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Gary G. Borisy - One of the best experts on this subject based on the ideXlab platform.
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Intrinsic dynamic behavior of fascin in Filopodia.
Molecular biology of the cell, 2007Co-Authors: Yvonne S. Aratyn, Thomas E. Schaus, Edwin W. Taylor, Gary G. BorisyAbstract:Recent studies showed that the actin cross-linking protein, fascin, undergoes rapid cycling between Filopodial filaments. Here, we used an experimental and computational approach to dissect features of fascin exchange and incorporation in Filopodia. Using expression of phosphomimetic fascin mutants, we determined that fascin in the phosphorylated state is primarily freely diffusing, whereas actin bundling in Filopodia is accomplished by fascin dephosphorylated at serine 39. Fluorescence recovery after photobleaching analysis revealed that fascin rapidly dissociates from Filopodial filaments with a kinetic off-rate of 0.12 s−1 and that it undergoes diffusion at moderate rates with a coefficient of 6 μm2s−1. This kinetic off-rate was recapitulated in vitro, indicating that dynamic behavior is intrinsic to the fascin cross-linker. A computational reaction–diffusion model showed that reversible cross-linking is required for the delivery of fascin to growing Filopodial tips at sufficient rates. Analysis of fascin bundling indicated that Filopodia are semiordered bundles with one bound fascin per 25–60 actin monomers.
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ena vasp proteins have an anti capping independent function in Filopodia formation
Molecular Biology of the Cell, 2007Co-Authors: Derek A. Applewhite, Gary G. Borisy, Tatyana Svitkina, Frank B. Gertler, Melanie Barzik, Shin Ichiro KojimaAbstract:Filopodia have been implicated in a number of diverse cellular processes including growth-cone path finding, wound healing, and metastasis. The Ena/VASP family of proteins has emerged as key to Filopodia formation but the exact mechanism for how they function has yet to be fully elucidated. Using cell spreading as a model system in combination with small interfering RNA depletion of Capping Protein, we determined that Ena/VASP proteins have a role beyond anticapping activity in Filopodia formation. Analysis of mutant Ena/VASP proteins demonstrated that the entire EVH2 domain was the minimal domain required for Filopodia formation. Fluorescent recovery after photobleaching data indicate that Ena/VASP proteins rapidly exchange at the leading edge of lamellipodia, whereas virtually no exchange occurred at Filopodial tips. Mutation of the G-actin-binding motif (GAB) partially compromised stabilization of Ena/VASP at Filopodia tips. These observations led us to propose a model where the EVH2 domain of Ena/VASP induces and maintains clustering of the barbed ends of actin filaments, which putatively corresponds to a transition from lamellipodial to Filopodial localization. Furthermore, the EVH1 domain, together with the GAB motif in the EVH2 domain, helps to maintain Ena/VASP at the growing barbed ends.
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Ena/VASP Proteins Have an Anti-Capping Independent Function in Filopodia Formation
Molecular biology of the cell, 2007Co-Authors: Derek A. Applewhite, Tatyana Svitkina, Frank B. Gertler, Melanie Barzik, Shin Ichiro Kojima, Gary G. BorisyAbstract:Filopodia have been implicated in a number of diverse cellular processes including growth-cone path finding, wound healing, and metastasis. The Ena/VASP family of proteins has emerged as key to Filopodia formation but the exact mechanism for how they function has yet to be fully elucidated. Using cell spreading as a model system in combination with small interfering RNA depletion of Capping Protein, we determined that Ena/VASP proteins have a role beyond anticapping activity in Filopodia formation. Analysis of mutant Ena/VASP proteins demonstrated that the entire EVH2 domain was the minimal domain required for Filopodia formation. Fluorescent recovery after photobleaching data indicate that Ena/VASP proteins rapidly exchange at the leading edge of lamellipodia, whereas virtually no exchange occurred at Filopodial tips. Mutation of the G-actin-binding motif (GAB) partially compromised stabilization of Ena/VASP at Filopodia tips. These observations led us to propose a model where the EVH2 domain of Ena/VASP induces and maintains clustering of the barbed ends of actin filaments, which putatively corresponds to a transition from lamellipodial to Filopodial localization. Furthermore, the EVH1 domain, together with the GAB motif in the EVH2 domain, helps to maintain Ena/VASP at the growing barbed ends.
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Role of fascin in Filopodial protrusion
The Journal of cell biology, 2006Co-Authors: Danijela Vignjevic, Tatyana Svitkina, Yvonne S. Aratyn, Shin Ichiro Kojima, Oana Danciu, Gary G. BorisyAbstract:In this study, the mechanisms of actin-bundling in Filopodia were examined. Analysis of cellular localization of known actin cross-linking proteins in mouse melanoma B16F1 cells revealed that fascin was specifically localized along the entire length of all Filopodia, whereas other actin cross-linkers were not. RNA interference of fascin reduced the number of Filopodia, and remaining Filopodia had abnormal morphology with wavy and loosely bundled actin organization. Dephosphorylation of serine 39 likely determined cellular Filopodia frequency. The constitutively active fascin mutant S39A increased the number and length of Filopodia, whereas the inactive fascin mutant S39E reduced Filopodia frequency. Fluorescence recovery after photobleaching of GFP-tagged wild-type and S39A fascin showed that dephosphorylated fascin underwent rapid cycles of association to and dissociation from actin filaments in Filopodia, with t1/2 < 10 s. We propose that fascin is a key specific actin cross-linker, providing stiffness for Filopodial bundles, and that its dynamic behavior allows for efficient coordination between elongation and bundling of Filopodial actin filaments.
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lamellipodial versus Filopodial mode of the actin nanomachinery pivotal role of the filament barbed end
Cell, 2004Co-Authors: Marisan R Mejillano, Tatyana Svitkina, Frank B. Gertler, Shin Ichiro Kojima, Derek A. Applewhite, Gary G. BorisyAbstract:Understanding how a particular cell type expresses the lamellipodial or Filopodial form of the actin machinery is essential to understanding a cell's functional interactions. To determine how a cell "chooses" among these alternative modes of "molecular hardware," we tested the role of key proteins that affect actin filament barbed ends. Depletion of capping protein (CP) by short hairpin RNA (shRNA) caused loss of lamellipodia and explosive formation of Filopodia. The knockdown phenotype was rescued by a CP mutant refractory to shRNA, but not by another barbed-end capper, gelsolin, demonstrating that the phenotype was specific for CP. In Ena/VASP deficient cells, CP depletion resulted in ruffling instead of Filopodia. We propose a model for selection of lamellipodial versus Filopodial organization in which CP is a negative regulator of Filopodia formation and Ena/VASP has recruiting/activating functions downstream of actin filament elongation in addition to its previously suggested anticapping and antibranching activities.
Tatyana Svitkina - One of the best experts on this subject based on the ideXlab platform.
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a minimal actomyosin based model predicts the dynamics of Filopodia on neuronal dendrites
Molecular Biology of the Cell, 2017Co-Authors: Olena Marchenko, Tatyana Svitkina, Sulagna Das, Igor L Novak, Vladimir Rodionov, Nadia Efimova, Charles W Wolgemuth, Leslie M LoewAbstract:Dendritic Filopodia are actin-filled dynamic subcellular structures that sprout on neuronal dendrites during neurogenesis. The exploratory motion of the Filopodia is crucial for synaptogenesis, but the underlying mechanisms are poorly understood. To study Filopodial motility, we collected and analyzed image data on Filopodia in cultured rat hippocampal neurons. We hypothesized that mechanical feedback among the actin retrograde flow, myosin activity, and substrate adhesion gives rise to various Filopodial behaviors. We formulated a minimal one-dimensional partial differential equation model that reproduced the range of observed motility. To validate our model, we systematically manipulated experimental correlates of parameters in the model: substrate adhesion strength, actin polymerization rate, myosin contractility, and the integrity of the putative microtubule-based barrier at the filopodium base. The model predicts the response of the system to each of these experimental perturbations, supporting the hypothesis that our actomyosin-driven mechanism controls dendritic Filopodia dynamics.
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actomyosin dynamics determine the extension and retraction of Filopodia on neuronal dendrites
bioRxiv, 2016Co-Authors: Olena Marchenko, Tatyana Svitkina, Sulagna Das, Igor L Novak, Vladimir Rodionov, Nadia Efimova, Charles W Wolgemuth, Leslie M LoewAbstract:Dendritic Filopodia are actin-filled dynamic subcellular structures that sprout on neuronal dendrites during neurogenesis. The exploratory motion of the Filopodia is crucial for synaptogenesis but the underlying mechanisms are poorly understood. To study the Filopodial motility, we collected and analyzed image data on Filopodia in cultured rat hippocampal neurons. We hypothesized that mechanical feedback among the actin retrograde flow, myosin activity and substrate adhesion gives rise to various Filopodial behaviors. We have formulated a minimal one-dimensional partial differential equation model that reproduced the range of observed motility. To validate our model, we systematically manipulated experimental correlates of parameters in the model: substrate adhesion strength, actin polymerization rate, myosin contractility and the integrity of the putative microtubule-based barrier at the filopodium base. The model predicts the response of the system to each of these experimental perturbations, supporting the hypothesis that our actomyosin-driven mechanism controls dendritic Filopodia dynamics.
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The Cytoskeletal Mechanisms of Cell-Cell Junction Formation in Endothelial Cells
Molecular biology of the cell, 2011Co-Authors: Matthew K. Hoelzle, Tatyana SvitkinaAbstract:The actin cytoskeleton and associated proteins play a vital role in cell-cell adhesion. However, the procedure by which cells establish adherens junctions remains unclear. We investigated the dynamics of cell-cell junction formation and the corresponding architecture of the underlying cytoskeleton in cultured human umbilical vein endothelial cells. We show that the initial interaction between cells is mediated by protruding lamellipodia. On their retraction, cells maintain contact through thin bridges formed by Filopodia-like protrusions connected by VE-cadherin-rich junctions. Bridges share multiple features with conventional Filopodia, such as an internal actin bundle associated with fascin along the length and vasodilator-stimulated phosphoprotein at the tip. It is striking that, unlike conventional Filopodia, transformation of actin organization from the lamellipodial network to Filopodial bundle during bridge formation occurs in a proximal-to-distal direction and is accompanied by recruitment of fascin in the same direction. Subsequently, bridge bundles recruit nonmuscle myosin II and mature into stress fibers. Myosin II activity is important for bridge formation and accumulation of VE-cadherin in nascent adherens junctions. Our data reveal a mechanism of cell-cell junction formation in endothelial cells using lamellipodia as the initial protrusive contact, subsequently transforming into Filopodia-like bridges connected through adherens junctions. Moreover, a novel lamellipodia-to-Filopodia transition is used in this context.
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coordination of membrane and actin cytoskeleton dynamics during Filopodia protrusion
PLOS ONE, 2009Co-Authors: Changsong Yang, Matthew K. Hoelzle, Andrea Disanza, Giorgio Scita, Tatyana SvitkinaAbstract:Leading edge protrusion of migrating cells involves tightly coordinated changes in the plasma membrane and actin cytoskeleton. It remains unclear whether polymerizing actin filaments push and deform the membrane, or membrane deformation occurs independently and is subsequently stabilized by actin filaments. To address this question, we employed an ability of the membrane-binding I-BAR domain of IRSp53 to uncouple the membrane and actin dynamics and to induce Filopodia in expressing cells. Using time-lapse imaging and electron microscopy of IRSp53-I-BAR-expressing B16F1 melanoma cells, we demonstrate that cells are not able to protrude or maintain durable long extensions without actin filaments in their interior, but I-BAR-dependent membrane deformation can create a small and transient space at Filopodial tips that is subsequently filled with actin filaments. Moreover, the expressed I-BAR domain forms a submembranous coat that may structurally support these transient actin-free protrusions until they are further stabilized by the actin cytoskeleton. Actin filaments in the I-BAR-induced Filopodia, in contrast to normal Filopodia, do not have a uniform length, are less abundant, poorly bundled, and display erratic dynamics. Such unconventional structural organization and dynamics of actin in I-BAR-induced Filopodia suggests that a typical bundle of parallel actin filaments is not necessary for generation and mechanical support of the highly asymmetric Filopodial geometry. Together, our data suggest that actin filaments may not directly drive the protrusion, but only stabilize the space generated by the membrane deformation; yet, such stabilization is necessary for efficient protrusion.
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ena vasp proteins have an anti capping independent function in Filopodia formation
Molecular Biology of the Cell, 2007Co-Authors: Derek A. Applewhite, Gary G. Borisy, Tatyana Svitkina, Frank B. Gertler, Melanie Barzik, Shin Ichiro KojimaAbstract:Filopodia have been implicated in a number of diverse cellular processes including growth-cone path finding, wound healing, and metastasis. The Ena/VASP family of proteins has emerged as key to Filopodia formation but the exact mechanism for how they function has yet to be fully elucidated. Using cell spreading as a model system in combination with small interfering RNA depletion of Capping Protein, we determined that Ena/VASP proteins have a role beyond anticapping activity in Filopodia formation. Analysis of mutant Ena/VASP proteins demonstrated that the entire EVH2 domain was the minimal domain required for Filopodia formation. Fluorescent recovery after photobleaching data indicate that Ena/VASP proteins rapidly exchange at the leading edge of lamellipodia, whereas virtually no exchange occurred at Filopodial tips. Mutation of the G-actin-binding motif (GAB) partially compromised stabilization of Ena/VASP at Filopodia tips. These observations led us to propose a model where the EVH2 domain of Ena/VASP induces and maintains clustering of the barbed ends of actin filaments, which putatively corresponds to a transition from lamellipodial to Filopodial localization. Furthermore, the EVH1 domain, together with the GAB motif in the EVH2 domain, helps to maintain Ena/VASP at the growing barbed ends.
Frank B. Gertler - One of the best experts on this subject based on the ideXlab platform.
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Ena/VASP regulates mDia2-initiated Filopodial length, dynamics, and function.
Molecular biology of the cell, 2014Co-Authors: Melanie Barzik, Stephanie L. Gupton, Leslie Marie Mcclain, Frank B. GertlerAbstract:Filopodia are long plasma membrane extensions involved in the formation of adhesive, contractile, and protrusive actin-based structures in spreading and migrating cells. Whether Filopodia formed by different molecular mechanisms equally support these cellular functions is unresolved. We used Enabled/vasodilator-stimulated phosphoprotein (Ena/VASP)-deficient MV(D7) fibroblasts, which are also devoid of endogenous mDia2, as a model system to investigate how these different actin regulatory proteins affect Filopodia morphology and dynamics independently of one another. Filopodia initiated by either Ena/VASP or mDia2 contained similar molecular inventory but differed significantly in parameters such as number, length, F-actin organization, lifetime, and protrusive persistence. Moreover, in the absence of Ena/VASP, Filopodia generated by mDia2 did not support initiation of integrin-dependent signaling cascades required for adhesion and subsequent lamellipodial extension, thereby causing a defect in early cell spreading. Coexpression of VASP with constitutively active mDia2(M/A) rescued these early adhesion defects. We conclude that Ena/VASP and mDia2 support the formation of Filopodia with significantly distinct properties and that Ena/VASP regulates mDia2-initiated Filopodial morphology, dynamics, and function.
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Characterization of EVL-I as a protein kinase D substrate
Cellular signalling, 2008Co-Authors: Katrien Janssens, Line De Kimpe, Frank B. Gertler, Jackie R. Vandenheede, Sandy Vandoninck, Michele Balsamo, Johan Van LintAbstract:EVL-I is a splice variant of EVL (Ena/VASP like protein), whose in vivo function and regulation are still poorly understood. We found that Protein Kinase D (PKD) interacts in vitro and in vivo with EVL-I and phosphorylates EVL-I in a 21 amino acid alternately-included insert in the EVH2 domain. Following knockdown of the capping protein CPβ and spreading on laminin, phosphorylated EVL-I can support Filopodia formation and the phosphorylated EVL-I is localized at Filopodial tips. Furthermore, we found that the lamellipodial localization of EVL-I is unaffected by phosphorylation, but that impairment of EVL-I phosphorylation is associated with ruffling of lamellipodia upon PDBu stimulation. Besides the lamellipodial and Filopodial localization of phosphorylated EVL-I in fibroblasts, we determined that EVL-I is hyperphosphorylated and localized in the cell–cell contacts of certain breast cancer cells and mouse embryo keratinocytes. Taken together, our results show that phosphorylated EVL-I is present in lamellipodia, Filopodia and cell–cell contacts and suggest the existence of signaling pathways that may affect EVL-I via phosphorylation of its EVH2 domain.
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ena vasp proteins have an anti capping independent function in Filopodia formation
Molecular Biology of the Cell, 2007Co-Authors: Derek A. Applewhite, Gary G. Borisy, Tatyana Svitkina, Frank B. Gertler, Melanie Barzik, Shin Ichiro KojimaAbstract:Filopodia have been implicated in a number of diverse cellular processes including growth-cone path finding, wound healing, and metastasis. The Ena/VASP family of proteins has emerged as key to Filopodia formation but the exact mechanism for how they function has yet to be fully elucidated. Using cell spreading as a model system in combination with small interfering RNA depletion of Capping Protein, we determined that Ena/VASP proteins have a role beyond anticapping activity in Filopodia formation. Analysis of mutant Ena/VASP proteins demonstrated that the entire EVH2 domain was the minimal domain required for Filopodia formation. Fluorescent recovery after photobleaching data indicate that Ena/VASP proteins rapidly exchange at the leading edge of lamellipodia, whereas virtually no exchange occurred at Filopodial tips. Mutation of the G-actin-binding motif (GAB) partially compromised stabilization of Ena/VASP at Filopodia tips. These observations led us to propose a model where the EVH2 domain of Ena/VASP induces and maintains clustering of the barbed ends of actin filaments, which putatively corresponds to a transition from lamellipodial to Filopodial localization. Furthermore, the EVH1 domain, together with the GAB motif in the EVH2 domain, helps to maintain Ena/VASP at the growing barbed ends.
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Ena/VASP function in retinal axons is required for terminal arborization but not pathway navigation
Development (Cambridge England), 2007Co-Authors: Asha Dwivedy, Frank B. Gertler, Jeffrey Boone Miller, Christine E. Holt, Cecile LebrandAbstract:The Enabled/vasodilator-stimulated phosphoprotein (Ena/VASP) family of proteins is required for Filopodia formation in growth cones and plays a crucial role in guidance cue-induced remodeling of the actin cytoskeleton. In vivo studies with pharmacological inhibitors of actin polymerization have previously provided evidence for the view that Filopodia are needed for growth cone navigation in the developing visual pathway. Here we have re-examined this issue using an alternative strategy to generate growth cones without Filopodia in vivo by artificially targeting Xena/XVASP (Xenopus homologs of Ena/VASP) proteins to mitochondria in retinal ganglion cells (RGCs). We used the specific binding of the EVH1 domain of the Ena/VASP family of proteins with the ligand motif FP4 to sequester the protein at the mitochondria surface. RGCs with reduced function of Xena/XVASP proteins extended fewer axons out of the eye and possessed dynamic lamellipodial growth cones missing Filopodia that advanced slowly in the optic tract. Surprisingly, despite lacking Filopodia, the axons navigated along the optic pathway without obvious guidance errors, indicating that the Xena/XVASP family of proteins and Filopodial protrusions are non-essential for pathfinding in retinal axons. However, depletion of Xena/XVASP proteins severely impaired the ability of growth cones to form branches within the optic tectum, suggesting that this protein family, and probably Filopodia, plays a key role in establishing terminal arborizations.
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Ena/VASP Proteins Have an Anti-Capping Independent Function in Filopodia Formation
Molecular biology of the cell, 2007Co-Authors: Derek A. Applewhite, Tatyana Svitkina, Frank B. Gertler, Melanie Barzik, Shin Ichiro Kojima, Gary G. BorisyAbstract:Filopodia have been implicated in a number of diverse cellular processes including growth-cone path finding, wound healing, and metastasis. The Ena/VASP family of proteins has emerged as key to Filopodia formation but the exact mechanism for how they function has yet to be fully elucidated. Using cell spreading as a model system in combination with small interfering RNA depletion of Capping Protein, we determined that Ena/VASP proteins have a role beyond anticapping activity in Filopodia formation. Analysis of mutant Ena/VASP proteins demonstrated that the entire EVH2 domain was the minimal domain required for Filopodia formation. Fluorescent recovery after photobleaching data indicate that Ena/VASP proteins rapidly exchange at the leading edge of lamellipodia, whereas virtually no exchange occurred at Filopodial tips. Mutation of the G-actin-binding motif (GAB) partially compromised stabilization of Ena/VASP at Filopodia tips. These observations led us to propose a model where the EVH2 domain of Ena/VASP induces and maintains clustering of the barbed ends of actin filaments, which putatively corresponds to a transition from lamellipodial to Filopodial localization. Furthermore, the EVH1 domain, together with the GAB motif in the EVH2 domain, helps to maintain Ena/VASP at the growing barbed ends.
Stephanie L. Gupton - One of the best experts on this subject based on the ideXlab platform.
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A pair of E3 ubiquitin ligases compete to regulate Filopodial dynamics and axon guidance.
The Journal of cell biology, 2019Co-Authors: Nicholas P. Boyer, Shalini Menon, Laura E. Mccormick, Fabio L. Urbina, Stephanie L. GuptonAbstract:Appropriate axon guidance is necessary to form accurate neuronal connections. Axon guidance cues that stimulate cytoskeletal reorganization within the growth cone direct axon navigation. Filopodia at the growth cone periphery have long been considered sensors for axon guidance cues, yet how they respond to extracellular cues remains ill defined. Our previous work found that the Filopodial actin polymerase VASP and consequently Filopodial stability are negatively regulated via nondegradative TRIM9-dependent ubiquitination. Appropriate VASP ubiquitination and deubiquitination are required for axon turning in response to the guidance cue netrin-1. Here we show that the TRIM9-related protein TRIM67 outcompetes TRIM9 for interacting with VASP and antagonizes TRIM9-dependent VASP ubiquitination. The surprising antagonistic roles of two closely related E3 ubiquitin ligases are required for netrin-1-dependent Filopodial responses, axon turning and branching, and fiber tract formation. We suggest a novel model in which coordinated regulation of VASP ubiquitination by a pair of interfering ligases is a critical element of VASP dynamics, Filopodial stability, and axon guidance.
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The E3 Ubiquitin Ligase TRIM9 Is a Filopodia Off Switch Required for Netrin-Dependent Axon Guidance.
Developmental Cell, 2015Co-Authors: Shalini Menon, Nicholas P. Boyer, Cortney C. Winkle, Leslie Marie Mcclain, Christopher Carey Hanlin, Dharmendra Pandey, Simon Rothenfußer, Anne Marion Taylor, Stephanie L. GuptonAbstract:Neuronal growth cone Filopodia contain guidance receptors and contribute to axon guidance; however, the mechanism by which the guidance cue netrin increases Filopodia density is unknown. Here, we demonstrate that TRIM9, an E3 ubiquitin ligase that localizes to Filopodia tips and binds the netrin receptor DCC, interacts with and ubiquitinates the barbed-end polymerase VASP to modulate Filopodial stability during netrin-dependent axon guidance. Studies with murine Trim9(+/+) and Trim9(-/-) cortical neurons, along with a non-ubiquitinatable VASP mutant, demonstrate that TRIM9-mediated ubiquitination of VASP reduces VASP Filopodial tip localization, VASP dynamics at tips, and Filopodial stability. Upon netrin treatment, VASP is deubiquitinated, which promotes VASP tip localization and Filopodial stability. Trim9 deletion induces axon guidance defects in vitro and in vivo, whereas a gradient of deubiquitinase inhibition promotes axon turning in vitro. We conclude that a gradient of TRIM9-mediated ubiquitination of VASP creates a Filopodial stability gradient during axon turning.
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Ena/VASP regulates mDia2-initiated Filopodial length, dynamics, and function.
Molecular biology of the cell, 2014Co-Authors: Melanie Barzik, Stephanie L. Gupton, Leslie Marie Mcclain, Frank B. GertlerAbstract:Filopodia are long plasma membrane extensions involved in the formation of adhesive, contractile, and protrusive actin-based structures in spreading and migrating cells. Whether Filopodia formed by different molecular mechanisms equally support these cellular functions is unresolved. We used Enabled/vasodilator-stimulated phosphoprotein (Ena/VASP)-deficient MV(D7) fibroblasts, which are also devoid of endogenous mDia2, as a model system to investigate how these different actin regulatory proteins affect Filopodia morphology and dynamics independently of one another. Filopodia initiated by either Ena/VASP or mDia2 contained similar molecular inventory but differed significantly in parameters such as number, length, F-actin organization, lifetime, and protrusive persistence. Moreover, in the absence of Ena/VASP, Filopodia generated by mDia2 did not support initiation of integrin-dependent signaling cascades required for adhesion and subsequent lamellipodial extension, thereby causing a defect in early cell spreading. Coexpression of VASP with constitutively active mDia2(M/A) rescued these early adhesion defects. We conclude that Ena/VASP and mDia2 support the formation of Filopodia with significantly distinct properties and that Ena/VASP regulates mDia2-initiated Filopodial morphology, dynamics, and function.
Jan Faix - One of the best experts on this subject based on the ideXlab platform.
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Filopodia formation induced by active mDia2/Drf3
Journal of microscopy, 2008Co-Authors: Jennifer Block, Theresia E B Stradal, Jan Hänisch, Robert Geffers, S.a. Köstler, Edit Urban, J.v. Small, Klemens Rottner, Jan FaixAbstract:Filopodia are rod-shaped cell surface protrusions composed of a parallel bundle of actin filaments. Since Filopodia frequently emanate from lamellipodia, it has been proposed that they form exclusively by the convergence and elongation of actin filaments generated in lamellipodia networks. However, Filopodia form without Arp2/3-complex, which is essential for lamellipodia formation, indicating that actin filaments in Filopodia may be generated by other nucleators. Here we analyzed the effects of ectopic expression of GFP-tagged full length or a constitutively active variant of the human formin mDia2/Drf3. By contrast to the full-length molecule, which did not affect cell behaviour and was entirely cytosolic, active Drf3 lacking the C-terminal regulatory region (Drf3DeltaDAD) induced the formation of Filopodia and accumulated at their tips. Low expression of Drf3DeltaDAD induced rod-shaped or tapered Filopodia, whereas over-expression resulted in multiple, club-shaped Filopodia. The clubs were filled with densely bundled actin filaments, whose number but not packing density decreased further away from the tip. Interestingly, clubs frequently increased in width after protrusion beyond the cell periphery, which correlated with increased amounts of Drf3DeltaDAD at their tips. These data suggest Drf3-induced Filopodia form and extend by de novo nucleation of actin filaments instead of convergent elongation. Finally, Drf3DeltaDAD also induced the formation of unusual, lamellipodia-like structures, which contained both lamellipodial markers and the prominent Filopodial protein fascin. Microarray analyses revealed highly variable Drf3 expression levels in different commonly used cell lines, reflecting the need for more detailed analyses of the functions of distinct formins in actin cytoskeleton turnover and different cell types.
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the bundling activity of vasodilator stimulated phosphoprotein is required for filopodium formation
Proceedings of the National Academy of Sciences of the United States of America, 2006Co-Authors: Antje Schirenbeck, Rajesh Arasada, Till Bretschneider, Theresia E B Stradal, Michael Schleicher, Jan FaixAbstract:Filopodia are highly dynamic finger-like cell protrusions filled with parallel bundles of actin filaments. Previously we have shown that Diaphanous-related formin dDia2 is involved in the formation of Filopodia. Another key player for the formation of Filopodia across many species is vasodilator-stimulated phosphoprotein (VASP). It has been proposed that the essential role of VASP for formation of Filopodia is its competition with capping proteins for filament barbed-end interaction. To better understand the function of VASP in filopodium formation, we analyzed the in vitro and in vivo properties of Dictyostelium VASP (DdVASP) and extended our findings to human VASP. Recombinant VASP from both species nucleated and bundled actin filaments, but did not compete with capping proteins or block depolymerization from barbed ends. Together with the finding that DdVASP binds to the FH2 domain of dDia2, these data indicate that the crucial role of VASP in filopodium formation is different from uncapping of actin filaments. To identify the activity of DdVASP required in this process, rescue experiments of DdVASP-null cells with mutant DdVASP constructs were performed. Only WT DdVASP, but not a mutant lacking the F-actin bundling activity, could rescue the ability of these cells to form WT-like Filopodia. Our data suggest that DdVASP is complexed with dDia2 in Filopodial tips and support formin-mediated filament elongation by bundling nascent actin filaments.
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the diaphanous related formin ddia2 is required for the formation and maintenance of Filopodia
Nature Cell Biology, 2005Co-Authors: Antje Schirenbeck, Rajesh Arasada, Till Bretschneider, Jan Faix, Michael SchleicherAbstract:Formins have important roles in the nucleation of actin and the formation of linear actin filaments, but their role in filopodium formation has remained elusive. Dictyostelium discoideum Diaphanous-related formin dDia2 is enriched at the tips of Filopodia and interacts with profilin II and Rac1. An FH1FH2 fragment of dDia2 nucleated actin polymerization and removed capping protein from capped filament ends. Genetic studies showed that dDia2 is important for cell migration as well as the formation, elongation and maintenance of Filopodia. Here we provide evidence that dDia2 specifically controls Filopodial dynamics by regulating actin turnover at the barbed ends of actin filaments.