between the N and I states) but thermodynamically unstable structure, which, in the absence of Ca(2+) or other bivalent metal ions, spontaneously converts to the thermodynamically stable I state. It was noted that native actin has much in common with intrinsically disordered proteins: it has functionally important disordered regions; it is constantly in complex with one of its numerous partners; and it plays key roles in many cellular processes, in a manner similar to disordered hub proteins. By analyzing actin folding in vivo and unfolding in vitro, we advanced the hypothesis that proteins in a native state may have a thermodynamically unstable quasi-stationary structure. The kinetically stable native state of these proteins appears forcibly under the influence of intracellular folding machinery. The denaturation of such proteins is always irreversible because the inactivated state, for which the structure is determined by the amino acid sequence of a protein, comprises the thermodynamically stable state under physiological conditions. PMID:26460158

  20. 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

    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

  1. 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

    刘军楼; 沈洪; 徐力; 杨继兵; 于希忠; 孙志岭

    2015-01-01

    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法检测细胞生长抑制率,DAPI染色法染色后荧光显微镜观察细胞核凋亡、流式细胞仪检测细胞凋亡率,激光共聚焦显微镜观察F-actin形态学变化。结果:冬凌草甲素对人胰腺癌SW1990细胞具有明显的增殖抑制作用,荧光显微镜见到典型的凋亡形态学改变。流式细胞仪检测结果显示,25、50μmol/L冬凌草甲素给药组早期凋亡的百分率显著高于对照组(3.78±0.46,9.51±0.63 vs 0.73±0.06,P<0.05),晚期凋亡和坏死细胞的百分率也显著高于未给药组(14.40±1.78,20.53±2.54 vs 4.16±0.31,P<0.05)。细胞骨架蛋白F-actin呈现解聚状态。结论:冬凌草甲素可抑制胰腺癌SW1990细胞增殖,促进肿瘤细胞凋亡,其作用机制可能是药物引起了细胞骨架蛋白F-actin解聚。%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

  2. Crystallization and preliminary structural characterization of the two actin-depolymerization factors of the malaria parasite

    The expression, purification and crystallization of Plasmodium actin-depolymerization factors 1 and 2 are described. X-ray diffraction data were collected to 2.0 and 2.1 Å resolution, respectively, and the structures of both proteins in solution were characterized. The malaria parasite Plasmodium depends on its actin-based motor system for motility and host-cell invasion. Actin-depolymerization factors are important regulatory proteins that affect the rate of actin turnover. Plasmodium has two actin-depolymerization factors which seem to have different functions and display low sequence homology to the higher eukaryotic family members. Plasmodium actin-depolymerization factors 1 and 2 have been crystallized. The crystals diffracted X-rays to maximum resolutions of 2.0 and 2.1 Å and belonged to space groups P3121 or P3221, with unit-cell parameters a = b = 68.8, c = 76.0 Å, and P21212, with unit-cell parameters a = 111.6, b = 57.9, c = 40.5 Å, respectively, indicating the presence of one or two molecules per asymmetric unit in both cases

  3. Antenna mechanism of length control of actin cables

    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...

  4. Towards the Structure Determination of a Modulated Protein Crystal: The Semicrystalline State of Profilin:Actin

    Borgstahl, G.; Lovelace, J.; Snell, E. H.; Bellamy, H.

    2003-01-01

    One of the remaining challenges to structural biology is the solution of modulated structures. While small molecule crystallographers have championed this type of structure, to date, no modulated macromolecular structures have been determined. Modulation of the molecular structures within the crystal can produce satellite reflections or a superlattice of reflections in reciprocal space. We have developed the data collection methods and strategies that are needed to collect and analyze these data. If the macromolecule's crystal lattice is composed of physiologically relevant packing contacts, structural changes induced under physiological conditions can cause distortion relevant to the function and biophysical processes of the molecule making up the crystal. By careful measurement of the distortion, and the corresponding three-dimensional structure of the distorted molecule, we will visualize the motion and mechanism of the biological macromolecule(s). We have measured the modulated diffraction pattern produced by the semicrystalline state of profilin:actin crystals using highly parallel and highly monochromatic synchrotron radiation coupled with fine phi slicing (0.001-0.010 degrees) for structure determination. These crystals present these crystals present a unique opportunity to address an important question in structural biology. The modulation is believed to be due to the formation of actin helical filaments from the actin beta ribbon upon the pH-induced dissociation of profilin. To date, the filamentous state of actin has resisted crystallization and no detailed structures are available. The semicrystalline state profilin:actin crystals provides a unique opportunity to understand the many conformational states of actin. This knowledge is essential for understanding the dynamics underlying shape changes and motility of eukaryotic cells. Many essential processes, such as cytokinesis, phagocytosis, and cellular migration depend upon the capacity of the actin

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

    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.

  6. Cations Stiffen Actin Filaments by Adhering a Key Structural Element to Adjacent Subunits.

    Hocky, Glen M; Baker, Joseph L; Bradley, Michael J; Sinitskiy, Anton V; De La Cruz, Enrique M; Voth, Gregory A

    2016-05-26

    Ions regulate the assembly and mechanical properties of actin filaments. Recent work using structural bioinformatics and site-specific mutagenesis favors the existence of two discrete and specific divalent cation binding sites on actin filaments, positioned in the long axis between actin subunits. Cation binding at one site drives polymerization, while the other modulates filament stiffness and plays a role in filament severing by the regulatory protein, cofilin. Existing structural methods have not been able to resolve filament-associated cations, and so in this work we turn to molecular dynamics simulations to suggest a candidate binding pocket geometry for each site and to elucidate the mechanism by which occupancy of the "stiffness site" affects filament mechanical properties. Incorporating a magnesium ion in the "polymerization site" does not seem to require any large-scale change to an actin subunit's conformation. Binding of a magnesium ion in the "stiffness site" adheres the actin DNase-binding loop (D-loop) to its long-axis neighbor, which increases the filament torsional stiffness and bending persistence length. Our analysis shows that bound D-loops occupy a smaller region of accessible conformational space. Cation occupancy buries key conserved residues of the D-loop, restricting accessibility to regulatory proteins and enzymes that target these amino acids. PMID:27146246

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

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

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

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

    2015-08-21

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

  9. Myopathy-inducing mutation H40Y in ACTA1 hampers actin filament structure and function.

    Chan, Chun; Fan, Jun; Messer, Andrew E; Marston, Steve B; Iwamoto, Hiroyuki; Ochala, Julien

    2016-08-01

    In humans, more than 200 missense mutations have been identified in the ACTA1 gene. The exact molecular mechanisms by which, these particular mutations become toxic and lead to muscle weakness and myopathies remain obscure. To address this, here, we performed a molecular dynamics simulation, and we used a broad range of biophysical assays to determine how the lethal and myopathy-related H40Y amino acid substitution in actin affects the structure, stability, and function of this protein. Interestingly, our results showed that H40Y severely disrupts the DNase I-binding-loop structure and actin filaments. In addition, we observed that normal and mutant actin monomers are likely to form distinctive homopolymers, with mutant filaments being very stiff, and not supporting proper myosin binding. These phenomena underlie the toxicity of H40Y and may be considered as important triggering factors for the contractile dysfunction, muscle weakness and disease phenotype seen in patients. PMID:27112274

  10. Structure-based analysis of high pressure adaptation of alpha-actin.

    Morita, Takami

    2003-07-25

    Deep-sea fishes occur to depths of several thousand meters, and at these abyssal depths encounter pressures that shallower living fishes cannot tolerate. Tolerance of abyssal pressures by deep-sea fish is likely to depend in part on adaptive modifications of proteins. However, the types of structural modifications to proteins that allow function at high pressure have not been discovered. To elucidate the mechanisms of protein adaptation to high pressure, we cloned the alpha-skeletal actin cDNAs from two abyssal Coryphaenoides species, C. armatus and C. yaquinae, and identified three amino acid substitutions, V54A or L67P, Q137K, and A155S, that distinguish these abyssal actins from orthologs of alpha-actin from non-abyssal Coryphaenoides. These substitutions, Q137K and A155S, prevent the dissociation reactions of ATP and Ca2+ from being influenced by high pressure. In particular, the lysine residue at position 137 results in a much smaller apparent volume change in the Ca2+ dissociation reaction. The V54A or L67P substitution reduces the volume change associated with actin polymerization and has a role in maintaining the DNase I activity of actin at high pressure. Together, these results indicate that a few amino acid substitutions in key functional positions can adaptively alter the pressure sensitivity of a protein. PMID:12740368

  11. Hierarchical self-organization of cytoskeletal active networks

    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 on the mechanisms involved in processes such as cytokinesis and cellular contractility, where myosin motors organized in clusters operate cooperatively to induce the structural organization of cytoskeletal networks. (paper)

  12. Cytoskeletal Interactions at the Nuclear Envelope Mediated by Nesprins

    Surayya Taranum

    2012-01-01

    Full Text Available Nesprin-1 is a giant tail-anchored nuclear envelope protein composed of an N-terminal F-actin binding domain, a long linker region formed by multiple spectrin repeats and a C-terminal transmembrane domain. Based on this structure, it connects the nucleus to the actin cytoskeleton. Earlier reports had shown that Nesprin-1 binds to nuclear envelope proteins emerin and lamin through C-terminal spectrin repeats. These repeats can also self-associate. We focus on the N-terminal Nesprin-1 sequences and show that they interact with Nesprin-3, a further member of the Nesprin family, which connects the nucleus to the intermediate filament network. We show that upon ectopic expression of Nesprin-3 in COS7 cells, which are nearly devoid of Nesprin-3 in vitro, vimentin filaments are recruited to the nucleus and provide evidence for an F-actin interaction of Nesprin-3 in vitro. We propose that Nesprins through interactions amongst themselves and amongst the various Nesprins form a network around the nucleus and connect the nucleus to several cytoskeletal networks of the cell.

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

    Mehrbod, Mehrdad; Mofrad, Mohammad R K

    2011-01-01

    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 exceed the axial energy

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

    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

  15. Antibodies to cytoskeletal proteins as evidenced by immunofluorescence microscopy and radioimmunoassay

    In patients suffering from chronic hepatitis, collagenosis and infectious mononucleosis, resp., as well as in blood donors antibodies against cytoskeletal antigens such as actin, myosin, actinin, desmin, keratin, and tubulin were determined by radioimmunoassay

  16. Dynamics of active actin networks

    Koehler, Simone

    2014-03-01

    Local mechanical and structural properties of a eukaryotic cell are determined by its cytoskeleton. To adapt to their environment, cells rely on constant self-organized rearrangement processes of their actin cytoskeleton. To shed light on the principles underlying these dynamic self-organization processes we investigate a minimal reconstituted active system consisting of actin filaments, crosslinking molecules and molecular motor filaments. Using quantitative fluorescence microscopy and image analysis, we show, that these minimal model systems exhibit a generic structure formation mechanism. The competition between force generation by molecular motors and the stabilization of the network by crosslinking proteins results in a highly dynamic reorganization process which is characterized by anomalous transport dynamics with a superdiffusive behavior also found in intracellular dynamics. In vitro, these dynamics are governed by chemical and physical parameters that alter the balance of motor and crosslinking proteins, such as pH. These findings can be expected to have broad implications in our understanding of cytoskeletal regulation in vivo.

  17. Cytoskeletal regulation of dermal regeneration.

    Strudwick, Xanthe L; Cowin, Allison J

    2012-01-01

    Wound healing results in the repair of injured tissues however fibrosis and scar formation are, more often than not the unfortunate consequence of this process. The ability of lower order vertebrates and invertebrates to regenerate limbs and tissues has been all but lost in mammals; however, there are some instances where glimpses of mammalian regenerative capacity do exist. Here we describe the unlocked potential that exists in mammals that may help us understand the process of regeneration post-injury and highlight the potential role of the actin cytoskeleton in this process. The precise function and regulation of the cytoskeleton is critical to the success of the healing process and its manipulation may therefore facilitate regenerative healing. The gelsolin family of actin remodelling proteins in particular has been shown to have important functions in wound healing and family member Flightless I (Flii) is involved in both regeneration and repair. Understanding the interactions between different cytoskeletal proteins and their dynamic control of processes including cellular adhesion, contraction and motility may assist the development of therapeutics that will stimulate regeneration rather than repair. PMID:24710556

  18. Biotechnological aspects of cytoskeletal regulation in plants.

    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. PMID:25784147

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

    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. PMID:21949650

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

    Kathleen A Estes

    2011-09-01

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

  1. Sex Hormones Regulate Cytoskeletal Proteins Involved in Brain Plasticity

    Hansberg-Pastor, Valeria; González-Arenas, Aliesha; Piña-Medina, Ana Gabriela; Camacho-Arroyo, Ignacio

    2015-01-01

    In the brain of female mammals, including humans, a number of physiological and behavioral changes occur as a result of sex hormone exposure. Estradiol and progesterone regulate several brain functions, including learning and memory. Sex hormones contribute to shape the central nervous system by modulating the formation and turnover of the interconnections between neurons as well as controlling the function of glial cells. The dynamics of neuron and glial cells morphology depends on the cytoskeleton and its associated proteins. Cytoskeletal proteins are necessary to form neuronal dendrites and dendritic spines, as well as to regulate the diverse functions in astrocytes. The expression pattern of proteins, such as actin, microtubule-associated protein 2, Tau, and glial fibrillary acidic protein, changes in a tissue-specific manner in the brain, particularly when variations in sex hormone levels occur during the estrous or menstrual cycles or pregnancy. Here, we review the changes in structure and organization of neurons and glial cells that require the participation of cytoskeletal proteins whose expression and activity are regulated by estradiol and progesterone. PMID:26635640

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

    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.

  3. Rho, nuclear actin, and actin-binding proteins in the regulation of transcription and gene expression.

    Rajakylä, Eeva Kaisa; Vartiainen, Maria K

    2014-01-01

    Actin cytoskeleton is one of the main targets of Rho GTPases, which act as molecular switches on many signaling pathways. During the past decade, actin has emerged as an important regulator of gene expression. Nuclear actin plays a key role in transcription, chromatin remodeling, and pre-mRNA processing. In addition, the "status" of the actin cytoskeleton is used as a signaling intermediate by at least the MKL1-SRF and Hippo-pathways, which culminate in the transcriptional regulation of cytoskeletal and growth-promoting genes, respectively. Rho GTPases may therefore regulate gene expression by controlling either cytoplasmic or nuclear actin dynamics. Although the regulation of nuclear actin polymerization is still poorly understood, many actin-binding proteins, which are downstream effectors of Rho, are found in the nuclear compartment. In this review, we discuss the possible mechanisms and key proteins that may mediate the transcriptional regulation by Rho GTPases through actin. PMID:24603113

  4. The role of actin networks in cellular mechanosensing

    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.

  5. Antenna Mechanism of Length Control of Actin Cables.

    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.

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

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

    2015-05-26

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

  7. Cytoskeletal and functional changes in bioreactor assembled thyroid tissue organoids exposed to gamma radiation

    Fischer rat thyroid cells were grown under low-shear stress in a bioreactor to a stage of organization composed of integrated follicles resembling small thyroid glands prior to exposure to 3 Gray-gamma radiation. Bioreactor tissues and controls (both irradiated and non-irradiated) were harvested at 24, 48, 96 and 144 hours post-exposure. Tissue samples were fixed and fluorescently labeled for actin and microtubules. Tissues were assessed for changes in cytoskeletal components induced by radiation and quantified by laser scanning cytometry. Enzyme-linked immunosorbent assay (ELISA's) were used to quantify transforming growth factor-beta and thyroxin released from cells to the culture supernatant. Tissue architecture was disrupted by exposure to radiation with the structural organization of actin and loss of follicular content the most obviously affected. With time post-irradiation the actin appeared disordered and the levels of fluorescence associated with filamentous-actin and microtubules cycled in the tissue analogs, but not in the flask-grown cultures. Active transforming growth factor-beta was higher in supernatants from the irradiated bioreactor tissue. Thyroxin release paralleled cell survival in the bioreactors and control cultures. Thus, the engineered tissue responses to radiation differed from those of conventional tissue culture making it a potentially better mimic of the in vivo situation. (author)

  8. Actinic Cheilitis

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

  9. Actin gene family in Branchiostoma belched

    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. Distinct structural changes detected by X-ray fiber diffraction in stabilization of F-actin by lowering pH and increasing ionic strength.

    Oda, T.; Makino, K.; Yamashita, I; Namba, K.; Maéda, Y

    2001-01-01

    Lowering pH or raising salt concentration stabilizes the F-actin structure by increasing the free energy change associated with its polymerization. To understand the F-actin stabilization mechanism, we studied the effect of pH, salt concentration, and cation species on the F-actin structure. X-ray fiber diffraction patterns recorded from highly ordered F-actin sols at high density enabled us to detect minute changes of diffraction intensities and to precisely determine the helical parameters....

  11. Pharmacologic regulation of AMPK in breast cancer affects cytoskeletal properties involved with microtentacle formation and re-attachment.

    Chakrabarti, Kristi R; Whipple, Rebecca A; Boggs, Amanda E; Hessler, Lindsay K; Bhandary, Lekhana; Vitolo, Michele I; Thompson, Keyata; Martin, Stuart S

    2015-11-01

    The presence of tumor cells in the circulation is associated with a higher risk of metastasis in patients with breast cancer. Circulating breast tumor cells use tubulin-based structures known as microtentacles (McTNs) to re-attach to endothelial cells and arrest in distant organs. McTN formation is dependent on the opposing cytoskeletal forces of stable microtubules and the actin network. AMP-activated protein kinase (AMPK) is a cellular metabolic regulator that can alter actin and microtubule organization in epithelial cells. We report that AMPK can regulate the cytoskeleton of breast cancer cells in both attached and suspended conditions. We tested the effects of AMPK on microtubule stability and the actin-severing protein, cofilin. AMPK inhibition with compound c increased both microtubule stability and cofilin activation, which also resulted in higher McTN formation and re-attachment. Conversely, AMPK activation with A-769662 decreased microtubule stability and cofilin activation with concurrent decreases in McTN formation and cell re-attachment. This data shows for the first time that AMPK shifts the balance of cytoskeletal forces in suspended breast cancer cells, which affect their ability to form McTNs and re-attach. These results support a model where AMPK activators may be used therapeutically to reduce the metastatic efficiency of breast tumor cells. PMID:26431377

  12. Cytoskeletal Dynamics: Concepts in Measles Virus Replication and Immunomodulation

    Sibylle Schneider-Schaulies

    2011-01-01

    Full Text Available In common with most viruses, measles virus (MV relies on the integrity of the cytoskeleton of its host cells both with regard to efficient replication in these cells, but also retention of their motility which favors viral dissemination. It is, however, the surface interaction of the viral glycoprotein (gp complex with receptors present on lymphocytes and dendritic cells (DCs, that signals effective initiation of host cell cytoskeletal dynamics. For DCs, these may act to regulate processes as diverse as viral uptake and sorting, but also the ability of these cells to successfully establish and maintain functional immune synapses (IS with T cells. In T cells, MV signaling causes actin cytoskeletal paralysis associated with a loss of polarization, adhesion and motility, which has been linked to activation of sphingomyelinases and subsequent accumulation of membrane ceramides. MV modulation of both DC and T cell cytoskeletal dynamics may be important for the understanding of MV immunosuppression at the cellular level.

  13. Modification of Experimental Protocols for a Space Shuttle Flight and Applications for the Analysis of Cytoskeletal Structures During Fertilization, Cell Division , and Development in Sea Urchin Embryos

    Chakrabarti, Amitabha; Stoecker, Andrew; Schatten, Heide

    1995-01-01

    To explore the role of microgravity on cytoskeletal organization and skeletal calcium deposition during fertilization, cell division, and early development, the sea urchin was chosen as a model developmental system. Methods were developed to employ light, immunofluorescence, and electron microscopy on cultures being prepared for flight on the Space Shuttle. For analysis of microfilaments, microtubules, centrosomes, and calcium-requiring events, our standard laboratory protocols had to be modified substantially for experimentation on the Space Shuttle. All manipulations were carried out in a closed culture chamber containing 35 ml artificial sea water as a culture fluid. Unfertilized eggs stored for 24 hours in these chambers were fertilized with sperm diluted in sea water and fixed with concentrated fixatives for final fixation in formaldehyde, taxol, EGTA, and MgCl2(exp -6)H2O for 1 cell to 16 cell stages to preserve cytoskeletal structures for simultaneous analysis with light, immunofluorescence, and electron microscopy, and 1.5 percent glutaraldehyde and 0.4 percent formaldehyde for blastula and plueus stages. The fixed samples wre maintained in chambers without degradation for up to two weeks after which the specimens were processed and analyzed with routine methods. Since complex manipulations are not possible in the closed chambers, the fertilization coat was removed from fixation using 0.5 percent freshly prepared sodium thioglycolate solution at pH 10.0 which provided reliable immunofluorescence staining for microtubules. Sperm/egg fusion, mitosis, cytokinesis, and calcium deposition during spicule formatin in early embryogenesis were found to be without artificial alterations when compared to cells fixed fresh and processed with conventional methods.

  14. The structural dynamics of α-tropomyosin on F-actin shape the overlap complex between adjacent tropomyosin molecules.

    Lehman, William; Li, Xiaochuan Edward; Orzechowski, Marek; Fischer, Stefan

    2014-06-15

    Coiled-coil tropomyosin, localized on actin filaments in virtually all eukaryotic cells, serves as a gatekeeper regulating access of the motor protein myosin and other actin-binding proteins onto the thin filament surface. Tropomyosin's modular pseudo-repeating pattern of approximately 39 amino acid residues is designed to allow binding of the coiled-coil to successive actin subunits along thin filaments. Even though different tropomyosin isoforms contain varying numbers of repeat modules, the pseudo-repeat length, in all cases, matches that of a single actin subunit. Thus, the seven pseudo-repeats of 42nm long muscle tropomyosin bind to seven successive actin subunits along thin filaments, while simultaneously bending into a super-helical conformation that is preshaped to the actin filament helix. In order to form a continuous cable on thin filaments that is free of gaps, adjacent tropomyosin molecules polymerize head-to-tail by means of a short (∼9 residue) overlap. Several laboratories have engineered peptides to mimic the N- and C-terminal tropomyosin association and to characterize the overlap structure. All overlapping domains examined show a compact N-terminal coiled-coil inserting into a partially opened C-terminal partner, where the opposing coiled-coils at the overlap junction face each other at up to ∼90° twist angles. Here, Molecular Dynamics (MD) simulations were carried out to determine constraints on the formation of the tropomyosin overlap complex and to assess the amount of twisting exhibited by full-length tropomyosin when bound to actin. With the exception of the last 20-40 C- and N-terminal residues, we find that the average tropomyosin structure closely resembles a "canonical" model proposed in the classic work of McLachlan and Stewart, displaying perfectly symmetrical supercoil geometry matching the F-actin helix with an integral number of coiled-coil turns, a coiled-coil helical pitch of 137Å, a superhelical pitch of 770Å, and no

  15. Hierarchical Cross-linked F-actin Networks: Understanding Structure and Assembly

    Hirst, Linda; Nguyen, Lam

    2009-11-01

    The protein, F-actin provides us with an interesting system in which to investigate the assembly properties of semi-flexible filaments in the presence of cross-linkers. Recently it was observed that F-actin, in the presence of the cross-linker alpha-actinin at high molar ratios will generate a novel hierarchical network of filament bundles. We investigate this system using coarse-grained molecular dynamics (MD) simulation, confocal microscopy and x-ray scattering. We have studied the F-actin/alpha-actinin system in detail with different actin conc. (C) and alpha-actinin/actin molar ratios (gamma). Confocal microscopy and analysis shows that the assembled systems fall into one of 3 phases depending on C and gamma: (1) loosely connected network of F-actin and bundles, (2) loosely connected network of dense domains and (3) uniform network of bundles. This can be explained and replicated using MD simulation. We have also examined different types of cross-linkers to represent the proteins, fascin and filamin. Results show that phase formation is related to the flexibility in binding between F-actin and cross-linkers. This degree of freedom, possible with longer cross-linkers allows the formation of branch points and thus bundle networks.

  16. MAPK uncouples cell cycle progression from cell spreading and cytoskeletal organization in cycling cells

    Margadant, Coert; Cremers, Lobke; Sonnenberg, Arnoud; Boonstra, Johannes

    2012-01-01

    Integrin-mediated cytoskeletal tension supports growth-factor-induced proliferation, and disruption of the actin cytoskeleton in growth factor-stimulated cells prevents the re-expression of cyclin D and cell cycle re-entry from quiescence. In contrast to cells that enter the cell cycle from G0, cycling cells continuously express cyclin D, and are subject to major cell shape changes during the cell cycle. Here, we investigated the cell cycle requirements for cytoskeletal tension and cell sprea...

  17. Actinic keratosis

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

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

    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

  19. Rk1, a ginsenoside, is a new blocker of vascular leakage acting through actin structure remodeling.

    Maeng, Yong-Sun; Maharjan, Sony; Kim, Jeong-Hun; Park, Jeong-Hill; Suk Yu, Young; Kim, Young-Myoung; Kwon, Young-Guen

    2013-01-01

    Endothelial barrier integrity is essential for vascular homeostasis and increased vascular permeability and has been implicated in many pathological processes, including diabetic retinopathy. Here, we investigated the effect of Rk1, a ginsenoside extracted from sun ginseng, on regulation of endothelial barrier function. In human retinal endothelial cells, Rk1 strongly inhibited permeability induced by VEGF, advanced glycation end-product, thrombin, or histamine. Furthermore, Rk1 significantly reduced the vessel leakiness of retina in a diabetic mouse model. This anti-permeability activity of Rk1 is correlated with enhanced stability and positioning of tight junction proteins at the boundary between cells. Signaling experiments revealed that Rk1 induces phosphorylation of myosin light chain and cortactin, which are critical regulators for the formation of the cortical actin ring structure and endothelial barrier. These findings raise the possibility that ginsenoside Rk1 could be exploited as a novel prototype compound for the prevention of human diseases that are characterized by vascular leakage. PMID:23894330

  20. Experimental and computational assessment of F-actin influence in regulating cellular stiffness and relaxation behaviour of fibroblasts.

    Fallqvist, Björn; Fielden, Matthew L; Pettersson, Torbjörn; Nordgren, Niklas; Kroon, Martin; Gad, Annica K B

    2016-06-01

    In biomechanics, a complete understanding of the structures and mechanisms that regulate cellular stiffness at a molecular level remain elusive. In this paper, we have elucidated the role of filamentous actin (F-actin) in regulating elastic and viscous properties of the cytoplasm and the nucleus. Specifically, we performed colloidal-probe atomic force microscopy (AFM) on BjhTERT fibroblast cells incubated with Latrunculin B (LatB), which results in depolymerisation of F-actin, or DMSO control. We found that the treatment with LatB not only reduced cellular stiffness, but also greatly increased the relaxation rate for the cytoplasm in the peripheral region and in the vicinity of the nucleus. We thus conclude that F-actin is a major determinant in not only providing elastic stiffness to the cell, but also in regulating its viscous behaviour. To further investigate the interdependence of different cytoskeletal networks and cell shape, we provided a computational model in a finite element framework. The computational model is based on a split strain energy function of separate cellular constituents, here assumed to be cytoskeletal components, for which a composite strain energy function was defined. We found a significant influence of cell geometry on the predicted mechanical response. Importantly, the relaxation behaviour of the cell can be characterised by a material model with two time constants that have previously been found to predict mechanical behaviour of actin and intermediate filament networks. By merely tuning two effective stiffness parameters, the model predicts experimental results in cells with a partly depolymerised actin cytoskeleton as well as in untreated control. This indicates that actin and intermediate filament networks are instrumental in providing elastic stiffness in response to applied forces, as well as governing the relaxation behaviour over shorter and longer time-scales, respectively. PMID:26766328

  1. Post-polymerization crosstalk between the actin cytoskeleton and microtubule network.

    Joo, E Emily; Yamada, Kenneth M

    2016-05-01

    Cellular cytoskeletal systems play many pivotal roles in living organisms by controlling cell shape, division, and migration, which ultimately govern morphology, physiology, and functions of animals. Although the cytoskeletal systems are distinct and play different roles, there is growing evidence that these diverse cytoskeletal systems coordinate their functions with each other. This coordination between cytoskeletal systems, often termed cytoskeletal crosstalk, has been identified when the dynamic state of one individual system affects the other system. In this review, we briefly describe some well-established examples of crosstalk between cytoskeletal systems and then introduce a newly discovered form of crosstalk between the actin cytoskeleton and microtubule network that does not appear to directly alter polymerization or depolymerization of either system. The biological impact and possible significance of this post-polymerization crosstalk between actin and microtubules will be discussed in detail. PMID:27058810

  2. The structural basis for the intrinsic disorder of the actin filament: the "lateral slipping" model.

    Bremer, A; Millonig, R C; Sütterlin, R; Engel, A; Pollard, T D; Aebi, U

    1991-11-01

    Three-dimensional (3-D) helical reconstructions computed from electron micrographs of negatively stained dispersed F-actin filaments invariably revealed two uninterrupted columns of mass forming the "backbone" of the double-helical filament. The contact between neighboring subunits along the thus defined two long-pitch helical strands was spatially conserved and of high mass density, while the intersubunit contact between them was of lower mass density and varied among reconstructions. In contrast, phalloidinstabilized F-actin filaments displayed higher and spatially more conserved mass density between the two long-pitch helical strands, suggesting that this bicyclic hepta-peptide toxin strengthens the intersubunit contact between the two strands. Consistent with this distinct intersubunit bonding pattern, the two long-pitch helical strands of unstabilized filaments were sometimes observed separated from each other over a distance of two to six subunits, suggesting that the intrastrand intersubunit contact is also physically stronger than the interstrand contact. The resolution of the filament reconstructions, extending to 2.5 nm axially and radially, enabled us to reproducibly "cut out" the F-actin subunit which measured 5.5 nm axially by 6.0 nm tangentially by 3.2 nm radially. The subunit is distinctly polar with a massive "base" pointing towards the "barbed" end of the filament, and a slender "tip" defining its "pointed" end (i.e., relative to the "arrowhead" pattern revealed after stoichiometric decoration of the filaments with myosin subfragment 1). Concavities running approximately parallel to the filament axis both on the inner and outer face of the subunit define a distinct cleft separating the subunit into two domains of similar size: an inner domain confined to radii less than or equal to 2.5-nm forms the uninterrupted backbone of the two long-pitch helical strands, and an outer domain placed at radii of 2-5-nm protrudes radially and thus predominantly

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

    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

  4. Fibronectin in cultured rat keratinocytes: distribution, synthesis, and relationship to cytoskeletal proteins

    Gibson, W T; Couchman, J R; Badley, R A; Saunders, H J; Smith, C G

    1983-01-01

    The aim of this study was to investigate whether epidermal cells can synthesise fibronectin and whether the distribution of this glycoprotein is related to the adhesion and cytoskeletal organisation of these cells. The production of fibronectin by newborn rat epidermal cells was shown by indirect...... immunofluorescence staining of cultures grown in the absence of a feeder layer using an antiserum which had been cross-adsorbed with foetal calf serum proteins to remove antibodies which recognised serum fibronectin. The distribution of fibronectin in areas of cell-cell and cell-substratum contact...... revealed evidence of a relationship between external fibronectin and internal structure in epidermal cells. Immunofluorescence showed that tonofilaments (keratin) and microtubules were present as fibrillar arrays but were not related to fibronectin distribution. Vimentin and desmin were absent. Actin was...

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

    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.

  6. Cytoskeletal reorganizations in human umbilical vein endothelial cells as a result of cytokine exposure

    Treatment of HUVECs in culture with several cytokines and phorbol esters caused reorganizations of the actin and microtubule networks, as well as a redistribution of focal contract proteins. However, expression of the cytoskeletal proteins which link cells, via integrins, to the substrate, was not significantly affected. Indirect immunofluorescence microscopy of endothelial cells after treatment with interleukin-1 alpha and beta, gamma-interferon, tumor necrosis factor (TNF), phorbol 12-myristate 13-acetate, and phorbol 12,13-dibutyrate allowed us to observe reductions in the areas of cell-cell contact, redistribution of the stress fiber network, and concomitant changes in focal contacts. Microtubule arrays in TNF-treated cells became bundled. Phorbol esters induced formation of microtubule organizing centers not seen in resting or TNF-treated HUVECs. Talin was distributed along stress fibers and not exclusively in focal contacts. Vitronectin receptor was observed in focal contacts, occasionally at cell-cell contacts, and in vesicular structures close to the lumenal surface, after both types of treatment. Although these morphological changes were easily observed by indirect immunofluorescence, no quantitative differences in specific cytoskeletal proteins were detected by immunoblots and [35S]cysteine metabolic labeling experiments

  7. Cytoskeletal elements in the bacterium Mycoplasma pneumoniae

    Hegermann, Jan; Herrmann, Richard; Mayer, Frank

    2002-09-01

    Mycoplasma pneumoniae is a pathogenic eubacterium lacking a cell wall. Three decades ago, a "rod", an intracellular cytoskeletal structure, was discovered that was assumed to define and stabilize the elongated cell shape. Later, by treatment with detergent, a "Triton shell" (i.e. a fraction of detergent-insoluble cell material) could be obtained, believed to contain additional cytoskeletal elements. Now, by application of a modified Triton X-100 treatment, we are able to demonstrate that M. pneumoniae possesses a cytoskeleton consisting of a blade-like rod and a peripheral lining located close to the inner face of the cytoplasmic membrane, exhibiting features of a highly regular network. Attached "stalks" may support the cytoplasmic membrane. The rod was connected to the cell periphery by "spokes" and showed a defined ultrastructure. Its proximal end was found to be attached to a wheel-like complex. Fibrils extended from the proximal end of the rod into the cytoplasm.

  8. The structural basis for the intrinsic disorder of the actin filament: the "lateral slipping" model

    1991-01-01

    Three-dimensional (3-D) helical reconstructions computed from electron micrographs of negatively stained dispersed F-actin filaments invariably revealed two uninterrupted columns of mass forming the "backbone" of the double-helical filament. The contact between neighboring subunits along the thus defined two long-pitch helical strands was spatially conserved and of high mass density, while the intersubunit contact between them was of lower mass density and varied among reconstructions. In con...

  9. Cytoskeletal binding proteins distinguish cultured dental follicle cells and periodontal ligament cells.

    Li, Jie; Li, Hui; Tian, Ye; Yang, Yaling; Chen, Guoqing; Guo, Weihua; Tian, Weidong

    2016-07-01

    Human dental follicle cells (DFCs) and periodontal ligament cells (PDLCs) derived from the ectomesenchymal tissue, have been shown to exhibit stem/progenitor cell properties and the ability to induce tissue regeneration. Stem cells in dental follicle differentiate into cementoblasts, periodontal ligament fibroblasts and osteoblasts, these cells form cementum, periodontal ligament and alveolar bone, respectively. While stem cells in dental follicle are a precursor to periodontal ligament fibroblasts, the molecular changes that distinguish cultured DFCs from PDLCs are still unknown. In this study, we have compared the immunophenotypic features and cell cycle status of the two cell lines. The results suggest that DFCs and PDLCs displayed similar features related to immunophenotype and cell cycle. Then we employed an isobaric tag for relative and absolute quantitation (iTRAQ) proteomics strategy to reveal the molecular differences between the two cell types. A total of 2138 proteins were identified and 39 of these proteins were consistently differentially expressed between DFCs and PDLCs. Gene ontology analyses revealed that the protein subsets expressed higher in PDLCs were related to actin binding, cytoskeletal protein binding, and structural constituent of muscle. Upon validation by real-time PCR, western blotting, and immunofluorescence staining. Tropomyosin 1 (TPM1) and caldesmon 1 (CALD1) were expressed higher in PDLCs than in DFCs. Our results suggested that PDLCs display enhanced actin cytoskeletal dynamics relative to DFCs while DFCs may exhibit a more robust antioxidant defense ability relative to PDLCs. This study expands our knowledge of the cultured DFCs and PDLCs proteome and provides new insights into possible mechanisms responsible for the different biological features observed in each cell type. PMID:26708290

  10. Forcing it on: Cytoskeletal dynamics during lymphocyte activation

    Upadhyaya, Arpita

    2012-02-01

    Formation of the immune synapse during lymphocyte activation involves cell spreading driven by large scale physical rearrangements of the actin cytoskeleton and the cell membrane. Several recent observations suggest that mechanical forces are important for efficient T cell activation. How forces arise from the dynamics of the cytoskeleton and the membrane during contact formation, and their effect on signaling activation is not well understood. We have imaged membrane topography, actin dynamics and the spatiotemporal localization of signaling clusters during the very early stages of spreading. Formation of signaling clusters was closely correlated with the movement and topography of the membrane in contact with the activating surface. Further, we observed membrane waves driven by actin polymerization originating at these signaling clusters. Actin-driven membrane protrusions likely play an important role in force generation at the immune synapse. In order to study cytoskeletal forces during T-cell activation, we studied cell spreading on elastic gels. We found that gel stiffness influences cell morphology, actin dynamics and receptor activation. Efforts to determine the quantitative relationships between cellular forces and signaling are underway. Our results suggest a role for cytoskeleton driven forces during signaling activation in lymphocytes.

  11. Regulation of cytoskeletal dynamics by redox signaling and oxidative stress: implications for neuronal development and trafficking

    Christian Gonzalez-Billault

    2015-09-01

    Full Text Available A proper balance between chemical reduction and oxidation (known as redox balance is essential for normal cellular physiology. Deregulation in the production of oxidative species leads to DNA damage, lipid peroxidation and aberrant post-translational modification of proteins, which in most cases induces injury, cell death and disease. However, physiological concentrations of oxidative species are necessary to support important cell functions, such as chemotaxis, hormone synthesis, immune response, cytoskeletal remodeling, Ca2+ homeostasis and others. Recent evidence suggests that redox balance regulates actin and microtubule dynamics in both physiological and pathological contexts. Microtubules and actin microfilaments contain certain amino acid residues that are susceptible to oxidation, which reduces the ability of microtubules to polymerize and causes severing of actin microfilaments in neuronal and non-neuronal cells. In contrast, inhibited production of reactive oxygen species (e.g., due to NOXs leads to aberrant actin polymerization, decreases neurite outgrowth and affects the normal development and polarization of neurons. In this review, we summarize emerging evidence suggesting that both general and specific enzymatic sources of redox species exert diverse effects on cytoskeletal dynamics. Considering the intimate relationship between cytoskeletal dynamics and trafficking, we also discuss the potential effects of redox balance on intracellular transport via regulation of the components of the microtubule and actin cytoskeleton as well as cytoskeleton-associated proteins, which may directly impact localization of proteins and vesicles across the soma, dendrites and axon of neurons.

  12. Imaging the fine-scale structure of the cellular actin cytoskeleton by Single Particle Tracking and Atomic Force Microscopy

    Mustata, Gina-Mirela

    It has been proposed that diffusion in the plasma membrane of eukaryotic cells it is compartmentalized due to the interaction with the underlying actin-based membrane skeleton that comes into close proximity to the lipid bilayer. The cytoskeleton is a dynamic structure that maintains cell shape, enables cell motion, and plays important roles in both intra-cellular transport and cellular division. We show here the evidence of plasma membrane compartmentalization using Single Particle Tracking (SPT) and Atomic Force Microscopy (AFM) imaging. SPT of Quantum dot labeled lipid in the plasma membrane of live normal rat kidney cells show compartments ranging from 325 nm to 391 nm depending on the sampling time. Using AFM imaging of live NRK cell in the presence of phalloidin, the membrane compartmentalization it is visible with the average size of the compartments of 325 +/- 10 nm (the main peak is centered at 260 nm). Further, the underlying membrane skeleton in fixed cells was directly imaged after partial removal of the plasma membrane to reveal size of the membrane skeleton meshwork of 339 +/- 10 nm. A new method of measuring the characteristics of the actin meshwork was proposed. Probing the local compliance of the plasma membrane through the deflection of a soft AFM cantilever we can expect that the stiffness of the membrane will be higher at locations directly above a cortical actin. This new method provided information about the structure of the skeletal meshwork of neuronal cell body predicting an average compartment size of about 132 nm. This was confirmed through SPT of QD-lipid incorporated into the neuronal cell membrane.

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

    Kitamura, H.; Matsumori, H.; Kalendová, Alžběta; Hozák, Pavel; Goldberg, I.G.; Nakao, M.; Saitoh, N.; Harata, M.

    2015-01-01

    Roč. 464, č. 2 (2015), s. 554-560. ISSN 0006-291X R&D Projects: GA MŠk EE2.3.30.0050; GA MŠk(CZ) ED1.1.00/02.0109 Grant ostatní: Human Frontier in Science programe(FR) RGP0017/2013 Institutional support: RVO:68378050 Keywords : Actin-related protein * ARP6 * Histone H2A.Z * Nucleolus * Wndchrm Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.297, year: 2014

  14. Probing mechanics and activity of cytoskeletal networks using carbon nanotubes

    Fakhri, Nikta

    2013-03-01

    We use single-walled carbon nanotubes (SWNTs) as multi-scale micro-probes to monitor transport and fluctuations in cytoskeletal networks. SWNTs are nanometer-diameter hollow carbon filaments with micrometer lengths and a tunable bending stiffness. Their persistence length varies between 20-100 microns. We study the motion of individual SWNTs in reconstituted actin networks by near-infrared fluorescence microscopy. At long times, SWNTs reptate through the networks. At short times, SWNTs sample the spectrum of thermal fluctuations in the networks. We can calculate complex shear moduli from recorded fluctuations and observe power-law scaling in equilibrium actin networks. In the non-equilibrium cytoskeleton of cells we have targeted SWNTs to kinesin motors and thereby to their microtubule tracks. We observe both transport along the tracks as well as active fluctuations of the tracks themselves. Human Frontier Science Program Cross-Disciplinary Fellow

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

    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

  16. Regulation of the formation of osteoclastic actin rings by proline-rich tyrosine kinase 2 interacting with gelsolin

    Wang, Qiang; Xie, Yi; Du, Quan-Sheng; Wu, Xiao-Jun; FENG, XU; Mei, Lin; McDonald, Jay M.; Xiong, Wen-Cheng

    2003-01-01

    Osteoclast activation is important for bone remodeling and is altered in multiple bone disorders. This process requires cell adhesion and extensive actin cytoskeletal reorganization. Proline-rich tyrosine kinase 2 (PYK2), a major cell adhesion–activated tyrosine kinase in osteoclasts, plays an important role in regulating this event. The mechanisms by which PYK2 regulates actin cytoskeletal organization and osteoclastic activation remain largely unknown. In this paper, we provide evidence tha...

  17. Regulation of T cell receptor signaling by the actin cytoskeleton and poroelastic cytoplasm

    Beemiller, Peter; Krummel, Matthew F.

    2013-01-01

    The actin cytoskeleton plays essential roles in modulating T-cell activation. Most models of T-cell receptor (TCR) triggering, signalosome assembl, y and immune synapse formation invoke actin-dependent mechanisms. As T cells are constitutively motile cells, TCR triggering and signaling occur against a cytoskeletal backdrop that is constantly remodeling. While the interplay between actin dynamics and TCR signaling have been the focus of research for many years, much of the work in T cells has ...

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

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

    2016-09-01

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

  19. A radioimmunoassay for determination of anti-actin antibodies

    The reaction of spontaneously occurring human anti-actin antibodies and experimentally produced rabbit anti-actin antibodies was investigated in a solid-phase radioimmunoassay (RIA). Three structurally different in vitro forms of actin, monomeric G-actin, filamentous F-actin and aggregated denatured actin were used as antigens. Human anti-actin antibodies reacted with F- and G-actin but not with aggregated actin, while rabbit anti-actin antibodies gave a strong reaction with all 3 forms of actin indicating differences in antibody specificities. The results of the anti-actin RIA were compared with those obtained by indirect immunofluorescence (IFL) on cryostat sections of rat stomach. The anti-actin RIA discriminated between patients' sera and control sera in most cases, although the indirect IFL test gave more conclusive results. The seemingly low sensitivity of the anti-actin RIA compared with that of indirect IFL test for detection of human anti-actin antibodies is probably due to favourable antigen distribution in tissue, not available in the solid phase. The anti-actin RIA was able to detect anti-actin antibodies in 8 out of 8 immunized rabbits although only two produced antibodies detectable by indirect IFL. The differences in reactivity between the two methods may depend on the presence of aggregated denatured actin in the antigen preparation used for immunization and exposure of the corresponding antigenic determinants of actin on the solid phase. (Auth.)

  20. Role of active contraction and tropomodulins in regulating actin filament length and sarcomere structure in developing zebrafish skeletal muscle

    Lise eMazelet

    2016-03-01

    Full Text Available Whilst it is recognised that contraction plays an important part in maintaining the structure and function of mature skeletal muscle, its role during development remains undefined. In this study the role of movement in skeletal muscle maturation was investigated in intact zebrafish embryos using a combination of genetic and pharmacological approaches. An immotile mutant line (cacnb1ts25 which lacks functional voltage-gated calcium channels (dihydropyridine receptors in the muscle and pharmacological immobilisation of embryos with a reversible anaesthetic (Tricaine, allowed the study of paralysis (in mutants and anaesthetised fish and recovery of movement (reversal of anaesthetic treatment. The effect of paralysis in early embryos (aged between 17-24 hours post fertilisation, hpf on skeletal muscle structure at both myofibrillar and myofilament level was determined using both immunostaining with confocal microscopy and small angle X-ray diffraction. The consequences of paralysis and subsequent recovery on the localisation of the actin capping proteins Tropomodulin 1 &4 (Tmod in fish aged from 17hpf until 42hpf was also assessed. The functional consequences of early paralysis were investigated by examining the mechanical properties of the larval muscle. The length-force relationship, active and passive tension, was measured in immotile, recovered and control skeletal muscle at 5 and 7 day post fertilisation (dpf. Recovery of muscle function was also assessed by examining swimming patterns in recovered and control fish. Inhibition of the initial embryonic movements (up to 24 hpf resulted in an increase in myofibril length and a decrease in width followed by almost complete recovery in both moving and paralysed fish by 42hpf. In conclusion, myofibril organisation is regulated by a dual mechanism involving movement-dependent and movement-independent processes. The initial contractile event itself drives the localisation of Tmod1 to its sarcomeric

  1. Initiation of Chondrocyte Self-Assembly Requires an Intact Cytoskeletal Network.

    Lee, Jennifer K; Hu, Jerry C Y; Yamada, Soichiro; Athanasiou, Kyriacos A

    2016-02-01

    Self-assembly and self-organization have recently emerged as robust scaffold-free tissue engineering methodologies that can be used to generate various tissues, including cartilage, vessel, and liver. Self-assembly, in particular, is a scaffold-free platform for tissue engineering that does not require the input of exogenous energy to the system. Although self-assembly can generate functional tissues, most notably neocartilage, the mechanisms of self-assembly remain unclear. To study the self-assembling process, we used articular chondrocytes as a model to identify parameters that can affect this process. Specifically, the roles of cell-cell and cell-matrix adhesion molecules, surface-bound collagen, and the actin cytoskeletal network were investigated. Using time-lapse imaging, we analyzed the early stages of chondrocyte self-assembly. Within hours, chondrocytes rapidly coalesced into cell clusters before compacting to form tight cellular structures. Chondrocyte self-assembly was found to depend primarily on integrin function and secondarily on cadherin function. In addition, actin or myosin II inhibitors prevented chondrocyte self-assembly, suggesting that cell adhesion alone is not sufficient, but rather the active contractile actin cytoskeleton is essential for proper chondrocyte self-assembly and the formation of neocartilage. Better understanding of the self-assembly mechanisms allows for the rational modulation of this process toward generating neocartilages with improved properties. These findings are germane to understanding self-assembly, an emerging platform for tissue engineering of a plethora of tissues, especially as these neotissues are poised for translation. PMID:26729374

  2. The structural dynamics of α-tropomyosin on F-actin shape the overlap complex between adjacent tropomyosin molecules

    Lehman, William; Li, Xiaochuan; Orzechowski, Marek; Fischer, Stefan

    2013-01-01

    Coiled-coil tropomyosin, localized on actin filaments in virtually all eukaryotic cells, serves as a gatekeeper regulating access of the motor protein myosin and other actin-binding proteins onto the thin filament surface. Tropomyosin's modular pseudo-repeating pattern of approximately 39 amino acid residues is designed to allow binding of the coiled-coil to successive actin subunits along thin filaments. Even though different tropomyosin isoforms contain varying numbers of repeat modules, th...

  3. Spiral actin-polymerization waves can generate amoeboidal cell crawling

    Amoeboidal cell crawling on solid substrates is characterized by protrusions that seemingly appear randomly along the cell periphery and drive the cell forward. For many cell types, it is known that the protrusions result from polymerization of the actin cytoskeleton. However, little is known about how the formation of protrusions is triggered and whether the appearance of subsequent protrusions is coordinated. Recently, the spontaneous formation of actin-polymerization waves was observed. These waves have been proposed to orchestrate the cytoskeletal dynamics during cell crawling. Here, we study the impact of cytoskeletal polymerization waves on cell migration using a phase-field approach. In addition to directionally moving cells, we find states reminiscent of amoeboidal cell crawling. In this framework, new protrusions are seen to emerge from a nucleation process, generating spiral actin waves in the cell interior. Nucleation of new spirals does not require noise, but occurs in a state that is apparently displaying spatio-temporal chaos. (paper)

  4. Monitoring the cytoskeletal EGF response in live gastric carcinoma cells.

    Marco Felkl

    Full Text Available Altered cell motility is considered to be a key factor in determining tumor invasion and metastasis. Epidermal growth factor (EGF signaling has been implicated in this process by affecting cytoskeletal organization and dynamics in multiple ways. To sort the temporal and spatial regulation of EGF-dependent cytoskeletal re-organization in relation to a cell's motile behavior time-lapse microscopy was performed on EGF-responsive gastric carcinoma-derived MKN1 cells co-expressing different fluorescently labeled cytoskeletal filaments and focal adhesion components in various combinations. The experiments showed that EGF almost instantaneously induces a considerable increase in membrane ruffling and lamellipodial activity that can be inhibited by Cetuximab EGF receptor antibodies and is not elicited in non-responsive gastric carcinoma Hs746T cells. The transient cell extensions are rich in actin but lack microtubules and keratin intermediate filaments. We show that this EGF-induced increase in membrane motility can be measured by a simple image processing routine. Microtubule plus-ends subsequently invade growing cell extensions, which start to accumulate focal complexes at the lamellipodium-lamellum junction. Such paxillin-positive complexes mature into focal adhesions by tyrosine phosphorylation and recruitment of zyxin. These adhesions then serve as nucleation sites for keratin filaments which are used to enlarge the neighboring peripheral keratin network. Focal adhesions are either disassembled or give rise to stable zyxin-rich fibrillar adhesions which disassemble in the presence of EGF to support formation of new focal adhesion sites in the cell periphery. Taken together the results serve as a basis for modeling the early cytoskeletal EGF response as a tightly coordinated and step-wise process which is relevant for the prediction of the effectiveness of anti-EGF receptor-based tumor therapy.

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

    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.

  6. Photoelectron microscopy and immunofluorescence microscopy of cytoskeletal elements in the same cells.

    Nadakavukaren, K K; Chen, L. B.; Habliston, D L; Griffith, O. H.

    1983-01-01

    Pt K2 rat kangaroo epithelial cells and Rat-1 fibroblasts were grown on conductive glass discs, fixed, and permeabilized, and the cytoskeletal elements actin, keratin, and vimentin were visualized by indirect immunofluorescence. After the fluorescence microscopy, the cells were postfixed and dehydrated for photoelectron microscopy. The contrast in these photoelectron micrographs is primarily topographical in origin, and the presence of fluorescent dyes at low density does not contribute signi...

  7. PTP-PEST controls EphA3 activation and ephrin-induced cytoskeletal remodelling.

    Mansour, Mariam; Nievergall, Eva; Gegenbauer, Kristina; Llerena, Carmen; Atapattu, Lakmali; Hallé, Maxime; Tremblay, Michel L; Janes, Peter W; Lackmann, Martin

    2016-01-15

    Eph receptors and their corresponding membrane-bound ephrin ligands regulate cell positioning and establish tissue patterns during embryonic and oncogenic development. Emerging evidence suggests that assembly of polymeric Eph signalling clusters relies on cytoskeletal reorganisation and underlies regulation by protein tyrosine phosphatases (PTPs). PTP-PEST (also known as PTPN12) is a central regulator of actin cytoskeletal dynamics. Here, we demonstrate that an N-terminal fragment of PTP-PEST, generated through an ephrinA5-triggered and spatially confined cleavage mediated by caspase-3, attenuates EphA3 receptor activation and its internalisation. Isolation of EphA3 receptor signalling clusters within intact plasma membrane fragments obtained by detergent-free cell fractionation reveals that stimulation of cells with ephrin triggers effective recruitment of this catalytically active truncated form of PTP-PEST together with key cytoskeletal and focal adhesion proteins. Importantly, modulation of actin polymerisation using pharmacological and dominant-negative approaches affects EphA3 phosphorylation in a similar manner to overexpression of PTP-PEST. We conclude that PTP-PEST regulates EphA3 activation both by affecting cytoskeletal remodelling and through its direct action as a PTP controlling EphA3 phosphorylation, indicating its multifaceted regulation of Eph signalling. PMID:26644181

  8. Conservation and divergence between cytoplasmic and muscle-specific actin capping proteins: insights from the crystal structure of cytoplasmic Cap32/34 from Dictyostelium discoideum

    Eckert Christian

    2012-06-01

    Full Text Available Abstract Background Capping protein (CP, also known as CapZ in muscle cells and Cap32/34 in Dictyostelium discoideum, plays a major role in regulating actin filament dynamics. CP is a ubiquitously expressed heterodimer comprising an α- and β-subunit. It tightly binds to the fast growing end of actin filaments, thereby functioning as a “cap” by blocking the addition and loss of actin subunits. Vertebrates contain two somatic variants of CP, one being primarily found at the cell periphery of non-muscle tissues while the other is mainly localized at the Z-discs of skeletal muscles. Results To elucidate structural and functional differences between cytoplasmic and sarcomercic CP variants, we have solved the atomic structure of Cap32/34 (32 = β- and 34 = α-subunit from the cellular slime mold Dictyostelium at 2.2 Å resolution and compared it to that of chicken muscle CapZ. The two homologs display a similar overall arrangement including the attached α-subunit C-terminus (α-tentacle and the flexible β-tentacle. Nevertheless, the structures exhibit marked differences suggesting considerable structural flexibility within the α-subunit. In the α-subunit we observed a bending motion of the β-sheet region located opposite to the position of the C-terminal β-tentacle towards the antiparallel helices that interconnect the heterodimer. Recently, a two domain twisting attributed mainly to the β-subunit has been reported. At the hinge of these two domains Cap32/34 contains an elongated and highly flexible loop, which has been reported to be important for the interaction of cytoplasmic CP with actin and might contribute to the more dynamic actin-binding of cytoplasmic compared to sarcomeric CP (CapZ. Conclusions The structure of Cap32/34 from Dictyostelium discoideum allowed a detailed analysis and comparison between the cytoplasmic and sarcomeric variants of CP. Significant structural flexibility could particularly be found within the

  9. Molecular Mechanotransduction: how forces trigger cytoskeletal dynamics

    Ehrlicher, Allen

    2012-02-01

    Mechanical stresses elicit cellular reactions mediated by chemical signals. Defective responses to forces underlie human medical disorders, such as cardiac failure and pulmonary injury. Despite detailed knowledge of the cytoskeleton's structure, the specific molecular switches that convert mechanical stimuli into chemical signals have remained elusive. Here we identify the actin-binding protein, filamin A (FLNa) as a central mechanotransduction element of the cytoskeleton by using Fluorescence Loss After photoConversion (FLAC), a novel high-speed alternative to FRAP. We reconstituted a minimal system consisting of actin filaments, FLNa and two FLNa-binding partners: the cytoplasmic tail of ß-integrin, and FilGAP. Integrins form an essential mechanical linkage between extracellular and intracellular environments, with ß integrin tails connecting to the actin cytoskeleton by binding directly to filamin. FilGAP is a FLNa-binding GTPase-activating protein specific for Rac, which in vivo regulates cell spreading and bleb formation. We demonstrate that both externally-imposed bulk shear and myosin II driven forces differentially regulate the binding of integrin and FilGAP to FLNa. Consistent with structural predictions, strain increases ß-integrin binding to FLNa, whereas it causes FilGAP to dissociate from FLNa, providing a direct and specific molecular basis for cellular mechanotransduction. These results identify the first molecular mechanotransduction element within the actin cytoskeleton, revealing that mechanical strain of key proteins regulates the binding of signaling molecules. Moreover, GAP activity has been shown to switch cell movement from mesenchymal to amoeboid motility, suggesting that mechanical forces directly impact the invasiveness of cancer.

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

    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. PMID:23644458

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

    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

  12. Head-neck domain of Arabidopsis myosin XI, MYA2, fused with GFP produces F-actin patterns that coincide with fast organelle streaming in different plant cells

    Holweg Carola L

    2008-07-01

    Full Text Available Abstract Background The cytoskeletal mechanisms that underlie organelle transport in plants are intimately linked to acto-myosin function. This function is mediated by the attachment of myosin heads to F-actin and the binding of cargo to the tails. Acto-myosin also powers vigorous cytoplasmic streaming in plant cells. Class XI myosins exhibit strikingly fast velocities and may have extraordinary roles in cellular motility. Studies of the structural basis of organelle transport have focused on the cargo-binding tails of myosin XI, revealing a close relationship with the transport of peroxisomes, mitochondria, and Golgi-vesicles. Links between myosin heads and F-actin-based motility have been less investigated. To address this function, we performed localization studies using the head-neck domain of AtMYA2, a myosin XI from Arabidopsis. Results We expressed the GFP-fused head-neck domain of MYA2 in epidermal cells of various plant species and found that it associated with F-actin. By comparison to other markers such as fimbrin and talin, we revealed that the myosin-labeled F-actin was of a lower quality and absent from the fine microfilament arrays at the cell cortex. However, it colocalized with cytoplasmic (transvacuolar F-actin in areas coinciding with the tracks of fast organelles. This observation correlates well with the proposed function of myosin XI in organelle trafficking. The fact that organelle streaming was reduced in cells expressing the GFP-MYA2-head6IQ indicated that the functionless motor protein inhibits endogenous myosins. Furthermore, co-expression of the GFP-MYA2-head6IQ with other F-actin markers disrupted its attachment to F-actin. In nuclei, the GFP-myosin associated with short bundles of F-actin. Conclusion The localization of the head of MYA2 in living plant cells, as investigated here for the first time, suggests a close linkage between this myosin XI and cytoplasmic microfilaments that support the rapid streaming of

  13. From pollen actin to crop male sterility

    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.

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

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

    2012-01-01

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

  15. Calponin 3 Regulates Actin Cytoskeleton Rearrangement in Trophoblastic Cell Fusion

    Shibukawa, Yukinao; Yamazaki, Natsuko; Kumasawa, Keiichi; Daimon, Etsuko; Tajiri, Michiko; Okada, Yuka; Ikawa, Masahito; Wada, Yoshinao

    2010-01-01

    Cell–cell fusion is an intriguing differentiation process, essential for placental development and maturation. A proteomic approach identified a cytoplasmic protein, calponin 3 (CNN3), related to the fusion of BeWo choriocarcinoma cells. CNN3 was expressed in cytotrophoblasts in human placenta. CNN3 gene knockdown promoted actin cytoskeletal rearrangement and syncytium formation in BeWo cells, suggesting CNN3 to be a negative regulator of trophoblast fusion. Indeed, CNN3 depletion promoted Be...

  16. Architecture and Connectivity Govern Actin Network Contractility.

    Ennomani, Hajer; Letort, Gaëlle; Guérin, Christophe; Martiel, Jean-Louis; Cao, Wenxiang; Nédélec, François; De La Cruz, Enrique M; Théry, Manuel; Blanchoin, Laurent

    2016-03-01

    Actomyosin contractility plays a central role in a wide range of cellular processes, including the establishment of cell polarity, cell migration, tissue integrity, and morphogenesis during development. The contractile response is variable and depends on actomyosin network architecture and biochemical composition. To determine how this coupling regulates actomyosin-driven contraction, we used a micropatterning method that enables the spatial control of actin assembly. We generated a variety of actin templates and measured how defined actin structures respond to myosin-induced forces. We found that the same actin filament crosslinkers either enhance or inhibit the contractility of a network, depending on the organization of actin within the network. Numerical simulations unified the roles of actin filament branching and crosslinking during actomyosin contraction. Specifically, we introduce the concept of "network connectivity" and show that the contractions of distinct actin architectures are described by the same master curve when considering their degree of connectivity. This makes it possible to predict the dynamic response of defined actin structures to transient changes in connectivity. We propose that, depending on the connectivity and the architecture, network contraction is dominated by either sarcomeric-like or buckling mechanisms. More generally, this study reveals how actin network contractility depends on its architecture under a defined set of biochemical conditions. PMID:26898468

  17. The interplay of nonlinearity and architecture in equilibrium cytoskeletal mechanics.

    Wang, Shenshen; Shen, Tongye; Wolynes, Peter G

    2011-01-01

    The interplay between cytoskeletal architecture and the nonlinearity of the interactions due to bucklable filaments plays a key role in modulating the cell's mechanical stability and affecting its structural rearrangements. We study a model of cytoskeletal structure treating it as an amorphous network of hard centers rigidly cross-linked by nonlinear elastic strings, neglecting the effects of motorization. Using simulations along with a self-consistent phonon method, we show that this minimal model exhibits diverse thermodynamically stable mechanical phases that depend on excluded volume, cross-link concentration, filament length, and stiffness. Within the framework set by the free energy functional formulation and making use of the random first order transition theory of structural glasses, we further estimate the characteristic densities for a kinetic glass transition to occur in this model system. Network connectivity strongly modulates the transition boundaries between various equilibrium phases, as well as the kinetic glass transition density. PMID:21219010

  18. Regulation of T cell receptor signaling by the actin cytoskeleton and poroelastic cytoplasm

    Beemiller, Peter; Krummel, Matthew F.

    2013-01-01

    Summary The actin cytoskeleton plays essential roles in modulating T-cell activation. Most models of T-cell receptor (TCR) triggering, signalosome assembl, y and immune synapse formation invoke actin-dependent mechanisms. As T cells are constitutively motile cells, TCR triggering and signaling occur against a cytoskeletal backdrop that is constantly remodeling. While the interplay between actin dynamics and TCR signaling have been the focus of research for many years, much of the work in T cells has considered actin largely for its ‘scaffolding’ function. We examine the roles of the actin cytoskeleton in TCR signaling and immune synapse formation with an emphasis on how poroelasticity, an ensemble feature of actin dynamics with the cytosol, relates to how T cells respond to stimulation. PMID:24117819

  19. Regulation of T-cell receptor signaling by the actin cytoskeleton and poroelastic cytoplasm.

    Beemiller, Peter; Krummel, Matthew F

    2013-11-01

    The actin cytoskeleton plays essential roles in modulating T-cell activation. Most models of T-cell receptor (TCR) triggering signalosome assembly and immune synapse formation invoke actin-dependent mechanisms. As T cells are constitutively motile cells, TCR triggering and signaling occur against a cytoskeletal backdrop that is constantly remodeling. While the interplay between actin dynamics and TCR signaling have been the focus of research for many years, much of the work in T cells has considered actin largely for its 'scaffolding' function. We examine the roles of the actin cytoskeleton in TCR signaling and immune synapse formation with an emphasis on how poroelasticity, an ensemble feature of actin dynamics with the cytosol, relates to how T cells respond to stimulation. PMID:24117819

  20. Cell stress promotes the association of phosphorylated HspB1 with F-actin.

    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.

  1. The Cytoskeletal Regulatory Scaffold Protein GIT2 Modulates Mesenchymal Stem Cell Differentiation and Osteoblastogenesis

    Wang, Xiaojuan; Liao, Shaoxi; Nelson, Erik R.; Schmalzigaug, Robert; Spurney, Robert F.; Guilak, Farshid; Premont, Richard T.; Gesty-Palmer, Diane

    2012-01-01

    G protein-coupled receptor kinase interacting protein 2 (GIT2) is a signaling scaffold protein involved in the regulation of cytoskeletal structure, membrane trafficking, and G protein-coupled receptor internalization. Since dynamic cytoskeletal reorganization plays key roles both in osteoblast differentiation and in the maintenance of osteoclast polarity during bone resorption, we hypothesized that skeletal physiology would be altered in GIT2−/− mice. We found that adult GIT2−/− mice have de...

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

    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

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

    Nadia D'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.

  4. Stiffening of Red Blood Cells Induced by Disordered Cytoskeleton Structures: A Joint Theory-experiment Study

    Lai, Lipeng; Lim, Chwee Teck; Cao, Jianshu

    2015-01-01

    The functions and elasticities of the cell are largely related to the structures of the cytoskeletons underlying the lipid bi-layer. Among various cell types, the Red Blood Cell (RBC) possesses a relatively simple cytoskeletal structure. Underneath the membrane, the RBC cytoskeleton takes the form of a two dimensional triangular network, consisting of nodes of actins (and other proteins) and edges of spectrins. Recent experiments focusing on the malaria infected RBCs (iRBCs) showed that there is a correlation between the elongation of spectrins in the cytoskeletal network and the stiffening of the iRBCs. Here we rationalize the correlation between these two observations by combining the worm-like chain (WLC) model for single spectrins and the Effective Medium Theory (EMT) for the network elasticity. We specifically focus on how the disorders in the cytoskeletal network affect its macroscopic elasticity. Analytical and numerical solutions from our model reveal that the stiffness of the membrane increases with ...

  5. Orexin A reverses propofol and thiopental induced cytoskeletal rearrangement in rat neurons.

    Turina, D; Gerhardsson, H; Bjornstrom, K

    2014-08-01

    Orexin A (OA) is an endogenous peptide regulating awakefulness, known to reduce anaesthesia in animals, but on cellular level its mechanisms to reverse anaesthetics are unknown. Primary cortical cell cultures from newborn rat brains are used and live cell light microscopy is performed to measure 1) neurite retraction after propofol, thiopental, barbituric acid and ketamine exposure and 2) the effect of OA application either before or after anaesthetics. Cytoskeletal reorganization is evaluated with fluorescence microscopy, protein changes are detected with Western blots and mass spectrometry is used to identify proteins after treatment with anaesthetics and/or OA. Adult rats are anaesthesized with propofol, and the cytoskeletal morphology is studied. Orexin A reverses and inhibits neurite retraction and actin ring formation induced by propofol and thiopental. No effect on retraction or actin rings was seen for ketamine (not active on gamma-aminobutiric acid A (GABA(A)) receptors), the non-anaesthetic barbituric acid, OA or solvents used. OA increases the tyrosine phosphorylation of a 50 kDa protein, identified as vimentin. Propofol induces an immediate granular appearance of vimentin, which OAreverses to a smooth distribution. Cytoskeletal morphology changes are also induced by propofol in vivo. All OA effects are blocked with an orexin receptor1 (OX1) antagonist. We conclude that OA reverses the GABAA receptor mediated cellular effects of both propofol and thiopental in rat brain cells. The morphologic changes of actin and vimentin caused by propofol and thiopental, and the subsequent reversal by OA, deepens our understanding of the mechanisms of anaesthesia. PMID:25179085

  6. Reinforcement versus fluidization in cytoskeletal mechanoresponsiveness.

    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.

  7. Hierarchical self-organization of cytoskeletal active networks

    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...

  8. Shear-induced reorganization of renal proximal tubule cell actin cytoskeleton and apical junctional complexes.

    Duan, Yi; Gotoh, Nanami; Yan, Qingshang; Du, Zhaopeng; Weinstein, Alan M; Wang, Tong; Weinbaum, Sheldon

    2008-08-12

    In this study, we demonstrate that fluid shear stress (FSS)-induced actin cytoskeletal reorganization and junctional formation in renal epithelial cells are nearly completely opposite the corresponding changes in vascular endothelial cells (ECs) [Thi MM et al. (2004) Proc Natl Acad Sci USA 101:16483-16488]. Mouse proximal tubule cells (PTCs) were subjected to 5 h of FSS (1 dyn/cm(2)) to investigate the dynamic responses of the cytoskeletal distribution of filamentous actin (F-actin), ZO-1, E-cadherin, vinculin, and paxillin to FSS. Immunofluorescence analysis revealed that FSS caused basal stress fiber disruption, more densely distributed peripheral actin bands (DPABs), and the formation of both tight junctions (TJs) and adherens junctions (AJs). A dramatic reinforcement of vinculin staining was found at the cell borders as well as the cell interior. These responses were abrogated by the actin-disrupting drug, cytochalasin D. To interpret these results, we propose a "junctional buttressing" model for PTCs in which FSS enables the DPABs, TJs, and AJs to become more tightly connected. In contrast, in the "bumper-car" model for ECs, all junctional connections were severely disrupted by FSS. This "junctional buttressing" model explains why a FSS of only 1/10 of that used in the EC study can cause a similarly dramatic, cytoskeletal response in these tall, cuboidal epithelial cells; and why junctional buttressing between adjacent cells may benefit renal epithelium in maximizing flow-activated, brush border-dependent, transcellular salt and water reabsorption. PMID:18685100

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

    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.

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

    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.

  11. Structures of the nucleoid occlusion protein SlmA bound to DNA and the C-terminal domain of the cytoskeletal protein FtsZ.

    Schumacher, Maria A; Zeng, Wenjie

    2016-05-01

    Cell division in most prokaryotes is mediated by FtsZ, which polymerizes to create the cytokinetic Z ring. Multiple FtsZ-binding proteins regulate FtsZ polymerization to ensure the proper spatiotemporal formation of the Z ring at the division site. The DNA-binding protein SlmA binds to FtsZ and prevents Z-ring formation through the nucleoid in a process called "nucleoid occlusion" (NO). As do most FtsZ-accessory proteins, SlmA interacts with the conserved C-terminal domain (CTD) that is connected to the FtsZ core by a long, flexible linker. However, SlmA is distinct from other regulatory factors in that it must be DNA-bound to interact with the FtsZ CTD. Few structures of FtsZ regulator-CTD complexes are available, but all reveal the CTD bound as a helix. To deduce the molecular basis for the unique SlmA-DNA-FtsZ CTD regulatory interaction and provide insight into FtsZ-regulator protein complex formation, we determined structures of Escherichia coli, Vibrio cholera, and Klebsiella pneumonia SlmA-DNA-FtsZ CTD ternary complexes. Strikingly, the FtsZ CTD does not interact with SlmA as a helix but binds as an extended conformation in a narrow, surface-exposed pocket formed only in the DNA-bound state of SlmA and located at the junction between the DNA-binding and C-terminal dimer domains. Binding studies are consistent with the structure and underscore key interactions in complex formation. Combined, these data reveal the molecular basis for the SlmA-DNA-FtsZ interaction with implications for SlmA's NO function and underscore the ability of the FtsZ CTD to adopt a wide range of conformations, explaining its ability to bind diverse regulatory proteins. PMID:27091999

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

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

    2014-01-01

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

  13. A role for the actin-bundling protein l-plastin in the regulation of leukocyte integrin function

    Jones, Samuel L.; Wang, Jun; Turck, Christoph W; Brown, Eric J.

    1998-01-01

    Regulation of leukocyte integrin avidity is a crucial aspect of inflammation and immunity. The actin cytoskeleton has an important role in the regulation of integrin function, but the cytoskeletal proteins involved are largely unknown. Because inflammatory stimuli that activate integrin-mediated adhesion in human polymorphonuclear neutrophils (PMN) and monocytes cause phosphorylation of the actin-bundling protein l-plastin, we tested whether l-plastin phosphorylation was involved in integrin ...

  14. ACF7 Is a Hair-Bundle Antecedent, Positioned to Integrate Cuticular Plate Actin and Somatic Tubulin

    Antonellis, Patrick J.; Pollock, Lana M.; Chou, Shih-Wei; Hassan, Ahmed; Geng, Ruishuang; Chen, Xi; Fuchs, Elaine; Alagramam, Kumar N.; Auer, Manfred; McDermott, Brian M., Jr.

    2014-01-01

    The precise morphology of the mechanosensitive hair bundle requires seamless integration of actin and microtubule networks. Here, we identify Acf7a (actin crosslinking family protein 7a) as a protein positioned to bridge these distinct cytoskeletal networks in hair cells. By imaging Acf7a–Citrine fusion protein in zebrafish and immunolabeling of vestibular and cochlear mouse hair cells, we show that Acf7a and ACF7 circumscribe, underlie, and are interwoven into the cuticular plate (CP), and t...

  15. The actin-binding ERM protein Moesin directly regulates spindle assembly and function during mitosis.

    Vilmos, Péter; Kristó, Ildikó; Szikora, Szilárd; Jankovics, Ferenc; Lukácsovich, Tamás; Kari, Beáta; Erdélyi, Miklós

    2016-06-01

    Ezrin-Radixin-Moesin proteins are highly conserved, actin-binding cytoskeletal proteins that play an essential role in microvilli formation, T-cell activation, and tumor metastasis by linking actin filaments to the plasma membrane. Recent studies demonstrated that the only Ezrin-Radixin-Moesin protein of Drosophila melanogaster, Moesin, is involved in mitotic spindle function through stabilizing cell shape and microtubules at the cell cortex. We previously observed that Moesin localizes to the mitotic spindle; hence, we tested for the biological significance of this surprising localization and investigated whether it plays a direct role in spindle function. To separate the cortical and spindle functions of Moesin during mitosis we combined cell biological and genetic methods. We used early Drosophila embryos, in which mitosis occurs in the absence of a cell cortex, and found in vivo evidence for the direct requirement of Moesin in mitotic spindle assembly and function. We also found that the accumulation of Moesin precedes the construction of the microtubule spindle, and the fusiform structure formed by Moesin persists even after the microtubules have disassembled. PMID:27006187

  16. Composition and structure of nucleolar skeleton (nucleolar matrix)——Actin and fibrillarin are two main protein components of nucleolar skeleton

    陈建明; 沈延; 焦仁杰; 翟中和

    1999-01-01

    Purified nucleoli of HeLa cells were treated sequentially with nonionic detergent, nucleic acid enzyme, low salt and high salt. The residual nucleolar structure termed nucleolar skeleton (nucleolar matrix) was shown as a fine network under electron microscope with DGD embedding-unembedding technique. Such structures of BHK-21 cell and mouse liver cell are similar to that of HeLa cell. The protein composition of the nucleolar skeleton of HeLa cells was analyzed. The protein composition of such nucleolar residual shows obvious difference from the compositions of nuclear matrix and chromosome scaffold. The major protein composition of the nucleolar skeleton of HeLa cells contains 6-7 polypeptides. Their molecular weights are about 48, 43, 36 and 33 ku. Further studies show that actin and fib-rillarin are two major protein components of nucleolar skeleton of HeLa cells.

  17. Impact of the phase defect structure on an actinic dark-field blank inspection signal and wafer printability

    Amano, Tsuyoshi; Murachi, Tetsunori; Yamane, Takeshi; Arisawa, Yukiyasu; Terasawa, Tsuneo

    2012-03-01

    A variety of phase defects (PDs) such as programmed bump and pit PDs, and native bump and pit PDs were detected by a dark-field ABI (Actinic Blank Inspection) tool. Among the PDs, some of them seemed to grow and propagate in an angular direction, away from substrate surface as was found by a TEM analysis. This presentation reports on the influence of 3-D phase defect on wafer printability, and on defect detection signals of an ABI tool. The result shows that the impact of the inclination angle on printing performance was quite significant when the PDs were not covered with the absorber pattern. On the other hand, the defect detection signal intensity was negligibly small in the case where the inclination angle was less than 9 degrees. However, ABI with its high magnification optics can pinpoint the PD's actual location as defined by the EUV light, rather than the ones that are not so clearly define by the surface topography.

  18. Cofilin-induced cooperative conformational changes of actin subunits revealed using cofilin-actin fusion protein

    Umeki, Nobuhisa; Hirose, Keiko; Uyeda, Taro Q. P.

    2016-01-01

    To investigate cooperative conformational changes of actin filaments induced by cofilin binding, we engineered a fusion protein made of Dictyostelium cofilin and actin. The filaments of the fusion protein were functionally similar to actin filaments bound with cofilin in that they did not bind rhodamine-phalloidin, had quenched fluorescence of pyrene attached to Cys374 and showed enhanced susceptibility of the DNase loop to cleavage by subtilisin. Quantitative analyses of copolymers made of different ratios of the fusion protein and control actin further demonstrated that the fusion protein affects the structure of multiple neighboring actin subunits in copolymers. Based on these and other recent related studies, we propose a mechanism by which conformational changes induced by cofilin binding is propagated unidirectionally to the pointed ends of the filaments, and cofilin clusters grow unidirectionally to the pointed ends following this path. Interestingly, the fusion protein was unable to copolymerize with control actin at pH 6.5 and low ionic strength, suggesting that the structural difference between the actin moiety in the fusion protein and control actin is pH-sensitive. PMID:26842224

  19. Dynamic Actin Controls Polarity Induction de novo in Protoplasts

    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.

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

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

    2016-07-18

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

  1. Calponin 3 regulates actin cytoskeleton rearrangement in trophoblastic cell fusion.

    Shibukawa, Yukinao; Yamazaki, Natsuko; Kumasawa, Keiichi; Daimon, Etsuko; Tajiri, Michiko; Okada, Yuka; Ikawa, Masahito; Wada, Yoshinao

    2010-11-15

    Cell-cell fusion is an intriguing differentiation process, essential for placental development and maturation. A proteomic approach identified a cytoplasmic protein, calponin 3 (CNN3), related to the fusion of BeWo choriocarcinoma cells. CNN3 was expressed in cytotrophoblasts in human placenta. CNN3 gene knockdown promoted actin cytoskeletal rearrangement and syncytium formation in BeWo cells, suggesting CNN3 to be a negative regulator of trophoblast fusion. Indeed, CNN3 depletion promoted BeWo cell fusion. CNN3 at the cytoplasmic face of cytoskeleton was dislocated from F-actin with forskolin treatment and diffused into the cytoplasm in a phosphorylation-dependent manner. Phosphorylation sites were located at Ser293/296 in the C-terminal region, and deletion of this region or site-specific disruption of Ser293/296 suppressed syncytium formation. These CNN3 mutants were colocalized with F-actin and remained there after forskolin treatment, suggesting that dissociation of CNN3 from F-actin is modulated by the phosphorylation status of the C-terminal region unique to CNN3 in the CNN family proteins. The mutant missing these phosphorylation sites displayed a dominant negative effect on cell fusion, while replacement of Ser293/296 with aspartic acid enhanced syncytium formation. These results indicated that CNN3 regulates actin cytoskeleton rearrangement which is required for the plasma membranes of trophoblasts to become fusion competent. PMID:20861310

  2. Cyclin G2 Promotes Hypoxia- Driven Local Invasion of Glioblastoma by Orchestrating Cytoskeletal Dynamics

    Atsushi Fujimura

    2013-11-01

    Full Text Available Microenvironmental conditions such as hypoxia potentiate the local invasion of malignant tumors including glioblastomas by modulating signal transduction and protein modification, yet the mechanism by which hypoxia controls cytoskeletal dynamics to promote the local invasion is not well defined. Here, we show that cyclin G2 plays pivotal roles in the cytoskeletal dynamics in hypoxia-driven invasion by glioblastoma cells. Cyclin G2 is a hypoxia-induced and cytoskeleton-associated protein and is required for glioblastoma expansion. Mechanistically, cyclin G2 recruits cortactin to the juxtamembrane through its SH3 domain-binding motif and consequently promotes the restricted tyrosine phosphorylation of cortactin in concert with src. Moreover, cyclin G2 interacts with filamentous actin to facilitate the formation of membrane ruffles. In primary glioblastoma, cyclin G2 is abundantly expressed in severely hypoxic regions such as pseudopalisades, which consist of actively migrating glioma cells. Furthermore, we show the effectiveness of dasatinib against hypoxia-driven, cyclin G2-involved invasion in vitro and in vivo. Our findings elucidate the mechanism of cytoskeletal regulation by which severe hypoxia promotes the local invasion and may provide a therapeutic target in glioblastoma.

  3. Loss of cytoskeletal proteins and lens cell opacification in the selenite cataract model.

    Matsushima, H; David, L L; Hiraoka, T; Clark, J I

    1997-03-01

    This study of lens protein composition found that some cytoskeletal proteins were degraded during the earliest stages of cataract formation. Cataract was induced in 13-14 day old rats by a single subcutaneous injection of sodium selenite (19 mumol kg-1). By 24 hr after the injection of selenite, the ratio of insoluble to soluble protein increased as lens opacification began. The increase in insoluble protein aggregates was correlated with an accelerated loss of proteins having molecular weights of 42, 55/57 and 235 kDa which reacted with antibodies to the cytoskeletal proteins actin, tubulin/vimentin and spectrin, respectively. We observed the loss of 49, 60 and 90 kDa proteins which were not identified. In the lenses of animals protected from protein aggregation and opacification by administration of 1.5 mmol kg-1 pantethine, the pattern of proteins in SDS-PAGE gels resembled the pattern for proteins from transparent lenses of normal untreated animals and loss of cytoskeletal proteins was prevented. PMID:9196390

  4. Epigenetic repression of ribosomal RNA transcription by ROCK-dependent aberrant cytoskeletal organization

    Wu, Tse-Hsiang; Kuo, Yuan-Yeh; Lee, Hsiao-Hui; Kuo, Jean-Cheng; Ou, Meng-Hsin; Chang, Zee-Fen

    2016-01-01

    It is known that ribosomal RNA (rRNA) synthesis is regulated by cellular energy and proliferation status. In this study, we investigated rRNA gene transcription in response to cytoskeletal stress. Our data revealed that the cell shape constrained by isotropic but not elongated micropatterns in HeLa cells led to a significant reduction in rRNA transcription dependent on ROCK. Expression of a dominant-active form of ROCK also repressed rRNA transcription. Isotropic constraint and ROCK over-activation led to different types of aberrant F-actin organization, but their suppression effects on rRNA transcription were similarly reversed by inhibition of histone deacetylase (HDAC) or overexpression of a dominant negative form of Nesprin, which shields the signal transmitted from actin filament to the nuclear interior. We further showed that the binding of HDAC1 to the active fraction of rDNA genes is increased by ROCK over-activation, thus reducing H3K9/14 acetylation and suppressing transcription. Our results demonstrate an epigenetic control of active rDNA genes that represses rRNA transcription in response to the cytoskeletal stress. PMID:27350000

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

    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

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

    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 32P-orthophosphate and/or protein kinase A (PKA), protein kinase dependent of Ca2+/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 Ca2+ quelators (1 mM EGTA; 10 µM BAPTA-AM, respectively) prevented QUIN-mediated effect, while Ca2+ influx through voltage-dependent Ca2+ 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 disorders. - Highlights:

  7. Cytoskeletal logic: a model for molecular computation via Boolean operations in microtubules and microtubule-associated proteins.

    Lahoz-Beltra, R; Hameroff, S R; Dayhoff, J E

    1993-01-01

    Adaptive behaviors and dynamic activities within living cells are organized by the cytoskeleton: intracellular networks of interconnected protein polymers which include microtubules (MTs), actin, intermediate filaments, microtubule associated proteins (MAPs) and other protein structures. Cooperative interactions among cytoskeletal protein subunit conformational states have been used to model signal transmission and information processing. In the present work we present a theoretical model for molecular computing in which Boolean logic is implemented in parallel networks of individual MTs interconnected by MAPs. Conformational signals propagate on MTs as in data buses and in the model MAPs are considered as Boolean operators, either as bit-lines (like MTs) where a signal can be transported unchanged between MTs ('BUS-MAP'), or as bit-lines where a Boolean operation is performed in one of the two MAP-MT attachments ('LOGIC-MAP'). Three logic MAPs have been defined ('NOT-MAP, 'AND-MAP', 'XOR-MAP') and used to demonstrate addition, subtraction and other arithmetic operations. Although our choice of Boolean logic is arbitrary, the simulations demonstrate symbolic manipulation in a connectionist system and suggest that MT-MAP networks can perform computation in living cells and are candidates for future molecular computing devices. PMID:8318677

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

    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

  9. Duplication in the microtubule-actin cross-linking factor 1 gene causes a novel neuromuscular condition

    Jørgensen, Louise H; Mosbech, Mai-Britt; Færgeman, Nils J;

    2014-01-01

    Spectrins and plakins are important communicators linking cytoskeletal components to each other and to cellular junctions. Microtubule-actin cross-linking factor 1 (MACF1) belongs to the spectraplakin family and is involved in control of microtubule dynamics. Complete knock out of MACF1 in mice is...

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

    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

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

    Highlights: ► Vitreous induces morphological changes and cytoskeletal rearrangements in RPE cells. ► Rac1 is activated in vitreous-transformed RPE cells. ► Rac inhibition prevents morphological changes in vitreous-transformed RPE cells. ► Rac inhibition suppresses cytoskeletal rearrangements in vitreous-transformed RPE cells. ► 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-transformed human RPE cells undergo cytoskeletal rearrangements via Rac1 GTPase-dependent pathways that modulate LIMK1 and

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

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

    2014-01-01

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

  13. Cytoskeletal network morphology regulates intracellular transport dynamics

    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...

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

    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.

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

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

    2016-01-01

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

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

    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...

  17. Abelson tyrosine kinase links PDGFbeta receptor activation to cytoskeletal regulation of NMDA receptors in CA1 hippocampal neurons

    Beazely Michael A

    2008-12-01

    Full Text Available Abstract Background We have previously demonstrated that PDGF receptor activation indirectly inhibits N-methyl-D-aspartate (NMDA currents by modifying the cytoskeleton. PDGF receptor ligand is also neuroprotective in hippocampal slices and cultured neurons. PDGF receptors are tyrosine kinases that control a variety of signal transduction pathways including those mediated by PLCγ. In fibroblasts Src and another non-receptor tyrosine kinase, Abelson kinase (Abl, control PDGF receptor regulation of cytoskeletal dynamics. The mechanism whereby PDGF receptor regulates cytoskeletal dynamics in central neurons remains poorly understood. Results Intracellular applications of active Abl, but not heat-inactivated Abl, decreased NMDA-evoked currents in isolated hippocampal neurons. This mimics the effects of PDGF receptor activation in these neurons. The Abl kinase inhibitor, STI571, blocked the inhibition of NMDA currents by Abl. We demonstrate that PDGF receptors can activate Abl kinase in hippocampal neurons via mechanisms similar to those observed previously in fibroblasts. Furthermore, PDGFβ receptor activation alters the subcellular localization of Abl. Abl kinase is linked to actin cytoskeletal dynamics in many systems. We show that the inhibition of NMDA receptor currents by Abl kinase is blocked by the inclusion of the Rho kinase inhibitor, Y-27632, and that activation of Abl correlates with an increase in ROCK tyrosine phosphorylation. Conclusion This study demonstrates that PDGFβ receptors act via an interaction with Abl kinase and Rho kinase to regulated cytoskeletal regulation of NMDA receptor channels in CA1 pyramidal neurons.

  18. β-Actin protein expression differs in the submandibular glands of male and female mice.

    Chen, Gang; Zou, Ye; Zhang, Xuan; Xu, Lingfei; Hu, Qiaoyun; Li, Ting; Yao, Chenjuan; Yu, Shali; Wang, Xiaoke; Wang, Chun

    2016-07-01

    β-actin, a cytoskeletal protein, is the most widely used housekeeping gene. Although housekeeping genes are expressed in all tissues, the β-actin gene is expressed in certain cell types because of differential binding of transcriptional factors to the regulatory elements of the gene. The expression and localization of β-actin protein in the submandibular glands (SMG) of mice were investigated in this study, using Western blot analysis and immunohistochemistry. In ICR and C57BL/6J mice, the levels of β-actin protein in the SMG of females are significantly higher than those in the SMG of males. β-actin protein is majorly distributed in acinar cells of SMG. There is no significant difference in the expression level of β-actin protein between females and castrated males. After castrated male ICR mice are treated with 10 mg/kg/day testosterone propionate (TP) for 3 weeks, the levels of β-actin protein in SMG decrease. The numbers of duct per unit area increase, whereas the numbers of acinus per unit area decrease after TP administration. These data suggest that β-actin protein is mainly distributed in acinar cells of SMG and results in a marked sexual dimorphism in mice. PMID:27079296

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

    Ioana Alesutan

    2013-09-01

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

  20. Mechanism of Actin Filament Bundling by Fascin

    Jansen, Silvia; Collins, Agnieszka; Yang, Changsong; Rebowski, Grzegorz; Svitkina, Tatyana; Dominguez, Roberto (UPENN); (UPENN-MED)

    2013-03-07

    Fascin is the main actin filament bundling protein in filopodia. Because of the important role filopodia play in cell migration, fascin is emerging as a major target for cancer drug discovery. However, an understanding of the mechanism of bundle formation by fascin is critically lacking. Fascin consists of four {beta}-trefoil domains. Here, we show that fascin contains two major actin-binding sites, coinciding with regions of high sequence conservation in {beta}-trefoil domains 1 and 3. The site in {beta}-trefoil-1 is located near the binding site of the fascin inhibitor macroketone and comprises residue Ser-39, whose phosphorylation by protein kinase C down-regulates actin bundling and formation of filopodia. The site in {beta}-trefoil-3 is related by pseudo-2-fold symmetry to that in {beta}-trefoil-1. The two sites are {approx}5 nm apart, resulting in a distance between actin filaments in the bundle of {approx}8.1 nm. Residue mutations in both sites disrupt bundle formation in vitro as assessed by co-sedimentation with actin and electron microscopy and severely impair formation of filopodia in cells as determined by rescue experiments in fascin-depleted cells. Mutations of other areas of the fascin surface also affect actin bundling and formation of filopodia albeit to a lesser extent, suggesting that, in addition to the two major actin-binding sites, fascin makes secondary contacts with other filaments in the bundle. In a high resolution crystal structure of fascin, molecules of glycerol and polyethylene glycol are bound in pockets located within the two major actin-binding sites. These molecules could guide the rational design of new anticancer fascin inhibitors.

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

    Rothenfluh, Adrian; Cowan, Christopher W.

    2013-01-01

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

  2. The plant actin cytoskeleton responds to signals from microbe-associated molecular patterns.

    Jessica L Henty-Ridilla

    Full Text Available Plants are constantly exposed to a large and diverse array of microbes; however, most plants are immune to the majority of potential invaders and susceptible to only a small subset of pathogens. The cytoskeleton comprises a dynamic intracellular framework that responds rapidly to biotic stresses and supports numerous fundamental cellular processes including vesicle trafficking, endocytosis and the spatial distribution of organelles and protein complexes. For years, the actin cytoskeleton has been assumed to play a role in plant innate immunity against fungi and oomycetes, based largely on static images and pharmacological studies. To date, however, there is little evidence that the host-cell actin cytoskeleton participates in responses to phytopathogenic bacteria. Here, we quantified the spatiotemporal changes in host-cell cytoskeletal architecture during the immune response to pathogenic and non-pathogenic strains of Pseudomonas syringae pv. tomato DC3000. Two distinct changes to host cytoskeletal arrays were observed that correspond to distinct phases of plant-bacterial interactions i.e. the perception of microbe-associated molecular patterns (MAMPs during pattern-triggered immunity (PTI and perturbations by effector proteins during effector-triggered susceptibility (ETS. We demonstrate that an immediate increase in actin filament abundance is a conserved and novel component of PTI. Notably, treatment of leaves with a MAMP peptide mimic was sufficient to elicit a rapid change in actin organization in epidermal cells, and this actin response required the host-cell MAMP receptor kinase complex, including FLS2, BAK1 and BIK1. Finally, we found that actin polymerization is necessary for the increase in actin filament density and that blocking this increase with the actin-disrupting drug latrunculin B leads to enhanced susceptibility of host plants to pathogenic and non-pathogenic bacteria.

  3. Sequence and comparative genomic analysis of actin-related proteins.

    Muller, Jean; Oma, Yukako; Vallar, Laurent; Friederich, Evelyne; Poch, Olivier; Winsor, Barbara

    2005-12-01

    Actin-related proteins (ARPs) are key players in cytoskeleton activities and nuclear functions. Two complexes, ARP2/3 and ARP1/11, also known as dynactin, are implicated in actin dynamics and in microtubule-based trafficking, respectively. ARP4 to ARP9 are components of many chromatin-modulating complexes. Conventional actins and ARPs codefine a large family of homologous proteins, the actin superfamily, with a tertiary structure known as the actin fold. Because ARPs and actin share high sequence conservation, clear family definition requires distinct features to easily and systematically identify each subfamily. In this study we performed an in depth sequence and comparative genomic analysis of ARP subfamilies. A high-quality multiple alignment of approximately 700 complete protein sequences homologous to actin, including 148 ARP sequences, allowed us to extend the ARP classification to new organisms. Sequence alignments revealed conserved residues, motifs, and inserted sequence signatures to define each ARP subfamily. These discriminative characteristics allowed us to develop ARPAnno (http://bips.u-strasbg.fr/ARPAnno), a new web server dedicated to the annotation of ARP sequences. Analyses of sequence conservation among actins and ARPs highlight part of the actin fold and suggest interactions between ARPs and actin-binding proteins. Finally, analysis of ARP distribution across eukaryotic phyla emphasizes the central importance of nuclear ARPs, particularly the multifunctional ARP4. PMID:16195354

  4. DNA segregation by the bacterial actin AlfA during Bacillus subtilis growth and development

    Becker, Eric; Herrera, Nick C; Gunderson, Felizza Q.; Derman, Alan I.; Dance, Amber L; Sims, Jennifer; Larsen, Rachel A.; Pogliano, Joe

    2006-01-01

    We here identify a protein (AlfA; actin like filament) that defines a new family of actins that are only distantly related to MreB and ParM. AlfA is required for segregation of Bacillus subtilis plasmid pBET131 (a mini pLS32-derivative) during growth and sporulation. A 3-kb DNA fragment encoding alfA and a downstream gene (alfB) is necessary and sufficient for plasmid stability. AlfA-GFP assembles dynamic cytoskeletal filaments that rapidly turn over (t1/2

  5. Actin depolymerization mediated loss of SNTA1 phosphorylation and Rac1 activity has implications on ROS production, cell migration and apoptosis.

    Bhat, Sehar Saleem; Parray, Arif Ali; Mushtaq, Umar; Fazili, Khalid Majid; Khanday, Firdous Ahmad

    2016-06-01

    Alpha-1-syntrophin (SNTA1) and Rac1 are part of a signaling pathway via the dystrophin glycoprotein complex (DGC). Both SNTA1 and Rac1 proteins are over-expressed in various carcinomas. It is through the DGC signaling pathway that SNTA1 has been shown to act as a link between the extra cellular matrix, the internal cell signaling apparatus and the actin cytoskeleton. SNTA1 is involved in the modulation of the actin cytoskeleton and actin reorganization. Rac1 also controls actin cytoskeletal organization in the cell. In this study, we present the interplay between f-actin, SNTA1 and Rac1. We analyzed the effect of actin depolymerization on SNTA1 tyrosine phosphorylation and Rac1 activity using actin depolymerizing drugs, cytochalasin D and latrunculin A. Our results indicate a marked decrease in the tyrosine phosphorylation of SNTA1 upon actin depolymerization. Results suggest that actin depolymerization mediated loss of SNTA1 phosphorylation leads to loss of interaction between SNTA1 and Rac1, with a concomitant loss of Rac1 activation. The loss of SNTA1tyrosine phosphorylation and Rac1 activity by actin depolymerization results in increased apoptosis, decreased cell migration and decreased reactive oxygen species (ROS) levels in breast carcinoma cells. Collectively, our results present a possible role of f-actin in the SNTA1-Rac1 signaling pathway and implications of actin depolymerization on cell migration, ROS production and apoptosis. PMID:27048259

  6. Coupled actin-lamin biopolymer networks and protecting DNA

    Zhang, Tao; Rocklin, D. Zeb; Mao, Xiaoming; Schwarz, J. M.

    The mechanical properties of cells are largely determined by networks of semiflexible biopolymers forming the cytoskeleton. Similarly, the mechanical properties of cell nuclei are also largely determined by networks of semiflexible biopolymers forming the nuclear cytoskeleton. In particular, a network of filamentous lamin sits just inside the inner nuclear membrane to presumably protect the heart of the cell nucleus--the DNA. It has been demonstrated over the past decade that the actin cytoskeletal biopolymer network and the lamin biopolymer network are coupled via a sequence of proteins bridging the outer and inner nuclear membranes, known as the LINC complex. We, therefore, probe the consequences of such a coupling in a model biopolymer network system via numerical simulations to understand the resulting deformations in the lamin network in response to perturbations in the actin cytoskeletal network. We find, for example, that the force transmission across the coupled system can depend sensitively on the concentration of LINC complexes. Such study could have implications for mechanical mechanisms of the regulation of transcription since DNA couples to lamin via lamin-binding domains so that deformations in the lamin network may result in deformations in the DNA.

  7. Cytoskeletal organization and collagen orientation in the fish scales.

    Zylberberg, L; Bereiter-Hahn, J; Sire, J Y

    1988-09-01

    Immunofluorescence and electron microscopy were used to analyze the relationships between the organization of collagen fibrils in elasmoid scales, and the orientation of microtubules and actin microfilaments in the scleroblasts producing this collagenous stroma. Attention was focused on the basal plate of the scales because of the highly ordered three-dimensional arrangement of the collagen fibrils in superimposed plies forming an acellular plywood-like structure. The collagen fibrils are synthesized by the scleroblasts forming a monolayered pseudo-epithelium, the hyposquama, at the lowest surface of the scale. Fully developed scales with a low collagen deposition rate were compared with regenerating scales active in fibrillogenesis. When an ordered array of the collagen fibrils is found, the innermost collagen fibrils are coaligned with microtubules and actin microfilaments. Thus, because of this coalignment, microtubules and actin microfilaments of the hyposquamal scleroblasts are subjected to consecutive alterations during the formation of the plies of the basal plate. The sequence of events when the collagen fibrils change their direction from one ply to the other in the basal plate is deduced from immunofluorescence and phase-contrast-microscopic observations. During the formation of the orthogonal plywood-like structure in the regenerating scales, first microtubules may change their curse with a rotating angle of about 90 degrees; then, actin microfilaments are disorganized and reorganized by interacting mechanically with the microtubules with which they are coaligned. Collagen fibrils are synthesized in a direction that is roughly perpendicular to that of the preceding ply. The unknown signals inducing the change in direction of the cytoskeleton may be transmitted throughout the hyposquama via gap junctions. PMID:3052849

  8. Spontaneous actin dynamics in contractile rings

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

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

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

    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.

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

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

  11. A quantitative analysis of contractility in active cytoskeletal protein networks.

    Bendix, Poul M; Koenderink, Gijsje H; Cuvelier, Damien; Dogic, Zvonimir; Koeleman, Bernard N; Brieher, William M; Field, Christine M; Mahadevan, L; Weitz, David A

    2008-04-15

    Cells actively produce contractile forces for a variety of processes including cytokinesis and motility. Contractility is known to rely on myosin II motors which convert chemical energy from ATP hydrolysis into forces on actin filaments. However, the basic physical principles of cell contractility remain poorly understood. We reconstitute contractility in a simplified model system of purified F-actin, muscle myosin II motors, and alpha-actinin cross-linkers. We show that contractility occurs above a threshold motor concentration and within a window of cross-linker concentrations. We also quantify the pore size of the bundled networks and find contractility to occur at a critical distance between the bundles. We propose a simple mechanism of contraction based on myosin filaments pulling neighboring bundles together into an aggregated structure. Observations of this reconstituted system in both bulk and low-dimensional geometries show that the contracting gels pull on and deform their surface with a contractile force of approximately 1 microN, or approximately 100 pN per F-actin bundle. Cytoplasmic extracts contracting in identical environments show a similar behavior and dependence on myosin as the reconstituted system. Our results suggest that cellular contractility can be sensitively regulated by tuning the (local) activity of molecular motors and the cross-linker density and binding affinity. PMID:18192374

  12. Actin polymerisation at the cytoplasmic face of eukaryotic nuclei

    David-Watine Brigitte

    2006-05-01

    Full Text Available Abstract Background There exists abundant molecular and ultra-structural evidence to suggest that cytoplasmic actin can physically interact with the nuclear envelope (NE membrane system. However, this interaction has yet to be characterised in living interphase cells. Results Using a fluorescent conjugate of the actin binding drug cytochalasin D (CD-BODIPY we provide evidence that polymerising actin accumulates in vicinity to the NE. In addition, both transiently expressed fluorescent actin and cytoplasmic micro-injection of fluorescent actin resulted in accumulation of actin at the NE-membrane. Consistent with the idea that the cytoplasmic phase of NE-membranes can support this novel pool of perinuclear actin polymerisation we show that isolated, intact, differentiated primary hepatocyte nuclei support actin polymerisation in vitro. Further this phenomenon was inhibited by treatments hindering steric access to outer-nuclear-membrane proteins (e.g. wheat germ agglutinin, anti-nesprin and anti-nucleoporin antibodies. Conclusion We conclude that actin polymerisation occurs around interphase nuclei of living cells at the cytoplasmic phase of NE-membranes.

  13. N-WASp is required for Schwann cell cytoskeletal dynamics, normal myelin gene expression and peripheral nerve myelination

    Jin, Fuzi; Dong, Baoxia; Georgiou, John; Jiang, Qiuhong; Zhang, Jinyi; Bharioke, Arjun; Qiu, Frank; Lommel, Silvia; Feltri, M. Laura; Wrabetz, Lawrence; Roder, John C.; Eyer, Joel; Chen, Xiequn; Peterson, Alan C.; Siminovitch, Katherine A.

    2011-01-01

    Schwann cells elaborate myelin sheaths around axons by spirally wrapping and compacting their plasma membranes. Although actin remodeling plays a crucial role in this process, the effectors that modulate the Schwann cell cytoskeleton are poorly defined. Here, we show that the actin cytoskeletal regulator, neural Wiskott-Aldrich syndrome protein (N-WASp), is upregulated in myelinating Schwann cells coincident with myelin elaboration. When N-WASp is conditionally deleted in Schwann cells at the onset of myelination, the cells continue to ensheath axons but fail to extend processes circumferentially to elaborate myelin. Myelin-related gene expression is also severely reduced in the N-WASp-deficient cells and in vitro process and lamellipodia formation are disrupted. Although affected mice demonstrate obvious motor deficits these do not appear to progress, the mutant animals achieving normal body weights and living to advanced age. Our observations demonstrate that N-WASp plays an essential role in Schwann cell maturation and myelin formation. PMID:21385763

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

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

    2013-11-29

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

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

    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

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

    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

  17. Role of active contraction and tropomodulins in regulating actin filament length and sarcomere structure in developing zebrafish skeletal muscle

    Mazelet, Lize; Parker, Matthew; Li, Mei; Anders, Arner; Ashworth, Rachel

    2016-01-01

    Whilst it is recognised that contraction plays an important part in maintaining the structure and function of mature skeletal muscle, its role during development remains undefined. In this study the role of movement in skeletal muscle maturation was investigated in intact zebrafish embryos using a combination of genetic and pharmacological approaches. An immotile mutant line (cacnb1ts25) which lacks functional voltage-gated calcium channels (dihydropyridine receptors) in the muscle and pharma...

  18. Immunohistochemical study of cytoskeletal and extracellular matrix components in the notochord and notochordal sheath of amphioxus

    2006-04-01

    Full Text Available A major cytoskeletal and extracellular matrix proteins of the amphioxus notochordal cells and sheath were detected by immunohistochemical techniques. The three-layered amphioxus notochordal sheath strongly expressed fish collagen type I in its outer and middle layers, while in the innermost layer expression did not occur. The amphioxus notochordal sheath was reactive to applied anti-human antibodies for intermediate filament proteins such as cytokeratins, desmin and vimentin, as well as to microtubule components (ß-tubulin, particularly in the area close to the epipharyngeal groove. Alpha-smooth muscle actin was expressed in some notochordal cells and in the area of the notochordal attachment to the sheath. Thus muscular nature of notochordal cells was shown by immunohistochemistry in tissue section. Our results confirm that genes encoding intermediate filament proteins, microtubules and microfilaments are highly conserved during evolution. Collagen type I was proven to be the key extracellular matrix protein that forms the amphioxus notochordal sheath.

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

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

    2016-01-01

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

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

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

    2016-01-01

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

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

    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...

  2. Towards Experimental Tests of Quantum Effects in Cytoskeletal Proteins

    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...

  3. Cytoskeletal inhibitors, anti-adhesion molecule antibodies, and lectins inhibit hepatocyte spheroid formation.

    Nakamura M

    2002-02-01

    Full Text Available We investigated the role of cytoskeletons, adhesion molecules, membrane-glycosylations, and proteoglycans in forming the shape of adult rat hepatocyte spheroids. Isolated hepatocytes were cultured on dishes coated with chondroitin sulfate phosphatidyl ethanolamine (CS-PE. Spheroid-forming ability was observed after adding cytoskeletal inhibitors (cytochalasin D, colchicine, okadaic acid, mycalolide B, anti-adhesion molecule antibodies (anti-E-cadherin, anti-connexin 32, anti-zo-1, a glycosphingolipid synthetic inhibitor (N-butyldeoxynojirimycin, a proteoglycan synthetic inhibitor (p-nitrophenyl-beta-D-xylopyranoside, and several lectins. Localization of actin was studied using confocal microscopy after rhodamine-phalloidin staining. Adding cytoskeletal inhibitors on the initial day resulted in weakly clustered cell aggregates rather than smoothly formed spheroids. These effects disappeared at lower reagent concentrations. When reagents were added on day 3, after the formation of spheroids, only mycalolide B was associated with an irregular spheroid surface; the others had no effect. Adding the anti-E-cadherin, anti-connexin 32 on the initial day showed inhibition of spheroid formation, but anti-zo-1 and proteoglycan synthetic inhibitor had no effects. Among the several lectins, only Wheat Germ Agglutinin (WGA, Ricinus communis Agglutinin I (RCA-I, and Concanavalin A (ConA showed inhibition. These results suggest that cytoskeletal conformation and some adhesion molecules are necessary to form spheroids. Based on the interactions between lectins and hepatocytes in the present study, hepatocytes appear to contain an N-linked complex or N-linked hybrid glycosylated chains.

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

    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

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

    Highlights: ► Estradiol induced stiffness changes of osteoblasts were quantified using AFM. ► Estradiol causes significant decrease in the stiffness of osteoblasts. ► Decreased stiffness was caused by decreased density of f-actin network. ► Stiffness changes were not associated with mineralized matrix of osteoblasts. ► 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∗) and corresponding cytoskeletal changes of human fetal osteoblasts (hFOB 1.19). The cells were cultured in either medium alone or medium supplemented with β-estradiol and then subjected to Atomic Force Microscopy indentation (AFM) to determine E∗. 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∗ 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 changes of osteoblasts were not associated with changes in the synthesized mineralized matrix of the cells. Thus, a decrease in osteoblast stiffness with estrogen treatment was

  6. Probing cytoplasmic organization and the actin cytoskeleton of plant cells with optical tweezers

    Ketelaar, T.; Honing, van der H.S.; Emons, A.M.C.

    2010-01-01

    In interphase plant cells, the actin cytoskeleton is essential for intracellular transport and organization. To fully understand how the actin cytoskeleton functions as the structural basis for cytoplasmic organization, both molecular and physical aspects of the actin organization have to be conside

  7. Force of an actin spring

    Shin, Jennifer; Mahadevan, L.; Matsudaira, Paul

    2003-03-01

    The acrosomal process of the horseshoe crab sperm is a novel mechanochemical molecular spring that converts its elastic stain energy to mechanical work upon the chemical activation by Ca2+. Twisted and bent, the initial state of the acrosomal bundle features a high degree of complexity in its structure and the energy is believed to be stored in the highly strained actin filaments as an elastic potential energy. When activated, the bundle relaxes from the coil of the highly twisted and bent filaments to its straight conformation at a mean velocity of 15um/s. The mean extension velocity increases dramatically from 3um/s to 27um/s when temperature of the medium is changed from 9.6C to 32C (respective viscosities of 1.25-0.75cp), yet it exhibits a very weak dependence on changes in the medium viscosity (1cp-33cp). These experiments suggest that the uncoiling of the actin spring should be limited not by the viscosity of the medium but by the unlatching events of involved proteins at a molecular level. Unlike the viscosity-limited processes, where force is directly related to the rate of the reaction, a direct measurement is required to obtain the spring force of the acrosomal process. The extending acrosomal bundle is forced to push against a barrier and its elastic buckling response is analyzed to measure the force generated during the uncoiling.

  8. Orientational Order of the Lamellipodial Actin Network as Demonstrated in Living Motile CellsV⃞

    Alexander B. Verkhovsky; Chaga, Oleg Y.; Schaub, Sébastien; Svitkina, Tatyana M.; Meister, Jean-Jacques; Borisy, Gary G.

    2003-01-01

    Lamellipodia of crawling cells represent both the motor for cell advance and the primary building site for the actin cytoskeleton. The organization of actin in the lamellipodium reflects actin dynamics and is of critical importance for the mechanism of cell motility. In previous structural studies, the lamellipodial actin network was analyzed primarily by electron microscopy (EM). An understanding of lamellipodial organization would benefit significantly if the EM data were complemented and p...

  9. Nanodiamonds coupled with 5,7-dimethoxycoumarin, a plant bioactive metabolite, interfere with the mitotic process in B16F10 cells altering the actin organization

    Gismondi A

    2016-02-01

    Full Text Available Angelo Gismondi,1 Valentina Nanni,1 Giacomo Reina,2 Silvia Orlanducci,2 Maria Letizia Terranova,2 Antonella Canini1 1Department of Biology, 2Department of Chemical Science and Technology, University of Rome “Tor Vergata”, Rome, Italy Abstract: For the first time, we coupled reduced detonation nanodiamonds (NDs with a plant secondary metabolite, citropten (5,7-dimethoxycoumarin, and demonstrated how this complex was able to reduce B16F10 tumor cell growth more effectively than treatment with the pure molecule. These results encouraged us to find out the specific mechanism underlying this phenomenon. Internalization kinetics and quantification of citropten in cells after treatment with its pure or ND-conjugated form were measured, and it was revealed that the coupling between NDs and citropten was essential for the biological properties of the complex. We showed that the adduct was not able to induce apoptosis, senescence, or differentiation, but it determined cell cycle arrest, morphological changes, and alteration of mRNA levels of the cytoskeletal-related genes. The identification of metaphasic nuclei and irregular disposition of β-actin in the cell cytoplasm supported the hypothesis that citropten conjugated with NDs showed antimitotic properties in B16F10 cells. This work can be considered a pioneering piece of research that could promote and support the biomedical use of plant drug-functionalized NDs in cancer therapy. Keywords: citropten, cytoskeletal structure, plant secondary metabolite, melanoma, internalization kinetics

  10. Computational analysis of viscoelastic properties of crosslinked actin networks.

    Taeyoon Kim

    2009-07-01

    Full Text Available Mechanical force plays an important role in the physiology of eukaryotic cells whose dominant structural constituent is the actin cytoskeleton composed mainly of actin and actin crosslinking proteins (ACPs. Thus, knowledge of rheological properties of actin networks is crucial for understanding the mechanics and processes of cells. We used Brownian dynamics simulations to study the viscoelasticity of crosslinked actin networks. Two methods were employed, bulk rheology and segment-tracking rheology, where the former measures the stress in response to an applied shear strain, and the latter analyzes thermal fluctuations of individual actin segments of the network. It was demonstrated that the storage shear modulus (G' increases more by the addition of ACPs that form orthogonal crosslinks than by those that form parallel bundles. In networks with orthogonal crosslinks, as crosslink density increases, the power law exponent of G' as a function of the oscillation frequency decreases from 0.75, which reflects the transverse thermal motion of actin filaments, to near zero at low frequency. Under increasing prestrain, the network becomes more elastic, and three regimes of behavior are observed, each dominated by different mechanisms: bending of actin filaments, bending of ACPs, and at the highest prestrain tested (55%, stretching of actin filaments and ACPs. In the last case, only a small portion of actin filaments connected via highly stressed ACPs support the strain. We thus introduce the concept of a 'supportive framework,' as a subset of the full network, which is responsible for high elasticity. Notably, entropic effects due to thermal fluctuations appear to be important only at relatively low prestrains and when the average crosslinking distance is comparable to or greater than the persistence length of the filament. Taken together, our results suggest that viscoelasticity of the actin network is attributable to different mechanisms depending on

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

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

    1999-01-01

    control cells and cells treated with VPA, indicating that VPA affected the cytoskeletal determinants of cell morphology. Furthermore, VPA treatment induced an increase of F-actin, and of FAK, paxillin, vinculin, and phosphotyrosine in focal adhesion complexes. These changes were accompanied by increased...... adhesion of VPA-treated cells to the extracellular matrix. Treatment with an RGD-containing peptide reducing integrin binding to components of the extracellular matrix partially reverted the motility inhibition induced by VPA, indicating that altered adhesion contributed to, but was not the sole reason for......, VPA caused a redistribution of the actin severing protein gelsolin, and left the cells unable to respond to treatment with a gelsolin-peptide known to reduce the amount of gelsolin bound to phosphatidylinositol bisphosphate (PIP2), leaving a larger amount of the protein in a potential actin binding...

  12. Papaverine Prevents Vasospasm by Regulation of Myosin Light Chain Phosphorylation and Actin Polymerization in Human Saphenous Vein

    Hocking, Kyle M.; Putumbaka, Gowthami; Wise, Eric S.; Cheung-Flynn, Joyce; Brophy, Colleen M.; Komalavilas, Padmini

    2016-01-01

    Objective Papaverine is used to prevent vasospasm in human saphenous veins (HSV) during vein graft preparation prior to implantation as a bypass conduit. Papaverine is a nonspecific inhibitor of phosphodiesterases, leading to increases in both intracellular cGMP and cAMP. We hypothesized that papaverine reduces force by decreasing intracellular calcium concentrations ([Ca2+]i) and myosin light chain phosphorylation, and increasing actin depolymerization via regulation of actin regulatory protein phosphorylation. Approach and Results HSV was equilibrated in a muscle bath, pre-treated with 1 mM papaverine followed by 5 μM norepinephrine, and force along with [Ca2+]i levels were concurrently measured. Filamentous actin (F-actin) level was measured by an in vitro actin assay. Tissue was snap frozen to measure myosin light chain and actin regulatory protein phosphorylation. Pre-treatment with papaverine completely inhibited norepinephrine-induced force generation, blocked increases in [Ca2+]i and led to a decrease in the phosphorylation of myosin light chain. Papaverine pre-treatment also led to increased phosphorylation of the heat shock-related protein 20 (HSPB6) and the vasodilator stimulated phosphoprotein (VASP), as well as decreased filamentous actin (F-actin) levels suggesting depolymerization of actin. Conclusions These results suggest that papaverine-induced force inhibition of HSV involves [Ca2+]i-mediated inhibition of myosin light chain phosphorylation and actin regulatory protein phosphorylation-mediated actin depolymerization. Thus, papaverine induces sustained inhibition of contraction of HSV by the modulation of both myosin cross-bridge formation and actin cytoskeletal dynamics and is a pharmacological alternative to high pressure distention to prevent vasospasm. PMID:27136356

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

    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.

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

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

    2016-04-01

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

  15. Short Stop provides an essential link between F-actin and microtubules during axon extension.

    Lee, Seungbok; Kolodziej, Peter A

    2002-03-01

    Coordination of F-actin and microtubule dynamics is important for cellular motility and morphogenesis, but little is known about underlying mechanisms. short stop (shot) encodes an evolutionarily conserved, neuronally expressed family of rod-like proteins required for sensory and motor axon extension in Drosophila melanogaster. We identify Shot isoforms that contain N-terminal F-actin and C-terminal microtubule-binding domains, and that crosslink F-actin and microtubules in cultured cells. The F-actin- and microtubule-binding domains of Shot are required in the same molecule for axon extension, though the length of the connecting rod domain can be dramatically reduced without affecting activity. Shot therefore functions as a cytoskeletal crosslinker in axon extension, rather than mediating independent interactions with F-actin and microtubules. A Ca(2+)-binding motif located adjacent to the microtubule-binding domain is also required for axon extension, suggesting that intracellular Ca(2+) release may regulate Shot activity. These results suggest that Shot coordinates regulated interactions between F-actin and microtubules that are crucial for neuronal morphogenesis. PMID:11874915

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

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

    2016-07-01

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

  17. Effects of fixation protocol and gravistimulation on cytoskeletal organization in Brassica rapa roots

    Edge, Andrea; Hasenstein, Karl H.

    2012-07-01

    In preparation for a flight experiment we have studied the optimization of the staining protocols for microtubules and actin filaments in Brassica rapa seedlings. Microtubules (MT) were stained with monoclonal antibody (mAb) YOL 1/34. F-actin (FA) staining was achieved with C4 mAb antibody. Fixative prepared more than three weeks before use produces specimens that stained poorly. Storage in fixative for more than four weeks resulted in noticeably poorer staining. Staining was best in cortical cells but more difficult and less consistent in cap cells, especially for FA. In addition, the quality of staining of root cap cells was dependent on the age of the formaldehyde. The organization of the MTs corresponded with previously published descriptions; FA was prominent in the stele with thick and numerous parallel bundles; cortical cells showed less dense and less directional organization of mostly thinner filaments. FA organization was determined by tissue rather than by differential elongation. The organization of MTs in cortical cells of curving roots was uniformly circular and perpendicular to the long cell axis despite different cell length. The effect of clinorotation around the horizontal axis and centrifugation on the cytoskeletal organization was inconsistent. (Supported by NASA grant NNX10AP91G)

  18. Quantitative analysis of cytoskeletal reorganization during epithelial tissue sealing by large-volume electron tomography.

    Eltsov, Mikhail; Dubé, Nadia; Yu, Zhou; Pasakarnis, Laurynas; Haselmann-Weiss, Uta; Brunner, Damian; Frangakis, Achilleas S

    2015-05-01

    The closure of epidermal openings is an essential biological process that causes major developmental problems such as spina bifida in humans if it goes awry. At present, the mechanism of closure remains elusive. Therefore, we reconstructed a model closure event, dorsal closure in fly embryos, by large-volume correlative electron tomography. We present a comprehensive, quantitative analysis of the cytoskeletal reorganization, enabling separated epidermal cells to seal the epithelium. After establishing contact through actin-driven exploratory filopodia, cells use a single lamella to generate 'roof tile'-like overlaps. These shorten to produce the force, 'zipping' the tissue closed. The shortening overlaps lack detectable actin filament ensembles but are crowded with microtubules. Cortical accumulation of shrinking microtubule ends suggests a force generation mechanism in which cortical motors pull on microtubule ends as for mitotic spindle positioning. In addition, microtubules orient filopodia and lamellae before zipping. Our 4D electron microscopy picture describes an entire developmental process and provides fundamental insight into epidermal closure. PMID:25893916

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

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

    2003-06-11

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

  20. Regulation of actin cytoskeleton architecture by Eps8 and Abi1

    Miller Jeffrey R

    2005-10-01

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

  1. Dynamics of an actin spring

    Riera, Christophe; Mahadevan, L.; Shin, Jennifer; Matsudaira, Paul

    2003-03-01

    The acrosome of the sperm of the horseshoe crab (Limulus Polyphemus) is an unusual actin based system that shows a spectacular dynamical transition in the presence of Ca++ that is present in abundance in the neighborhood of the egg. During this process, the bundle, which is initially bent and twisted uncoils and becomes straight in a matter of a few seconds. Based on microstructural data, we propose a model for the dynamics of uncoiling that is best represented by a triple-well potential corresponding to the different structural arrangements of the supertwisted filaments. Each of the false, true and coiled states corresponds to a local minimum of the energy, with the true state being the one with the lowest energy. Using an evolution equation derived by balancing torques, we investigate the nucleation and propagation of the phase transition and compare the results with those of experiments. Our model quantifies the hypothesis that the acrosomal bundle behaves like a mechano-chemical spring.

  2. Simultaneous tracking of 3D actin and microtubule strains in individual MLO-Y4 osteocytes under oscillatory flow.

    Baik, Andrew D; Qiu, Jun; Hillman, Elizabeth M C; Dong, Cheng; Guo, X Edward

    2013-02-22

    Osteocytes in vivo experience complex fluid shear flow patterns to activate mechanotransduction pathways. The actin and microtubule (MT) cytoskeletons have been shown to play an important role in the osteocyte's biochemical response to fluid shear loading. The dynamic nature of physiologically relevant fluid flow profiles (i.e., 1Hz oscillatory flow) impedes the ability to image and study both actin and MT cytoskeletons simultaneously in the same cell with high spatiotemporal resolution. To overcome these limitations, a multi-channel quasi-3D microscopy technique was developed to track the actin and MT networks simultaneously under steady and oscillatory flow. Cells displayed high intercellular variability and intracellular cytoskeletal variability in strain profiles. Shear Exz was the predominant strain in both steady and oscillatory flows in the form of viscoelastic creep and elastic oscillations, respectively. Dramatic differences were seen in oscillatory flow, however. The actin strains displayed an oscillatory strain profile more often than the MT networks in all the strains tested and had a higher peak-to-trough strain magnitude. Taken together, the actin networks are the more responsive cytoskeletal networks in osteocytes under oscillatory flow and may play a bigger role in mechanotransduction pathway activation and regulation. PMID:23352617

  3. Cucurbitacin covalent bonding to cysteine thiols: the filamentous-actin severing protein Cofilin1 as an exemplary target

    Gabrielsen, M.; Schuldt, M.; Munro, J; Borucka, D.; Cameron, J.; Baugh, M.; Mleczak, A; Lilla, S.; Morrice, N.; Olson, M.F.

    2013-01-01

    Background: Cucurbitacins are a class of triterpenoid natural compounds with potent bioactivities that led to their use as traditional remedies, and which continue to attract considerable attention as chemical biology tools and potential therapeutics. One obvious target is the actin-cytoskeleton; treatment with cucurbitacins results in cytoskeletal rearrangements that impact upon motility and cell morphology. Findings: Cucurbitacin reacted with protein cysteine thiols as well as dithiothr...

  4. Visualisation of microtubules and actin filaments in fixed BY-2 suspension cells using an optimised whole mount immunolabelling protocol

    Szechynska-Hebda, M.; Wedzony, M.; Dubas, E.; Kieft, H; Lammeren, van, ACAP Andre

    2006-01-01

    Excellent visualisation of microtubules and actin filaments was obtained in fixed tobacco BY-2 suspension cells after optimising a protocol for whole mount immunolabelling. The procedure is based on modification of fixation, cell wall digestion, dimethyl sulfoxide (DMSO) treatment, post fixation, and blocking. The most critical aspects of successful preservation and visualization of cytoskeletal elements appeared to be: a two-step fixation with paraformaldehyde and glutaraldehyde before enzym...

  5. The Actin-Cytoskeleton Pathway and Its Potential Role in Inflammatory Bowel Disease-Associated Human Colorectal Cancer

    Kanaan, Ziad; Qadan, Motaz; Eichenberger, Maurice Robert; Galandiuk, Susan

    2010-01-01

    Introduction: To improve our understanding of the various clinical phenotypes in inflammatory bowel disease (IBD)-associated colorectal cancer (CRC) and provide potential targets for early diagnosis and future therapy, we sought to identify new candidate genes and molecular pathways involved in the pathogenesis and progression of this disorder. Recent evidence has implicated the actin-cytoskeleton pathway in the development of metastatic sporadic CRC through cytoskeletal proteins such as fasc...

  6. Methods for modeling cytoskeletal and DNA filaments

    This review summarizes the models that researchers use to represent the conformations and dynamics of cytoskeletal and DNA filaments. It focuses on models that address individual filaments in continuous space. Conformation models include the freely jointed, Gaussian, angle-biased chain (ABC), and wormlike chain (WLC) models, of which the first three bend at discrete joints and the last bends continuously. Predictions from the WLC model generally agree well with experiment. Dynamics models include the Rouse, Zimm, stiff rod, dynamic WLC, and reptation models, of which the first four apply to isolated filaments and the last to entangled filaments. Experiments show that the dynamic WLC and reptation models are most accurate. They also show that biological filaments typically experience strong hydrodynamic coupling and/or constrained motion. Computer simulation methods that address filament dynamics typically compute filament segment velocities from local forces using the Langevin equation and then integrate these velocities with explicit or implicit methods; the former are more versatile and the latter are more efficient. Much remains to be discovered in biological filament modeling. In particular, filament dynamics in living cells are not well understood, and current computational methods are too slow and not sufficiently versatile. Although primarily a review, this paper also presents new statistical calculations for the ABC and WLC models. Additionally, it corrects several discrepancies in the literature about bending and torsional persistence length definitions, and their relations to flexural and torsional rigidities. (topical review)

  7. Mechanical models of the cellular cytoskeletal network for the analysis of intracellular mechanical properties and force distributions: a review.

    Chen, Ting-Jung; Wu, Chia-Ching; Su, Fong-Chin

    2012-12-01

    The cytoskeleton, which is the major mechanical component of cells, supports the cell body and regulates the cellular motility to assist the cell in performing its biological functions. Several cytoskeletal network models have been proposed to investigate the mechanical properties of cells. This review paper summarizes these models with a focus on the prestressed cable network, the semi-flexible chain network, the open-cell foam, the tensegrity, and the granular models. The components, material parameters, types of connection joints, tension conditions, and the advantages and disadvantages of each model are evaluated from a structural and biological point of view. The underlying mechanisms that are associated with the morphological changes of spreading cells are expected to be simulated using a cytoskeletal model; however, it is still paid less attention most likely due to the lack of a suitable cytoskeletal model that can accurately model the spreading process. In this review article, the established cytoskeletal models are hoped to provide useful information for the development of future cytoskeletal models with different degrees of cell attachment for the study of the mechanical mechanisms underlying the cellular behaviors in response to external stimulations. PMID:23062682

  8. Phosphoproteome Profiling of SH-SY5y Neuroblastoma Cells Treated with Anesthetics: Sevoflurane and Isoflurane Affect the Phosphorylation of Proteins Involved in Cytoskeletal Regulation.

    Lee, Joomin; Ahn, Eunsook; Park, Wyun Kon; Park, Seyeon

    2016-01-01

    Inhalation anesthetics are used to decrease the spinal cord transmission of painful stimuli. However, the molecular or biochemical processes within cells that regulate anesthetic-induced responses at the cellular level are largely unknown. Here, we report the phosphoproteome profile of SH-SY5y human neuroblastoma cells treated with sevoflurane, a clinically used anesthetic. Phosphoproteins were isolated from cell lysates and analyzed using two-dimensional gel electrophoresis. The phosphorylation of putative anesthetic-responsive marker proteins was validated using western blot analysis in cells treated with both sevoflurane and isoflurane. A total of 25 phosphoproteins were identified as differentially phosphorylated proteins. These included key regulators that signal cytoskeletal remodeling steps in pathways related to vesicle trafficking, axonal growth, and cell migration. These proteins included the Rho GTPase, Ras-GAP SH3 binding protein, Rho GTPase activating protein, actin-related protein, and actin. Sevoflurane and isoflurane also resulted in the dissolution of F-actin fibers in SH-SY5y cells. Our results show that anesthetics affect the phosphorylation of proteins involved in cytoskeletal remodeling pathways. PMID:27611435

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

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

    2016-01-01

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

  10. Expression of cytoskeletal and matrix genes following exposure to ionizing radiation: Dose-rate effects and protein synthesis requirements

    Experiments were designed to examine the effects of radiation dose-rate and of the protein synthesis inhibitor cycloheximide on expression of cytoskeletal elements (γ- and β-actin and α-tubulin) and matrix elements (fibronectin) in Syrian hamster embryo cells. Past work from our laboratory had already demonstrated optimum time points and doses for examination of radiation effects on accumulation of specific transcripts. Our results here demonstrated little effect of dose-rate for JANUS fission spectrum neutrons when comparing expression of either α-tubulin or fibronectin genes. Past work had already documented similar results for expression of actin transcripts. Effects of cycloheximide, however, revealed several interesting and novel findings: (1) Cycloheximide repressed accumulation of α-tubulin following exposure to high dose-rate neutrons or γ rays; this did not occur following similar low dose-rate exposure (2) Cycloheximide did not affect accumulation of mRNA for actin genes. Cycloheximide abrogated the moderate induction of fibronectin-mRNA which occurred following exposure to γ rays and high dose-rate neutrons. These results suggest a role for labile proteins in the maintenance of α-tubulin and fibronectin mRNA accumulation following exposure to ionizing radiation. In addition, they suggest that the cellular/molecular response to low dose-rate neutrons may be different from the response to high dose-rate neutrons

  11. Expression of cytoskeletal and matrix genes following exposure to ionizing radiation: Dose-rate effects and protein synthesis requirements

    Experiments were designed to examine the effects Of radiation dose-rate and of the protein synthesis inhibitor cycloheximide on expression of cytoskeletal elements (γ- and β-actin and α-tubulin) and matrix elements (fibronectin) in Syrian hamster embryo cells. Past work from our laboratory had already demonstrated optimum time points and doses for examination of radiation effects on accumulation of specific transcripts. Our results here demonstrated little effect of dose-rate for JANUS fission spectrum neutrons when comparing expression of either α-tubulin or fibronectin genes. Past work had already documented similar results for expression of actin transcripts. Effects of cycloheximide revealed that cycloheximide repressed accumulation of α-tubulin following exposure to high dose-rate neutrons or γ rays; this did not occur following similar low dose-rate exposure. (2) Cycloheximide did not affect accumulation of MRNA for actin genes; and that cycloheximide abrogated the moderate induction of fibronectin-mRNA which occurred following exposure to γ rays and high dose-rate neutrons. These results suggest a role for labile proteins in the maintenance of α-tubulin and fibronectin MRNA accumulation following exposure to ionizing radiation. in addition, they suggest that the cellular/molecular response to low dose-rate neutrons may be different from the response to high dose-rate neutrons

  12. Actin marker lines in grapevine reveal a gatekeeper function of guard cells.

    Guan, Xin; Buchholz, Günther; Nick, Peter

    2014-08-15

    Resistance to abiotic and biotic stress is a central topic for sustainable agriculture, especially in grapevine, one of the field crops with the highest economic output per acreage. As early cellular factors for plant defense, actin microfilaments (AF) are of high relevance. We therefore generated a transgenic actin marker line for grapevine by expressing a fusion protein between green fluorescent protein and the second actin-binding domain of Arabidopsis (Arabidopsis thaliana) fimbrin, AtFIM1. Based on this first cytoskeletal-marker line in grapevine, the response of AFs to phytopathogenic microorganisms could be followed in vivo. Upon inoculation with fluorescently labeled strains of phytopathogenic bacteria, actin responses were confined to the guard cells. In contrast, upon contact with zoospores of Plasmopara viticola, not only the guard cells, but also epidermal pavement cells, where no zoospores had attached responded with the formation of a perinuclear actin basket. Our data support the hypothesis that guard cells act as pacemakers of defense, dominating the responses of the remaining epidermal cells. PMID:24973589

  13. Study of the influence of actin-binding proteins using linear analyses of cell deformability.

    Plaza, Gustavo R; Uyeda, Taro Q P; Mirzaei, Zahra; Simmons, Craig A

    2015-07-21

    The actin cytoskeleton plays a key role in the deformability of the cell and in mechanosensing. Here we analyze the contributions of three major actin cross-linking proteins, myosin II, α-actinin and filamin, to cell deformability, by using micropipette aspiration of Dictyostelium cells. We examine the applicability of three simple mechanical models: for small deformation, linear viscoelasticity and drop of liquid with a tense cortex; and for large deformation, a Newtonian viscous fluid. For these models, we have derived linearized equations and we provide a novel, straightforward methodology to analyze the experiments. This methodology allowed us to differentiate the effects of the cross-linking proteins in the different regimes of deformation. Our results confirm some previous observations and suggest important relations between the molecular characteristics of the actin-binding proteins and the cell behavior: the effect of myosin is explained in terms of the relation between the lifetime of the bond to actin and the resistive force; the presence of α-actinin obstructs the deformation of the cytoskeleton, presumably mainly due to the higher molecular stiffness and to the lower dissociation rate constants; and filamin contributes critically to the global connectivity of the network, possibly by rapidly turning over cross-links during the remodeling of the cytoskeletal network, thanks to the higher rate constants, flexibility and larger size. The results suggest a sophisticated relationship between the expression levels of actin-binding proteins, deformability and mechanosensing. PMID:26059185

  14. Altered Cell Mechanics from the Inside: Dispersed Single Wall Carbon Nanotubes Integrate with and Restructure Actin

    Mohammad F. Islam

    2012-05-01

    Full Text Available With a range of desirable mechanical and optical properties, single wall carbon nanotubes (SWCNTs are a promising material for nanobiotechnologies. SWCNTs also have potential as biomaterials for modulation of cellular structures. Previously, we showed that highly purified, dispersed SWCNTs grossly alter F-actin inside cells. F-actin plays critical roles in the maintenance of cell structure, force transduction, transport and cytokinesis. Thus, quantification of SWCNT-actin interactions ranging from molecular, sub-cellular and cellular levels with both structure and function is critical for developing SWCNT-based biotechnologies. Further, this interaction can be exploited, using SWCNTs as a unique actin-altering material. Here, we utilized molecular dynamics simulations to explore the interactions of SWCNTs with actin filaments. Fluorescence lifetime imaging microscopy confirmed that SWCNTs were located within ~5 nm of F-actin in cells but did not interact with G-actin. SWCNTs did not alter myosin II sub-cellular localization, and SWCNT treatment in cells led to significantly shorter actin filaments. Functionally, cells with internalized SWCNTs had greatly reduced cell traction force. Combined, these results demonstrate direct, specific SWCNT alteration of F-actin structures which can be exploited for SWCNT-based biotechnologies and utilized as a new method to probe fundamental actin-related cellular processes and biophysics.

  15. Conditioning nerve crush accelerates cytoskeletal protein transport in sprouts that form after a subsequent crush

    To examine the relationship between axonal outgrowth and the delivery of cytoskeletal proteins to the growing axon tip, outgrowth was accelerated by using a conditioning nerve crush. Because slow component b (SCb) of axonal transport is the most rapid vehicle for carrying cytoskeletal proteins to the axon tip, the rate of SCb was measured in conditioned vs. sham-conditioned sprouts. In young Sprague-Dawley rats, the conditioning crush was made to sciatic nerve branches at the knee; 14 days later, the test crush was made where the L4 and L5 spinal nerves join to form the sciatic nerve in the flank. Newly synthesized proteins were labeled in motor neurons by injecting 35S-methionine into the lumbar spinal cord 7 days before the test crush. The wave of pulse-labeled SCb proteins reached the crush by the time it was made and subsequently entered sprouts. The nerve was removed and sectioned for SDS-PAGE and fluorography 4-12 days after the crush. Tubulins, neurofilament proteins, and representative 'cytomatrix' proteins (actin, calmodulin, and putative microtubule-associated proteins) were removed from gels for liquid scintillation counting. Labeled SCb proteins entered sprouts without first accumulating in parent axon stumps, presumably because sprouts begin to grow within hours after axotomy. The peak of SCb moved 11% faster in conditioned than in sham-conditioned sprouts: 3.0 vs. 2.7 mm/d (p less than 0.05). To confirm that sprouts elongate more rapidly when a test crush is preceded by a conditioning crush, outgrowth distances were measured in a separate group of rats by labeling fast axonal transport with 3H-proline 24 hours before nerve retrieval

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

    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.

  17. 25 Years of Tension over Actin Binding to the Cadherin Cell Adhesion Complex: The Devil is in the Details.

    Nelson, W James; Weis, William I

    2016-07-01

    Over the past 25 years, there has been a conceptual (re)evolution in understanding how the cadherin cell adhesion complex, which contains F-actin-binding proteins, binds to the actin cytoskeleton. There is now good synergy between structural, biochemical, and cell biological results that the cadherin-catenin complex binds to F-actin under force. PMID:27166091

  18. Mechanical Properties of Re-constituted Actin Networks at an Oil/Water Interface Determined by Microrheology

    Ershov, D.S.; Cohen Stuart, M.A.; Gucht, van der J.

    2012-01-01

    There have been various attempts to investigate the mechanical properties of the actin cortex in cells, but the factors that control them remain poorly understood. To make progress, we develop a reconstituted model of the actin cortex that mimics its structure. We attach actin filaments to lipids li

  19. Progresses in studies of nuclear actin

    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.

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

    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

  1. The Actin Binding Protein Adseverin Regulates Osteoclastogenesis

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

    2014-01-01

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

  2. Resveratrol and Estradiol Exert Disparate Effects on Cell Migration, Cell Surface Actin Structures, and Focal Adhesion Assembly in MDA-MB-231 Human Breast Cancer Cells

    Nicolas G. Azios

    2005-02-01

    Full Text Available Resveratrol, a grape polyphenol, is thought to be a cancer preventive, yet its effects on metastatic breast cancer are relatively unknown. Since cancer cell invasion is dependent on cell migration, the chemotactic response of MDA-MB-231 metastatic human breast cancer cells to resveratrol, estradiol (E2, or epidermal growth factor (EGF was investigated. Resveratrol decreased while E2 and EGF increased directed cell migration. Resveratrol may inhibit cell migration by altering the cytoskeleton. Resveratrol induced a rapid global array of filopodia and decreased focal adhesions and focal adhesion kinase (FAK activity. E2 or EGF treatment did not affect filopodia extension but increased lamellipodia and associated focal adhesions that are integral for cell migration. Combined resveratrol and E2 treatment resulted in a filopodia and focal adhesion response similar to resveratrol alone. Combined resveratrol and EGF resulted in a lamellipodia and focal adhesion response similar to EGF alone. E2 and to a lesser extent resveratrol increased EGFR activity. The cytoskeletal changes and EGFR activity in response to E2 were blocked by EGFR1 inhibitor indicating that E2 may increase cell migration via crosstalk with EGFR signaling. These data suggest a promotional role for E2 in breast cancer cell migration but an antiestrogenic, preventative role for resveratrol.

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

    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.

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

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

    2008-08-21

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

  5. The pathogen-actin connection: A platform for defense signaling in plants

    Day, B; Henty, Jessica L; Porter, K J; Staiger, Chris J

    2011-09-08

    The cytoskeleton, a dynamic network of cytoplasmic polymers, plays a central role in numerous fundamental processes, such as development, reproduction, and cellular responses to biotic and abiotic stimuli. As a platform for innate immune responses in mammalian cells, the actin cytoskeleton is a central component in the organization and activation of host defenses, including signaling and cellular repair. In plants, our understanding of the genetic and biochemical responses in both pathogen and host that are required for virulence and resistance has grown enormously. Additional advances in live-cell imaging of cytoskeletal dynamics have markedly altered our view of actin turnover in plants. In this review, we outline current knowledge of host resistance following pathogen perception, both in terms of the genetic interactions that mediate defense signaling, as well as the biochemical and cellular processes that are required for defense signaling.

  6. Effect of collagen I and fibronectin on the adhesion, elasticity and cytoskeletal organization of prostate cancer cells

    Research highlights: → Depending on the metastatic origin, prostate cancer cells differ in their affinity to COL1. → COL1 affects specifically the F-actin and cell elasticity of bone-derived prostate cancer cells. → Cell elasticity can be used as a biomarker for cancer cells from different metastases. -- Abstract: Despite of intensive research efforts, the precise mechanism of prostate cancer metastasis in bone is still not fully understood. Several studies have suggested that specific matrix production by the bone cells, such as collagen I, supports cancer cell invasion. The aim of this study was to investigate the effect of collagen I (COL1) and fibronectin (FN) on cell adhesion, cell elasticity and cytoskeletal organization of prostate cancer cells. Two cell lines, bone marrow- (PC3) and lymph node-derived (LNCaP) were cultivated on COL1 and FN (control protein). By using a quantitative adhesion assay and time-lapse analysis, it was found that PC3, but not LNCaP, adhered strongly and were more spread on COL1. Next, PC3 and LNCaP were evaluated by atomic force microscopy (AFM) and flatness shape factor and cellular Young's modulus were calculated. The shape analysis revealed that PC3 were significantly flatter when grown on COL1 in comparison to LNCaP. In general, PC3 were also significantly stiffer than LNCaP and furthermore, their stiffness increased upon interaction with COL1. Since cell stiffness is strongly dependent on actin organization, phalloidin-based actin staining was performed and revealed that, of the two cell types as well as the two different matrix proteins, only PC3 grown on COL1 formed robust actin cytoskeleton. In conclusion, our study showed that PC3 cells have a strong affinity towards COL1. On this matrix protein, the cells adhered strongly and underwent a specific cell flattening. Moreover, with the establishment of PC3 contact to COL1 a significant increase of PC3 stiffness was observed due to a profound cytoskeletal rearrangement.

  7. Freezing tolerance of sea urchin embryonic cells: Differentiation commitment and cytoskeletal disturbances in culture.

    Odintsova, Nelly A; Ageenko, Natalya V; Kipryushina, Yulia O; Maiorova, Mariia A; Boroda, Andrey V

    2015-08-01

    This study focuses on the freezing tolerance of sea urchin embryonic cells. To significantly reduce the loss of physiological activity of these cells that occurs after cryopreservation and to study the effects of ultra-low temperatures on sea urchin embryonic cells, we tested the ability of the cells to differentiate into spiculogenic or pigment directions in culture, including an evaluation of the expression of some genes involved in pigment differentiation. A morphological analysis of cytoskeletal disturbances after freezing in a combination of penetrating (dimethyl sulfoxide and ethylene glycol) and non-penetrating (trehalose and polyvinylpyrrolidone) cryoprotectants revealed that the distribution pattern of filamentous actin and tubulin was similar to that in the control cultures. In contrast, very rare spreading cells and a small number of cells with filamentous actin and tubulin were detected after freezing in the presence of only non-penetrating cryoprotectants. The largest number of pigment cells was found in cultures frozen with trehalose or trehalose and dimethyl sulfoxide. The ability to induce the spicule formation was lost in the cells frozen only with non-penetrating cryoprotectants, while it was maximal in cultures frozen in a cryoprotective mixture containing both non-penetrating and penetrating cryoprotectants (particularly, when ethylene glycol was present). Using different markers for cell state assessment, an effective cryopreservation protocol for sea urchin cells was developed: three-step freezing with a low cooling rate (1-2°C/min) and a combination of non-penetrating and penetrating cryoprotectants made it possible to obtain a high level of cell viability (up to 65-80%). PMID:26049089

  8. The Formin Diaphanous Regulates Myoblast Fusion through Actin Polymerization and Arp2/3 Regulation.

    Su Deng

    2015-08-01

    Full Text Available The formation of multinucleated muscle cells through cell-cell fusion is a conserved process from fruit flies to humans. Numerous studies have shown the importance of Arp2/3, its regulators, and branched actin for the formation of an actin structure, the F-actin focus, at the fusion site. This F-actin focus forms the core of an invasive podosome-like structure that is required for myoblast fusion. In this study, we find that the formin Diaphanous (Dia, which nucleates and facilitates the elongation of actin filaments, is essential for Drosophila myoblast fusion. Following cell recognition and adhesion, Dia is enriched at the myoblast fusion site, concomitant with, and having the same dynamics as, the F-actin focus. Through analysis of Dia loss-of-function conditions using mutant alleles but particularly a dominant negative Dia transgene, we demonstrate that reduction in Dia activity in myoblasts leads to a fusion block. Significantly, no actin focus is detected, and neither branched actin regulators, SCAR or WASp, accumulate at the fusion site when Dia levels are reduced. Expression of constitutively active Dia also causes a fusion block that is associated with an increase in highly dynamic filopodia, altered actin turnover rates and F-actin distribution, and mislocalization of SCAR and WASp at the fusion site. Together our data indicate that Dia plays two roles during invasive podosome formation at the fusion site: it dictates the level of linear F-actin polymerization, and it is required for appropriate branched actin polymerization via localization of SCAR and WASp. These studies provide new insight to the mechanisms of cell-cell fusion, the relationship between different regulators of actin polymerization, and invasive podosome formation that occurs in normal development and in disease.

  9. The Role of Actin Cytoskeleton in Memory Formation in Amygdala.

    Lamprecht, Raphael

    2016-01-01

    The central, lateral and basolateral amygdala (BLA) nuclei are essential for the formation of long-term memories including emotional and drug-related memories. Studying cellular and molecular mechanisms of memory in amygdala may lead to better understanding of how memory is formed and of fear and addiction-related disorders. A challenge is to identify molecules activated by learning that subserve cellular changes needed for memory formation and maintenance in amygdala. Recent studies show that activation of synaptic receptors during fear and drug-related learning leads to alteration in actin cytoskeleton dynamics and structure in amygdala. Such changes in actin cytoskeleton in amygdala are essential for fear and drug-related memories formation. Moreover, the actin cytoskeleton subserves, after learning, changes in neuronal morphogenesis and glutamate receptors trafficking in amygdala. These cellular events are involved in fear and drug-related memories formation. Actin polymerization is also needed for the maintenance of drug-associated memories in amygdala. Thus, the actin cytoskeleton is a key mediator between receptor activation during learning and cellular changes subserving long-term memory (LTM) in amygdala. The actin cytoskeleton may serve as a target for pharmacological treatment of fear memory associated with fear and anxiety disorders and drug addiction to prevent the debilitating consequences of these diseases. PMID:27065800

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

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

    1992-01-01

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