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Sample records for actin cytoskeletal dynamics

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

    PubMed

    Groen, Christopher M; Tootle, Tina L

    2015-01-01

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

  2. Actin dynamics in living mammalian cells.

    PubMed

    Ballestrem, C; Wehrle-Haller, B; Imhof, B A

    1998-06-01

    The actin cytoskeleton maintains the cellular architecture and mediates cell movements. To explore actin cytoskeletal dynamics, the enhanced green fluorescent protein (EGFP) was fused to human &bgr ;-actin. The fusion protein was incorporated into actin fibers which became depolymerized upon cytochalasin B treatment. This functional EGFP-actin construct enabled observation of the actin cytoskeleton in living cells by time lapse fluorescence microscopy. Stable expression of the construct was obtained in mammalian cell lines of different tissue origins. In stationary cells, actin rich, ring-like structured 'actin clouds' were observed in addition to stress fibers. These ruffle-like structures were found to be involved in the reorganization of the actin cytoskeleton. In migratory cells, EGFP-actin was found in the advancing lamellipodium. Immobile actin spots developed in the lamellipodium and thin actin fibers formed parallel to the leading edge. Thus EGFP-actin expressed in living cells unveiled structures involved in the dynamics of the actin cytoskeleton.

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

    PubMed

    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. Actin Grips: Circular Actin-Rich Cytoskeletal Structures that Mediate the Wrapping of Polymeric Microfibers by Endothelial Cells

    PubMed Central

    Jones, Desiree; Park, DoYoung; Anghelina, Mirela; Pecot, Thierry; Machiraju, Raghu; Xue, Ruipeng; Lannutti, John; Thomas, Jessica; Cole, Sara; Moldovan, Leni; Moldovan, Nicanor I.

    2015-01-01

    Interaction of endothelial-lineage cells with three-dimensional substrates was much less studied than that with flat culture surfaces. We investigated the in vitro attachment of both mature endothelial cells (ECs) and of less differentiated EC colony-forming cells to poly-e-capro-lactone (PCL) fibers with diameters in 5–20 μm range (‘scaffold microfibers’, SMFs). We found that notwithstanding the poor intrinsic adhesiveness to PCL, both cell types completely wrapped the SMFs after long-term cultivation, thus attaining a cylindrical morphology. In this system, both EC types grew vigorously for more than a week and became increasingly more differentiated, as shown by multiplexed gene expression. Three-dimensional reconstructions from multiphoton confocal microscopy images using custom software showed that the filamentous (F) actin bundles took a conspicuous ring-like organization around the SMFs. Unlike the classical F-actin-containing stress fibers, these rings were not associated with either focal adhesions or intermediate filaments. We also demonstrated that plasma membrane boundaries adjacent to these circular cytoskeletal structures were tightly yet dynamically apposed to the SMFs, for which reason we suggest to call them ‘actin grips’. In conclusion, we describe a particular form of F-actin assembly with relevance for cytoskeletal organization in response to biomaterials, for endothelial-specific cell behavior in vitro and in vivo, and for tissue engineering. PMID:25818446

  5. Defining a core set of actin cytoskeletal proteins critical for actin-based motility of Rickettsia.

    PubMed

    Serio, Alisa W; Jeng, Robert L; Haglund, Cat M; Reed, Shawna C; Welch, Matthew D

    2010-05-20

    Many Rickettsia species are intracellular bacterial pathogens that use actin-based motility for spread during infection. However, while other bacteria assemble actin tails consisting of branched networks, Rickettsia assemble long parallel actin bundles, suggesting the use of a distinct mechanism for exploiting actin. To identify the underlying mechanisms and host factors involved in Rickettsia parkeri actin-based motility, we performed an RNAi screen targeting 115 actin cytoskeletal genes in Drosophila cells. The screen delineated a set of four core proteins-profilin, fimbrin/T-plastin, capping protein, and cofilin--as crucial for determining actin tail length, organizing filament architecture, and enabling motility. In mammalian cells, these proteins were localized throughout R. parkeri tails, consistent with a role in motility. Profilin and fimbrin/T-plastin were critical for the motility of R. parkeri but not Listeria monocytogenes. Our results highlight key distinctions between the evolutionary strategies and molecular mechanisms employed by bacterial pathogens to assemble and organize actin. PMID:20478540

  6. Nuclear actin modulates cell motility via transcriptional regulation of adhesive and cytoskeletal genes

    PubMed Central

    Sharili, Amir S.; Kenny, Fiona N.; Vartiainen, Maria K.; Connelly, John T.

    2016-01-01

    The actin cytoskeleton is a classic biomechanical mediator of cell migration. While it is known that actin also shuttles in and out of the nucleus, its functions within this compartment remain poorly understood. In this study, we investigated how nuclear actin regulates keratinocyte gene expression and cell behavior. Gene expression profiling of normal HaCaT keratinocytes compared to HaCaTs over-expressing wild-type β-actin or β-actin tagged with a nuclear localization sequence (NLS-actin), identified multiple adhesive and cytoskeletal genes, such as MYL9, ITGB1, and VCL, which were significantly down-regulated in keratinocytes with high levels of nuclear actin. In addition, genes associated with transcriptional regulation and apoptosis were up-regulated in cells over expressing NLS-actin. Functionally, accumulation of actin in the nucleus altered cytoskeletal and focal adhesion organization and inhibited cell motility. Exclusion of endogenous actin from the nucleus by knocking down Importin 9 reversed this phenotype and enhanced cell migration. Based on these findings, we conclude that the level of actin in the nucleus is a transcriptional regulator for tuning keratinocyte migration. PMID:27650314

  7. Nuclear actin modulates cell motility via transcriptional regulation of adhesive and cytoskeletal genes.

    PubMed

    Sharili, Amir S; Kenny, Fiona N; Vartiainen, Maria K; Connelly, John T

    2016-01-01

    The actin cytoskeleton is a classic biomechanical mediator of cell migration. While it is known that actin also shuttles in and out of the nucleus, its functions within this compartment remain poorly understood. In this study, we investigated how nuclear actin regulates keratinocyte gene expression and cell behavior. Gene expression profiling of normal HaCaT keratinocytes compared to HaCaTs over-expressing wild-type β-actin or β-actin tagged with a nuclear localization sequence (NLS-actin), identified multiple adhesive and cytoskeletal genes, such as MYL9, ITGB1, and VCL, which were significantly down-regulated in keratinocytes with high levels of nuclear actin. In addition, genes associated with transcriptional regulation and apoptosis were up-regulated in cells over expressing NLS-actin. Functionally, accumulation of actin in the nucleus altered cytoskeletal and focal adhesion organization and inhibited cell motility. Exclusion of endogenous actin from the nucleus by knocking down Importin 9 reversed this phenotype and enhanced cell migration. Based on these findings, we conclude that the level of actin in the nucleus is a transcriptional regulator for tuning keratinocyte migration. PMID:27650314

  8. Hes6 is required for actin cytoskeletal organization in differentiating C2C12 myoblasts

    SciTech Connect

    Malone, Caroline M.P.; Domaschenz, Renae; Amagase, Yoko; Dunham, Ian; Murai, Kasumi; Jones, Philip H.

    2011-07-01

    Hes6 is a member of the hairy-enhancer-of-split family of transcription factors that regulate proliferating cell fate in development and is known to be expressed in developing muscle. Here we investigate its function in myogenesis in vitro. We show that Hes6 is a direct transcriptional target of the myogenic transcription factors MyoD and Myf5, indicating that it is integral to the myogenic transcriptional program. The localization of Hes6 protein changes during differentiation, becoming predominantly nuclear. Knockdown of Hes6 mRNA levels by siRNA has no effect on cell cycle exit or induction of myosin heavy chain expression in differentiating C2C12 myoblasts, but F-actin filament formation is disrupted and both cell motility and myoblast fusion are reduced. The knockdown phenotype is rescued by expression of Hes6 cDNA resistant to siRNA. These results define a novel role for Hes6 in actin cytoskeletal dynamics in post mitotic myoblasts.

  9. γ-Actin is required for cytoskeletal maintenance but not development

    PubMed Central

    Belyantseva, Inna A.; Perrin, Benjamin J.; Sonnemann, Kevin J.; Zhu, Mei; Stepanyan, Ruben; McGee, JoAnn; Frolenkov, Gregory I.; Walsh, Edward J.; Friderici, Karen H.; Friedman, Thomas B.; Ervasti, James M.

    2009-01-01

    βcyto-Actin and γcyto-actin are ubiquitous proteins thought to be essential building blocks of the cytoskeleton in all non-muscle cells. Despite this widely held supposition, we show that γcyto-actin null mice (Actg1−/−) are viable. However, they suffer increased mortality and show progressive hearing loss during adulthood despite compensatory up-regulation of βcyto-actin. The surprising viability and normal hearing of young Actg1−/− mice means that βcyto-actin can likely build all essential non-muscle actin-based cytoskeletal structures including mechanosensory stereocilia of hair cells that are necessary for hearing. Although γcyto-actin–deficient stereocilia form normally, we found that they cannot maintain the integrity of the stereocilia actin core. In the wild-type, γcyto-actin localizes along the length of stereocilia but re-distributes to sites of F-actin core disruptions resulting from animal exposure to damaging noise. In Actg1−/− stereocilia similar disruptions are observed even without noise exposure. We conclude that γcyto-actin is required for reinforcement and long-term stability of F-actin–based structures but is not an essential building block of the developing cytoskeleton. PMID:19497859

  10. Mechanical force mobilizes zyxin from focal adhesions to actin filaments and regulates cytoskeletal reinforcement.

    PubMed

    Yoshigi, Masaaki; Hoffman, Laura M; Jensen, Christopher C; Yost, H Joseph; Beckerle, Mary C

    2005-10-24

    Organs and tissues adapt to acute or chronic mechanical stress by remodeling their actin cytoskeletons. Cells that are stimulated by cyclic stretch or shear stress in vitro undergo bimodal cytoskeletal responses that include rapid reinforcement and gradual reorientation of actin stress fibers; however, the mechanism by which cells respond to mechanical cues has been obscure. We report that the application of either unidirectional cyclic stretch or shear stress to cells results in robust mobilization of zyxin from focal adhesions to actin filaments, whereas many other focal adhesion proteins and zyxin family members remain at focal adhesions. Mechanical stress also induces the rapid zyxin-dependent mobilization of vasodilator-stimulated phosphoprotein from focal adhesions to actin filaments. Thickening of actin stress fibers reflects a cellular adaptation to mechanical stress; this cytoskeletal reinforcement coincides with zyxin mobilization and is abrogated in zyxin-null cells. Our findings identify zyxin as a mechanosensitive protein and provide mechanistic insight into how cells respond to mechanical cues. PMID:16247023

  11. Topography design concept of a tissue engineering scaffold for controlling cell function and fate through actin cytoskeletal modulation.

    PubMed

    Miyoshi, Hiromi; Adachi, Taiji

    2014-12-01

    The physiological role of the actin cytoskeleton is well known: it provides mechanical support and endogenous force generation for formation of a cell shape and for migration. Furthermore, a growing number of studies have demonstrated another significant role of the actin cytoskeleton: it offers dynamic epigenetic memory for guiding cell fate, in particular, proliferation and differentiation. Because instantaneous imbalance in the mechanical homeostasis is adjusted through actin remodeling, a synthetic extracellular matrix (ECM) niche as a source of topographical and mechanical cues is expected to be effective at modulation of the actin cytoskeleton. In this context, the synthetic ECM niche determines cell migration, proliferation, and differentiation, all of which have to be controlled in functional tissue engineering scaffolds to ensure proper regulation of tissue/organ formation, maintenance of tissue integrity and repair, and regeneration. Here, with an emphasis on the epigenetic role of the actin cytoskeletal system, we propose a design concept of micro/nanotopography of a tissue engineering scaffold for control of cell migration, proliferation, and differentiation in a stable and well-defined manner, both in vitro and in vivo. PMID:24720435

  12. Accumulation of Glucosylceramide in the Absence of the Beta-Glucosidase GBA2 Alters Cytoskeletal Dynamics

    PubMed Central

    Raju, Diana; Schonauer, Sophie; Hamzeh, Hussein; Flynn, Kevin C.; Bradke, Frank; vom Dorp, Katharina; Dörmann, Peter; Yildiz, Yildiz; Trötschel, Christian; Poetsch, Ansgar; Breiden, Bernadette; Sandhoff, Konrad; Körschen, Heinz G.; Wachten, Dagmar

    2015-01-01

    Glycosphingolipids are key elements of cellular membranes, thereby, controlling a variety of cellular functions. Accumulation of the simple glycosphingolipid glucosylceramide results in life-threatening lipid storage-diseases or in male infertility. How glucosylceramide regulates cellular processes is ill defined. Here, we reveal that glucosylceramide accumulation in GBA2 knockout-mice alters cytoskeletal dynamics due to a more ordered lipid organization in the plasma membrane. In dermal fibroblasts, accumulation of glucosylceramide augments actin polymerization and promotes microtubules persistence, resulting in a higher number of filopodia and lamellipodia and longer microtubules. Similar cytoskeletal defects were observed in male germ and Sertoli cells from GBA2 knockout-mice. In particular, the organization of F-actin structures in the ectoplasmic specialization and microtubules in the sperm manchette is affected. Thus, glucosylceramide regulates cytoskeletal dynamics, providing mechanistic insights into how glucosylceramide controls signaling pathways not only during sperm development, but also in other cell types. PMID:25803043

  13. Accumulation of glucosylceramide in the absence of the beta-glucosidase GBA2 alters cytoskeletal dynamics.

    PubMed

    Raju, Diana; Schonauer, Sophie; Hamzeh, Hussein; Flynn, Kevin C; Bradke, Frank; Vom Dorp, Katharina; Dörmann, Peter; Yildiz, Yildiz; Trötschel, Christian; Poetsch, Ansgar; Breiden, Bernadette; Sandhoff, Konrad; Körschen, Heinz G; Wachten, Dagmar

    2015-03-01

    Glycosphingolipids are key elements of cellular membranes, thereby, controlling a variety of cellular functions. Accumulation of the simple glycosphingolipid glucosylceramide results in life-threatening lipid storage-diseases or in male infertility. How glucosylceramide regulates cellular processes is ill defined. Here, we reveal that glucosylceramide accumulation in GBA2 knockout-mice alters cytoskeletal dynamics due to a more ordered lipid organization in the plasma membrane. In dermal fibroblasts, accumulation of glucosylceramide augments actin polymerization and promotes microtubules persistence, resulting in a higher number of filopodia and lamellipodia and longer microtubules. Similar cytoskeletal defects were observed in male germ and Sertoli cells from GBA2 knockout-mice. In particular, the organization of F-actin structures in the ectoplasmic specialization and microtubules in the sperm manchette is affected. Thus, glucosylceramide regulates cytoskeletal dynamics, providing mechanistic insights into how glucosylceramide controls signaling pathways not only during sperm development, but also in other cell types.

  14. Cytoskeletal proteins in cortical development and disease: actin associated proteins in periventricular heterotopia

    PubMed Central

    Lian, Gewei; Sheen, Volney L.

    2015-01-01

    The actin cytoskeleton regulates many important cellular processes in the brain, including cell division and proliferation, migration, and cytokinesis and differentiation. These developmental processes can be regulated through actin dependent vesicle and organelle movement, cell signaling, and the establishment and maintenance of cell junctions and cell shape. Many of these processes are mediated by extensive and intimate interactions of actin with cellular membranes and proteins. Disruption in the actin cytoskeleton in the brain gives rise to periventricular heterotopia (PH), a malformation of cortical development, characterized by abnormal neurons clustered deep in the brain along the lateral ventricles. This disorder can give rise to seizures, dyslexia and psychiatric disturbances. Anatomically, PH is characterized by a smaller brain (impaired proliferation), heterotopia (impaired initial migration) and disruption along the neuroependymal lining (impaired cell-cell adhesion). Genes causal for PH have also been implicated in actin-dependent processes. The current review provides mechanistic insight into actin cytoskeletal regulation of cortical development in the context of this malformation of cortical development. PMID:25883548

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

    PubMed Central

    Wilson, Carlos; González-Billault, Christian

    2015-01-01

    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 (ROS; 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. PMID:26483635

  16. The role of Nox-mediated oxidation in the regulation of cytoskeletal dynamics.

    PubMed

    Valdivia, Alejandra; Duran, Charity; San Martin, Alejandra

    2015-01-01

    Nox generated ROS, particularly those derived from Nox1, Nox2 and Nox4, have emerged as important regulators of the actin cytoskeleton and cytoskeleton-supported cell functions, such as migration and adhesion. The effects of Nox-derived ROS on cytoskeletal remodeling may be largely attributed to the ability of ROS to directly modify proteins that constitute or are associated with the cytoskeleton. Additionally, Nox-derived ROS may participate in signaling pathways governing cytoskeletal remodeling. In addition to these more extensively studied signaling pathways involving Nox-derived ROS, there also exist redox sensitive pathways for which the source of ROS is unclear. ROS from as of yet undetermined sources play a role in modifying, and thus regulating, the activity of several proteins critical for remodeling of the actin cytoskeleton. In this review we discuss ROS sensitive targets that are likely to affect cytoskeletal dynamics, as well as the potential involvement of Nox proteins.

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2014-09-15

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

  19. Actin3 promoter reveals undulating F-actin bundles at shanks and dynamic F-actin meshworks at tips of tip-growing pollen tubes.

    PubMed

    Jásik, Ján; Mičieta, Karol; Siao, Wei; Voigt, Boris; Stuchlík, Stanislav; Schmelzer, Elmon; Turňa, Ján; Baluška, František

    2016-01-01

    The dynamic actin cytoskeleton of pollen tubes is both the driver of the tip growth and the organizer of cell polarity. In order to understand this fast re-arranging cytoskeletal system, we need reliable constructs expressed under relevant promoters. Here we are reporting that the Lifeact reporter, expressed under the pollen-specific Actin3 promoter, visualizes very dynamic F-actin elements both in germinating pollen grains and tip-growing pollen tubes. Importantly, we have documented very active actin polymerization at the cell periphery, especially in the bulging area during pollen germination and in the apical clear zone. Expression of the Lifeact reporter under control of the pollen-specific Actin3 promoter revealed 2 new aspects: (i) long F-actin bundles in pollen tube shanks are dynamic, showing undulating movements, (ii) subapical 'actin collars' or 'fringes' are absent.

  20. Actin3 promoter reveals undulating F-actin bundles at shanks and dynamic F-actin meshworks at tips of tip-growing pollen tubes

    PubMed Central

    Jásik, Ján; Mičieta, Karol; Siao, Wei; Voigt, Boris; Stuchlík, Stanislav; Schmelzer, Elmon; Turňa, Ján; Baluška, František

    2016-01-01

    ABSTRACT The dynamic actin cytoskeleton of pollen tubes is both the driver of the tip growth and the organizer of cell polarity. In order to understand this fast re-arranging cytoskeletal system, we need reliable constructs expressed under relevant promoters. Here we are reporting that the Lifeact reporter, expressed under the pollen-specific Actin3 promoter, visualizes very dynamic F-actin elements both in germinating pollen grains and tip-growing pollen tubes. Importantly, we have documented very active actin polymerization at the cell periphery, especially in the bulging area during pollen germination and in the apical clear zone. Expression of the Lifeact reporter under control of the pollen-specific Actin3 promoter revealed 2 new aspects: (i) long F-actin bundles in pollen tube shanks are dynamic, showing undulating movements, (ii) subapical ‘actin collars’ or ‘fringes’ are absent. PMID:26980067

  1. Neuroprotective effects of hypothermia on synaptic actin cytoskeletal changes induced by perinatal asphyxia.

    PubMed

    Muñiz, Javier; Romero, Juan; Holubiec, Mariana; Barreto, George; González, Janneth; Saint-Martin, Madeleine; Blanco, Eduardo; Carlos Cavicchia, Juan; Castilla, Rocío; Capani, Francisco

    2014-05-14

    Cerebral hypoxia-ischemia damages synaptic proteins, resulting in cytoskeletal alterations, protein aggregation and neuronal death. In the previous works, we have shown neuronal and synaptic changes in rat neostriatum subjected to hypoxia that leads to ubi-protein accumulation. Recently, we also showed that, changes in F-actin organization could be related to early alterations induced by hypoxia in the Central Nervous System. However, little is known about effective treatment to diminish the damage. The main aim of this work is to study the effects of birth hypothermia on the actin cytoskeleton of neostriatal post-synaptic densities (PSD) in 60 days olds rats by immunohistochemistry, photooxidation and western blot. We used 2 different protocols of hypothermia: (a) intrahypoxic hypothermia at 15°C and (b) post-hypoxia hypothermia at 32°C. Consistent with previous data at 30 days, staining with phalloidin-Alexa(488) followed by confocal microscopy analysis showed an increase of F-actin fluorescent staining in the neostriatum of hypoxic animals. Correlative photooxidation electron microscopy confirmed these observations showing an increment in the number of mushroom-shaped F-actin staining spines in neostriatal excitatory synapses in rats subjected to hypoxia. In addition, western blot revealed β-actin increase in PSDs in hypoxic animals. The optic relative density measurement showed a significant difference between controls and hypoxic animals. When hypoxia was induced under hypothermic conditions, the changes observed in actin cytoskeleton were blocked. Post-hypoxic hypothermia showed similar answer but actin cytoskeleton modifications were not totally reverted as we observed at 15°C. These data suggest that the decrease of the body temperature decreases the actin modifications in dendritic spines preventing the neuronal death.

  2. Neuroprotective effects of hypothermia on synaptic actin cytoskeletal changes induced by perinatal asphyxia.

    PubMed

    Muñiz, Javier; Romero, Juan; Holubiec, Mariana; Barreto, George; González, Janneth; Saint-Martin, Madeleine; Blanco, Eduardo; Carlos Cavicchia, Juan; Castilla, Rocío; Capani, Francisco

    2014-05-14

    Cerebral hypoxia-ischemia damages synaptic proteins, resulting in cytoskeletal alterations, protein aggregation and neuronal death. In the previous works, we have shown neuronal and synaptic changes in rat neostriatum subjected to hypoxia that leads to ubi-protein accumulation. Recently, we also showed that, changes in F-actin organization could be related to early alterations induced by hypoxia in the Central Nervous System. However, little is known about effective treatment to diminish the damage. The main aim of this work is to study the effects of birth hypothermia on the actin cytoskeleton of neostriatal post-synaptic densities (PSD) in 60 days olds rats by immunohistochemistry, photooxidation and western blot. We used 2 different protocols of hypothermia: (a) intrahypoxic hypothermia at 15°C and (b) post-hypoxia hypothermia at 32°C. Consistent with previous data at 30 days, staining with phalloidin-Alexa(488) followed by confocal microscopy analysis showed an increase of F-actin fluorescent staining in the neostriatum of hypoxic animals. Correlative photooxidation electron microscopy confirmed these observations showing an increment in the number of mushroom-shaped F-actin staining spines in neostriatal excitatory synapses in rats subjected to hypoxia. In addition, western blot revealed β-actin increase in PSDs in hypoxic animals. The optic relative density measurement showed a significant difference between controls and hypoxic animals. When hypoxia was induced under hypothermic conditions, the changes observed in actin cytoskeleton were blocked. Post-hypoxic hypothermia showed similar answer but actin cytoskeleton modifications were not totally reverted as we observed at 15°C. These data suggest that the decrease of the body temperature decreases the actin modifications in dendritic spines preventing the neuronal death. PMID:24685534

  3. Phosphatidic acid regulation of PIPKI is critical for actin cytoskeletal reorganization.

    PubMed

    Roach, Akua N; Wang, Ziqing; Wu, Ping; Zhang, Feng; Chan, Robin B; Yonekubo, Yoshiya; Di Paolo, Gilbert; Gorfe, Alemayehu A; Du, Guangwei

    2012-12-01

    Type I phosphatidylinositol-4-phosphate 5-kinase (PIPKI) is the main enzyme generating the lipid second messenger phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2], which has critical functions in many cellular processes, such as cytoskeletal reorganization, membrane trafficking, and signal transduction. All three members of the PIPKI family are activated by phosphatidic acid (PA). However, how PA regulates the activity and functions of PIPKI have not been fully elucidated. In this study, we identify a PA-binding site on PIPKIγ. Mutation of this site inhibited the PA-stimulated activity and membrane localization of PIPKIγ as well as the formation of actin comets and foci induced by PIPKIγ. We also demonstrate that phospholipase D (PLD) generates a pool of PA involved in PIPKIγ regulation by showing that PLD inhibitors blocked the membrane localization of PIPKIγ and its ability to induce actin cytoskeletal reorganization. Targeting the PIPKIγ PA-binding-deficient mutant to membranes by a membrane localization sequence failed to restore the actin reorganization activity of PIPKIγ, suggesting that PA binding is not only involved in recruiting PIPKIγ to membranes but also may induce a conformational change. Taken together, these results reveal a new molecular mechanism through which PA regulates PIPKI and provides direct evidence that PA is important for the localization and functions of PIPKI in intact cells. PMID:22991193

  4. A Legionella effector modulates host cytoskeletal structure by inhibiting actin polymerization

    PubMed Central

    Guo, Zhenhua; Stephenson, Robert; Qiu, Jiazhang; Zheng, Shijun; Luo, Zhao-Qing

    2014-01-01

    Successful infection by the opportunistic pathogen Legionella pneumophila requires the collective activity of hundreds of virulence proteins delivered into the host cell by the Dot/Icm type IV secretion system. These virulence proteins, also called effectors modulate distinct host cellular processes to create a membrane-bound niche called the Legionella containing vacuole (LCV) supportive of bacterial growth. We found that Ceg14(Lpg0437), a Dot/Icm substrate is toxic to yeast and such toxicity can be alleviated by overexpression of profilin, a protein involved in cytoskeletal structure in eukaryotes. We further showed that mutations in profilin affect actin binding but not other functions such as interactions with poly-L-proline or phosphatidylinositol, abolish its suppressor activity. Consistent with the fact the profilin suppresses its toxicity, expression of Ceg14 but not its non-toxic mutants in yeast affects actin distribution and budding of daughter cells. Although Ceg14 does not detectably interact with profilin, it co-sediments with filamentous actin and inhibits actin polymerization, causing the accumulation of short actin filaments. These results reveal that multiple L. pneumophila effectors target components of the host cytoskeleton. PMID:24286927

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

    SciTech Connect

    Choong, Grace; Liu, Ying; Xiao, Weiqun; Templeton, Douglas M.

    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.

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

    PubMed Central

    Ahn, Young-Ho; Gibbons, Don L.; Chakravarti, Deepavali; Creighton, Chad J.; Rizvi, Zain H.; Adams, Henry P.; Pertsemlidis, Alexander; Gregory, Philip A.; Wright, Josephine A.; Goodall, Gregory J.; Flores, Elsa R.; Kurie, Jonathan M.

    2012-01-01

    Metastatic cancer is extremely difficult to treat, and the presence of metastases greatly reduces a cancer patient’s likelihood of long-term survival. The ZEB1 transcriptional repressor promotes metastasis through downregulation of microRNAs (miRs) that are strong inducers of epithelial differentiation and inhibitors of stem cell factors. Given that each miR can target multiple genes with diverse functions, we posited that the prometastatic network controlled by ZEB1 extends beyond these processes. We tested this hypothesis using a mouse model of human lung adenocarcinoma metastasis driven by ZEB1, human lung carcinoma cells, and human breast carcinoma cells. Transcriptional profiling studies revealed that ZEB1 controls the expression of numerous oncogenic and tumor-suppressive miRs, including miR-34a. Ectopic expression of miR-34a decreased tumor cell invasion and metastasis, inhibited the formation of promigratory cytoskeletal structures, suppressed activation of the RHO GTPase family, and regulated a gene expression signature enriched in cytoskeletal functions and predictive of outcome in human lung adenocarcinomas. We identified several miR-34a target genes, including Arhgap1, which encodes a RHO GTPase activating protein that was required for tumor cell invasion. These findings demonstrate that ZEB1 drives prometastatic actin cytoskeletal remodeling by downregulating miR-34a expression and provide a compelling rationale to develop miR-34a as a therapeutic agent in lung cancer patients. PMID:22850877

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

    NASA Astrophysics Data System (ADS)

    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

  8. Cytoskeletal social networking in the growth cone: How +TIPs mediate microtubule-actin cross-linking to drive axon outgrowth and guidance.

    PubMed

    Cammarata, Garrett M; Bearce, Elizabeth A; Lowery, Laura Anne

    2016-09-01

    The growth cone is a unique structure capable of guiding axons to their proper destinations. Within the growth cone, extracellular guidance cues are interpreted and then transduced into physical changes in the actin filament (F-actin) and microtubule cytoskeletons, providing direction and movement. While both cytoskeletal networks individually possess important growth cone-specific functions, recent data over the past several years point towards a more cooperative role between the two systems. Facilitating this interaction between F-actin and microtubules, microtubule plus-end tracking proteins (+TIPs) have been shown to link the two cytoskeletons together. Evidence suggests that many +TIPs can couple microtubules to F-actin dynamics, supporting both microtubule advance and retraction in the growth cone periphery. In addition, growing in vitro and in vivo data support a secondary role for +TIPs in which they may participate as F-actin nucleators, thus directly influencing F-actin dynamics and organization. This review focuses on how +TIPs may link F-actin and microtubules together in the growth cone, and how these interactions may influence axon guidance. © 2016 Wiley Periodicals, Inc.

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

    PubMed

    Arous, Caroline; Halban, Philippe A

    2015-10-01

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

  10. A dynamic formin-dependent deep F-actin network in axons

    PubMed Central

    Ganguly, Archan; Tang, Yong; Wang, Lina; Ladt, Kelsey; Loi, Jonathan; Dargent, Bénédicte; Leterrier, Christophe

    2015-01-01

    Although actin at neuronal growth cones is well-studied, much less is known about actin organization and dynamics along axon shafts and presynaptic boutons. Using probes that selectively label filamentous-actin (F-actin), we found focal “actin hotspots” along axons—spaced ∼3–4 µm apart—where actin undergoes continuous assembly/disassembly. These foci are a nidus for vigorous actin polymerization, generating long filaments spurting bidirectionally along axons—a phenomenon we call “actin trails.” Super-resolution microscopy reveals intra-axonal deep actin filaments in addition to the subplasmalemmal “actin rings” described recently. F-actin hotspots colocalize with stationary axonal endosomes, and blocking vesicle transport diminishes the actin trails, suggesting mechanistic links between vesicles and F-actin kinetics. Actin trails are formin—but not Arp2/3—dependent and help enrich actin at presynaptic boutons. Finally, formin inhibition dramatically disrupts synaptic recycling. Collectively, available data suggest a two-tier F-actin organization in axons, with stable “actin rings” providing mechanical support to the plasma membrane and dynamic "actin trails" generating a flexible cytoskeletal network with putative physiological roles. PMID:26216902

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

    PubMed

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

    2016-06-01

    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.

  12. Arp2/3 complex and actin dynamics are required for actin-based mitochondrial motility in yeast.

    PubMed

    Boldogh, I R; Yang, H C; Nowakowski, W D; Karmon, S L; Hays, L G; Yates, J R; Pon, L A

    2001-03-13

    The Arp2/3 complex is implicated in actin polymerization-driven movement of Listeria monocytogenes. Here, we find that Arp2p and Arc15p, two subunits of this complex, show tight, actin-independent association with isolated yeast mitochondria. Arp2p colocalizes with mitochondria. Consistent with this result, we detect Arp2p-dependent formation of actin clouds around mitochondria in intact yeast. Cells bearing mutations in ARP2 or ARC15 genes show decreased velocities of mitochondrial movement, loss of all directed movement and defects in mitochondrial morphology. Finally, we observe a decrease in the velocity and extent of mitochondrial movement in yeast in which actin dynamics are reduced but actin cytoskeletal structure is intact. These results support the idea that the movement of mitochondria in yeast is actin polymerization driven and that this movement requires Arp2/3 complex.

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

    SciTech Connect

    Hirano, Hidemi; Matsuura, Yoshiyuki

    2011-10-22

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

  14. Cytoskeletal Network Morphology Regulates Intracellular Transport Dynamics.

    PubMed

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

    2015-10-20

    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 timescales, 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 redirect cargo back to the nucleus caused large variations in network transport. Filament polarity was more important than filament orientation in reducing average transit times, and transport properties were optimized in networks with intermediate motor on and off rates. Our results provide important insights into the functional constraints on intracellular transport under which cells have evolved cytoskeletal structures, and have potential applications for enhancing reactions in biomimetic systems through rational transport network design.

  15. Cytoskeletal dynamics and lung fluid balance.

    PubMed

    Vogel, Stephen M; Malik, Asrar B

    2012-01-01

    This article examines the role of the endothelial cytoskeleton in the lung's ability to restrict fluid and protein to vascular space at normal vascular pressures and thereby to protect lung alveoli from lethal flooding. The barrier properties of microvascular endothelium are dependent on endothelial cell contact with other vessel-wall lining cells and with the underlying extracellular matrix (ECM). Focal adhesion complexes are essential for attachment of endothelium to ECM. In quiescent endothelial cells, the thick cortical actin rim helps determine cell shape and stabilize endothelial adherens junctions and focal adhesions through protein bridges to actin cytoskeleton. Permeability-increasing agonists signal activation of "small GTPases" of the Rho family to reorganize the actin cytoskeleton, leading to endothelial cell shape change, disassembly of cortical actin rim, and redistribution of actin into cytoplasmic stress fibers. In association with calcium- and Src-regulated myosin light chain kinase (MLCK), stress fibers become actinomyosin-mediated contractile units. Permeability-increasing agonists stimulate calcium entry and induce tyrosine phosphorylation of VE-cadherin (vascular endothelial cadherin) and β-catenins to weaken or pull apart endothelial adherens junctions. Some permeability agonists cause latent activation of the small GTPases, Cdc42 and Rac1, which facilitate endothelial barrier recovery and eliminate interendothelial gaps. Under the influence of Cdc42 and Rac1, filopodia and lamellipodia are generated by rearrangements of actin cytoskeleton. These motile evaginations extend endothelial cell borders across interendothelial gaps, and may initiate reannealing of endothelial junctions. Endogenous barrier protective substances, such as sphingosine-1-phosphate, play an important role in maintaining a restrictive endothelial barrier and counteracting the effects of permeability-increasing agonists.

  16. Molecular Mechanotransduction: how forces trigger cytoskeletal dynamics

    NASA Astrophysics Data System (ADS)

    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.

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

    PubMed

    Zaban, Beatrix; Maisch, Jan; Nick, Peter

    2013-02-01

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

  18. Regulators of Actin Dynamics in Gastrointestinal Tract Tumors.

    PubMed

    Steinestel, Konrad; Wardelmann, Eva; Hartmann, Wolfgang; Grünewald, Inga

    2015-01-01

    Reorganization of the actin cytoskeleton underlies cell migration in a wide variety of physiological and pathological processes, such as embryonic development, wound healing, and tumor cell invasion. It has been shown that actin assembly and disassembly are precisely regulated by intracellular signaling cascades that respond to changes in the cell microenvironment, ligand binding to surface receptors, or oncogenic transformation of the cell. Actin-nucleating and actin-depolymerizing (ANFs/ADFs) and nucleation-promoting factors (NPFs) regulate cytoskeletal dynamics at the leading edge of migrating cells, thereby modulating cell shape; these proteins facilitate cellular movement and mediate degradation of the surrounding extracellular matrix by secretion of lytic proteases, thus eliminating barriers for tumor cell invasion. Accordingly, expression and activity of these actin-binding proteins have been linked to enhanced metastasis and poor prognosis in a variety of malignancies. In this review, we will summarize what is known about expression patterns and the functional role of actin regulators in gastrointestinal tumors and evaluate first pharmacological approaches to prevent invasion and metastatic dissemination of malignant cells. PMID:26345720

  19. Regulators of Actin Dynamics in Gastrointestinal Tract Tumors

    PubMed Central

    Steinestel, Konrad; Wardelmann, Eva; Hartmann, Wolfgang; Grünewald, Inga

    2015-01-01

    Reorganization of the actin cytoskeleton underlies cell migration in a wide variety of physiological and pathological processes, such as embryonic development, wound healing, and tumor cell invasion. It has been shown that actin assembly and disassembly are precisely regulated by intracellular signaling cascades that respond to changes in the cell microenvironment, ligand binding to surface receptors, or oncogenic transformation of the cell. Actin-nucleating and actin-depolymerizing (ANFs/ADFs) and nucleation-promoting factors (NPFs) regulate cytoskeletal dynamics at the leading edge of migrating cells, thereby modulating cell shape; these proteins facilitate cellular movement and mediate degradation of the surrounding extracellular matrix by secretion of lytic proteases, thus eliminating barriers for tumor cell invasion. Accordingly, expression and activity of these actin-binding proteins have been linked to enhanced metastasis and poor prognosis in a variety of malignancies. In this review, we will summarize what is known about expression patterns and the functional role of actin regulators in gastrointestinal tumors and evaluate first pharmacological approaches to prevent invasion and metastatic dissemination of malignant cells. PMID:26345720

  20. Cytoskeletal Mechanics

    NASA Astrophysics Data System (ADS)

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

    2006-10-01

    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.

  1. Cytoskeletal Mechanics

    NASA Astrophysics Data System (ADS)

    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.

  2. Hic-5 Regulates Actin Cytoskeletal Reorganization and Expression of Fibrogenic Markers and Myocilin in Trabecular Meshwork Cells

    PubMed Central

    Pattabiraman, Padmanabhan Paranji; Rao, Ponugoti Vasantha

    2015-01-01

    Purpose To explore the role of inducible focal adhesion (FA) protein Hic-5 in actin cytoskeletal reorganization, FA formation, fibrogenic activity, and expression of myocilin in trabecular meshwork (TM) cells. Methods Using primary cultures of human TM (HTM) cells, the effects of various external factors on Hic-5 protein levels, as well as the effects of recombinant Hic-5 and Hic-5 small interfering RNA (siRNA) on actin cytoskeleton, FAs, myocilin, α-smooth muscle actin (αSMA), and collagen-1 were determined by immunofluorescence and immunoblot analyses. Results Hic-5 distributes discretely to the FAs in HTM cells and throughout the TM and Schlemm's canal of the human aqueous humor (AH) outflow pathway. Transforming growth factor-β2 (TGF-β2), endothelin-1, lysophosphatidic acid, hydrogen peroxide, and RhoA significantly increased Hic-5 protein levels in HTM cells in association with reorganization of actin cytoskeleton and FAs. While recombinant Hic-5 induced actin stress fibers, FAs, αv integrin redistribution to the FAs, increased levels of αSMA, collagen-1, and myocilin, Hic-5 siRNA suppressed most of these responses in HTM cells. Hic-5 siRNA also suppressed TGF-β2-induced fibrogenic activity and dexamethasone-induced myocilin expression in HTM cells. Conclusions Taken together, these results reveal that Hic-5, whose levels were increased by various external factors implicated in elevated intraocular pressure, induces actin cytoskeletal reorganization, FAs, expression of fibrogenic markers, and myocilin in HTM cells. These characteristics of Hic-5 in TM cells indicate its importance in regulation of AH outflow through the TM in both normal and glaucomatous eyes. PMID:26313302

  3. Dynamic actin structures stabilized by profilin.

    PubMed Central

    Finkel, T; Theriot, J A; Dise, K R; Tomaselli, G F; Goldschmidt-Clermont, P J

    1994-01-01

    We describe the production and analysis of clonal cell lines in which we have overexpressed human profilin, a small ubiquitous actin monomer binding protein, to assess the role of profilin on actin function in vivo. The concentration of filamentous actin is increased in cells with higher profilin levels, and actin filament half-life measured in these cells is directly proportional to the steady-state profilin concentration. The distribution of actin filaments is altered by profilin overexpression. While parallel actin bundles crossing the cells are virtually absent in cells overexpressing profilin, the submembranous actin network of these cells is denser than in control cells. These results suggest that in vivo profilin regulates the stability, and thereby distribution, of specific dynamic actin structures. Images PMID:8108438

  4. Actomyosin-dependent dynamic spatial patterns of cytoskeletal components drive mesoscale podosome organization

    PubMed Central

    Meddens, Marjolein B. M.; Pandzic, Elvis; Slotman, Johan A.; Guillet, Dominique; Joosten, Ben; Mennens, Svenja; Paardekooper, Laurent M.; Houtsmuller, Adriaan B.; van den Dries, Koen; Wiseman, Paul W.; Cambi, Alessandra

    2016-01-01

    Podosomes are cytoskeletal structures crucial for cell protrusion and matrix remodelling in osteoclasts, activated endothelial cells, macrophages and dendritic cells. In these cells, hundreds of podosomes are spatially organized in diversely shaped clusters. Although we and others established individual podosomes as micron-sized mechanosensing protrusive units, the exact scope and spatiotemporal organization of podosome clustering remain elusive. By integrating a newly developed extension of Spatiotemporal Image Correlation Spectroscopy with novel image analysis, we demonstrate that F-actin, vinculin and talin exhibit directional and correlated flow patterns throughout podosome clusters. Pattern formation and magnitude depend on the cluster actomyosin machinery. Indeed, nanoscopy reveals myosin IIA-decorated actin filaments interconnecting multiple proximal podosomes. Extending well-beyond podosome nearest neighbours, the actomyosin-dependent dynamic spatial patterns reveal a previously unappreciated mesoscale connectivity throughout the podosome clusters. This directional transport and continuous redistribution of podosome components provides a mechanistic explanation of how podosome clusters function as coordinated mechanosensory area. PMID:27721497

  5. Dynamics of an actin spring

    NASA Astrophysics Data System (ADS)

    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.

  6. Thermally Driven and Cytoskeletal-Assisted Dynamics of the Mitochondrial Reticulum

    NASA Astrophysics Data System (ADS)

    Knowles, Michelle K.; Marcus, Andrew H.

    2003-05-01

    We report Fourier imaging correlation spectroscopy (FICS) and digital video fluorescence microscopy (DVFM) measurements of the dynamics of the mitochondrial reticulum in living osteosarcoma cells. Mitochondrial dynamics are strongly influenced by interactions with cytoskeletal filaments and their associated motor proteins, which lead to complex multi-exponential relaxations that occur over a wide range of spatial and temporal scales. The cytoskeleton consists of an interconnected polymer network whose primary components are microfilaments (actin) and microtubules (tubulin). These filaments work with motor proteins to translate organelles through the cell. We studied the dynamics of osteosarcoma cells labeled with red fluorescent protein in the mitochondrial matrix space using DVFM and FICS. Cells were then treated with cytoskeletal destabilizing drugs. Analysis of microscopy data allows for us to determine whether dynamic processes are diffusive or driven (by the cytoskeleton or collective dynamics). In FICS experiments, the control cells exhibit a unique pattern of dynamics that are then simplified when the cytoskeleton is depolymerized. Upon depolymerization, the dynamics of the organelle appear primarily diffusive.

  7. Collective dynamics of processive cytoskeletal motors.

    PubMed

    McLaughlin, R Tyler; Diehl, Michael R; Kolomeisky, Anatoly B

    2016-01-01

    Major cellular processes are supported by various biomolecular motors that usually operate together as teams. We present an overview of the collective dynamics of processive cytokeletal motor proteins based on recent experimental and theoretical investigations. Experimental studies show that multiple motors function with different degrees of cooperativity, ranging from negative to positive. This effect depends on the mechanical properties of individual motors, the geometry of their connections, and the surrounding cellular environment. Theoretical models based on stochastic approaches underline the importance of intermolecular interactions, the properties of single motors, and couplings with cellular medium in predicting the collective dynamics. We discuss several features that specify the cooperativity in motor proteins. Based on this approach a general picture of collective dynamics of motor proteins is formulated, and the future directions and challenges are discussed.

  8. Collective Dynamics of Processive Cytoskeletal Motors

    PubMed Central

    McLaughlin, R. Tyler; Diehl, Michael R.; Kolomeisky, Anatoly B.

    2015-01-01

    Major cellular processes are supported by various biomolecular motors that usually operate together as teams. We present an overview of the collective dynamics of processive cytokeletal motor proteins based on recent experimental and theoretical investigations. Experimental studies show that multiple motors function with different degrees of cooperativity, ranging from negative to positive. This effect depends on the mechanical properties of individual motors, the geometry of their connections, and the surrounding cellular environment. Theoretical models based on stochastic approaches underline the importance of intermolecular interactions, the properties of single motors, and couplings with cellular medium in predicting the collective dynamics. We discuss several features that specify the cooperativity in motor proteins. Based on this approach a general picture of collective dynamics of motor proteins is formulated, and the future directions and challenges are discussed. PMID:26444155

  9. Simultaneous Visualization of Peroxisomes and Cytoskeletal Elements Reveals Actin and Not Microtubule-Based Peroxisome Motility in Plants1[w

    PubMed Central

    Mathur, Jaideep; Mathur, Neeta; Hülskamp, Martin

    2002-01-01

    Peroxisomes were visualized in living plant cells using a yellow fluorescent protein tagged with a peroxisomal targeting signal consisting of the SKL motif. Simultaneous visualization of peroxisomes and microfilaments/microtubules was accomplished in onion (Allium cepa) epidermal cells transiently expressing the yellow fluorescent protein-peroxi construct, a green fluorescent protein-mTalin construct that labels filamentous-actin filaments, and a green fluorescent protein-microtubule-binding domain construct that labels microtubules. The covisualization of peroxisomes and cytoskeletal elements revealed that, contrary to the reports from animal cells, peroxisomes in plants appear to associate with actin filaments and not microtubules. That peroxisome movement is actin based was shown by pharmacological studies. For this analysis we used onion epidermal cells and various cell types of Arabidopsis including trichomes, root hairs, and root cortex cells exhibiting different modes of growth. In transient onion epidermis assay and in transgenic Arabidopsis plants, an interference with the actin cytoskeleton resulted in progressive loss of saltatory movement followed by the aggregation and a complete cessation of peroxisome motility within 30 min of drug application. Microtubule depolymerization or stabilization had no effect. PMID:11891258

  10. Widespread mRNA Association with Cytoskeletal Motor Proteins and Identification and Dynamics of Myosin-Associated mRNAs in S. cerevisiae

    PubMed Central

    Casolari, Jason M.; Thompson, Michael A.; Salzman, Julia; Champion, Lowry M.; Moerner, W. E.; Brown, Patrick O.

    2012-01-01

    Programmed mRNA localization to specific subcellular compartments for localized translation is a fundamental mechanism of post-transcriptional regulation that affects many, and possibly all, mRNAs in eukaryotes. We describe her e a systematic approach to identify the RNA cargoes associated with the cytoskeletal motor proteins of Saccharomyces cerevisiae in combination with live-cell 3D super-localization microscopy of endogenously tagged mRNAs. Our analysis identified widespread association of mRNAs with cytoskeletal motor proteins, including association of Myo3 with mRNAs encoding key regulators of actin branching and endocytosis such as WASP and WIP. Using conventional fluorescence microscopy and expression of MS2-tagged mRNAs from endogenous loci, we observed a strong bias for actin patch nucleator mRNAs to localize to the cell cortex and the actin patch in a Myo3- and F-actin dependent manner. Use of a double-helix point spread function (DH-PSF) microscope allowed super-localization measurements of single mRNPs at a spatial precision of 25 nm in x and y and 50 nm in z in live cells with 50 ms exposure times, allowing quantitative profiling of mRNP dynamics. The actin patch mRNA exhibited distinct and characteristic diffusion coefficients when compared to a control mRNA. In addition, disruption of F-actin significantly expanded the 3D confinement radius of an actin patch nucleator mRNA, providing a quantitative assessment of the contribution of the actin cytoskeleton to mRNP dynamic localization. Our results provide evidence for specific association of mRNAs with cytoskeletal motor proteins in yeast, suggest that different mRNPs have distinct and characteristic dynamics, and lend insight into the mechanism of actin patch nucleator mRNA localization to actin patches. PMID:22359641

  11. Arabidopsis Actin Depolymerizing Factor4 Modulates the Stochastic Dynamic Behavior of Actin Filaments in the Cortical Array of Epidermal Cells[C][W

    PubMed Central

    Henty, Jessica L.; Bledsoe, Samuel W.; Khurana, Parul; Meagher, Richard B.; Day, Brad; Blanchoin, Laurent; Staiger, Christopher J.

    2011-01-01

    Actin filament arrays are constantly remodeled as the needs of cells change as well as during responses to biotic and abiotic stimuli. Previous studies demonstrate that many single actin filaments in the cortical array of living Arabidopsis thaliana epidermal cells undergo stochastic dynamics, a combination of rapid growth balanced by disassembly from prolific severing activity. Filament turnover and dynamics are well understood from in vitro biochemical analyses and simple reconstituted systems. However, the identification in living cells of the molecular players involved in controlling actin dynamics awaits the use of model systems, especially ones where the power of genetics can be combined with imaging of individual actin filaments at high spatial and temporal resolution. Here, we test the hypothesis that actin depolymerizing factor (ADF)/cofilin contributes to stochastic filament severing and facilitates actin turnover. A knockout mutant for Arabidopsis ADF4 has longer hypocotyls and epidermal cells when compared with wild-type seedlings. This correlates with a change in actin filament architecture; cytoskeletal arrays in adf4 cells are significantly more bundled and less dense than in wild-type cells. Several parameters of single actin filament turnover are also altered. Notably, adf4 mutant cells have a 2.5-fold reduced severing frequency as well as significantly increased actin filament lengths and lifetimes. Thus, we provide evidence that ADF4 contributes to the stochastic dynamic turnover of actin filaments in plant cells. PMID:22010035

  12. The Switch-associated Protein 70 (SWAP-70) Bundles Actin Filaments and Contributes to the Regulation of F-actin Dynamics*

    PubMed Central

    Chacón-Martínez, Carlos Andrés; Kiessling, Nadine; Winterhoff, Moritz; Faix, Jan; Müller-Reichert, Thomas; Jessberger, Rolf

    2013-01-01

    Coordinated assembly and disassembly of actin into filaments and higher order structures such as stress fibers and lamellipodia are fundamental for cell migration and adhesion. However, the precise spatiotemporal regulation of F-actin structures is not completely understood. SWAP-70, a phosphatidylinositol 3,4,5-trisphosphate-interacting, F-actin-binding protein, participates in actin rearrangements through yet unknown mechanisms. Here, we show that SWAP-70 is an F-actin-bundling protein that oligomerizes through a Gln/Glu-rich stretch within a coiled-coil region. SWAP-70 bundles filaments in parallel and anti-parallel fashion through its C-terminal F-actin binding domain and delays dilution-induced F-actin depolymerization. We further demonstrate that SWAP-70 co-localizes and directly interacts with cofilin, an F-actin severing and depolymerization factor, and contributes to the regulation of cofilin activity in vivo. In line with these activities, upon stem cell factor stimulation, murine bone marrow-derived mast cells lacking SWAP-70 display aberrant regulation of F-actin and actin free barbed ends dynamics. Moreover, proper stem cell factor-dependent cofilin activation via dephosphorylation and subcellular redistribution into a detergent-resistant cytoskeletal compartment also require SWAP-70. Together, these findings reveal an important role of SWAP-70 in the dynamic spatiotemporal regulation of F-actin networks. PMID:23921380

  13. Profilin connects actin assembly with microtubule dynamics.

    PubMed

    Nejedla, Michaela; Sadi, Sara; Sulimenko, Vadym; de Almeida, Francisca Nunes; Blom, Hans; Draber, Pavel; Aspenström, Pontus; Karlsson, Roger

    2016-08-01

    Profilin controls actin nucleation and assembly processes in eukaryotic cells. Actin nucleation and elongation promoting factors (NEPFs) such as Ena/VASP, formins, and WASP-family proteins recruit profilin:actin for filament formation. Some of these are found to be microtubule associated, making actin polymerization from microtubule-associated platforms possible. Microtubules are implicated in focal adhesion turnover, cell polarity establishment, and migration, illustrating the coupling between actin and microtubule systems. Here we demonstrate that profilin is functionally linked to microtubules with formins and point to formins as major mediators of this association. To reach this conclusion, we combined different fluorescence microscopy techniques, including superresolution microscopy, with siRNA modulation of profilin expression and drug treatments to interfere with actin dynamics. Our studies show that profilin dynamically associates with microtubules and this fraction of profilin contributes to balance actin assembly during homeostatic cell growth and affects micro-tubule dynamics. Hence profilin functions as a regulator of microtubule (+)-end turnover in addition to being an actin control element.

  14. Profilin connects actin assembly with microtubule dynamics

    PubMed Central

    Nejedla, Michaela; Sadi, Sara; Sulimenko, Vadym; de Almeida, Francisca Nunes; Blom, Hans; Draber, Pavel; Aspenström, Pontus; Karlsson, Roger

    2016-01-01

    Profilin controls actin nucleation and assembly processes in eukaryotic cells. Actin nucleation and elongation promoting factors (NEPFs) such as Ena/VASP, formins, and WASP-family proteins recruit profilin:actin for filament formation. Some of these are found to be microtubule associated, making actin polymerization from microtubule-associated platforms possible. Microtubules are implicated in focal adhesion turnover, cell polarity establishment, and migration, illustrating the coupling between actin and microtubule systems. Here we demonstrate that profilin is functionally linked to microtubules with formins and point to formins as major mediators of this association. To reach this conclusion, we combined different fluorescence microscopy techniques, including superresolution microscopy, with siRNA modulation of profilin expression and drug treatments to interfere with actin dynamics. Our studies show that profilin dynamically associates with microtubules and this fraction of profilin contributes to balance actin assembly during homeostatic cell growth and affects micro­tubule dynamics. Hence profilin functions as a regulator of microtubule (+)-end turnover in addition to being an actin control element. PMID:27307590

  15. Nucleotide Regulation of the Structure and Dynamics of G-Actin

    PubMed Central

    Saunders, Marissa G.; Tempkin, Jeremy; Weare, Jonathan; Dinner, Aaron R.; Roux, Benoît; Voth, Gregory A.

    2014-01-01

    Actin, a highly conserved cytoskeletal protein found in all eukaryotic cells, facilitates cell motility and membrane remodeling via a directional polymerization cycle referred to as treadmilling. The nucleotide bound at the core of each actin subunit regulates this process. Although the biochemical kinetics of treadmilling has been well characterized, the atomistic details of how the nucleotide affects polymerization remain to be definitively determined. There is increasing evidence that the nucleotide regulation (and other characteristics) of actin cannot be fully described from the minimum energy structure, but rather depends on a dynamic equilibrium between conformations. In this work we explore the conformational mobility of the actin monomer (G-actin) in a coarse-grained subspace using umbrella sampling to bias all-atom molecular-dynamics simulations along the variables of interest. The results reveal that ADP-bound actin subunits are more conformationally mobile than ATP-bound subunits. We used a multiscale analysis method involving coarse-grained and atomistic representations of these simulations to characterize how the nucleotide affects the low-energy states of these systems. The interface between subdomains SD2–SD4, which is important for polymerization, is stabilized in an actin filament-like (F-actin) conformation in ATP-bound G-actin. Additionally, the nucleotide modulates the conformation of the SD1-SD3 interface, a region involved in the binding of several actin-binding proteins. PMID:24739170

  16. Cell adhesion: integrating cytoskeletal dynamics and cellular tension

    PubMed Central

    Parsons, J. Thomas; Horwitz, Alan Rick; Schwartz, Martin A.

    2010-01-01

    Cell migration affects all morphogenetic processes and contributes to numerous diseases, including cancer and cardiovascular disease. For most cells in most environments, movement begins with protrusion of the cell membrane followed by the formation of new adhesions at the cell front that link the actin cytoskeleton to the substratum, generation of traction forces that move the cell forwards and disassembly of adhesions at the cell rear. Adhesion formation and disassembly drive the migration cycle by activating Rho GTPases, which in turn regulate actin polymerization and myosin II activity, and therefore adhesion dynamics. PMID:20729930

  17. A Secreted Ankyrin-Repeat Protein from Clinical Stenotrophomonas maltophilia Isolates Disrupts Actin Cytoskeletal Structure.

    PubMed

    MacDonald, Logan C; O'Keefe, Sean; Parnes, Mei-Fan; MacDonald, Hanlon; Stretz, Lindsey; Templer, Suzanne J; Wong, Emily L; Berger, Bryan W

    2016-01-01

    Stenotrophomonas maltophilia is an emerging, multidrug-resistant pathogen of increasing importance for the immunocompromised, including cystic fibrosis patients. Despite its significance as an emerging pathogen, relatively little is known regarding the specific factors and mechanisms that contribute to its pathogenicity. We identify and characterize a putative ankyrin-repeat protein (Smlt3054) unique to clinical S. maltophilia isolates that binds F-actin in vitro and co-localizes with actin in transfected HEK293a cells. Smlt3054 is endogenously expressed and secreted from clinical S. maltophilia isolates, but not an environmental isolate (R551-3). The in vitro binding of Smlt3054 to F-actin resulted in a thickening of the filaments as observed by TEM. Ectopic expression of Smlt3054-GFP exhibits strong co-localization with F-actin, with distinct, retrograde F-actin waves specifically associated with Smlt3054 in individual cells as well as formation of dense, internal inclusions at the expense of retrograde F-actin waves. Collectively, our results point to an interaction between Smlt3054 and F-actin. Furthermore, as a potentially secreted protein unique to clinical S. maltophilia isolates, Smlt3054 may serve as a starting point for understanding the mechanisms by which S. maltophilia has become an emergent pathogen. PMID:27622948

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

    PubMed Central

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

    2016-01-01

    The actin bundle is an array of linear actin filaments cross-linked by actin-bundling proteins, but its assembly and dynamics are not as well understood as those of the branched actin network. Here we used the Drosophila bristle as a model system to study actin bundle formation. We found that cofilin, a major actin disassembly factor of the branched actin network, promotes the formation and positioning of actin bundles in the developing bristles. Loss of function of cofilin or AIP1, a cofactor of cofilin, each resulted in increased F-actin levels and severe defects in actin bundle organization, with the defects from cofilin deficiency being more severe. Further analyses revealed that cofilin likely regulates actin bundle formation and positioning by the following means. First, cofilin promotes a large G-actin pool both locally and globally, likely ensuring rapid actin polymerization for bundle initiation and growth. Second, cofilin limits the size of a nonbundled actin-myosin network to regulate the positioning of actin bundles. Third, cofilin prevents incorrect assembly of branched and myosin-associated actin filament into bundles. Together these results demonstrate that the interaction between the dynamic dendritic actin network and the assembling actin bundles is critical for actin bundle formation and needs to be closely regulated. PMID:27385345

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

    PubMed

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

    2016-08-15

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

  20. Retraction in amoeboid cell motility powered by cytoskeletal dynamics.

    PubMed

    Miao, Long; Vanderlinde, Orion; Stewart, Murray; Roberts, Thomas M

    2003-11-21

    Cells crawl by coupling protrusion of their leading edge with retraction of their cell body. Protrusion is generated by the polymerization and bundling of filaments, but the mechanism of retraction is less clear. We have reconstituted retraction in vitro by adding Yersinia tyrosine phosphatase to the major sperm protein-based motility apparatus assembled from Ascaris sperm extracts. Retraction in vitro parallels that observed in vivo and is generated primarily by disassembly and rearrangement of the cytoskeleton. Therefore, cytoskeletal dynamics alone, unassisted by conventional motors, are able to generate both of these central components of amoeboid locomotion.

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

    PubMed

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

    2015-11-01

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

  2. Dynamic reorganization of the actin cytoskeleton

    PubMed Central

    Gressin, Laurène; Théry, Manuel; Blanchoin, Laurent

    2015-01-01

    Cellular processes, including morphogenesis, polarization, and motility, rely on a variety of actin-based structures. Although the biochemical composition and filament organization of these structures are different, they often emerge from a common origin. This is possible because the actin structures are highly dynamic. Indeed, they assemble, grow, and disassemble in a time scale of a second to a minute. Therefore, the reorganization of a given actin structure can promote the formation of another. Here, we discuss such transitions and illustrate them with computer simulations. PMID:26989473

  3. Shaping up to divide: coordinating actin and microtubule cytoskeletal remodelling during mitosis.

    PubMed

    Lancaster, Oscar M; Baum, Buzz

    2014-10-01

    Cell division requires the wholesale reorganization of cell architecture. At the same time as the microtubule network is remodelled to generate a bipolar spindle, animal cells entering mitosis replace their interphase actin cytoskeleton with a contractile mitotic actomyosin cortex that is tightly coupled to the plasma membrane--driving mitotic cell rounding. Here, we consider how these two processes are coordinated to couple chromosome segregation and cell division. In doing so we explore the relative roles of cell shape and the actin cortex in spindle morphogenesis, orientation and positioning.

  4. Structural Dynamics of an Actin Spring

    PubMed Central

    Mahadevan, L.; Riera, C.S.; Shin, Jennifer H.

    2011-01-01

    Actin-based motility in cells is usually associated with either polymerization/depolymerization in the presence of cross-linkers or contractility in the presence of myosin motors. Here, we focus on a third distinct mechanism involving actin in motility, seen in the dynamics of an active actin spring that powers the acrosomal reaction of the horseshoe crab (Limulus polyphemus) sperm. During this process, a 60-μm bent and twisted bundle of cross-linked actin uncoils and becomes straight in a few seconds in the presence of Ca2+. This straightening, which occurs at a constant velocity, allows the acrosome to forcefully penetrate the egg. Synthesizing ultrastructural information with the kinetics, energetics, and imaging of calcium binding allows us to construct a dynamical theory for this mechanochemical engine consistent with our experimental observations. It also illuminates the general mechanism by which energy may be stored in conformational changes and released cooperatively in ordered macromolecular assemblies. PMID:21320427

  5. Dynamics of Cytoskeletal Proteins during Fcγ Receptor-mediated Phagocytosis in MacrophagesV⃞

    PubMed Central

    Diakonova, Maria; Bokoch, Gary; Swanson, Joel A.

    2002-01-01

    Particle ingestion by phagocytosis results from sequential rearrangements of the actin cytoskeleton and overlying membrane. To assemble a chronology of molecular events during phagosome formation and to examine the contributions of phosphoinositide 3-kinase (PI 3-kinase) to these dynamics, a method was developed for synchronizing Fcγ receptor-mediated phagocytosis by murine macrophages. Erythrocytes opsonized with complement component C3bi were bound to macrophages at 37°C, a condition that does not favor particle phagocytosis. Addition of soluble anti-erythrocyte IgG resulted in rapid opsonization of the bound erythrocytes, followed by their immediate internalization via phagocytosis. Cellular content of F-actin, as measured by binding of rhodamine-phalloidin, increased transiently during phagocytosis, and this increase was not diminished by inhibitors of PI 3-kinase. Immunofluorescence localization of myosins in macrophages fixed at various times during phagocytosis indicated that myosins II and IXb were concentrated in early phagosomes, myosin IC increased later, and myosin V appeared after phagosome closure. Other cytoskeletal proteins showed similar variations in the timing of their appearance in phagosomes. The PI 3-kinase inhibitor wortmannin did not change the dynamics of PI 3-kinase or ezrin localization but prevented the loss of PAK1 from phagosomes. These results suggest that PI 3-kinase deactivates PAK1, and that this may be needed for phagosome closure. PMID:11854399

  6. VEGF-A, cytoskeletal dynamics, and the pathological vascular phenotype

    SciTech Connect

    Nagy, Janice A. . E-mail: jnagy@bidmc.harvard.edu; Senger, Donald R. . E-mail: dsenger@bidmc.harvard.edu

    2006-03-10

    Normal angiogenesis is a complex process involving the organization of proliferating and migrating endothelial cells (ECs) into a well-ordered and highly functional vascular network. In contrast, pathological angiogenesis, which is a conspicuous feature of tumor growth, ischemic diseases, and chronic inflammation, is characterized by vessels with aberrant angioarchitecture and compromised barrier function. Herein we review the subject of pathological angiogenesis, particularly that driven by vascular endothelial growth factor (VEGF-A), from a new perspective. We propose that the serious structural and functional anomalies associated with VEGF-A-elicited neovessels, reflect, at least in part, imbalances in the internal molecular cues that govern the ordered assembly of ECs into three dimensional vascular networks and preserve vessel barrier function. Adopting such a viewpoint widens the focus from solely on specific pro-angiogenic stimuli such as VEGF-A to include a key set of cytoskeletal regulatory molecules, the Rho GTPases, which are known to direct multiple aspects of vascular morphogenesis including EC motility, alignment, multi-cellular organization, as well as intercellular junction integrity. We offer this perspective to draw attention to the importance of endothelial cytoskeletal dynamics for proper neovascularization and to suggest new therapeutic strategies with the potential to improve the pathological vascular phenotype.

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

    PubMed

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

    2015-01-01

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

  8. Actin Filament Segmentation Using Dynamic Programming

    PubMed Central

    Li, Hongsheng; Shen, Tian; Huang, Xiaolei

    2011-01-01

    We introduce a novel algorithm for actin filament segmentation in 2D TIRFM image sequences. This problem is difficult because actin filaments dynamically change shapes during their growth, and the TIRFM images are usually noisy. We ask a user to specify the two tips of a filament of interest in the first frame. We then model the segmentation problem in an image sequence as a temporal chain, where its states are tip locations; given candidate tip locations, actin filaments' body points are inferred by a dynamic programming method, which adaptively generates candidate solutions. Combining candidate tip locations and their inferred body points, the temporal chain model is efficiently optimized using another dynamic programming method. Evaluation on noisy TIRFM image sequences demonstrates the accuracy and robustness of this approach. PMID:21761674

  9. Pdlim7 Regulates Arf6-Dependent Actin Dynamics and Is Required for Platelet-Mediated Thrombosis in Mice

    PubMed Central

    Miller, Kaylie P.; Krcmery, Jennifer; Simon, Hans-Georg

    2016-01-01

    Upon vessel injury, platelets become activated and rapidly reorganize their actin cytoskeleton to adhere to the site of endothelial damage, triggering the formation of a fibrin-rich plug to prevent further blood loss. Inactivation of Pdlim7 provides the new perspective that regulation of actin cytoskeletal changes in platelets is dependent on the encoded PDZ-LIM protein. Loss-of-function of Pdlim7 triggers hypercoagulopathy and causes significant perinatal lethality in mice. Our in vivo and in vitro studies reveal that Pdlim7 is dynamically distributed along actin fibers, and lack of Pdlim7 leads to a marked inability to rearrange the actin cytoskeleton. Specifically, the absence of Pdlim7 prevents platelets from bundling actin fibers into a concentric ring that defines the round spread shape of activated platelets. Similarly, in mouse embryonic fibroblasts, loss of Pdlim7 abolishes the formation of stress fibers needed to adopt the typical elongated fibroblast shape. In addition to revealing a fundamental cell biological role in actin cytoskeletal organization, we also demonstrate a function of Pdlim7 in regulating the cycling between the GTP/GDP-bound states of Arf6. The small GTPase Arf6 is an essential factor required for actin dynamics, cytoskeletal rearrangements, and platelet activation. Consistent with our findings of significantly elevated initial F-actin ratios and subsequent morphological aberrations, loss of Pdlim7 causes a shift in balance towards an increased Arf6-GTP level in resting platelets. These findings identify a new Pdlim7-Arf6 axis controlling actin dynamics and implicate Pdlim7 as a primary endogenous regulator of platelet-dependent hemostasis. PMID:27792740

  10. The alternatively-included 11a sequence modifies the effects of Mena on actin cytoskeletal organization and cell behavior

    PubMed Central

    Balsamo, Michele; Mondal, Chandrani; Carmona, Guillaume; McClain, Leslie M.; Riquelme, Daisy N.; Tadros, Jenny; Ma, Duan; Vasile, Eliza; Condeelis, John S.; Lauffenburger, Douglas A.; Gertler, Frank B.

    2016-01-01

    During tumor progression, alternative splicing gives rise to different Mena protein isoforms. We analyzed how Mena11a, an isoform enriched in epithelia and epithelial-like cells, affects Mena-dependent regulation of actin dynamics and cell behavior. While other Mena isoforms promote actin polymerization and drive membrane protrusion, we find that Mena11a decreases actin polymerization and growth factor-stimulated membrane protrusion at lamellipodia. Ectopic Mena11a expression slows mesenchymal-like cell motility, while isoform-specific depletion of endogenous Mena11a in epithelial-like tumor cells perturbs cell:cell junctions and increases membrane protrusion and overall cell motility. Mena11a can dampen membrane protrusion and reduce actin polymerization in the absence of other Mena isoforms, indicating that it is not simply an inactive Mena isoform. We identify a phosphorylation site within 11a that is required for some Mena11a-specific functions. RNA-seq data analysis from patient cohorts demonstrates that the difference between mRNAs encoding constitutive Mena sequences and those containing the 11a exon correlates with metastasis in colorectal cancer, suggesting that 11a exon exclusion contributes to invasive phenotypes and leads to poor clinical outcomes. PMID:27748415

  11. Interior decoration: tropomyosin in actin dynamics and cell migration.

    PubMed

    Lees, Justin G; Bach, Cuc T T; O'Neill, Geraldine M

    2011-01-01

    Cell migration and invasion requires the precise temporal and spatial orchestration of a variety of biological processes. Filaments of polymerized actin are critical players in these diverse processes, including the regulation of cell anchorage points (both cell-cell and cell-extracellular matrix), the uptake and delivery of molecules via endocytic pathways and the generation of force for both membrane protrusion and retraction. How the actin filaments are specialized for each of these discrete functions is yet to be comprehensively elucidated. The cytoskeletal tropomyosins are a family of actin associating proteins that form head-to-tail polymers which lay in the major groove of polymerized actin filaments. In the present review we summarize the emerging isoform-specific functions of tropomyosins in cell migration and invasion and discuss their potential roles in the specialization of actin filaments for the diverse cellular processes that together regulate cell migration and invasion.

  12. Force-Induced Dynamical Properties of Multiple Cytoskeletal Filaments Are Distinct from that of Single Filaments

    PubMed Central

    Das, Dipjyoti; Das, Dibyendu; Padinhateeri, Ranjith

    2014-01-01

    How cytoskeletal filaments collectively undergo growth and shrinkage is an intriguing question. Collective properties of multiple bio-filaments (actin or microtubules) undergoing hydrolysis have not been studied extensively earlier within simple theoretical frameworks. In this paper, we study the collective dynamical properties of multiple filaments under force, and demonstrate the distinct properties of a multi-filament system in comparison to a single filament. Comparing stochastic simulation results with recent experimental data, we show that multi-filament collective catastrophes are slower than catastrophes of single filaments. Our study also shows further distinctions as follows: (i) force-dependence of the cap-size distribution of multiple filaments are quantitatively different from that of single filaments, (ii) the diffusion constant associated with the system length fluctuations is distinct for multiple filaments, and (iii) switching dynamics of multiple filaments between capped and uncapped states and the fluctuations therein are also distinct. We build a unified picture by establishing interconnections among all these collective phenomena. Additionally, we show that the collapse times during catastrophes can be sharp indicators of collective stall forces exceeding the additive contributions of single filaments. PMID:25531397

  13. Dynamic Regulation of Sarcomeric Actin Filaments in Striated Muscle

    PubMed Central

    Ono, Shoichiro

    2010-01-01

    In striated muscle, the actin cytoskeleton is differentiated into myofibrils. Actin and myosin filaments are organized in sarcomeres and specialized for producing contractile forces. Regular arrangement of actin filaments with uniform length and polarity is critical for the contractile function. However, the mechanisms of assembly and maintenance of sarcomeric actin filaments in striated muscle are not completely understood. Live imaging of actin in striated muscle has revealed that actin subunits within sarcomeric actin filaments are dynamically exchanged without altering overall sarcomeric structures. A number of regulators for actin dynamics have been identified, and malfunction of these regulators often result in disorganization of myofibril structures or muscle diseases. Therefore, proper regulation of actin dynamics in striated muscle is critical for assembly and maintenance of functional myofibrils. Recent studies have suggested that both enhancers of actin dynamics and stabilizers of actin filaments are important for sarcomeric actin organization. Further investigation of the regulatory mechanism of actin dynamics in striated muscle should be a key to understanding how myofibrils develop and operate. © 2010 Wiley-Liss, Inc. PMID:20737540

  14. Actin dynamics in papilla cells of Brassica rapa during self- and cross-pollination.

    PubMed

    Iwano, Megumi; Shiba, Hiroshi; Matoba, Kyoko; Miwa, Teruhiko; Funato, Miyuki; Entani, Tetsuyuki; Nakayama, Pulla; Shimosato, Hiroko; Takaoka, Akio; Isogai, Akira; Takayama, Seiji

    2007-05-01

    The self-incompatibility system of the plant species Brassica is controlled by the S-locus, which contains S-RECEPTOR KINASE (SRK) and S-LOCUS PROTEIN11 (SP11). SP11 binding to SRK induces SRK autophosphorylation and initiates a signaling cascade leading to the rejection of self pollen. However, the mechanism controlling hydration and germination arrest during self-pollination is unclear. In this study, we examined the role of actin, a key cytoskeletal component regulating the transport system for hydration and germination in the papilla cell during pollination. Using rhodamine-phalloidin staining, we showed that cross-pollination induced actin polymerization, whereas self-pollination induced actin reorganization and likely depolymerization. By monitoring transiently expressed green fluorescent protein fused to the actin-binding domain of mouse talin, we observed the concentration of actin bundles at the cross-pollen attachment site and actin reorganization and likely depolymerization at the self-pollen attachment site; the results correspond to those obtained by rhodamine-phalloidin staining. We further showed that the coat of self pollen is sufficient to mediate this response. The actin-depolymerizing drug cytochalasin D significantly inhibited pollen hydration and germination during cross-pollination, further emphasizing a role for actin in these processes. Additionally, three-dimensional electron microscopic tomography revealed the close association of the actin cytoskeleton with an apical vacuole network. Self-pollination disrupted the vacuole network, whereas cross-pollination led to vacuolar rearrangements toward the site of pollen attachment. Taken together, our data suggest that self- and cross-pollination differentially affect the dynamics of the actin cytoskeleton, leading to changes in vacuolar structure associated with hydration and germination.

  15. Actin-binding proteins: the long road to understanding the dynamic landscape of cellular actin networks.

    PubMed

    Lappalainen, Pekka

    2016-08-15

    The actin cytoskeleton supports a vast number of cellular processes in nonmuscle cells. It is well established that the organization and dynamics of the actin cytoskeleton are controlled by a large array of actin-binding proteins. However, it was only 40 years ago that the first nonmuscle actin-binding protein, filamin, was identified and characterized. Filamin was shown to bind and cross-link actin filaments into higher-order structures and contribute to phagocytosis in macrophages. Subsequently many other nonmuscle actin-binding proteins were identified and characterized. These proteins regulate almost all steps of the actin filament assembly and disassembly cycles, as well as the arrangement of actin filaments into diverse three-dimensional structures. Although the individual biochemical activities of most actin-regulatory proteins are relatively well understood, knowledge of how these proteins function together in a common cytoplasm to control actin dynamics and architecture is only beginning to emerge. Furthermore, understanding how signaling pathways and mechanical cues control the activities of various actin-binding proteins in different cellular, developmental, and pathological processes will keep researchers busy for decades. PMID:27528696

  16. Changes in cytoskeletal dynamics and nonlinear rheology with metastatic ability in cancer cell lines

    NASA Astrophysics Data System (ADS)

    Coughlin, Mark F.; Fredberg, Jeffrey J.

    2013-12-01

    Metastatic outcome is impacted by the biophysical state of the primary tumor cell. To determine if changes in cancer cell biophysical properties facilitate metastasis, we quantified cytoskeletal biophysics in well-characterized human skin, bladder, prostate and kidney cell line pairs that differ in metastatic ability. Using magnetic twisting cytometry with optical detection, cytoskeletal dynamics was observed through spontaneous motion of surface bound marker beads and nonlinear rheology was characterized through large amplitude forced oscillations of probe beads. Measurements of cytoskeletal dynamics and nonlinear rheology differed between strongly and weakly metastatic cells. However, no set of biophysical parameters changed systematically with metastatic ability across all cell lines. Compared to their weakly metastatic counterparts, the strongly metastatic kidney cancer cells exhibited both increased cytoskeletal dynamics and stiffness at large deformation which are thought to facilitate the process of vascular invasion.

  17. Cortactin Branches Out: Roles in Regulating Protrusive Actin Dynamics

    PubMed Central

    Ammer, Amanda Gatesman; Weed, Scott A.

    2008-01-01

    Since its discovery in the early 1990’s, cortactin has emerged as a key signaling protein in many cellular processes, including cell adhesion, migration, endocytosis, and tumor invasion. While the list of cellular functions influenced by cortactin grows, the ability of cortactin to interact with and alter the cortical actin network is central to its role in regulating these processes. Recently, several advances have been made in our understanding of the interaction between actin and cortactin, providing insight into how these two proteins work together to provide a framework for normal and altered cellular function. This review examines how regulation of cortactin through post-translational modifications and interactions with multiple binding partners elicits changes in cortical actin cytoskeletal organization, impacting the regulation and formation of actin-rich motility structures. PMID:18615630

  18. AKAP-Independent Localization of Type-II Protein Kinase A to Dynamic Actin Microspikes

    PubMed Central

    Rivard, Robert L.; Birger, Monique; Gaston, Kara J.; Howe, Alan K.

    2010-01-01

    Regulation of the cyclic AMP-dependent protein kinase (PKA) in subcellular space is required for cytoskeletal dynamics and chemotaxis. Currently, spatial regulation of PKA is thought to require the association of PKA regulatory (R) subunits with A-kinase anchoring proteins (AKAPs). Here, we show that the regulatory RIIα subunit of PKA associates with dynamic actin microspikes in an AKAP-independent manner. Both endogenous RIIα and a GFP-RIIα fusion protein co-localize with F-actin in microspikes within hippocampal neuron growth cones and the leading edge lamellae of NG108-15 cells. Live-cell imaging demonstrates that RIIα-associated microspikes are highly dynamic and that the coupling of RIIα to actin is tight, as the movement of both actin and RIIα are immediately and coincidently stopped by low-dose cytochalasin D. Importantly, co-localization of RIIα and actin in these structures is resistant to displacement by a cell-permeable disrupter of PKA-AKAP interactions. Biochemical fractionation confirms that a substantial pool of PKA RIIα is associated with the detergent-insoluble cytoskeleton and is resistant to extraction by a peptide inhibitor of AKAP interactions. Finally, mutation of the AKAP-binding domain of RIIα fails to disrupt its association with actin microspikes. These data provide the first demonstration of the physical association of a kinase with such dynamic actin structures, as well as the first demonstration of the ability of type-II PKA to localize to discrete subcellular structures independently of canonical AKAP function. This association is likely to be important for microfilament dynamics and cell migration and may prime the investigation of novel mechanisms for localizing PKA activity. PMID:19536823

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

    SciTech Connect

    Morita, Tsuyoshi; Mayanagi, Taira; Sobue, Kenji

    2007-10-01

    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.

  20. Partial Depletion of Gamma-Actin Suppresses Microtubule Dynamics

    PubMed Central

    Po'uha, Sela T; Honore, Stephane; Braguer, Diane; Kavallaris, Maria

    2013-01-01

    Actin and microtubule interactions are important for many cellular events, however these interactions are poorly described. Alterations in γ-actin are associated with diseases such as hearing loss and cancer. Functional investigations demonstrated that partial depletion of γ-actin affects cell polarity and induces resistance to microtubule-targeted agents. To determine whether γ-actin alterations directly affect microtubule dynamics, microtubule dynamic instability was analyzed in living cells following partial siRNA depletion of γ-actin. Partial depletion of γ-actin suppresses interphase microtubule dynamics by 17.5% due to a decrease in microtubule shortening rates and an increase in microtubule attenuation. γ-Actin partial depletion also increased distance-based microtubule catastrophe and rescue frequencies. In addition, knockdown of γ-actin delayed mitotic progression, partially blocking metaphase–anaphase transition and inhibiting cell proliferation. Interestingly, in the presence of paclitaxel, interphase microtubule dynamics were further suppressed by 24.4% in the γ-actin knockdown cells, which is comparable to 28.8% suppression observed in the control siRNA treated cells. Paclitaxel blocked metaphase–anaphase transition in both the γ-actin knockdown cells and the control siRNA cells. However, the extent of mitotic arrest was much higher in the control cells (28.4%), compared to the γ-actin depleted cells (8.5%). Therefore, suppression of microtubule dynamics by partial depletion of γ-actin is associated with marked delays in metaphase-anaphase transition and not mitotic arrest. This is the first demonstration that γ-actin can modulate microtubule dynamics by reducing the microtubule shortening rate, promoting paused/attenuated microtubules, and increasing transition frequencies suggesting a mechanistic link between γ-actin and microtubules. © 2013 Wiley Periodicals, Inc PMID:23335583

  1. CP beta3, a novel isoform of an actin-binding protein, is a component of the cytoskeletal calyx of the mammalian sperm head.

    PubMed

    von Bülow, M; Rackwitz, H R; Zimbelmann, R; Franke, W W

    1997-05-25

    In the mammalian sperm head, the nucleus is tightly associated with the calyx, a cell type-specific cytoskeletal structure. Previously, we have identified and characterized some basic proteins such as calicin and cylicins I and II as major calyx components of bovine and human spermatids and spermatozoa. Surprisingly we have now discovered another calyx constituent which by amino acid sequencing and cDNA cloning was recognized as a novel isoform of the widespread beta subunit of the heterodimeric actin-binding "capping protein" (CP). This polypeptide, CP beta3, of sperm calices, is identical with the beta2 subunit present in diverse somatic cell types, except that it shows an amino-terminal extension of 29 amino acids and its mRNA is detected only in testis and, albeit in trace amounts, brain. This CP beta3 mRNA contains the additional sequence, encoded by exon 1 of the gene, which is missing in beta2 mRNAs. Antibodies specific for the beta3 amino-terminal addition have been used to identify the protein by immunoblotting and to localize it to the calyx structure by immunofluorescence microscopy. We conclude that in spermiogenesis the transcription of the gene encoding the beta1, beta2, and beta3 CP subunits is regulated specifically to include exon 1 and to give rise to the testis isoform CP beta3, which is integrated into the calyx structure of the forming sperm head. This surprising finding of an actin-binding protein isoform in an insoluble cytoskeletal structure is discussed in relation to the demonstrated roles of actin and certain actin-binding proteins, such as Limulus alpha-scruin, in spermiogenesis and spermatozoa.

  2. Texture sensing of cytoskeletal dynamics in cell migration

    NASA Astrophysics Data System (ADS)

    Das, Satarupa; Lee, Rachel; Hourwitz, Matthew J.; Sun, Xiaoyu; Parent, Carole; Fourkas, John T.; Losert, Wolfgang

    Migrating cells can be directed towards a target by gradients in properties such as chemical concentration or mechanical properties of the surrounding microenvironment. In previous studies we have shown that micro/nanotopographical features on scales comparable to those of natural collagen fibers can guide fast migrating amoeboid cells by aligning actin polymerization waves to such nanostructures. We find that actin microfilaments and microtubules are aligned along the nanoridge topographies, modulating overall cell polarity and directional migration in epithelial cells. This work shows that topographic features on a biologically relevant length scale can modulate migration outcomes by affecting the texture sensing property of the cytoskeleton.

  3. Mechanical Response of Cytoskeletal Networks

    PubMed Central

    Gardel, Margaret L.; Kasza, Karen E.; Brangwynne, Clifford P.; Liu, Jiayu; Weitz, David A.

    2015-01-01

    The cellular cytoskeleton is a dynamic network of filamentous proteins, consisting of filamentous actin (F-actin), microtubules, and intermediate filaments. However, these networks are not simple linear, elastic solids; they can exhibit highly nonlinear elasticity and athermal dynamics driven by ATP-dependent processes. To build quantitative mechanical models describing complex cellular behaviors, it is necessary to understand the underlying physical principles that regulate force transmission and dynamics within these networks. In this chapter, we review our current understanding of the physics of networks of cytoskeletal proteins formed in vitro. We introduce rheology, the technique used to measure mechanical response. We discuss our current understanding of the mechanical response of F-actin networks, and how the biophysical properties of F-actin and actin cross-linking proteins can dramatically impact the network mechanical response. We discuss how incorporating dynamic and rigid microtubules into F-actin networks can affect the contours of growing microtubules and composite network rigidity. Finally, we discuss the mechanical behaviors of intermediate filaments. PMID:19118688

  4. Excitable actin dynamics in lamellipodial protrusion and retraction.

    PubMed

    Ryan, Gillian L; Petroccia, Heather M; Watanabe, Naoki; Vavylonis, Dimitrios

    2012-04-01

    Many animal cells initiate crawling by protruding lamellipodia, consisting of a dense network of actin filaments, at their leading edge. We imaged XTC cells that exhibit flat lamellipodia on poly-L-lysine-coated coverslips. Using active contours, we tracked the leading edge and measured the total amount of F-actin by summing the pixel intensities within a 5-μm band. We observed protrusion and retraction with period 130-200 s and local wavelike features. Positive (negative) velocities correlated with minimum (maximum) integrated actin concentration. Approximately constant retrograde flow indicated that protrusions and retractions were driven by fluctuations of the actin polymerization rate. We present a model of these actin dynamics as an excitable system in which a diffusive, autocatalytic activator causes actin polymerization; F-actin accumulation in turn inhibits further activator accumulation. Simulations of the model reproduced the pattern of actin polymerization seen in experiments. To explore the model's assumption of an autocatalytic activation mechanism, we imaged cells expressing markers for both F-actin and the p21 subunit of the Arp2/3 complex. We found that integrated Arp2/3-complex concentrations spike several seconds before spikes of F-actin concentration. This suggests that the Arp2/3 complex participates in an activation mechanism that includes additional diffuse components. Response of cells to stimulation by fetal calf serum could be reproduced by the model, further supporting the proposed dynamical picture.

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

    PubMed

    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

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

    PubMed

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

    2003-06-01

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

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

    PubMed

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

    2013-01-01

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

  8. Cytoskeletal F-actin patterns in whole-mounted larval and adult salivary glands of the fleshfly, Sarcophaga bullata.

    PubMed

    Meulemans, W; De Loof, A

    1991-01-01

    The patterns of filamentous actin were analysed in different larval, pupal and adult stages in the salivary glands of the fleshfly Sarcophaga bullata. Using the rhodamine labelled phalloidin staining method in combination with detergent extraction specific actin filament distribution was detected. The salivary glands which are histolysed during the process of metamorphosis show distinct cellular morphology and actin filament patterns in larvae and adults. The large third instar larval salivary gland cells contain a well developed apicolateral microvillar zone. In third instar larvae this microvillar zone invaginates and expands in the basal part of the lateral membranes. Larval salivary gland cells also contain numerous parallel basal actin bundles. The larval glands are histolysed during metamorphosis and adult glands are formed out of the imaginal cell group. At the onset of metamorphosis these basal actin bundles form a network of crossing bundles. The filamentous actin patterns of the proximal part of adult gland cells is confined to the apicolateral microvillar membranes. The cells in the distal, tubular part of the adult salivary glands show intense staining of their folded lateral membranes.

  9. Actin in dendritic spines: connecting dynamics to function

    PubMed Central

    2010-01-01

    Dendritic spines are small actin-rich protrusions from neuronal dendrites that form the postsynaptic part of most excitatory synapses and are major sites of information processing and storage in the brain. Changes in the shape and size of dendritic spines are correlated with the strength of excitatory synaptic connections and heavily depend on remodeling of its underlying actin cytoskeleton. Emerging evidence suggests that most signaling pathways linking synaptic activity to spine morphology influence local actin dynamics. Therefore, specific mechanisms of actin regulation are integral to the formation, maturation, and plasticity of dendritic spines and to learning and memory. PMID:20457765

  10. Side-binding proteins modulate actin filament dynamics.

    PubMed

    Crevenna, Alvaro H; Arciniega, Marcelino; Dupont, Aurélie; Mizuno, Naoko; Kowalska, Kaja; Lange, Oliver F; Wedlich-Söldner, Roland; Lamb, Don C

    2015-01-01

    Actin filament dynamics govern many key physiological processes from cell motility to tissue morphogenesis. A central feature of actin dynamics is the capacity of filaments to polymerize and depolymerize at their ends in response to cellular conditions. It is currently thought that filament kinetics can be described by a single rate constant for each end. In this study, using direct visualization of single actin filament elongation, we show that actin polymerization kinetics at both filament ends are strongly influenced by the binding of proteins to the lateral filament surface. We also show that the pointed-end has a non-elongating state that dominates the observed filament kinetic asymmetry. Estimates of flexibility as well as effects on fragmentation and growth suggest that the observed kinetic diversity arises from structural alteration. Tuning elongation kinetics by exploiting the malleability of the filament structure may be a ubiquitous mechanism to generate a rich variety of cellular actin dynamics. PMID:25706231

  11. Cytoskeletal Dynamics and Fluid Flow in Drosophila Oocytes

    NASA Astrophysics Data System (ADS)

    de Canio, Gabriele; Goldstein, Raymond; Lauga, Eric

    2015-11-01

    The biological world includes a broad range of phenomena in which transport in a fluid plays a central role. Among these is the fundamental issue of cell polarity arising during development, studied historically using the model organism Drosophila melanogaster. The polarity of the oocyte is known to be induced by the translocation of mRNAs by kinesin motor proteins along a dense microtubule cytoskeleton, a process which also induces cytoplasmic streaming. Recent experimental observations have revealed the remarkable fluid-structure interactions that occur as the streaming flows back-react on the microtubules. In this work we use a combination of theory and simulations to address the interplay between the fluid flow and the configuration of cytoskeletal filaments leading to the directed motion inside the oocyte. We show in particular that the mechanical coupling between the fluid motion and the orientation of the microtubules can lead to a transition to coherent motion within the oocyte, as observed. Supported by EPSRC and ERC Advanced Investigator Grant 247333.

  12. Endothelial actin-binding proteins and actin dynamics in leukocyte transendothelial migration.

    PubMed

    Schnoor, Michael

    2015-04-15

    The endothelium is the first barrier that leukocytes have to overcome during recruitment to sites of inflamed tissues. The leukocyte extravasation cascade is a complex multistep process that requires the activation of various adhesion molecules and signaling pathways, as well as actin remodeling, in both leukocytes and endothelial cells. Endothelial adhesion molecules, such as E-selectin or ICAM-1, are connected to the actin cytoskeleton via actin-binding proteins (ABPs). Although the contribution of receptor-ligand interactions to leukocyte extravasation has been studied extensively, the contribution of endothelial ABPs to the regulation of leukocyte adhesion and transendothelial migration remains poorly understood. This review focuses on recently published evidence that endothelial ABPs, such as cortactin, myosin, or α-actinin, regulate leukocyte extravasation by controlling actin dynamics, biomechanical properties of endothelia, and signaling pathways, such as GTPase activation, during inflammation. Thus, ABPs may serve as targets for novel treatment strategies for disorders characterized by excessive leukocyte recruitment.

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

    PubMed

    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.

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

    PubMed Central

    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

  15. The bacterial cell division proteins FtsA and FtsZ self-organize into dynamic cytoskeletal patterns

    PubMed Central

    Loose, Martin; Mitchison, Timothy J.

    2014-01-01

    Bacterial cytokinesis is commonly initiated by the Z-ring, a cytoskeletal structure assembling at the site of division. Its primary component is FtsZ, a tubulin superfamily GTPase, which is recruited to the membrane by the actin-related protein FtsA. Both proteins are required for the formation of the Z-ring, but if and how they influence each other’s assembly dynamics is not known. Here, we reconstituted FtsA-dependent recruitment of FtsZ polymers to supported membranes, where both proteins self-organize into complex patterns, such as fast-moving filament bundles and chirally rotating rings. Using fluorescence microscopy and biochemical perturbations, we found that these large-scale rearrangements of FtsZ emerge from its polymerization dynamics and a dual, antagonistic role of FtsA: recruitment of FtsZ filaments to the membrane and a negative regulation on FtsZ organization. Our findings provide a model for the initial steps of bacterial cell division and illustrate how dynamic polymers can self-organize into large-scale structures. PMID:24316672

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

    SciTech Connect

    Splettstoesser, Thomas; Holmes, Kenneth; Noe, Frank; Smith, Jeremy C

    2011-01-01

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

  17. Quantitative fluorescent speckle microscopy (QFSM) to measure actin dynamics.

    PubMed

    Mendoza, Michelle C; Besson, Sebastien; Danuser, Gaudenz

    2012-10-01

    Quantitative fluorescent speckle microscopy (QFSM) is a live-cell imaging method to analyze the dynamics of macromolecular assemblies with high spatial and temporal resolution. Its greatest successes were in the analysis of actin filament and adhesion dynamics in the context of cell migration and microtubule dynamics in interphase and the meiotic/mitotic spindle. Here, focus is on the former application to illustrate the procedures of FSM imaging and the computational image processing that extracts quantitative information from these experiments. QFSM is advantageous over other methods because it measures the movement and turnover kinetics of the actin filament (F-actin) network in living cells across the entire field of view. Experiments begin with the microinjection of fluorophore-labeled actin into cells, which generate a low ratio of fluorescently labeled to endogenously unlabeled actin monomers. Spinning disk confocal or wide-field imaging then visualizes fluorophore clusters (two to eight actin monomers) within the assembled F-actin network as speckles. QFSM software identifies and computationally tracks and utilizes the location, appearance, and disappearance of speckles to derive network flows and maps of the rate of filament assembly and disassembly. PMID:23042526

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

    NASA Astrophysics Data System (ADS)

    Wang, Ruizhe

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

  19. Accelerators, Brakes, and Gears of Actin Dynamics in Dendritic Spines

    PubMed Central

    Pontrello, Crystal G.; Ethell, Iryna M.

    2010-01-01

    Dendritic spines are actin-rich structures that accommodate the postsynaptic sites of most excitatory synapses in the brain. Although dendritic spines form and mature as synaptic connections develop, they remain plastic even in the adult brain, where they can rapidly grow, change, or collapse in response to normal physiological changes in synaptic activity that underlie learning and memory. Pathological stimuli can adversely affect dendritic spine shape and number, and this is seen in neurodegenerative disorders and some forms of mental retardation and autism as well. Many of the molecular signals that control these changes in dendritic spines act through the regulation of filamentous actin (F-actin), some through direct interaction with actin, and others via downstream effectors. For example, cortactin, cofilin, and gelsolin are actin-binding proteins that directly regulate actin dynamics in dendritic spines. Activities of these proteins are precisely regulated by intracellular signaling events that control their phosphorylation state and localization. In this review, we discuss how actin-regulating proteins maintain the balance between F-actin assembly and disassembly that is needed to stabilize mature dendritic spines, and how changes in their activities may lead to rapid remodeling of dendritic spines. PMID:20463852

  20. Spontaneous actin dynamics in contractile rings

    NASA Astrophysics Data System (ADS)

    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.

  1. Cytoskeletal-assisted dynamics of the mitochondrial reticulum in living cells

    PubMed Central

    Knowles, Michelle K.; Guenza, Marina G.; Capaldi, Roderick A.; Marcus, Andrew H.

    2002-01-01

    Subcellular organelle dynamics are strongly influenced by interactions with cytoskeletal filaments and their associated motor proteins, and lead to complex multiexponential relaxations that occur over a wide range of spatial and temporal scales. Here we report spatio-temporal measurements of the fluctuations of the mitochondrial reticulum in osteosarcoma cells by using Fourier imaging correlation spectroscopy, over time and distance scales of 10−2 to 103 s and 0.5–2.5 μm. We show that the method allows a more complete description of mitochondrial dynamics, through the time- and length-scale-dependent collective diffusion coefficient D(k,τ), than available by other means. Addition of either nocodazole to disrupt microtubules or cytochalasin D to disassemble microfilaments simplifies the intermediate scattering function. When both drugs are used, the reticulum morphology of mitochondria is retained even though the cytoskeletal elements have been de-polymerized. The dynamics of the organelle are then primarily diffusive and can be modeled as a collection of friction points interconnected by elastic springs. This study quantitatively characterizes organelle dynamics in terms of collective cytoskeletal interactions in living cells. PMID:12417764

  2. Proline-rich region of non-muscle myosin light chain kinase modulates kinase activity and endothelial cytoskeletal dynamics.

    PubMed

    Belvitch, Patrick; Adyshev, Djanybek; Elangovan, Venkateswaran R; Brown, Mary E; Naureckas, Caitlin; Rizzo, Alicia N; Siegler, Jessica H; Garcia, Joe G N; Dudek, Steven M

    2014-09-01

    Disruption of the pulmonary endothelial barrier and subsequent vascular leak is a hallmark of acute lung injury. Dynamic rearrangements in the endothelial cell (EC) peripheral membrane and underlying cytoskeleton are critical determinants of barrier function. The cytoskeletal effector protein non-muscle myosin light chain kinase (nmMLCK) and the actin-binding regulatory protein cortactin are important regulators of the endothelial barrier. In the present study we functionally characterize a proline-rich region of nmMLCK previously identified as the possible site of interaction between nmMLCK and cortactin. A mutant nmMLCK construct deficient in proline residues at the putative sites of cortactin binding (amino acids 973, 976, 1019, 1022) was generated. Co-immunoprecipitation studies in human lung EC transfected with wild-type or mutant nmMLCK demonstrated similar levels of cortactin interaction at baseline and after stimulation with the barrier-enhancing agonist, sphingosine 1-phosphate (S1P). In contrast, binding studies utilizing recombinant nmMLCK fragments containing the wild-type or proline-deficient sequence demonstrated a two-fold increase in cortactin binding (p<0.01) to the mutant construct. Immunofluorescent microscopy revealed an increased stress fiber density in ECs expressing GFP-labeled mutant nmMLCK at baseline (p=0.02) and after thrombin (p=0.01) or S1P (p=0.02) when compared to wild-type. Mutant nmMLCK demonstrated an increase in kinase activity in response to thrombin (p<0.01). Kymographic analysis demonstrated an increased EC membrane retraction distance and velocity (p<0.01) in response to the barrier disrupting agent thrombin in cells expressing the mutant vs. the wild-type nmMLCK construct. These results provide evidence that critical prolines within nmMLCK (amino acids 973, 976, 1019, 1022) regulate cytoskeletal and membrane events associated with pulmonary endothelial barrier function. PMID:25072537

  3. Modulation of the extracellular matrix patterning of thrombospondins by actin dynamics and thrombospondin oligomer state

    PubMed Central

    Hellewell, Andrew L.; Gong, Xianyun; Schärich, Karsten; Christofidou, Elena D.; Adams, Josephine C.

    2015-01-01

    Thrombospondins (TSPs) are evolutionarily-conserved, secreted glycoproteins that interact with cell surfaces and extracellular matrix (ECM) and have complex roles in cell interactions. Unlike the structural components of the ECM that form networks or fibrils, TSPs are deposited into ECM as arrays of nanoscale puncta. The cellular and molecular mechanisms for the patterning of TSPs in ECM are poorly understood. In the present study, we investigated whether the mechanisms of TSP patterning in cell-derived ECM involves actin cytoskeletal pathways or TSP oligomer state. From tests of a suite of pharmacological inhibitors of small GTPases, actomyosin-based contractility, or actin microfilament integrity and dynamics, cytochalasin D and jasplakinolide treatment of cells were identified to result in altered ECM patterning of a model TSP1 trimer. The strong effect of cytochalasin D indicated that mechanisms controlling puncta patterning depend on global F-actin dynamics. Similar spatial changes were obtained with endogenous TSPs after cytochalasin D treatment, implicating physiological relevance. Under matched experimental conditions with ectopically-expressed TSPs, the magnitude of the effect was markedly lower for pentameric TSP5 and Drosophila TSP, than for trimeric TSP1 or dimeric Ciona TSPA. To distinguish between the variables of protein sequence or oligomer state, we generated novel, chimeric pentamers of TSP1. These proteins accumulated within ECM at higher levels than TSP1 trimers, yet the effect of cytochalasin D on the spatial distribution of puncta was reduced. These findings introduce a novel concept that F-actin dynamics modulate the patterning of TSPs in ECM and that TSP oligomer state is a key determinant of this process. PMID:26182380

  4. Dynacortin is a novel actin bundling protein that localizes to dynamic actin structures.

    PubMed

    Robinson, Douglas N; Ocon, Stephani S; Rock, Ronald S; Spudich, James A

    2002-03-15

    Dynacortin is a novel protein that was discovered in a genetic suppressor screen of a Dictyostelium discoideum cytokinesis-deficient mutant cell line devoid of the cleavage furrow actin bundling protein, cortexillin I. While dynacortin is highly enriched in the cortex, particularly in cell-surface protrusions, it is excluded from the cleavage furrow cortex during cytokinesis. Here, we describe the biochemical characterization of this new protein. Purified dynacortin is an 80-kDa dimer with a large 5.7-nm Stokes radius. Dynacortin cross-links actin filaments into parallel arrays with a mole ratio of one dimer to 1.3 actin monomers and a 3.1 microm K(d). Using total internal reflection fluorescence microscopy, GFP-dynacortin and the actin bundling protein coronin-GFP are seen to concentrate in highly dynamic cortical structures with assembly and disassembly half-lives of about 15 s. These results indicate that cells have evolved different actin-filament cross-linking proteins with complementary cellular distributions that collaborate to orchestrate complex cell shape changes.

  5. Identification of a Putative Network of Actin-Associated Cytoskeletal Proteins in Glomerular Podocytes Defined by Co-Purified mRNAs

    PubMed Central

    Nabet, Behnam; Tsai, Arthur; Tobias, John W.; Carstens, Russ P.

    2009-01-01

    The glomerular podocyte is a highly specialized and polarized kidney cell type that contains major processes and foot processes that extend from the cell body. Foot processes from adjacent podocytes form interdigitations with those of adjacent cells, thereby creating an essential intercellular junctional domain of the renal filtration barrier known as the slit diaphragm. Interesting parallels have been drawn between the slit diaphragm and other sites of cell-cell contact by polarized cells. Notably mutations in several genes encoding proteins localized to the foot processes can lead to proteinuria and kidney failure. Mutations in the Wilm's tumor gene (WT1) can also lead to kidney disease and one isoform of WT1, WT1(+KTS), has been proposed to regulate gene expression post-transcriptionally. We originally sought to identify mRNAs associated with WT1(+KTS) through an RNA immunoprecipitation and microarray approach, hypothesizing that the proteins encoded by these mRNAs might be important for podocyte morphology and function. We identified a subset of mRNAs that were remarkably enriched for transcripts encoding actin-binding proteins and other cytoskeletal proteins including several that are localized at or near the slit diaphragm. Interestingly, these mRNAs included those of α-actinin-4 and non-muscle myosin IIA that are mutated in genetic forms of kidney disease. However, isolation of the mRNAs occurred independently of the expression of WT1, suggesting that the identified mRNAs were serendipitously co-purified on the basis of co-association in a common subcellular fraction. Mass spectroscopy revealed that other components of the actin cytoskeleton co-purified with these mRNAs, namely actin, tubulin, and elongation factor 1α. We propose that these mRNAs encode a number of proteins that comprise a highly specialized protein interactome underlying the slit diaphragm. Collectively, these gene products and their interactions may prove to be important for the

  6. p21-activated kinase regulates mast cell degranulation via effects on calcium mobilization and cytoskeletal dynamics

    PubMed Central

    Allen, Jayme D.; Jaffer, Zahara M.; Park, Su-Jung; Burgin, Sarah; Hofmann, Clemens; Sells, Mary Ann; Chen, Shi; Derr-Yellin, Ethel; Michels, Elizabeth G.; McDaniel, Andrew; Bessler, Waylan K.; Ingram, David A.; Atkinson, Simon J.; Travers, Jeffrey B.

    2009-01-01

    Mast cells are key participants in allergic diseases via activation of high-affinity IgE receptors (FcϵRI) resulting in release of proinflammatory mediators. The biochemical pathways linking IgE activation to calcium influx and cytoskeletal changes required for intracellular granule release are incompletely understood. We demonstrate, genetically, that Pak1 is required for this process. In a passive cutaneous anaphylaxis experiment, Wsh/Wsh mast cell–deficient mice locally reconstituted with Pak1−/− bone marrow–derived mast cells (BMMCs) experienced strikingly decreased allergen-induced vascular permeability compared with controls. Consistent with the in vivo phenotype, Pak1−/− BMMCs exhibited a reduction in FcϵRI-induced degranulation. Further, Pak1−/− BMMCs demonstrated diminished calcium mobilization and altered depolymerization of cortical filamentous actin (F-actin) in response to FcϵRI stimulation. These data implicate Pak1 as an essential molecular target for modulating acute mast cell responses that contribute to allergic diseases. PMID:19124833

  7. Tracking Cytoskeletal Dynamics in Living Neurons via Combined Atomic Force and Fluorescence Microscopy

    NASA Astrophysics Data System (ADS)

    Spedden, Elise; Kaplan, David; Staii, Cristian

    2013-03-01

    Living cells are active mechanical structures which evolve within and in response to their local microenvironments. Various cell types possess different mechanical properties and respond uniquely to growth, environmental changes, and the application of chemical stimuli. Here we present a powerful approach which combines high resolution Atomic Force Microscopy with Fluorescence Microscopy to systematically obtain real-time micrometer and sub-micrometer resolution elasticity maps for live neuronal cells cultured on glass substrates. Through this approach we measure the topography, the elastic properties, and the dynamics of neuronal cells, and identify changes in cytoskeletal components during axonal growth, chemical modification, and changes in ambient temperature. We will also show high resolution elasticity measurements of the cell body and of axons/dendrites during growth, as well as identification of cytoskeletal components during cell growth and environmental changes.

  8. Meiotic chromosomes move by linkage to dynamic actin cables with transduction of force through the nuclear envelope.

    PubMed

    Koszul, R; Kim, K P; Prentiss, M; Kleckner, N; Kameoka, S

    2008-06-27

    Chromosome movement is prominent during meiosis. Here, using a combination of in vitro and in vivo approaches, we elucidate the basis for dynamic mid-prophase telomere-led chromosome motion in budding yeast. Diverse findings reveal a process in which, at the pachytene stage, individual telomere/nuclear envelope (NE) ensembles attach passively to, and then move in concert with, nucleus-hugging actin cables that are continuous with the global cytoskeletal actin network. Other chromosomes move in concert with lead chromosome(s). The same process, in modulated form, explains the zygotene "bouquet" configuration in which, immediately preceding pachytene, chromosome ends colocalize dynamically in a restricted region of the NE. Mechanical properties of the system and biological roles of mid-prophase movement for meiosis, including recombination, are discussed. PMID:18585353

  9. Measuring actin dynamics during phagocytosis using photo-switchable fluorescence

    NASA Astrophysics Data System (ADS)

    Kovari, Daniel T.; Curtis, Jennifer E.

    2013-03-01

    Phagocytosis has traditionally been investigated in terms of the relevant biochemical signaling pathways. However, a growing number of studies investigating the physical aspects of phagocytosis have demonstrated that several distinct forces are exerted throughout particle ingestion. We use variations on FRAP (Fluorescence Recovery After Photobleaching) in combination with photo-switchable fluorescent protein to investigate actin dynamics as a phagocyte attempts to engulf its prey. The goal of our actin studies are to determine the recruitment and polymerization rate of actin in the forming phagosome and whether an organized contractile actin ring is present and responsible for phagosome closure, as proposed in the literature. These experiments are ongoing and contribute to our long term effort of developing a physics based model of phagocytosis.

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

    NASA Technical Reports Server (NTRS)

    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.

  11. Comparison of actin and cell surface dynamics in motile fibroblasts

    PubMed Central

    1992-01-01

    We have investigated the dynamic behavior of actin in fibroblast lamellipodia using photoactivation of fluorescence. Activated regions of caged resorufin (CR)-labeled actin in lamellipodia of IMR 90 and MC7 3T3 fibroblasts were observed to move centripetally over time. Thus in these cells, actin filaments move centripetally relative to the substrate. Rates were characteristic for each cell type; 0.66 +/- 0.27 microns/min in IMR 90 and 0.36 +/- 0.16 microns/min in MC7 3T3 cells. In neither case was there any correlation between the rate of actin movement and the rate of lamellipodial protrusion. The half-life of the activated CR-actin filaments was approximately 1 min in IMR 90 lamellipodia, and approximately 3 min in MC7 3T3 lamellipodia. Thus continuous filament turnover accompanies centripetal movement. In both cell types, the length of time required for a section of the actin meshwork to traverse the lamellipodium was several times longer than the filament half-life. The dynamic behavior of the dorsal surface of the cell was also observed by tracking lectin-coated beads on the surface and phase-dense features within lamellipodia of MC7 3T3 cells. The movement of these dorsal features occurred at rates approximately three times faster than the rate of movement of the underlying bulk actin cytoskeleton, even when measured in the same individual cells. Thus the transport of these dorsal features must occur by some mechanism other than simple attachment to the moving bulk actin cytoskeleton. PMID:1400580

  12. F-actin-myosin II inhibitors affect chromaffin granule plasma membrane distance and fusion kinetics by retraction of the cytoskeletal cortex.

    PubMed

    Villanueva, José; Torres, Vanesa; Torregrosa-Hetland, Cristina J; Garcia-Martinez, Virginia; López-Font, Inmaculada; Viniegra, Salvador; Gutiérrez, Luis M

    2012-10-01

    Chromaffin cell catecholamines are released when specialized secretory vesicles undergo exocytotic membrane fusion. Evidence indicates that vesicle supply and fusion are controlled by the activity of the cortical F-actin-myosin II network. To study in detail cell cortex and vesicle interactions, we use fluorescent labeling with GFP-lifeact and acidotropic dyes in confocal and evanescent wave microscopy. These techniques provide structural details and dynamic images of chromaffin granules caged in a complex cortical structure. Both the movement of cortical structures and granule motion appear to be linked, and this motion can be restricted by the myosin II-specific inhibitor, blebbistatin, and the F-actin stabilizer, jasplakinolide. These treatments also affect the position of the vesicles in relation to the plasma membrane, increasing the distance between them and the fusion sites. Consequently, we observed slower single vesicle fusion kinetics in treated cells after neutralization of acridine orange-loaded granules during exocytosis. Increasing the distance between the granules and the fusion sites appears to be linked to the retraction of the F-actin cytoskeleton when treated with jasplakinolide. Thus, F-actin-myosin II inhibitors appear to slow granule fusion kinetics by altering the position of vesicles after relaxation of the cortical network.

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

    PubMed Central

    Holzinger, Andreas; Blaas, Kathrin

    2016-01-01

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

  14. Extended-resolution structured illumination imaging of endocytic and cytoskeletal dynamics

    PubMed Central

    Li, Dong; Shao, Lin; Chen, Bi-Chang; Zhang, Xi; Zhang, Mingshu; Moses, Brian; Milkie, Daniel E.; Beach, Jordan R.; Hammer, John A.; Pasham, Mithun; Kirchhausen, Tomas; Baird, Michelle A.; Davidson, Michael W.; Xu, Pingyong; Betzig, Eric

    2015-01-01

    Super-resolution fluorescence microscopy is distinct among nanoscale imaging tools in its ability to image protein dynamics in living cells. Structured illumination microscopy (SIM) stands out in this regard because of its high speed and low illumination intensities, but typically offers only a twofold resolution gain. We extended the resolution of live-cell SIM through two approaches: ultrahigh numerical aperture SIM at 84-nanometer lateral resolution for more than 100 multicolor frames, and nonlinear SIM with patterned activation at 45- to 62-nanometer resolution for approximately 20 to 40 frames. We applied these approaches to image dynamics near the plasma membrane of spatially resolved assemblies of clathrin and caveolin, Rab5a in early endosomes, and a-actinin, often in relationship to cortical actin. In addition, we examined mitochondria, actin, and the Golgi apparatus dynamics in three dimensions. PMID:26315442

  15. Endoplasmic Reticulum Dynamics, Inheritance, and Cytoskeletal Interactions in Budding YeastV⃞

    PubMed Central

    Fehrenbacher, K. L.; Davis, D.; Wu, M.; Boldogh, I.; Pon, Liza A.

    2002-01-01

    The endoplasmic reticulum (ER) in Saccharomyces cerevisiae consists of a reticulum underlying the plasma membrane (cortical ER) and ER associated with the nuclear envelope (nuclear ER). We used a Sec63p-green fluorescent protein fusion protein to study motility events associated with inheritance of cortical ER and nuclear ER in living yeast cells. During M phase before nuclear migration, we observed thick, apparently rigid tubular extensions emanating from the nuclear ER that elongate, undergo sweeping motions along the cell cortex, and shorten. Two findings support a role for microtubules in this process. First, extension of tubular structures from the nuclear ER is inhibited by destabilization of microtubules. Second, astral microtubules, structures that undergo similar patterns of extension, cortical surveillance and retraction, colocalize with nuclear ER extensions. During S and G2 phases of the cell cycle, we observed anchorage of the cortical ER at the site of bud emergence and apical bud growth. Thin tubules of the ER that extend from the anchored cortical ER display undulating, apparently random movement and move into the bud as it grows. Finally, we found that cortical ER morphology is sensitive to a filamentous actin–destabilizing drug, latrunculin-A, and to mutations in the actin-encoding ACT1 gene. Our observations support 1) different mechanisms and cytoskeletal mediators for the inheritance of nuclear and cortical ER elements and 2) a mechanism for cortical ER inheritance that is cytoskeleton dependent but relies on anchorage, not directed movement. PMID:11907267

  16. Dynamic Localization of G-actin During Membrane Protrusion in Neuronal Motility

    PubMed Central

    Lee, Chi Wai; Vitriol, Eric A.; Shim, Sangwoo; Wise, Ariel L.; Velayutham, Radhi P.; Zheng, James Q.

    2013-01-01

    Summary Background Actin-based cell motility is fundamental for the development, function, and malignant events of eukaryotic organisms. During neural development, axonal growth cones depend on rapid assembly and disassembly of actin filaments (F-actin) for their guided extension to specific targets for wiring. Monomeric globular actin (G-actin) is the building block for F-actin but is not considered to play a direct role in spatiotemporal control of actin dynamics in cell motility. Results Here we report that a pool of G-actin dynamically localizes to the leading edge of growth cones and neuroblastoma cells to spatially elevate the G-/F-actin ratio that drives membrane protrusion and cell movement. Loss of G-actin localization leads to the cessation and retraction of membrane protrusions. Moreover, G-actin localization occurs asymmetrically in growth cones during attractive turning. Finally, we identify the actin monomer binding proteins profilin and thymosin β4 as key molecules that localize actin monomers to the leading edge of lamellipodia for their motility. Conclusions Our results suggest that dynamic localization of G-actin provides a novel mechanism to regulate the spatiotemporal actin dynamics underlying membrane protrusion in cell locomotion and growth cone chemotaxis. PMID:23746641

  17. Actin filament dynamics impacts keratinocyte stem cell maintenance

    PubMed Central

    Nanba, Daisuke; Toki, Fujio; Matsushita, Natsuki; Matsushita, Sachi; Higashiyama, Shigeki; Barrandon, Yann

    2013-01-01

    Cultured human epidermal keratinocyte stem cells (holoclones) are crucial for regenerative medicine for burns and genetic disorders. In serial culture, holoclones progressively lose their proliferative capacity to become transient amplifying cells with limited growth (paraclones), a phenomenon termed clonal conversion. Although it negatively impacts the culture lifespan and the success of cell transplantation, little is known on the molecular mechanism underlying clonal conversion. Here, we show that holoclones and paraclones differ in their actin filament organization, with actin bundles distributed radially in holoclones and circumferentially in paraclones. Moreover, actin organization sets the stage for a differing response to epidermal growth factor (EGF), since EGF signalling induces a rapid expansion of colony size in holoclones and a significant reduction in paraclones. Furthermore, inhibition of PI3K or Rac1 in holoclones results in the reorganization of actin filaments in a pattern that is similar to that of paraclones. Importantly, continuous Rac1 inhibition in holoclones results in clonal conversion and reduction of growth potential. Together, our data connect loss of stem cells to EGF-induced colony dynamics governed by Rac1. PMID:23554171

  18. Alternative cytoskeletal landscapes: cytoskeletal novelty and evolution in basal excavate protists.

    PubMed

    Dawson, Scott C; Paredez, Alexander R

    2013-02-01

    Microbial eukaryotes encompass the majority of eukaryotic evolutionary and cytoskeletal diversity. The cytoskeletal complexity observed in multicellular organisms appears to be an expansion of components present in genomes of diverse microbial eukaryotes such as the basal lineage of flagellates, the Excavata. Excavate protists have complex and diverse cytoskeletal architectures and life cycles-essentially alternative cytoskeletal 'landscapes'-yet still possess conserved microtubule-associated and actin-associated proteins. Comparative genomic analyses have revealed that a subset of excavates, however, lack many canonical actin-binding proteins central to actin cytoskeleton function in other eukaryotes. Overall, excavates possess numerous uncharacterized and 'hypothetical' genes, and may represent an undiscovered reservoir of novel cytoskeletal genes and cytoskeletal mechanisms. The continued development of molecular genetic tools in these complex microbial eukaryotes will undoubtedly contribute to our overall understanding of cytoskeletal diversity and evolution.

  19. Interplay of cytoskeletal activity and lipid phase stability in dynamic protein recruitment and clustering

    PubMed Central

    Gómez-Llobregat, Jordi; Buceta, Javier; Reigada, Ramon

    2013-01-01

    Recent experiments have revealed that some membrane proteins aggregate to form clusters. This type of process has been proven to be dynamic and to be actively maintained by external kinetics. Additionally, this dynamic recruiting is cholesterol- and actin-dependent, suggesting that raft organization and cytoskeleton rearrangement play a crucial role. In the present study, we propose a simple model that provides a general framework to describe the dynamical behavior of lipid-protein assemblies. Our results suggest that lipid-mediated interactions and cytoskeleton-anchored proteins contribute to the modulation of such behavior. In particular, we find a resonant condition between the membrane protein and cytoskeleton dynamics that results in the invariance of the ratio of clustered proteins that is found in in vivo experimental observations. PMID:24018870

  20. Dynamic actin cycling through mitochondrial subpopulations locally regulates the fission–fusion balance within mitochondrial networks

    PubMed Central

    Moore, Andrew S.; Wong, Yvette C.; Simpson, Cory L.; Holzbaur, Erika L. F.

    2016-01-01

    Mitochondria form interconnected networks that dynamically remodel in response to cellular needs. Using live-cell imaging, we investigate the role of the actin cytoskeleton in regulating mitochondrial fission and fusion. We identify cycling of actin filaments onto and off of subsets of cellular mitochondria. The association of actin filaments with mitochondrial subpopulations is transient; actin quickly disassembles, then reassembles around a distinct subpopulation, efficiently cycling through all cellular mitochondria within 14 min. The focal assembly of actin induces local, Drp1-dependent fragmentation of the mitochondrial network. On actin disassembly, fragmented mitochondria undergo rapid fusion, leading to regional recovery of the tubular mitochondrial network. Cycling requires dynamic actin polymerization and is blocked by inhibitors of both Arp2/3 and formins. We propose that cyclic assembly of actin onto mitochondria modulates the fission/fusion balance, promotes network remodelling and content mixing, and thus may serve as an essential mechanism regulating mitochondrial network homeostasis. PMID:27686185

  1. Biotechnological aspects of cytoskeletal regulation in plants.

    PubMed

    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.

  2. Biotechnological aspects of cytoskeletal regulation in plants.

    PubMed

    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

  3. Internal dynamics of F-actin and myosin subfragment-1 studied by quasielastic neutron scattering

    SciTech Connect

    Matsuo, Tatsuhito; Arata, Toshiaki; Oda, Toshiro; Nakajima, Kenji; Ohira-Kawamura, Seiko; Kikuchi, Tatsuya; Fujiwara, Satoru

    2015-04-10

    Various biological functions related to cell motility are driven by the interaction between the partner proteins, actin and myosin. To obtain insights into how this interaction occurs, the internal dynamics of F-actin and myosin subfragment-1 (S1) were characterized by the quasielastic neutron scattering measurements on the solution samples of F-actin and S1. Contributions of the internal motions of the proteins to the scattering spectra were separated from those of the global macromolecular diffusion. Analysis of the spectra arising from the internal dynamics showed that the correlation times of the atomic motions were about two times shorter for F-actin than for S1, suggesting that F-actin fluctuates more rapidly than S1. It was also shown that the fraction of the immobile atoms is larger for S1 than for F-actin. These results suggest that F-actin actively facilitates the binding of myosin by utilizing the more frequent conformational fluctuations than those of S1. - Highlights: • We studied the internal dynamics of F-actin and myosin S1 by neutron scattering. • The correlation times of the atomic motions were smaller for F-actin than for S1. • The fraction of the immobile atoms was also smaller for F-actin than for S1. • Our results suggest that mobility of atoms in F-actin is higher than that in S1. • We propose that high flexibility of F-actin facilitates the binding of myosin.

  4. Integrin-cytoskeletal interactions in migrating fibroblasts are dynamic, asymmetric, and regulated

    PubMed Central

    1993-01-01

    We have used laser optical trapping and nanometer-level motion analysis to investigate the cytoskeletal associations and surface dynamics of beta 1 integrin, a cell-substrate adhesion molecule, on the dorsal surfaces of migrating fibroblast cells. A single-beam optical gradient trap (laser tweezers) was used to restrain polystyrene beads conjugated with anti-beta 1 integrin mAbs and place them at desired locations on the cell exterior. This technique was used to demonstrate a spatial difference in integrin-cytoskeleton interactions in migrating cells. We found a distinct increase in the stable attachment of beads, and subsequent rearward flow, on the lamellipodia of locomoting cells compared with the retracting portions. Complementary to the enhanced linkage of integrin at the cell lamellipodium, the membrane was more deformable at the rear versus the front of moving cells while nonmotile cells did not exhibit this asymmetry in membrane architecture. Video microscopy and nanometer-precision tracking routines were used to study the surface dynamics of integrin on the lamellipodia of migrating cells by monitoring the displacements of colloidal gold particles coated with anti-beta 1 integrin mAbs. Small gold aggregates were rapidly transported preferentially to the leading edge of the lamellipod where they resumed diffusion restricted along the edge. This fast transport was characterized by brief periods of directed movement ("jumps") having an instantaneous velocity of 37 +/- 15 microns/min (SD), separated by periods of diffusion. In contrast, larger aggregates of gold particles and the large latex beads underwent slow, steady rearward movement (0.85 +/- 0.44 micron/min) (SD) at a rate similar to that reported for other capping events and for migration of these cells. Cell lines containing mutated beta 1 integrins were used to show that the cytoplasmic domain is essential for an asymmetry in attachment of integrin to the underlying cytoskeletal network and is also

  5. Dynamics of Actin Cables in Polarized Growth of the Filamentous Fungus Aspergillus nidulans

    PubMed Central

    Bergs, Anna; Ishitsuka, Yuji; Evangelinos, Minoas; Nienhaus, G. U.; Takeshita, Norio

    2016-01-01

    Highly polarized growth of filamentous fungi requires a continuous supply of proteins and lipids to the hyphal tip. This transport is managed by vesicle trafficking via the actin and microtubule cytoskeletons and their associated motor proteins. Particularly, actin cables originating from the hyphal tip are essential for hyphal growth. Although, specific marker proteins have been developed to visualize actin cables in filamentous fungi, the exact organization and dynamics of actin cables has remained elusive. Here, we observed actin cables using tropomyosin (TpmA) and Lifeact fused to fluorescent proteins in living Aspergillus nidulans hyphae and studied the dynamics and regulation. GFP tagged TpmA visualized dynamic actin cables formed from the hyphal tip with cycles of elongation and shrinkage. The elongation and shrinkage rates of actin cables were similar and approximately 0.6 μm/s. Comparison of actin markers revealed that high concentrations of Lifeact reduced actin dynamics. Simultaneous visualization of actin cables and microtubules suggests temporally and spatially coordinated polymerization and depolymerization between the two cytoskeletons. Our results provide new insights into the molecular mechanism of ordered polarized growth regulated by actin cables and microtubules. PMID:27242709

  6. Tau co-organizes dynamic microtubule and actin networks

    PubMed Central

    Elie, Auréliane; Prezel, Elea; Guérin, Christophe; Denarier, Eric; Ramirez-Rios, Sacnicte; Serre, Laurence; Andrieux, Annie; Fourest-Lieuvin, Anne; Blanchoin, Laurent; Arnal, Isabelle

    2015-01-01

    The crosstalk between microtubules and actin is essential for cellular functions. However, mechanisms underlying the microtubule-actin organization by cross-linkers remain largely unexplored. Here, we report that tau, a neuronal microtubule-associated protein, binds to microtubules and actin simultaneously, promoting in vitro co-organization and coupled growth of both networks. By developing an original assay to visualize concomitant microtubule and actin assembly, we show that tau can induce guided polymerization of actin filaments along microtubule tracks and growth of single microtubules along actin filament bundles. Importantly, tau mediates microtubule-actin co-alignment without changing polymer growth properties. Mutagenesis studies further reveal that at least two of the four tau repeated motifs, primarily identified as tubulin-binding sites, are required to connect microtubules and actin. Tau thus represents a molecular linker between microtubule and actin networks, enabling a coordination of the two cytoskeletons that might be essential in various neuronal contexts. PMID:25944224

  7. SelR reverses Mical-mediated oxidation of actin to regulate F-actin dynamics.

    PubMed

    Hung, Ruei-Jiun; Spaeth, Christopher S; Yesilyurt, Hunkar Gizem; Terman, Jonathan R

    2013-12-01

    Actin's polymerization properties are markedly altered by oxidation of its conserved Met 44 residue. Mediating this effect is a specific oxidation-reduction (redox) enzyme, Mical, that works with Semaphorin repulsive guidance cues and selectively oxidizes Met 44. We now find that this actin-regulatory process is reversible. Employing a genetic approach, we identified a specific methionine sulfoxide reductase (MsrB) enzyme SelR that opposes Mical redox activity and Semaphorin-Plexin repulsion to direct multiple actin-dependent cellular behaviours in vivo. SelR specifically catalyses the reduction of the R isomer of methionine sulfoxide (methionine-R-sulfoxide) to methionine, and we found that SelR directly reduced Mical-oxidized actin, restoring its normal polymerization properties. These results indicate that Mical oxidizes actin stereospecifically to generate actin Met-44-R-sulfoxide (actin(Met(R)O-44)), and also implicate the interconversion of specific Met/Met(R)O residues as a precise means to modulate protein function. Our results therefore uncover a specific reversible redox actin regulatory system that controls cell and developmental biology.

  8. Differences in structural dynamics of muscle and yeast actin accompany differences in functional interactions with myosin.

    PubMed

    Prochniewicz, E; Thomas, D D

    1999-11-01

    We have used spectroscopic probes ErIA and IAEDANS attached to Cys374 to compare the structural dynamics of yeast actin filaments with that of muscle actin, to understand the structural basis of the less productive interaction of yeast actin with myosin. Time-resolved phosphorescence anisotropy (TPA) of ErIA and steady-state fluorescence of IAEDANS were measured. TPA indicated more rapid rotational motion and more restricted angular amplitude in yeast actin. The fluorescence spectrum was less intense and more red-shifted in yeast actin, suggesting more exposure of the probe to solvent. These results indicate that the two actins differ substantially in the conformational dynamics of the C-terminal region. Binding of myosin S1 induced significantly different spectroscopic changes in TPA and fluorescence of muscle and yeast actin. As a result, the spectroscopic differences between the two actins were decreased by the addition of S1. These results suggest that yeast actin is less effective at activating myosin because of larger changes required in the structure of actin upon strong myosin binding. These results provide insight into the relationship between actomyosin dynamics and function, and they provide a useful framework for structure-function analysis of mutant yeast actin.

  9. Coupling of the hydration water dynamics and the internal dynamics of actin detected by quasielastic neutron scattering

    SciTech Connect

    Fujiwara, Satoru; Plazanet, Marie; Oda, Toshiro

    2013-02-15

    Highlights: ► Quasielastic neutron scattering spectra of F-actin and G-actin were measured. ► Analysis of the samples in D{sub 2}O and H{sub 2}O provided the spectra of hydration water. ► The first layer hydration water around F-actin is less mobile than around G-actin. ► This difference in hydration water is in concert with the internal dynamics of actin. ► Water outside the first layer behaves bulk-like but influenced by the first layer. -- Abstract: In order to characterize dynamics of water molecules around F-actin and G-actin, quasielastic neutron scattering experiments were performed on powder samples of F-actin and G-actin, hydrated either with D{sub 2}O or H{sub 2}O, at hydration ratios of 0.4 and 1.0. By combined analysis of the quasielastic neutron scattering spectra, the parameter values characterizing the dynamics of the water molecules in the first hydration layer and those of the water molecules outside of the first layer were obtained. The translational diffusion coefficients (D{sub T}) of the hydration water in the first layer were found to be 1.2 × 10{sup −5} cm{sup 2}/s and 1.7 × 10{sup −5} cm{sup 2}/s for F-actin and G-actin, respectively, while that for bulk water was 2.8 × 10{sup −5} cm{sup 2}/s. The residence times were 6.6 ps and 5.0 ps for F-actin and G-actin, respectively, while that for bulk water was 0.62 ps. These differences between F-actin and G-actin, indicating that the hydration water around G-actin is more mobile than that around F-actin, are in concert with the results of the internal dynamics of F-actin and G-actin, showing that G-actin fluctuates more rapidly than F-actin. This implies that the dynamics of the hydration water is coupled to the internal dynamics of the actin molecules. The D{sub T} values of the water molecules outside of the first hydration layer were found to be similar to that of bulk water though the residence times are strongly affected by the first hydration layer. This supports the

  10. Computational model of polarized actin cables and cytokinetic actin ring formation in budding yeast

    PubMed Central

    Tang, Haosu; Bidone, Tamara C.

    2015-01-01

    The budding yeast actin cables and contractile ring are important for polarized growth and division, revealing basic aspects of cytoskeletal function. To study these formin-nucleated structures, we built a 3D computational model with actin filaments represented as beads connected by springs. Polymerization by formins at the bud tip and bud neck, crosslinking, severing, and myosin pulling, are included. Parameter values were estimated from prior experiments. The model generates actin cable structures and dynamics similar to those of wild type and formin deletion mutant cells. Simulations with increased polymerization rate result in long, wavy cables. Simulated pulling by type V myosin stretches actin cables. Increasing the affinity of actin filaments for the bud neck together with reduced myosin V pulling promotes the formation of a bundle of antiparallel filaments at the bud neck, which we suggest as a model for the assembly of actin filaments to the contractile ring. PMID:26538307

  11. MamK, a bacterial actin, forms dynamic filaments in vivo that are regulated by the acidic proteins MamJ and LimJ

    PubMed Central

    Draper, Olga; Byrne, Meghan E.; Li, Zhuo; Keyhani, Sepehr; Cueto Barrozo, Joyce; Jensen, Grant; Komeili, Arash

    2011-01-01

    SUMMARY Bacterial actins, in contrast to their eukaryotic counterparts, are highly divergent proteins whose wide-ranging functions are thought to correlate with their evolutionary diversity. One clade, represented by the MamK protein of magnetotactic bacteria, is required for the subcellular organization of magnetosomes, membrane-bound organelles that aid in navigation along the earth’s magnetic field. Using a fluorescence recovery after photobleaching assay in Magnetospirillum magneticum AMB-1, we find that, like traditional actins, MamK forms dynamic filaments that require an intact NTPase motif for their turnover in vivo. We also uncover two proteins, MamJ and LimJ, which perform a redundant function to promote the dynamic behavior of MamK filaments in wildtype cells. The absence of both MamJ and LimJ leads to static filaments, a disrupted magnetosome chain, and an anomalous build-up of cytoskeletal filaments between magnetosomes. Our results suggest that MamK filaments, like eukaryotic actins, are intrinsically stable and rely on regulators for their dynamic behavior, a feature that stands in contrast to some classes of bacterial actins characterized to date. PMID:21883528

  12. Probing the flexibility of tropomyosin and its binding to filamentous actin using molecular dynamics simulations.

    PubMed

    Zheng, Wenjun; Barua, Bipasha; Hitchcock-DeGregori, Sarah E

    2013-10-15

    Tropomyosin (Tm) is a coiled-coil protein that binds to filamentous actin (F-actin) and regulates its interactions with actin-binding proteins like myosin by moving between three positions on F-actin (the blocked, closed, and open positions). To elucidate the molecular details of Tm flexibility in relation to its binding to F-actin, we conducted extensive molecular dynamics simulations for both Tm alone and Tm-F-actin complex in the presence of explicit solvent (total simulation time >400 ns). Based on the simulations, we systematically analyzed the local flexibility of the Tm coiled coil using multiple parameters. We found a good correlation between the regions with high local flexibility and a number of destabilizing regions in Tm, including six clusters of core alanines. Despite the stabilization by F-actin binding, the distribution of local flexibility in Tm is largely unchanged in the absence and presence of F-actin. Our simulations showed variable fluctuations of individual Tm periods from the closed position toward the open position. In addition, we performed Tm-F-actin binding calculations based on the simulation trajectories, which support the importance of Tm flexibility to Tm-F-actin binding. We identified key residues of Tm involved in its dynamic interactions with F-actin, many of which have been found in recent mutational studies to be functionally important, and the rest of which will make promising targets for future mutational experiments.

  13. Cleavage of human and mouse cytoskeletal and sarcomeric proteins by human immunodeficiency virus type 1 protease. Actin, desmin, myosin, and tropomyosin.

    PubMed Central

    Shoeman, R. L.; Sachse, C.; Höner, B.; Mothes, E.; Kaufmann, M.; Traub, P.

    1993-01-01

    HeLa cell actin was cleaved by human immunodeficiency virus type 1 protease when in its soluble, globular form (G-actin). No cleavage of the polymerized, filamentous form of actin (F-actin) was observed when examined by denaturing gel electrophoresis; however, electron microscopy revealed a low level of cleavage of F-actin. Immunoblotting of mouse skeletal and human pectoral muscle myofibrils treated in vitro with human immunodeficiency virus type 1 protease showed that myosin heavy chain, desmin, tropomyosin, and a fraction of the actin were all cleaved. Electron microscopy of these myofibrils demonstrated changes consistent with cleavage of these proteins: Z-lines were rapidly lost, the length of the A bands was shortened, and the thick filaments (myosin filaments) were often laterally frayed such that the structures disintegrated. Nonmuscle myosin heavy chains were also cleaved by this enzyme in vitro. These data demonstrate that this protease can cause alterations in muscle cell ultrastructure in vitro that may be of clinical relevance in infected individuals. Images Figure 1 Figure 3 Figure 4 Figure 5 PMID:8424456

  14. Learning, AMPA receptor mobility and synaptic plasticity depend on n-cofilin-mediated actin dynamics

    PubMed Central

    Rust, Marco B; Gurniak, Christine B; Renner, Marianne; Vara, Hugo; Morando, Laura; Görlich, Andreas; Sassoè-Pognetto, Marco; Banchaabouchi, Mumna Al; Giustetto, Maurizio; Triller, Antoine; Choquet, Daniel; Witke, Walter

    2010-01-01

    Neuronal plasticity is an important process for learning, memory and complex behaviour. Rapid remodelling of the actin cytoskeleton in the postsynaptic compartment is thought to have an important function for synaptic plasticity. However, the actin-binding proteins involved and the molecular mechanisms that in vivo link actin dynamics to postsynaptic physiology are not well understood. Here, we show that the actin filament depolymerizing protein n-cofilin is controlling dendritic spine morphology and postsynaptic parameters such as late long-term potentiation and long-term depression. Loss of n-cofilin-mediated synaptic actin dynamics in the forebrain specifically leads to impairment of all types of associative learning, whereas exploratory learning is not affected. We provide evidence for a novel function of n-cofilin function in synaptic plasticity and in the control of extrasynaptic excitatory AMPA receptors diffusion. These results suggest a critical function of actin dynamics in associative learning and postsynaptic receptor availability. PMID:20407421

  15. Myosin II Motors and F-Actin Dynamics Drive the Coordinated Movement of the Centrosome and Soma during CNS Glial-Guided Neuronal Migration

    SciTech Connect

    Solecki, Dr. David; Trivedi, Dr. Niraj; Govek, Eve-Ellen; Kerekes, Ryan A; Gleason, Shaun Scott; Hatten, Mary E

    2009-01-01

    Lamination of cortical regions of the vertebrate brain depends on glial-guided neuronal migration. The conserved polarity protein Par6{alpha} localizes to the centrosome and coordinates forward movement of the centrosome and soma in migrating neurons. The cytoskeletal components that produce this unique form of cell polarity and their relationship to polarity signaling cascades are unknown. We show that F-actin and Myosin II motors are enriched in the neuronal leading process and that Myosin II activity is necessary for leading process actin dynamics. Inhibition of Myosin II decreased the speed of centrosome and somal movement, whereas Myosin II activation increased coordinated movement. Ectopic expression or silencing of Par6{alpha} inhibited Myosin II motors by decreasing Myosin light-chain phosphorylation. These findings suggest leading-process Myosin II may function to 'pull' the centrosome and soma forward during glial-guided migration by a mechanism involving the conserved polarity protein Par6{alpha}.

  16. Heterogeneous Porphyromonas gingivalis LPS modulates immuno-inflammatory response, antioxidant defense and cytoskeletal dynamics in human gingival fibroblasts

    PubMed Central

    Herath, Thanuja D. K.; Darveau, Richard P.; Seneviratne, Chaminda J.; Wang, Cun-Yu; Wang, Yu; Jin, Lijian

    2016-01-01

    Periodontal (gum) disease is a highly prevalent infection and inflammation accounting for the majority of tooth loss in adult population worldwide. Porphyromonas gingivalis is a keystone periodontal pathogen and its lipopolysaccharide (PgLPS) acts as a major virulence attribute to the disease. Herein, we deciphered the overall host response of human gingival fibroblasts (HGFs) to two featured isoforms of tetra-acylated PgLPS1435/1449 and penta-acylated PgLPS1690 with reference to E. coli LPS through quantitative proteomics. This study unraveled differentially expressed novel biomarkers of immuno-inflammatory response, antioxidant defense and cytoskeletal dynamics in HGFs. PgLPS1690 greatly upregulated inflammatory proteins (e.g. cyclophilin, inducible nitric oxide synthase, annexins, galectin, cathepsins and heat shock proteins), whereas the anti-inflammatory proteins (e.g. Annexin A2 and Annexin A6) were significantly upregulated by PgLPS1435/1449. Interestingly, the antioxidants proteins such as mitochondrial manganese-containing superoxide dismutase and peroxiredoxin 5 were only upregulated by PgLPS1690. The cytoskeletal rearrangement-related proteins like myosin were differentially regulated by these PgLPS isoforms. The present study gives new insight into the biological properties of P. gingivalis LPS lipid A moiety that could critically modulate immuno-inflammatory response, antioxidant defense and cytoskeletal dynamics in HGFs, and thereby enhances our understanding of periodontal pathogenesis. PMID:27538450

  17. Diffusion of GPI-anchored proteins is influenced by the activity of dynamic cortical actin.

    PubMed

    Saha, Suvrajit; Lee, Il-Hyung; Polley, Anirban; Groves, Jay T; Rao, Madan; Mayor, Satyajit

    2015-11-01

    Molecular diffusion at the surface of living cells is believed to be predominantly driven by thermal kicks. However, there is growing evidence that certain cell surface molecules are driven by the fluctuating dynamics of cortical cytoskeleton. Using fluorescence correlation spectroscopy, we measure the diffusion coefficient of a variety of cell surface molecules over a temperature range of 24-37 °C. Exogenously incorporated fluorescent lipids with short acyl chains exhibit the expected increase of diffusion coefficient over this temperature range. In contrast, we find that GPI-anchored proteins exhibit temperature-independent diffusion over this range and revert to temperature-dependent diffusion on cell membrane blebs, in cells depleted of cholesterol, and upon acute perturbation of actin dynamics and myosin activity. A model transmembrane protein with a cytosolic actin-binding domain also exhibits the temperature-independent behavior, directly implicating the role of cortical actin. We show that diffusion of GPI-anchored proteins also becomes temperature dependent when the filamentous dynamic actin nucleator formin is inhibited. However, changes in cortical actin mesh size or perturbation of branched actin nucleator Arp2/3 do not affect this behavior. Thus cell surface diffusion of GPI-anchored proteins and transmembrane proteins that associate with actin is driven by active fluctuations of dynamic cortical actin filaments in addition to thermal fluctuations, consistent with expectations from an "active actin-membrane composite" cell surface.

  18. Diffusion of GPI-anchored proteins is influenced by the activity of dynamic cortical actin

    PubMed Central

    Saha, Suvrajit; Lee, Il-Hyung; Polley, Anirban; Groves, Jay T.; Rao, Madan; Mayor, Satyajit

    2015-01-01

    Molecular diffusion at the surface of living cells is believed to be predominantly driven by thermal kicks. However, there is growing evidence that certain cell surface molecules are driven by the fluctuating dynamics of cortical cytoskeleton. Using fluorescence correlation spectroscopy, we measure the diffusion coefficient of a variety of cell surface molecules over a temperature range of 24–37°C. Exogenously incorporated fluorescent lipids with short acyl chains exhibit the expected increase of diffusion coefficient over this temperature range. In contrast, we find that GPI-anchored proteins exhibit temperature-independent diffusion over this range and revert to temperature-dependent diffusion on cell membrane blebs, in cells depleted of cholesterol, and upon acute perturbation of actin dynamics and myosin activity. A model transmembrane protein with a cytosolic actin-binding domain also exhibits the temperature-independent behavior, directly implicating the role of cortical actin. We show that diffusion of GPI-anchored proteins also becomes temperature dependent when the filamentous dynamic actin nucleator formin is inhibited. However, changes in cortical actin mesh size or perturbation of branched actin nucleator Arp2/3 do not affect this behavior. Thus cell surface diffusion of GPI-anchored proteins and transmembrane proteins that associate with actin is driven by active fluctuations of dynamic cortical actin filaments in addition to thermal fluctuations, consistent with expectations from an “active actin-membrane composite” cell surface. PMID:26378258

  19. Capping protein integrates multiple MAMP signalling pathways to modulate actin dynamics during plant innate immunity.

    PubMed

    Li, Jiejie; Henty-Ridilla, Jessica L; Staiger, Benjamin H; Day, Brad; Staiger, Christopher J

    2015-01-01

    Plants and animals perceive diverse microbe-associated molecular patterns (MAMPs) via pattern recognition receptors and activate innate immune signalling. The actin cytoskeleton has been suggested as a target for innate immune signalling and a key transducer of cellular responses. However, the molecular mechanisms underlying actin remodelling and the precise functions of these rearrangements during innate immunity remain largely unknown. Here we demonstrate rapid actin remodelling in response to several distinct MAMP signalling pathways in plant epidermal cells. The regulation of actin dynamics is a convergence point for basal defence machinery, such as cell wall fortification and transcriptional reprogramming. Our quantitative analyses of actin dynamics and genetic studies reveal that MAMP-stimulated actin remodelling is due to the inhibition of capping protein (CP) by the signalling lipid, phosphatidic acid. In addition, CP promotes resistance against bacterial and fungal phytopathogens. These findings demonstrate that CP is a central target for the plant innate immune response. PMID:26018794

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

    PubMed Central

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

    2015-01-01

    Recent studies have indicated that the serotonin receptor subtype 7 (5-HT7R) plays a crucial role in shaping neuronal morphology during embryonic and early postnatal life. Here we show that pharmacological stimulation of 5-HT7R using a highly selective agonist, LP-211, enhances neurite outgrowth in neuronal primary cultures from the cortex, hippocampus and striatal complex of embryonic mouse brain, through multiple signal transduction pathways. All these signaling systems, involving mTOR, the Rho GTPase Cdc42, Cdk5, and ERK, are known to converge on the reorganization of cytoskeletal proteins that subserve neurite outgrowth. Indeed, our data indicate that neurite elongation stimulated by 5-HT7R is modulated by drugs affecting actin polymerization. In addition, we show, by 2D Western blot analyses, that treatment of neuronal cultures with LP-211 alters the expression profile of cofilin, an actin binding protein involved in microfilaments dynamics. Furthermore, by using microfluidic chambers that physically separate axons from the soma and dendrites, we demonstrate that agonist-dependent activation of 5-HT7R stimulates axonal elongation. Our results identify for the first time several signal transduction pathways, activated by stimulation of 5-HT7R, that converge to promote cytoskeleton reorganization and consequent modulation of axonal elongation. Therefore, the activation of 5-HT7R might represent one of the key elements regulating CNS connectivity and plasticity during development. PMID:25814944

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

    PubMed

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

    2015-01-01

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

  2. Synthetic polyamines: new compounds specific to actin dynamics for mammalian cell and fission yeast.

    PubMed

    Riveline, Daniel; Thiagarajan, Raghavan; Lehn, Jean-Marie; Carlier, Marie-France

    2014-01-01

    Actin is a major actor in the determination of cell shape. On the one hand, site-directed assembly/disassembly cycles of actin filaments drive protrusive force leading to lamellipodia and filopodia dynamics. Force produced by actin similarly contributes in membrane scission in endocytosis or Golgi remodeling. On the other hand, cellular processes like adhesion, immune synapse, cortex dynamics or cytokinesis are achieved by combining acto-myosin contractility and actin assembly in a complex and not fully understood manner. New chemical compounds are therefore needed to disentangle acto-myosin and actin dynamics. We have found that synthetic, cell permeant, short polyamines are promising new actin regulators in this context. They generate growth and stabilization of lamellipodia within minutes by slowing down the actin assembly/disassembly cycle and facilitating nucleation. We now report that these polyamines also slow down cytokinetic ring closure in fission yeast. This shows that these synthetic compounds are active also in yeasts, and these experiments specifically highlight that actin depolymerization is involved in the ring closure. Thus, synthetic polyamines appear to be potentially powerful agents in a quantitative approach to the role of actin in complex processes in cell biology, developmental biology and potentially cancer research.

  3. Cytoskeletal remodeling during growth cone-target interactions

    PubMed Central

    1993-01-01

    Reorganization of the cytoskeleton of neuronal growth cones in response to environmental cues underlies the process of axonal guidance. Most previous studies addressing cytoskeletal changes during growth cone pathfinding have focused on the dynamics of a single cytoskeletal component. We report here an investigation of homophilic growth cone- target interactions between Aplysia bag cell neurons using digitally enhanced video microscopy, which addresses dynamic interactions between actin filaments and microtubules. After physical contact of a growth cone with a physiological target, mechanical coupling occurred after a delay; and then the growth cone exerted forces on and displaced the target object. Subsequent to coupling, F-actin accumulation was observed at the target contact zone, followed by preferential microtubule extension to the same site. After successful target interactions, growth cones typically moved off highly adhesive poly-L- lysine substrates into native target cell surfaces. These events were associated with modulation of both the direction and rate of neurite outgrowth: growth cone migration was typically reoriented to a trajectory along the target interaction axis and rates of advance increased by about one order of magnitude. Directed microtubule movements toward the contact site appeared to be F-actin dependent as target site-specific microtubule extension and bundling could be reversibly randomized by micromolar levels of cytochalasin B in a characteristic manner. Our results suggest that target contacts can induce focal F-actin assembly and reorganization which, in turn, guides target site-directed microtubule redistribution. PMID:8509456

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

    PubMed

    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

  5. Dystrophin and utrophin have distinct effects on the structural dynamics of actin

    PubMed Central

    Prochniewicz, Ewa; Henderson, Davin; Ervasti, James M.; Thomas, David D.

    2009-01-01

    We have used time-resolved spectroscopy to investigate the structural dynamics of actin interaction with dystrophin and utrophin in relationship to the pathology of muscular dystrophy. Dystrophin and utrophin bind actin in vitro with similar affinities, but the molecular contacts of these two proteins with actin are different. It has been hypothesized that the presence of two low-affinity actin-binding sites in dystrophin allows more elastic response of the actin–dystrophin–sarcolemma linkage to muscle stretches, compared with utrophin, which binds via one contiguous actin-binding domain. We have directly tested this hypothesis by determining the effects of dystrophin and utrophin on the microsecond rotational dynamics of a phosphorescent dye attached to C374 on actin, as detected by transient phosphorescence anisotropy (TPA). Binding of dystrophin or utrophin to actin resulted in significant changes in the TPA decay, increasing the final anisotropy (restricting the rotational amplitude) and decreasing the rotational correlation times (increasing the rotational rates and the torsional flexibility). This paradoxical combination of effects on actin dynamics (decreased amplitude but increased rate) has not been observed for other actin-binding proteins. Thus, when dystrophin or utrophin binds, actin becomes less like cast iron (strong but brittle) and more like steel (stronger and more resilient). At low levels of saturation, the binding of dystrophin and utrophin has similar effects, but at higher levels, utrophin caused much greater restrictions in amplitude and increases in rate. The effects of dystrophin and utrophin on actin dynamics provide molecular insight into the pathology of muscular dystrophy. PMID:19416869

  6. TWISTED DWARF1 Mediates the Action of Auxin Transport Inhibitors on Actin Cytoskeleton Dynamics.

    PubMed

    Zhu, Jinsheng; Bailly, Aurelien; Zwiewka, Marta; Sovero, Valpuri; Di Donato, Martin; Ge, Pei; Oehri, Jacqueline; Aryal, Bibek; Hao, Pengchao; Linnert, Miriam; Burgardt, Noelia Inés; Lücke, Christian; Weiwad, Matthias; Michel, Max; Weiergräber, Oliver H; Pollmann, Stephan; Azzarello, Elisa; Mancuso, Stefano; Ferro, Noel; Fukao, Yoichiro; Hoffmann, Céline; Wedlich-Söldner, Roland; Friml, Jiří; Thomas, Clément; Geisler, Markus

    2016-04-01

    Plant growth and architecture is regulated by the polar distribution of the hormone auxin. Polarity and flexibility of this process is provided by constant cycling of auxin transporter vesicles along actin filaments, coordinated by a positive auxin-actin feedback loop. Both polar auxin transport and vesicle cycling are inhibited by synthetic auxin transport inhibitors, such as 1-N-naphthylphthalamic acid (NPA), counteracting the effect of auxin; however, underlying targets and mechanisms are unclear. Using NMR, we map the NPA binding surface on the Arabidopsis thaliana ABCB chaperone TWISTED DWARF1 (TWD1). We identify ACTIN7 as a relevant, although likely indirect, TWD1 interactor, and show TWD1-dependent regulation of actin filament organization and dynamics and that TWD1 is required for NPA-mediated actin cytoskeleton remodeling. The TWD1-ACTIN7 axis controls plasma membrane presence of efflux transporters, and as a consequence act7 and twd1 share developmental and physiological phenotypes indicative of defects in auxin transport. These can be phenocopied by NPA treatment or by chemical actin (de)stabilization. We provide evidence that TWD1 determines downstream locations of auxin efflux transporters by adjusting actin filament debundling and dynamizing processes and mediating NPA action on the latter. This function appears to be evolutionary conserved since TWD1 expression in budding yeast alters actin polarization and cell polarity and provides NPA sensitivity. PMID:27053424

  7. Biophysical models of length control of cytoskeletal structures

    NASA Astrophysics Data System (ADS)

    Mohapatra, Lishibanya

    Cells contain elaborate and interconnected networks of protein polymers which make up the cytoskeleton. The cytoskeleton governs the internal positioning and movement of vesicles and organelles, and controls dynamic changes in cell polarity, shape and movement. Many of these processes require tight control of the size and shape of these cytoskeletal structures. A key question in cell biology is how these structures maintain a particular size and shape despite the rapid turnover of their components. In this thesis I show that the emerging mechanisms by which cells control and regulate the size of filamentous cytoskeletal structures can be classified using key parameters related to their assembly and disassembly kinetics. First, I examine quantitative models based on these specific molecular mechanisms of length control and make experimentally testable predictions that can be used to distinguish different mechanisms of length-control. Second, I study the length control of actin cables in budding yeast cells. Inspired by recent experimental observations in cells, I propose a novel antenna mechanism for cable length control which 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. My results provide testable predictions of the antenna mechanism of actin-cable length control. Next I consider the question of how different sized structures can co-exist in the same cytoplasm while making use of the same building blocks. Using theory, I discover limitations imposed by physics on the finite monomer pool as a mechanism of size control and conclude that additional length control mechanisms are required if a cell is to maintain multiple structures. While the primary focus of this thesis is on cytoskeletal structures, the broader principles and mechanisms discussed herein will apply to a range of

  8. Actin-cytoskeleton dynamics in non-monotonic cell spreading

    PubMed Central

    Heinrich, Doris; Youssef, Simon; Schroth-Diez, Britta; Engel, Ulrike; Aydin, Daniel; Blümmel, Jacques; Spatz, Joachim P

    2008-01-01

    The spreading of motile cells on a substrate surface is accompanied by reorganization of their actin network. We show that spreading in the highly motile cells of Dictyostelium is non-monotonic, and thus differs from the passage of spreading cells through a regular series of stages. Quantification of the gain and loss of contact area revealed fluctuating forces of protrusion and retraction that dominate the interaction of Dictyostelium cells with a substrate. The molecular basis of these fluctuations is elucidated by dual-fluorescence labeling of filamentous actin together with proteins that highlight specific activities in the actin system. Front-to-tail polarity is established by the sorting out of myosin-II from regions where dense actin assemblies are accumulating. Myosin-IB identifies protruding front regions, and the Arp2/3 complex localizes to lamellipodia protruded from the fronts. Coronin is used as a sensitive indicator of actin disassembly to visualize the delicate balance of polymerization and depolymerization in spreading cells. Short-lived actin patches that co-localize with clathrin suggest that membrane internalization occurs even when the substrate-attached cell surface expands. We conclude that non-monotonic cell spreading is characterized by spatiotemporal patterns formed by motor proteins together with regulatory proteins that either promote or terminate actin polymerization on the scale of seconds. PMID:19262103

  9. Dynamic F-actin movement is essential for fertilization in Arabidopsis thaliana.

    PubMed

    Kawashima, Tomokazu; Maruyama, Daisuke; Shagirov, Murat; Li, Jing; Hamamura, Yuki; Yelagandula, Ramesh; Toyama, Yusuke; Berger, Frédéric

    2014-10-10

    In animals, microtubules and centrosomes direct the migration of gamete pronuclei for fertilization. By contrast, flowering plants have lost essential components of the centrosome, raising the question of how flowering plants control gamete nuclei migration during fertilization. Here, we use Arabidopsis thaliana to document a novel mechanism that regulates F-actin dynamics in the female gametes and is essential for fertilization. Live imaging shows that F-actin structures assist the male nucleus during its migration towards the female nucleus. We identify a female gamete-specific Rho-GTPase that regulates F-actin dynamics and further show that actin-myosin interactions are also involved in male gamete nucleus migration. Genetic analyses and imaging indicate that microtubules are dispensable for migration and fusion of male and female gamete nuclei. The innovation of a novel actin-based mechanism of fertilization during plant evolution might account for the complete loss of the centrosome in flowering plants.

  10. Segmentation and Tracking of Cytoskeletal Filaments Using Open Active Contours

    PubMed Central

    Smith, Matthew B.; Li, Hongsheng; Shen, Tian; Huang, Xiaolei; Yusuf, Eddy; Vavylonis, Dimitrios

    2010-01-01

    We use open active contours to quantify cytoskeletal structures imaged by fluorescence microscopy in two and three dimensions. We developed an interactive software tool for segmentation, tracking, and visualization of individual fibers. Open active contours are parametric curves that deform to minimize the sum of an external energy derived from the image and an internal bending and stretching energy. The external energy generates (i) forces that attract the contour toward the central bright line of a filament in the image, and (ii) forces that stretch the active contour toward the ends of bright ridges. Images of simulated semiflexible polymers with known bending and torsional rigidity are analyzed to validate the method. We apply our methods to quantify the conformations and dynamics of actin in two examples: actin filaments imaged by TIRF microscopy in vitro, and actin cables in fission yeast imaged by spinning disk confocal microscopy. PMID:20814909

  11. Measurements and models of cytoskeletal rheology

    NASA Astrophysics Data System (ADS)

    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.

  12. Calcium influx through CRAC channels controls actin organization and dynamics at the immune synapse

    PubMed Central

    Hartzell, Catherine A; Jankowska, Katarzyna I; Burkhardt, Janis K; Lewis, Richard S

    2016-01-01

    T cell receptor (TCR) engagement opens Ca2+ release-activated Ca2+ (CRAC) channels and triggers formation of an immune synapse between T cells and antigen-presenting cells. At the synapse, actin reorganizes into a concentric lamellipod and lamella with retrograde actin flow that helps regulate the intensity and duration of TCR signaling. We find that Ca2+ influx is required to drive actin organization and dynamics at the synapse. Calcium acts by promoting actin depolymerization and localizing actin polymerization and the actin nucleation promotion factor WAVE2 to the periphery of the lamellipod while suppressing polymerization elsewhere. Ca2+-dependent retrograde actin flow corrals ER tubule extensions and STIM1/Orai1 complexes to the synapse center, creating a self-organizing process for CRAC channel localization. Our results demonstrate a new role for Ca2+ as a critical regulator of actin organization and dynamics at the synapse, and reveal potential feedback loops through which Ca2+ influx may modulate TCR signaling. DOI: http://dx.doi.org/10.7554/eLife.14850.001 PMID:27440222

  13. Changes in actin dynamics are involved in salicylic acid signaling pathway.

    PubMed

    Matoušková, Jindřiška; Janda, Martin; Fišer, Radovan; Sašek, Vladimír; Kocourková, Daniela; Burketová, Lenka; Dušková, Jiřina; Martinec, Jan; Valentová, Olga

    2014-06-01

    Changes in actin cytoskeleton dynamics are one of the crucial players in many physiological as well as non-physiological processes in plant cells. Positioning of actin filament arrays is necessary for successful establishment of primary lines of defense toward pathogen attack, depolymerization leads very often to the enhanced susceptibility to the invading pathogen. On the other hand it was also shown that the disruption of actin cytoskeleton leads to the induction of defense response leading to the expression of PATHOGENESIS RELATED proteins (PR). In this study we show that pharmacological actin depolymerization leads to the specific induction of genes in salicylic acid pathway but not that involved in jasmonic acid signaling. Life imaging of leafs of Arabidopsis thaliana with GFP-tagged fimbrin (GFP-fABD2) treated with 1 mM salicylic acid revealed rapid disruption of actin filaments resembling the pattern viewed after treatment with 200 nM latrunculin B. The effect of salicylic acid on actin filament fragmentation was prevented by exogenous addition of phosphatidic acid, which binds to the capping protein and thus promotes actin polymerization. The quantitative evaluation of actin filament dynamics is also presented.

  14. Dynamic in vivo analysis of drug induced actin cytoskeleton degradation by digital holographic microscopy

    NASA Astrophysics Data System (ADS)

    Schnekenburger, Juergen; Bredebusch, Ilona; Langehanenberg, Patrik; Domschke, Wolfram; von Bally, Gert; Kemper, Björn

    2007-07-01

    The actin cytoskeleton mediates a variety of crucial cellular functions as migration, intracellular transport, exocytosis, endocytosis and force generation. The highly dynamic actin fibers are therefore targets for several drugs and toxins. However the study of actin interfering processes by standard microscopy techniques fails in the detailed resolution of dynamic spatial alterations required for a deeper understanding of toxic effects. Here we applied digital holographic microscopy in the online functional analysis of the actin cytoskeleton disrupting marine toxin Latrunculin B. SEM and fluorescence microscopy showed rapid Latrunculin B induced alterations in cell morphology and actin fiber degradation in pancreas tumor cells. The dynamic digital holographic in vivo analysis of the drug dependent cellular processes demonstrated differences in the actin cytoskeleton stability of highly differentiated and dedifferentiated pancreas tumor cell lines. The spatial resolution of the morphological alterations revealed unequal changes in cell morphology. While cells with a low metastatic potential showed Latrunculin B induced cell collapse within 4 h the metastatic tumor cells were increased in cell volume indicating Latrunculin B effects also on cell water content. These data demonstrate that marker free, non-destructive online analysis of cellular morphology and dynamic spatial processes in living cells by digital holography offers new insights in actin dependent cellular mechanisms. Digital holographic microscopy was shown to be a versatile tool in the screening of toxic drug effects and cancer cell biology.

  15. Actin gene family dynamics in cryptomonads and red algae.

    PubMed

    Tanifuji, Goro; Archibald, John M

    2010-09-01

    Here we present evidence for a complex evolutionary history of actin genes in red algae and cryptomonads, a group that acquired photosynthesis secondarily through the engulfment of a red algal endosymbiont. Four actin genes were found in the nuclear genome of the cryptomonad, Guillardia theta, and in the genome of the red alga, Galdieria sulphuraria, a member of the Cyanidiophytina. Phylogenetic analyses reveal that the both organisms possess two distinct sequence types, designated "type-1" and "type-2." A weak but consistent phylogenetic affinity between the cryptomonad type-2 sequences and the type-2 sequences of G. sulphuraria and red algae belonging to the Rhodophytina was observed. This is consistent with the possibility that the cryptomonad type-2 sequences are derived from the red algal endosymbiont that gave rise to the cryptomonad nucleomorph and plastid. Red algae as a whole possess two very different actin sequence types, with G. sulphuraria being the only organism thus far known to possess both. The common ancestor of Rhodophytina and Cyanidiophytina may have had two actin genes, with differential loss explaining the distribution of these genes in modern-day groups. Our study provides new insight into the evolution and divergence of actin genes in cryptomonads and red algae, and in doing so underscores the challenges associated with heterogeneity in actin sequence evolution and ortholog/paralog detection.

  16. Probing the dynamic responses of individual actin filaments under fluidic mechanical stimulation via microfluidics

    NASA Astrophysics Data System (ADS)

    Cheng, Chao-Min; Yang, Chung-Yao; Kim, YongTae; LeDuc, Philip R.

    2013-05-01

    Herein, we demonstrate an easy-to-handle approach that employs a combination of microcurvilinear flow and fluorescence microscopy for probing the dynamic responses of individual synthesized actin filaments. We observed morphological changes of single actin filaments with different spatiotemporal responses when they were elongated with rotation or underwent significant bending during fluidic shear stress, and found that they may initially increase their curvature but then start releasing the external force immediately thereafter. Our approach allowed us to visibly examine the dynamic responses of individual actin filaments under simultaneous forces of rotation and elongation, as well as bending resulting from fluidic shear stress.

  17. Elevated Glucose Levels Promote Contractile and Cytoskeletal Gene Expression in Vascular Smooth Muscle via Rho/Protein Kinase C and Actin Polymerization.

    PubMed

    Hien, Tran Thi; Turczyńska, Karolina M; Dahan, Diana; Ekman, Mari; Grossi, Mario; Sjögren, Johan; Nilsson, Johan; Braun, Thomas; Boettger, Thomas; Garcia-Vaz, Eliana; Stenkula, Karin; Swärd, Karl; Gomez, Maria F; Albinsson, Sebastian

    2016-02-12

    Both type 1 and type 2 diabetes are associated with increased risk of cardiovascular disease. This is in part attributed to the effects of hyperglycemia on vascular endothelial and smooth muscle cells, but the underlying mechanisms are not fully understood. In diabetic animal models, hyperglycemia results in hypercontractility of vascular smooth muscle possibly due to increased activation of Rho-kinase. The aim of the present study was to investigate the regulation of contractile smooth muscle markers by glucose and to determine the signaling pathways that are activated by hyperglycemia in smooth muscle cells. Microarray, quantitative PCR, and Western blot analyses revealed that both mRNA and protein expression of contractile smooth muscle markers were increased in isolated smooth muscle cells cultured under high compared with low glucose conditions. This effect was also observed in hyperglycemic Akita mice and in diabetic patients. Elevated glucose activated the protein kinase C and Rho/Rho-kinase signaling pathways and stimulated actin polymerization. Glucose-induced expression of contractile smooth muscle markers in cultured cells could be partially or completely repressed by inhibitors of advanced glycation end products, L-type calcium channels, protein kinase C, Rho-kinase, actin polymerization, and myocardin-related transcription factors. Furthermore, genetic ablation of the miR-143/145 cluster prevented the effects of glucose on smooth muscle marker expression. In conclusion, these data demonstrate a possible link between hyperglycemia and vascular disease states associated with smooth muscle contractility.

  18. Cucurbitacin I Inhibits Cell Motility by Indirectly Interfering with Actin Dynamics

    PubMed Central

    Knecht, David A.; LaFleur, Rebecca A.; Kahsai, Alem W.; Argueta, Christian E.; Beshir, Anwar B.; Fenteany, Gabriel

    2010-01-01

    Background Cucurbitacins are plant natural products that inhibit activation of the Janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) pathway by an unknown mechanism. They are also known to cause changes in the organization of the actin cytoskeleton. Methodology/Principal Findings We show that cucurbitacin I potently inhibits the migration of Madin-Darby canine kidney (MDCK) cell sheets during wound closure, as well as the random motility of B16-F1 mouse melanoma cells, but has no effect on movement of Dictyostelium discoideum amoebae. Upon treatment of MDCK or B16-F1 cells with cucurbitacin I, there is a very rapid cessation of motility and gradual accumulation of filamentous actin aggregates. The cellular effect of the compound is similar to that observed when cells are treated with the actin filament-stabilizing agent jasplakinolide. However, we found that, unlike jasplakinolide or phallacidin, cucurbitacin I does not directly stabilize actin filaments. In in vitro actin depolymerization experiments, cucurbitacin I had no effect on the rate of actin filament disassembly at the nanomolar concentrations that inhibit cell migration. At elevated concentrations, the depolymerization rate was also unaffected, although there was a delay in the initiation of depolymerization. Therefore, cucurbitacin I targets some factor involved in cellular actin dynamics other than actin itself. Two candidate proteins that play roles in actin depolymerization are the actin-severing proteins cofilin and gelsolin. Cucurbitacin I possesses electrophilic reactivity that may lead to chemical modification of its target protein, as suggested by structure-activity relationship data. However, mass spectrometry revealed no evidence for modification of purified cofilin or gelsolin by cucurbitacin I. Conclusions/Significance Cucurbitacin I results in accumulation of actin filaments in cells by a unique indirect mechanism. Furthermore, the proximal target of

  19. Dynamic regimes and bifurcations in a model of actin-based motility

    NASA Astrophysics Data System (ADS)

    Enculescu, Mihaela; Gholami, Azam; Falcke, Martin

    2008-09-01

    Propulsion by actin polymerization is widely used in cell motility. Here, we investigate a model of the brush range of an actin gel close to a propelled object, describing the force generation and the dynamics of the propagation velocity. We find transitions between stable steady states and relaxation oscillations when the attachment rate of actin filaments to the obstacle is varied. The oscillations set in at small values of the attachment rate via a homoclinic bifurcation. A second transition from a stable steady state to relaxation oscillations, found for higher values of the attachment rate, occurs via a supercritical Hopf bifurcation. The behavior of the model near the second transition is similar that of a system undergoing a canard explosion. Consequently, we observe excitable dynamics also. The model further exhibits bistability between stationary states or stationary states and limit cycles. Therefore, the brush of actin filament ends appears to have a much richer dynamics than was assumed until now.

  20. Actin dynamics tune the integrated stress response by regulating eukaryotic initiation factor 2α dephosphorylation

    PubMed Central

    Chambers, Joseph E; Dalton, Lucy E; Clarke, Hanna J; Malzer, Elke; Dominicus, Caia S; Patel, Vruti; Moorhead, Greg; Ron, David; Marciniak, Stefan J

    2015-01-01

    Four stress-sensing kinases phosphorylate the alpha subunit of eukaryotic translation initiation factor 2 (eIF2α) to activate the integrated stress response (ISR). In animals, the ISR is antagonised by selective eIF2α phosphatases comprising a catalytic protein phosphatase 1 (PP1) subunit in complex with a PPP1R15-type regulatory subunit. An unbiased search for additional conserved components of the PPP1R15-PP1 phosphatase identified monomeric G-actin. Like PP1, G-actin associated with the functional core of PPP1R15 family members and G-actin depletion, by the marine toxin jasplakinolide, destabilised the endogenous PPP1R15A-PP1 complex. The abundance of the ternary PPP1R15-PP1-G-actin complex was responsive to global changes in the polymeric status of actin, as was its eIF2α-directed phosphatase activity, while localised G-actin depletion at sites enriched for PPP1R15 enhanced eIF2α phosphorylation and the downstream ISR. G-actin's role as a stabilizer of the PPP1R15-containing holophosphatase provides a mechanism for integrating signals regulating actin dynamics with stresses that trigger the ISR. DOI: http://dx.doi.org/10.7554/eLife.04872.001 PMID:25774599

  1. Actin dynamics tune the integrated stress response by regulating eukaryotic initiation factor 2α dephosphorylation.

    PubMed

    Chambers, Joseph E; Dalton, Lucy E; Clarke, Hanna J; Malzer, Elke; Dominicus, Caia S; Patel, Vruti; Moorhead, Greg; Ron, David; Marciniak, Stefan J

    2015-01-01

    Four stress-sensing kinases phosphorylate the alpha subunit of eukaryotic translation initiation factor 2 (eIF2α) to activate the integrated stress response (ISR). In animals, the ISR is antagonised by selective eIF2α phosphatases comprising a catalytic protein phosphatase 1 (PP1) subunit in complex with a PPP1R15-type regulatory subunit. An unbiased search for additional conserved components of the PPP1R15-PP1 phosphatase identified monomeric G-actin. Like PP1, G-actin associated with the functional core of PPP1R15 family members and G-actin depletion, by the marine toxin jasplakinolide, destabilised the endogenous PPP1R15A-PP1 complex. The abundance of the ternary PPP1R15-PP1-G-actin complex was responsive to global changes in the polymeric status of actin, as was its eIF2α-directed phosphatase activity, while localised G-actin depletion at sites enriched for PPP1R15 enhanced eIF2α phosphorylation and the downstream ISR. G-actin's role as a stabilizer of the PPP1R15-containing holophosphatase provides a mechanism for integrating signals regulating actin dynamics with stresses that trigger the ISR.

  2. Protein phosphatase 2A, a potential regulator of actin dynamics and actin-based organelle motility in the green alga Acetabularia.

    PubMed

    Menzel, D; Vugrek, O; Frank, S; Elsner-Menzel, C

    1995-06-01

    The giant, unicellular alga Acetabularia is a well known experimental model for the study of actin-dependent intracellular organelle motility. In the cyst stage, however, which is equivalent to the gametophytic stage, organelles are immobile, even though an actin cytoskeleton is present. The reason for the lack of organelle motility at this stage has not been known. To test the hypothesis that organelle motility could be under the control of posttranslational modification by protein phosphorylation, we have treated cysts with submicromolar concentrations of okadaic acid or calyculin A, both potent inhibitors of serine/threonine protein phosphatases (ser/thr-PPases). The effects were dramatic: Instead of linear actin bundles typical for control cysts, circular arrays of actin bundles formed in the cortical cyst cytoplasm. Concomitant with the formation of these action rings, the cytoplasmic layers beneath the rings began to slowly rotate in a continuous and uniform counter-clockwise fashion. This effect suggests that protein phosphorylation acts on the actin cytoskeleton at two levels: (1) It changes the assembly properties of the actin filament system to the extent that novel cytoskeletal configurations are formed and (2) it raises the activity of putative motor proteins involved in the rotational movements to levels sufficiently high to support motility at a stage when organelle motility does not normally occur. Northern blot analysis of cyst stage-mRNA using probes specific to protein phosphatase type 1 (PP1) and type 2A (PP2A) reveals that PP2A is strongly expressed at this developmental stage whereas PP1 is not detectable, suggesting that PP2A is the likely target to the protein phosphatase inhibitors.(ABSTRACT TRUNCATED AT 250 WORDS)

  3. Some distinctive features of zebrafish myogenesis based on unexpected distributions of the muscle cytoskeletal proteins actin, myosin, desmin, alpha-actinin, troponin and titin.

    PubMed

    Costa, Manoel L; Escaleira, Roberta C; Rodrigues, Viviane B; Manasfi, Muhamed; Mermelstein, Claudia S

    2002-08-01

    The current myofibrillogenesis model is based mostly on in vitro cell cultures and on avian and mammalian embryos in situ. We followed the expression of actin, myosin, desmin, alpha-actinin, titin, and troponin using immunofluorescence microscopy of zebrafish (Danio rerio) embryos. We could see young mononucleated myoblasts with sharp striations. The striations were positive for all the sarcomeric proteins. Desmin distribution during muscle maturation changes from dispersed aggregates to a perinuclear concentration to striated afterwards. We could not observe desmin-positive, myofibrillar-proteins-negative cells, and we could not find any non-striated distribution of sarcomeric proteins, such as stress fiber-like structures. Some steps, like fusion before striation, seem to be different in the zebrafish when compared with the previously described myogenesis sequences.

  4. Synergistic action of dendritic mitochondria and creatine kinase maintains ATP homeostasis and actin dynamics in growing neuronal dendrites.

    PubMed

    Fukumitsu, Kansai; Fujishima, Kazuto; Yoshimura, Azumi; Wu, You Kure; Heuser, John; Kengaku, Mineko

    2015-04-01

    The distribution of mitochondria within mature, differentiated neurons is clearly adapted to their regional physiological needs and can be perturbed under various pathological conditions, but the function of mitochondria in developing neurons has been less well studied. We have studied mitochondrial distribution within developing mouse cerebellar Purkinje cells and have found that active delivery of mitochondria into their dendrites is a prerequisite for proper dendritic outgrowth. Even when mitochondria in the Purkinje cell bodies are functioning normally, interrupting the transport of mitochondria into their dendrites severely disturbs dendritic growth. Additionally, we find that the growth of atrophic dendrites lacking mitochondria can be rescued by activating ATP-phosphocreatine exchange mediated by creatine kinase (CK). Conversely, inhibiting cytosolic CKs decreases dendritic ATP levels and also disrupts dendrite development. Mechanistically, this energy depletion appears to perturb normal actin dynamics and enhance the aggregation of cofilin within growing dendrites, reminiscent of what occurs in neurons overexpressing the dephosphorylated form of cofilin. These results suggest that local ATP synthesis by dendritic mitochondria and ATP-phosphocreatine exchange act synergistically to sustain the cytoskeletal dynamics necessary for dendritic development.

  5. [Cytoskeletal control of cell length regulation].

    PubMed

    Kharitonova, M A; Levina, C M; Rovenskii, I A

    2002-01-01

    It was shown that mouse embryo fibroblasts and human foreskin diploid fibroblasts of AGO 1523 line cultivated on specially prepared substrates with narrow (15 +/- 3 microns) linear adhesive strips were elongated and oriented along the strips, but the mean lengths of the fibroblasts of each type on the strips differed from those on the standard culture substrates. In contrast to the normal fibroblasts, the length of mouse embryonic fibroblasts with inactivated gene-suppresser Rb responsible for negative control of cell proliferation (MEF Rb-/-), ras-transformed mouse embryonic fibroblasts (MEF Rb-/-ras), or normal rat epitheliocytes of IAR2 line significantly exceeded those of the same cells on the standard culture substrates. The results of experiments with the drugs specifically affecting the cytoskeleton (colcemid and cytochalasin D) suggest that the constant mean length of normal fibroblasts is controlled by a dynamic equilibrium between two forces: centripetal tension of contractile actin-myosin microfilaments and centrifugal force generated by growing microtubules. This cytoskeletal mechanism is disturbed in MEF Rb-/- or MEF Rb-/-ras, probably, because of an impaired actin cytoskeleton and also in IAR2 epitheliocytes due to the different organization of the actin-myosin system in these cells, as compared to that in the fibroblasts. PMID:11862697

  6. Mathematical modelling and numerical simulations of actin dynamics in the eukaryotic cell.

    PubMed

    George, Uduak Z; Stéphanou, Angélique; Madzvamuse, Anotida

    2013-02-01

    The aim of this article is to study cell deformation and cell movement by considering both the mechanical and biochemical properties of the cortical network of actin filaments and its concentration. Actin is a polymer that can exist either in filamentous form (F-actin) or in monometric form (G-actin) (Chen et al. in Trends Biochem Sci 25:19-23, 2000) and the filamentous form is arranged in a paired helix of two protofilaments (Ananthakrishnan et al. in Recent Res Devel Biophys 5:39-69, 2006). By assuming that cell deformations are a result of the cortical actin dynamics in the cell cytoskeleton, we consider a continuum mathematical model that couples the mechanics of the network of actin filaments with its bio-chemical dynamics. Numerical treatment of the model is carried out using the moving grid finite element method (Madzvamuse et al. in J Comput Phys 190:478-500, 2003). Furthermore, by assuming slow deformations of the cell, we use linear stability theory to validate the numerical simulation results close to bifurcation points. Far from bifurcation points, we show that the mathematical model is able to describe the complex cell deformations typically observed in experimental results. Our numerical results illustrate cell expansion, cell contraction, cell translation and cell relocation as well as cell protrusions. In all these results, the contractile tonicity formed by the association of actin filaments to the myosin II motor proteins is identified as a key bifurcation parameter. PMID:22434394

  7. Neuronal Actin Dynamics, Spine Density and Neuronal Dendritic Complexity Are Regulated by CAP2.

    PubMed

    Kumar, Atul; Paeger, Lars; Kosmas, Kosmas; Kloppenburg, Peter; Noegel, Angelika A; Peche, Vivek S

    2016-01-01

    Actin remodeling is crucial for dendritic spine development, morphology and density. CAP2 is a regulator of actin dynamics through sequestering G-actin and severing F-actin. In a mouse model, ablation of CAP2 leads to cardiovascular defects and delayed wound healing. This report investigates the role of CAP2 in the brain using Cap2(gt/gt) mice. Dendritic complexity, the number and morphology of dendritic spines were altered in Cap2(gt/gt) with increased number of excitatory synapses. This was accompanied by increased F-actin content and F-actin accumulation in cultured Cap2(gt/gt) neurons. Moreover, reduced surface GluA1 was observed in mutant neurons under basal condition and after induction of chemical LTP. Additionally, we show an interaction between CAP2 and n-cofilin, presumably mediated through the C-terminal domain of CAP2 and dependent on cofilin Ser3 phosphorylation. In vivo, the consequences of this interaction were altered phosphorylated cofilin levels and formation of cofilin aggregates in the neurons. Thus, our studies identify a novel role of CAP2 in neuronal development and neuronal actin dynamics. PMID:27507934

  8. Neuronal Actin Dynamics, Spine Density and Neuronal Dendritic Complexity Are Regulated by CAP2

    PubMed Central

    Kumar, Atul; Paeger, Lars; Kosmas, Kosmas; Kloppenburg, Peter; Noegel, Angelika A.; Peche, Vivek S.

    2016-01-01

    Actin remodeling is crucial for dendritic spine development, morphology and density. CAP2 is a regulator of actin dynamics through sequestering G-actin and severing F-actin. In a mouse model, ablation of CAP2 leads to cardiovascular defects and delayed wound healing. This report investigates the role of CAP2 in the brain using Cap2gt/gt mice. Dendritic complexity, the number and morphology of dendritic spines were altered in Cap2gt/gt with increased number of excitatory synapses. This was accompanied by increased F-actin content and F-actin accumulation in cultured Cap2gt/gt neurons. Moreover, reduced surface GluA1 was observed in mutant neurons under basal condition and after induction of chemical LTP. Additionally, we show an interaction between CAP2 and n-cofilin, presumably mediated through the C-terminal domain of CAP2 and dependent on cofilin Ser3 phosphorylation. In vivo, the consequences of this interaction were altered phosphorylated cofilin levels and formation of cofilin aggregates in the neurons. Thus, our studies identify a novel role of CAP2 in neuronal development and neuronal actin dynamics. PMID:27507934

  9. Live Cell Imaging of F-actin Dynamics via Fluorescent Speckle Microscopy (FSM)

    PubMed Central

    Lim, James; Danuser, Gaudenz

    2009-01-01

    In this protocol we describe the use of Fluorescent Speckle Microscopy (FSM) to capture high-resolution images of actin dynamics in PtK1 cells. A unique advantage of FSM is its ability to capture the movement and turnover kinetics (assembly/disassembly) of the F-actin network within living cells. This technique is particularly useful in deriving quantitative measurements of F-actin dynamics when paired with computer vision software (qFSM). We describe the selection, microinjection and visualization of fluorescent actin probes in living cells. Importantly, similar procedures are applicable to visualizing other macomolecular assemblies. FSM has been demonstrated for microtubules, intermediate filaments, and adhesion complexes. PMID:19684563

  10. Structural, Mechanical, and Dynamical Variability of the Actin Cortex in Living Cells

    PubMed Central

    Eghiaian, Frédéric; Rigato, Annafrancesca; Scheuring, Simon

    2015-01-01

    In eukaryotic cells, an actin-based cortex lines the inner leaflet of the plasma membrane, endowing the cells with crucial mechanical and functional properties. Unfortunately, it has not been possible to study the structural dynamics of the actin cortex at high lateral resolution in living cells. Here, we performed atomic force microscopy time-lapse imaging and mechanical mapping of actin in the cortex of living cells at high lateral and temporal resolution. Cortical actin filaments adopted discernible arrangements, ranging from large parallel bundles with low connectivity to a tight meshwork of short filaments. Mixing of these architectures resulted in attuned cortex networks with specific connectivity, mechanical responses, and marked differences in their dynamic behavior. PMID:25809247

  11. Profilin Interaction with Actin Filament Barbed End Controls Dynamic Instability, Capping, Branching, and Motility

    PubMed Central

    Pernier, Julien; Shekhar, Shashank; Jegou, Antoine; Guichard, Bérengère; Carlier, Marie-France

    2016-01-01

    Summary Cell motility and actin homeostasis depend on the control of polarized growth of actin filaments. Profilin, an abundant regulator of actin dynamics, supports filament assembly at barbed ends by binding G-actin. Here, we demonstrate how, by binding and destabilizing filament barbed ends at physiological concentrations, profilin also controls motility, cell migration, and actin homeostasis. Profilin enhances filament length fluctuations. Profilin competes with Capping Protein at barbed ends, which generates a lower amount of profilin-actin than expected if barbed ends were tightly capped. Profilin competes with barbed end polymerases, such as formins and VopF, and inhibits filament branching by WASP-Arp2/3 complex by competition for filament barbed ends, accounting for its as-yet-unknown effects on motility and metastatic cell migration observed in this concentration range. In conclusion, profilin is a major coordinator of polarized growth of actin filaments, controlled by competition between barbed end cappers, trackers, destabilizers, and filament branching machineries. PMID:26812019

  12. The interplay between neuronal activity and actin dynamics mimic the setting of an LTD synaptic tag.

    PubMed

    Szabó, Eszter C; Manguinhas, Rita; Fonseca, Rosalina

    2016-01-01

    Persistent forms of plasticity, such as long-term depression (LTD), are dependent on the interplay between activity-dependent synaptic tags and the capture of plasticity-related proteins. We propose that the synaptic tag represents a structural alteration that turns synapses permissive to change. We found that modulation of actin dynamics has different roles in the induction and maintenance of LTD. Inhibition of either actin depolymerisation or polymerization blocks LTD induction whereas only the inhibition of actin depolymerisation blocks LTD maintenance. Interestingly, we found that actin depolymerisation and CaMKII activation are involved in LTD synaptic-tagging and capture. Moreover, inhibition of actin polymerisation mimics the setting of a synaptic tag, in an activity-dependent manner, allowing the expression of LTD in non-stimulated synapses. Suspending synaptic activation also restricts the time window of synaptic capture, which can be restored by inhibiting actin polymerization. Our results support our hypothesis that modulation of the actin cytoskeleton provides an input-specific signal for synaptic protein capture. PMID:27650071

  13. AIP1 acts with cofilin to control actin dynamics during epithelial morphogenesis.

    PubMed

    Chu, Dandan; Pan, Hanshuang; Wan, Ping; Wu, Jing; Luo, Jun; Zhu, Hong; Chen, Jiong

    2012-10-01

    During epithelial morphogenesis, cells not only maintain tight adhesion for epithelial integrity but also allow dynamic intercellular movement to take place within cell sheets. How these seemingly opposing processes are coordinated is not well understood. Here, we report that the actin disassembly factors AIP1 and cofilin are required for remodeling of adherens junctions (AJs) during ommatidial precluster formation in Drosophila eye epithelium, a highly stereotyped cell rearrangement process which we describe in detail in our live imaging study. AIP1 is enriched together with F-actin in the apical region of preclusters, whereas cofilin displays a diffuse and uniform localization pattern. Cofilin overexpression completely rescues AJ remodeling defects caused by AIP1 loss of function, and cofilin physically interacts with AIP1. Pharmacological reduction of actin turnover results in similar AJ remodeling defects and decreased turnover of E-cadherin, which also results from AIP1 deficiency, whereas an F-actin-destabilizing drug affects AJ maintenance and epithelial integrity. Together with other data on actin polymerization, our results suggest that AIP1 enhances cofilin-mediated actin disassembly in the apical region of precluster cells to promote remodeling of AJs and thus intercellular movement, but also that robust actin polymerization promotes AJ general adhesion and integrity during the remodeling process.

  14. The interplay between neuronal activity and actin dynamics mimic the setting of an LTD synaptic tag.

    PubMed

    Szabó, Eszter C; Manguinhas, Rita; Fonseca, Rosalina

    2016-09-21

    Persistent forms of plasticity, such as long-term depression (LTD), are dependent on the interplay between activity-dependent synaptic tags and the capture of plasticity-related proteins. We propose that the synaptic tag represents a structural alteration that turns synapses permissive to change. We found that modulation of actin dynamics has different roles in the induction and maintenance of LTD. Inhibition of either actin depolymerisation or polymerization blocks LTD induction whereas only the inhibition of actin depolymerisation blocks LTD maintenance. Interestingly, we found that actin depolymerisation and CaMKII activation are involved in LTD synaptic-tagging and capture. Moreover, inhibition of actin polymerisation mimics the setting of a synaptic tag, in an activity-dependent manner, allowing the expression of LTD in non-stimulated synapses. Suspending synaptic activation also restricts the time window of synaptic capture, which can be restored by inhibiting actin polymerization. Our results support our hypothesis that modulation of the actin cytoskeleton provides an input-specific signal for synaptic protein capture.

  15. MLN64 Is Involved in Actin-mediated Dynamics of Late Endocytic OrganellesD⃞V⃞

    PubMed Central

    Hölttä-Vuori, Maarit; Alpy, Fabien; Tanhuanpää, Kimmo; Jokitalo, Eija; Mutka, Aino-Liisa; Ikonen, Elina

    2005-01-01

    MLN64 is a late endosomal cholesterol-binding membrane protein of an unknown function. Here, we show that MLN64 depletion results in the dispersion of late endocytic organelles to the cell periphery similarly as upon pharmacological actin disruption. The dispersed organelles in MLN64 knockdown cells exhibited decreased association with actin and the Arp2/3 complex subunit p34-Arc. MLN64 depletion was accompanied by impaired fusion of late endocytic organelles and delayed cargo degradation. MLN64 overexpression increased the number of actin and p34-Arc-positive patches on late endosomes, enhanced the fusion of late endocytic organelles in an actin-dependent manner, and stimulated the deposition of sterol in late endosomes harboring the protein. Overexpression of wild-type MLN64 was capable of rescuing the endosome dispersion in MLN64-depleted cells, whereas mutants of MLN64 defective in cholesterol binding were not, suggesting a functional connection between MLN64-mediated sterol transfer and actin-dependent late endosome dynamics. We propose that local sterol enrichment by MLN64 in the late endosomal membranes facilitates their association with actin, thereby governing actin-dependent fusion and degradative activity of late endocytic organelles. PMID:15930133

  16. Auxin transport inhibitors impair vesicle motility and actin cytoskeleton dynamics in diverse eukaryotes

    PubMed Central

    Dhonukshe, Pankaj; Grigoriev, Ilya; Fischer, Rainer; Tominaga, Motoki; Robinson, David G.; Hašek, Jiří; Paciorek, Tomasz; Petrášek, Jan; Seifertová, Daniela; Tejos, Ricardo; Meisel, Lee A.; Zažímalová, Eva; Gadella, Theodorus W. J.; Stierhof, York-Dieter; Ueda, Takashi; Oiwa, Kazuhiro; Akhmanova, Anna; Brock, Roland; Spang, Anne; Friml, Jiří

    2008-01-01

    Many aspects of plant development, including patterning and tropisms, are largely dependent on the asymmetric distribution of the plant signaling molecule auxin. Auxin transport inhibitors (ATIs), which interfere with directional auxin transport, have been essential tools in formulating this concept. However, despite the use of ATIs in plant research for many decades, the mechanism of ATI action has remained largely elusive. Using real-time live-cell microscopy, we show here that prominent ATIs such as 2,3,5-triiodobenzoic acid (TIBA) and 2-(1-pyrenoyl) benzoic acid (PBA) inhibit vesicle trafficking in plant, yeast, and mammalian cells. Effects on micropinocytosis, rab5-labeled endosomal motility at the periphery of HeLa cells and on fibroblast mobility indicate that ATIs influence actin cytoskeleton. Visualization of actin cytoskeleton dynamics in plants, yeast, and mammalian cells show that ATIs stabilize actin. Conversely, stabilizing actin by chemical or genetic means interferes with endocytosis, vesicle motility, auxin transport, and plant development, including auxin transport-dependent processes. Our results show that a class of ATIs act as actin stabilizers and advocate that actin-dependent trafficking of auxin transport components participates in the mechanism of auxin transport. These studies also provide an example of how the common eukaryotic process of actin-based vesicle motility can fulfill a plant-specific physiological role. PMID:18337510

  17. The interplay between neuronal activity and actin dynamics mimic the setting of an LTD synaptic tag

    PubMed Central

    Szabó, Eszter C.; Manguinhas, Rita; Fonseca, Rosalina

    2016-01-01

    Persistent forms of plasticity, such as long-term depression (LTD), are dependent on the interplay between activity-dependent synaptic tags and the capture of plasticity-related proteins. We propose that the synaptic tag represents a structural alteration that turns synapses permissive to change. We found that modulation of actin dynamics has different roles in the induction and maintenance of LTD. Inhibition of either actin depolymerisation or polymerization blocks LTD induction whereas only the inhibition of actin depolymerisation blocks LTD maintenance. Interestingly, we found that actin depolymerisation and CaMKII activation are involved in LTD synaptic-tagging and capture. Moreover, inhibition of actin polymerisation mimics the setting of a synaptic tag, in an activity-dependent manner, allowing the expression of LTD in non-stimulated synapses. Suspending synaptic activation also restricts the time window of synaptic capture, which can be restored by inhibiting actin polymerization. Our results support our hypothesis that modulation of the actin cytoskeleton provides an input-specific signal for synaptic protein capture. PMID:27650071

  18. Asymmetric Nano/Microtopography Biases Cytoskeletal Dynamics and Promotes Unidirectional Cell Guidance

    NASA Astrophysics Data System (ADS)

    Sun, Xiaoyu; Driscoll, Meghan; Guven, Can; Das, Satarupa; Parent, Carole; Fourkas, John; Losert, Wolfgang

    Many biological and physiological processes depend upon directed migration of cells, which is typically mediated by chemical or physical gradients or by signal relay. Here we show that cells can be guided in a single preferred direction based solely on local asymmetries in nano/microtopography on subcellular scales. These asymmetries can be repeated, and thereby provide directional guidance, over arbitrarily large areas. The direction and strength of the guidance is sensitive to the details of the nano/microtopography, suggesting that this phenomenon plays a context-dependent role in vivo. We demonstrate that asymmetric nano/microtopography guides the direction of internal actin polymerization waves (esotaxis), and that cells move in the same direction as these waves (microthigmotaxis). This phenomenon is observed both for the pseudopod-dominated migration of the amoeboid Dictyostelium discoideum and for the lamellipod-driven migration of human neutrophils. The conservation of this mechanism across cell types and the asymmetric shape of many natural scaffolds suggests that actin-wave-based guidance is important in biology and physiology.

  19. F-actin waves, actin cortex disassembly and focal exocytosis driven by actin-phosphoinositide positive feedback.

    PubMed

    Masters, Thomas A; Sheetz, Michael P; Gauthier, Nils C

    2016-04-01

    Actin polymerization is controlled by the phosphoinositide composition of the plasma membrane. However, the molecular mechanisms underlying the spatiotemporal regulation of actin network organization over extended length scales are still unclear. To observe phosphoinositide-dependent cytoskeletal dynamics we combined the model system of frustrated phagocytosis, total internal reflection microscopy and manipulation of the buffer tonicity. We found that macrophages interacting with IgG-coated glass substrates formed circular F-actin waves on their ventral surface enclosing a region of plasma membrane devoid of cortical actin. Plasma membrane free of actin cortex was strongly depleted of PI(4,5)P2 , but enriched in PI(3,4)P2 and displayed a fivefold increase in exocytosis. Wave formation could be promoted by application of a hypotonic shock. The actin waves were characteristic of a bistable wavefront at the boundary between the regions of membrane containing and lacking cortical actin. Phosphoinositide modifiers and RhoGTPase activities dramatically redistributed with respect to the wavefronts, which often exhibited spatial oscillations. Perturbation of either lipid or actin cytoskeleton-related pathways led to rapid loss of both the polarized lipid distribution and the wavefront. As waves travelled over the plasma membrane, wavefront actin was seen to rapidly polymerize and depolymerize at pre-existing clusters of FcγRIIA, coincident with rapid changes in lipid composition. Thus the potential of receptors to support rapid F-actin polymerization appears to depend acutely on the local concentrations of multiple lipid species. We propose that interdependence through positive feedback from the cytoskeleton to lipid modifiers leads to coordinated local cortex remodeling, focal exocytosis, and organizes extended actin networks. PMID:26915738

  20. F-actin waves, actin cortex disassembly and focal exocytosis driven by actin-phosphoinositide positive feedback.

    PubMed

    Masters, Thomas A; Sheetz, Michael P; Gauthier, Nils C

    2016-04-01

    Actin polymerization is controlled by the phosphoinositide composition of the plasma membrane. However, the molecular mechanisms underlying the spatiotemporal regulation of actin network organization over extended length scales are still unclear. To observe phosphoinositide-dependent cytoskeletal dynamics we combined the model system of frustrated phagocytosis, total internal reflection microscopy and manipulation of the buffer tonicity. We found that macrophages interacting with IgG-coated glass substrates formed circular F-actin waves on their ventral surface enclosing a region of plasma membrane devoid of cortical actin. Plasma membrane free of actin cortex was strongly depleted of PI(4,5)P2 , but enriched in PI(3,4)P2 and displayed a fivefold increase in exocytosis. Wave formation could be promoted by application of a hypotonic shock. The actin waves were characteristic of a bistable wavefront at the boundary between the regions of membrane containing and lacking cortical actin. Phosphoinositide modifiers and RhoGTPase activities dramatically redistributed with respect to the wavefronts, which often exhibited spatial oscillations. Perturbation of either lipid or actin cytoskeleton-related pathways led to rapid loss of both the polarized lipid distribution and the wavefront. As waves travelled over the plasma membrane, wavefront actin was seen to rapidly polymerize and depolymerize at pre-existing clusters of FcγRIIA, coincident with rapid changes in lipid composition. Thus the potential of receptors to support rapid F-actin polymerization appears to depend acutely on the local concentrations of multiple lipid species. We propose that interdependence through positive feedback from the cytoskeleton to lipid modifiers leads to coordinated local cortex remodeling, focal exocytosis, and organizes extended actin networks.

  1. Actin-associated Proteins in the Pathogenesis of Podocyte Injury

    PubMed Central

    He, Fang-Fang; Chen, Shan; Su, Hua; Meng, Xian-Fang; Zhang, Chun

    2013-01-01

    Podocytes have a complex cellular architecture with interdigitating processes maintained by a precise organization of actin filaments. The actin-based foot processes of podocytes and the interposed slit diaphragm form the final barrier to proteinuria. The function of podocytes is largely based on the maintenance of the normal foot process structure with actin cytoskeleton. Cytoskeletal dynamics play important roles during normal podocyte development, in maintenance of the healthy glomerular filtration barrier, and in the pathogenesis of glomerular diseases. In this review, we focused on recent findings on the mechanisms of organization and reorganization of these actin-related molecules in the pathogenesis of podocyte injury and potential therapeutics targeting the regulation of actin cytoskeleton in podocytopathies. PMID:24396279

  2. Live cell imaging of actin dynamics in dexamethasone-treated porcine trabecular meshwork cells.

    PubMed

    Fujimoto, Tomokazu; Inoue, Toshihiro; Inoue-Mochita, Miyuki; Tanihara, Hidenobu

    2016-04-01

    The regulation of the actin cytoskeleton in trabecular meshwork (TM) cells is important for controlling outflow of the aqueous humor. In some reports, dexamethasone (DEX) increased the aqueous humor outflow resistance and induced unusual actin structures, such as cross-linked actin networks (CLAN), in TM cells. However, the functions and dynamics of CLAN in TM cells are not completely known, partly because actin stress fibers have been observed only in fixed cells. We conducted live-cell imaging of the actin dynamics in TM cells with or without DEX treatment. An actin-green fluorescent protein (GFP) fusion construct with a modified insect virus was transfected into porcine TM cells. Time-lapse imaging of live TM cells treated with 25 μM Y-27632 and 100 nM DEX was performed using an inverted fluorescence microscope. Fluorescent images were recorded every 15 s for 30 min after Y-27632 treatment or every 30 min for 72 h after DEX treatment. The GFP-actin was expressed in 22.7 ± 10.9% of the transfected TM cells. In live TM cells, many actin stress fibers were observed before the Y-27632 treatment. Y-27632 changed the cell shape and decreased stress fibers in a time-dependent manner. In fixed cells, CLAN-like structures were seen in 26.5 ± 1.7% of the actin-GFP expressed PTM cells treated with DEX for 72 h. In live imaging, there was 28% CLAN-like structure formation at 72 h after DEX treatment, and the lifetime of CLAN-like structures increased after DEX treatment. The DEX-treated cells with CLAN-like structures showed less migration than DEX-treated cells without CLAN-like structures. Furthermore, the control cells (without DEX treatment) with CLAN-like structures also showed less migration than the control cells without CLAN-like structures. These results suggested that CLAN-like structure formation was correlated with cell migration in TM cells. Live cell imaging of the actin cytoskeleton provides valuable information on the actin dynamics in TM

  3. Channel-interacting PDZ protein, 'CIPP', interacts with proteins involved in cytoskeletal dynamics.

    PubMed

    Alpi, Emanuele; Landi, Elena; Barilari, Manuela; Serresi, Michela; Salvadori, Piero; Bachi, Angela; Dente, Luciana

    2009-04-15

    Neuronal CIPP (channel-interacting PDZ protein) is a multivalent PDZ protein that interacts with specific channels and receptors highly expressed in the brain. It is composed of four PDZ domains that behave as a scaffold to clusterize functionally connected proteins. In the present study, we selected a set of potential CIPP interactors that are involved directly or indirectly in mechanisms of cytoskeletal remodelling and membrane protrusion formation. For some of these, we first proved the direct binding to specific CIPP PDZ domains considered as autonomous elements, and then confirmed the interaction with the whole protein. In particular, the small G-protein effector IRSp53 (insulin receptor tyrosine kinase substrate protein p53) specifically interacts with the second PDZ domain of CIPP and, when co-transfected in cultured mammalian cells with a tagged full-length CIPP, it induces a marked reorganization of CIPP cytoplasmic localization. Large punctate structures are generated as a consequence of CIPP binding to the IRSp53 C-terminus. Analysis of the puncta nature, using various endocytic markers, revealed that they are not related to cytoplasmic vesicles, but rather represent multi-protein assemblies, where CIPP can tether other potential interactors.

  4. Depolymerization dynamics of individual filaments of bacterial cytoskeletal protein FtsZ

    PubMed Central

    Mateos-Gil, Pablo; Paez, Alfonso; Hörger, Ines; Rivas, Germán; Vicente, Miguel; Tarazona, Pedro; Vélez, Marisela

    2012-01-01

    We report observation and analysis of the depolymerization filaments of the bacterial cytoskeletal protein FtsZ (filament temperature-sensitive Z) formed on a mica surface. At low concentration, proteins adsorbed on the surface polymerize forming curved filaments that close into rings that remain stable for some time before opening irreversibly and fully depolymerizing. The distribution of ring lifetimes (T) as a function of length (N), shows that the rate of ring aperture correlates with filament length. If this ring lifetime is expressed as a bond survival time, (Tb ≡ NT), this correlation is abolished, indicating that these rupture events occur randomly and independently at each monomer interface. After rings open irreversibly, depolymerization of the remaining filaments is fast, but can be slowed down and followed using a nonhydrolyzing GTP analogue. The histogram of depolymerization velocities of individual filaments has an asymmetric distribution that can be fit with a computer model that assumes two rupture rates, a slow one similar to the one observed for ring aperture, affecting monomers in the central part of the filaments, and a faster one affecting monomers closer to the open ends. From the quantitative analysis, we conclude that the depolymerization rate is affected both by nucleotide hydrolysis rate and by its exchange along the filament, that all monomer interfaces are equally competent for hydrolysis, although depolymerization is faster at the open ends than in central filament regions, and that all monomer–monomer interactions, regardless of the nucleotide present, can adopt a curved configuration. PMID:22566654

  5. In vivo dynamics of the F-actin-binding protein neurabin-II.

    PubMed Central

    Stephens, D J; Banting, G

    2000-01-01

    Neurabin-II (spinophilin) is a ubiquitously expressed F-actin-binding protein containing an N-terminal actin-binding domain, a PDZ (PSD95/discs large/ZO-1) domain and a C-terminal domain predicted to form a coiled-coil structure. We have stably expressed a green fluorescent protein (GFP)-tagged version of neurabin-II in PC12 cells, and characterized the in vivo dynamics of this actin-binding protein using confocal fluorescence microscopy. We show that GFP-neurabin-II localizes to actin filaments, especially at cortical sites and areas underlying sites of active membrane remodelling. GFP-neurabin-II labels only a subset of F-actin within these cells, as indicated by rhodamine-phalloidin staining. Both actin filaments and small, highly motile structures within the cell body are seen. Photobleaching experiments show that GFP-neurabin-II also exhibits highly dynamic behaviour when bound to actin filaments. Latrunculin B treatment results in rapid relocalization of GFP-neurabin-II to the cytosol, whereas cytochalasin D treatment causes the collapse of GFP-neurabin-II fluorescence to intensely fluorescent foci of F-actin within the cell body. This collapse is reversed on cytochalasin D removal, recovery from which is greatly accelerated by stimulation of cells with epidermal growth factor (EGF). Furthermore, we show that this EGF-induced relocalization of GFP-neurabin-II is dependent on the activity of the small GTPase Rac1 but not the activity of ADP-ribosylation factor 6. PMID:10620493

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

    PubMed

    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

  7. Unconventional myosins, actin dynamics and endocytosis: a ménage à trois?

    PubMed

    Soldati, Thierry

    2003-06-01

    Ever since the discovery of class I myosins, the first nonmuscle myosins, about 30 years ago, the history of unconventional myosins has been linked to the organization and working of actin filaments. It slowly emerged from studies of class I myosins in lower eukaryotes that they are involved in mechanisms of endocytosis. Most interestingly, a flurry of recent findings assign a more active role to class I myosins in regulating the spatial and temporal organization of actin filament nucleation and elongation. The results highlight the multiple links between class I myosins and the major actin nucleator, the Arp2/3 complex, and its newly described activators. Two additional types of unconventional myosins, myosinIX, and Dictyostelium discoideum MyoM, have recently been tied to the signaling pathways controlling actin cytoskeleton remodeling. The present review surveys the links between these three classes of molecular motors and the complex cellular processes of endocytosis and actin dynamics, and concentrates on a working model accounting for the function of class I myosins via recruitment of the machinery responsible for actin nucleation and elongation. PMID:12753645

  8. Simiate is an Actin binding protein involved in filopodia dynamics and arborization of neurons

    PubMed Central

    Derlig, Kristin; Ehrhardt, Toni; Gießl, Andreas; Brandstätter, Johann H.; Enz, Ralf; Dahlhaus, Regina

    2014-01-01

    The Actin cytoskeleton constitutes the functional base for a multitude of cellular processes extending from motility and migration to cell mechanics and morphogenesis. The latter is particularly important to neuronal cells since the accurate functioning of the brain crucially depends on the correct arborization of neurons, a process that requires the formation of several dozens to hundreds of dendritic branches. Recently, a model was proposed where different transcription factors are detailed to distinct facets and phases of dendritogenesis and exert their function by acting on the Actin cytoskeleton, however, the proteins involved as well as the underlying molecular mechanisms are largely unknown. Here, we demonstrate that Simiate, a protein previously indicated to activate transcription, directly associates with both, G- and F-Actin and in doing so, affects Actin polymerization and Actin turnover in living cells. Imaging studies illustrate that Simiate particularly influences filopodia dynamics and specifically increases the branching of proximal, but not distal dendrites of developing neurons. The data suggests that Simiate functions as a direct molecular link between transcription regulation on one side, and dendritogenesis on the other, wherein Simiate serves to coordinate the development of proximal and distal dendrites by acting on the Actin cytoskeleton of filopodia and on transcription regulation, hence supporting the novel model. PMID:24782708

  9. Molecular dynamics simulation of a myosin subfragment-1 docking with an actin filament.

    PubMed

    Masuda, Tadashi

    2013-09-01

    Myosins are typical molecular motor proteins, which convert the chemical energy of ATP into mechanical work. The fundamental mechanism of this energy conversion is still unknown. To explain the experimental results observed in molecular motors, Masuda has proposed a theory called the "Driven by Detachment (DbD)" mechanism for the working principle of myosins. Based on this theory, the energy used during the power stroke of the myosins originates from the attractive force between a detached myosin head and an actin filament, and does not directly arise from the energy of ATP. According to this theory, every step in the myosin working process may be reproduced by molecular dynamics (MD) simulations, except for the ATP hydrolysis step. Therefore, MD simulations were conducted to reproduce the docking process of a myosin subfragment-1 (S1) against an actin filament. A myosin S1 directed toward the barbed end of an actin filament was placed at three different positions by shifting it away from the filament axis. After 30 ns of MD simulations, in three cases out of ten trials on average, the myosin made a close contact with two actin monomers by changing the positions and the orientation of both the myosin and the actin as predicted in previous studies. Once the docking was achieved, the distance between the myosin and the actin showed smaller fluctuations, indicating that the docking is stable over time. If the docking was not achieved, the myosin moved randomly around the initial position or moved away from the actin filament. MD simulations thus successfully reproduced the docking of a myosin S1 with an actin filament. By extending the similar MD simulations to the other steps of the myosin working process, the validity of the DbD theory may be computationally demonstrated.

  10. Regulation of actin dynamics by WNT-5A: implications for human airway smooth muscle contraction

    PubMed Central

    Koopmans, Tim; Kumawat, Kuldeep; Halayko, Andrew J; Gosens, Reinoud

    2016-01-01

    A defining feature of asthma is airway hyperresponsiveness (AHR), which underlies the exaggerated bronchoconstriction response of asthmatics. The role of the airway smooth muscle (ASM) in AHR has garnered increasing interest over the years, but how asthmatic ASM differs from healthy ASM is still an active topic of debate. WNT-5A is increasingly expressed in asthmatic ASM and has been linked with Th2-high asthma. Due to its link with calcium and cytoskeletal remodelling, we propose that WNT-5A may modulate ASM contractility. We demonstrated that WNT-5A can increase maximum isometric tension in bovine tracheal smooth muscle strips. In addition, we show that WNT-5A is preferentially expressed in contractile human airway myocytes compared to proliferative cells, suggesting an active role in maintaining contractility. Furthermore, WNT-5A treatment drives actin polymerisation, but has no effect on intracellular calcium flux. Next, we demonstrated that WNT-5A directly regulates TGF-β1-induced expression of α-SMA via ROCK-mediated actin polymerization. These findings suggest that WNT-5A modulates fundamental mechanisms that affect ASM contraction and thus may be of relevance for AHR in asthma. PMID:27468699

  11. The WAVE Regulatory Complex Links Diverse Receptors to the Actin Cytoskeleton

    PubMed Central

    Chen, Baoyu; Chen, Zhucheng; Brinkmann, Klaus; Pak, Chi W.; Liao, Yuxing; Shi, Shuoyong; Henry, Lisa; Grishin, Nick V.; Bogdan, Sven; Rosen, Michael K.

    2014-01-01

    SUMMARY The WAVE regulatory complex (WRC) controls actin cytoskeletal dynamics throughout the cell by stimulating the actin nucleating activity of the Arp2/3 complex at distinct membrane sites. However, the factors that recruit the WRC to specific locations remain poorly understood. Here we have identified a large family of potential WRC ligands, consisting of ~120 diverse membrane proteins including protocadherins, ROBOs, netrin receptors, Neuroligins, GPCRs and channels. Structural, biochemical and cellular studies reveal that a novel sequence motif that defines these ligands binds to a highly conserved interaction surface of the WRC formed by the Sra and Abi subunits. Mutating this binding surface in flies resulted in defects in actin cytoskeletal organization and egg morphology during oogenesis, leading to female sterility. Our findings directly link diverse membrane proteins to the WRC and actin cytoskeleton, and have broad physiological and pathological ramifications in metazoans. PMID:24439376

  12. Stable Force Balance between Epithelial Cells Arises from F-Actin Turnover.

    PubMed

    Jodoin, Jeanne N; Coravos, Jonathan S; Chanet, Soline; Vasquez, Claudia G; Tworoger, Michael; Kingston, Elena R; Perkins, Lizabeth A; Perrimon, Norbert; Martin, Adam C

    2015-12-21

    The propagation of force in epithelial tissues requires that the contractile cytoskeletal machinery be stably connected between cells through E-cadherin-containing adherens junctions. In many epithelial tissues, the cells' contractile network is positioned at a distance from the junction. However, the mechanism or mechanisms that connect the contractile networks to the adherens junctions, and thus mechanically connect neighboring cells, are poorly understood. Here, we identified the role for F-actin turnover in regulating the contractile cytoskeletal network's attachment to adherens junctions. Perturbing F-actin turnover via gene depletion or acute drug treatments that slow F-actin turnover destabilized the attachment between the contractile actomyosin network and adherens junctions. Our work identifies a critical role for F-actin turnover in connecting actomyosin to intercellular junctions, defining a dynamic process required for the stability of force balance across intercellular contacts in tissues.

  13. Dynamics of the actin cytoskeleton mediates receptor cross talk: An emerging concept in tuning receptor signaling

    PubMed Central

    Mattila, Pieta K.; Batista, Facundo D.

    2016-01-01

    Recent evidence implicates the actin cytoskeleton in the control of receptor signaling. This may be of particular importance in the context of immune receptors, such as the B cell receptor, where dysregulated signaling can result in autoimmunity and malignancy. Here, we discuss the role of the actin cytoskeleton in controlling receptor compartmentalization, dynamics, and clustering as a means to regulate receptor signaling through controlling the interactions with protein partners. We propose that the actin cytoskeleton is a point of integration for receptor cross talk through modulation of protein dynamics and clustering. We discuss the implication of this cross talk via the cytoskeleton for both ligand-induced and low-level constitutive (tonic) signaling necessary for immune cell survival. PMID:26833785

  14. Distinct Functional Interactions between Actin Isoforms and Nonsarcomeric Myosins

    PubMed Central

    Müller, Mirco; Diensthuber, Ralph P.; Chizhov, Igor; Claus, Peter; Heissler, Sarah M.; Preller, Matthias; Taft, Manuel H.; Manstein, Dietmar J.

    2013-01-01

    Despite their near sequence identity, actin isoforms cannot completely replace each other in vivo and show marked differences in their tissue-specific and subcellular localization. Little is known about isoform-specific differences in their interactions with myosin motors and other actin-binding proteins. Mammalian cytoplasmic β- and γ-actin interact with nonsarcomeric conventional myosins such as the members of the nonmuscle myosin-2 family and myosin-7A. These interactions support a wide range of cellular processes including cytokinesis, maintenance of cell polarity, cell adhesion, migration, and mechano-electrical transduction. To elucidate differences in the ability of isoactins to bind and stimulate the enzymatic activity of individual myosin isoforms, we characterized the interactions of human skeletal muscle α-actin, cytoplasmic β-actin, and cytoplasmic γ-actin with human myosin-7A and nonmuscle myosins-2A, -2B and -2C1. In the case of nonmuscle myosins-2A and -2B, the interaction with either cytoplasmic actin isoform results in 4-fold greater stimulation of myosin ATPase activity than was observed in the presence of α-skeletal muscle actin. Nonmuscle myosin-2C1 is most potently activated by β-actin and myosin-7A by γ-actin. Our results indicate that β- and γ-actin isoforms contribute to the modulation of nonmuscle myosin-2 and myosin-7A activity and thereby to the spatial and temporal regulation of cytoskeletal dynamics. FRET-based analyses show efficient copolymerization abilities for the actin isoforms in vitro. Experiments with hybrid actin filaments show that the extent of actomyosin coupling efficiency can be regulated by the isoform composition of actin filaments. PMID:23923011

  15. Remodeling of cytoskeletal architecture of nonneuronal cells induced by synapsin.

    PubMed Central

    Han, H Q; Greengard, P

    1994-01-01

    The synapsins, a family of neuron-specific phosphoproteins, have been implicated in the functional and structural maturation of synapses. The cell biological basis for these effects is unknown. In vitro, the synapsins interact with cytoskeletal elements including actin. To examine, in vivo, the possible effect of the synapsins on cytoskeletal organization and cell morphology, we have transfected each of the four known members of the synapsin family into nonneuronal cells. We report here that synapsin expression in fibroblast cells gives rise to an alteration in cell morphology that is associated with formation of highly elongated processes. This morphological change is accompanied by a reorganization of filamentous actin (F-actin) characterized by disruption of existing stress fibers and formation of bundles of actin cables in the elongated processes. These results suggest that interactions of the synapsins with actin, and possibly with other cytoskeletal elements, may play a role in the morphological differentiation of neurons. Images PMID:8078922

  16. Analysis of the local organization and dynamics of cellular actin networks

    PubMed Central

    Luo, Weiwei; Yu, Cheng-han; Lieu, Zi Zhao; Allard, Jun; Mogilner, Alex; Sheetz, Michael P.

    2013-01-01

    A ctin filaments, with the aid of multiple accessory proteins, self-assemble into a variety of network patterns. We studied the organization and dynamics of the actin network in nonadhesive regions of cells bridging fibronectin-coated adhesive strips. The network was formed by actin nodes associated with and linked by myosin II and containing the formin disheveled-associated activator of morphogenesis 1 (DAAM1) and the cross-linker filamin A (FlnA). After Latrunculin A (LatA) addition, actin nodes appeared to be more prominent and demonstrated drift-diffusion motion. Superresolution microscopy revealed that, in untreated cells, DAAM1 formed patches with a similar spatial arrangement to the actin nodes. Node movement (diffusion coefficient and velocity) in LatA-treated cells was dependent on the level and activity of myosin IIA, DAAM1, and FlnA. Based on our results, we developed a computational model of the dynamic formin-filamin-actin asters that can self-organize into a contractile actomyosin network. We suggest that such networks are critical for connecting distant parts of the cell to maintain the mechanical coherence of the cytoplasm. PMID:24081490

  17. Collapsin response mediator protein 4 regulates growth cone dynamics through the actin and microtubule cytoskeleton.

    PubMed

    Khazaei, Mohamad R; Girouard, Marie-Pier; Alchini, Ricardo; Ong Tone, Stephan; Shimada, Tadayuki; Bechstedt, Susanne; Cowan, Mitra; Guillet, Dominique; Wiseman, Paul W; Brouhard, Gary; Cloutier, Jean Francois; Fournier, Alyson E

    2014-10-24

    Coordinated control of the growth cone cytoskeleton underlies axon extension and guidance. Members of the collapsin response mediator protein (CRMP) family of cytosolic phosphoproteins regulate the microtubule and actin cytoskeleton, but their roles in regulating growth cone dynamics remain largely unexplored. Here, we examine how CRMP4 regulates the growth cone cytoskeleton. Hippocampal neurons from CRMP4-/- mice exhibited a selective decrease in axon extension and reduced growth cone area, whereas overexpression of CRMP4 enhanced the formation and length of growth cone filopodia. Biochemically, CRMP4 can impact both microtubule assembly and F-actin bundling in vitro. Through a structure function analysis of CRMP4, we found that the effects of CRMP4 on axon growth and growth cone morphology were dependent on microtubule assembly, whereas filopodial extension relied on actin bundling. Intriguingly, anterograde movement of EB3 comets, which track microtubule protrusion, slowed significantly in neurons derived from CRMP4-/- mice, and rescue of microtubule dynamics required CRMP4 activity toward both the actin and microtubule cytoskeleton. Together, this study identified a dual role for CRMP4 in regulating the actin and microtubule growth cone cytoskeleton. PMID:25225289

  18. In vitro analysis of rod composition and actin dynamics in inherited myopathies.

    PubMed

    Vandebrouck, Aurélie; Domazetovska, Ana; Mokbel, Nancy; Cooper, Sandra T; Ilkovski, Biljana; North, Kathryn N

    2010-05-01

    Rods are the pathological hallmark of nemaline myopathy, but they can also occur as a secondary phenomenon in other disorders, including mitochondrial myopathies such as complex I deficiency. The mechanisms of rod formation are not well understood, particularly when rods occur in diverse disorders with very different structural and metabolic defects. We compared the characteristics of rods associated with abnormalities in structural components of skeletal muscle thin filament (3 mutations in the skeletal actin gene ACTA1) with those of rods induced by the metabolic cell stress of adenosine triphosphate depletion. C2C12 and NIH/3T3 cell culture models and immunocytochemistry were used to study rod composition and conformation. Fluorescent recovery after photobleaching was used to measure actin dynamics inside the rods. We demonstrate that not all rods are the same. Rods formed under different conditions contain a unique fingerprint of actin-binding proteins (cofilin and alpha-actinin) and display differences in actin dynamics that are specific to the mutation, to the cellular location of the rods (intranuclear vs cytoplasmic), and/or to the underlying pathological process (i.e. mutant actin or adenosine triphosphate depletion). Thus, rods likely represent a common morphological end point of a variety of different pathological processes, either structural or metabolic. PMID:20418783

  19. Tuba stimulates intracellular N-WASP-dependent actin assembly.

    PubMed

    Kovacs, Eva M; Makar, Robert S; Gertler, Frank B

    2006-07-01

    Tuba is a multidomain scaffolding protein that links cytoskeletal dynamics and membrane trafficking pathways. The N-terminus of Tuba binds dynamin1, and the C-terminus contains domains that can interact with signaling pathways and cytoskeletal regulatory elements. We investigated Tuba localization, distribution and function in B16 melanoma cells. Tuba overexpression stimulated dorsal ruffles that occurred independently of dynamin function. Tuba expression induced actin-driven motility of small puncta that required the C-terminal SH3, GEF and BAR domains. Additionally, Tuba was recruited to lipid vesicles generated by overexpression of phosphatidylinositol-4-phosphate 5-kinase type Ialpha (PIP5Kalpha), localizing prominently to the head of the comets and at lower levels along the actin tail. We propose that Tuba facilitates dorsal ruffling of melanoma cells through direct interaction with actin-regulatory proteins and the recruitment of signaling molecules to lipid microdomains for the coordinated assembly of a cytoskeletal network. Knockdown of Tuba by RNA interference (RNAi) attenuated PIP5Kalpha-generated comet formation and the invasive behavior of B16 cells, implying that Tuba function is required for certain aspects of these processes. These results suggest first that Tuba-stimulated dorsal ruffling might represent a novel mechanism for the coordination of N-WASP-dependent cytoskeletal rearrangements and second that Tuba function is implicated in motility processes. PMID:16757518

  20. A small molecule inhibitor of tropomyosin dissociates actin binding from tropomyosin-directed regulation of actin dynamics

    PubMed Central

    Bonello, Teresa T.; Janco, Miro; Hook, Jeff; Byun, Alex; Appaduray, Mark; Dedova, Irina; Hitchcock-DeGregori, Sarah; Hardeman, Edna C.; Stehn, Justine R.; Böcking, Till; Gunning, Peter W.

    2016-01-01

    The tropomyosin family of proteins form end-to-end polymers along the actin filament. Tumour cells rely on specific tropomyosin-containing actin filament populations for growth and survival. To dissect out the role of tropomyosin in actin filament regulation we use the small molecule TR100 directed against the C terminus of the tropomyosin isoform Tpm3.1. TR100 nullifies the effect of Tpm3.1 on actin depolymerisation but surprisingly Tpm3.1 retains the capacity to bind F-actin in a cooperative manner. In vivo analysis also confirms that, in the presence of TR100, fluorescently tagged Tpm3.1 recovers normally into stress fibers. Assembling end-to-end along the actin filament is thereby not sufficient for tropomyosin to fulfil its function. Rather, regulation of F-actin stability by tropomyosin requires fidelity of information communicated at the barbed end of the actin filament. This distinction has significant implications for perturbing tropomyosin-dependent actin filament function in the context of anti-cancer drug development. PMID:26804624

  1. Nodal signaling regulates endodermal cell motility and actin dynamics via Rac1 and Prex1

    PubMed Central

    Housley, Michael P.; Weiner, Orion D.

    2012-01-01

    Embryo morphogenesis is driven by dynamic cell behaviors, including migration, that are coordinated with fate specification and differentiation, but how such coordination is achieved remains poorly understood. During zebrafish gastrulation, endodermal cells sequentially exhibit first random, nonpersistent migration followed by oriented, persistent migration and finally collective migration. Using a novel transgenic line that labels the endodermal actin cytoskeleton, we found that these stage-dependent changes in migratory behavior correlated with changes in actin dynamics. The dynamic actin and random motility exhibited during early gastrulation were dependent on both Nodal and Rac1 signaling. We further identified the Rac-specific guanine nucleotide exchange factor Prex1 as a Nodal target and showed that it mediated Nodal-dependent random motility. Reducing Rac1 activity in endodermal cells caused them to bypass the random migration phase and aberrantly contribute to mesodermal tissues. Together, our results reveal a novel role for Nodal signaling in regulating actin dynamics and migration behavior, which are crucial for endodermal morphogenesis and cell fate decisions. PMID:22945937

  2. SelR/MsrB Reverses Mical-mediated Oxidation of Actin to Regulate F-actin Dynamics

    PubMed Central

    Hung, Ruei-Jiun; Spaeth, Christopher S.; Yesilyurt, Hunkar Gizem; Terman, Jonathan R.

    2014-01-01

    Actin's polymerization properties are dramatically altered by oxidation of its conserved methionine (Met)-44 residue. Mediating this effect is a specific oxidation-reduction (Redox) enzyme, Mical, that works with Semaphorin repulsive guidance cues and selectively oxidizes Met-44. We now find that this actin regulatory process is reversible. Employing a genetic approach, we identified a specific methionine sulfoxide reductase enzyme SelR that opposes Mical Redox activity and Semaphorin/Plexin repulsion to direct multiple actin-dependent cellular behaviors in vivo. SelR specifically catalyzes the reduction of the R-isomer of methionine sulfoxide (methionine-R-sulfoxide) to methionine, and we found that SelR directly reduced Mical-oxidized actin, restoring its normal polymerization properties. These results indicate that Mical oxidizes actin stereo-specifically to generate actin Met-44-R-sulfoxide (actinMet(R)O-44) – and they also implicate the interconversion of specific Met/Met(R)O residues as a precise means to modulate protein function. Our results therefore uncover a specific reversible Redox actin regulatory system that controls cell and developmental biology. PMID:24212093

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

    PubMed

    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

  4. Force-velocity relation for actin-polymerization-driven motility from Brownian dynamics simulations.

    PubMed

    Lee, Kun-Chun; Liu, Andrea J

    2009-09-01

    We report numerical simulation results for the force-velocity relation for actin-polymerization-driven motility. We use Brownian dynamics to solve a physically consistent formulation of the dendritic nucleation model with semiflexible filaments that self-assemble and push a disk. We find that at small loads, the disk speed is independent of load, whereas at high loads, the speed decreases and vanishes at a characteristic stall pressure. Our results demonstrate that at small loads, the velocity is controlled by the reaction rates, whereas at high loads the stall pressure is determined by the mechanical properties of the branched actin network. The behavior is consistent with experiments and with our recently proposed self-diffusiophoretic mechanism for actin-polymerization-driven motility. New in vitro experiments to measure the force-velocity relation are proposed.

  5. CASEIN KINASE1-LIKE PROTEIN2 Regulates Actin Filament Stability and Stomatal Closure via Phosphorylation of Actin Depolymerizing Factor.

    PubMed

    Zhao, Shuangshuang; Jiang, Yuxiang; Zhao, Yang; Huang, Shanjin; Yuan, Ming; Zhao, Yanxiu; Guo, Yan

    2016-06-01

    The opening and closing of stomata are crucial for plant photosynthesis and transpiration. Actin filaments undergo dynamic reorganization during stomatal closure, but the underlying mechanism for this cytoskeletal reorganization remains largely unclear. In this study, we identified and characterized Arabidopsis thaliana casein kinase 1-like protein 2 (CKL2), which responds to abscisic acid (ABA) treatment and participates in ABA- and drought-induced stomatal closure. Although CKL2 does not bind to actin filaments directly and has no effect on actin assembly in vitro, it colocalizes with and stabilizes actin filaments in guard cells. Further investigation revealed that CKL2 physically interacts with and phosphorylates actin depolymerizing factor 4 (ADF4) and inhibits its activity in actin filament disassembly. During ABA-induced stomatal closure, deletion of CKL2 in Arabidopsis alters actin reorganization in stomata and renders stomatal closure less sensitive to ABA, whereas deletion of ADF4 impairs the disassembly of actin filaments and causes stomatal closure to be more sensitive to ABA Deletion of ADF4 in the ckl2 mutant partially recues its ABA-insensitive stomatal closure phenotype. Moreover, Arabidopsis ADFs from subclass I are targets of CKL2 in vitro. Thus, our results suggest that CKL2 regulates actin filament reorganization and stomatal closure mainly through phosphorylation of ADF. PMID:27268429

  6. Live-cell imaging of actin dynamics reveals mechanisms of stereocilia length regulation in the inner ear

    PubMed Central

    Drummond, Meghan C.; Barzik, Melanie; Bird, Jonathan E.; Zhang, Duan-Sun; Lechene, Claude P.; Corey, David P.; Cunningham, Lisa L.; Friedman, Thomas B.

    2015-01-01

    The maintenance of sensory hair cell stereocilia is critical for lifelong hearing; however, mechanisms of structural homeostasis remain poorly understood. Conflicting models propose that stereocilia F-actin cores are either continually renewed every 24–48 h via a treadmill or are stable, exceptionally long-lived structures. Here to distinguish between these models, we perform an unbiased survey of stereocilia actin dynamics in more than 500 utricle hair cells. Live-imaging EGFP-β-actin or dendra2-β-actin reveal stable F-actin cores with turnover and elongation restricted to stereocilia tips. Fixed-cell microscopy of wild-type and mutant β-actin demonstrates that incorporation of actin monomers into filaments is required for localization to stereocilia tips. Multi-isotope imaging mass spectrometry and live imaging of single differentiating hair cells capture stereociliogenesis and explain uniform incorporation of 15N-labelled protein and EGFP-β-actin into nascent stereocilia. Collectively, our analyses support a model in which stereocilia actin cores are stable structures that incorporate new F-actin only at the distal tips. PMID:25898120

  7. Continuum modeling of forces in growing viscoelastic cytoskeletal networks.

    PubMed

    Kim, Jin Seob; Sun, Sean X

    2009-02-21

    Mechanical properties of the living cell are important in cell movement, cell division, cancer development and cell signaling. There is considerable interest in measuring local mechanical properties of living materials and the living cytoskeleton using micromechanical techniques. However, living materials are constantly undergoing internal dynamics such as growth and remodeling. A modeling framework that combines mechanical deformations with cytoskeletal growth dynamics is necessary to describe cellular shape changes. The present paper develops a general finite deformation modeling approach that can treat the viscoelastic cytoskeleton. Given the growth dynamics in the cytoskeletal network and the relationship between deformation and stress, the shape of the network is computed in an incremental fashion. The growth dynamics of the cytoskeleton can be modeled as stress dependent. The result is a consistent treatment of overall cell deformation. The framework is applied to a growing 1-d bundle of actin filaments against an elastic cantilever, and a 2-d cell undergoing wave-like protrusion dynamics. In the latter example, mechanical forces on the cell adhesion are examined as a function of the protrusion dynamics. PMID:19041329

  8. The role of Arp2/3 in growth cone actin dynamics and guidance is substrate dependent.

    PubMed

    San Miguel-Ruiz, José E; Letourneau, Paul C

    2014-04-23

    During development extrinsic guidance cues modulate the peripheral actin network in growth cones to direct axons to their targets. We wanted to understand the role of the actin nucleator Arp2/3 in growth cone actin dynamics and guidance. Since growth cones migrate in association with diverse adhesive substrates during development, we probed the hypothesis that the functional significance of Arp2/3 is substrate dependent. We report that Arp2/3 inhibition led to a reduction in the number of filopodia and growth cone F-actin content on laminin and L1. However, we found substrate-dependent differences in growth cone motility, actin retrograde flow, and guidance after Arp2/3 inhibition, suggesting that its role, and perhaps that of other actin binding proteins, in growth cone motility is substrate dependent. PMID:24760849

  9. Signaling and Dynamic Actin Responses of B Cells on Topographical Substrates

    NASA Astrophysics Data System (ADS)

    Ketchum, Christina; Sun, Xiaoyu; Fourkas, John; Song, Wenxia; Upadhyaya, Arpita

    B cells become activated upon physical contact with antigen on the surface of antigen presenting cells, such as dendritic cells. Binding of the B cell receptor with antigen initiates actin-mediated spreading of B cells, signaling cascades and eventually infection fighting antibodies. Lymphocytes, including B cells and T cells, have been shown to be responsive to the physical parameters of the contact surface, such as antigen mobility and substrate stiffness. However the roll of surface topography on lymphocyte function is unknown. Here we investigate the degree to which substrate topography controls actin-mediated spreading and B cell activation using nano-fabricated surfaces and live cell imaging. The model topographical system consists of 600 nanometer tall ridges with spacing varying between 800 nanometers and 5 micrometers. Using TIRF imaging we observe actin dynamics, B cell receptor motion and calcium signaling of B cells as they spread on the ridged substrates. We show that the spacing between ridges had a strong effect on the dynamics of actin and calcium influx on B cells. Our results indicate that B cells are highly sensitive to surface topography during cell spreading and signaling activation.

  10. Dynamic F-actin movement is essential for fertilization in Arabidopsis thaliana

    PubMed Central

    Kawashima, Tomokazu; Maruyama, Daisuke; Shagirov, Murat; Li, Jing; Hamamura, Yuki; Yelagandula, Ramesh; Toyama, Yusuke; Berger, Frédéric

    2014-01-01

    In animals, microtubules and centrosomes direct the migration of gamete pronuclei for fertilization. By contrast, flowering plants have lost essential components of the centrosome, raising the question of how flowering plants control gamete nuclei migration during fertilization. Here, we use Arabidopsis thaliana to document a novel mechanism that regulates F-actin dynamics in the female gametes and is essential for fertilization. Live imaging shows that F-actin structures assist the male nucleus during its migration towards the female nucleus. We identify a female gamete-specific Rho-GTPase that regulates F-actin dynamics and further show that actin–myosin interactions are also involved in male gamete nucleus migration. Genetic analyses and imaging indicate that microtubules are dispensable for migration and fusion of male and female gamete nuclei. The innovation of a novel actin-based mechanism of fertilization during plant evolution might account for the complete loss of the centrosome in flowering plants. DOI: http://dx.doi.org/10.7554/eLife.04501.001 PMID:25303363

  11. Refilins are short-lived Actin-bundling proteins that regulate lamellipodium protrusion dynamics

    PubMed Central

    Gay, Olivia; Gilquin, Benoît; Assard, Nicole; Stuelsatz, Pascal; Delphin, Christian; Lachuer, Joël; Gidrol, Xavier; Baudier, Jacques

    2016-01-01

    ABSTRACT Refilins (RefilinA and RefilinB) are members of a novel family of Filamin binding proteins that function as molecular switches to conformationally alter the Actin filament network into bundles. We show here that Refilins are extremely labile proteins. An N-terminal PEST/DSG(X)2-4S motif mediates ubiquitin-independent rapid degradation. A second degradation signal is localized within the C-terminus. Only RefilinB is protected from rapid degradation by an auto-inhibitory domain that masks the PEST/DSG(X)2-4S motif. Dual regulation of RefilinA and RefilinB stability was confirmed in rat brain NG2 precursor cells (polydendrocyte). Using loss- and gain-of-function approaches we show that in these cells, and in U373MG cells, Refilins contribute to the dynamics of lamellipodium protrusion by catalysing Actin bundle formation within the lamella Actin network. These studies extend the Actin bundling function of the Refilin-Filamin complex to dynamic regulation of cell membrane remodelling. PMID:27744291

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

    PubMed

    Jiu, Yaming; Lehtimäki, Jaakko; Tojkander, Sari; Cheng, Fang; Jäälinoja, Harri; Liu, Xiaonan; Varjosalo, Markku; Eriksson, John E; Lappalainen, Pekka

    2015-06-16

    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.

  13. Mathematical modeling of the dynamic mechanical behavior of neighboring sarcomeres in actin stress fibers

    PubMed Central

    Chapin, L.M.; Edgar, L.T.; Blankman, E.; Beckerle, M.C.; Shiu, Y T

    2014-01-01

    Actin stress fibers (SFs) in live cells consist of series of dynamic individual sarcomeric units. Within a group of consecutive SF sarcomeres, individual sarcomeres can spontaneously shorten or lengthen without changing the overall length of this group, but the underlying mechanism is unclear. We used a computational model to test our hypothesis that this dynamic behavior is inherent to the heterogeneous mechanical properties of the sarcomeres and the cytoplasmic viscosity. Each sarcomere was modeled as a discrete element consisting of an elastic spring, a viscous dashpot and an active contractile unit all connected in parallel, and experiences forces as a result of actin filament elastic stiffness, myosin II contractility, internal viscoelasticity, or cytoplasmic drag. When all four types of forces are considered, the simulated dynamic behavior closely resembles the experimental observations, which include a low-frequency fluctuation in individual sarcomere length and compensatory lengthening and shortening of adjacent sarcomeres. Our results suggest that heterogeneous stiffness and viscoelasticity of actin fibers, heterogeneous myosin II contractility, and the cytoplasmic drag are sufficient to cause spontaneous fluctuations in SF sarcomere length. Our results shed new light to the dynamic behavior of SF and help design experiments to further our understanding of SF dynamics. PMID:25110525

  14. Morphology, cytoskeletal organization, and myosin dynamics of mouse embryonic fibroblasts cultured on nanofibrillar surfaces.

    PubMed

    Ahmed, Ijaz; Ponery, Abdul S; Nur-E-Kamal, Alam; Kamal, Jabeen; Meshel, Adam S; Sheetz, Michael P; Schindler, Melvin; Meiners, Sally

    2007-07-01

    Growth of cells in tissue culture is generally performed on two-dimensional (2D) surfaces composed of polystyrene or glass. Recent work, however, has shown that such 2D cultures are incomplete and do not adequately represent the physical characteristics of native extracellular matrix (ECM)/basement membrane (BM), namely dimensionality, compliance, fibrillarity, and porosity. In the current study, a three-dimensional (3D) nanofibrillar surface composed of electrospun polyamide nanofibers was utilized to mimic the topology and physical structure of ECM/BM. Additional chemical cues were incorporated into the nanofibrillar matrix by coating the surfaces with fibronectin, collagen I, or laminin-1. Results from the current study show an enhanced response of primary mouse embryonic fibroblasts (MEFs) to culture on nanofibrillar surfaces with more dramatic changes in cell spreading and reorganization of the cytoskeleton than previously observed for established cell lines. In addition, the cells cultured on nanofibrillar and 2D surfaces exhibited differential responses to the specific ECM/BM coatings. The localization and activity of myosin II-B for MEFs cultured on nanofibers was also compared. A dynamic redistribution of myosin II-B was observed within membrane protrusions. This was previously described for cells associated with nanofibers composed of collagen I but not for cells attached to 2D surfaces coated with monomeric collagen. These results provide further evidence that nanofibrillar surfaces offer a significantly different environment for cells than 2D substrates. PMID:17294137

  15. Cell Mechanics From cytoskeletal dynamics to tissue-scale mechanical phenomena

    NASA Astrophysics Data System (ADS)

    Banerjee, Shiladitya

    This dissertation explores the mechanics of living cells, integrating the role of intracellular activity to capture the emergent mechanical behavior of cells. The topics covered in this dissertation fall into three broad categories : (a) intracellular mechanics, (b) interaction of cells with the extracellular matrix and (c) collective mechanics of multicellular colonies. In part (a) I propose theoretical models for motor-filament interactions in the cell cytoskeleton, which is the site for mechanical force generation in cells. The models predict in a unified manner how contractility, dynamic instabilities and mechanical waves arise in the cytoskeleton by tuning the activity of molecular motors. The results presented in (a) holds relevance to a variety of cellular systems that behave elastically at long time scales, such as muscle sarcomeres, actomyosin stress fibers, adherent cells. In part (b) I introduce a continuum mechanical model for cells adherent to two-dimensional extracellular matrix, and discuss how cells can sense mechanical and geometrical cues from its surrounding matrix. The model provides an important step towards a unified theoretical description of the dependence of traction forces on cell size, actomyosin activity, matrix depth and stiffness, strength of focal adhesions and makes experimentally testable predictions. In part (c) we combine experiment and theory to reveal how intercellular adhesions modulate forces transmitted to the extracellular matrix. We find that In the absence of cadherin-based adhesions, cells within a colony appear to act independently, whereas with strong cadherin-based adhesions, the cell colony behaves like a liquid droplet wetting the substrate underneath. This work defines the importance of intercellular adhesions in coordinating mechanical activity of cell monolayers and has implications for the mechanical regulation of tissues during development, homeostasis, and disease.

  16. Computational spatiotemporal analysis identifies WAVE2 and Cofilin as joint regulators of costimulation-mediated T cell actin dynamics

    PubMed Central

    Roybal, Kole T.; Buck, Taráz E.; Ruan, Xiongtao; Cho, Baek Hwan; Clark, Danielle J.; Ambler, Rachel; Tunbridge, Helen M.; Zhang, Jianwei; Verkade, Paul; Wülfing, Christoph; Murphy, Robert F.

    2016-01-01

    Fluorescence microscopy is one of the most important tools in cell biology research and it provides spatial and temporal information to investigate regulatory systems inside cells. This technique can generate data in the form of signal intensities at thousands of positions resolved inside individual live cells; however, given extensive cell-to-cell variation, methods do not currently exist to assemble these data into three- or four-dimensional maps of protein concentration that can be compared across different cells and conditions. Here, we have developed one such method and applied it to investigate actin dynamics in T cell activation. Antigen recognition in T cells by the T cell receptor (TCR) is amplified by engagement of the costimulatory receptor CD28 and we have determined how CD28 modulates actin dynamics. We imaged actin and eight core actin regulators under conditions where CD28 in the context of a strong TCR signal was engaged or blocked to yield over a thousand movies. Our computational analysis identified diminished recruitment of the activator of actin nucleation WAVE2 and the actin severing protein cofilin to F-actin as the dominant difference upon costimulation blockade. Reconstitution of WAVE2 and cofilin activity restored the defect in actin signaling dynamics upon costimulation blockade. Thus we have developed and validated an approach to quantify protein distributions in time and space for analysis of complex regulatory systems. PMID:27095595

  17. Actin dynamics at the living cell submembrane imaged by total internal reflection fluorescence photobleaching.

    PubMed Central

    Sund, S E; Axelrod, D

    2000-01-01

    Although reversible chemistry is crucial to dynamical processes in living cells, relatively little is known about relevant chemical kinetic rates in vivo. Total internal reflection/fluorescence recovery after photobleaching (TIR/FRAP), an established technique previously demonstrated to measure reversible biomolecular kinetic rates at surfaces in vitro, is extended here to measure reversible biomolecular kinetic rates of actin at the cytofacial (subplasma membrane) surface of living cells. For the first time, spatial imaging (with a charge-coupled device camera) is used in conjunction with TIR/FRAP. TIR/FRAP imaging produces both spatial maps of kinetic parameters (off-rates and mobile fractions) and estimates of kinetic correlation distances, cell-wide kinetic gradients, and dependences of kinetic parameters on initial fluorescence intensity. For microinjected rhodamine actin in living cultured smooth muscle (BC3H1) cells, the unbinding rate at or near the cytofacial surface of the plasma membrane (averaged over the entire cell) is measured at 0.032 +/- 0.007 s(-1). The corresponding rate for actin marked by microinjected rhodamine phalloidin is very similar, 0.033 +/- 0.013 s(-1), suggesting that TIR/FRAP is reporting the dynamics of entire filaments or protofilaments. For submembrane fluorescence-marked actin, the intensity, off-rate, and mobile fraction show a positive correlation over a characteristic distance of 1-3 microm and a negative correlation over larger distances greater than approximately 7-14 microm. Furthermore, the kinetic parameters display a statistically significant cell-wide gradient, with the cell having a "fast" and "slow" end with respect to actin kinetics. PMID:10969025

  18. Cytoskeletal regulation of calcium-permeable cation channels in the human syncytiotrophoblast: role of gelsolin

    PubMed Central

    Montalbetti, Nicolás; Li, Qiang; Timpanaro, Gustavo A; González-Perrett, Silvia; Dai, Xiao-Qing; Chen, Xing-Zhen; Cantiello, Horacio F

    2005-01-01

    The human syncytiotrophoblast (hST) is the most apical epithelial barrier that covers the villous tree of the human placenta. An intricate and highly organized network of cytoskeletal structures supports the hST. Recently, polycystin-2 (PC2), a TRP-type nonselective cation channel, was functionally observed in hST, where it may be an important player to Ca2+ transport. Little is known, however, about channel regulation in hST. In this report, the regulatory role of actin dynamics on PC2 channels reconstituted from hST apical membranes was explored. Acute addition of cytochalasin D (CD, 5 μg ml−1) to reconstituted hST apical membranes transiently increased K+-permeable channel activity. The actin-binding proteins α-actinin and gelsolin, as well as PC2, were observed by Western blot and immunofluorescence analyses in hST vesicles. CD treatment of hST vesicles resulted in a re-distribution of actin filaments, in agreement with the effect of CD on K+ channel activity. In contrast, addition of exogenous monomeric actin, but not prepolymerized actin, induced a rapid inhibition of channel function in hST. This inhibition was obliterated by the presence of CD in the medium. The acute (<15 min) CD stimulation of K+ channel activity was mimicked by addition of the actin-severing protein gelsolin in the presence, but not in the absence, of micromolar Ca2+. Ca2+ transport through PC2 triggers a regulatory feedback mechanism, which is based on the severing and re-formation of filamentous actin near the channels. Cytoskeletal structures may thus be relevant to ion transport regulation in the human placenta. PMID:15845576

  19. Dynamics of actin waves on patterned substrates: a quantitative analysis of circular dorsal ruffles.

    PubMed

    Bernitt, Erik; Koh, Cheng Gee; Gov, Nir; Döbereiner, Hans-Günther

    2015-01-01

    Circular Dorsal Ruffles (CDRs) have been known for decades, but the mechanism that organizes these actin waves remains unclear. In this article we systematically analyze the dynamics of CDRs on fibroblasts with respect to characteristics of current models of actin waves. We studied CDRs on heterogeneously shaped cells and on cells that we forced into disk-like morphology. We show that CDRs exhibit phenomena such as periodic cycles of formation, spiral patterns, and mutual wave annihilations that are in accord with an active medium description of CDRs. On cells of controlled morphologies, CDRs exhibit extremely regular patterns of repeated wave formation and propagation, whereas on random-shaped cells the dynamics seem to be dominated by the limited availability of a reactive species. We show that theoretical models of reaction-diffusion type incorporating conserved species capture partially the behavior we observe in our data. PMID:25574668

  20. Caldesmon regulates actin dynamics to influence cranial neural crest migration in Xenopus.

    PubMed

    Nie, Shuyi; Kee, Yun; Bronner-Fraser, Marianne

    2011-09-01

    Caldesmon (CaD) is an important actin modulator that associates with actin filaments to regulate cell morphology and motility. Although extensively studied in cultured cells, there is little functional information regarding the role of CaD in migrating cells in vivo. Here we show that nonmuscle CaD is highly expressed in both premigratory and migrating cranial neural crest cells of Xenopus embryos. Depletion of CaD with antisense morpholino oligonucleotides causes cranial neural crest cells to migrate a significantly shorter distance, prevents their segregation into distinct migratory streams, and later results in severe defects in cartilage formation. Demonstrating specificity, these effects are rescued by adding back exogenous CaD. Interestingly, CaD proteins with mutations in the Ca(2+)-calmodulin-binding sites or ErK/Cdk1 phosphorylation sites fail to rescue the knockdown phenotypes, whereas mutation of the PAK phosphorylation site is able to rescue them. Analysis of neural crest explants reveals that CaD is required for the dynamic arrangements of actin and, thus, for cell shape changes and process formation. Taken together, these results suggest that the actin-modulating activity of CaD may underlie its critical function and is regulated by distinct signaling pathways during normal neural crest migration. PMID:21795398

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

    PubMed

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

    2015-02-13

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

  2. The PAR complex controls the spatiotemporal dynamics of F-actin and the MTOC in directionally migrating leukocytes

    PubMed Central

    Crespo, Carolina Lage; Vernieri, Claudio; Keller, Philipp J.; Garrè, Massimiliano; Bender, Jeffrey R.; Wittbrodt, Joachim; Pardi, Ruggero

    2014-01-01

    ABSTRACT Inflammatory cells acquire a polarized phenotype to migrate towards sites of infection or injury. A conserved polarity complex comprising PAR-3, PAR-6 and atypical protein kinase C (aPKC) relays extracellular polarizing cues to control cytoskeletal and signaling networks affecting morphological and functional polarization. However, there is no evidence that myeloid cells use PAR signaling to migrate vectorially in three-dimensional (3D) environments in vivo. Using genetically encoded bioprobes and high-resolution live imaging, we reveal the existence of F-actin oscillations in the trailing edge and constant repositioning of the microtubule organizing center (MTOC) to direct leukocyte migration in wounded medaka fish larvae (Oryzias latipes). Genetic manipulation in live myeloid cells demonstrates that the catalytic activity of aPKC and the regulated interaction with PAR-3 and PAR-6 are required for consistent F-actin oscillations, MTOC perinuclear mobility, aPKC repositioning and wound-directed migration upstream of Rho kinase (also known as ROCK or ROK) activation. We propose that the PAR complex coordinately controls cytoskeletal changes affecting both the generation of traction force and the directionality of leukocyte migration to sites of injury. PMID:25179599

  3. STED Nanoscopy of Actin Dynamics in Synapses Deep Inside Living Brain Slices

    PubMed Central

    Urban, Nicolai T.; Willig, Katrin I.; Hell, Stefan W.; Nägerl, U. Valentin

    2011-01-01

    It is difficult to investigate the mechanisms that mediate long-term changes in synapse function because synapses are small and deeply embedded inside brain tissue. Although recent fluorescence nanoscopy techniques afford improved resolution, they have so far been restricted to dissociated cells or tissue surfaces. However, to study synapses under realistic conditions, one must image several cell layers deep inside more-intact, three-dimensional preparations that exhibit strong light scattering, such as brain slices or brains in vivo. Using aberration-reducing optics, we demonstrate that it is possible to achieve stimulated emission depletion superresolution imaging deep inside scattering biological tissue. To illustrate the power of this novel (to our knowledge) approach, we resolved distinct distributions of actin inside dendrites and spines with a resolution of 60–80 nm in living organotypic brain slices at depths up to 120 μm. In addition, time-lapse stimulated emission depletion imaging revealed changes in actin-based structures inside spines and spine necks, and showed that these dynamics can be modulated by neuronal activity. Our approach greatly facilitates investigations of actin dynamics at the nanoscale within functionally intact brain tissue. PMID:21889466

  4. Super-Resolution Dynamic Imaging of Dendritic Spines Using a Low-Affinity Photoconvertible Actin Probe

    PubMed Central

    Lelek, Mickaël; Darzacq, Xavier; Triller, Antoine; Zimmer, Christophe; Dahan, Maxime

    2011-01-01

    The actin cytoskeleton of dendritic spines plays a key role in morphological aspects of synaptic plasticity. The detailed analysis of the spine structure and dynamics in live neurons, however, has been hampered by the diffraction-limited resolution of conventional fluorescence microscopy. The advent of nanoscopic imaging techniques thus holds great promise for the study of these processes. We implemented a strategy for the visualization of morphological changes of dendritic spines over tens of minutes at a lateral resolution of 25 to 65 nm. We have generated a low-affinity photoconvertible probe, capable of reversibly binding to actin and thus allowing long-term photoactivated localization microscopy of the spine cytoskeleton. Using this approach, we resolve structural parameters of spines and record their long-term dynamics at a temporal resolution below one minute. Furthermore, we have determined changes in the spine morphology in response to pharmacologically induced synaptic activity and quantified the actin redistribution underlying these changes. By combining PALM imaging with quantum dot tracking, we could also simultaneously visualize the cytoskeleton and the spine membrane, allowing us to record complementary information on the morphological changes of the spines at super-resolution. PMID:21264214

  5. Structural dynamics of actin during active interaction with myosin: different effects of weakly and strongly bound myosin heads.

    PubMed

    Prochniewicz, Ewa; Walseth, Timothy F; Thomas, David D

    2004-08-24

    We have used optical spectroscopy (transient phosphorescence anisotropy, TPA, and fluorescence resonance energy transfer, FRET) to detect the effects of weakly bound myosin S1 on actin during the actomyosin ATPase cycle. The changes in actin were reported by (a) a phosphorescent probe (ErIA) attached to Cys 374 and (b) a FRET donor-acceptor pair, IAEDANS attached to Cys 374 and a nucleotide analogue (TNPADP) in the nucleotide-binding cleft. Strong interactions were detected in the absence of ATP, and weak interactions were detected in the presence of ATP or its slowly hydrolyzed analogue ATP-gamma-S, under conditions where a significant fraction of weakly bound acto-S1 complex was present and the rate of nucleotide hydrolysis was low enough to enable steady-state measurements. The results show that actin in the weakly bound complex with S1 assumes a new structural state in which (a) the actin filament has microsecond rotational dynamics intermediate between that of free actin and the strongly bound complex and (b) S1-induced changes are not propagated along the actin filament, in contrast to the highly cooperative changes due to the strongly bound complex. We propose that the transition on the acto-myosin interface from weak to strong binding is accompanied by transitions in the structural dynamics of actin parallel to transitions in the dynamics of interacting myosin heads.

  6. Live imaging provides new insights on dynamic F-actin filopodia and differential endocytosis during myoblast fusion in Drosophila.

    PubMed

    Haralalka, Shruti; Shelton, Claude; Cartwright, Heather N; Guo, Fengli; Trimble, Rhonda; Kumar, Ram P; Abmayr, Susan M

    2014-01-01

    The process of myogenesis includes the recognition, adhesion, and fusion of committed myoblasts into multinucleate syncytia. In the larval body wall muscles of Drosophila, this elaborate process is initiated by Founder Cells and Fusion-Competent Myoblasts (FCMs), and cell adhesion molecules Kin-of-IrreC (Kirre) and Sticks-and-stones (Sns) on their respective surfaces. The FCMs appear to provide the driving force for fusion, via the assembly of protrusions associated with branched F-actin and the WASp, SCAR and Arp2/3 pathways. In the present study, we utilize the dorsal pharyngeal musculature that forms in the Drosophila embryo as a model to explore myoblast fusion and visualize the fusion process in live embryos. These muscles rely on the same cell types and genes as the body wall muscles, but are amenable to live imaging since they do not undergo extensive morphogenetic movement during formation. Time-lapse imaging with F-actin and membrane markers revealed dynamic FCM-associated actin-enriched protrusions that rapidly extend and retract into the myotube from different sites within the actin focus. Ultrastructural analysis of this actin-enriched area showed that they have two morphologically distinct structures: wider invasions and/or narrow filopodia that contain long linear filaments. Consistent with this, formin Diaphanous (Dia) and branched actin nucleator, Arp3, are found decorating the filopodia or enriched at the actin focus, respectively, indicating that linear actin is present along with branched actin at sites of fusion in the FCM. Gain-of-function Dia and loss-of-function Arp3 both lead to fusion defects, a decrease of F-actin foci and prominent filopodia from the FCMs. We also observed differential endocytosis of cell surface components at sites of fusion, with actin reorganizing factors, WASp and SCAR, and Kirre remaining on the myotube surface and Sns preferentially taken up with other membrane proteins into early endosomes and lysosomes in the

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

    PubMed

    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

  8. Identification of actin as a 15-deoxy-Delta12,14-prostaglandin J2 target in neuroblastoma cells: mass spectrometric, computational, and functional approaches to investigate the effect on cytoskeletal derangement.

    PubMed

    Aldini, Giancarlo; Carini, Marina; Vistoli, Giulio; Shibata, Takahiro; Kusano, Yuri; Gamberoni, Luca; Dalle-Donne, Isabella; Milzani, Aldo; Uchida, Koji

    2007-03-13

    A proteomic approach was used to identify 15-deoxy-Delta12,14-prostaglandin J2 (15d-PGJ2) protein targets in human neuroblastoma SH-SY5Y cells. By using biotinylated 15d-PGJ2, beta-actin was found as the major adducted protein; at least 12 proteins were also identified as minor biotin-positive spots, falling in different functional classes, including glycolytic enzymes (enolase and lactate dehydrogenase), redox enzymes (biliverdin reductase), and a eukaryotic regulatory protein (14-3-3gamma). 15d-PGJ2 induced marked morphological changes in the actin filament network and in particular promoted F-actin depolymerization as confirmed by Western blot analysis. By using a mass spectrometric approach, we found that 15d-PGJ2 reacts with isolated G-actin in a 1:1 stoichiometric ratio and selectively binds the Cys374 site through a Michael adduction mechanism. Computational studies showed that the covalent binding of 15d-PGJ2 induces a significant unfolding of actin structure and in particular that 15d-PGJ2 distorts the actin subdomains 2 and 4, which define the nucleotide binding sites impeding the nucleotide exchange. The functional effect of 15d-PGJ2 on G-actin was studied by polymerization measurement: in the presence of 15d-PGJ2, a lower amount of F-actin forms, as followed by the increase in pyrenyl-actin fluorescence intensity, as the major effect of increasing 15d-PGJ2 concentrations occurs on the maximum extent of actin polymerization, whereas it is negligible on the initial rate of reaction. In summary, the results here reported give an insight into the role of 15d-PGJ2 as a cytotoxic compound in neuronal cell dysfunction. Actin is the main protein cellular target of 15d-PGJ2, which specifically binds through a Michael adduction to Cys374, leading to a protein conformational change that can explain the disruption of the actin cytoskeleton, F-actin depolymerization, and impairment of G-actin polymerization. PMID:17297918

  9. High expression of Lifeact in Arabidopsis thaliana reduces dynamic reorganization of actin filaments but does not affect plant development.

    PubMed

    van der Honing, Hannie S; van Bezouwen, Laura S; Emons, Anne Mie C; Ketelaar, Tijs

    2011-10-01

    Lifeact is a novel probe that labels actin filaments in a wide range of organisms. We compared the localization and reorganization of Lifeact:Venus-labeled actin filaments in Arabidopsis root hairs and root epidermal cells of lines that express different levels of Lifeact: Venus with that of actin filaments labeled with GFP:FABD2, a commonly used probe in plants. Unlike GFP:FABD2, Lifeact:Venus labeled the highly dynamic fine F-actin in the subapical region of tip-growing root hairs. Lifeact:Venus expression at varying levels was not observed to affect plant development. However, at expression levels comparable to those of GFP:FABD2 in a well-characterized marker line, Lifeact:Venus reduced reorganization rates of bundles of actin filaments in root epidermal cells. Reorganization rates of cytoplasmic strands, which reflect the reorganization of the actin cytoskeleton, were also reduced in these lines. Moreover, in the same line, Lifeact:Venus-decorated actin filaments were more resistant to depolymerization by latrunculin B than those in an equivalent GFP:FABD2-expressing line. In lines where Lifeact: Venus is expressed at lower levels, these effects are less prominent or even absent. We conclude that Lifeact: Venus reduces remodeling of the actin cytoskeleton in Arabidopsis in a concentration-dependent manner. Since this reduction occurs at expression levels that do not cause defects in plant development, selection of normally growing plants is not sufficient to determine optimal Lifeact expression levels. When correct expression levels of Lifeact have been determined, it is a valuable probe that labels dynamic populations of actin filaments such as fine F-actin, better than FABD2 does.

  10. Hierarchical self-organization of cytoskeletal active networks

    NASA Astrophysics Data System (ADS)

    Gordon, Daniel; Bernheim-Groswasser, Anne; Keasar, Chen; Farago, Oded

    2012-04-01

    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 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.

  11. Ornithine decarboxylase and extracellular polyamines regulate microvascular sprouting and actin cytoskeleton dynamics in endothelial cells

    SciTech Connect

    Kucharzewska, Paulina; Welch, Johanna E.; Svensson, Katrin J.; Belting, Mattias

    2010-10-01

    The polyamines are essential for cancer cell proliferation during tumorigenesis. Targeted inhibition of ornithine decarboxylase (ODC), i.e. a key enzyme of polyamine biosynthesis, by {alpha}-difluoromethylornithine (DFMO) has shown anti-neoplastic activity in various experimental models. This activity has mainly been attributed to the anti-proliferative effect of DFMO in cancer cells. Here, we provide evidence that unperturbed ODC activity is a requirement for proper microvessel sprouting ex vivo as well as the migration of primary human endothelial cells. DFMO-mediated ODC inhibition was reversed by extracellular polyamine supplementation, showing that anti-angiogenic effects of DFMO were specifically related to polyamine levels. ODC inhibition was associated with an abnormal morphology of the actin cytoskeleton during cell spreading and migration. Moreover, our data suggest that de-regulated actin cytoskeleton dynamics in DFMO treated endothelial cells may be related to constitutive activation of the small GTPase CDC42, i.e. a well-known regulator of cell motility and actin cytoskeleton remodeling. These insights into the potential role of polyamines in angiogenesis should stimulate further studies testing the combined anti-tumor effect of polyamine inhibition and established anti-angiogenic therapies in vivo.

  12. Dynamic and elastic properties of F-actin: a normal-modes analysis.

    PubMed Central

    ben-Avraham, D; Tirion, M M

    1995-01-01

    We examine the dynamic, elastic, and mechanical consequences of the proposed atomic models of F-actin, using a normal mode analysis. This initial analysis is done in vacuo and assumes that all monomers are rigid and equivalent. Our computation proceeds from the atomic level and, relying on a single fitting parameter, reproduces various experimental results, including persistence lengths, elastic moduli, and contact energies. The computations reveal modes of motion characteristic to all polymers, such as longitudinal pressure waves, torsional waves, and bending, as well as motions unique to F-actin. Motions typical to actin include a "groove-swinging" motion of the two long-pitch helices, as well as an axial slipping motion of the two strands. We prepare snapshots of thermally activated filaments and quantify the accumulation of azimuthal angular "disorder," variations in cross-over lengths, and various other fluctuations. We find that the orientation of a small number of select residues has a surprisingly large effect on the filament flexibility and elasticity characteristics. PMID:7787015

  13. Actin dynamics provides membrane tension to merge fusing vesicles into the plasma membrane

    PubMed Central

    Wen, Peter J.; Grenklo, Staffan; Arpino, Gianvito; Tan, Xinyu; Liao, Hsien-Shun; Heureaux, Johanna; Peng, Shi-Yong; Chiang, Hsueh-Cheng; Hamid, Edaeni; Zhao, Wei-Dong; Shin, Wonchul; Näreoja, Tuomas; Evergren, Emma; Jin, Yinghui; Karlsson, Roger; Ebert, Steven N.; Jin, Albert; Liu, Allen P.; Shupliakov, Oleg; Wu, Ling-Gang

    2016-01-01

    Vesicle fusion is executed via formation of an Ω-shaped structure (Ω-profile), followed by closure (kiss-and-run) or merging of the Ω-profile into the plasma membrane (full fusion). Although Ω-profile closure limits release but recycles vesicles economically, Ω-profile merging facilitates release but couples to classical endocytosis for recycling. Despite its crucial role in determining exocytosis/endocytosis modes, how Ω-profile merging is mediated is poorly understood in endocrine cells and neurons containing small ∼30–300 nm vesicles. Here, using confocal and super-resolution STED imaging, force measurements, pharmacology and gene knockout, we show that dynamic assembly of filamentous actin, involving ATP hydrolysis, N-WASP and formin, mediates Ω-profile merging by providing sufficient plasma membrane tension to shrink the Ω-profile in neuroendocrine chromaffin cells containing ∼300 nm vesicles. Actin-directed compounds also induce Ω-profile accumulation at lamprey synaptic active zones, suggesting that actin may mediate Ω-profile merging at synapses. These results uncover molecular and biophysical mechanisms underlying Ω-profile merging. PMID:27576662

  14. Actin dynamics provides membrane tension to merge fusing vesicles into the plasma membrane.

    PubMed

    Wen, Peter J; Grenklo, Staffan; Arpino, Gianvito; Tan, Xinyu; Liao, Hsien-Shun; Heureaux, Johanna; Peng, Shi-Yong; Chiang, Hsueh-Cheng; Hamid, Edaeni; Zhao, Wei-Dong; Shin, Wonchul; Näreoja, Tuomas; Evergren, Emma; Jin, Yinghui; Karlsson, Roger; Ebert, Steven N; Jin, Albert; Liu, Allen P; Shupliakov, Oleg; Wu, Ling-Gang

    2016-01-01

    Vesicle fusion is executed via formation of an Ω-shaped structure (Ω-profile), followed by closure (kiss-and-run) or merging of the Ω-profile into the plasma membrane (full fusion). Although Ω-profile closure limits release but recycles vesicles economically, Ω-profile merging facilitates release but couples to classical endocytosis for recycling. Despite its crucial role in determining exocytosis/endocytosis modes, how Ω-profile merging is mediated is poorly understood in endocrine cells and neurons containing small ∼30-300 nm vesicles. Here, using confocal and super-resolution STED imaging, force measurements, pharmacology and gene knockout, we show that dynamic assembly of filamentous actin, involving ATP hydrolysis, N-WASP and formin, mediates Ω-profile merging by providing sufficient plasma membrane tension to shrink the Ω-profile in neuroendocrine chromaffin cells containing ∼300 nm vesicles. Actin-directed compounds also induce Ω-profile accumulation at lamprey synaptic active zones, suggesting that actin may mediate Ω-profile merging at synapses. These results uncover molecular and biophysical mechanisms underlying Ω-profile merging. PMID:27576662

  15. Shank–cortactin interactions control actin dynamics to maintain flexibility of neuronal spines and synapses

    PubMed Central

    MacGillavry, Harold D.; Kerr, Justin M.; Kassner, Josh; Frost, Nicholas A.; Blanpied, Thomas A.

    2016-01-01

    The family of Shank scaffolding molecules (comprising Shank1, 2 and 3) are core components of the postsynaptic density (PSD) in neuronal synapses. Shanks link surface receptors to other scaffolding molecules within the PSD, as well as to the actin cytoskeleton. However, determining the function of Shank proteins in neurons has been complicated because the different Shank isoforms share a very high degree of sequence and domain homology. Therefore, to control Shank content while minimizing potential compensatory effects, a miRNA-based knockdown strategy was developed to reduce the expression of all synaptically targeted Shank isoforms simultaneously in rat hippocampal neurons. Using this approach, a strong (>75%) reduction in total Shank protein levels was achieved at individual dendritic spines, prompting an approximately 40% decrease in mushroom spine density. Furthermore, Shank knockdown reduced spine actin levels and increased sensitivity to the actin depolymerizing agent Latrunculin A. A SHANK2 mutant lacking the proline-rich cortactin-binding motif (SHANK2-ΔPRO) was unable to rescue these defects. Furthermore, Shank knockdown reduced cortactin levels in spines and increased the mobility of spine cortactin as measured by single-molecule tracking photoactivated localization microscopy, suggesting that Shank proteins recruit and stabilize cortactin at the synapse. Furthermore, it was found that Shank knockdown significantly reduced spontaneous remodelling of synapse morphology that could not be rescued by the SHANK2-ΔPRO mutant. It was concluded that Shank proteins are key intermediates between the synapse and the spine interior that, via cortactin, permit the actin cytoskeleton to dynamically regulate synapse morphology and function. PMID:26547831

  16. Viscoelastic properties of actin-coated membranes

    NASA Astrophysics Data System (ADS)

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

    2001-02-01

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

  17. Heterotypic and homotypic associations between ezrin and moesin, two putative membrane-cytoskeletal linking proteins.

    PubMed Central

    Gary, R; Bretscher, A

    1993-01-01

    Ezrin and moesin are components of actin-rich cell surface structures that are thought to function as membrane-cytoskeletal linking proteins. Here we show that a stable complex of ezrin and moesin can be isolated from cultured cells by immunoprecipitation with specific antibodies. The capacity of these two proteins to interact directly was confirmed with a blot-overlay procedure in which biotin-tagged proteins in solution were incubated with immobilized binding partners. In addition to the heterotypic association of ezrin and moesin, homotypic binding of ezrin to ezrin and of moesin to moesin was also demonstrated in vitro. These results suggest mechanisms by which ezrin and moesin might participate in dynamic aspects of cortical cytoskeletal structure. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 PMID:8248180

  18. Identification of a new actin binding surface on vinculin that mediates mechanical cell and focal adhesion properties

    PubMed Central

    Thompson, Peter M.; Tolbert, Caitlin E.; Shen, Kai; Kota, Pradeep; Palmer, Sean M.; Plevock, Karen M.; Orlova, Albina; Galkin, Vitold E.; Burridge, Keith; Egelman, Edward H.; Dokholyan, Nikolay V.; Superfine, Richard; Campbell, Sharon L.

    2014-01-01

    SUMMARY Vinculin, a cytoskeletal scaffold protein essential for embryogenesis and cardiovascular function, localizes to focal adhesions and adherens junctions, connecting cell surface receptors to the actin cytoskeleton. While vinculin interacts with many adhesion proteins, its interaction with filamentous actin regulates cell morphology, motility, and mechanotransduction. Disruption of this interaction lowers cell traction forces and enhances actin flow rates. Although a model for the vinculin:actin complex exists, we recently identified actin-binding deficient mutants of vinculin outside sites predicted to bind actin, and developed an alternative model to better define this novel actin-binding surface, using negative-stain EM, discrete molecular dynamics, and mutagenesis. Actin-binding deficient vinculin variants expressed in vinculin knockout fibroblasts fail to rescue cell-spreading defects and reduce cellular response to external force. These findings highlight the importance of this new actin-binding surface and provide the molecular basis for elucidating additional roles of this interaction, including actin-induced conformational changes which promote actin bundling. PMID:24685146

  19. ROP Gtpase–Dependent Dynamics of Tip-Localized F-Actin Controls Tip Growth in Pollen Tubes

    PubMed Central

    Fu, Ying; Wu, Guang; Yang, Zhenbiao

    2001-01-01

    Tip-growing pollen tubes provide a useful model system to study polar growth. Although roles for tip-focused calcium gradient and tip-localized Rho-family GTPase in pollen tube growth is established, the existence and function of tip-localized F-actin have been controversial. Using the green fluorescent protein–tagged actin-binding domain of mouse talin, we found a dynamic form of tip-localized F-actin in tobacco pollen tubes, termed short actin bundles (SABs). The dynamics of SABs during polar growth in pollen tubes is regulated by Rop1At, a Rop GTPase belonging to the Rho family. When overexpressed, Rop1At transformed SAB into a network of fine filaments and induced a transverse actin band behind the tip, leading to depolarized growth. These changes were due to ectopic Rop1At localization to the apical region of the plasma membrane and were suppressed by guanine dissociation inhibitor overexpression, which removed ectopically localized Rop1At. Rop GTPase–activating protein (RopGAP1) overexpression, or Latrunculin B treatments, also recovered normal actin organization and tip growth in Rop1At-overexpressing tubes. Moreover, overexpression of RopGAP1 alone disrupted SABs and inhibited growth. Finally, SAB oscillates and appears at the tip before growth. Together, these results indicate that the dynamics of tip actin are essential for tip growth and provide the first direct evidence to link Rho GTPase to actin organization in controlling cell polarity and polar growth in plants. PMID:11238457

  20. Oscillatory increases in alkalinity anticipate growth and may regulate actin dynamics in pollen tubes of lily.

    PubMed

    Lovy-Wheeler, Alenka; Kunkel, Joseph G; Allwood, Ellen G; Hussey, Patrick J; Hepler, Peter K

    2006-09-01

    Lily (Lilium formosanum or Lilium longiflorum) pollen tubes, microinjected with a low concentration of the pH-sensitive dye bis-carboxyethyl carboxyfluorescein dextran, show oscillating pH changes in their apical domain relative to growth. An increase in pH in the apex precedes the fastest growth velocities, whereas a decline follows growth, suggesting a possible relationship between alkalinity and cell extension. A target for pH may be the actin cytoskeleton, because the apical cortical actin fringe resides in the same region as the alkaline band in lily pollen tubes and elongation requires actin polymerization. A pH-sensitive actin binding protein, actin-depolymerizing factor (ADF), together with actin-interacting protein (AIP) localize to the cortical actin fringe region. Modifying intracellular pH leads to reorganization of the actin cytoskeleton, especially in the apical domain. Acidification causes actin filament destabilization and inhibits growth by 80%. Upon complete growth inhibition, the actin fringe is the first actin cytoskeleton component to disappear. We propose that during normal growth, the pH increase in the alkaline band stimulates the fragmenting activity of ADF/AIP, which in turn generates more sites for actin polymerization. Increased actin polymerization supports faster growth rates and a proton influx, which inactivates ADF/AIP, decreases actin polymerization, and retards growth. As pH stabilizes and increases, the activity of ADF/AIP again increases, repeating the cycle of events. PMID:16920777

  1. Quantitative Evaluation of Stomatal Cytoskeletal Patterns during the Activation of Immune Signaling in Arabidopsis thaliana

    PubMed Central

    Shimono, Masaki; Higaki, Takumi; Kaku, Hanae; Shibuya, Naoto; Hasezawa, Seiichiro

    2016-01-01

    Historically viewed as primarily functioning in the regulation of gas and water vapor exchange, it is now evident that stomata serve an important role in plant immunity. Indeed, in addition to classically defined functions related to cell architecture and movement, the actin cytoskeleton has emerged as a central component of the plant immune system, underpinning not only processes related to cell shape and movement, but also receptor activation and signaling. Using high resolution quantitative imaging techniques, the temporal and spatial changes in the actin microfilament array during diurnal cycling of stomatal guard cells has revealed a highly orchestrated transition from random arrays to ordered bundled filaments. While recent studies have demonstrated that plant stomata close in response to pathogen infection, an evaluation of stimulus-induced changes in actin cytoskeletal dynamics during immune activation in the guard cell, as well as the relationship of these changes to the function of the actin cytoskeleton and stomatal aperture, remains undefined. In the current study, we employed quantitative cell imaging and hierarchical clustering analyses to define the response of the guard cell actin cytoskeleton to pathogen infection and the elicitation of immune signaling. Using this approach, we demonstrate that stomatal-localized actin filaments respond rapidly, and specifically, to both bacterial phytopathogens and purified pathogen elicitors. Notably, we demonstrate that higher order temporal and spatial changes in the filament array show distinct patterns of organization during immune activation, and that changes in the naïve diurnal oscillations of guard cell actin filaments are perturbed by pathogens, and that these changes parallel pathogen-induced stomatal gating. The data presented herein demonstrate the application of a highly tractable and quantifiable method to assign transitions in actin filament organization to the activation of immune signaling in

  2. Quantitative Evaluation of Stomatal Cytoskeletal Patterns during the Activation of Immune Signaling in Arabidopsis thaliana.

    PubMed

    Shimono, Masaki; Higaki, Takumi; Kaku, Hanae; Shibuya, Naoto; Hasezawa, Seiichiro; Day, Brad

    2016-01-01

    Historically viewed as primarily functioning in the regulation of gas and water vapor exchange, it is now evident that stomata serve an important role in plant immunity. Indeed, in addition to classically defined functions related to cell architecture and movement, the actin cytoskeleton has emerged as a central component of the plant immune system, underpinning not only processes related to cell shape and movement, but also receptor activation and signaling. Using high resolution quantitative imaging techniques, the temporal and spatial changes in the actin microfilament array during diurnal cycling of stomatal guard cells has revealed a highly orchestrated transition from random arrays to ordered bundled filaments. While recent studies have demonstrated that plant stomata close in response to pathogen infection, an evaluation of stimulus-induced changes in actin cytoskeletal dynamics during immune activation in the guard cell, as well as the relationship of these changes to the function of the actin cytoskeleton and stomatal aperture, remains undefined. In the current study, we employed quantitative cell imaging and hierarchical clustering analyses to define the response of the guard cell actin cytoskeleton to pathogen infection and the elicitation of immune signaling. Using this approach, we demonstrate that stomatal-localized actin filaments respond rapidly, and specifically, to both bacterial phytopathogens and purified pathogen elicitors. Notably, we demonstrate that higher order temporal and spatial changes in the filament array show distinct patterns of organization during immune activation, and that changes in the naïve diurnal oscillations of guard cell actin filaments are perturbed by pathogens, and that these changes parallel pathogen-induced stomatal gating. The data presented herein demonstrate the application of a highly tractable and quantifiable method to assign transitions in actin filament organization to the activation of immune signaling in

  3. Enabled negatively regulates diaphanous-driven actin dynamics in vitro and in vivo.

    PubMed

    Bilancia, Colleen G; Winkelman, Jonathan D; Tsygankov, Denis; Nowotarski, Stephanie H; Sees, Jennifer A; Comber, Kate; Evans, Iwan; Lakhani, Vinal; Wood, Will; Elston, Timothy C; Kovar, David R; Peifer, Mark

    2014-02-24

    Actin regulators facilitate cell migration by controlling cell protrusion architecture and dynamics. As the behavior of individual actin regulators becomes clear, we must address why cells require multiple regulators with similar functions and how they cooperate to create diverse protrusions. We characterized Diaphanous (Dia) and Enabled (Ena) as a model, using complementary approaches: cell culture, biophysical analysis, and Drosophila morphogenesis. We found that Dia and Ena have distinct biochemical properties that contribute to the different protrusion morphologies each induces. Dia is a more processive, faster elongator, paralleling the long, stable filopodia it induces in vivo, while Ena promotes filopodia with more dynamic changes in number, length, and lifetime. Acting together, Ena and Dia induce protrusions distinct from those induced by either alone, with Ena reducing Dia-driven protrusion length and number. Consistent with this, EnaEVH1 binds Dia directly and inhibits DiaFH1FH2-mediated nucleation in vitro. Finally, Ena rescues hemocyte migration defects caused by activated Dia.

  4. Muscle Lim Protein isoform negatively regulates striated muscle actin dynamics and differentiation

    PubMed Central

    Vafiadaki, Elizabeth; Arvanitis, Demetrios A.; Papalouka, Vasiliki; Terzis, Gerasimos; Roumeliotis, Theodoros I.; Spengos, Konstantinos; Garbis, Spiros D.; Manta, Panagiota; Kranias, Evangelia G.; Sanoudou, Despina

    2015-01-01

    Muscle Lim Protein (MLP) has emerged as a critical regulator of striated muscle physiology and pathophysiology. Mutations in cysteine and glycine-rich protein 3 (CSRP3), the gene encoding MLP, have been directly associated with human cardiomyopathies, while aberrant expression patterns are reported in human cardiac and skeletal muscle diseases. Increasing evidence suggests that MLP has an important role in both myogenic differentiation and myocyte cytoarchitecture, although the full spectrum of its intracellular roles has not been delineated. We report the discovery of an alternative splice variant of MLP, designated as MLP-b, showing distinct expression in neuromuscular disease and direct roles in actin dynamics and muscle differentiation. This novel isoform originates by alternative splicing of exons 3 and 4. At the protein level, it contains the N-terminus first half LIM domain of MLP and a unique sequence of 22 amino acids. Physiologically it is expressed during early differentiation, whereas its overexpression reduces C2C12 differentiation and myotube formation. This may be mediated through its inhibition of MLP/CFL2-mediated F-actin dynamics. In differentiated striated muscles, MLP-b localizes to the sarcomeres and binds directly to Z-disc components including α-actinin, T-cap and MLP. Our findings unveil a novel player in muscle physiology and pathophysiology that is implicated in myogenesis as a negative regulator of myotube formation, and in differentiated striated muscles as a contributor to sarcomeric integrity. PMID:24860983

  5. The degree of resistance of erythrocyte membrane cytoskeletal proteins to supra-physiologic concentrations of calcium: an in vitro study.

    PubMed

    Mostafavi, Ebrahim; Nargesi, Arash Aghajani; Ghazizadeh, Zaniar; Larry, Mehrdad; Farahani, Roya Horabad; Morteza, Afsaneh; Esteghamati, Alireza; Vigneron, Claude; Nakhjavani, Manouchehr

    2014-08-01

    Calcium is a key regulator of cell dynamics. Dysregulation of its cytosolic concentration is implicated in the pathophysiology of several diseases. This study aimed to assess the effects of calcium on the network of membrane cytoskeletal proteins. Erythrocyte membranes were obtained from eight healthy donors and incubated with 250 µM and 1.25 mM calcium solutions. Membrane cytoskeletal proteins were quantified using SDS-PAGE at baseline and after 3 and 5 days of incubation. Supra-physiologic concentrations of calcium (1.25 mM) induced a significant proteolysis in membrane cytoskeletal proteins, compared with magnesium (p < 0.001). Actin exhibited the highest sensitivity to calcium-induced proteolysis (6.8 ± 0.3 vs. 5.3 ± 0.6, p < 0.001), while spectrin (39.9 ± 1.0 vs. 40.3 ± 2.0, p = 0.393) and band-6 (6.3 ± 0.3 vs. 6.8 ± 0.8, p = 0.191) were more resistant to proteolysis after incubation with calcium in the range of endoplasmic reticulum concentrations (250 µM). Aggregation of membrane cytoskeletal proteins was determined after centrifugation and was significantly higher after incubation with calcium ions compared with control, EDTA and magnesium solutions (p < 0.001). In a supra-physiologic range of 1.25-10 mM of calcium ions, there was a nearly perfect linear relationship between calcium concentration and aggregation of erythrocyte membrane cytoskeletal proteins (R(2) = 0.971, p < 0.001). Our observation suggests a strong interaction between calcium ions and membrane cytoskeletal network. Cumulative effects of disrupted calcium homeostasis on cytoskeletal proteins need to be further investigated at extended periods of time in disease states.

  6. The degree of resistance of erythrocyte membrane cytoskeletal proteins to supra-physiologic concentrations of calcium: an in vitro study.

    PubMed

    Mostafavi, Ebrahim; Nargesi, Arash Aghajani; Ghazizadeh, Zaniar; Larry, Mehrdad; Farahani, Roya Horabad; Morteza, Afsaneh; Esteghamati, Alireza; Vigneron, Claude; Nakhjavani, Manouchehr

    2014-08-01

    Calcium is a key regulator of cell dynamics. Dysregulation of its cytosolic concentration is implicated in the pathophysiology of several diseases. This study aimed to assess the effects of calcium on the network of membrane cytoskeletal proteins. Erythrocyte membranes were obtained from eight healthy donors and incubated with 250 µM and 1.25 mM calcium solutions. Membrane cytoskeletal proteins were quantified using SDS-PAGE at baseline and after 3 and 5 days of incubation. Supra-physiologic concentrations of calcium (1.25 mM) induced a significant proteolysis in membrane cytoskeletal proteins, compared with magnesium (p < 0.001). Actin exhibited the highest sensitivity to calcium-induced proteolysis (6.8 ± 0.3 vs. 5.3 ± 0.6, p < 0.001), while spectrin (39.9 ± 1.0 vs. 40.3 ± 2.0, p = 0.393) and band-6 (6.3 ± 0.3 vs. 6.8 ± 0.8, p = 0.191) were more resistant to proteolysis after incubation with calcium in the range of endoplasmic reticulum concentrations (250 µM). Aggregation of membrane cytoskeletal proteins was determined after centrifugation and was significantly higher after incubation with calcium ions compared with control, EDTA and magnesium solutions (p < 0.001). In a supra-physiologic range of 1.25-10 mM of calcium ions, there was a nearly perfect linear relationship between calcium concentration and aggregation of erythrocyte membrane cytoskeletal proteins (R(2) = 0.971, p < 0.001). Our observation suggests a strong interaction between calcium ions and membrane cytoskeletal network. Cumulative effects of disrupted calcium homeostasis on cytoskeletal proteins need to be further investigated at extended periods of time in disease states. PMID:24930024

  7. Sex Hormones Regulate Cytoskeletal Proteins Involved in Brain Plasticity

    PubMed Central

    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

  8. Sex Hormones Regulate Cytoskeletal Proteins Involved in Brain Plasticity.

    PubMed

    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

  9. Secretagogin affects insulin secretion in pancreatic β-cells by regulating actin dynamics and focal adhesion

    PubMed Central

    Yang, Seo-Yun; Lee, Jae-Jin; Lee, Jin-Hee; Lee, Kyungeun; Oh, Seung Hoon; Lim, Yu-Mi; Lee, Myung-Shik; Lee, Kong-Joo

    2016-01-01

    Secretagogin (SCGN), a Ca2+-binding protein having six EF-hands, is selectively expressed in pancreatic β-cells and neuroendocrine cells. Previous studies suggested that SCGN enhances insulin secretion by functioning as a Ca2+-sensor protein, but the underlying mechanism has not been elucidated. The present study explored the mechanism by which SCGN enhances glucose-induced insulin secretion in NIT-1 insulinoma cells. To determine whether SCGN influences the first or second phase of insulin secretion, we examined how SCGN affects the kinetics of insulin secretion in NIT-1 cells. We found that silencing SCGN suppressed the second phase of insulin secretion induced by glucose and H2O2, but not the first phase induced by KCl stimulation. Recruitment of insulin granules in the second phase of insulin secretion was significantly impaired by knocking down SCGN in NIT-1 cells. In addition, we found that SCGN interacts with the actin cytoskeleton in the plasma membrane and regulates actin remodelling in a glucose-dependent manner. Since actin dynamics are known to regulate focal adhesion, a critical step in the second phase of insulin secretion, we examined the effect of silencing SCGN on focal adhesion molecules, including FAK (focal adhesion kinase) and paxillin, and the cell survival molecules ERK1/2 (extracellular-signal-regulated kinase 1/2) and Akt. We found that glucose- and H2O2-induced activation of FAK, paxillin, ERK1/2 and Akt was significantly blocked by silencing SCGN. We conclude that SCGN controls glucose-stimulated insulin secretion and thus may be useful in the therapy of Type 2 diabetes. PMID:27095850

  10. LlSR28 is involved in pollen germination by affecting filamentous actin dynamics.

    PubMed

    Cao, Li-Juan; Zhao, Meng-Meng; Liu, Chang; Dong, Huai-Jian; Li, Wang-Cheng; Ren, Hai-Yun

    2013-07-01

    Alternative splicing plays important roles in gene regulation and contributes to protein complexity. Previous studies suggest that alternative splicing exists in members of the villin/gelsolin/fragmin superfamily. In this study, a serine/argine-rich (SR) protein cDNA with 28 kDa protein (LlSR28) was isolated from a lily (Lilium longiflorum) expression library. Protein domain analysis showed that LlSR28 had similar structures to Arabidopsis SR45 (AtSR45), and LlSR28 could complement the phenotype of loss of AtSR45 function. Therefore, overexpression of LlSR28 and AtSR45 mutant (atsr45-1) were used in the following experiments. Overexpression of LlSR28 in Arabidopsis completely inhibited pollen germination. In contrast, the pollen germination of atsr45-1 was earlier than that of wild-type. In addition, pollen of atsr45-1 contained less F-actin at the corresponding hydration stage during pollen germination compared to that of wild-type. Alternative splicing analysis showed that Arabidopsis villin1 (AtVLN1) transcript encoding the full-length protein was increased, and that encoding the truncated protein was decreased in atst45-1. Moreover, the mRNA expression level of other actin-binding proteins (ABPs) abundant in Arabidopsis pollen was also changed in atsr45-1. In conclusion, we hypothesize that LlSR28 alters F-actin dynamics probably through its alternative splicing activities to affect directly or indirectly the alternative splicing of AtVLN1 and the expression of different ABPs, which then affects the pollen germination. PMID:23741063

  11. Secretagogin affects insulin secretion in pancreatic β-cells by regulating actin dynamics and focal adhesion.

    PubMed

    Yang, Seo-Yun; Lee, Jae-Jin; Lee, Jin-Hee; Lee, Kyungeun; Oh, Seung Hoon; Lim, Yu-Mi; Lee, Myung-Shik; Lee, Kong-Joo

    2016-06-15

    Secretagogin (SCGN), a Ca(2+)-binding protein having six EF-hands, is selectively expressed in pancreatic β-cells and neuroendocrine cells. Previous studies suggested that SCGN enhances insulin secretion by functioning as a Ca(2+)-sensor protein, but the underlying mechanism has not been elucidated. The present study explored the mechanism by which SCGN enhances glucose-induced insulin secretion in NIT-1 insulinoma cells. To determine whether SCGN influences the first or second phase of insulin secretion, we examined how SCGN affects the kinetics of insulin secretion in NIT-1 cells. We found that silencing SCGN suppressed the second phase of insulin secretion induced by glucose and H2O2, but not the first phase induced by KCl stimulation. Recruitment of insulin granules in the second phase of insulin secretion was significantly impaired by knocking down SCGN in NIT-1 cells. In addition, we found that SCGN interacts with the actin cytoskeleton in the plasma membrane and regulates actin remodelling in a glucose-dependent manner. Since actin dynamics are known to regulate focal adhesion, a critical step in the second phase of insulin secretion, we examined the effect of silencing SCGN on focal adhesion molecules, including FAK (focal adhesion kinase) and paxillin, and the cell survival molecules ERK1/2 (extracellular-signal-regulated kinase 1/2) and Akt. We found that glucose- and H2O2-induced activation of FAK, paxillin, ERK1/2 and Akt was significantly blocked by silencing SCGN. We conclude that SCGN controls glucose-stimulated insulin secretion and thus may be useful in the therapy of Type 2 diabetes. PMID:27095850

  12. A variational approach to the growth dynamics of pre-stressed actin filament networks.

    PubMed

    John, Karin; Stöter, Thomas; Misbah, Chaouqi

    2016-09-21

    In order to model the growth dynamics of elastic bodies with residual stresses a thermodynamically consistent approach is needed such that the cross-coupling between growth and mechanics can be correctly described. In the present work we apply a variational principle to the formulation of the interfacial growth dynamics of dendritic actin filament networks growing from biomimetic beads, an experimentally well studied system, where the buildup of residual stresses governs the network growth. We first introduce the material model for the network via a strain energy density for an isotropic weakly nonlinear elastic material and then derive consistently from this model the dynamic equations for the interfaces, i.e. for a polymerizing internal interface in contact with the bead and a depolymerizing external interface directed towards the solvent. We show that (i) this approach automatically preserves thermodynamic symmetry-properties, which is not the case for the often cited 'rubber-band-model' (Sekimoto et al 2004 Eur. Phys. J. E 13 247-59, Plastino et al 2004 Eur. Biophys. J. 33 310-20) and (ii) leads to a robust morphological instability of the treadmilling network interfaces. The nature of the instability depends on the interplay of the two dynamic interfaces. Depending on the biochemical conditions the network envelope evolves into a comet-like shape (i.e. the actin envelope thins out at one side and thickens on the opposite side of the bead) via a varicose instability or it breaks the symmetry via higher order zigzag modes. We conclude that morphological instabilities due to mechano-chemical coupling mechanisms and the presences of mechancial pre-stresses can play a major role in locally organizing the cytoskeleton of living cells. PMID:27420637

  13. A variational approach to the growth dynamics of pre-stressed actin filament networks

    NASA Astrophysics Data System (ADS)

    John, Karin; Stöter, Thomas; Misbah, Chaouqi

    2016-09-01

    In order to model the growth dynamics of elastic bodies with residual stresses a thermodynamically consistent approach is needed such that the cross-coupling between growth and mechanics can be correctly described. In the present work we apply a variational principle to the formulation of the interfacial growth dynamics of dendritic actin filament networks growing from biomimetic beads, an experimentally well studied system, where the buildup of residual stresses governs the network growth. We first introduce the material model for the network via a strain energy density for an isotropic weakly nonlinear elastic material and then derive consistently from this model the dynamic equations for the interfaces, i.e. for a polymerizing internal interface in contact with the bead and a depolymerizing external interface directed towards the solvent. We show that (i) this approach automatically preserves thermodynamic symmetry-properties, which is not the case for the often cited ‘rubber-band-model’ (Sekimoto et al 2004 Eur. Phys. J. E 13 247–59, Plastino et al 2004 Eur. Biophys. J. 33 310–20) and (ii) leads to a robust morphological instability of the treadmilling network interfaces. The nature of the instability depends on the interplay of the two dynamic interfaces. Depending on the biochemical conditions the network envelope evolves into a comet-like shape (i.e. the actin envelope thins out at one side and thickens on the opposite side of the bead) via a varicose instability or it breaks the symmetry via higher order zigzag modes. We conclude that morphological instabilities due to mechano-chemical coupling mechanisms and the presences of mechancial pre-stresses can play a major role in locally organizing the cytoskeleton of living cells.

  14. A variational approach to the growth dynamics of pre-stressed actin filament networks

    NASA Astrophysics Data System (ADS)

    John, Karin; Stöter, Thomas; Misbah, Chaouqi

    2016-09-01

    In order to model the growth dynamics of elastic bodies with residual stresses a thermodynamically consistent approach is needed such that the cross-coupling between growth and mechanics can be correctly described. In the present work we apply a variational principle to the formulation of the interfacial growth dynamics of dendritic actin filament networks growing from biomimetic beads, an experimentally well studied system, where the buildup of residual stresses governs the network growth. We first introduce the material model for the network via a strain energy density for an isotropic weakly nonlinear elastic material and then derive consistently from this model the dynamic equations for the interfaces, i.e. for a polymerizing internal interface in contact with the bead and a depolymerizing external interface directed towards the solvent. We show that (i) this approach automatically preserves thermodynamic symmetry-properties, which is not the case for the often cited ‘rubber-band-model’ (Sekimoto et al 2004 Eur. Phys. J. E 13 247-59, Plastino et al 2004 Eur. Biophys. J. 33 310-20) and (ii) leads to a robust morphological instability of the treadmilling network interfaces. The nature of the instability depends on the interplay of the two dynamic interfaces. Depending on the biochemical conditions the network envelope evolves into a comet-like shape (i.e. the actin envelope thins out at one side and thickens on the opposite side of the bead) via a varicose instability or it breaks the symmetry via higher order zigzag modes. We conclude that morphological instabilities due to mechano-chemical coupling mechanisms and the presences of mechancial pre-stresses can play a major role in locally organizing the cytoskeleton of living cells.

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

    PubMed

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

    2016-01-01

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

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

    PubMed

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

    2016-01-01

    Actin cables, composed of actin filament bundles nucleated by formins, mediate intracellular transport for cell polarity establishment and maintenance. We previously observed that metaphase cells preferentially promote actin cable assembly through cyclin-dependent kinase 1 (Cdk1) activity. However, the relevant metaphase Cdk1 targets were not known. Here we show that the highly conserved actin filament crosslinking protein fimbrin is a critical Cdk1 target for actin cable assembly regulation in budding yeast. Fimbrin is specifically phosphorylated on threonine 103 by the metaphase cyclin-Cdk1 complex, in vivo and in vitro. On the basis of conformational simulations, we suggest that this phosphorylation stabilizes fimbrin's N-terminal domain, and modulates actin filament binding to regulate actin cable assembly and stability in cells. Overall, this work identifies fimbrin as a key target for cell cycle regulation of actin cable assembly in budding yeast, and suggests an underlying mechanism. PMID:27068241

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

    PubMed Central

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

    2016-01-01

    Actin cables, composed of actin filament bundles nucleated by formins, mediate intracellular transport for cell polarity establishment and maintenance. We previously observed that metaphase cells preferentially promote actin cable assembly through cyclin-dependent kinase 1 (Cdk1) activity. However, the relevant metaphase Cdk1 targets were not known. Here we show that the highly conserved actin filament crosslinking protein fimbrin is a critical Cdk1 target for actin cable assembly regulation in budding yeast. Fimbrin is specifically phosphorylated on threonine 103 by the metaphase cyclin–Cdk1 complex, in vivo and in vitro. On the basis of conformational simulations, we suggest that this phosphorylation stabilizes fimbrin's N-terminal domain, and modulates actin filament binding to regulate actin cable assembly and stability in cells. Overall, this work identifies fimbrin as a key target for cell cycle regulation of actin cable assembly in budding yeast, and suggests an underlying mechanism. PMID:27068241

  18. 2,4-Dichlorophenoxyacetic acid promotes S-nitrosylation and oxidation of actin affecting cytoskeleton and peroxisomal dynamics.

    PubMed

    Rodríguez-Serrano, M; Pazmiño, D M; Sparkes, I; Rochetti, A; Hawes, C; Romero-Puertas, M C; Sandalio, L M

    2014-09-01

    2,4-Dichlorophenoxyacetic acid (2,4-D) is a synthetic auxin used as a herbicide to control weeds in agriculture. A high concentration of 2,4-D promotes leaf epinasty and cell death. In this work, the molecular mechanisms involved in the toxicity of this herbicide are studied by analysing in Arabidopsis plants the accumulation of reactive oxygen species (ROS) and nitric oxide (NO), and their effect on cytoskeleton structure and peroxisome dynamics. 2,4-D (23 mM) promotes leaf epinasty, whereas this process was prevented by EDTA, which can reduce ·OH accumulation. The analysis of ROS accumulation by confocal microscopy showed a 2,4-D-dependent increase in both H2O2 and O2·(-), whereas total NO was not affected by the treatment. The herbicide promotes disturbances on the actin cytoskeleton structure as a result of post-translational modification of actin by oxidation and S-nitrosylation, which could disturb actin polymerization, as suggested by the reduction of the F-actin/G-actin ratio. These effects were reduced by EDTA, and the reduction of ROS production in Arabidopsis mutants deficient in xanthine dehydrogenase (Atxdh) gave rise to a reduction in actin oxidation. Also, 2,4-D alters the dynamics of the peroxisome, slowing the speed and shortening the distances by which these organelles are displaced. It is concluded that 2,4-D promotes oxidative and nitrosative stress, causing disturbances in the actin cytoskeleton, thereby affecting the dynamics of peroxisomes and some other organelles such as the mitochondria, with xanthine dehydrogenase being involved in ROS production under these conditions. These structural changes in turn appear to be responsible for the leaf epinasty.

  19. Live cell imaging reveals structural associations between the actin and microtubule cytoskeleton in Arabidopsis.

    PubMed

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

    2011-06-01

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

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

    PubMed Central

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

    2011-01-01

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

  1. Immunological Responses and Actin Dynamics in Macrophages Are Controlled by N-Cofilin but Are Independent from ADF

    PubMed Central

    Jönsson, Friederike; Gurniak, Christine B.; Fleischer, Bernhard; Kirfel, Gregor; Witke, Walter

    2012-01-01

    Dynamic changes in the actin cytoskeleton are essential for immune cell function and a number of immune deficiencies have been linked to mutations, which disturb the actin cytoskeleton. In macrophages and dendritic cells, actin remodelling is critical for motility, phagocytosis and antigen presentation, however the actin binding proteins, which control antigen presentation have been poorly characterized. Here we dissect the specific roles of the family of ADF/cofilin F-actin depolymerizing factors in macrophages and in local immune responses. Macrophage migration, cell polarization and antigen presentation to T-cells require n-cofilin mediated F-actin remodelling. Using a conditional mouse model, we show that n-cofilin also controls MHC class II-dependent antigen presentation. Other cellular processes such as phagocytosis and antigen processing were found to be independent of n-cofilin. Our data identify n-cofilin as a novel regulator of antigen presentation, while ADF on the other hand is dispensable for macrophage motility and antigen presentation. PMID:22558315

  2. Regimes of wave type patterning driven by refractory actin feedback: transition from static polarization to dynamic wave behaviour

    NASA Astrophysics Data System (ADS)

    Holmes, W. R.; Carlsson, A. E.; Edelstein-Keshet, L.

    2012-08-01

    Patterns of waves, patches, and peaks of actin are observed experimentally in many living cells. Models of this phenomenon have been based on the interplay between filamentous actin (F-actin) and its nucleation promoting factors (NPFs) that activate the Arp2/3 complex. Here we present an alternative biologically-motivated model for F-actin-NPF interaction based on properties of GTPases acting as NPFs. GTPases (such as Cdc42, Rac) are known to promote actin nucleation, and to have active membrane-bound and inactive cytosolic forms. The model is a natural extension of a previous mathematical mini-model of small GTPases that generates static cell polarization. Like other modellers, we assume that F-actin negative feedback shapes the observed patterns by suppressing the trailing edge of NPF-generated wave-fronts, hence localizing the activity spatially. We find that our NPF-actin model generates a rich set of behaviours, spanning a transition from static polarization to single pulses, reflecting waves, wave trains, and oscillations localized at the cell edge. The model is developed with simplicity in mind to investigate the interaction between nucleation promoting factor kinetics and negative feedback. It explains distinct types of pattern initiation mechanisms, and identifies parameter regimes corresponding to distinct behaviours. We show that weak actin feedback yields static patterning, moderate feedback yields dynamical behaviour such as travelling waves, and strong feedback can lead to wave trains or total suppression of patterning. We use a recently introduced nonlinear bifurcation analysis to explore the parameter space of this model and predict its behaviour with simulations validating those results.

  3. Docking, molecular dynamics and QM/MM studies to delineate the mode of binding of CucurbitacinE to F-actin.

    PubMed

    Kumar, R Pravin; Roopa, L; Nongthomba, Upendra; Sudheer Mohammed, M M; Kulkarni, Naveen

    2016-01-01

    CucurbitacinE (CurE) has been known to bind covalently to F-actin and inhibit depolymerization. However, the mode of binding of CurE to F-actin and the consequent changes in the F-actin dynamics have not been studied. Through quantum mechanical/molecular mechanical (QM/MM) and density function theory (DFT) simulations after the molecular dynamics (MD) simulations of the docked complex of F-actin and CurE, a detailed transition state (TS) model for the Michael reaction is proposed. The TS model shows nucleophilic attack of the sulphur of Cys257 at the β-carbon of Michael Acceptor of CurE producing an enol intermediate that forms a covalent bond with CurE. The MD results show a clear difference between the structure of the F-actin in free form and F-actin complexed with CurE. CurE affects the conformation of the nucleotide binding pocket increasing the binding affinity between F-actin and ADP, which in turn could affect the nucleotide exchange. CurE binding also limits the correlated displacement of the relatively flexible domain 1 of F-actin causing the protein to retain a flat structure and to transform into a stable "tense" state. This structural transition could inhibit depolymerization of F-actin. In conclusion, CurE allosterically modulates ADP and stabilizes F-actin structure, thereby affecting nucleotide exchange and depolymerization of F-actin.

  4. Characterization of Actin Filament Dynamics during Mitosis in Wheat Protoplasts under UV-B Radiation

    PubMed Central

    Chen, Huize; Han, Rong

    2016-01-01

    Enhanced ultraviolet-B (UV-B) radiation is caused by the thinning ozone and affects photosynthesis and crop yield. Recently, UV-B radiation has been considered as an environmental signal that regulates plant growth. Elucidating the downstream effectors in UV-B-triggered pathways is of particular interest. Previous studies have shown that actin filaments (AFs) play many roles during cell physiological processes. However, the underlying response of AFs to UV-B radiation remains unclear. In this study, wheat protoplasts were isolated from 7-d-old leaves. The dynamics of AFs during mitosis were observed under different treatments. The protoplasts were treated with UV-B radiation, cytochalasin B (CB) and jasplakinolide (JAS). Ph-FITC labelling results revealed typical actin filament structures in the control group; AFs were rearranged under UV-B radiation. AFs polymerized into bundles during interphase, the preprophase band (PPB) structure was destroyed during prophase, and the AFs gathered into plaques during metaphase in response to UV-B radiation. During anaphase and telophase, the distribution of AFs was dispersed. Pharmacologic experiments revealed that CB induced apoptosis and JAS induced nuclear division without cytokinesis in wheat protoplasts. These results indicated that AFs respond to UV-B radiation during mitosis, supplying evidence of UV-B signal transduction in plants. PMID:26823006

  5. Characterization of Actin Filament Dynamics during Mitosis in Wheat Protoplasts under UV-B Radiation.

    PubMed

    Chen, Huize; Han, Rong

    2016-01-01

    Enhanced ultraviolet-B (UV-B) radiation is caused by the thinning ozone and affects photosynthesis and crop yield. Recently, UV-B radiation has been considered as an environmental signal that regulates plant growth. Elucidating the downstream effectors in UV-B-triggered pathways is of particular interest. Previous studies have shown that actin filaments (AFs) play many roles during cell physiological processes. However, the underlying response of AFs to UV-B radiation remains unclear. In this study, wheat protoplasts were isolated from 7-d-old leaves. The dynamics of AFs during mitosis were observed under different treatments. The protoplasts were treated with UV-B radiation, cytochalasin B (CB) and jasplakinolide (JAS). Ph-FITC labelling results revealed typical actin filament structures in the control group; AFs were rearranged under UV-B radiation. AFs polymerized into bundles during interphase, the preprophase band (PPB) structure was destroyed during prophase, and the AFs gathered into plaques during metaphase in response to UV-B radiation. During anaphase and telophase, the distribution of AFs was dispersed. Pharmacologic experiments revealed that CB induced apoptosis and JAS induced nuclear division without cytokinesis in wheat protoplasts. These results indicated that AFs respond to UV-B radiation during mitosis, supplying evidence of UV-B signal transduction in plants. PMID:26823006

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

    PubMed

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

    2013-04-01

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

  7. Transport of cytoskeletal elements in the squid giant axon.

    PubMed Central

    Terasaki, M; Schmidek, A; Galbraith, J A; Gallant, P E; Reese, T S

    1995-01-01

    In order to explore how cytoskeletal proteins are moved by axonal transport, we injected fluorescent microtubules and actin filaments as well as exogenous particulates into squid giant axons and observed their movements by confocal microscopy. The squid giant axon is large enough to allow even cytoskeletal assemblies to be injected without damaging the axon or its transport mechanisms. Negatively charged, 10- to 500-nm beads and large dextrans moved down the axon, whereas small (70 kDa) dextrans diffused in all directions and 1000-nm beads did not move. Only particles with negative charge were transported. Microtubules and actin filaments, which have net negative charges, made saltatory movements down the axon, resulting in a net rate approximating that previously shown for slow transport of cytoskeletal elements. The present observations suggest that particle size and charge determine which materials are transported down the axon. Images Fig. 1 Fig. 2 Fig. 3 PMID:8524791

  8. Transport of cytoskeletal elements in the squid giant axon.

    PubMed

    Terasaki, M; Schmidek, A; Galbraith, J A; Gallant, P E; Reese, T S

    1995-12-01

    In order to explore how cytoskeletal proteins are moved by axonal transport, we injected fluorescent microtubules and actin filaments as well as exogenous particulates into squid giant axons and observed their movements by confocal microscopy. The squid giant axon is large enough to allow even cytoskeletal assemblies to be injected without damaging the axon or its transport mechanisms. Negatively charged, 10- to 500-nm beads and large dextrans moved down the axon, whereas small (70 kDa) dextrans diffused in all directions and 1000-nm beads did not move. Only particles with negative charge were transported. Microtubules and actin filaments, which have net negative charges, made saltatory movements down the axon, resulting in a net rate approximating that previously shown for slow transport of cytoskeletal elements. The present observations suggest that particle size and charge determine which materials are transported down the axon.

  9. Analysis of the human cofilin 1 structure reveals conformational changes required for actin binding

    PubMed Central

    Klejnot, Marta; Gabrielsen, Mads; Cameron, Jenifer; Mleczak, Andrzej; Talapatra, Sandeep K.; Kozielski, Frank; Pannifer, Andrew; Olson, Michael F.

    2013-01-01

    The actin cytoskeleton is the chassis that gives a cell its shape and structure, and supplies the power for numerous dynamic processes including motility, endocytosis, intracellular transport and division. To perform these activities, the cytoskeleton undergoes constant remodelling and reorganization. One of the major actin-remodelling families are the cofilin proteins, made up of cofilin 1, cofilin 2 and actin-depolymerizing factor (ADF), which sever aged ADP-associated actin filaments to reduce filament length and provide new potential nucleation sites. Despite the significant interest in cofilin as a central node in actin-cytoskeleton dynamics, to date the only forms of cofilin for which crystal structures have been solved are from the yeast, Chromalveolata and plant kingdoms; none have previously been reported for an animal cofilin protein. Two distinct regions in animal cofilin are significantly larger than in the forms previously crystallized, suggesting that they would be uniquely organized. Therefore, it was sought to determine the structure of human cofilin 1 by X-ray crystallography to elucidate how it could interact with and regulate dynamic actin-cytoskeletal structures. Although wild-type human cofilin 1 proved to be recalcitrant, a C147A point mutant yielded crystals that diffracted to 2.8 Å resolution. These studies revealed how the actin-binding helix undergoes a conformational change that increases the number of potential hydrogen bonds available for substrate binding. PMID:23999301

  10. High-resolution elasticity maps and cytoskeletal dynamics of neurons measured by combined fluorescence and atomic force microscopy

    NASA Astrophysics Data System (ADS)

    Staii, Cristian

    2014-03-01

    Detailed knowledge of mechanical parameters such as cell elasticity, stiffness of the growth substrate, or traction stresses generated during axonal extensions is essential for understanding the mechanisms that control neuronal growth. Here I present results obtained in my research group, which combine Atomic Force Microscopy and Fluorescence Microscopy measurements to produce systematic, high-resolution elasticity maps for different types of live neuronal cells cultured on glass or biopolymer-based substrates. We measure how the stiffness of neurons changes both during neurite outgrowth and upon chemical modification (disruption of the cytoskeleton) of the cell. We find a reversible local stiffening of the cell during growth, and show that the increase in local elastic modulus is primarily due to the formation of microtubules in the cell soma. We also report a reversible shift in the elastic modulus of the cortical neurons cytoskeleton with temperature, from tubulin dominated regions at 37C to actin dominated regions at 25C. We demonstrate that the dominant mechanism by which the elasticity of the neuronal soma changes in response to temperature is the contractile stiffening of the actin component of the cytoskeleton induced by the activity of myosin II motors. We acknowledge financial support from NSF grant CBET 1067093.

  11. MicroRNA-34a Modulates Cytoskeletal Dynamics through Regulating RhoA/Rac1 Cross-talk in Chondroblasts*

    PubMed Central

    Kim, Dongkyun; Song, Jinsoo; Kim, Sunhyo; Park, Hyang Mi; Chun, Churl-Hong; Sonn, Jongkyung; Jin, Eun-Jung

    2012-01-01

    MicroRNAs (miRNAs) have been implicated in various cellular processes, such as cell fate determination, cell death, and tumorigenesis. In the present study, we investigated the role of miRNA-34a (miR-34a) in the reorganization of the actin cytoskeleton, which is essential for chondrocyte differentiation. miRNA arrays to identify genes that appeared to be up-regulated or down-regulated during chondrogenesis were applied with chondrogenic progenitors treated with JNK inhibitor. PNA-based antisense oligonucleotides and miRNA precursor were used for investigation of the functional roles of miR-34a. We found that, in chick chondroprogenitors treated with JNK inhibitor, which suppresses chondrogenic differentiation, the expression levels of miR-34a and RhoA1 are up-regulated through modulation of Rac1 expression. Blockade of miR-34a via the use of PNA-based antisense oligonucleotides was associated with decreased protein expression of RhoA (a known modulator of stress fiber expression), down-regulation of stress fibers, up-regulation of Rac1, and recovery of protein level of type II collagen. miR-34a regulates RhoA/Rac1 cross-talk and negatively modulates reorganization of the actin cytoskeleton, which is one of the essential processes for establishing chondrocyte-specific morphology. PMID:22351754

  12. Multiscale modeling of cell shape from the actin cytoskeleton.

    PubMed

    Rangamani, Padmini; Xiong, Granville Yuguang; Iyengar, Ravi

    2014-01-01

    The actin cytoskeleton is a dynamic structure that constantly undergoes complex reorganization events during many cellular processes. Mathematical models and simulations are powerful tools that can provide insight into the physical mechanisms underlying these processes and make predictions that can be experimentally tested. Representation of the interactions of the actin filaments with the plasma membrane and the movement of the plasma membrane for computation remains a challenge. Here, we provide an overview of the different modeling approaches used to study cytoskeletal dynamics and highlight the differential geometry approach that we have used to implement the interactions between the plasma membrane and the cytoskeleton. Using cell spreading as an example, we demonstrate how this approach is able to successfully capture in simulations, experimentally observed behavior. We provide a perspective on how the differential geometry approach can be used for other biological processes. PMID:24560144

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

    PubMed

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

    2012-12-01

    Although prostaglandins (PGs)-lipid signals produced downstream of cyclooxygenase (COX) enzymes-regulate actin cytoskeletal dynamics, their mechanisms of action are unknown. We previously established Drosophila oogenesis, in particular nurse cell dumping, as a new model to determine how PGs regulate actin remodeling. PGs, and thus the Drosophila COX-like enzyme Pxt, are required for both the parallel actin filament bundle formation and the cortical actin strengthening required for dumping. Here we provide the first link between Fascin (Drosophila Singed, Sn), an actin-bundling protein, and PGs. Loss of either pxt or fascin results in similar actin defects. Fascin interacts, both pharmacologically and genetically, with PGs, as reduced Fascin levels enhance the effects of COX inhibition and synergize with reduced Pxt levels to cause both parallel bundle and cortical actin defects. Conversely, overexpression of Fascin in the germline suppresses the effects of COX inhibition and genetic loss of Pxt. These data lead to the conclusion that PGs regulate Fascin to control actin remodeling. This novel interaction has implications beyond Drosophila, as both PGs and Fascin-1, in mammalian systems, contribute to cancer cell migration and invasion.

  14. Distinct cytoskeletal domains revealed in sperm cells

    PubMed Central

    1984-01-01

    Antibodies against different cytoskeletal proteins were used to study the cytoskeletal organization of human spermatozoa. A positive staining with actin antibodies was seen in both the acrosomal cap region and the principal piece region of the tail. However, no staining was obtained with nitrobenzoxadiazol-phallacidin, suggesting that most of the actin was in the nonpolymerized form. Most of the myosin immunoreactivity was confirmed to a narrow band in the neck region of spermatozoa. Tubulin was located to the entire tail, whereas vimentin was only seen in a discrete band-like structure encircling the sperm head, apparently coinciding with the equatorial segment region. Surface staining of the spermatozoa with fluorochrome-coupled Helix pomatia agglutinin revealed a similar band-like structure that co-distributed with the vimentin- specific staining. Instead, other lectin conjugates used labeled either the acrosomal cap region (peanut and soybean agglutinins), both the acrosomal cap and the postacrosomal region of the head (concanavalin A), or the whole sperm cell surface membrane (wheat germ and lens culinaris agglutinins and ricinus communis agglutinin l). In lectin blotting experiments, the Helix pomatia agglutinin-binding was assigned to a 80,000-mol-wt polypeptide which, together with vimentin, also resisted treatment with Triton X-100. Only the acrosomal cap and the principal piece of the tail were decorated with rabbit and hydridoma antibodies against an immunoanalogue of erythrocyte alpha-spectrin (p230). p230 appeared to be the major calmodulin-binding polypeptide in spermatozoa, as shown by a direct overlay assay of electrophoretic blots of spermatozoa with 125I-calmodulin. The results indicate that spermatozoa have a highly specialized cytoskeletal organization and that the distribution of actin, spectrin, and vimentin can be correlated with distinct surface specializations of the sperm cells. This suggest that cytoskeleton may regulate the maintenance

  15. Modulation of the interaction between G-actin and thymosin beta 4 by the ATP/ADP ratio: possible implication in the regulation of actin dynamics.

    PubMed Central

    Carlier, M F; Jean, C; Rieger, K J; Lenfant, M; Pantaloni, D

    1993-01-01

    The interaction of G-actin with thymosin beta 4 (T beta 4), the major G-actin-sequestering protein in motile and proliferating cells, has been analyzed in vitro. T beta 4 is found to have a 50-fold higher affinity for MgATP-actin than for MgADP-actin. These results imply that in resting platelets and neutrophils, actin is sequestered by T beta 4 as MgATP-G-actin. Kinetic experiments and theoretical calculations demonstrate that this ATP/ADP dependence of T beta 4 affinity for G-actin can generate a mechanism of desequestration of G-actin by ADP, in the presence of physiological concentrations of T beta 4 (approximately 0.1 mM). The desequestration of G-actin by ADP is kinetically enhanced by profilin, which accelerates the dissociation of ATP from G-actin. Whether a local drop in the ATP/ADP ratio can allow local, transient desequestration and polymerization of actin either close to the plasma membrane, following platelet or neutrophil stimulation, or behind the Listeria bacterium in the host cell, while the surrounding cytoplasm contains sequestered ATP-G-actin, is an open issue raised by the present work. PMID:8506348

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

    NASA Astrophysics Data System (ADS)

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

    1999-06-01

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

  17. Actinic Keratosis

    MedlinePlus

    ... rashes clinical tools newsletter | contact Share | Actinic Keratosis (Solar Keratosis) Information for adults A A A Actinic ... the touch. Overview Actinic keratoses, also known as solar keratoses, are small rough or scaly areas of ...

  18. Clathrin is Important for Normal Actin Dynamics and Progression of Sla2p-Containing Patches During Endocytosis in Yeast

    PubMed Central

    Newpher, Thomas M.; Lemmon, Sandra K.

    2010-01-01

    Clathrin is a major vesicle coat protein involved in receptor-mediated endocytosis. In yeast and higher eukaryotes, clathrin is recruited to the plasma membrane during the early stage of endocytosis along with clathrin-associated adaptors. As coated pits undergo maturation, a burst of actin polymerization accompanies and helps drive vesicle internalization. Here, we investigate the dynamics of clathrin relative to the early endocytic patch protein Sla2p. We find that clathrin is recruited to the cortex prior to Sla2p. In the absence of clathrin, normal numbers of Sla2p patches form, but many do not internalize or are dramatically delayed in completion of endocytosis. Patches that do internalize receive Sla1p late, which is followed by Abp1, which appears near the end of Sla2p lifetime. In addition, clathrin mutants develop actin comet tails, suggesting an important function in actin patch organization/dynamics. Similar to its mammalian counterparts, the light chain (LC) subunit of yeast clathrin interacts directly with the coiled-coil domain of Sla2p. A mutant of Sla2p that no longer interacts with LC (sla2Δ376-573) results in delayed progression of endocytic patches and aberrant actin dynamics. These data demonstrate an important role for clathrin in organization and progression of early endocytic patches to the late stages of endocytosis. PMID:16643280

  19. Assembly Kinetics Determine the Architecture of α-actinin Crosslinked F-actin Networks

    PubMed Central

    Falzone, Tobias T.; Lenz, Martin; Kovar, David R.; Gardel, Margaret L.

    2013-01-01

    The actin cytoskeleton is organized into diverse meshworks and bundles that support many aspects of cell physiology. Understanding the self-assembly of these actin-based structures is essential for developing predictive models of cytoskeletal organization. Here we show that the competing kinetics of bundle formation with the onset of dynamic arrest arising from filament entanglements and cross-linking determine the architecture of reconstituted actin networks formed with α-actinin cross-links. Cross-link mediated bundle formation only occurs in dilute solutions of highly mobile actin filaments. As actin polymerization proceeds, filament mobility and bundle formation are arrested concomitantly. By controlling the onset of dynamic arrest, perturbations to actin assembly kinetics dramatically alter the architecture of biochemically identical samples. Thus, the morphology of reconstituted F-actin networks is a kinetically determined structure similar to those formed by physical gels and glasses. These results establish mechanisms controlling the structure and mechanics in diverse semi-flexible biopolymer networks. PMID:22643888

  20. Actin and myosin function in directed vacuole movement during cell division in Saccharomyces cerevisiae

    PubMed Central

    1996-01-01

    During cell division, cytoplasmic organelles are not synthesized de novo, rather they are replicated and partitioned between daughter cells. Partitioning of the vacuole in the budding yeast Saccharomyces cerevisiae is coordinated with the cell cycle and involves a dramatic translocation of a portion of the parental organelle from the mother cell into the bud. While the molecular mechanisms that mediate this event are unknown, the vacuole's rapid and directed movements suggest cytoskeleton involvement. To identify cytoskeletal components that function in this process, vacuole inheritance was examined in a collection of actin mutants. Six strains were identified as being defective in vacuole inheritance. Tetrad analysis verified that the defect cosegregates with the mutant actin gene. One strain with a deletion in a myosin-binding region was analyzed further. The vacuole inheritance defect in this strain appears to result from the loss of a specific actin function; the actin cytoskeleton is intact and protein targeting to the vacuole is normal. Consistent with these findings, a mutation in the actin-binding domain of Myo2p, a class V unconventional myosin, abolishes vacuole inheritance. This suggests that Myo2p serves as a molecular motor for vacuole transport along actin filaments. The location of actin and Myo2p relative to the vacuole membrane is consistent with this model. Additional studies suggest that the actin filaments used for vacuole transport are dynamic, and that profilin plays a critical role in regulating their assembly. These results present the first demonstration that specific cytoskeletal proteins function in vacuole inheritance. PMID:8978821

  1. Triggering signaling pathways using F-actin self-organization

    PubMed Central

    Colin, A.; Bonnemay, L.; Gayrard, C.; Gautier, J.; Gueroui, Z.

    2016-01-01

    The spatiotemporal organization of proteins within cells is essential for cell fate behavior. Although it is known that the cytoskeleton is vital for numerous cellular functions, it remains unclear how cytoskeletal activity can shape and control signaling pathways in space and time throughout the cell cytoplasm. Here we show that F-actin self-organization can trigger signaling pathways by engineering two novel properties of the microfilament self-organization: (1) the confinement of signaling proteins and (2) their scaffolding along actin polymers. Using in vitro reconstitutions of cellular functions, we found that both the confinement of nanoparticle-based signaling platforms powered by F-actin contractility and the scaffolding of engineered signaling proteins along actin microfilaments can drive a signaling switch. Using Ran-dependent microtubule nucleation, we found that F-actin dynamics promotes the robust assembly of microtubules. Our in vitro assay is a first step towards the development of novel bottom-up strategies to decipher the interplay between cytoskeleton spatial organization and signaling pathway activity. PMID:27698406

  2. Dynamic Filament Formation by a Divergent Bacterial Actin-Like ParM Protein

    PubMed Central

    Brzoska, Anthony J.; Jensen, Slade O.; Barton, Deborah A.; Davies, Danielle S.; Overall, Robyn L.; Skurray, Ronald A.; Firth, Neville

    2016-01-01

    Actin-like proteins (Alps) are a diverse family of proteins whose genes are abundant in the chromosomes and mobile genetic elements of many bacteria. The low-copy-number staphylococcal multiresistance plasmid pSK41 encodes ParM, an Alp involved in efficient plasmid partitioning. pSK41 ParM has previously been shown to form filaments in vitro that are structurally dissimilar to those formed by other bacterial Alps. The mechanistic implications of these differences are not known. In order to gain insights into the properties and behavior of the pSK41 ParM Alp in vivo, we reconstituted the parMRC system in the ectopic rod-shaped host, E. coli, which is larger and more genetically amenable than the native host, Staphylococcus aureus. Fluorescence microscopy showed a functional fusion protein, ParM-YFP, formed straight filaments in vivo when expressed in isolation. Strikingly, however, in the presence of ParR and parC, ParM-YFP adopted a dramatically different structure, instead forming axial curved filaments. Time-lapse imaging and selective photobleaching experiments revealed that, in the presence of all components of the parMRC system, ParM-YFP filaments were dynamic in nature. Finally, molecular dissection of the parMRC operon revealed that all components of the system are essential for the generation of dynamic filaments. PMID:27310470

  3. Filopodial retraction force is generated by cortical actin dynamics and controlled by reversible tethering at the tip

    PubMed Central

    Bornschlögl, Thomas; Romero, Stéphane; Vestergaard, Christian L.; Joanny, Jean-François; Van Nhieu, Guy Tran; Bassereau, Patricia

    2013-01-01

    Filopodia are dynamic, finger-like plasma membrane protrusions that sense the mechanical and chemical surroundings of the cell. Here, we show in epithelial cells that the dynamics of filopodial extension and retraction are determined by the difference between the actin polymerization rate at the tip and the retrograde flow at the base of the filopodium. Adhesion of a bead to the filopodial tip locally reduces actin polymerization and leads to retraction via retrograde flow, reminiscent of a process used by pathogens to invade cells. Using optical tweezers, we show that filopodial retraction occurs at a constant speed against counteracting forces up to 50 pN. Our measurements point toward retrograde flow in the cortex together with frictional coupling between the filopodial and cortical actin networks as the main retraction-force generator for filopodia. The force exerted by filopodial retraction, however, is limited by the connection between filopodial actin filaments and the membrane at the tip. Upon mechanical rupture of the tip connection, filopodia exert a passive retraction force of 15 pN via their plasma membrane. Transient reconnection at the tip allows filopodia to continuously probe their surroundings in a load-and-fail manner within a well-defined force range. PMID:24198333

  4. Effect of Flumorph on F-Actin Dynamics in the Potato Late Blight Pathogen Phytophthora infestans.

    PubMed

    Hua, Chenlei; Kots, Kiki; Ketelaar, Tijs; Govers, Francine; Meijer, Harold J G

    2015-04-01

    Oomycetes are fungal-like pathogens that cause notorious diseases. Protecting crops against oomycetes requires regular spraying with chemicals, many with an unknown mode of action. In the 1990s, flumorph was identified as a novel crop protection agent. It was shown to inhibit the growth of oomycete pathogens including Phytophthora spp., presumably by targeting actin. We recently generated transgenic Phytophthora infestans strains that express Lifeact-enhanced green fluorescent protein (eGFP), which enabled us to monitor the actin cytoskeleton during hyphal growth. For analyzing effects of oomicides on the actin cytoskeleton in vivo, the P. infestans Lifeact-eGFP strain is an excellent tool. Here, we confirm that flumorph is an oomicide with growth inhibitory activity. Microscopic analyses showed that low flumorph concentrations provoked hyphal tip swellings accompanied by accumulation of actin plaques in the apex, a feature reminiscent of tips of nongrowing hyphae. At higher concentrations, swelling was more pronounced and accompanied by an increase in hyphal bursting events. However, in hyphae that remained intact, actin filaments were indistinguishable from those in nontreated, nongrowing hyphae. In contrast, in hyphae treated with the actin depolymerizing drug latrunculin B, no hyphal bursting was observed but the actin filaments were completely disrupted. This difference demonstrates that actin is not the primary target of flumorph. PMID:25496300

  5. All-atom molecular dynamics simulations of actin-myosin interactions: a comparative study of cardiac α myosin, β myosin, and fast skeletal muscle myosin.

    PubMed

    Li, Minghui; Zheng, Wenjun

    2013-11-26

    Myosins are a superfamily of actin-binding motor proteins with significant variations in kinetic properties (such as actin binding affinity) between different isoforms. It remains unknown how such kinetic variations arise from the structural and dynamic tuning of the actin-myosin interface at the amino acid residue level. To address this key issue, we have employed molecular modeling and simulations to investigate, with atomistic details, the isoform dependence of actin-myosin interactions in the rigor state. By combining electron microscopy-based docking with homology modeling, we have constructed three all-atom models for human cardiac α and β and rabbit fast skeletal muscle myosin in complex with three actin subunits in the rigor state. Starting from these models, we have performed extensive all-atom molecular dynamics (MD) simulations (total of 100 ns per system) and then used the MD trajectories to calculate actin-myosin binding free energies with contributions from both electrostatic and nonpolar forces. Our binding calculations are in good agreement with the experimental finding of isoform-dependent differences in actin binding affinity between these myosin isoforms. Such differences are traced to changes in actin-myosin electrostatic interactions (i.e., hydrogen bonds and salt bridges) that are highly dynamic and involve several flexible actin-binding loops. By partitioning the actin-myosin binding free energy to individual myosin residues, we have also identified key myosin residues involved in the actin-myosin interactions, some of which were previously validated experimentally or implicated in cardiomyopathy mutations, and the rest make promising targets for future mutational experiments. PMID:24224850

  6. The role of actin networks in cellular mechanosensing

    NASA Astrophysics Data System (ADS)

    Azatov, Mikheil

    Physical processes play an important role in many biological phenomena, such as wound healing, organ development, and tumor metastasis. During these processes, cells constantly interact with and adapt to their environment by exerting forces to mechanically probe the features of their surroundings and generating appropriate biochemical responses. The mechanisms underlying how cells sense the physical properties of their environment are not well understood. In this thesis, I present my studies to investigate cellular responses to the stiffness and topography of the environment. In order to sense the physical properties of their environment, cells dynamically reorganize the structure of their actin cytoskeleton, a dynamic network of biopolymers, altering the shape and spatial distribution of protein assemblies. Several observations suggest that proteins that crosslink actin filaments may play an important role in cellular mechanosensitivity. Palladin is an actin-crosslinking protein that is found in the lamellar actin network, stress fibers and focal adhesions, cellular structures that are critical for mechanosensing of the physical environment. By virtue of its close interactions with these structures in the cell, palladin may play an important role in cell mechanics. However, the role of actin crosslinkers in general, and palladin in particular, in cellular force generation and mechanosensing is not well known. I have investigated the role of palladin in regulating the plasticity of the actin cytoskeleton and cellular force generation in response to alterations in substrate stiffness. I have shown that the expression levels of palladin modulate the forces exerted by cells and their ability to sense substrate stiffness. Perturbation experiments also suggest that palladin levels in cells altered myosin motor activity. These results suggest that the actin crosslinkers, such as palladin, and myosin motors coordinate for optimal cell function and to prevent aberrant

  7. Viscoelastic properties of actin-coated membranes.

    PubMed

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

    2001-02-01

    In living cells, cytoskeletal filaments interact with the plasma membrane to form structures that play a key role in cell shape and mechanical properties. To study the interaction between these basic components, we designed an in vitro self-assembled network of actin filaments attached to the outer surface of giant unilamellar vesicles. Optical tweezers and single-particle tracking experiments are used to study the rich dynamics of these actin-coated membranes (ACM). We show that microrheology studies can be carried out on such an individual microscopic object. The principle of the experiment consists in measuring the thermally excited position fluctuations of a probe bead attached biochemically to the membrane. We propose a model that relates the power spectrum of these thermal fluctuations to the viscoelastic properties of the membrane. The presence of the actin network modifies strongly the membrane dynamics with respect to a fluid, lipid bilayer one. It induces first a finite (omega=0) two-dimensional (2D) shear modulus G(0)(2D) approximately 0.5 to 5 microN/m in the membrane plane. Moreover, the frequency dependence at high frequency of the shear modulus [G(')(2D)(f ) approximately f(0.85+/-0.07)] and of the bending modulus (kappa(ACM)(f) approximately f(0.55+/-0.21)) demonstrate the viscoelastic behavior of the composite membrane. These results are consistent with a common exponent of 0.75 for both moduli as expected from our model and from prior measurements on actin solutions.

  8. Cortical instability drives periodic supracellular actin pattern formation in epithelial tubes.

    PubMed

    Hannezo, Edouard; Dong, Bo; Recho, Pierre; Joanny, Jean-François; Hayashi, Shigeo

    2015-07-14

    An essential question of morphogenesis is how patterns arise without preexisting positional information, as inspired by Turing. In the past few years, cytoskeletal flows in the cell cortex have been identified as a key mechanism of molecular patterning at the subcellular level. Theoretical and in vitro studies have suggested that biological polymers such as actomyosin gels have the property to self-organize, but the applicability of this concept in an in vivo setting remains unclear. Here, we report that the regular spacing pattern of supracellular actin rings in the Drosophila tracheal tubule is governed by a self-organizing principle. We propose a simple biophysical model where pattern formation arises from the interplay of myosin contractility and actin turnover. We validate the hypotheses of the model using photobleaching experiments and report that the formation of actin rings is contractility dependent. Moreover, genetic and pharmacological perturbations of the physical properties of the actomyosin gel modify the spacing of the pattern, as the model predicted. In addition, our model posited a role of cortical friction in stabilizing the spacing pattern of actin rings. Consistently, genetic depletion of apical extracellular matrix caused strikingly dynamic movements of actin rings, mirroring our model prediction of a transition from steady to chaotic actin patterns at low cortical friction. Our results therefore demonstrate quantitatively that a hydrodynamical instability of the actin cortex can trigger regular pattern formation and drive morphogenesis in an in vivo setting.

  9. Cortical instability drives periodic supracellular actin pattern formation in epithelial tubes

    PubMed Central

    Hannezo, Edouard; Dong, Bo; Recho, Pierre; Joanny, Jean-François; Hayashi, Shigeo

    2015-01-01

    An essential question of morphogenesis is how patterns arise without preexisting positional information, as inspired by Turing. In the past few years, cytoskeletal flows in the cell cortex have been identified as a key mechanism of molecular patterning at the subcellular level. Theoretical and in vitro studies have suggested that biological polymers such as actomyosin gels have the property to self-organize, but the applicability of this concept in an in vivo setting remains unclear. Here, we report that the regular spacing pattern of supracellular actin rings in the Drosophila tracheal tubule is governed by a self-organizing principle. We propose a simple biophysical model where pattern formation arises from the interplay of myosin contractility and actin turnover. We validate the hypotheses of the model using photobleaching experiments and report that the formation of actin rings is contractility dependent. Moreover, genetic and pharmacological perturbations of the physical properties of the actomyosin gel modify the spacing of the pattern, as the model predicted. In addition, our model posited a role of cortical friction in stabilizing the spacing pattern of actin rings. Consistently, genetic depletion of apical extracellular matrix caused strikingly dynamic movements of actin rings, mirroring our model prediction of a transition from steady to chaotic actin patterns at low cortical friction. Our results therefore demonstrate quantitatively that a hydrodynamical instability of the actin cortex can trigger regular pattern formation and drive morphogenesis in an in vivo setting. PMID:26077909

  10. Actin-Like Protein 1 (ALP1) is a component of dynamic, high molecular weight complexes in Toxoplasma gondii

    PubMed Central

    Gordon, Jennifer L.; Buguliskis, Jeffrey S.; Buske, Paul J.; David Sibley, L

    2009-01-01

    Apicomplexan parasites, such as Toxoplasma gondii, rely on actin-based motility for cell invasion, yet conventional actin does not appear to be required for cell division in these parasites. Apicomplexans also contain a variety of actin-related proteins (Arps); however, most of these not directly orthologous to Arps in well-studied systems. We recently identified an apicomplexan-specific member of this family called Actin-Like Protein 1, (ALP1), which plays a role in the assembly of vesicular components recruited to the inner membrane complex (IMC) of daughter cells during cell division. In addition to its enrichment at daughter cell membranes, ALP1 is localized throughout the cytoplasm both diffusely distributed and concentrated in clusters that are detected by fluorescence microscopy, suggesting it forms complexes. Using quantitative optical imaging methods, including fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP), we demonstrated that ALP1 is a component of a large complex, and that it readily exchanges between diffusible and complex-bound forms. Sedimentation and density gradient analyses revealed that ALP1 is found in a freely soluble state as well as high molecular weight complexes. During cell division, ALP1 was dynamically associated with the IMC, suggesting it rapidly cycles between freely diffusible and complex forms during daughter cell assembly. PMID:19701930

  11. Cytoskeletal Mechanics Regulating Amoeboid Cell Locomotion

    PubMed Central

    Álvarez-González, Begoña; Meili, Ruedi; Firtel, Richard; Bastounis, Effie; del Álamo, Juan C.; Lasheras, Juan C.

    2014-01-01

    Migrating cells exert traction forces when moving. Amoeboid cell migration is a common type of cell migration that appears in many physiological and pathological processes and is performed by a wide variety of cell types. Understanding the coupling of the biochemistry and mechanics underlying the process of migration has the potential to guide the development of pharmacological treatment or genetic manipulations to treat a wide range of diseases. The measurement of the spatiotemporal evolution of the traction forces that produce the movement is an important aspect for the characterization of the locomotion mechanics. There are several methods to calculate the traction forces exerted by the cells. Currently the most commonly used ones are traction force microscopy methods based on the measurement of the deformation induced by the cells on elastic substrate on which they are moving. Amoeboid cells migrate by implementing a motility cycle based on the sequential repetition of four phases. In this paper we review the role that specific cytoskeletal components play in the regulation of the cell migration mechanics. We investigate the role of specific cytoskeletal components regarding the ability of the cells to perform the motility cycle effectively and the generation of traction forces. The actin nucleation in the leading edge of the cell, carried by the ARP2/3 complex activated through the SCAR/WAVE complex, has shown to be fundamental to the execution of the cyclic movement and to the generation of the traction forces. The protein PIR121, a member of the SCAR/WAVE complex, is essential to the proper regulation of the periodic movement and the protein SCAR, also included in the SCAR/WAVE complex, is necessary for the generation of the traction forces during migration. The protein Myosin II, an important F-actin cross-linker and motor protein, is essential to cytoskeletal contractility and to the generation and proper organization of the traction forces during

  12. Interplay of active processes modulates tension and drives phase transition in self-renewing, motor-driven cytoskeletal networks

    PubMed Central

    Mak, Michael; Zaman, Muhammad H.; Kamm, Roger D.; Kim, Taeyoon

    2016-01-01

    The actin cytoskeleton—a complex, nonequilibrium network consisting of filaments, actin-crosslinking proteins (ACPs) and motors—confers cell structure and functionality, from migration to morphogenesis. While the core components are recognized, much less is understood about the behaviour of the integrated, disordered and internally active system with interdependent mechano-chemical component properties. Here we use a Brownian dynamics model that incorporates key and realistic features—specifically actin turnover, ACP (un)binding and motor walking—to reveal the nature and underlying regulatory mechanisms of overarching cytoskeletal states. We generate multi-dimensional maps that show the ratio in activity of these microscopic elements determines diverse global stress profiles and the induction of nonequilibrium morphological phase transition from homogeneous to aggregated networks. In particular, actin turnover dynamics plays a prominent role in tuning stress levels and stabilizing homogeneous morphologies in crosslinked, motor-driven networks. The consequence is versatile functionality, from dynamic steady-state prestress to large, pulsed constrictions. PMID:26744226

  13. Actin Mechanics and Fragmentation*

    PubMed Central

    De La Cruz, Enrique M.; Gardel, Margaret L.

    2015-01-01

    Cell physiological processes require the regulation and coordination of both mechanical and dynamical properties of the actin cytoskeleton. Here we review recent advances in understanding the mechanical properties and stability of actin filaments and how these properties are manifested at larger (network) length scales. We discuss how forces can influence local biochemical interactions, resulting in the formation of mechanically sensitive dynamic steady states. Understanding the regulation of such force-activated chemistries and dynamic steady states reflects an important challenge for future work that will provide valuable insights as to how the actin cytoskeleton engenders mechanoresponsiveness of living cells. PMID:25957404

  14. Regulation of the Postsynaptic Compartment of Excitatory Synapses by the Actin Cytoskeleton in Health and Its Disruption in Disease

    PubMed Central

    Stefen, Holly; Chaichim, Chanchanok

    2016-01-01

    Disruption of synaptic function at excitatory synapses is one of the earliest pathological changes seen in wide range of neurological diseases. The proper control of the segregation of neurotransmitter receptors at these synapses is directly correlated with the intact regulation of the postsynaptic cytoskeleton. In this review, we are discussing key factors that regulate the structure and dynamics of the actin cytoskeleton, the major cytoskeletal building block that supports the postsynaptic compartment. Special attention is given to the complex interplay of actin-associated proteins that are found in the synaptic specialization. We then discuss our current understanding of how disruption of these cytoskeletal elements may contribute to the pathological events observed in the nervous system under disease conditions with a particular focus on Alzheimer's disease pathology. PMID:27127658

  15. A close relationship between Cercozoa and Foraminifera supported by phylogenetic analyses based on combined amino acid sequences of three cytoskeletal proteins (actin, alpha-tubulin, and beta-tubulin).

    PubMed

    Takishita, Kiyotaka; Inagaki, Yuji; Tsuchiya, Masashi; Sakaguchi, Miako; Maruyama, Tadashi

    2005-12-01

    Recently, there has been increasing molecular evidence of phylogenetic affinity between Cercozoa and Foraminifera in the eukaryotic lineage. We performed phylogenetic analyses based on the combined (concatenated) amino acid sequence data of actin, alpha-tubulin, and beta-tubulin from a wide variety of eukaryotes, including the foraminifers Planoglabratella opercularis and Reticulomyxa filosa, as well as cercomonad and chlorarachniophyte members of Cercozoa. A monophyletic lineage composed of two foraminiferan species branched with the centroheliozoan species Raphidiophrys contractilis was reconstructed in both Bayesian and maximum-likelihood (ML) analyses under 'linked' models, enforcing a single set of the parameters (the parameter for among-site rate variation and branch lengths) on the entire combined alignment. Considering the extremely divergent nature of Foraminifera and Raphidiophyrs tubulins, the union of these lineages recovered is most probably a long-branch attraction artifact due to ignoring gene-specific evolutionary processes. On the other hand, the foraminiferan lineage was within the radiation of Cercozoa in Bayesian analyses under 'unlinked' model conditions, accommodating differences in evolutionary processes across the three genes in the combined alignment. The Foraminifera+Cercozoa affinity recovered in the latter multi-gene analyses is most likely genuine, and thus our data presented here provide further support for the close relationship between these two protist lineages.

  16. Rictor Regulates Spermatogenesis by Controlling Sertoli Cell Cytoskeletal Organization and Cell Polarity in the Mouse Testis.

    PubMed

    Dong, Heling; Chen, Zhenguo; Wang, Caixia; Xiong, Zhi; Zhao, Wanlu; Jia, Chunhong; Lin, Jun; Lin, Yan; Yuan, Weiping; Zhao, Allan Z; Bai, Xiaochun

    2015-11-01

    Maintenance of cell polarity is essential for Sertoli cell and blood-testis barrier (BTB) function and spermatogenesis; however, the signaling mechanisms that regulate the integrity of the cytoskeleton and polarity of Sertoli cells are not fully understood. Here, we demonstrate that rapamycin-insensitive component of target of rapamycin (TOR) (Rictor), a core component of mechanistic TOR complex 2 (mTORC2), was expressed in the seminiferous epithelium during testicular development, and was down-regulated in a cadmium chloride-induced BTB damage model. We then conditionally deleted the Rictor gene in Sertoli cells and mutant mice exhibited azoospermia and were sterile as early as 3 months old. Further study revealed that Rictor may regulate actin organization via both mTORC2-dependent and mTORC2-independent mechanisms, in which the small GTPase, ras-related C3 botulinum toxin substrate 1, and phosphorylation of the actin filament regulatory protein, Paxillin, are involved, respectively. Loss of Rictor in Sertoli cells perturbed actin dynamics and caused microtubule disarrangement, both of which accumulatively disrupted Sertoli cell polarity and BTB integrity, accompanied by testicular developmental defects, spermiogenic arrest and excessive germ cell loss in mutant mice. Together, these findings establish the importance of Rictor/mTORC2 signaling in Sertoli cell function and spermatogenesis through the maintenance of Sertoli cell cytoskeletal dynamics, BTB integrity, and cell polarity. PMID:26360620

  17. Actin dynamics affect mitochondrial quality control and aging in budding yeast.

    PubMed

    Higuchi, Ryo; Vevea, Jason D; Swayne, Theresa C; Chojnowski, Robert; Hill, Vanessa; Boldogh, Istvan R; Pon, Liza A

    2013-12-01

    Actin cables of budding yeast are bundles of F-actin that extend from the bud tip or neck to the mother cell tip, serve as tracks for bidirectional cargo transport, and undergo continuous movement from buds toward mother cells [1]. This movement, retrograde actin cable flow (RACF), is similar to retrograde actin flow in lamellipodia, growth cones, immunological synapses, dendritic spines, and filopodia [2-5]. In all cases, actin flow is driven by the push of actin polymerization and assembly at the cell cortex, and myosin-driven pulling forces deeper within the cell [6-10]. Therefore, for movement and inheritance from mothers to buds, mitochondria must "swim upstream" against the opposing force of RACF [11]. We find that increasing RACF rates results in increased fitness of mitochondria inherited by buds and that the increase in mitochondrial fitness leads to extended replicative lifespan and increased cellular healthspan. The sirtuin SIR2 is required for normal RACF and mitochondrial fitness, and increasing RACF rates in sir2Δ cells increases mitochondrial fitness and cellular healthspan but does not affect replicative lifespan. These studies support the model that RACF serves as a filter for segregation of fit from less-fit mitochondria during inheritance, which controls cellular lifespan and healthspan. They also support a role for Sir2p in these processes.

  18. Vault-poly-ADP-ribose polymerase in the Octopus vulgaris brain: a regulatory factor of actin polymerization dynamic.

    PubMed

    De Maio, Anna; Natale, Emiliana; Rotondo, Sergio; Di Cosmo, Anna; Faraone-Mennella, Maria Rosaria

    2013-09-01

    Our previous behavioural, biochemical and immunohistochemical analyses conducted in selected regions (supra/sub oesophageal masses) of the Octopus vulgaris brain detected a cytoplasmic poly-ADP-ribose polymerase (more than 90% of total enzyme activity). The protein was identified as the vault-free form of vault-poly-ADP-ribose polymerase. The present research extends and integrates the biochemical characterization of poly-ADP-ribosylation system, namely, reaction product, i.e., poly-ADP-ribose, and acceptor proteins, in the O. vulgaris brain. Immunochemical analyses evidenced that the sole poly-ADP-ribose acceptor was the octopus cytoskeleton 50-kDa actin. It was present in both free, endogenously poly-ADP-ribosylated form (70kDa) and in complex with V-poly-ADP-ribose polymerase and poly-ADP-ribose (260kDa). The components of this complex, alkali and high salt sensitive, were purified and characterized. The kind and the length of poly-ADP-ribose corresponded to linear chains of 30-35 ADP-ribose units, in accordance with the features of the polymer synthesized by the known vault-poly-ADP-ribose polymerase. In vitro experiments showed that V-poly-ADP-ribose polymerase activity of brain cytoplasmic fraction containing endogenous actin increased upon the addition of commercial actin and was highly reduced by ATP. Anti-actin immunoblot of the mixture in the presence and absence of ATP showed that the poly-ADP-ribosylation of octopus actin is a dynamic process balanced by the ATP-dependent polymerization of the cytoskeleton protein, a fundamental mechanism for synaptic plasticity.

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

    PubMed Central

    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

  20. Crumbs is an essential regulator of cytoskeletal dynamics and cell-cell adhesion during dorsal closure in Drosophila

    PubMed Central

    Flores-Benitez, David; Knust, Elisabeth

    2015-01-01

    The evolutionarily conserved Crumbs protein is required for epithelial polarity and morphogenesis. Here we identify a novel role of Crumbs as a negative regulator of actomyosin dynamics during dorsal closure in the Drosophila embryo. Embryos carrying a mutation in the FERM (protein 4.1/ezrin/radixin/moesin) domain-binding motif of Crumbs die due to an overactive actomyosin network associated with disrupted adherens junctions. This phenotype is restricted to the amnioserosa and does not affect other embryonic epithelia. This function of Crumbs requires DMoesin, the Rho1-GTPase, class-I p21-activated kinases and the Arp2/3 complex. Data presented here point to a critical role of Crumbs in regulating actomyosin dynamics, cell junctions and morphogenesis. DOI: http://dx.doi.org/10.7554/eLife.07398.001 PMID:26544546

  1. Crumbs is an essential regulator of cytoskeletal dynamics and cell-cell adhesion during dorsal closure in Drosophila.

    PubMed

    Flores-Benitez, David; Knust, Elisabeth

    2015-11-06

    The evolutionarily conserved Crumbs protein is required for epithelial polarity and morphogenesis. Here we identify a novel role of Crumbs as a negative regulator of actomyosin dynamics during dorsal closure in the Drosophila embryo. Embryos carrying a mutation in the FERM (protein 4.1/ezrin/radixin/moesin) domain-binding motif of Crumbs die due to an overactive actomyosin network associated with disrupted adherens junctions. This phenotype is restricted to the amnioserosa and does not affect other embryonic epithelia. This function of Crumbs requires DMoesin, the Rho1-GTPase, class-I p21-activated kinases and the Arp2/3 complex. Data presented here point to a critical role of Crumbs in regulating actomyosin dynamics, cell junctions and morphogenesis.

  2. Cytoskeletal Expression and Remodeling in Pluripotent Stem Cells

    PubMed Central

    Boraas, Liana C.; Guidry, Julia B.; Pineda, Emma T.; Ahsan, Tabassum

    2016-01-01

    Many emerging cell-based therapies are based on pluripotent stem cells, though complete understanding of the properties of these cells is lacking. In these cells, much is still unknown about the cytoskeletal network, which governs the mechanoresponse. The objective of this study was to determine the cytoskeletal state in undifferentiated pluripotent stem cells and remodeling with differentiation. Mouse embryonic stem cells (ESCs) and reprogrammed induced pluripotent stem cells (iPSCs), as well as the original un-reprogrammed embryonic fibroblasts (MEFs), were evaluated for expression of cytoskeletal markers. We found that pluripotent stem cells overall have a less developed cytoskeleton compared to fibroblasts. Gene and protein expression of smooth muscle cell actin, vimentin, lamin A, and nestin were markedly lower for ESCs than MEFs. Whereas, iPSC samples were heterogeneous with most cells expressing patterns of cytoskeletal proteins similar to ESCs with a small subpopulation similar to MEFs. This indicates that dedifferentiation during reprogramming is associated with cytoskeletal remodeling to a less developed state. In differentiation studies, it was found that shear stress-mediated differentiation resulted in an increase in expression of cytoskeletal intermediate filaments in ESCs, but not in iPSC samples. In the embryoid body model of spontaneous differentiation of pluripotent stem cells, however, both ESCs and iPSCs had similar gene expression for cytoskeletal proteins during early differentiation. With further differentiation, however, gene levels were significantly higher for iPSCs compared to ESCs. These results indicate that reprogrammed iPSCs more readily reacquire cytoskeletal proteins compared to the ESCs that need to form the network de novo. The strategic selection of the parental phenotype is thus critical not only in the context of reprogramming but also the ultimate functionality of the iPSC-differentiated cell population. Overall, this

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

    PubMed

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

    2008-05-01

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

  4. Formin-Dependent Synaptic Growth; Evidence that Dlar Signals via Diaphanous to Modulate Synaptic Actin and Dynamic Pioneer Microtubules

    PubMed Central

    Pawson, Catherine; Eaton, Benjamin A.; Davis, Graeme W.

    2008-01-01

    The diaphanous gene is the founding member of a family of Diaphanous Related Formin proteins (DRF). We identified diaphanous in a screen for genes that are necessary for the normal growth and stabilization of the Drosophila neuromuscular junction (NMJ). Here we demonstrate that diaphanous mutations perturb synaptic growth at the NMJ. Diaphanous protein is present both pre- and postsynaptically. However, genetic rescue experiments in combination with additional genetic interaction experiments support the conclusion that dia is necessary presynaptically for normal NMJ growth. We then document defects in both the actin and microtubule cytoskeletons in dia mutant nerve terminals. In so doing, we define and characterize a population of dynamic pioneer microtubules within the NMJ that are distinct from the bundled core of microtubules identified by the MAP1b-like protein Futsch. Defects in both synaptic actin and dynamic pioneer MTs are correlated with impaired synaptic growth in dia mutants. Finally, we present genetic evidence that Dia functions downstream of the presynaptic receptor tyrosine phosphatase Dlar and the Rho-type GEF trio to control NMJ growth. Based upon the established function of DRFs as Rho-GTPase dependent regulators of the cell cytoskeleton, we propose a model in which Diaphanous links receptor tyrosine phosphatase signaling at the plasma membrane to growth-dependent modulation of the synaptic actin and microtubule cytoskeletons. PMID:18971454

  5. Comparative transcriptomics reveals RhoE as a novel regulator of actin dynamics in bone-resorbing osteoclasts.

    PubMed

    Georgess, Dan; Mazzorana, Marlène; Terrado, José; Delprat, Christine; Chamot, Christophe; Guasch, Rosa M; Pérez-Roger, Ignacio; Jurdic, Pierre; Machuca-Gayet, Irma

    2014-02-01

    The function of osteoclasts (OCs), multinucleated giant cells (MGCs) of the monocytic lineage, is bone resorption. To resorb bone, OCs form podosomes. These are actin-rich adhesive structures that pattern into rings that drive OC migration and into "sealing-zones" (SZs) that confine the resorption lacuna. Although changes in actin dynamics during podosome patterning have been documented, the mechanisms that regulate these changes are largely unknown. From human monocytic precursors, we differentiated MGCs that express OC degradation enzymes but are unable to resorb the mineral matrix. We demonstrated that, despite exhibiting bona fide podosomes, these cells presented dysfunctional SZs. We then performed two-step differential transcriptomic profiling of bone-resorbing OCs versus nonresorbing MGCs to generate a list of genes implicated in bone resorption. From this list of candidate genes, we investigated the role of Rho/Rnd3. Using primary RhoE-deficient OCs, we demonstrated that RhoE is indispensable for OC migration and bone resorption by maintaining fast actin turnover in podosomes. We further showed that RhoE activates podosome component cofilin by inhibiting its Rock-mediated phosphorylation. We conclude that the RhoE-Rock-cofilin pathway, by promoting podosome dynamics and patterning, is central for OC migration, SZ formation, and, ultimately, bone resorption.

  6. Comparative transcriptomics reveals RhoE as a novel regulator of actin dynamics in bone-resorbing osteoclasts

    PubMed Central

    Georgess, Dan; Mazzorana, Marlène; Terrado, José; Delprat, Christine; Chamot, Christophe; Guasch, Rosa M.; Pérez-Roger, Ignacio; Jurdic, Pierre; Machuca-Gayet, Irma

    2014-01-01

    The function of osteoclasts (OCs), multinucleated giant cells (MGCs) of the monocytic lineage, is bone resorption. To resorb bone, OCs form podosomes. These are actin-rich adhesive structures that pattern into rings that drive OC migration and into “sealing-zones” (SZs) that confine the resorption lacuna. Although changes in actin dynamics during podosome patterning have been documented, the mechanisms that regulate these changes are largely unknown. From human monocytic precursors, we differentiated MGCs that express OC degradation enzymes but are unable to resorb the mineral matrix. We demonstrated that, despite exhibiting bona fide podosomes, these cells presented dysfunctional SZs. We then performed two-step differential transcriptomic profiling of bone-resorbing OCs versus nonresorbing MGCs to generate a list of genes implicated in bone resorption. From this list of candidate genes, we investigated the role of Rho/Rnd3. Using primary RhoE-deficient OCs, we demonstrated that RhoE is indispensable for OC migration and bone resorption by maintaining fast actin turnover in podosomes. We further showed that RhoE activates podosome component cofilin by inhibiting its Rock-mediated phosphorylation. We conclude that the RhoE-Rock-cofilin pathway, by promoting podosome dynamics and patterning, is central for OC migration, SZ formation, and, ultimately, bone resorption. PMID:24284899

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

    PubMed

    Busiek, Kimberly K; Margolin, William

    2015-03-16

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

  8. Cytoskeletal proteins inside human immunodeficiency virus type 1 virions.

    PubMed Central

    Ott, D E; Coren, L V; Kane, B P; Busch, L K; Johnson, D G; Sowder, R C; Chertova, E N; Arthur, L O; Henderson, L E

    1996-01-01

    We have identified three types of cytoskeletal proteins inside human immunodeficiency virus type 1 (HIV-1) virions by analyzing subtilisin-digested particles. HIV-1 virions were digested with protease, and the treated particles were isolated by sucrose density centrifugation. This method removes both exterior viral proteins and proteins associated with microvesicles that contaminate virion preparations. Since the proteins inside the virion are protected from digestion by the viral lipid envelope, they can be isolated and analyzed after treatment. Experiments presented here demonstrated that this procedure removed more than 95% of the protein associated with microvesicles. Proteins in digested HIV-1(MN) particles from infected H9 and CEM(ss) cell lines were analyzed by high-pressure liquid chromatography, protein sequencing, and immunoblotting. The data revealed that three types of cytoskeletal proteins are present in virions at different concentrations relative to the molar level of Gag: actin (approximately 10 to 15%), ezrin and moesin (approximately 2%), and cofilin (approximately 2 to 10%). Our analysis of proteins within virus particles detected proteolytic fragments of alpha-smooth muscle actin and moesin that were cleaved at sites which might be recognized by HIV-1 protease. These cleavage products are not present in microvesicles from uninfected cells. Therefore, these processed proteins are most probably produced by HIV-1 protease digestion. The presence of these fragments, as well as the incorporation of a few specific cytoskeletal proteins into virions, suggests an active interaction between cytoskeletal and viral proteins. PMID:8892894

  9. KCC2 regulates actin dynamics in dendritic spines via interaction with β-PIX.

    PubMed

    Llano, Olaya; Smirnov, Sergey; Soni, Shetal; Golubtsov, Andrey; Guillemin, Isabelle; Hotulainen, Pirta; Medina, Igor; Nothwang, Hans Gerd; Rivera, Claudio; Ludwig, Anastasia

    2015-06-01

    Chloride extrusion in mature neurons is largely mediated by the neuron-specific potassium-chloride cotransporter KCC2. In addition, independently of its chloride transport function, KCC2 regulates the development and morphology of dendritic spines through structural interactions with the actin cytoskeleton. The mechanism of this effect remains largely unknown. In this paper, we show a novel pathway for KCC2-mediated regulation of the actin cytoskeleton in neurons. We found that KCC2, through interaction with the b isoform of Rac/Cdc42 guanine nucleotide exchange factor β-PIX, regulates the activity of Rac1 GTPase and the phosphorylation of one of the major actin-regulating proteins, cofilin-1. KCC2-deficient neurons had abnormally high levels of phosphorylated cofilin-1. Consistently, dendritic spines of these neurons exhibited a large pool of stable actin, resulting in reduced spine motility and diminished density of functional synapses. In conclusion, we describe a novel signaling pathway that couples KCC2 to the cytoskeleton and regulates the formation of glutamatergic synapses. PMID:26056138

  10. Interdependence of endomembrane trafficking and actin dynamics during polarized growth of Arabidopsis pollen tubes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    During polarized growth of pollen tubes, endomembrane trafficking and actin polymerization are two critical processes that establish membrane/wall homeostasis and maintain growth polarity. Fine-tuned interactions between these two processes are therefore necessary but poorly understood. To better un...

  11. KCC2 regulates actin dynamics in dendritic spines via interaction with β-PIX

    PubMed Central

    Llano, Olaya; Smirnov, Sergey; Soni, Shetal; Golubtsov, Andrey; Guillemin, Isabelle; Hotulainen, Pirta; Medina, Igor; Nothwang, Hans Gerd

    2015-01-01

    Chloride extrusion in mature neurons is largely mediated by the neuron-specific potassium-chloride cotransporter KCC2. In addition, independently of its chloride transport function, KCC2 regulates the development and morphology of dendritic spines through structural interactions with the actin cytoskeleton. The mechanism of this effect remains largely unknown. In this paper, we show a novel pathway for KCC2-mediated regulation of the actin cytoskeleton in neurons. We found that KCC2, through interaction with the b isoform of Rac/Cdc42 guanine nucleotide exchange factor β-PIX, regulates the activity of Rac1 GTPase and the phosphorylation of one of the major actin-regulating proteins, cofilin-1. KCC2-deficient neurons had abnormally high levels of phosphorylated cofilin-1. Consistently, dendritic spines of these neurons exhibited a large pool of stable actin, resulting in reduced spine motility and diminished density of functional synapses. In conclusion, we describe a novel signaling pathway that couples KCC2 to the cytoskeleton and regulates the formation of glutamatergic synapses. PMID:26056138

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

    PubMed

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

    2013-09-01

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

  13. Expression of a dynamin 2 mutant associated with Charcot-Marie-Tooth disease leads to aberrant actin dynamics and lamellipodia formation.

    PubMed

    Yamada, Hiroshi; Kobayashi, Kinue; Zhang, Yubai; Takeda, Tetsuya; Takei, Kohji

    2016-08-15

    Specific mutations in dynamin 2 are linked to Charcot-Marie-Tooth disease (CMT), an inherited peripheral neuropathy. However, the effects of these mutations on dynamin function, particularly in relation to the regulation of the actin cytoskeleton remain unclear. Here, selected CMT-associated dynamin mutants were expressed to examine their role in the pathogenesis of CMT in U2OS cells. Ectopic expression of the dynamin CMT mutants 555Δ3 and K562E caused an approximately 50% decrease in serum stimulation-dependent lamellipodia formation; however, only K562E caused aberrations in the actin cytoskeleton. Immunofluorescence analysis showed that the K562E mutation resulted in the disappearance of radially aligned actin bundles and the simultaneous appearance of F-actin clusters. Live-cell imaging analyses showed F-actin polymers of decreased length assembled into immobile clusters in K562E-expressing cells. The K562E dynamin mutant colocalized with the F-actin clusters, whereas its colocalization with clathrin-coated pit marker proteins was decreased. Essentially the same results were obtained using another cell line, HeLa and NG108-15 cells. The present study is the first to show the association of dynamin CMT mutations with aberrant actin dynamics and lamellipodia, which may contribute to defective endocytosis and myelination in Schwann cells in CMT.

  14. Nuclear actin and lamins in viral infections.

    PubMed

    Cibulka, Jakub; Fraiberk, Martin; Forstova, Jitka

    2012-03-01

    Lamins are the best characterized cytoskeletal components of the cell nucleus that help to maintain the nuclear shape and participate in diverse nuclear processes including replication or transcription. Nuclear actin is now widely accepted to be another cytoskeletal protein present in the nucleus that fulfills important functions in the gene expression. Some viruses replicating in the nucleus evolved the ability to interact with and probably utilize nuclear actin for their replication, e.g., for the assembly and transport of capsids or mRNA export. On the other hand, lamins play a role in the propagation of other viruses since nuclear lamina may represent a barrier for virions entering or escaping the nucleus. This review will summarize the current knowledge about the roles of nuclear actin and lamins in viral infections.

  15. Coordinated integrin activation by actin-dependent force during T-cell migration

    PubMed Central

    Nordenfelt, Pontus; Elliott, Hunter L.; Springer, Timothy A.

    2016-01-01

    For a cell to move forward it must convert chemical energy into mechanical propulsion. Force produced by actin polymerization can generate traction across the plasma membrane by transmission through integrins to their ligands. However, the role this force plays in integrin activation is unknown. Here we show that integrin activity and cytoskeletal dynamics are reciprocally linked, where actin-dependent force itself appears to regulate integrin activity. We generated fluorescent tension-sensing constructs of integrin αLβ2 (LFA-1) to visualize intramolecular tension during cell migration. Using quantitative imaging of migrating T cells, we correlate tension in the αL or β2 subunit with cell and actin dynamics. We find that actin engagement produces tension within the β2 subunit to induce and stabilize an active integrin conformational state and that this requires intact talin and kindlin motifs. This supports a general mechanism where localized actin polymerization can coordinate activation of the complex machinery required for cell migration. PMID:27721490

  16. Cryptococcus neoformans is internalized by receptor-mediated or 'triggered' phagocytosis, dependent on actin recruitment.

    PubMed

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

    2014-01-01

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

  17. Attenuation of LDHA expression in cancer cells leads to redox-dependent alterations in cytoskeletal structure and cell migration.

    PubMed

    Arseneault, Robert; Chien, Andrew; Newington, Jordan T; Rappon, Tim; Harris, Richard; Cumming, Robert C

    2013-09-28

    Aerobic glycolysis, the preferential use of glycolysis even in the presence of oxygen to meet cellular metabolic demands, is a near universal feature of cancer. This unique type of metabolism is thought to protect cancer cells from damaging reactive oxygen species (ROS) produced in the mitochondria. Using the cancer cell line MDA-MB-435 it is shown that shRNA mediated knockdown of lactate dehydrogenase A (LDHA), a key mediator of aerobic glycolysis, results in elevated mitochondrial ROS production and a concomitant decrease in cell proliferation and motility. Redox-sensitive proteins affected by oxidative stress associated with LDHA knockdown were identified by Redox 2D-PAGE and mass spectrometry. In particular, tropomyosin (Tm) isoforms Tm4, Tm5NM1 and Tm5NM5, proteins involved in cell migration and cytoskeletal dynamics, exhibited changes in disulfide bonding and co-localized with peri-nuclear actin aggregates in LDHA knockdown cells. In contrast, treatment with the thiol-based antioxidant N-acetylcysteine promoted the relocalization of Tms to cortical actin microfilaments and partially rescued the migration defects associated with attenuated LDHA expression. These results suggest that aerobic glycolysis and reduced mitochondrial ROS production create an environment conducive to cytoskeletal remodeling; key events linked to the high cell motility associated with cancer.

  18. Networks Models of Actin Dynamics during Spermatozoa Postejaculatory Life: A Comparison among Human-Made and Text Mining-Based Models

    PubMed Central

    Ordinelli, Alessandra; Ramal Sanchez, Marina; Mattioli, Mauro; Barboni, Barbara

    2016-01-01

    Here we realized a networks-based model representing the process of actin remodelling that occurs during the acquisition of fertilizing ability of human spermatozoa (HumanMade_ActinSpermNetwork, HM_ASN). Then, we compared it with the networks provided by two different text mining tools: Agilent Literature Search (ALS) and PESCADOR. As a reference, we used the data from the online repository Kyoto Encyclopaedia of Genes and Genomes (KEGG), referred to the actin dynamics in a more general biological context. We found that HM_ALS and the networks from KEGG data shared the same scale-free topology following the Barabasi-Albert model, thus suggesting that the information is spread within the network quickly and efficiently. On the contrary, the networks obtained by ALS and PESCADOR have a scale-free hierarchical architecture, which implies a different pattern of information transmission. Also, the hubs identified within the networks are different: HM_ALS and KEGG networks contain as hubs several molecules known to be involved in actin signalling; ALS was unable to find other hubs than “actin,” whereas PESCADOR gave some nonspecific result. This seems to suggest that the human-made information retrieval in the case of a specific event, such as actin dynamics in human spermatozoa, could be a reliable strategy.

  19. Networks Models of Actin Dynamics during Spermatozoa Postejaculatory Life: A Comparison among Human-Made and Text Mining-Based Models

    PubMed Central

    Ordinelli, Alessandra; Ramal Sanchez, Marina; Mattioli, Mauro; Barboni, Barbara

    2016-01-01

    Here we realized a networks-based model representing the process of actin remodelling that occurs during the acquisition of fertilizing ability of human spermatozoa (HumanMade_ActinSpermNetwork, HM_ASN). Then, we compared it with the networks provided by two different text mining tools: Agilent Literature Search (ALS) and PESCADOR. As a reference, we used the data from the online repository Kyoto Encyclopaedia of Genes and Genomes (KEGG), referred to the actin dynamics in a more general biological context. We found that HM_ALS and the networks from KEGG data shared the same scale-free topology following the Barabasi-Albert model, thus suggesting that the information is spread within the network quickly and efficiently. On the contrary, the networks obtained by ALS and PESCADOR have a scale-free hierarchical architecture, which implies a different pattern of information transmission. Also, the hubs identified within the networks are different: HM_ALS and KEGG networks contain as hubs several molecules known to be involved in actin signalling; ALS was unable to find other hubs than “actin,” whereas PESCADOR gave some nonspecific result. This seems to suggest that the human-made information retrieval in the case of a specific event, such as actin dynamics in human spermatozoa, could be a reliable strategy. PMID:27642606

  20. Networks Models of Actin Dynamics during Spermatozoa Postejaculatory Life: A Comparison among Human-Made and Text Mining-Based Models.

    PubMed

    Bernabò, Nicola; Ordinelli, Alessandra; Ramal Sanchez, Marina; Mattioli, Mauro; Barboni, Barbara

    2016-01-01

    Here we realized a networks-based model representing the process of actin remodelling that occurs during the acquisition of fertilizing ability of human spermatozoa (HumanMade_ActinSpermNetwork, HM_ASN). Then, we compared it with the networks provided by two different text mining tools: Agilent Literature Search (ALS) and PESCADOR. As a reference, we used the data from the online repository Kyoto Encyclopaedia of Genes and Genomes (KEGG), referred to the actin dynamics in a more general biological context. We found that HM_ALS and the networks from KEGG data shared the same scale-free topology following the Barabasi-Albert model, thus suggesting that the information is spread within the network quickly and efficiently. On the contrary, the networks obtained by ALS and PESCADOR have a scale-free hierarchical architecture, which implies a different pattern of information transmission. Also, the hubs identified within the networks are different: HM_ALS and KEGG networks contain as hubs several molecules known to be involved in actin signalling; ALS was unable to find other hubs than "actin," whereas PESCADOR gave some nonspecific result. This seems to suggest that the human-made information retrieval in the case of a specific event, such as actin dynamics in human spermatozoa, could be a reliable strategy. PMID:27642606

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

    SciTech Connect

    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.

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

    PubMed Central

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

    2006-01-01

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

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

    PubMed

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

    2006-03-01

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

  4. Enabled Negatively Regulates Diaphanous-Driven Actin Dynamics In Vitro and In Vivo

    PubMed Central

    Bilancia, Colleen G.; Winkelman, Jonathan D.; Tsygankov, Denis; Nowotarski, Stephanie H.; Sees, Jennifer A.; Comber, Kate; Evans, Iwan; Lakhani, Vinal; Wood, Will; Elston, Timothy C.; Kovar, David R.; Peifer, Mark

    2014-01-01

    Summary Actin regulators facilitate cell migration by controlling cell protrusion architecture and dynamics. As the behavior of individual actin regulators becomes clear, we must address why cells require multiple regulators with similar functions and how they cooperate to create diverse protrusions. We characterized Diaphanous (Dia) and Enabled (Ena) as a model, using complementary approaches: cell culture, biophysical analysis, and Drosophila morphogenesis. We found that Dia and Ena have distinct biochemical properties that contribute to the different protrusion morphologies each induces. Dia is a more processive, faster elongator, paralleling the long, stable filopodia it induces in vivo, while Ena promotes filopodia with more dynamic changes in number, length, and lifetime. Acting together, Ena and Dia induce protrusions distinct from those induced by either alone, with Ena reducing Dia-driven protrusion length and number. Consistent with this, EnaEVH1 binds Dia directly and inhibits DiaFH1FH2-mediated nucleation in vitro. Finally, Ena rescues hemocyte migration defects caused by activated Dia. PMID:24576424

  5. Dynamin at actin tails.

    PubMed

    Lee, Eunkyung; De Camilli, Pietro

    2002-01-01

    Dynamin, the product of the shibire gene of Drosophila, is a GTPase critically required for endocytosis. Some studies have suggested a functional link between dynamin and the actin cytoskeleton. This link is of special interest, because there is evidence implicating actin dynamics in endocytosis. Here we show that endogenous dynamin 2, as well as green fluorescence protein fusion proteins of both dynamin 1 and 2, is present in actin comets generated by Listeria or by type I PIP kinase (PIPK) overexpression. In PIPK-induced tails, dynamin is further enriched at the interface between the tails and the moving organelles. Dynamin mutants harboring mutations in the GTPase domain inhibited nucleation of actin tails induced by PIPK and moderately reduced their speed. Although dynamin localization to the tails required its proline-rich domain, expression of a dynamin mutant lacking this domain also diminished tail formation. In addition, this mutant disrupted a membrane-associated actin scaffold (podosome rosette) previously shown to include dynamin. These findings suggest that dynamin is part of a protein network that controls nucleation of actin from membranes. At endocytic sites, dynamin may couple the fission reaction to the polymerization of an actin pool that functions in the separation of the endocytic vesicles from the plasma membrane. PMID:11782545

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

    PubMed

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

    2016-09-15

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

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

    PubMed

    Banigan, Edward J; Lee, Kun-Chun; Liu, Andrea J

    2013-12-01

    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.

  8. Disentangling Membrane Dynamics and Cell Migration; Differential Influences of F-actin and Cell-Matrix Adhesions.

    PubMed

    Kowalewski, Jacob M; Shafqat-Abbasi, Hamdah; Jafari-Mamaghani, Mehrdad; Endrias Ganebo, Bereket; Gong, Xiaowei; Strömblad, Staffan; Lock, John G

    2015-01-01

    Cell migration is heavily interconnected with plasma membrane protrusion and retraction (collectively termed "membrane dynamics"). This makes it difficult to distinguish regulatory mechanisms that differentially influence migration and membrane dynamics. Yet such distinctions may be valuable given evidence that cancer cell invasion in 3D may be better predicted by 2D membrane dynamics than by 2D cell migration, implying a degree of functional independence between these processes. Here, we applied multi-scale single cell imaging and a systematic statistical approach to disentangle regulatory associations underlying either migration or membrane dynamics. This revealed preferential correlations between membrane dynamics and F-actin features, contrasting with an enrichment of links between cell migration and adhesion complex properties. These correlative linkages were often non-linear and therefore context-dependent, strengthening or weakening with spontaneous heterogeneity in cell behavior. More broadly, we observed that slow moving cells tend to increase in area, while fast moving cells tend to shrink, and that the size of dynamic membrane domains is independent of cell area. Overall, we define macromolecular features preferentially associated with either cell migration or membrane dynamics, enabling more specific interrogation and targeting of these processes in future.

  9. Actinic keratosis

    MedlinePlus

    Solar keratosis; Sun-induced skin changes - keratosis; Keratosis - actinic (solar) ... Actinic keratosis is caused by exposure to sunlight. You are more likely to develop it if you: Have fair skin, blue or green eyes, or blond or red hair Had a ...

  10. Bidirectional interactions between NOX2-type NADPH oxidase and the F-actin cytoskeleton in neuronal growth cones.

    PubMed

    Munnamalai, Vidhya; Weaver, Cory J; Weisheit, Corinne E; Venkatraman, Prahatha; Agim, Zeynep Sena; Quinn, Mark T; Suter, Daniel M

    2014-08-01

    NADPH oxidases are important for neuronal function but detailed subcellular localization studies have not been performed. Here, we provide the first evidence for the presence of functional NADPH oxidase 2 (NOX2)-type complex in neuronal growth cones and its bidirectional relationship with the actin cytoskeleton. NADPH oxidase inhibition resulted in reduced F-actin content, retrograde F-actin flow, and neurite outgrowth. Stimulation of NADPH oxidase via protein kinase C activation increased levels of hydrogen peroxide in the growth cone periphery. The main enzymatic NADPH oxidase subunit NOX2/gp91(phox) localized to the growth cone plasma membrane and showed little overlap with the regulatory subunit p40(phox) . p40(phox) itself exhibited colocalization with filopodial actin bundles. Differential subcellular fractionation revealed preferential association of NOX2/gp91(phox) and p40(phox) with the membrane and the cytoskeletal fraction, respectively. When neurite growth was evoked with beads coated with the cell adhesion molecule apCAM, we observed a significant increase in colocalization of p40(phox) with NOX2/gp91(phox) at apCAM adhesion sites. Together, these findings suggest a bidirectional functional relationship between NADPH oxidase activity and the actin cytoskeleton in neuronal growth cones, which contributes to the control of neurite outgrowth. We have previously shown that reactive oxygen species (ROS) are critical for actin organization and dynamics in neuronal growth cones as well as neurite outgrowth. Here, we report that the cytosolic subunit p40(phox) of the NOX2-type NADPH oxidase complex is partially associated with F-actin in neuronal growth cones, while ROS produced by this complex regulates F-actin dynamics and neurite growth. These findings provide evidence for a bidirectional relationship between NADPH oxidase activity and the actin cytoskeleton in neuronal growth cones. PMID:24702317

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

    PubMed

    Cook, Mandy; Bolkan, Bonnie J; Kretzschmar, Doris

    2014-01-01

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

  12. Rab1 recruits WHAMM during membrane remodeling but limits actin nucleation

    PubMed Central

    Russo, Ashley J.; Mathiowetz, Alyssa J.; Hong, Steven; Welch, Matthew D.; Campellone, Kenneth G.

    2016-01-01

    Small G-proteins are key regulatory molecules that activate the actin nucleation machinery to drive cytoskeletal rearrangements during plasma membrane remodeling. However, the ability of small G-proteins to interact with nucleation factors on internal membranes to control trafficking processes has not been well characterized. Here we investigated roles for members of the Rho, Arf, and Rab G-protein families in regulating WASP homologue associated with actin, membranes, and microtubules (WHAMM), an activator of Arp2/3 complex–mediated actin nucleation. We found that Rab1 stimulated the formation and elongation of WHAMM-associated membrane tubules in cells. Active Rab1 recruited WHAMM to dynamic tubulovesicular structures in fibroblasts, and an active prenylated version of Rab1 bound directly to an N-terminal domain of WHAMM in vitro. In contrast to other G-protein–nucleation factor interactions, Rab1 binding inhibited WHAMM-mediated actin assembly. This ability of Rab1 to regulate WHAMM and the Arp2/3 complex represents a distinct strategy for membrane remodeling in which a Rab G-protein recruits the actin nucleation machinery but dampens its activity. PMID:26823012

  13. Phylogenetic Analysis Identifies Many Uncharacterized Actin-like Proteins (Alps) in Bacteria: Regulated Polymerization, Dynamic Instability, and Treadmilling in Alp7A

    PubMed Central

    Derman, Alan I.; Becker, Eric C.; Truong, Bao D.; Fujioka, Akina; Tucey, Timothy M.; Erb, Marcella L.; Patterson, Paula C.; Pogliano, Joe

    2010-01-01

    Summary Actin, one of the most abundant proteins in the eukaryotic cell, also has an abundance of relatives in the eukaryotic proteome. To date though, only five families of actins have been characterized in bacteria. We have conducted a phylogenetic search and uncovered more than 35 highly divergent families of actin-like proteins (Alps) in bacteria. Their genes are found primarily on phage genomes, on plasmids, and on integrating conjugative elements, and are likely to be involved in a variety of functions. We characterize three Alps and find that all form filaments in the cell. The filaments of Alp7A, a plasmid partitioning protein and one of the most divergent of the Alps, display dynamic instability and also treadmill. Alp7A requires other elements from the plasmid to assemble into dynamic polymers in the cell. Our findings suggest that most if not all of the Alps are indeed actin relatives, and that actin is very well represented in bacteria. PMID:19602153

  14. Symbiont-Induced Changes in Host Actin during the Onset of a Beneficial Animal-Bacterial Association

    PubMed Central

    Kimbell, Jennifer R.; McFall-Ngai, Margaret J.

    2004-01-01

    The influence of bacteria on the cytoskeleton of animal cells has been studied extensively only in pathogenic associations. We characterized changes in host cytoskeletal actin induced by the bacterial partner during the onset of a cooperative animal-bacteria association using the squid-vibrio model. Two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis revealed that Vibrio fischeri induced a dramatic increase in actin protein abundance in the bacteria-associated host tissues during the onset of the symbiosis. Immunocytochemistry revealed that this change in actin abundance correlated with a two- to threefold increase in actin in the apical cell surface of the epithelium-lined ducts, the route of entry of symbionts into host tissues. Real-time reverse transcriptase PCR and in situ hybridization did not detect corresponding changes in actin mRNA. Temporally correlated with the bacteria-induced changes in actin levels was a two- to threefold decrease in duct circumference, a 20% loss in the average number of cells interfacing with the duct lumina, and dramatic changes in duct cell shape. When considered with previous studies of the biomechanical and biochemical characteristics of the duct, these findings suggest that the bacterial symbionts, upon colonizing the host organ, induce modifications that physically and chemically limit the opportunity for subsequent colonizers to pass through the ducts. Continued study of the squid-vibrio system will allow further comparisons of the mechanisms by which pathogenic and cooperative bacteria influence cytoskeleton dynamics in host cells. PMID:15006763

  15. Actin Automata with Memory

    NASA Astrophysics Data System (ADS)

    Alonso-Sanz, Ramón; Adamatzky, Andy

    Actin is a globular protein which forms long polar filaments in eukaryotic. The actin filaments play the roles of cytoskeleton, motility units, information processing and learning. We model actin filament as a double chain of finite state machines, nodes, which take states “0” and “1”. The states are abstractions of absence and presence of a subthreshold charge on actin units corresponding to the nodes. All nodes update their state in parallel to discrete time. A node updates its current state depending on states of two closest neighbors in the node chain and two closest neighbors in the complementary chain. Previous models of actin automata consider momentary state transitions of nodes. We enrich the actin automata model by assuming that states of nodes depend not only on the current states of neighboring node but also on their past states. Thus, we assess the effect of memory of past states on the dynamics of acting automata. We demonstrate in computational experiments that memory slows down propagation of perturbations, decrease entropy of space-time patterns generated, transforms traveling localizations to stationary oscillators, and stationary oscillations to still patterns.

  16. Energetic modeling and single-molecule verification of dynamic regulation on receptor complexes by actin corrals and lipid raft domains

    NASA Astrophysics Data System (ADS)

    Lin, Chien Y.; Huang, Jung Y.; Lo, Leu-Wei

    2014-12-01

    We developed an energetic model by integrating the generalized Langevin equation with the Cahn-Hilliard equation to simulate the diffusive behaviors of receptor proteins in the plasma membrane of a living cell. Simulation results are presented to elaborate the confinement effects from actin corrals and protein-induced lipid domains. Single-molecule tracking data of epidermal growth factor receptors (EGFR) acquired on live HeLa cells agree with the simulation results and the mechanism that controls the diffusion of single-molecule receptors is clarified. We discovered that after ligand binding, EGFR molecules move into lipid nanodomains. The transition rates between different diffusion states of liganded EGFR molecules are regulated by the lipid domains. Our method successfully captures dynamic interactions of receptors at the single-molecule level and provides insight into the functional architecture of both the diffusing EGFR molecules and their local cellular environment.

  17. Energetic modeling and single-molecule verification of dynamic regulation on receptor complexes by actin corrals and lipid raft domains.

    PubMed

    Lin, Chien Y; Huang, Jung Y; Lo, Leu-Wei

    2014-12-01

    We developed an energetic model by integrating the generalized Langevin equation with the Cahn-Hilliard equation to simulate the diffusive behaviors of receptor proteins in the plasma membrane of a living cell. Simulation results are presented to elaborate the confinement effects from actin corrals and protein-induced lipid domains. Single-molecule tracking data of epidermal growth factor receptors (EGFR) acquired on live HeLa cells agree with the simulation results and the mechanism that controls the diffusion of single-molecule receptors is clarified. We discovered that after ligand binding, EGFR molecules move into lipid nanodomains. The transition rates between different diffusion states of liganded EGFR molecules are regulated by the lipid domains. Our method successfully captures dynamic interactions of receptors at the single-molecule level and provides insight into the functional architecture of both the diffusing EGFR molecules and their local cellular environment. PMID:25481171

  18. Implementing cell contractility in filament-based cytoskeletal models.

    PubMed

    Fallqvist, B

    2016-02-01

    Cells are known to respond over time to mechanical stimuli, even actively generating force at longer times. In this paper, a microstructural filament-based cytoskeletal network model is extended to incorporate this active response, and a computational study to assess the influence on relaxation behaviour was performed. The incorporation of an active response was achieved by including a strain energy function of contractile activity from the cross-linked actin filaments. A four-state chemical model and strain energy function was adopted, and generalisation to three dimensions and the macroscopic deformation field was performed by integration over the unit sphere. Computational results in MATLAB and ABAQUS/Explicit indicated an active cellular response over various time-scales, dependent on contractile parameters. Important features such as force generation and increasing cell stiffness due to prestress are qualitatively predicted. The work in this paper can easily be extended to encompass other filament-based cytoskeletal models as well. PMID:26899417

  19. Actinic Cheilitis

    MedlinePlus

    ... is a precancerous condition related to cumulative lifetime sun exposure. The lower lip is most often affected. Individuals ... Wearing barrier clothing (eg, wide-brimmed hats) and sunscreen-containing lip balms can aid in preventing actinic ...

  20. Actin Filaments at the Leading Edge of Cancer Cells Are Characterized by a High Mobile Fraction and Turnover Regulation by Profilin I

    PubMed Central

    Lorente, Gisela; Syriani, Emilio; Morales, Miguel

    2014-01-01

    Cellular motility is the basis for cancer cell invasion and metastasis. In the case of breast cancer, the most common type of cancer among women, metastasis represents the most devastating stage of the disease. The central role of cellular motility in cancer development emphasizes the importance of understanding the specific mechanisms involved in this process. In this context, tumor development and metastasis would be the consequence of a loss or defect of the mechanisms that control cytoskeletal remodeling. Profilin I belongs to a family of small actin binding proteins that are thought to assist in actin filament elongation at the leading edge of migrating cells. Traditionally, Profilin I has been considered to be an essential control element for actin polymerization and cell migration. Expression of Profilin I is down-regulated in breast and various other cancer cells. In MDA-MB-231 cells, a breast cancer cell line, further inhibition of Profilin I expression promotes hypermotility and metastatic spread, a finding that contrasts with the proposed role of Profilin in enhancing polymerization. In this report, we have taken advantage of the fluorescence recovery after photobleaching (FRAP) of GFP-actin to quantify and compare actin dynamics at the leading edge level in both cancer and non-cancer cell models. Our results suggest that (i) a high level of actin dynamics (i.e., a large mobile fraction of actin filaments and a fast turnover) is a common characteristic of some cancer cells; (ii) actin polymerization shows a high degree of independence from the presence of extracellular growth factors; and (iii) our results also corroborate the role of Profilin I in regulating actin polymerization, as raising the intracellular levels of Profilin I decreased the mobile fraction ratio of actin filaments and slowed their polymerization rate; furthermore, increased Profilin levels also led to reduced individual cell velocity and directionality. PMID:24465723

  1. Regulation of cellular actin architecture by S100A10.

    PubMed

    Jung, M Juliane; Murzik, Ulrike; Wehder, Liane; Hemmerich, Peter; Melle, Christian

    2010-04-15

    Actin structures are involved in several biological processes and the disruption of actin polymerisation induces impaired motility of eukaryotic cells. Different factors are involved in regulation and maintenance of the cytoskeletal actin architecture. Here we show that S100A10 participates in the particular organisation of actin filaments. Down-regulation of S100A10 by specific siRNA triggered a disorganisation of filamentous actin structures without a reduction of the total cellular actin concentration. In contrast, the formation of cytoskeleton structures containing tubulin was unhindered in S100A10 depleted cells. Interestingly, the cellular distribution of annexin A2, an interaction partner of S100A10, was unaffected in S100A10 depleted cells. Cells lacking S100A10 showed an impaired migration activity and were unable to close a scratched wound. Our data provide first insights of S100A10 function as a regulator of the filamentous actin network. PMID:20100475

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

    NASA Astrophysics Data System (ADS)

    Appert-Rolland, C.; Ebbinghaus, M.; Santen, L.

    2015-09-01

    Cells are the elementary units of living organisms, which are able to carry out many vital functions. These functions rely on active processes on a microscopic scale. Therefore, they are strongly out-of-equilibrium systems, which are driven by continuous energy supply. The tasks that have to be performed in order to maintain the cell alive require transportation of various ingredients, some being small, others being large. Intracellular transport processes are able to induce concentration gradients and to carry objects to specific targets. These processes cannot be carried out only by diffusion, as cells may be crowded, and quite elongated on molecular scales. Therefore active transport has to be organized. The cytoskeleton, which is composed of three types of filaments (microtubules, actin and intermediate filaments), determines the shape of the cell, and plays a role in cell motion. It also serves as a road network for a special kind of vehicles, namely the cytoskeletal motors. These molecules can attach to a cytoskeletal filament, perform directed motion, possibly carrying along some cargo, and then detach. It is a central issue to understand how intracellular transport driven by molecular motors is regulated. The interest for this type of question was enhanced when it was discovered that intracellular transport breakdown is one of the signatures of some neuronal diseases like the Alzheimer. We give a survey of the current knowledge on microtubule based intracellular transport. Our review includes on the one hand an overview of biological facts, obtained from experiments, and on the other hand a presentation of some modeling attempts based on cellular automata. We present some background knowledge on the original and variants of the TASEP (Totally Asymmetric Simple Exclusion Process), before turning to more application oriented models. After addressing microtubule based transport in general, with a focus on in vitro experiments, and on cooperative effects in the

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

    NASA Technical Reports Server (NTRS)

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

    2000-01-01

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

  4. Actin nucleators in the nucleus: an emerging theme.

    PubMed

    Weston, Louise; Coutts, Amanda S; La Thangue, Nicholas B

    2012-08-01

    Actin is an integral component of the cytoskeleton, forming a plethora of macromolecular structures that mediate various cellular functions. The formation of such structures relies on the ability of actin monomers to associate into polymers, and this process is regulated by actin nucleation factors. These factors use monomeric actin pools at specific cellular locations, thereby permitting rapid actin filament formation when required. It has now been established that actin is also present in the nucleus, where it is implicated in chromatin remodelling and the regulation of eukaryotic gene transcription. Notably, the presence of typical actin filaments in the nucleus has not been demonstrated directly. However, studies in recent years have provided evidence for the nuclear localisation of actin nucleation factors that promote cytoplasmic actin polymerisation. Their localisation to the nucleus suggests that these proteins mediate collaboration between the cytoskeleton and the nucleus, which might be dependent on their ability to promote actin polymerisation. The nature of this cooperation remains enigmatic and it will be important to elucidate the physiological relevance of the link between cytoskeletal actin networks and nuclear events. This Commentary explores the current evidence for the nuclear roles of actin nucleation factors. Furthermore, the implication of actin-associated proteins in relaying exogenous signals to the nucleus, particularly in response to cellular stress, will be considered.

  5. The dynamic distribution of fluorescent analogues of actin and myosin in protrusions at the leading edge of migrating Swiss 3T3 fibroblasts

    PubMed Central

    1988-01-01

    The formation of protrusions at the leading edge of the cell is an essential step in fibroblast locomotion. Using fluorescent analogue cytochemistry, ratio imaging, multiple parameter analysis, and fluorescence photobleaching recovery, the distribution of actin and myosin was examined in the same protrusions at the leading edge of live, locomoting cells during wound-healing in vitro. We have previously defined two temporal stages of the formation of protrusions: (a) initial protrusion and (b) established protrusion (Fisher et al., 1988). Actin was slightly concentrated in initial protrusions, while myosin was either totally absent or present at extremely low levels at the base of the initial protrusions. In contrast, established protrusions contained diffuse actin and actin microspikes, as well as myosin in both diffuse and structured forms. Actin and myosin were also localized along concave transverse fibers near the base of initial and established protrusions. The dynamics of myosin penetration into a relatively stable, established protrusion was demonstrated by recording sequential images over time. Myosin was shown to be absent from an initial protrusion, but diffuse and punctate myosin was detected in the same protrusion within 1-2 min. Fluorescence photobleaching recovery indicated that myosin was 100% immobile in the region behind the leading edge containing transverse fibers, in comparison to the 21% immobile fraction detected in the perinuclear region. Possible explanations of the delayed penetration of myosin into established protrusions and the implications on the mechanism of protrusion are discussed. PMID:3204122

  6. Simulated Microgravity Induced Cytoskeletal Rearrangements are Modulated by Protooncogenes

    NASA Technical Reports Server (NTRS)

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

    1998-01-01

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

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

    PubMed Central

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

    2014-01-01

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

  8. Vascular disease-causing mutation R258C in ACTA2 disrupts actin dynamics and interaction with myosin

    PubMed Central

    Lu, Hailong; Fagnant, Patricia M.; Bookwalter, Carol S.; Joel, Peteranne; Trybus, Kathleen M.

    2015-01-01

    Point mutations in vascular smooth muscle α-actin (SM α-actin), encoded by the gene ACTA2, are the most prevalent cause of familial thoracic aortic aneurysms and dissections (TAAD). Here, we provide the first molecular characterization, to our knowledge, of the effect of the R258C mutation in SM α-actin, expressed with the baculovirus system. Smooth muscles are unique in that force generation requires both interaction of stable actin filaments with myosin and polymerization of actin in the subcortical region. Both aspects of R258C function therefore need investigation. Total internal reflection fluorescence (TIRF) microscopy was used to quantify the growth of single actin filaments as a function of time. R258C filaments are less stable than WT and more susceptible to severing by cofilin. Smooth muscle tropomyosin offers little protection from cofilin cleavage, unlike its effect on WT actin. Unexpectedly, profilin binds tighter to the R258C monomer, which will increase the pool of globular actin (G-actin). In an in vitro motility assay, smooth muscle myosin moves R258C filaments more slowly than WT, and the slowing is exacerbated by smooth muscle tropomyosin. Under loaded conditions, small ensembles of myosin are unable to produce force on R258C actin-tropomyosin filaments, suggesting that tropomyosin occupies an inhibitory position on actin. Many of the observed defects cannot be explained by a direct interaction with the mutated residue, and thus the mutation allosterically affects multiple regions of the monomer. Our results align with the hypothesis that defective contractile function contributes to the pathogenesis of TAAD. PMID:26153420

  9. Bistability in the Rac1, PAK, and RhoA Signaling Network Drives Actin Cytoskeleton Dynamics and Cell Motility Switches

    PubMed Central

    Byrne, Kate M.; Monsefi, Naser; Dawson, John C.; Degasperi, Andrea; Bukowski-Wills, Jimi-Carlo; Volinsky, Natalia; Dobrzyński, Maciej; Birtwistle, Marc R.; Tsyganov, Mikhail A.; Kiyatkin, Anatoly; Kida, Katarzyna; Finch, Andrew J.; Carragher, Neil O.; Kolch, Walter; Nguyen, Lan K.; von Kriegsheim, Alex; Kholodenko, Boris N.

    2016-01-01

    Summary Dynamic interactions between RhoA and Rac1, members of the Rho small GTPase family, play a vital role in the control of cell migration. Using predictive mathematical modeling, mass spectrometry-based quantitation of network components, and experimental validation in MDA-MB-231 mesenchymal breast cancer cells, we show that a network containing Rac1, RhoA, and PAK family kinases can produce bistable, switch-like responses to a graded PAK inhibition. Using a small chemical inhibitor of PAK, we demonstrate that cellular RhoA and Rac1 activation levels respond in a history-dependent, bistable manner to PAK inhibition. Consequently, we show that downstream signaling, actin dynamics, and cell migration also behave in a bistable fashion, displaying switches and hysteresis in response to PAK inhibition. Our results demonstrate that PAK is a critical component in the Rac1-RhoA inhibitory crosstalk that governs bistable GTPase activity, cell morphology, and cell migration switches. PMID:27136688

  10. Identification of a hormone-regulated dynamic nuclear actin network associated with estrogen receptor alpha in human breast cancer cell nuclei.

    PubMed

    Ambrosino, Concetta; Tarallo, Roberta; Bamundo, Angela; Cuomo, Danila; Franci, Gianluigi; Nassa, Giovanni; Paris, Ornella; Ravo, Maria; Giovane, Alfonso; Zambrano, Nicola; Lepikhova, Tatiana; Jänne, Olli A; Baumann, Marc; Nyman, Tuula A; Cicatiello, Luigi; Weisz, Alessandro

    2010-06-01

    Estrogen receptor alpha (ERalpha) is a modular protein of the steroid/nuclear receptor family of transcriptional regulators that upon binding to the hormone undergoes structural changes, resulting in its nuclear translocation and docking to specific chromatin sites. In the nucleus, ERalpha assembles in multiprotein complexes that act as final effectors of estrogen signaling to the genome through chromatin remodeling and epigenetic modifications, leading to dynamic and coordinated regulation of hormone-responsive genes. Identification of the molecular partners of ERalpha and understanding their combinatory interactions within functional complexes is a prerequisite to define the molecular basis of estrogen control of cell functions. To this end, affinity purification was applied to map and characterize the ERalpha interactome in hormone-responsive human breast cancer cell nuclei. MCF-7 cell clones expressing human ERalpha fused to a tandem affinity purification tag were generated and used to purify native nuclear ER-containing complexes by IgG-Sepharose affinity chromatography and glycerol gradient centrifugation. Purified complexes were analyzed by two-dimensional DIGE and mass spectrometry, leading to the identification of a ligand-dependent multiprotein complex comprising beta-actin, myosins, and several proteins involved in actin filament organization and dynamics and/or known to participate in actin-mediated regulation of gene transcription, chromatin dynamics, and ribosome biogenesis. Time course analyses indicated that complexes containing ERalpha and actin are assembled in the nucleus early after receptor activation by ligands, and gene knockdown experiments showed that gelsolin and the nuclear isoform of myosin 1c are key determinants for assembly and/or stability of these complexes. Based on these results, we propose that the actin network plays a role in nuclear ERalpha actions in breast cancer cells, including coordinated regulation of target gene

  11. Motor-induced sliding of microtubule and actin bundles

    PubMed Central

    Zemel, Assaf; Mogilner, Alex

    2009-01-01

    Interactions of multiple molecular motors with bundles of actin and microtubule filaments form the basis for many cytoskeletal processes including axonal growth, muscle contraction, cell division and platelet formation. Continuum models based on generalized diffusion equations have been suggested to quantify the dynamics of such active bundles. In highly cross-linked and densely packed filament bundles, however, a major complication arises due to the multiple interactions that each filament forms with its neighbors. To explore the effects of these interactions, we used detailed computer simulations and studied the bundles with different types of motors at different densities and boundary conditions. We found that highly cross-linked bundles exhibit effects of long-ranged interactions that are sensitive to the boundary conditions. In open bundles, these give rise to ‘telescopic’ patterns resulting in significant acceleration of the filaments at the edges. In contrast, in ringed bundles, the long-ranged interactions ‘lock’ filaments and slow down their movements. The filaments in loosely connected bundles, on the other hand, undergo local diffusion-drift dynamics consistent with previous continuum models. Our simulations also demonstrate the sorting phenomena in the mixed-polarity bundles and reveal characteristic scales and conditions for spontaneous pattern formation in the bundle. We discuss the relevance of our results for cytoskeleton systems such as microtubules in axons, platelet formation, kinetochore fibers and actin bundles in motile cells. PMID:19506757

  12. Analysis of Cytoskeletal and Motility Proteins in the Sea Urchin Genome Assembly

    PubMed Central

    RL, Morris; MP, Hoffman; RA, Obar; SS, McCafferty; IR, Gibbons; AD, Leone; J, Cool; EL, Allgood; AM, Musante; KM, Judkins; BJ, Rossetti; AP, Rawson; DR, Burgess

    2007-01-01

    The sea urchin embryo is a classical model system for studying the role of the cytoskeleton in such events as fertilization, mitosis, cleavage, cell migration and gastrulation. We have conducted an analysis of gene models derived from the Strongylocentrotus purpuratus genome assembly and have gathered strong evidence for the existence of multiple gene families encoding cytoskeletal proteins and their regulators in sea urchin. While many cytoskeletal genes have been cloned from sea urchin with sequences already existing in public databases, genome analysis reveals a significantly higher degree of diversity within certain gene families. Furthermore, genes are described corresponding to homologs of cytoskeletal proteins not previously documented in sea urchins. To illustrate the varying degree of sequence diversity that exists within cytoskeletal gene families, we conducted an analysis of genes encoding actins, specific actin-binding proteins, myosins, tubulins, kinesins, dyneins, specific microtubule-associated proteins, and intermediate filaments. We conducted ontological analysis of select genes to better understand the relatedness of urchin cytoskeletal genes to those of other deuterostomes. We analyzed developmental expression (EST) data to confirm the existence of select gene models and to understand their differential expression during various stages of early development. PMID:17027957

  13. Conformational phases of membrane bound cytoskeletal filaments

    NASA Astrophysics Data System (ADS)

    Quint, David A.; Grason, Gregory; Gopinathan, Ajay

    2013-03-01

    Membrane bound cytoskeletal filaments found in living cells are employed to carry out many types of activities including cellular division, rigidity and transport. When these biopolymers are bound to a membrane surface they may take on highly non-trivial conformations as compared to when they are not bound. This leads to the natural question; What are the important interactions which drive these polymers to particular conformations when they are bound to a surface? Assuming that there are binding domains along the polymer which follow a periodic helical structure set by the natural monomeric handedness, these bound conformations must arise from the interplay of the intrinsic monomeric helicity and membrane binding. To probe this question, we study a continuous model of an elastic filament with intrinsic helicity and map out the conformational phases of this filament for various mechanical and structural parameters in our model, such as elastic stiffness and intrinsic twist of the filament. Our model allows us to gain insight into the possible mechanisms which drive real biopolymers such as actin and tubulin in eukaryotes and their prokaryotic cousins MreB and FtsZ to take on their functional conformations within living cells.

  14. Semaphorin3a Enhances Endocytosis at Sites of Receptor–F-Actin Colocalization during Growth Cone Collapse

    PubMed Central

    Fournier, Alyson E.; Nakamura, Fumio; Kawamoto, Susumu; Goshima, Yoshio; Kalb, Robert G.; Strittmatter, Stephen M.

    2000-01-01

    Axonal growth cone collapse is accompanied by a reduction in filopodial F-actin. We demonstrate here that semaphorin 3A (Sema3A) induces a coordinated rearrangement of Sema3A receptors and F-actin during growth cone collapse. Differential interference contrast microscopy reveals that some sites of Sema3A-induced F-actin reorganization correlate with discrete vacuoles, structures involved in endocytosis. Endocytosis of FITC-dextran by the growth cone is enhanced during Sema3A treatment, and sites of dextran accumulation colocalize with actin-rich vacuoles and ridges of membrane. Furthermore, the Sema3A receptor proteins, neuropilin-1 and plexin, and the Sema3A signaling molecule, rac1, also reorganize to vacuoles and membrane ridges after Sema3A treatment. These data support a model whereby Sema3A stimulates endocytosis by focal and coordinated rearrangement of receptor and cytoskeletal elements. Dextran accumulation is also increased in retinal ganglion cell (RGC) growth cones, in response to ephrin A5, and in RGC and DRG growth cones, in response to myelin and phorbol-ester. Therefore, enhanced endocytosis may be a general principle of physiologic growth cone collapse. We suggest that growth cone collapse is mediated by both actin filament rearrangements and alterations in membrane dynamics. PMID:10769032

  15. Actin foci facilitate activation of the phospholipase C-γ in primary T lymphocytes via the WASP pathway

    PubMed Central

    Kumari, Sudha; Depoil, David; Martinelli, Roberta; Judokusumo, Edward; Carmona, Guillaume; Gertler, Frank B; Kam, Lance C; Carman, Christopher V; Burkhardt, Janis K; Irvine, Darrell J; Dustin, Michael L

    2015-01-01

    Wiscott Aldrich Syndrome protein (WASP) deficiency results in defects in calcium ion signaling, cytoskeletal regulation, gene transcription and overall T cell activation. The activation of WASP constitutes a key pathway for actin filament nucleation. Yet, when WASP function is eliminated there is negligible effect on actin polymerization at the immunological synapse, leading to gaps in our understanding of the events connecting WASP and calcium ion signaling. Here, we identify a fraction of total synaptic F-actin selectively generated by WASP in the form of distinct F-actin ‘foci’. These foci are polymerized de novo as a result of the T cell receptor (TCR) proximal tyrosine kinase cascade, and facilitate distal signaling events including PLCγ1 activation and subsequent cytoplasmic calcium ion elevation. We conclude that WASP generates a dynamic F-actin architecture in the context of the immunological synapse, which then amplifies the downstream signals required for an optimal immune response. DOI: http://dx.doi.org/10.7554/eLife.04953.001 PMID:25758716

  16. Dynamin-2 regulates fusion pore expansion and quantal release through a mechanism that involves actin dynamics in neuroendocrine chromaffin cells.

    PubMed

    González-Jamett, Arlek M; Momboisse, Fanny; Guerra, María José; Ory, Stéphane; Báez-Matus, Ximena; Barraza, Natalia; Calco, Valerie; Houy, Sébastien; Couve, Eduardo; Neely, Alan; Martínez, Agustín D; Gasman, Stéphane; Cárdenas, Ana M

    2013-01-01

    Over the past years, dynamin has been implicated in tuning the amount and nature of transmitter released during exocytosis. However, the mechanism involved remains poorly understood. Here, using bovine adrenal chromaffin cells, we investigated whether this mechanism rely on dynamin's ability to remodel actin cytoskeleton. According to this idea, inhibition of dynamin GTPase activity suppressed the calcium-dependent de novo cortical actin and altered the cortical actin network. Similarly, expression of a small interfering RNA directed against dynamin-2, an isoform highly expressed in chromaffin cells, changed the cortical actin network pattern. Disruption of dynamin-2 function, as well as the pharmacological inhibition of actin polymerization with cytochalasine-D, slowed down fusion pore expansion and increased the quantal size of individual exocytotic events. The effects of cytochalasine-D and dynamin-2 disruption were not additive indicating that dynamin-2 and F-actin regulate the late steps of exocytosis by a common mechanism. Together our data support a model in which dynamin-2 directs actin polymerization at the exocytosis site where both, in concert, adjust the hormone quantal release to efficiently respond to physiological demands.

  17. Shape remodeling and blebbing of active cytoskeletal vesicles.

    PubMed

    Loiseau, Etienne; Schneider, Jochen A M; Keber, Felix C; Pelzl, Carina; Massiera, Gladys; Salbreux, Guillaume; Bausch, Andreas R

    2016-04-01

    Morphological transformations of living cells, such as shape adaptation to external stimuli, blebbing, invagination, or tethering, result from an intricate interplay between the plasma membrane and its underlying cytoskeleton, where molecular motors generate forces. Cellular complexity defies a clear identification of the competing processes that lead to such a rich phenomenology. In a synthetic biology approach, designing a cell-like model assembled from a minimal set of purified building blocks would allow the control of all relevant parameters. We reconstruct actomyosin vesicles in which the coupling of the cytoskeleton to the membrane, the topology of the cytoskeletal network, and the contractile activity can all be precisely controlled and tuned. We demonstrate that tension generation of an encapsulated active actomyosin network suffices for global shape transformation of cell-sized lipid vesicles, which are reminiscent of morphological adaptations in living cells. The observed polymorphism of our cell-like model, such as blebbing, tether extrusion, or faceted shapes, can be qualitatively explained by the protein concentration dependencies and a force balance, taking into account the membrane tension, the density of anchoring points between the membrane and the actin network, and the forces exerted by molecular motors in the actin network. The identification of the physical mechanisms for shape transformations of active cytoskeletal vesicles sets a conceptual and quantitative benchmark for the further exploration of the adaptation mechanisms of cells. PMID:27152328

  18. Shape remodeling and blebbing of active cytoskeletal vesicles

    PubMed Central

    Loiseau, Etienne; Schneider, Jochen A. M.; Keber, Felix C.; Pelzl, Carina; Massiera, Gladys; Salbreux, Guillaume; Bausch, Andreas R.

    2016-01-01

    Morphological transformations of living cells, such as shape adaptation to external stimuli, blebbing, invagination, or tethering, result from an intricate interplay between the plasma membrane and its underlying cytoskeleton, where molecular motors generate forces. Cellular complexity defies a clear identification of the competing processes that lead to such a rich phenomenology. In a synthetic biology approach, designing a cell-like model assembled from a minimal set of purified building blocks would allow the control of all relevant parameters. We reconstruct actomyosin vesicles in which the coupling of the cytoskeleton to the membrane, the topology of the cytoskeletal network, and the contractile activity can all be precisely controlled and tuned. We demonstrate that tension generation of an encapsulated active actomyosin network suffices for global shape transformation of cell-sized lipid vesicles, which are reminiscent of morphological adaptations in living cells. The observed polymorphism of our cell-like model, such as blebbing, tether extrusion, or faceted shapes, can be qualitatively explained by the protein concentration dependencies and a force balance, taking into account the membrane tension, the density of anchoring points between the membrane and the actin network, and the forces exerted by molecular motors in the actin network. The identification of the physical mechanisms for shape transformations of active cytoskeletal vesicles sets a conceptual and quantitative benchmark for the further exploration of the adaptation mechanisms of cells. PMID:27152328

  19. A dynamical systems approach to actin-based motility in Listeria monocytogenes

    NASA Astrophysics Data System (ADS)

    Hotton, S.

    2010-11-01

    A simple kinematic model for the trajectories of Listeria monocytogenes is generalized to a dynamical system rich enough to exhibit the resonant Hopf bifurcation structure of excitable media and simple enough to be studied geometrically. It is shown how L. monocytogenes trajectories and meandering spiral waves are organized by the same type of attracting set.

  20. Unconventional actin conformations localize on intermediate filaments in mitosis

    SciTech Connect

    Hubert, Thomas; Vandekerckhove, Joel; Gettemans, Jan

    2011-03-04

    Research highlights: {yields} Unconventional actin conformations colocalize with vimentin on a cage-like structure in metaphase HEK 293T cells. {yields} These conformations are detected with the anti-actin antibodies 1C7 ('lower dimer') and 2G2 ('nuclear actin'), but not C4 (monomeric actin). {yields} Mitotic unconventional actin cables are independent of filamentous actin or microtubules. {yields} Unconventional actin colocalizes with vimentin on a nocodazole-induced perinuclear dense mass of cables. -- Abstract: Different structural conformations of actin have been identified in cells and shown to reside in distinct subcellular locations of cells. In this report, we describe the localization of actin on a cage-like structure in metaphase HEK 293T cells. Actin was detected with the anti-actin antibodies 1C7 and 2G2, but not with the anti-actin antibody C4. Actin contained in this structure is independent of microtubules and actin filaments, and colocalizes with vimentin. Taking advantage of intermediate filament collapse into a perinuclear dense mass of cables when microtubules are depolymerized, we were able to relocalize actin to such structures. We hypothesize that phosphorylation of intermediate filaments at mitosis entry triggers the recruitment of different actin conformations to mitotic intermediate filaments. Storage and partition of the nuclear actin and antiparallel 'lower dimer' actin conformations between daughter cells possibly contribute to gene transcription and transient actin filament dynamics at G1 entry.

  1. GhCFE1A, a dynamic linker between the ER network and actin cytoskeleton, plays an important role in cotton fibre cell initiation and elongation.

    PubMed

    Lv, Fenni; Wang, Haihai; Wang, Xinyu; Han, Libo; Ma, Yinping; Wang, Sen; Feng, Zhidi; Niu, Xiaowei; Cai, Caiping; Kong, Zhaosheng; Zhang, Tianzhen; Guo, Wangzhen

    2015-04-01

    Fibre cell initiation and elongation is critical for cotton fibre development. However, little is known about the regulation of initiation and elongation during fibre cell development. Here, the regulatory role of a novel protein GhCFE1A was uncovered. GhCFE1A is preferentially expressed at initiation and rapid elongation stages during fibre development; in addition, much higher expression of GhCFE1A was detected at the fibre initiation stage in fibreless cotton mutants than in the fibre-bearing TM-1 wild-type. Importantly, overexpression of GhCFE1A in cotton not only delayed fibre cell elongation but also significantly reduced the density of lint and fuzz fibre initials and stem trichomes. Yeast two-hybrid assay showed that GhCFE1A interacted with several actin proteins, and the interaction was further confirmed by co-sedimentation assay. Interestingly, a subcellular localization assay showed that GhCFE1A resided on the cortical endoplasmic reticulum (ER) network and co-localized with actin cables. Moreover, the density of F-actin filaments was shown to be reduced in GhCFE1A-overexpressing fibres at the rapid elongation stage compared with the wild-type control. Taken together, the results demonstrate that GhCFE1A probably functions as a dynamic linker between the actin cytoskeleton and the ER network, and plays an important role in fibre cell initiation and elongation during cotton fibre development.

  2. Hijacking Host Cell Highways: Manipulation of the Host Actin Cytoskeleton by Obligate Intracellular Bacterial Pathogens

    PubMed Central

    Colonne, Punsiri M.; Winchell, Caylin G.; Voth, Daniel E.

    2016-01-01

    Intracellular bacterial pathogens replicate within eukaryotic cells and display unique adaptations that support key infection events including invasion, replication, immune evasion, and dissemination. From invasion to dissemination, all stages of the intracellular bacterial life cycle share the same three-dimensional cytosolic space containing the host cytoskeleton. For successful infection and replication, many pathogens hijack the cytoskeleton using effector proteins introduced into the host cytosol by specialized secretion systems. A subset of effectors contains eukaryotic-like motifs that mimic host proteins to exploit signaling and modify specific cytoskeletal components such as actin and microtubules. Cytoskeletal rearrangement promotes numerous events that are beneficial to the pathogen, including internalization of bacteria, structural support for bacteria-containing vacuoles, altered vesicular trafficking, actin-dependent bacterial movement, and pathogen dissemination. This review highlights a diverse group of obligate intracellular bacterial pathogens that manipulate the host cytoskeleton to thrive within eukaryotic cells and discusses underlying molecular mechanisms that promote these dynamic host-pathogen interactions. PMID:27713866

  3. Fibroblast cytoskeletal remodeling contributes to connective tissue tension.

    PubMed

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

    2011-05-01

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

  4. FIBROBLAST CYTOSKELETAL REMODELING CONTRIBUTES TO CONNECTIVE TISSUE TENSION

    PubMed Central

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

    2011-01-01

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

  5. The More the Tubular: Dynamic Bundling of Actin Filaments for Membrane Tube Formation

    PubMed Central

    Weichsel, Julian; Geissler, Phillip L.

    2016-01-01

    Tubular protrusions are a common feature of living cells, arising from polymerization of stiff protein filaments against a comparably soft membrane. Although this process involves many accessory proteins in cells, in vitro experiments indicate that similar tube-like structures can emerge without them, through spontaneous bundling of filaments mediated by the membrane. Using theory and simulation of physical models, we have elaborated how nonequilibrium fluctuations in growth kinetics and membrane shape can yield such protrusions. Enabled by a new grand canonical Monte Carlo method for membrane simulation, our work reveals a cascade of dynamical transitions from individually polymerizing filaments to highly cooperatively growing bundles as a dynamical bottleneck to tube formation. Filament network organization as well as adhesion points to the membrane, which bias filament bending and constrain membrane height fluctuations, screen the effective attractive interactions between filaments, significantly delaying bundling and tube formation. PMID:27384915

  6. Actin dynamics regulated by the balance of neuronal Wiskott-Aldrich syndrome protein (N-WASP) and cofilin activities determines the biphasic response of glucose-induced insulin secretion.

    PubMed

    Uenishi, Eita; Shibasaki, Tadao; Takahashi, Harumi; Seki, Chihiro; Hamaguchi, Hitomi; Yasuda, Takao; Tatebe, Masao; Oiso, Yutaka; Takenawa, Tadaomi; Seino, Susumu

    2013-09-01

    Actin dynamics in pancreatic β-cells is involved in insulin secretion. However, the molecular mechanisms of the regulation of actin dynamics by intracellular signals in pancreatic β-cells and its role in phasic insulin secretion are largely unknown. In this study, we elucidate the regulation of actin dynamics by neuronal Wiskott-Aldrich syndrome protein (N-WASP) and cofilin in pancreatic β-cells and demonstrate its role in glucose-induced insulin secretion (GIIS). N-WASP, which promotes actin polymerization through activation of the actin nucleation factor Arp2/3 complex, was found to be activated by glucose stimulation in insulin-secreting clonal pancreatic β-cells (MIN6-K8 β-cells). Introduction of a dominant-negative mutant of N-WASP, which lacks G-actin and Arp2/3 complex-binding region VCA, into MIN6-K8 β-cells or knockdown of N-WASP suppressed GIIS, especially the second phase. We also found that cofilin, which severs F-actin in its dephosphorylated (active) form, is converted to the phosphorylated (inactive) form by glucose stimulation in MIN6-K8 β-cells, thereby promoting F-actin remodeling. In addition, the dominant-negative mutant of cofilin, which inhibits activation of endogenous cofilin, or knockdown of cofilin reduced the second phase of GIIS. However, the first phase of GIIS occurs in the G-actin predominant state, in which cofilin activity predominates over N-WASP activity. Thus, actin dynamics regulated by the balance of N-WASP and cofilin activities determines the biphasic response of GIIS.

  7. Microtubule-dependent transport of vimentin filament precursors is regulated by actin and by the concerted action of Rho- and p21-activated kinases.

    PubMed

    Robert, Amélie; Herrmann, Harald; Davidson, Michael W; Gelfand, Vladimir I

    2014-07-01

    Intermediate filaments (IFs) form a dense and dynamic network that is functionally associated with microtubules and actin filaments. We used the GFP-tagged vimentin mutant Y117L to study vimentin-cytoskeletal interactions and transport of vimentin filament precursors. This mutant preserves vimentin interaction with other components of the cytoskeleton, but its assembly is blocked at the unit-length filament (ULF) stage. ULFs are easy to track, and they allow a reliable and quantifiable analysis of movement. Our results show that in cultured human vimentin-negative SW13 cells, 2% of vimentin-ULFs move along microtubules bidirectionally, while the majority are stationary and tightly associated with actin filaments. Rapid motor-dependent transport of ULFs along microtubules is enhanced ≥ 5-fold by depolymerization of actin cytoskeleton with latrunculin B. The microtubule-dependent transport of vimentin ULFs is further regulated by Rho-kinase (ROCK) and p21-activated kinase (PAK): ROCK inhibits ULF transport, while PAK stimulates it. Both kinases act on microtubule transport independently of their effects on actin cytoskeleton. Our study demonstrates the importance of the actin cytoskeleton to restrict IF transport and reveals a new role for PAK and ROCK in the regulation of IF precursor transport.-Robert, A., Herrmann, H., Davidson, M. W., and Gelfand, V. I. Microtubule-dependent transport of vimentin filament precursors is regulated by actin and by the concerted action of Rho- and p21-activated kinases.

  8. Microtubule-dependent transport of vimentin filament precursors is regulated by actin and by the concerted action of Rho- and p21-activated kinases

    PubMed Central

    Robert, Amélie; Herrmann, Harald; Davidson, Michael W.; Gelfand, Vladimir I.

    2014-01-01

    Intermediate filaments (IFs) form a dense and dynamic network that is functionally associated with microtubules and actin filaments. We used the GFP-tagged vimentin mutant Y117L to study vimentin-cytoskeletal interactions and transport of vimentin filament precursors. This mutant preserves vimentin interaction with other components of the cytoskeleton, but its assembly is blocked at the unit-length filament (ULF) stage. ULFs are easy to track, and they allow a reliable and quantifiable analysis of movement. Our results show that in cultured human vimentin-negative SW13 cells, 2% of vimentin-ULFs move along microtubules bidirectionally, while the majority are stationary and tightly associated with actin filaments. Rapid motor-dependent transport of ULFs along microtubules is enhanced ≥5-fold by depolymerization of actin cytoskeleton with latrunculin B. The microtubule-dependent transport of vimentin ULFs is further regulated by Rho-kinase (ROCK) and p21-activated kinase (PAK): ROCK inhibits ULF transport, while PAK stimulates it. Both kinases act on microtubule transport independently of their effects on actin cytoskeleton. Our study demonstrates the importance of the actin cytoskeleton to restrict IF transport and reveals a new role for PAK and ROCK in the regulation of IF precursor transport.—Robert, A., Herrmann, H., Davidson, M. W., and Gelfand, V. I. Microtubule-dependent transport of vimentin filament precursors is regulated by actin and by the concerted action of Rho- and p21-activated kinases. PMID:24652946

  9. Actinic Keratoses

    PubMed Central

    Brown, Marc D.

    2009-01-01

    Actinic keratoses are common intra-epidermal neoplasms that lie on a continuum with squamous cell carcinoma. Tightly linked to ultraviolet irradiation, they occur in areas of chronic sun exposure, and early treatment of these lesions may prevent their progression to invasive disease. A large variety of effective treatment modalities exist, and the optimal therapeutic choice is dependent on a variety of patient- and physician-associated variables. Many established and more recent approaches are discussed in this review with a focus on efficacy and administration techniques. Several previously experimental options, such as imiquimod and photodynamic therapy, have become incorporated as first-line options for the treatment of actinic keratoses, while combination treatment strategies have been gaining in popularity. The goal of all therapies is to ultimately limit the morbidity and mortality of squamous cell carcinoma. (J Clin Aesthetic Dermatol. 2009;2(7):43–48.) PMID:20729970

  10. Quantifying actin wave modulation on periodic topography

    NASA Astrophysics Data System (ADS)

    Guven, Can; Driscoll, Meghan; Sun, Xiaoyu; Parker, Joshua; Fourkas, John; Carlsson, Anders; Losert, Wolfgang

    2014-03-01

    Actin is the essential builder of the cell cytoskeleton, whose dynamics are responsible for generating the necessary forces for the formation of protrusions. By exposing amoeboid cells to periodic topographical cues, we show that actin can be directionally guided via inducing preferential polymerization waves. To quantify the dynamics of these actin waves and their interaction with the substrate, we modify a technique from computer vision called ``optical flow.'' We obtain vectors that represent the apparent actin flow and cluster these vectors to obtain patches of newly polymerized actin, which represent actin waves. Using this technique, we compare experimental results, including speed distribution of waves and distance from the wave centroid to the closest ridge, with actin polymerization simulations. We hypothesize the modulation of the activity of nucleation promotion factors on ridges (elevated regions of the surface) as a potential mechanism for the wave-substrate coupling. Funded by NIH grant R01GM085574.

  11. Entropic forces drive contraction of cytoskeletal networks.

    PubMed

    Braun, Marcus; Lansky, Zdenek; Hilitski, Feodor; Dogic, Zvonimir; Diez, Stefan

    2016-05-01

    The cytoskeleton is a network of interconnected protein filaments, which provide a three-dimensional scaffold for cells. Remodeling of the cytoskeleton is important for key cellular processes, such as cell motility, division, or morphogenesis. This remodeling is traditionally considered to be driven exclusively by processes consuming chemical energy, such as the dynamics of the filaments or the action of molecular motors. Here, we review two mechanisms of cytoskeletal network remodeling that are independent of the consumption of chemical energy. In both cases directed motion of overlapping filaments is driven by entropic forces, which arise from harnessing thermal energy present in solution. Entropic forces are induced either by macromolecular crowding agents or by diffusible crosslinkers confined to the regions where filaments overlap. Both mechanisms increase filament overlap length and lead to the contraction of filament networks. These force-generating mechanisms, together with the chemical energy-dependent mechanisms, need to be considered for the comprehensive quantitative picture of the remodeling of cytoskeletal networks in cells. PMID:26996935

  12. Keratin 8/18 Regulation of Cell Stiffness-Extracellular Matrix Interplay through Modulation of Rho-Mediated Actin Cytoskeleton Dynamics

    PubMed Central

    Bordeleau, François; Myrand Lapierre, Marie-Eve; Sheng, Yunlong; Marceau, Normand

    2012-01-01

    Cell mechanical activity generated from the interplay between the extracellular matrix (ECM) and the actin cytoskeleton is essential for the regulation of cell adhesion, spreading and migration during normal and cancer development. Keratins are the intermediate filament (IF) proteins of epithelial cells, expressed as pairs in a lineage/differentiation manner. Hepatic epithelial cell IFs are made solely of keratins 8/18 (K8/K18), hallmarks of all simple epithelia. Notably, our recent work on these epithelial cells has revealed a key regulatory function for K8/K18 IFs in adhesion/migration, through modulation of integrin interactions with ECM, actin adaptors and signaling molecules at focal adhesions. Here, using K8-knockdown rat H4 hepatoma cells and their K8/K18-containing counterparts seeded on fibronectin-coated substrata of different rigidities, we show that the K8/K18 IF-lacking cells lose their ability to spread and exhibit an altered actin fiber organization, upon seeding on a low-rigidity substratum. We also demonstrate a concomitant reduction in local cell stiffness at focal adhesions generated by fibronectin-coated microbeads attached to the dorsal cell surface. In addition, we find that this K8/K18 IF modulation of cell stiffness and actin fiber organization occurs through RhoA-ROCK signaling. Together, the results uncover a K8/K18 IF contribution to the cell stiffness-ECM rigidity interplay through a modulation of Rho-dependent actin organization and dynamics in simple epithelial cells. PMID:22685604

  13. Chemotaxis and Actin Oscillations

    NASA Astrophysics Data System (ADS)

    Bodenschatz, Eberhard; Hsu, Hsin-Fang; Negrete, Jose; Beta, Carsten; Pumir, Alain; Gholami, Azam; Tarantola, Marco; Westendorf, Christian; Zykov, Vladimir

    Recently, self-oscillations of the cytoskeletal actin have been observed in Dictyostelium, a model system for studying chemotaxis. Here we report experimental results on the self-oscillation mechanism and the role of regulatory proteins and myosin II. We stimulate cells rapidly and periodically by using photo un-caging of the chemoattractant in a micro-fluidic device and measured the cellular responses. We found that the response amplitude grows with stimulation strength only in a very narrow region of stimulation, after which the response amplitude reaches a plateau. Moreover, the frequency-response is not constant but rather varies with the strength of external stimuli. To understand the underlying mechanism, we analyzed the polymerization and de-polymerization time in the single cell level. Despite of the large cell-to-cell variability, we found that the polymerization time is independent of external stimuli and the de-polymerization time is prolonged as the stimulation strength increases. Our conclusions will be summarized and the role of noise in the signaling network will be discussed. German Science Foundation CRC 937.

  14. Steady-state helices of the actin homolog MreB inside bacteria: Dynamics without motors

    NASA Astrophysics Data System (ADS)

    Allard, Jun F.; Rutenberg, Andrew D.

    2007-09-01

    Within individual bacteria, we combine force-dependent polymerization dynamics of individual MreB protofilaments with an elastic model of protofilament bundles buckled into helical configurations. We use variational techniques and stochastic simulations to relate the pitch of the MreB helix, the total abundance of MreB, and the number of protofilaments. By comparing our simulations with mean-field calculations, we find that stochastic fluctuations are significant. We examine the quasistatic evolution of the helical pitch with cell growth, as well as time scales of helix turnover and de novo establishment. We find that while the body of a polarized MreB helix treadmills toward its slow-growing end, the fast-growing tips of laterally associated protofilaments move toward the opposite fast-growing end of the MreB helix. This offers a possible mechanism for targeted polar localization without cytoplasmic motor proteins.

  15. Mechanism of Actin-Based Motility

    NASA Astrophysics Data System (ADS)

    Pantaloni, Dominique; Le Clainche, Christophe; Carlier, Marie-France

    2001-05-01

    Spatially controlled polymerization of actin is at the origin of cell motility and is responsible for the formation of cellular protrusions like lamellipodia. The pathogens Listeria monocytogenes and Shigella flexneri, which undergo actin-based propulsion, are acknowledged models of the leading edge of lamellipodia. Actin-based motility of the bacteria or of functionalized microspheres can be reconstituted in vitro from only five pure proteins. Movement results from the regulated site-directed treadmilling of actin filaments, consistent with observations of actin dynamics in living motile cells and with the biochemical properties of the components of the synthetic motility medium.

  16. Formin' actin in the nucleus.

    PubMed

    Baarlink, Christian; Grosse, Robert

    2014-01-01

    Many if not most proteins can, under certain conditions, change cellular compartments, such as, for example, shuttling from the cytoplasm to the nucleus. Thus, many proteins may exert functions in various and very different subcellular locations, depending on the signaling context. A large amount of actin regulatory proteins has been detected in the mammalian cell nucleus, although their potential roles are much debated and are just beginning to emerge. Recently, members of the formin family of actin nucleators were also reported to dynamically localize to the nuclear environment. Here we discuss our findings that specific diaphanous-related formins can promote nuclear actin assembly in a signal-dependent manner.

  17. Phosphorylation of platelet actin-binding protein during platelet activation

    SciTech Connect

    Carroll, R.C.; Gerrard, J.M.

    1982-03-01

    In this study we have followed the 32P-labeling of actin-binding protein as a function of platelet activation. Utilizing polyacrylamide-sodium dodecyl sulfate gel electrophoresis to resolve total platelet protein samples, we found 2 to 3-fold labeling increases in actin-binding protein 30 to 60 sec after thrombin stimulation. Somewhat larger increases were observed for 40,000 and 20,000 apparent molecular weight peptides. The actin-binding protein was identified on the gels by coelectrophoresis with purified actin-binding protein, its presence in cytoskeletal cores prepared by detergent extraction of activated 32P-labeled platelets, and by direct immunoprecipitation with antibodies against guinea pig vas deferens filamin (actin-binding protein). In addition, these cytoskeletal cores indicated that the 32P-labeled actin-binding protein was closely associated with the activated platelet's cytoskeleton. Following the 32P-labeling of actin-binding protein over an 8-min time course revealed that in aggregating platelet samples rapid dephosphorylation to almost initial levels occurred between 3 and 5 min. A similar curve was obtained for the 20,000 apparent molecular weight peptide. However, rapid dephosphorylation was not observed if platelet aggregation was prevented by chelating external calcium or by using thrombasthenic platelets lacking the aggregation response. Thus, cell-cell contact would seem to be crucial in initiating the rapid dephosphorylation response.

  18. Formation of actin networks in microfluidic concentration gradients

    NASA Astrophysics Data System (ADS)

    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.

  19. Platelet adhesion: structural and functional diversity of short dystrophin and utrophins in the formation of dystrophin-associated-protein complexes related to actin dynamics

    PubMed Central

    Cerecedo, Doris; Martínez-Rojas, Dalila; Chávez, Oscar; Martínez-Pérez, Francisco; García-Sierra, Francisco; Rendon, Alvaro; Mornet, Dominique; Mondragón, Ricardo

    2005-01-01

    Summary Platelets are dynamic cell fragments that modify their shape during activation. Utrophin and dystrophins are minor actin-binding proteins present in muscle and non-muscle cytoskeleton. In the present study, we characterised the pattern of Dp71 isoforms and utrophin gene products by immunoblot in human platelets. Two new dystrophin isoforms were found, Dp71f and Dp71d, as well as the Up71 isoform and the dystrophin-associated proteins, α and β-dystrobrevins. Distribution of Dp71d/Dp71Δ110m, Up400/Up71 and dystrophin-associated proteins in relation to the actin cytoskeleton was evaluated by confocal microscopy in both resting and platelets adhered on glass. Formation of two dystrophin-associated protein complexes (Dp71d/Dp71Δ110m~DAPC and Up400/Up71~DAPC) was demonstrated by co-immunoprecipitation and their distribution in relation to the actin cytoskeleton was characterised during platelet adhesion. The Dp71d/Dp71Δ110m~DAPC is maintained mainly at the granulomere and is associated with dynamic structures during activation by adhesion to thrombin-coated surfaces. Participation of both Dp71d/Dp71Δ110m~DAPC and Up400/Up71~DAPC in the biological roles of the platelets is discussed. PMID:16411395

  20. Platelet adhesion: structural and functional diversity of short dystrophin and utrophins in the formation of dystrophin-associated-protein complexes related to actin dynamics.

    PubMed

    Cerecedo, Doris; Martínez-Rojas, Dalila; Chávez, Oscar; Martínez-Pérez, Francisco; García-Sierra, Francisco; Rendon, Alvaro; Mornet, Dominique; Mondragón, Ricardo

    2005-12-01

    Platelets are dynamic cell fragments that modify their shape during activation. Utrophin and dystrophins are minor actin-binding proteins present in muscle and non-muscle cytoskeleton. In the present study, we characterised the pattern of Dp71 isoforms and utrophin gene products by immunoblot in human platelets. Two new dystrophin isoforms were found, Dp71f and Dp71 d, as well as the Up71 isoform and the dystrophin-associated proteins, alpha and beta -dystrobrevins. Distribution of Dp71d/Dp71delta110m, Up400/Up71 and dystrophin-associated proteins in relation to the actin cytoskeleton was evaluated by confocal microscopy in both resting and platelets adhered on glass. Formation of two dystrophin-associated protein complexes (Dp71d/Dp71delta110m approximately DAPC and Up400/Up71 approximately DAPC) was demonstrated by co-immunoprecipitation and their distribution in relation to the actin cytoskeleton was characterised during platelet adhesion. The Dp71d/Dp71delta100m approximately DAPC is maintained mainly at the granulomere and is associated with dynamic structures during activation by adhesion to thrombin-coated surfaces. Participation of both Dp71d/Dp71delta110m approximately DAPC and Up400/Up71 approximately DAPC in the biological roles of the platelets is discussed.

  1. Cytoskeletal reorganization of human platelets after stimulation revealed by the quick-freeze deep-etch technique

    PubMed Central

    1987-01-01

    We studied the cytoskeletal reorganization of saponized human platelets after stimulation by using the quick-freeze deep-etch technique, and examined the localization of myosin in thrombin-treated platelets by immunocytochemistry at the electron microscopic level. In unstimulated saponized platelets we observed cross-bridges between: adjoining microtubules, adjoining actin filaments, microtubules and actin filaments, and actin filaments and plasma membranes. After activation with 1 U/ml thrombin for 3 min, massive arrays of actin filaments with mixed polarity were found in the cytoplasm. Two types of cross-bridges between actin filaments were observed: short cross-bridges (11 +/- 2 nm), just like those observed in the resting platelets, and longer ones (22 +/- 3 nm). Actin filaments were linked with the plasma membrane via fine short filaments and sometimes ended on the membrane. Actin filaments and microtubules frequently ran close to the membrane organelles. We also found that actin filaments were associated by end- on attachments with some organelles. Decoration with subfragment 1 of myosin revealed that all the actin filaments associated end-on with the membrane pointed away in their polarity. Immunocytochemical study revealed that myosin was present in the saponin-extracted cytoskeleton after activation and that myosin was localized on the filamentous network. The results suggest that myosin forms a gel with actin filaments in activated platelets. Close associations between actin filaments and organelles in activated platelets suggests that contraction of this actomyosin gel could bring about the observed centralization of organelles. PMID:3667697

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

    PubMed

    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.

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

    PubMed

    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

  4. Effects of ionizing radiation on expression of genes encoding cytoskeletal elements: Kinetics and dose effects

    SciTech Connect

    Woloschak, G.E.; Shearin-Jones, P.; Chang-Liu, C.M. )

    1990-01-01

    We examined the modulation in expression of genes encoding three cytoskeletal elements (beta-actin, gamma-actin, and alpha-tubulin) in Syrian hamster embryo (SHE) cells following exposure to ionizing radiations. Early-passage SHE cells were irradiated in plateau phase with various low doses (12-200 cGy) of neutrons, gamma-rays, or x-rays. RNA samples were prepared from cells at different times postexposure and were analyzed for levels of specific transcripts by northern blots. The results revealed that alpha-tubulin was induced by both high-linear energy of transfer (LET) (neutrons) and low-LET (gamma-rays and x-rays) radiations with similar kinetics. The peak in alpha-tubulin mRNA accumulation occurred between 1 and 3 h postexposure; for gamma-actin mRNA, accumulation was similarly induced. For both gamma-actin and alpha-tubulin, the higher the dose during the first hour postexposure (up to 200 cGy gamma-rays), the greater the level of mRNA induction. In contrast, mRNA specific for beta-actin showed decreased accumulation during the first hour following radiation exposure, and remained low up to 3 h postexposure. These results document the differential modulation of genes specific for cytoskeletal elements following radiation exposure. In addition, they demonstrate a decrease in the ratio of beta-actin:gamma-actin mRNA within the first 3 h following gamma-ray exposure. These changes in mRNA accumulation are similar to those reported in some transformed cell lines and in cells treated with tumor promoters, which suggests a role for changes in actin- and tubulin-mRNA expression in radiation-mediated transformation.

  5. Methods for modeling cytoskeletal and DNA filaments

    NASA Astrophysics Data System (ADS)

    Andrews, Steven S.

    2014-02-01

    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.

  6. Cosolvent and Crowding Effects on the Temperature and Pressure Dependent Conformational Dynamics and Stability of Globular Actin.

    PubMed

    Schummel, Paul Hendrik; Haag, Andreas; Kremer, Werner; Kalbitzer, Hans Robert; Winter, Roland

    2016-07-14

    Actin can be found in nearly all eukaryotic cells and is responsible for many different cellular functions. The polymerization process of actin has been found to be among the most pressure sensitive processes in vivo. In this study, we explored the effects of chaotropic and kosmotropic cosolvents, such as urea and the compatible osmolyte trimethylamine-N-oxide (TMAO), and, to mimic a more cell-like environment, crowding agents on the pressure and temperature stability of globular actin (G-actin). The temperature and pressure of unfolding as well as thermodynamic parameters upon unfolding, such as enthalpy and volume changes, have been determined by fluorescence spectroscopy over a wide range of temperatures and pressures, ranging from 10 to 80 °C and from 1 to 3000 bar, respectively. Complementary high-pressure NMR studies revealed additional information on the existence of native-like conformational substates of G-actin as well as a molten-globule-like state preceding the complete pressure denaturation. Different from the chaotropic agent urea, TMAO increases both the temperature and pressure stability for the protein most effectively. The Gibbs free energy differences of most of the native substates detected are not influenced significantly by TMAO. In mixtures of these osmolytes, urea counteracts the stabilizing effect of TMAO to some extent. Addition of the crowding agent Ficoll increases the temperature and pressure stability even further, thereby allowing sufficient stability of the protein at temperature and pressure conditions encountered under extreme environmental conditions on Earth. PMID:27314563

  7. Structure and Dynamics of an Arp2/3 Complex-independent Component of the Lamellipodial Actin Network

    PubMed Central

    Henson, John H.; Cheung, David; Fried, Christopher A.; Shuster, Charles B.; McClellan, Mary K.; Voss, Meagen K.; Sheridan, John T.; Oldenbourg, Rudolf

    2010-01-01

    Sea urchin coelomocytes contain an unusually broad lamellipodial region and have served as a useful model experimental system for studying the process of actin-based retrograde/centripetal flow. In the current study the small molecule drug 2,3-butanedione monoxime (BDM) was employed as a means of delocalizing the Arp2/3 complex from the cell edge in an effort to investigate the Arp2/3 complex-independent aspects of retrograde flow. Digitally-enhanced phase contrast, fluorescence and polarization light microscopy, along with rotary shadow TEM methods demonstrated that BDM treatment resulted in the centripetal displacement of the Arp2/3 complex and the associated dendritic lamellipodial (LP) actin network from the cell edge. In its wake there remained an array of elongate actin filaments organized into concave arcs that displayed retrograde flow at approximately one quarter the normal rate. Actin polymerization inhibitor experiments indicated that these arcs were generated by polymerization at the cell edge, while active myosin-based contraction in BDM treated cells was demonstrated by localization with anti-phospho-MRLC antibody, the retraction of the cytoskeleton in the presence of BDM, and the response of the BDM arcs to laser-based severing. The results suggest that BDM treatment reveals an Arp2/3 complex-independent actin structure in coelomocytes consisting of elongate filaments integrated into the LP network and that these filaments represent a potential connection between the LP network and the central cytoskeleton. PMID:19530177

  8. On the Significance of Microtubule Flexural Behavior in Cytoskeletal Mechanics

    PubMed Central

    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

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

    PubMed

    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

  10. Microrheology and micromechanics of actin-coated membranes

    NASA Astrophysics Data System (ADS)

    Bourdieu, Laurent

    2002-03-01

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

  11. Alpha 7 nicotinic receptor coupling to heterotrimeric G proteins modulates RhoA activation, cytoskeletal motility, and structural growth.

    PubMed

    King, Justin R; Kabbani, Nadine

    2016-08-01

    Nicotinic acetylcholine receptors (nAChRs) modulate the growth and structure of neurons throughout the nervous system. Ligand stimulation of the α7 nAChR has been shown to regulate the large heterotrimeric GTP-binding protein (G protein) signaling in various types of cells. Here, we demonstrate a role for α7 nAChR/G protein interaction in the activation of the small (monomeric) RhoA GTPase leading to cytoskeletal changes during neurite growth. Treatment of PC12 cells with the α7 nAChR agonist choline or PNU-282987 was associated with an increase in RhoA activity and an inhibition in neurite growth. Specifically, choline treatment was found to attenuate the velocity of microtubule growth at the growth cone and decrease the rate of actin polymerization throughout the cell. The effects of α7 nAChR activation were abolished by expression of a dominant negative α7 nAChR (α7345-348A ) deficient in G protein coupling. Proteomic analysis of immunoprecipitated α7 nAChR complexes from differentiating PC12 cells and synaptic fractions of the developing mouse hippocampus revealed the existence of Rho GTPase-regulating guanine nucleotide exchange factors within α7 nAChR interactomes. These findings underscore the role of α7 nAChR/G protein in cytoskeletal regulation during neurite growth. This image depicts the hypothesized interaction of the traditionally ionotropic α7 nicotinic acetylcholine receptor (α7 nAChR) and its ability to interact and signal through both large and small G proteins, leading to the regulation of cytoskeletal growth. Using differentiated PC12 cells, and the specific agonist choline, it was shown that α7 nAChR/G protein interactions mediate both short- and long-term neurite growth dynamics through increased RhoA activation. Activation of RhoA was shown to decrease actin polymerization, and lead to an overall decrease in neurite growth via regulation of the microtubule network. Cover Image for this issue: doi: 10.1111/jnc.13330.

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

    PubMed

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

    2012-11-01

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

  13. In vitro and in vivo evidence for actin association of the naphthylphthalamic acid-binding protein from zucchini hypocotyls

    NASA Technical Reports Server (NTRS)

    Butler, J. H.; Hu, S.; Brady, S. R.; Dixon, M. W.; Muday, G. K.

    1998-01-01

    The N-1-naphthylphthalamic acid (NPA)-binding protein is part of the auxin efflux carrier, the protein complex that controls polar auxin transport in plant tissues. This study tested the hypothesis that the NPA-binding protein (NBP) is associated with the actin cytoskeleton in vitro and that an intact actin cytoskeleton is required for polar auxin transport in vivo. Cytoskeletal polymerization was altered in extracts of zucchini hypocotyls with reagents that stabilized either the polymeric or monomeric forms of actin or tubulin. Phalloidin treatment altered actin polymerization, as demonstrated by immunoblot analyses following native and denaturing electrophoresis. Phalloidin increased both filamentous actin (F-actin) and NPA-binding activity, while cytochalasin D and Tris decreased both F-actin and NPA-binding activity in cytoskeletal pellets. The microtubule stabilizing drug taxol increased pelletable tubulin, but did not alter either the amount of pelletable actin or NPA-binding activity. Treatment of etiolated zucchini hypocotyls with cytochalasin D decreased the amount of auxin transport and its regulation by NPA. These experimental results are consistent with an in vitro actin cytoskeletal association of the NPA-binding protein and with the requirement of an intact actin cytoskeleton for maximal polar auxin transport in vivo.

  14. [Actinic Keratosis].

    PubMed

    Dejaco, D; Hauser, U; Zelger, B; Riechelmann, H

    2015-07-01

    Actinic keratosis is a cutaneous lesion characterized by proliferation of atypical epidermal keratinocytes due to prolonged exposure to exogenous factors such as ultraviolet radiation. AKs are in-situ-squamous cell carcinomas (PEC) of the skin. AK typically presents as erythematous, scaly patch or papule (classic AK), occasionally as thick, adherent scale on an erythematous base. Mostly fair-skinned adults are affected. AKs typically occur in areas of frequent sun exposure (balding scalp, face, "H-region", lateral neck, décolleté, dorsum of the hand and lower extremities). Actinic Cheilitis is the term used for AKs appearing on the lips. The diagnosis of AK is based on clinical examination including inspection and palpation. The typical palpable rough surface of AK often precedes a visible lesion. Dermoscopy may provide additional information. If diagnosis is uncertain and invasion suspected, biopsy and histopathologic evaluation should be performed. The potential for progression to invasive PECs mandates therapeutic intervention. Treatment options include topical and systemic therapies. Topical therapies are classified into physical, medical and combined physical-chemical approaches and a sequential combination of treatment modalities is possible. Topical-physical cryotherapy is the treatment of choice for isolated, non-hypertrophic AK. Topical-medical treatment, e. g. 5-fluoruracil (5FU) cream or Imiquomod or Ingenolmebutat application is used for multiple, non-hypertrophic AKs. For hypertrophic AKs, a dehorning pretreatment with salicinated vaseline is recommended. Isolated hypertrophic AKs often need cryotherapy with prolonged freezing time or several consecutive applications. Sequentially combined approaches are recommended for multiple, hypertrophic AKs. Photodynamic therapy (PDT) as example for a combined physical-chemical approach is an established treatment for multiple, non-hypertrophic and hypertrophic AKs. Prevention includes avoidance of sun and

  15. Integration of linear and dendritic actin nucleation in Nck-induced actin comets

    PubMed Central

    Borinskaya, Sofya; Velle, Katrina B.; Campellone, Kenneth G.; Talman, Arthur; Alvarez, Diego; Agaisse, Hervé; Wu, Yi I.; Loew, Leslie M.; Mayer, Bruce J.

    2016-01-01

    The Nck adaptor protein recruits cytosolic effectors such as N-WASP that induce localized actin polymerization. Experimental aggregation of Nck SH3 domains at the membrane induces actin comet tails—dynamic, elongated filamentous actin structures similar to those that drive the movement of microbial pathogens such as vaccinia virus. Here we show that experimental manipulation of the balance between unbranched/branched nucleation altered the morphology and dynamics of Nck-induced actin comets. Inhibition of linear, formin-based nucleation with the small-molecule inhibitor SMIFH2 or overexpression of the formin FH1 domain resulted in formation of predominantly circular-shaped actin structures with low mobility (actin blobs). These results indicate that formin-based linear actin polymerization is critical for the formation and maintenance of Nck-dependent actin comet tails. Consistent with this, aggregation of an exclusively branched nucleation-promoting factor (the VCA domain of N-WASP), with density and turnover similar to those of N-WASP in Nck comets, did not reconstitute dynamic, elongated actin comets. Furthermore, enhancement of branched Arp2/3-mediated nucleation by N-WASP overexpression caused loss of the typical actin comet tail shape induced by Nck aggregation. Thus the ratio of linear to dendritic nucleation activity may serve to distinguish the properties of actin structures induced by various viral and bacterial pathogens. PMID:26609071

  16. Integration of linear and dendritic actin nucleation in Nck-induced actin comets.

    PubMed

    Borinskaya, Sofya; Velle, Katrina B; Campellone, Kenneth G; Talman, Arthur; Alvarez, Diego; Agaisse, Hervé; Wu, Yi I; Loew, Leslie M; Mayer, Bruce J

    2016-01-15

    The Nck adaptor protein recruits cytosolic effectors such as N-WASP that induce localized actin polymerization. Experimental aggregation of Nck SH3 domains at the membrane induces actin comet tails--dynamic, elongated filamentous actin structures similar to those that drive the movement of microbial pathogens such as vaccinia virus. Here we show that experimental manipulation of the balance between unbranched/branched nucleation altered the morphology and dynamics of Nck-induced actin comets. Inhibition of linear, formin-based nucleation with the small-molecule inhibitor SMIFH2 or overexpression of the formin FH1 domain resulted in formation of predominantly circular-shaped actin structures with low mobility (actin blobs). These results indicate that formin-based linear actin polymerization is critical for the formation and maintenance of Nck-dependent actin comet tails. Consistent with this, aggregation of an exclusively branched nucleation-promoting factor (the VCA domain of N-WASP), with density and turnover similar to those of N-WASP in Nck comets, did not reconstitute dynamic, elongated actin comets. Furthermore, enhancement of branched Arp2/3-mediated nucleation by N-WASP overexpression caused loss of the typical actin comet tail shape induced by Nck aggregation. Thus the ratio of linear to dendritic nucleation activity may serve to distinguish the properties of actin structures induced by various viral and bacterial pathogens. PMID:26609071

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

    NASA Astrophysics Data System (ADS)

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

    2010-05-01

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

  18. Structural Rearrangements in CHO Cells After Disruption of Individual Cytoskeletal Elements and Plasma Membrane.

    PubMed

    Jokhadar, Špela Zemljič; Derganc, Jure

    2015-04-01

    Cellular structural integrity is provided primarily by the cytoskeleton, which comprises microtubules, actin filaments, and intermediate filaments. The plasma membrane has been also recognized as a mediator of physical forces, yet its contribution to the structural integrity of the cell as a whole is less clear. In order to investigate the relationship between the plasma membrane and the cytoskeleton, we selectively disrupted the plasma membrane and each of the cytoskeletal elements in Chinese hamster ovary cells and assessed subsequent changes in cellular structural integrity. Confocal microscopy was used to visualize cytoskeletal rearrangements, and optical tweezers were utilized to quantify membrane tether extraction. We found that cholesterol depletion from the plasma membrane resulted in rearrangements of all cytoskeletal elements. Conversely, the state of the plasma membrane, as assessed by tether extraction, was affected by disruption of any of the cytoskeletal elements, including microtubules and intermediate filaments, which are located mainly in the cell interior. The results demonstrate that, besides the cytoskeleton, the plasma membrane is an important contributor to cellular integrity, possibly by acting as an essential framework for cytoskeletal anchoring. In agreement with the tensegrity model of cell mechanics, our results support the notion of the cell as a prestressed structure. PMID:25395197

  19. Structural Differences Explain Diverse Functions of Plasmodium Actins

    PubMed Central

    Vahokoski, Juha; Martinez, Silvia Muñico; Ignatev, Alexander; Lepper, Simone; Frischknecht, Friedrich; Sidén-Kiamos, Inga; Sachse, Carsten; Kursula, Inari

    2014-01-01

    Actins are highly conserved proteins and key players in central processes in all eukaryotic cells. The two actins of the malaria parasite are among the most divergent eukaryotic actins and also differ from each other more than isoforms in any other species. Microfilaments have not been directly observed in Plasmodium and are presumed to be short and highly dynamic. We show that actin I cannot complement actin II in male gametogenesis, suggesting critical structural differences. Cryo-EM reveals that Plasmodium actin I has a unique filament structure, whereas actin II filaments resemble canonical F-actin. Both Plasmodium actins hydrolyze ATP more efficiently than α-actin, and unlike any other actin, both parasite actins rapidly form short oligomers induced by ADP. Crystal structures of both isoforms pinpoint several structural changes in the monomers causing the unique polymerization properties. Inserting the canonical D-loop to Plasmodium actin I leads to the formation of long filaments in vitro. In vivo, this chimera restores gametogenesis in parasites lacking actin II, suggesting that stable filaments are required for exflagellation. Together, these data underline the divergence of eukaryotic actins and demonstrate how structural differences in the monomers translate into filaments with different properties, implying that even eukaryotic actins have faced different evolutionary pressures and followed different paths for developing their polymerization properties. PMID:24743229

  20. Active gels: dynamics of patterning and self-organization

    NASA Astrophysics Data System (ADS)

    Backouche, F.; Haviv, L.; Groswasser, D.; Bernheim-Groswasser, A.

    2006-12-01

    The actin cytoskeleton is an active gel which constantly remodels during cellular processes such as motility and division. Myosin II molecular motors are involved in this active remodeling process and therefore control the dynamic self-organization of cytoskeletal structures. Due to the complexity of in vivo systems, it is hard to investigate the role of myosin II in the reorganization process which determines the resulting cytoskeletal structures. Here we use an in vitro model system to show that myosin II actively reorganizes actin into a variety of mesoscopic patterns, but only in the presence of bundling proteins. We find that the nature of the reorganization process is complex, exhibiting patterns and dynamical phenomena not predicted by current theoretical models and not observed in corresponding passive systems (excluding motors). This system generates active networks, asters and even rings depending on motor and bundling protein concentrations. Furthermore, the motors generate the formation of the patterns, but above a critical concentration they can also disassemble them and even totally prevent the polymerization and bundling of actin filaments. These results may suggest that tuning the assembly and disassembly of cytoskeletal structures can be obtained by tuning the local myosin II concentration/activity.

  1. F-actin bundles direct the initiation and orientation of lamellipodia through adhesion-based signaling

    PubMed Central

    Johnson, Heath E.; King, Samantha J.; Asokan, Sreeja B.; Rotty, Jeremy D.; Bear, James E.

    2015-01-01

    Mesenchymal cells such as fibroblasts are weakly polarized and reorient directionality by a lamellipodial branching mechanism that is stabilized by phosphoinositide 3-kinase (PI3K) signaling. However, the mechanisms by which new lamellipodia are initiated and directed are unknown. Using total internal reflection fluorescence microscopy to monitor cytoskeletal and signaling dynamics in migrating cells, we show that peripheral F-actin bundles/filopodia containing fascin-1 serve as templates for formation and orientation of lamellipodia. Accordingly, modulation of fascin-1 expression tunes cell shape, quantified as the number of morphological extensions. Ratiometric imaging reveals that F-actin bundles/filopodia play both structural and signaling roles, as they prime the activation of PI3K signaling mediated by integrins and focal adhesion kinase. Depletion of fascin-1 ablated fibroblast haptotaxis on fibronectin but not platelet-derived growth factor chemotaxis. Based on these findings, we conceptualize haptotactic sensing as an exploration, with F-actin bundles directing and lamellipodia propagating the process and with signaling mediated by adhesions playing the role of integrator. PMID:25666809

  2. Specific release of membrane-bound annexin II and cortical cytoskeletal elements by sequestration of membrane cholesterol.

    PubMed Central

    Harder, T; Kellner, R; Parton, R G; Gruenberg, J

    1997-01-01

    Annexin II is an abundant protein which is present in the cytosol and on the cytoplasmic face of plasma membrane and early endosomes. It is generally believed that this association occurs via Ca(2+)-dependent binding to lipids, a mechanism typical for the annexin protein family. Although previous studies have shown that annexin II is involved in early endosome dynamics and organization, the precise biological role of the protein is unknown. In this study, we found that approximately 50% of the total cellular annexin was associated with membranes in a Ca(2+)-independent manner. This binding was extremely tight, since it resisted high salt and, to some extent, high pH treatments. We found, however, that membrane-associated annexin II could be quantitatively released by low concentrations of the cholesterol-sequestering agents filipin and digitonin. Both treatments released an identical and limited set of proteins but had no effects on other membrane-associated proteins. Among the released proteins, we identified, in addition to annexin II itself, the cortical cytoskeletal proteins alpha-actinin, ezrin and moesin, and membrane-associated actin. Our biochemical and immunological observations indicate that these proteins are part of a complex containing annexin II and that stability of the complex is sensitive to cholesterol sequestering agents. Since annexin II is tightly membrane-associated in a cholesterol-dependent manner, and since it seems to interact physically with elements of the cortical actin cytoskeleton, we propose that the protein serves as interface between membranes containing high amounts of cholesterol and the actin cytoskeleton. Images PMID:9188103

  3. Cytoskeletal protein kinases: titin and its relations in mechanosensing.

    PubMed

    Gautel, Mathias

    2011-07-01

    Titin, the giant elastic ruler protein of striated muscle sarcomeres, contains a catalytic kinase domain related to a family of intrasterically regulated protein kinases. The most extensively studied member of this branch of the human kinome is the Ca(2+)-calmodulin (CaM)-regulated myosin light-chain kinases (MLCK). However, not all kinases of the MLCK branch are functional MLCKs, and about half lack a CaM binding site in their C-terminal autoinhibitory tail (AI). A unifying feature is their association with the cytoskeleton, mostly via actin and myosin filaments. Titin kinase, similar to its invertebrate analogue twitchin kinase and likely other "MLCKs", is not Ca(2+)-calmodulin-activated. Recently, local protein unfolding of the C-terminal AI has emerged as a common mechanism in the activation of CaM kinases. Single-molecule data suggested that opening of the TK active site could also be achieved by mechanical unfolding of the AI. Mechanical modulation of catalytic activity might thus allow cytoskeletal signalling proteins to act as mechanosensors, creating feedback mechanisms between cytoskeletal tension and tension generation or cellular remodelling. Similar to other MLCK-like kinases like DRAK2 and DAPK1, TK is linked to protein turnover regulation via the autophagy/lysosomal system, suggesting the MLCK-like kinases have common functions beyond contraction regulation. PMID:21416260

  4. Localizations of γ-Actins in Skin, Hair, Vibrissa, Arrector Pili Muscle and Other Hair Appendages of Developing Rats

    PubMed Central

    Morioka, Kiyokazu; Takano-Ohmuro, Hiromi

    2016-01-01

    Six isoforms of actins encoded by different genes have been identified in mammals including α-cardiac, α-skeletal, α-smooth muscle (α-SMA), β-cytoplasmic, γ-smooth muscle (γ-SMA), and γ-cytoplasmic actins (γ-CYA). In a previous study we showed the localization of α-SMA and other cytoskeletal proteins in the hairs and their appendages of developing rats (Morioka K., et al. (2011) Acta Histochem. Cytochem. 44, 141–153), and herein we determined the localization of γ type actins in the same tissues and organs by immunohistochemical staining. Our results indicate that the expression of γ-SMA and γ-CYA is suggested to be poor in actively proliferating tissues such as the basal layer of the epidermis and the hair matrix in the hair bulb, and as well as in highly keratinized tissues such as the hair cortex and hair cuticle. In contrast, the expression of γ-actins were high in the spinous layer, granular layer, hair shaft, and inner root sheath, during their active differentiations. In particular, the localization of γ-SMA was very similar to that of α-SMA. It was located not only in the arrector pili muscles and muscles in the dermis, but also in the dermal sheath and in a limited area of the outer root sheath in both the hair and vibrissal follicles. The γ-CYA was suggested to be co-localized with γ-SMA in the dermal sheath, outer root sheath, and arrector pili muscles. Sparsely distributed dermal cells expressed both types of γ-actin. The expression of γ-actins is suggested to undergo dynamic changes according to the proliferation and differentiation of the skin and hair-related cells. PMID:27222613

  5. The Effect of Ultrasound Stimulation on the Cytoskeletal Organization of Chondrocytes Seeded In 3D Matrices

    PubMed Central

    Noriega, Sandra; Hasanova, Gulnara; Subramanian, Anuradha

    2013-01-01

    The impact of low intensity diffuse ultrasound (LIDUS) stimulation on the cytoskeletal organization of chondrocytes seeded in 3D scaffolds was evaluated. Chondrocytes seeded on 3D chitosan matrices were exposed to LIDUS at 5.0 MHz (~15kPa, 51-secs, 4-applications/day) in order to study the organization of actin, tubulin and vimentin. The results showed that actin presented a cytosolic punctuated distribution, tubulin presented a quasi parallel organization of microtubules whereas vimentin distribution was unaffected. Chondrocytes seeded on 3D scaffolds responded to US stimulation by the disruption of actin stress fibers and were sensitive to the presence of ROCK inhibitor (Y27632). The gene expression of ROCK-I, a key element in the formation of stress fibers and mDia1, was significantly up-regulated under the application of US. We conclude that the results of both the cytoskeletal analyses and gene expression support the argument that the presence of punctuated actin upon US stimulation was accompanied by the up-regulation of the RhoA/ROCK pathway. PMID:22987069

  6. Fascin 1 is an actin filament-bundling protein that regulates ectoplasmic specialization dynamics in the rat testis.

    PubMed

    Gungor-Ordueri, N Ece; Celik-Ozenci, Ciler; Cheng, C Yan

    2014-11-01

    In the testis, spermatids are polarized cells, with their heads pointing toward the basement membrane during maturation. This polarity is crucial to pack the maximal number of spermatids in the seminiferous epithelium so that millions of sperms can be produced daily. A loss of spermatid polarity is detected after rodents are exposed to toxicants (e.g., cadmium) or nonhormonal male contraceptives (e.g., adjudin), which is associated with a disruption on the expression and/or localization of polarity proteins. In the rat testis, fascin 1, an actin-bundling protein found in mammalian cells, was expressed by Sertoli and germ cells. Fascin 1 was a component of the ectoplasmic specialization (ES), a testis-specific anchoring junction known to confer spermatid adhesion and polarity. Its expression in the seminiferous epithelium was stage specific. Fascin 1 was localized to the basal ES at the Sertoli cell-cell interface of the blood-testis barrier in all stages of the epithelial cycle, except it diminished considerably at late stage VIII. Fascin 1 was highly expressed at the apical ES at stage VII-early stage VIII and restricted to the step 19 spermatids. Its knockdown by RNAi that silenced fascin 1 by ~70% in Sertoli cells cultured in vitro was found to perturb the tight junction-permeability barrier via a disruption of F-actin organization. Knockdown of fascin 1 in vivo by ~60-70% induced defects in spermatid polarity, which was mediated by a mislocalization and/or downregulation of actin-bundling proteins Eps8 and palladin, thereby impeding F-actin organization and disrupting spermatid polarity. In summary, these findings provide insightful information on spermatid polarity regulation.

  7. Fascin 1 is an actin filament-bundling protein that regulates ectoplasmic specialization dynamics in the rat testis

    PubMed Central

    Gungor-Ordueri, N. Ece; Celik-Ozenci, Ciler

    2014-01-01

    In the testis, spermatids are polarized cells, with their heads pointing toward the basement membrane during maturation. This polarity is crucial to pack the maximal number of spermatids in the seminiferous epithelium so that millions of sperms can be produced daily. A loss of spermatid polarity is detected after rodents are exposed to toxicants (e.g., cadmium) or nonhormonal male contraceptives (e.g., adjudin), which is associated with a disruption on the expression and/or localization of polarity proteins. In the rat testis, fascin 1, an actin-bundling protein found in mammalian cells, was expressed by Sertoli and germ cells. Fascin 1 was a component of the ectoplasmic specialization (ES), a testis-specific anchoring junction known to confer spermatid adhesion and polarity. Its expression in the seminiferous epithelium was stage specific. Fascin 1 was localized to the basal ES at the Sertoli cell-cell interface of the blood-testis barrier in all stages of the epithelial cycle, except it diminished considerably at late stage VIII. Fascin 1 was highly expressed at the apical ES at stage VII–early stage VIII and restricted to the step 19 spermatids. Its knockdown by RNAi that silenced fascin 1 by ∼70% in Sertoli cells cultured in vitro was found to perturb the tight junction-permeability barrier via a disruption of F-actin organization. Knockdown of fascin 1 in vivo by ∼60–70% induced defects in spermatid polarity, which was mediated by a mislocalization and/or downregulation of actin-bundling proteins Eps8 and palladin, thereby impeding F-actin organization and disrupting spermatid polarity. In summary, these findings provide insightful information on spermatid polarity regulation. PMID:25159326

  8. Structural Polymorphism of the Actin-Espin System: A Prototypical System of Filaments and Linkers in Stereocilia

    SciTech Connect

    Purdy, Kirstin R.; Wong, Gerard C. L.; Bartles, James R.

    2007-02-02

    We examine the interaction between cytoskeletal F-actin and espin 3A, a prototypical actin bundling protein found in sensory cell microvilli, including ear cell stereocilia. Espin induces twist distortions in F-actin as well as facilitates bundle formation. Mutations in one of the two F-actin binding sites of espin, which have been implicated in deafness, can tune espin-actin interactions and radically transform the system's phase behavior. These results are compared to recent theoretical work on the general phase behavior linker-rod systems.

  9. Structural Polymorphism of the Actin-Espin System: A Prototypical System of Filaments and Linkers in Stereocilia

    NASA Astrophysics Data System (ADS)

    Purdy, Kirstin R.; Bartles, James R.; Wong, Gerard C. L.

    2007-02-01

    We examine the interaction between cytoskeletal F-actin and espin 3A, a prototypical actin bundling protein found in sensory cell microvilli, including ear cell stereocilia. Espin induces twist distortions in F-actin as well as facilitates bundle formation. Mutations in one of the two F-actin binding sites of espin, which have been implicated in deafness, can tune espin-actin interactions and radically transform the system’s phase behavior. These results are compared to recent theoretical work on the general phase behavior linker-rod systems.

  10. The actin cytoskeleton in endothelial cell phenotypes

    PubMed Central

    Prasain, Nutan; Stevens, Troy

    2009-01-01

    Endothelium forms a semi-permeable barrier that separates blood from the underlying tissue. Barrier function is largely determined by cell-cell and cell-matrix adhesions that define the limits of cell borders. Yet, such cell-cell and cell-matrix tethering is critically reliant upon the nature of adherence within the cell itself. Indeed, the actin cytoskeleton fulfills this essential function, to provide a strong, dynamic intracellular scaffold that organizes integral membrane proteins with the cell’s interior, and responds to environmental cues to orchestrate appropriate cell shape. The actin cytoskeleton is comprised of three distinct, but interrelated structures, including actin cross-linking of spectrin within the membrane skeleton, the cortical actin rim, and actomyosin-based stress fibers. This review addresses each of these actin-based structures, and discusses cellular signals that control the disposition of actin in different endothelial cell phenotypes. PMID:19028505

  11. Cytoskeletal Regulation of Inflammation and Its Impact on Skin Blistering Disease Epidermolysis Bullosa Acquisita.

    PubMed

    Kopecki, Zlatko; Ludwig, Ralf J; Cowin, Allison J

    2016-01-01

    Actin remodelling proteins regulate cytoskeletal cell responses and are important in both innate and adaptive immunity. These responses play a major role in providing a fine balance in a cascade of biological events that results in either protective acute inflammation or chronic inflammation that leads to a host of diseases including autoimmune inflammation mediated epidermolysis bullosa acquisita (EBA). This review describes the role of the actin cytoskeleton and in particular the actin remodelling protein called Flightless I (Flii) in regulating cellular inflammatory responses and its subsequent effect on the autoimmune skin blistering disease EBA. It also outlines the potential of an antibody based therapy for decreasing Flii expression in vivo to ameliorate the symptoms associated with EBA. PMID:27420054

  12. Cytoskeletal Regulation of Inflammation and Its Impact on Skin Blistering Disease Epidermolysis Bullosa Acquisita

    PubMed Central

    Kopecki, Zlatko; Ludwig, Ralf J.; Cowin, Allison J.

    2016-01-01

    Actin remodelling proteins regulate cytoskeletal cell responses and are important in both innate and adaptive immunity. These responses play a major role in providing a fine balance in a cascade of biological events that results in either protective acute inflammation or chronic inflammation that leads to a host of diseases including autoimmune inflammation mediated epidermolysis bullosa acquisita (EBA). This review describes the role of the actin cytoskeleton and in particular the actin remodelling protein called Flightless I (Flii) in regulating cellular inflammatory responses and its subsequent effect on the autoimmune skin blistering disease EBA. It also outlines the potential of an antibody based therapy for decreasing Flii expression in vivo to ameliorate the symptoms associated with EBA. PMID:27420054

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

    NASA Astrophysics Data System (ADS)

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

    2014-10-01

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

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

    PubMed Central

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

    2014-01-01

    Many cell movements occur via polymerization of the actin cytoskeleton beneath the plasma membrane at the front of the cell, forming a protrusion called a lamellipodium, while myosin contraction squeezes forward the back of the cell. In what is known as the “molecular clutch” description of cell motility, forward movement results from the engagement of the acto-myosin motor with cell-matrix adhesions, thus transmitting force to the substrate and producing movement. However during cell translocation, clutch engagement is not perfect, and as a result, the cytoskeleton slips with respect to the substrate, undergoing backward (retrograde) flow in the direction of the cell body. Retrograde flow is therefore inversely proportional to cell speed and depends on adhesion and acto-myosin dynamics. Here we asked whether the molecular clutch was a general mechanism by measuring motility and retrograde flow for the Caenorhabditis elegans sperm cell in different adhesive conditions. These cells move by adhering to the substrate and emitting a dynamic lamellipodium, but the sperm cell does not contain an acto-myosin cytoskeleton. Instead the lamellipodium is formed by the assembly of Major Sperm Protein (MSP), which has no biochemical or structural similarity to actin. We find that these cells display the same molecular clutch characteristics as acto-myosin containing cells. We further show that retrograde flow is produced both by cytoskeletal assembly and contractility in these cells. Overall this study shows that the molecular clutch hypothesis of how polymerization is transduced into motility via adhesions is a general description of cell movement regardless of the composition of the cytoskeleton. PMID:25383039

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

    PubMed

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

    2006-12-13

    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 (t(1/2)< approximately 45 s) in fluorescence recovery after photobleaching experiments. A point mutation (alfA D168A) that completely inhibits AlfA subunit exchange in vivo is strongly defective for plasmid segregation, demonstrating that dynamic polymerization of AlfA is necessary for function. During sporulation, plasmid segregation occurs before septation and independently of the DNA translocase SpoIIIE and the chromosomal Par proteins Soj and Spo0J. The absence of the RacA chromosome anchoring protein reduces the efficiency of plasmid segregation (by about two-fold), suggesting that it might contribute to anchoring the plasmid at the pole during sporulation. Our results suggest that the dynamic polymerization of AlfA mediates plasmid separation during both growth and sporulation.

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

    PubMed

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

    2006-12-13

    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 (t(1/2)< approximately 45 s) in fluorescence recovery after photobleaching experiments. A point mutation (alfA D168A) that completely inhibits AlfA subunit exchange in vivo is strongly defective for plasmid segregation, demonstrating that dynamic polymerization of AlfA is necessary for function. During sporulation, plasmid segregation occurs before septation and independently of the DNA translocase SpoIIIE and the chromosomal Par proteins Soj and Spo0J. The absence of the RacA chromosome anchoring protein reduces the efficiency of plasmid segregation (by about two-fold), suggesting that it might contribute to anchoring the plasmid at the pole during sporulation. Our results suggest that the dynamic polymerization of AlfA mediates plasmid separation during both growth and sporulation. PMID:17139259

  17. Actin Interacts with Dengue Virus 2 and 4 Envelope Proteins.

    PubMed

    Jitoboam, Kunlakanya; Phaonakrop, Narumon; Libsittikul, Sirikwan; Thepparit, Chutima; Roytrakul, Sittiruk; Smith, Duncan R

    2016-01-01

    Dengue virus (DENV) remains a significant public health problem in many tropical and sub-tropical countries worldwide. The DENV envelope (E) protein is the major antigenic determinant and the protein that mediates receptor binding and endosomal fusion. In contrast to some other DENV proteins, relatively few cellular interacting proteins have been identified. To address this issue a co-immuoprecipitation strategy was employed. The predominant co-immunoprecipitating proteins identified were actin and actin related proteins, however the results suggested that actin was the only bona fide interacting partner. Actin was shown to interact with the E protein of DENV 2 and 4, and the interaction between actin and DENV E protein was shown to occur in a truncated DENV consisting of only domains I and II. Actin was shown to decrease during infection, but this was not associated with a decrease in gene transcription. Actin-related proteins also showed a decrease in expression during infection that was not transcriptionally regulated. Cytoskeletal reorganization was not observed during infection, suggesting that the interaction between actin and E protein has a cell type specific component. PMID:27010925

  18. Actin Interacts with Dengue Virus 2 and 4 Envelope Proteins

    PubMed Central

    Jitoboam, Kunlakanya; Phaonakrop, Narumon; Libsittikul, Sirikwan; Thepparit, Chutima; Roytrakul, Sittiruk; Smith, Duncan R.

    2016-01-01

    Dengue virus (DENV) remains a significant public health problem in many tropical and sub-tropical countries worldwide. The DENV envelope (E) protein is the major antigenic determinant and the protein that mediates receptor binding and endosomal fusion. In contrast to some other DENV proteins, relatively few cellular interacting proteins have been identified. To address this issue a co-immuoprecipitation strategy was employed. The predominant co-immunoprecipitating proteins identified were actin and actin related proteins, however the results suggested that actin was the only bona fide interacting partner. Actin was shown to interact with the E protein of DENV 2 and 4, and the interaction between actin and DENV E protein was shown to occur in a truncated DENV consisting of only domains I and II. Actin was shown to decrease during infection, but this was not associated with a decrease in gene transcription. Actin-related proteins also showed a decrease in expression during infection that was not transcriptionally regulated. Cytoskeletal reorganization was not observed during infection, suggesting that the interaction between actin and E protein has a cell type specific component. PMID:27010925

  19. TREK-1 Regulates Cytokine Secretion from Cultured Human Alveolar Epithelial Cells Independently of Cytoskeletal Rearrangements

    PubMed Central

    Schwingshackl, Andreas; Roan, Esra; Teng, Bin; Waters, Christopher M.

    2015-01-01

    Background TREK-1 deficient alveolar epithelial cells (AECs) secrete less IL-6, more MCP-1, and contain less F-actin. Whether these alterations in cytokine secretion and F-actin content are related remains unknown. We now hypothesized that cytokine secretion from TREK-1-deficient AECs was regulated by cytoskeletal rearrangements. Methods We determined F-actin and α-tubulin contents of control, TREK-1-deficient and TREK-1-overexpressing human A549 cells by confocal microscopy and western blotting, and measured IL-6 and MCP-1 levels using real-time PCR and ELISA. Results Cytochalasin D decreased the F-actin content of control cells. Jasplakinolide increased the F-actin content of TREK-1 deficient cells, similar to the effect of TREK-1 overexpression in control cells. Treatment of control and TREK-1 deficient cells with TNF-α, a strong stimulus for IL-6 and MCP-1 secretion, had no effect on F-actin structures. The combination of TNF-α+cytochalasin D or TNF-α+jasplakinolide had no additional effect on the F-actin content or architecture when compared to cytochalasin D or jasplakinolide alone. Although TREK-1 deficient AECs contained less F-actin at baseline, quantified biochemically, they contained more α-tubulin. Exposure to nocodazole disrupted α-tubulin filaments in control and TREK-1 deficient cells, but left the overall amount of α-tubulin unchanged. Although TNF-α had no effect on the F-actin or α-tubulin contents, it increased IL-6 and MCP-1 production and secretion from control and TREK-1 deficient cells. IL-6 and MCP-1 secretions from control and TREK-1 deficient cells after TNF-α+jasplakinolide or TNF-α+nocodazole treatment was similar to the effect of TNF-α alone. Interestingly, cytochalasin D decreased TNF-α-induced IL-6 but not MCP-1 secretion from control but not TREK-1 deficient cells. Conclusion Although cytochalasin D, jasplakinolide and nocodazole altered the F-actin and α-tubulin structures of control and TREK-1 deficient AEC, the

  20. Mutations of the Mouse ELMO Domain Containing 1 Gene (Elmod1) Link Small GTPase Signaling to Actin Cytoskeleton Dynamics in Hair Cell Stereocilia

    PubMed Central

    Johnson, Kenneth R.; Longo-Guess, Chantal M.; Gagnon, Leona H.

    2012-01-01

    Stereocilia, the modified microvilli projecting from the apical surfaces of the sensory hair cells of the inner ear, are essential to the mechanoelectrical transduction process underlying hearing and balance. The actin-filled stereocilia on each hair cell are tethered together by fibrous links to form a highly patterned hair bundle. Although many structural components of hair bundles have been identified, little is known about the signaling mechanisms that regulate their development, morphology, and maintenance. Here, we describe two naturally occurring, allelic mutations that result in hearing and balance deficits in mice, named roundabout (rda) and roundabout-2J (rda2J). Positional cloning identified both as mutations of the mouse ELMO domain containing 1 gene (Elmod1), a poorly characterized gene with no previously reported mutant phenotypes. The rda mutation is a 138 kb deletion that includes exons 1–5 of Elmod1, and rda2J is an intragenic duplication of exons 3–8 of Elmod1. The deafness associated with these mutations is caused by cochlear hair cell dysfunction, as indicated by conspicuous elongations and fusions of inner hair cell stereocilia and progressive degeneration of outer hair cell stereocilia. Mammalian ELMO-family proteins are known to be involved in complexes that activate small GTPases to regulate the actin cytoskeleton during phagocytosis and cell migration. ELMOD1 and ELMOD2 recently were shown to function as GTPase-activating proteins (GAPs) for the Arf family of small G proteins. Our finding connecting ELMOD1 deficiencies with stereocilia dysmorphologies thus establishes a link between the Ras superfamily of small regulatory GTPases and the actin cytoskeleton dynamics of hair cell stereocilia. PMID:22558334

  1. Overexpression of Isoforms of Nitric Oxide Synthase 1 Adaptor Protein, Encoded by a Risk Gene for Schizophrenia, Alters Actin Dynamics and Synaptic Function.

    PubMed

    Hernandez, Kristina; Swiatkowski, Przemyslaw; Patel, Mihir V; Liang, Chen; Dudzinski, Natasha R; Brzustowicz, Linda M; Firestein, Bonnie L

    2016-01-01

    Proper communication between neurons depends upon appropriate patterning of dendrites and correct distribution and structure of spines. Schizophrenia is a neuropsychiatric disorder characterized by alterations in dendrite branching and spine density. Nitric oxide synthase 1 adaptor protein (NOS1AP), a risk gene for schizophrenia, encodes proteins that are upregulated in the dorsolateral prefrontal cortex (DLPFC) of individuals with schizophrenia. To elucidate the effects of NOS1AP overexpression observed in individuals with schizophrenia, we investigated changes in actin dynamics and spine development when a long (NOS1AP-L) or short (NOS1AP-S) isoform of NOS1AP is overexpressed. Increased NOS1AP-L protein promotes the formation of immature spines when overexpressed in rat cortical neurons from day in vitro (DIV) 14 to DIV 17 and reduces the amplitude of miniature excitatory postsynaptic currents (mEPSCs). In contrast, increased NOS1AP-S protein increases the rate of actin polymerization and the number of immature and mature spines, which may be attributed to a decrease in total Rac1 expression and a reduction in the levels of active cofilin. The increase in the number of mature spines by overexpression of NOS1AP-S is accompanied by an increase in the frequency of mEPSCs. Our findings show that overexpression of NOS1AP-L or NOS1AP-S alters the actin cytoskeleton and synaptic function. However, the mechanisms by which these isoforms induce these changes are distinct. These results are important for understanding how increased expression of NOS1AP isoforms can influence spine development and synaptic function. PMID:26869880

  2. Overexpression of Isoforms of Nitric Oxide Synthase 1 Adaptor Protein, Encoded by a Risk Gene for Schizophrenia, Alters Actin Dynamics and Synaptic Function

    PubMed Central

    Hernandez, Kristina; Swiatkowski, Przemyslaw; Patel, Mihir V.; Liang, Chen; Dudzinski, Natasha R.; Brzustowicz, Linda M.; Firestein, Bonnie L.

    2016-01-01

    Proper communication between neurons depends upon appropriate patterning of dendrites and correct distribution and structure of spines. Schizophrenia is a neuropsychiatric disorder characterized by alterations in dendrite branching and spine density. Nitric oxide synthase 1 adaptor protein (NOS1AP), a risk gene for schizophrenia, encodes proteins that are upregulated in the dorsolateral prefrontal cortex (DLPFC) of individuals with schizophrenia. To elucidate the effects of NOS1AP overexpression observed in individuals with schizophrenia, we investigated changes in actin dynamics and spine development when a long (NOS1AP-L) or short (NOS1AP-S) isoform of NOS1AP is overexpressed. Increased NOS1AP-L protein promotes the formation of immature spines when overexpressed in rat cortical neurons from day in vitro (DIV) 14 to DIV 17 and reduces the amplitude of miniature excitatory postsynaptic currents (mEPSCs). In contrast, increased NOS1AP-S protein increases the rate of actin polymerization and the number of immature and mature spines, which may be attributed to a decrease in total Rac1 expression and a reduction in the levels of active cofilin. The increase in the number of mature spines by overexpression of NOS1AP-S is accompanied by an increase in the frequency of mEPSCs. Our findings show that overexpression of NOS1AP-L or NOS1AP-S alters the actin cytoskeleton and synaptic function. However, the mechanisms by which these isoforms induce these changes are distinct. These results are important for understanding how increased expression of NOS1AP isoforms can influence spine development and synaptic function. PMID:26869880

  3. Dissecting the contribution of actin and vimentin intermediate filaments to mechanical phenotype of suspended cells using high-throughput deformability measurements and computational modeling.

    PubMed

    Gladilin, Evgeny; Gonzalez, Paula; Eils, Roland

    2014-08-22

    Mechanical cell properties play an important role in many basic biological functions, including motility, adhesion, proliferation and differentiation. There is a growing body of evidence that the mechanical cell phenotype can be used for detection and, possibly, treatment of various diseases, including cancer. Understanding of pathological mechanisms requires investigation of the relationship between constitutive properties and major structural components of cells, i.e., the nucleus and cytoskeleton. While the contribution of actin und microtubules to cellular rheology has been extensively studied in the past, the role of intermediate filaments has been scarcely investigated up to now. Here, for the first time we compare the effects of drug-induced disruption of actin and vimentin intermediate filaments on mechanical properties of suspended NK cells using high-throughput deformability measurements and computational modeling. Although, molecular mechanisms of actin and vimentin disruption by the applied cytoskeletal drugs, Cytochalasin-D and Withaferin-A, are different, cell softening in both cases can be attributed to reduction of the effective density and stiffness of filament networks. Our experimental data suggest that actin and vimentin deficient cells exhibit, in average, 41% and 20% higher deformability in comparison to untreated control. 3D Finite Element simulation is performed to quantify the contribution of cortical actin and perinuclear vimentin to mechanical phenotype of the whole cell. Our simulation provides quantitative estimates for decreased filament stiffness in drug-treated cells and predicts more than two-fold increase of the strain magnitude in the perinuclear vimentin layer of actin deficient cells relatively to untreated control. Thus, the mechanical function of vimentin becomes particularly essential in motile and proliferating cells that have to dynamically remodel the cortical actin network. These insights add functional cues to frequently

  4. 3D Actin Network Centerline Extraction with Multiple Active Contours

    PubMed Central

    Xu, Ting; Vavylonis, Dimitrios; Huang, Xiaolei

    2013-01-01

    Fluorescence microscopy is frequently used to study two and three dimensional network structures formed by cytoskeletal polymer fibers such as actin filaments and actin cables. While these cytoskeletal structures are often dilute enough to allow imaging of individual filaments or bundles of them, quantitative analysis of these images is challenging. To facilitate quantitative, reproducible and objective analysis of the image data, we propose a semi-automated method to extract actin networks and retrieve their topology in 3D. Our method uses multiple Stretching Open Active Contours (SOACs) that are automatically initialized at image intensity ridges and then evolve along the centerlines of filaments in the network. SOACs can merge, stop at junctions, and reconfigure with others to allow smooth crossing at junctions of filaments. The proposed approach is generally applicable to images of curvilinear networks with low SNR. We demonstrate its potential by extracting the centerlines of synthetic meshwork images, actin networks in 2D Total Internal Reflection Fluorescence Microscopy images, and 3D actin cable meshworks of live fission yeast cells imaged by spinning disk confocal microscopy. Quantitative evaluation of the method using synthetic images shows that for images with SNR above 5.0, the average vertex error measured by the distance between our result and ground truth is 1 voxel, and the average Hausdorff distance is below 10 voxels. PMID:24316442

  5. Xenopus egg cytoplasm with intact actin.

    PubMed

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

    2014-01-01

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

  6. Actin network architecture can determine myosin motor activity.

    PubMed

    Reymann, Anne-Cécile; Boujemaa-Paterski, Rajaa; Martiel, Jean-Louis; Guérin, Christophe; Cao, Wenxiang; Chin, Harvey F; De La Cruz, Enrique M; Théry, Manuel; Blanchoin, Laurent

    2012-06-01

    The organization of actin filaments into higher-ordered structures governs eukaryotic cell shape and movement. Global actin network size and architecture are maintained in a dynamic steady state through regulated assembly and disassembly. Here, we used experimentally defined actin structures in vitro to investigate how the activity of myosin motors depends on network architecture. Direct visualization of filaments revealed myosin-induced actin network deformation. During this reorganization, myosins selectively contracted and disassembled antiparallel actin structures, while parallel actin bundles remained unaffected. The local distribution of nucleation sites and the resulting orientation of actin filaments appeared to regulate the scalability of the contraction process. This "orientation selection" mechanism for selective contraction and disassembly suggests how the dynamics of the cellular actin cytoskeleton can be spatially controlled by actomyosin contractility.

  7. Actin Age Orchestrates Myosin-5 and Myosin-6 Runlengths

    PubMed Central

    Zimmermann, Dennis; Santos, Alicja; Kovar, David R.; Rock, Ronald S.

    2015-01-01

    Summary Unlike a static and immobile skeleton, the actin cytoskeleton is a highly dynamic network of filamentous actin (F-actin) polymers that continuously turn over. In addition to generating mechanical forces and sensing mechanical deformation, dynamic F-actin networks serve as cellular tracks for myosin motor traffic. However, much of our mechanistic understanding of processive myosins comes from in vitro studies where motility was studied on pre-assembled and artificially stabilized, static F-actin tracks. In this work, we examine the role of actin dynamics in single-molecule myosin motility using assembling F-actin and the two highly processive motors, myosin-5 and myosin-6. These two myosins have distinct functions in the cell and travel in opposite directions along actin filaments [1–3]. Myosin-5 walks towards the barbed ends of F-actin, traveling to sites of actin polymerization at the cell periphery [4]. Myosin-6 walks towards the pointed end of F-actin [5], traveling towards the cell center along older segments of the actin filament. We find that myosin-5 takes 1.3 to 1.5-fold longer runs on ADP•Pi (young) F-actin, while myosin-6 takes 1.7 to 3.6-fold longer runs along ADP (old) F-actin. These results suggest that conformational differences between ADP•Pi and ADP F-actin tailor these myosins to walk farther toward their preferred actin filament end. Taken together, these experiments define a new mechanism by which myosin traffic may sort to different F-actin networks depending on filament age. PMID:26190073

  8. Canonical and Noncanonical G-Protein Signaling Helps Coordinate Actin Dynamics To Promote Macrophage Phagocytosis of Zymosan

    PubMed Central

    Huang, Ning-Na; Becker, Steven; Boularan, Cedric; Kamenyeva, Olena; Vural, Ali; Hwang, Il-Young; Shi, Chong-Shan

    2014-01-01

    Both chemotaxis and phagocytosis depend upon actin-driven cell protrusions and cell membrane remodeling. While chemoattractant receptors rely upon canonical G-protein signaling to activate downstream effectors, whether such signaling pathways affect phagocytosis is contentious. Here, we report that Gαi nucleotide exchange and signaling helps macrophages coordinate the recognition, capture, and engulfment of zymosan bioparticles. We show that zymosan exposure recruits F-actin, Gαi proteins, and Elmo1 to phagocytic cups and early phagosomes. Zymosan triggered an increase in intracellular Ca2+ that was partially sensitive to Gαi nucleotide exchange inhibition and expression of GTP-bound Gαi recruited Elmo1 to the plasma membrane. Reducing GDP-Gαi nucleotide exchange, decreasing Gαi expression, pharmacologically interrupting Gβγ signaling, or reducing Elmo1 expression all impaired phagocytosis, while favoring the duration that Gαi remained GTP bound promoted it. Our studies demonstrate that targeting heterotrimeric G-protein signaling offers opportunities to enhance or retard macrophage engulfment of phagocytic targets such as zymosan. PMID:25225330

  9. Distribution of actin of the human erythrocyte membrane cytoskeleton after interaction with radiographic contrast media.

    PubMed

    Franke, R P; Scharnweber, T; Fuhrmann, R; Krüger, A; Wenzel, F; Mrowietz, C; Jung, F

    2013-01-01

    A type-dependent chemotoxic effect of radiographic contrast media on erythrocytes and endothelial cells was reported several times. While mechanisms of toxicity are still unclear the cellular reactions e.g. echinocyte formation in erythrocytes and the buckling of endothelial cells coincided with deterioration of capillary perfusion (in patients with coronary artery disease) and tissue oxygen tension (in the myocardium of pigs). Whether the shape changes in erythrocytes coincide with changes in the arrangement of actin, the core of the actin-spectrin cytoskeletal network and possible actor in membrane stresses and deformation is not known until now. To get specific informations actin was stained using two different staining methods (antibodies to β-actin staining oligomeric G-actin and polymeric F-actin and Phalloidin-Rhodamin staining polymeric F-actin only). In addition, an advanced version of confocal laser scanning microscopes was used enabling the display of the actin arrangement near substrate surfaces. Blood smears were produced after erythrocyte suspension in autologous plasma or in two different plasma/RCM mixtures. In this study an even homogenous distribution of fine grained globular actin in the normal human erythrocyte could be demonstrated. After suspension of erythrocytes in a plasma/Iodixanol mixture an increased number of membrane protrusions appeared densely filled with intensely stained actin similar to cells suspended in autologous plasma, however, there in less numbers. Suspension in Iopromide, in contrast, induced a complete reorganization of the cytoskeletal actin: the fine grained globular actin distribution disappeared and only few, long and thick actin filaments bundled and possibly polymerized appeared, instead, shown here for the first time.

  10. Architecture and Connectivity Govern Actin Network Contractility.

    PubMed

    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.

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

    PubMed

    Joseph, Jayabalan M; Fey, Petra; Ramalingam, Nagendran; Liu, Xiao I; Rohlfs, Meino; Noegel, Angelika A; Müller-Taubenberger, Annette; Glöckner, Gernot; Schleicher, Michael

    2008-07-09

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

  12. A kinematic description of the trajectories of Listeria monocytogenes propelled by actin comet tails

    NASA Astrophysics Data System (ADS)

    Tambe, Dhananjay; Shenoy, Vivek

    2007-03-01

    The bacterial pathogen Listeria monocytogenes propels itself in the cytoplasm of the infected cells by forming a filamentous comet tail assembled by the polymerization of the cytoskeletal protein, actin. While a great deal is known about the molecular processes that lead to actin based movement, most macroscale aspects of motion, including the nature of the trajectories traced out by the motile bacteria are not well understood. Listeria moving between a glass-slide and cover slip in a Xenopus frog egg extract motility assay is observed to display a number of geometrically fascinating trajectories including sine curves, serpentine shapes, circles, and a variety of spirals. We have developed a dynamic model that provides a unified description of these seemingly unrelated trajectories. A key ingredient of the model is a torque (not included in any microscopic models to date) that arises from the rotation of the propulsive force about the body-axis of the bacterium. The trajectories of bacteria executing both steady and saltatory motion are found to be in excellent agreement with the predictions of our dynamic model. When the constraints that lead to planar motion are removed, our model predicts motion along regular helical trajectories, observed in recent experiments. We discover from the analysis of the trajectories of spherical beads that the comet tail revolves around the bead.

  13. Utilization of paramagnetic relaxation enhancements for structural analysis of actin-binding proteins in complex with actin

    PubMed Central

    Huang, Shuxian; Umemoto, Ryo; Tamura, Yuki; Kofuku, Yutaka; Uyeda, Taro Q. P.; Nishida, Noritaka; Shimada, Ichio

    2016-01-01

    Actin cytoskeleton dynamics are controlled by various actin binding proteins (ABPs) that modulate the polymerization of the monomeric G-actin and the depolymerization of filamentous F-actin. Although revealing the structures of the actin/ABP complexes is crucial to understand how the ABPs regulate actin dynamics, the X-ray crystallography and cryoEM methods are inadequate to apply for the ABPs that interact with G- or F-actin with lower affinity or multiple binding modes. In this study, we aimed to establish the alternative method to build a structural model of G-actin/ABP complexes, utilizing the paramagnetic relaxation enhancement (PRE) experiments. Thymosin β4 (Tβ4) was used as a test case for validation, since its structure in complex with G-actin was reported recently. Recombinantly expressed G-actin, containing a cysteine mutation, was conjugated with a nitroxyl spin label at the specific site. Based on the intensity ratio of the 1H-15N HSQC spectra of Tβ4 in the complex with G-actin in the paramagnetic and diamagnetic states, the distances between the amide groups of Tβ4 and the spin label of G-actin were estimated. Using the PRE-derived distance constraints, we were able to compute a well-converged docking structure of the G-actin/Tβ4 complex that shows great accordance with the reference structure. PMID:27654858

  14. Drosophila pupal macrophages--a versatile tool for combined ex vivo and in vivo imaging of actin dynamics at high resolution.

    PubMed

    Sander, Moritz; Squarr, Anna Julia; Risse, Benjamin; Jiang, Xiaoyi; Bogdan, Sven

    2013-01-01

    Molecular understanding of actin dynamics requires a genetically traceable model system that allows live cell imaging together with high-resolution microscopy techniques. Here, we used Drosophila pupal macrophages that combine many advantages of cultured cells with a genetic in vivo model system. Using structured illumination microscopy together with advanced spinning disk confocal microscopy we show that these cells provide a powerful system for single gene analysis. It allows forward genetic screens to characterize the regulatory network controlling cell shape and directed cell migration in a physiological context. We knocked down components regulating lamellipodia formation, including WAVE, single subunits of Arp2/3 complex and CPA, one of the two capping protein subunits and demonstrate the advantages of this model system by imaging mutant macrophages ex vivo as well as in vivo upon laser-induced wounding.

  15. Addition of Phenylboronic Acid to Malus domestica Pollen Tubes Alters Calcium Dynamics, Disrupts Actin Filaments and Affects Cell Wall Architecture.

    PubMed

    Fang, Kefeng; Gao, Sai; Zhang, Weiwei; Xing, Yu; Cao, Qingqin; Qin, Ling

    2016-01-01

    A key role of boron in plants is to cross-link the cell wall pectic polysaccharide rhamnogalacturonan-II (RG-II) through borate diester linkages. Phenylboronic acid (PBA) can form the same reversible ester bonds but cannot cross-link two molecules, so can be used as an antagonist to study the function of boron. This study aimed to evaluate the effect of PBA on apple (Malus domestica) pollen tube growth and the underlying regulatory mechanism. We observed that PBA caused an inhibition of pollen germination, tube growth and led to pollen tube morphological abnormalities. Fluorescent labeling, coupled with a scanning ion-selective electrode technique, revealed that PBA induced an increase in extracellular Ca2+ influx, thereby elevating the cytosolic Ca2+ concentration [Ca2+]c and disrupting the [Ca2+]c gradient, which is critical for pollen tube growth. Moreover the organization of actin filaments was severely perturbed by the PBA treatment. Immunolocalization studies and fluorescent labeling, together with Fourier-transform infrared analysis (FTIR) suggested that PBA caused an increase in the abundance of callose, de-esterified pectins and arabinogalactan proteins (AGPs) at the tip. However, it had no effect on the deposition of the wall polymers cellulose. These effects are similar to those of boron deficiency in roots and other organs, indicating that PBA can induce boron deficiency symptoms. The results provide new insights into the roles of boron in pollen tube development, which likely include regulating [Ca2+]c and the formation of the actin cytoskeleton, in addition to the synthesis and assembly of cell wall components. PMID:26886907

  16. Addition of Phenylboronic Acid to Malus domestica Pollen Tubes Alters Calcium Dynamics, Disrupts Actin Filaments and Affects Cell Wall Architecture.

    PubMed

    Fang, Kefeng; Gao, Sai; Zhang, Weiwei; Xing, Yu; Cao, Qingqin; Qin, Ling

    2016-01-01

    A key role of boron in plants is to cross-link the cell wall pectic polysaccharide rhamnogalacturonan-II (RG-II) through borate diester linkages. Phenylboronic acid (PBA) can form the same reversible ester bonds but cannot cross-link two molecules, so can be used as an antagonist to study the function of boron. This study aimed to evaluate the effect of PBA on apple (Malus domestica) pollen tube growth and the underlying regulatory mechanism. We observed that PBA caused an inhibition of pollen germination, tube growth and led to pollen tube morphological abnormalities. Fluorescent labeling, coupled with a scanning ion-selective electrode technique, revealed that PBA induced an increase in extracellular Ca2+ influx, thereby elevating the cytosolic Ca2+ concentration [Ca2+]c and disrupting the [Ca2+]c gradient, which is critical for pollen tube growth. Moreover the organization of actin filaments was severely perturbed by the PBA treatment. Immunolocalization studies and fluorescent labeling, together with Fourier-transform infrared analysis (FTIR) suggested that PBA caused an increase in the abundance of callose, de-esterified pectins and arabinogalactan proteins (AGPs) at the tip. However, it had no effect on the deposition of the wall polymers cellulose. These effects are similar to those of boron deficiency in roots and other organs, indicating that PBA can induce boron deficiency symptoms. The results provide new insights into the roles of boron in pollen tube development, which likely include regulating [Ca2+]c and the formation of the actin cytoskeleton, in addition to the synthesis and assembly of cell wall components.

  17. Addition of Phenylboronic Acid to Malus domestica Pollen Tubes Alters Calcium Dynamics, Disrupts Actin Filaments and Affects Cell Wall Architecture

    PubMed Central

    Fang, Kefeng; Gao, Sai; Zhang, Weiwei; Xing, Yu; Cao, Qingqin; Qin, Ling

    2016-01-01

    A key role of boron in plants is to cross-link the cell wall pectic polysaccharide rhamnogalacturonan-II (RG-II) through borate diester linkages. Phenylboronic acid (PBA) can form the same reversible ester bonds but cannot cross-link two molecules, so can be used as an antagonist to study the function of boron. This study aimed to evaluate the effect of PBA on apple (Malus domestica) pollen tube growth and the underlying regulatory mechanism. We observed that PBA caused an inhibition of pollen germination, tube growth and led to pollen tube morphological abnormalities. Fluorescent labeling, coupled with a scanning ion-selective electrode technique, revealed that PBA induced an increase in extracellular Ca2+ influx, thereby elevating the cytosolic Ca2+ concentration [Ca2+]c and disrupting the [Ca2+]c gradient, which is critical for pollen tube growth. Moreover the organization of actin filaments was severely perturbed by the PBA treatment. Immunolocalization studies and fluorescent labeling, together with Fourier-transform infrared analysis (FTIR) suggested that PBA caused an increase in the abundance of callose, de-esterified pectins and arabinogalactan proteins (AGPs) at the tip. However, it had no effect on the deposition of the wall polymers cellulose. These effects are similar to those of boron deficiency in roots and other organs, indicating that PBA can induce boron deficiency symptoms. The results provide new insights into the roles of boron in pollen tube development, which likely include regulating [Ca2+]c and the formation of the actin cytoskeleton, in addition to the synthesis and assembly of cell wall components. PMID:26886907

  18. The cytoskeletal protein α-catenin unfurls upon binding to vinculin.

    PubMed

    Rangarajan, Erumbi S; Izard, Tina

    2012-05-25

    Adherens junctions (AJs) are essential for cell-cell contacts, morphogenesis, and the development of all higher eukaryotes. AJs are formed by calcium-dependent homotypic interactions of the ectodomains of single membrane-pass cadherin family receptors. These homotypic interactions in turn promote binding of the intracellular cytoplasmic tail domains of cadherin receptors with β-catenin, a multifunctional protein that plays roles in both transcription and AJs. The cadherin receptor-β-catenin complex binds to the cytoskeletal protein α-catenin, which is essential for both the formation and the stabilization of these junctions. Precisely how α-catenin contributes to the formation and stabilization of AJs is hotly debated, although the latter is thought to involve its interactions with the cytoskeletal protein vinculin. Here we report the crystal structure of the vinculin binding domain (VBD) of α-catenin in complex with the vinculin head domain (Vh1). This structure reveals that α-catenin is in a unique unfurled mode allowing dimer formation when bound to vinculin. Finally, binding studies suggest that vinculin must be in an activated state to bind to α-catenin and that this interaction is stabilized by the formation of a ternary α-catenin-vinculin-F-actin complex, which can be formed via the F-actin binding domain of either protein. We propose a feed-forward model whereby α-catenin-vinculin interactions promote their binding to the actin cytoskeleton to stabilize AJs. PMID:22493458

  19. Organized F-actin is essential for normal trichome morphogenesis in Arabidopsis.

    PubMed Central

    Szymanski, D B; Marks, M D; Wick, S M

    1999-01-01

    Actin microfilaments form a three-dimensional cytoskeletal network throughout the cell and constitute an essential throughway for organelle and vesicle transport. Development of Arabidopsis trichomes, unicellular structures derived from the epidermis, is being used as a genetic system in which to study actin-dependent growth in plant cells. The present study indicates that filamentous actin (F-actin) plays an important role during Arabidopsis trichome morphogenesis. For example, immunolocalization of actin filaments during trichome morphogenesis identified rearrangements of the cytoskeletal structure during the development of the mature cell. Moreover, pharmacological experiments indicate that there are distinct requirements for actin- and microtubule-dependent function during trichome morphogenesis. The F-actin-disrupting drug cytochalasin D does not affect the establishment of polarity during trichome development; however, maintenance and coordination of the normal pattern of cell growth are very sensitive to this drug. In contrast, oryzalin, an agent that depolymerizes microtubules, severely inhibits cell polarization. Furthermore, cytochalasin D treatment phenocopies a known class of mutations that cause distorted trichome morphology. Results of an analysis of cell shape and microfilament structure in wild-type, mutant, and drug-treated trichomes are consistent with a role for actin in the maintenance and coordination of an established growth pattern. PMID:10590162

  20. Actin depolymerizing factor controls actin turnover and gliding motility in Toxoplasma gondii

    PubMed Central

    Mehta, Simren; Sibley, L. David

    2011-01-01

    Apicomplexan parasites rely on actin-based gliding motility to move across the substratum, cross biological barriers, and invade their host cells. Gliding motility depends on polymerization of parasite actin filaments, yet ∼98% of actin is nonfilamentous in resting parasites. Previous studies suggest that the lack of actin filaments in the parasite is due to inherent instability, leaving uncertain the role of actin-binding proteins in controlling dynamics. We have previously shown that the single allele of Toxoplasma gondii actin depolymerizing factor (TgADF) has strong actin monomer–sequestering and weak filament-severing activities in vitro. Here we used a conditional knockout strategy to investigate the role of TgADF in vivo. Suppression of TgADF led to accumulation of actin-rich filaments that were detected by immunofluorescence and electron microscopy. Parasites deficient in TgADF showed reduced speed of motility, increased aberrant patterns of motion, and inhibition of sustained helical gliding. Lack of TgADF also led to severe defects in entry and egress from host cells, thus blocking infection in vitro. These studies establish that the absence of stable actin structures in the parasite are not simply the result of intrinsic instability, but that TgADF is required for the rapid turnover of parasite actin filaments, gliding motility, and cell invasion. PMID:21346192

  1. Cytoskeletal architecture and cell motility remain unperturbed in mouse embryonic fibroblasts from Plk3 knockout mice

    PubMed Central

    Michel, Daniel R; Mun, Kyu-Shik; Ho, Chia-Chi

    2016-01-01

    Polo-like kinase 3 (Plk3) is best known for its involvement in cell cycle checkpoint regulation following exposure to cytotoxicants or induction of DNA damage. Yet, Plk3 has also been implicated in roles beyond those of cellular responses to DNA damage. Here, we have investigated the proposition, suggested by the Plk literature, that Plk3 regulates cytoskeletal architecture and cell functions mediated by the cytoskeleton. To this end, we have assayed mouse embryonic fibroblasts (MEFs) generated from both Plk3 knockout and wild-type mice. In particular, we asked whether Plk3 is involved in actin fiber and microtubule integrity, cell migration, cell attachment, and/or cell invasion. Our results demonstrate that functional Plk3 is not critical for the regulation of cytoskeletal integrity, cell morphology, cell adhesion, or motility in MEFs. PMID:26843517

  2. Prostate Specific Membrane Antigen-Targeted Photodynamic Therapy Induces Rapid Cytoskeletal Disruption

    PubMed Central

    Liu, Tiancheng; Wu, Lisa Y.; Berkman, Clifford E.

    2010-01-01

    Prostate-specific membrane antigen (PSMA), an established enzyme-biomarker for prostate cancer, has attracted considerable attention as a target for imaging and therapeutic applications. We aimed to determine the effects of PSMA-targeted photodynamic therapy (PDT) on cytoskeletal networks in prostate cancer cells. PSMA-targeted PDT resulted in rapid disruption of microtubules (α-/β-tubulin), microfilaments (actin), and intermediate filaments (cytokeratin 8/18) in the cytoplasm of LNCaP cells. The collapse of cytoplasmic microtubules and the later nuclear translocation of α-/β-tubulin were the most dramatic alternation. It is likely that these early changes of cytoskeletal networks are partly involved in the initiation of cell death. PMID:20452720

  3. Cytotoxic effects of incense particles in relation to oxidative stress, the cell cycle and F-actin assembly.

    PubMed

    Chuang, Hsiao-Chi; Jones, Tim; Chen, Tzu-Tao; BéruBé, Kelly

    2013-07-18

    Epidemiological studies have suggested that combustion-derived smoke, such as that produced during incense burning, is a deleterious air pollutant. It is capable of initiating oxidative stress and mutation; however, the related apoptotic processes remain unclear. In order to elucidate the biological mechanisms of reactive oxygen species (ROS)-induced respiratory toxicology, alveolar epithelial A549 cells were exposed to incense particulate matter (PM), with and without antioxidant N-acetyl-l-cysteine (NAC). The cross-linking associations between oxidative capacity, cell cycle events, actin cytoskeletal dynamics and intracellular calcium signals were investigated. An incense PM suspension caused significant oxidative stress in A549 cells, as shown by inhibition of the cell cycle at G1 and G2/M check-points, and the induction of apoptosis at Sub-G1. At the same time, alterations in the F-actin filamentous assemblies were observed. The levels of intracellular Ca(2+) were increased after incense PM exposure. Antioxidant NAC treatment revealed that oxidative stress and F-actin remodelling was significantly mitigated. This suggests that ROS accumulation could alter cell cycle regulation and anomalous remodelling of the cortical cytoskeleton that allowed impaired cells to enter into apoptosis. This study has elucidated the integral patho-physiological interactions of incense PM and the potential mechanisms for the development of ROS-driven respiratory impairment.

  4. Bundling actin filaments from membranes: some novel players

    PubMed Central

    Thomas, Clément

    2012-01-01

    Progress in live-cell imaging of the cytoskeleton has significantly extended our knowledge about the organization and dynamics of actin filaments near the plasma membrane of plant cells. Noticeably, two populations of filamentous structures can be distinguished. On the one hand, fine actin filaments which exhibit an extremely dynamic behavior basically characterized by fast polymerization and prolific severing events, a process referred to as actin stochastic dynamics. On the other hand, thick actin bundles which are composed of several filaments and which are comparatively more stable although they constantly remodel as well. There is evidence that the actin cytoskeleton plays critical roles in trafficking and signaling at both the cell cortex and organelle periphery but the exact contribution of actin bundles remains unclear. A common view is that actin bundles provide the long-distance tracks used by myosin motors to deliver their cargo to growing regions and accordingly play a particularly important role in cell polarization. However, several studies support that actin bundles are more than simple passive highways and display multiple and dynamic roles in the regulation of many processes, such as cell elongation, polar auxin transport, stomatal and chloroplast movement, and defense against pathogens. The list of identified plant actin-bundling proteins is ever expanding, supporting that plant cells shape structurally and functionally different actin bundles. Here I review the most recently characterized actin-bundling proteins, with a particular focus on those potentially relevant to membrane trafficking and/or signaling. PMID:22936939

  5. Palladin interacts with SH3 domains of SPIN90 and Src and is required for Src-induced cytoskeletal remodeling

    PubMed Central

    Rönty, Mikko; Taivainen, Anu; Heiska, Leena; Otey, Carol; Ehler, Elisabeth; Song, Woo Keun; Carpen, Olli

    2007-01-01

    Palladin and SPIN90 are widely expressed proteins, which participate in modulation of actin cytoskeleton by binding to a variety of scaffold and signaling molecules. Cytoskeletal reorganization can induced by activation of signaling pathways, including the PDGF receptor and Src tyrosine kinase pathways. In this study we have analyzed the interplay between palladin, SPIN90 and Src, and characterized the role of palladin and SPIN90 in PDGF and Src-induced cytoskeletal remodeling. We show that the SH3 domains of SPIN90 and Src directly bind palladin’s poly-proline sequence and the interaction controls intracellular targeting of SPIN90. In PDGF-treated cells, palladin and SPIN90 co-localize in actin rich membrane ruffles and lamellipodia. The effect of PDGF on the cytoskeleton is at least partly mediated by the Src kinase, since PP2, a selective Src kinase family inhibitor, blocked PDGF-induced changes. Furthermore, expression of active Src kinase resulted in coordinated translocation of both palladin and SPIN90 to membrane protrusions. Knock-down of endogenous SPIN90 did not inhibit Src-induced cytoskeletal rearrangement, whereas knock-down of palladin resulted in cytoskeletal disorganization and inhibition of remodeling. Further studies showed that palladin is tyrosine phosphorylated in cells expressing active Src indicating bidirectional interplay between palladin and Src. These results may have implications in understanding the invasive and metastatic phenotype of neoplastic cells induced by Src. PMID:17537434

  6. The actin cytoskeleton as a sensor and mediator of apoptosis

    PubMed Central

    Desouza, Melissa; Gunning, Peter W.; Stehn, Justine R.

    2012-01-01

    Apoptosis is an important biological process required for the removal of unwanted or damaged cells. Mounting evidence implicates the actin cytoskeleton as both a sensor and mediator of apoptosis. Studies also suggest that actin binding proteins (ABPs) significantly contribute to apoptosis and that actin dynamics play a key role in regulating apoptosis signaling. Changes in the organization of the actin cytoskeleton has been attributed to the process of malignant transformation and it is hypothesized that remodeling of the actin cytoskeleton may enable tumor cells to evade normal apoptotic signaling. This review aims to illuminate the role of the actin cytoskeleton in apoptosis by systematically analyzing how actin and ABPs regulate different apoptosis pathways and to also highlight the potential for developing novel compounds that target tumor-specific actin filaments. PMID:22880146

  7. CNS myelin wrapping is driven by actin disassembly.

    PubMed

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

    2015-07-27

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

  8. Arabidopsis ACTIN-DEPOLYMERIZING FACTOR7 Severs Actin Filaments and Regulates Actin Cable Turnover to Promote Normal Pollen Tube Growth[W

    PubMed Central

    Zheng, Yiyan; Xie, Yurong; Jiang, Yuxiang; Qu, Xiaolu; Huang, Shanjin

    2013-01-01

    Actin filaments are often arranged into higher-order structures, such as the longitudinal actin cables that generate the reverse fountain cytoplasmic streaming pattern present in pollen tubes. While several actin binding proteins have been implicated in the generation of these cables, the mechanisms that regulate their dynamic turnover remain largely unknown. Here, we show that Arabidopsis thaliana ACTIN-DEPOLYMERIZING FACTOR7 (ADF7) is required for turnover of longitudinal actin cables. In vitro biochemical analyses revealed that ADF7 is a typical ADF that prefers ADP-G-actin over ATP-G-actin. ADF7 inhibits nucleotide exchange on actin and severs filaments, but its filament severing and depolymerizing activities are less potent than those of the vegetative ADF1. ADF7 primarily decorates longitudinal actin cables in the shanks of pollen tubes. Consistent with this localization pattern, the severing frequency and depolymerization rate of filaments significantly decreased, while their maximum lifetime significantly increased, in adf7 pollen tube shanks. Furthermore, an ADF7–enhanced green fluorescent protein fusion with defective severing activity but normal G-actin binding activity could not complement adf7, providing compelling evidence that the severing activity of ADF7 is vital for its in vivo functions. These observations suggest that ADF7 evolved to promote turnover of longitudinal actin cables by severing actin filaments in pollen tubes. PMID:24058157

  9. Cytoskeletal remodeling of rat aortic smooth muscle cells in vitro: relationships to culture conditions and analogies to in vivo situations.

    PubMed

    Skalli, O; Bloom, W S; Ropraz, P; Azzarone, B; Gabbiani, G

    1986-07-01

    Cytoskeletal features of arterial smooth muscle cells (SMC) vary characteristically during development and during atheromatous plaque formation (Gabbiani et al., 1984; Kocher et al., 1985). We have analyzed the cytoskeletal features of rat aortic SMC placed in culture in the presence of 10% foetal calf serum (thus containing growth factors probably playing a role in SMC development and atheroma formation), as compared to SMC freshly isolated from the rat aortic media. Under these conditions, SMC show a typical cytoskeletal remodeling characterized by: 1) increased content of vimentin per cell, increased number of cells containing only vimentin, and decreased number of vimentin plus desmin containing cells; 2) decreased contents of actin, tropomyosin and myosin; 3) a switch in the pattern of actin isoforms with the appearance of a beta-type predominance. Some of these changes (e.g. increase of vimentin and decrease of alpha-type actin) are seen already in cells entering for the first time in S-phase after plating. Pulse-chase experiments with 3H-thymidine (3H-TdR) indicate that vimentin containing SMC possess a higher replicative activity than vimentin plus desmin containing SMC, thus explaining the selection of vimentin containing cells during culture. Our results indicate that during culture SMC develop features similar to those observed in normal foetal SMC or in SMC present in atheromatous plaques; this model may be useful for the understanding of mechanisms leading to SMC differentiation and to atheroma formation. PMID:3528513

  10. Colchicine activates actin polymerization by microtubule depolymerization.

    PubMed

    Jung, H I; Shin, I; Park, Y M; Kang, K W; Ha, K S

    1997-06-30

    Swiss 3T3 fibroblasts were treated with the microtubule-disrupting agent colchicine to study any interaction between microtubule dynamics and actin polymerization. Colchicine increased the amount of filamentous actin (F-actin), in a dose- and time-dependent manner with a significant increase at 1 h by about 130% over control level. Confocal microscopic observation showed that colchicine increased F-actin contents by stress fiber formation without inducing membrane ruffling. Colchicine did not activate phospholipase C and phospholipase D, whereas lysophosphatidic acid did, indicating that colchicine may have a different mechanism of actin polymerization regulation from LPA. A variety of microtubule-disrupting agents stimulated actin polymerization in Swiss 3T3 and Rat-2 fibroblasts as did colchicine, but the microtubule-stabilizing agent taxol inhibited actin polymerization induced by the above microtubule-disrupting agents. In addition, colchicine-induced actin polymerization was blocked by two protein phosphatase inhibitors, okadaic acid and calyculin A. These results suggest that microtubule depolymerization activates stress fiber formation by serine/threonine dephosphorylation in fibroblasts. PMID:9264034

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

    PubMed

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

    2014-05-15

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

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

    NASA Technical Reports Server (NTRS)

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

    1995-01-01

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

  13. Dynamic actin interaction of cross-bridges during force generation: implications for cross-bridge action in muscle.

    PubMed

    Brenner, B

    1993-01-01

    The force response of Ca(2+)-activated, permeabilized segments of rabbit psoas muscle fibers to stretches and releases was studied. These length changes were imposed (i) during isometric steady state contraction, (ii) as a restretch at the end of a ramp-shaped prerelease, and (iii) during isotonic steady state shortening. The speed of the stretches/releases was varied between about 10 and 10(5) (nm/half-sarcomere)/s. At physiological ionic strength and at low temperature (5 degrees C), the force response to stretches apparently is neither affected by cross-bridges that occupy weak-binding states nor by redistribution among various attached force-generating states. Plots of force vs. imposed length change ("T-plots") and plots of apparent fiber stiffness vs. speed of the imposed length change ("stiffness-speed relations") recorded under all these conditions suggest that cross-bridges, even during force-generation, dissociate and reassociate from and to actin on a time scale that is fast compared to active cross-bridge cycling (> 50- 1000s-1 vs. 1-10s-1). This rapid dissociation/reassociation of force-generating cross-bridges may provide a mechanism to account for the unexpectedly low ATPase activity during high-speed shortening and for filament sliding exceeding 10-20nm while a cross-bridge passes through the force-generating states.

  14. Labial Salivary Glands in Infants: Histochemical Analysis of Cytoskeletal and Antimicrobial Proteins.

    PubMed

    Stoeckelhuber, Mechthild; Loeffelbein, Denys J; Olzowy, Bernhard; Schmitz, Christoph; Koerdt, Steffen; Kesting, Marco R

    2016-08-01

    Human labial glands secrete mucous and serous substances for maintaining oral health. The normal microbial flora of the oral cavity is regulated by the acquired and innate immune systems. The localization and distribution of proteins of the innate immune system were investigated in serous acinar cells and the ductal system by the method of immunohistochemistry. Numerous antimicrobial proteins could be detected in the labial glands: β-defensin-1, -2, -3; lysozyme; lactoferrin; and cathelicidin. Cytoskeletal components such as actin, myosin II, cytokeratins 7 and 19, α- and β-tubulin were predominantly observed in apical cell regions and may be involved in secretory activities. PMID:27439958

  15. Cell protrusions induced by hyaluronan synthase 3 (HAS3) resemble mesothelial microvilli and share cytoskeletal features of filopodia.

    PubMed

    Koistinen, Ville; Kärnä, Riikka; Koistinen, Arto; Arjonen, Antti; Tammi, Markku; Rilla, Kirsi

    2015-10-01

    Previous studies have shown that overexpression of enzymatically active GFP-HAS induces the growth of long, slender protrusions that share many features of both filopodia and microvilli. These protrusions are dependent on continuing hyaluronan synthesis, and disrupt upon digestion of hyaluronan by hyaluronidase. However, complete understanding of their nature is still missing. This work shows that the protrusions on rat peritoneal surface are ultrastructurally indistinguishable from those induced by GFP-HAS3 in MCF-7 cells. Analysis of the actin-associated proteins villin, ezrin, espin, fascin, and Myo10 indicated that the HAS3-induced protrusions share most cytoskeletal features with filopodia, but they do not require adherence to the substratum like traditional filopodia. GFP-HAS3 overexpression was found to markedly enhance filamentous actin in the protrusions and their cortical basis. Analysis of the protrusion dynamics after enzymatic digestion of hyaluronan revealed that while GFP-HAS3 escape from the protrusions and the protrusion collapse takes place immediately, the complete retraction of the protrusions occurs more slowly. This finding also suggests that hyaluronan chain maintains HAS3 in the plasma membrane. The results of this work suggest that protrusions similar to those of HAS3 overexpressing cells in vitro exist also in cells with active hyaluronan synthesis in vivo. These protrusions are similar to common filopodia but are independent of substratum attachment due to the extracellular scaffolding by the hyaluronan coat that accounts for the growth and maintenance of these structures, previously associated to invasion, adhesion and multidrug resistance. PMID:26162854