The Experts below are selected from a list of 12414 Experts worldwide ranked by ideXlab platform
Viswanathan Natarajan - One of the best experts on this subject based on the ideXlab platform.
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Role of Sphingosine Kinase 1 and S1P Transporter Spns2 in HGF-mediated Lamellipodia Formation in Lung Endothelium
Journal of Biological Chemistry, 2016Co-Authors: David L. Ebenezer, Evgeny Berdyshev, Irina Bronova, Mark Shaaya, Anantha Harijith, Viswanathan NatarajanAbstract:Hepatocyte growth factor (HGF) signaling via c-Met is known to promote endothelial cell motility and angiogenesis. We have previously reported that HGF stimulates Lamellipodia formation and motility of human lung microvascular endothelial cells (HLMVECs) via PI3K/Akt signal transduction and reactive oxygen species generation. Here, we report a role for HGF-induced intracellular sphingosine-1-phosphate (S1P) generation catalyzed by sphingosine kinase 1 (SphK1), S1P transporter, spinster homolog 2 (Spns2), and S1P receptor, S1P1, in Lamellipodia formation and perhaps motility of HLMVECs. HGF stimulated SphK1 phosphorylation and enhanced intracellular S1P levels in HLMVECs, which was blocked by inhibition of SphK1. HGF enhanced co-localization of SphK1/p-SphK1 with actin/cortactin in Lamellipodia and down-regulation or inhibition of SphK1 attenuated HGF-induced Lamellipodia formation in HLMVECs. In addition, down-regulation of Spns2 also suppressed HGF-induced Lamellipodia formation, suggesting a key role for inside-out S1P signaling. The HGF-mediated phosphorylation of SphK1 and its localization in Lamellipodia was dependent on c-Met and ERK1/2 signaling, but not the PI3K/Akt pathway; however, blocking PI3K/Akt signaling attenuated HGF-mediated phosphorylation of Spns2. Down-regulation of S1P1, but not S1P2 or S1P3, with specific siRNA attenuated HGF-induced Lamellipodia formation. Further, HGF enhanced association of Spns2 with S1P1 that was blocked by inhibiting SphK1 activity with PF-543. Moreover, HGF-induced migration of HLMVECs was attenuated by down-regulation of Spns2. Taken together, these results suggest that HGF/c-Met-mediated Lamellipodia formation, and perhaps motility is dependent on intracellular generation of S1P via activation and localization of SphK1 to cell periphery and Spns2-mediated extracellular transportation of S1P and its inside-out signaling via S1P1.
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ID: 111: THE S1P TRANSPORTER, SPNS2, MEDIATES HGF-INDUCED Lamellipodia FORMATION AND MIGRATION OF HUMAN LUNG ENDOTHELIAL CELLS
Journal of Investigative Medicine, 2016Co-Authors: Peter V. Usatyuk, David L. Ebenezer, Viswanathan NatarajanAbstract:Rationale We have demonstrated earlier that HGF-induced Lamellipodia formation in human lung microvascular endothelial cells (HLMVECs) was through c-Met receptor tyrosine kinase and PI3 kinase/Akt signal transduction. Here, we show that HGF-mediated Lamellipodia formation is dependent on intracellular S1P generation mediated by sphingosine kinase 1 (SphK1), the S1P transporter, Spns2 and S1P1 in HLMVECs. Methods HLMVECs were treated with HGF (20 ng/ml) for different time points. Lamellipodia were detected after HGF treatment by immunofluorescent staining of Spns2, cortactin and actin in Lamellipodia, and Lamellipodia were quantified by measuring cell periphery fluorescence intensity. Pearson9s correlation coefficient was used to statistically quantify co-localization of proteins in Lamellipodia. Endogenous SphK activity was blocked by SphK1 specific inhibitor PF-543, and expression of SphK1 in cells was down-regulated by siRNA. Cellular S1P levels were quantified by mass spectrometry. Results HGF stimulated phosphorylation of SphK1, and its localization to Lamellipodia of HLMVECs. Down-regulation of SphK1, but not SphK2, with siRNA or inhibition of SphK1 with PF-543 (1–5 µM) attenuated HGF-induced Lamellipodia formation in HLMVECs. The HGF-mediated phosphorylation of SphK1 and its localization in Lamellipodia was dependent on PI3K/Akt and ERK1/2 signaling apthways. HGF increased S1P levels in HLMVECs, which was blocked by inhibition of SphK1 with PF-543. Further, HGF induced serine phosphorylation and translocation of Spns2, the S1P transporter, to Lamellipodia, which was Akt dependent. The HGF-induced Lamellipodia formation in HLMVECs was blocked by down-regulation of Spns2, suggesting extracellular action of S1P in Lamellipodia formation. Down-regulation of S1P1, but not S1P2 or S1P3, with siRNA attenuated HGF-induced Lamellipodia formation. Further, HGF stimulation enhanced association of Spns2 with S1P1 and blocking SphK1 activty with PF-543 attenuated the association between Spns2 and S1P1. Additionally, HGF-induced migration of HLMVECs was attenuated by down-regulation of Spns2. Conclusion These results suggest that HGF/c-Met mediated Lamellipodia formation and motility is dependent on intracellular generation of S1P via activation and localization of SphK1 to cell periphery and Spns2 mediated transport of S1P to outside for signaling via S1P1 in HLMVECs. This work was supported by NIH/HLBI P01 HL98050 to VN.
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role played by paxillin and paxillin tyrosine phosphorylation in hepatocyte growth factor sphingosine 1 phosphate mediated reactive oxygen species generation Lamellipodia formation and endothelial barrier function
Pulmonary circulation, 2015Co-Authors: Peter V. Usatyuk, Jeffrey R Jacobson, Anne E Cress, Joe G N Garcia, Ravi Salgia, Viswanathan NatarajanAbstract:Paxillin is a multifunctional and multidomain focal adhesion adaptor protein. It serves as an important scaffolding protein at focal adhesions by recruiting and binding to structural and signaling molecules. Paxillin tyrosine phosphorylation at Y31 and Y118 is important for paxillin redistribution to focal adhesions and angiogenesis. Hepatocyte growth factor (HGF) and sphingosine-1-phosphate (S1P) are potent stimulators of Lamellipodia formation, a prerequisite for endothelial cell migration. The role played by paxillin and its tyrosine phosphorylated forms in HGF- or S1P-induced Lamellipodia formation and barrier function is unclear. HGF or S1P stimulated Lamellipodia formation, tyrosine phosphorylation of paxillin at Y31 and Y118, and c-Abl in human lung microvascular endothelial cells (HLMVECs). Knockdown of paxillin with small interfering RNA (siRNA) or transfection with paxillin mutants (Y31F or Y118F) mitigated HGF- or S1P-induced Lamellipodia formation, translocation of p47 (phox) to Lamellipodia, and reactive oxygen species (ROS) generation in HLMVECs. Furthermore, exposure of HLMVECs to HGF or S1P stimulated c-Abl-mediated tyrosine phosphorylation of paxillin at Y31 and Y118 in a time-dependent fashion, and down-regulation of c-Abl with siRNA attenuated HGF- or S1P-mediated Lamellipodia formation, translocation of p47 (phox) to Lamellipodia, and endothelial barrier enhancement. In vivo, knockdown of paxillin with siRNA in mouse lungs attenuated ventilator-induced lung injury. Together, these results suggest that c-Abl-mediated tyrosine phosphorylation of paxillin at Y31 and Y118 regulates HGF- or S1P-mediated Lamellipodia formation, ROS generation in Lamellipodia, and endothelial permeability.
Gary G. Borisy - One of the best experts on this subject based on the ideXlab platform.
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cascade pathway of filopodia formation downstream of scar
Journal of Cell Science, 2004Co-Authors: Assel Biyasheva, Buzz Baum, Tatyana Svitkina, Patricia Kunda, Gary G. BorisyAbstract:The protrusion of two distinct actin-containing organelles, Lamellipodia and filopodia, is thought to be regulated by two parallel pathways: from Rac1 through Scar/WAVEs to Lamellipodia, and from Cdc42 through N-WASP to filopodia. We tested this hypothesis in Drosophila, which contains a single gene for each WASP subfamilies, SCAR and WASp. We performed targeted depletion of SCAR or WASp by dsRNA-mediated interference in two Drosophila cultured cell lines expressing Lamellipodial and filopodial protrusion. Knockdown was verified by laser capture microdissection and RT-PCR, as well as western blotting. Morphometrical, kinetic and electron microscopy analyses of the SCAR-depleted phenotype in both cell types revealed strong inhibition of Lamellipodial formation and cell spreading, as expected. More importantly, filopodia formation was also strongly inhibited, which is not consistent with the parallel pathway hypothesis. By contrast, depletion of WASp did not produce any significant phenotype, except for a slight inhibition of spreading, showing that both Lamellipodia and filopodia in Drosophila cells are regulated predominantly by SCAR. We propose a new, cascade pathway model of filopodia regulation in which SCAR signals to Lamellipodia and then filopodia arise from Lamellipodia in response to additional signal(s).
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antagonism between ena vasp proteins and actin filament capping regulates fibroblast motility
Cell, 2002Co-Authors: James E Bear, Tatyana M Svitkina, Matthias Krause, Dorothy A Schafer, Joseph Loureiro, Geraldine A Strasser, Ivan V Maly, Oleg Y Chaga, John A Cooper, Gary G. BorisyAbstract:Abstract Cell motility requires Lamellipodial protrusion, a process driven by actin polymerization. Ena/VASP proteins accumulate in protruding Lamellipodia and promote the rapid actin-driven motility of the pathogen Listeria . In contrast, Ena/VASP negatively regulate cell translocation. To resolve this paradox, we analyzed the function of Ena/VASP during Lamellipodial protrusion. Ena/VASP-deficient Lamellipodia protruded slower but more persistently, consistent with their increased cell translocation rates. Actin networks in Ena/VASP-deficient Lamellipodia contained shorter, more highly branched filaments compared to controls. Lamellipodia with excess Ena/VASP contained longer, less branched filaments. In vitro, Ena/VASP promoted actin filament elongation by interacting with barbed ends, shielding them from capping protein. We conclude that Ena/VASP regulates cell motility by controlling the geometry of actin filament networks within Lamellipodia.
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arp2 3 complex and actin depolymerizing factor cofilin in dendritic organization and treadmilling of actin filament array in Lamellipodia
Journal of Cell Biology, 1999Co-Authors: Tatyana M Svitkina, Gary G. BorisyAbstract:The leading edge (∼1 μm) of Lamellipodia in Xenopus laevis keratocytes and fibroblasts was shown to have an extensively branched organization of actin filaments, which we term the dendritic brush. Pointed ends of individual filaments were located at Y-junctions, where the Arp2/3 complex was also localized, suggesting a role of the Arp2/3 complex in branch formation. Differential depolymerization experiments suggested that the Arp2/3 complex also provided protection of pointed ends from depolymerization. Actin depolymerizing factor (ADF)/cofilin was excluded from the distal 0.4 μm of the Lamellipodial network of keratocytes and in fibroblasts it was located within the depolymerization-resistant zone. These results suggest that ADF/cofilin, per se, is not sufficient for actin brush depolymerization and a regulatory step is required. Our evidence supports a dendritic nucleation model (Mullins, R.D., J.A. Heuser, and T.D. Pollard. 1998. Proc. Natl. Acad. Sci. USA. 95:6181–6186) for Lamellipodial protrusion, which involves treadmilling of a branched actin array instead of treadmilling of individual filaments. In this model, Arp2/3 complex and ADF/cofilin have antagonistic activities. Arp2/3 complex is responsible for integration of nascent actin filaments into the actin network at the cell front and stabilizing pointed ends from depolymerization, while ADF/cofilin promotes filament disassembly at the rear of the brush, presumably by pointed end depolymerization after dissociation of the Arp2/3 complex.
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Arp2/3 Complex and Actin Depolymerizing Factor/Cofilin in Dendritic Organization and Treadmilling of Actin Filament Array in Lamellipodia
Journal of Cell Biology, 1999Co-Authors: Tatyana M Svitkina, Gary G. BorisyAbstract:The leading edge (∼1 μm) of Lamellipodia in Xenopus laevis keratocytes and fibroblasts was shown to have an extensively branched organization of actin filaments, which we term the dendritic brush. Pointed ends of individual filaments were located at Y-junctions, where the Arp2/3 complex was also localized, suggesting a role of the Arp2/3 complex in branch formation. Differential depolymerization experiments suggested that the Arp2/3 complex also provided protection of pointed ends from depolymerization. Actin depolymerizing factor (ADF)/cofilin was excluded from the distal 0.4 μm of the Lamellipodial network of keratocytes and in fibroblasts it was located within the depolymerization-resistant zone. These results suggest that ADF/cofilin, per se, is not sufficient for actin brush depolymerization and a regulatory step is required. Our evidence supports a dendritic nucleation model (Mullins, R.D., J.A. Heuser, and T.D. Pollard. 1998. Proc. Natl. Acad. Sci. USA. 95:6181–6186) for Lamellipodial protrusion, which involves treadmilling of a branched actin array instead of treadmilling of individual filaments. In this model, Arp2/3 complex and ADF/cofilin have antagonistic activities. Arp2/3 complex is responsible for integration of nascent actin filaments into the actin network at the cell front and stabilizing pointed ends from depolymerization, while ADF/cofilin promotes filament disassembly at the rear of the brush, presumably by pointed end depolymerization after dissociation of the Arp2/3 complex.
Ali Badache - One of the best experts on this subject based on the ideXlab platform.
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memo rhoa mdia1 signaling controls microtubules the actin network and adhesion site formation in migrating cells
Journal of Cell Biology, 2008Co-Authors: Kossay Zaoui, Stephane Honore, Daniel Isnardon, Diane Braguer, Ali BadacheAbstract:Actin assembly at the cell front drives membrane protrusion and initiates the cell migration cycle. Microtubules (MTs) extend within forward protrusions to sustain cell polarity and promote adhesion site turnover. Memo is an effector of the ErbB2 receptor tyrosine kinase involved in breast carcinoma cell migration. However, its mechanism of action remained unknown. We report in this study that Memo controls ErbB2-regulated MT dynamics by altering the transition frequency between MT growth and shortening phases. Moreover, although Memo-depleted cells can assemble the Rac1-dependent actin meshwork and form Lamellipodia, they show defective localization of Lamellipodial markers such as α-actinin-1 and a reduced number of short-lived adhesion sites underlying the advancing edge of migrating cells. Finally, we demonstrate that Memo is required for the localization of the RhoA guanosine triphosphatase and its effector mDia1 to the plasma membrane and that Memo–RhoA–mDia1 signaling coordinates the organization of the Lamellipodial actin network, adhesion site formation, and MT outgrowth within the cell leading edge to sustain cell motility.
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Memo–RhoA–mDia1 signaling controls microtubules, the actin network, and adhesion site formation in migrating cells
The Journal of cell biology, 2008Co-Authors: Kossay Zaoui, Stephane Honore, Daniel Isnardon, Diane Braguer, Ali BadacheAbstract:Actin assembly at the cell front drives membrane protrusion and initiates the cell migration cycle. Microtubules (MTs) extend within forward protrusions to sustain cell polarity and promote adhesion site turnover. Memo is an effector of the ErbB2 receptor tyrosine kinase involved in breast carcinoma cell migration. However, its mechanism of action remained unknown. We report in this study that Memo controls ErbB2-regulated MT dynamics by altering the transition frequency between MT growth and shortening phases. Moreover, although Memo-depleted cells can assemble the Rac1-dependent actin meshwork and form Lamellipodia, they show defective localization of Lamellipodial markers such as α-actinin-1 and a reduced number of short-lived adhesion sites underlying the advancing edge of migrating cells. Finally, we demonstrate that Memo is required for the localization of the RhoA guanosine triphosphatase and its effector mDia1 to the plasma membrane and that Memo–RhoA–mDia1 signaling coordinates the organization of the Lamellipodial actin network, adhesion site formation, and MT outgrowth within the cell leading edge to sustain cell motility.
Matthias Krause - One of the best experts on this subject based on the ideXlab platform.
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Steering cell migration: lamellipodium dynamics and the regulation of directional persistence
Nature Reviews Molecular Cell Biology, 2014Co-Authors: Matthias Krause, Alexis GautreauAbstract:Lamellipodial protrusion depends on the force generated by actin polymerization. Actin polymerization is the sum of the activities of nucleators — for example, the actin-related protein 2/3 (ARP2/3) complex — and elongators — formins and ENA/VASP proteins. Small GTPases, such as RAC and CDC42, control both actin nucleators and actin elongators; RAC activates the WASP family verprolin-homologous protein (WAVE) complex upstream of the ARP2/3 complex independently of the activation of the formin FMNL2 by CDC42, but RAC may coordinate ARP2/3 with ENA/VASP proteins by inducing a complex between WAVE and lamellipodin. The speed of cell migration depends on the turnover of actin branched junctions and on the elongation of actin networks. An intrinsic instability of Lamellipodia is due to ARP2/3 inhibitory proteins, such as Arpin, which is also activated downstream of RAC. The persistence of Lamellipodia is the major controller of cell directionality. Directional persistence (that is, the characteristic time during which a cell sustains its migration in the same direction) is the combinatory result of several intertwined positive- and negative-feedback loops that sustain or stop actin polymerization at the leading edge. Membrane protrusions at the leading edge of cells, known as Lamellipodia, drive cell migration in many normal and pathological situations. Lamellipodial protrusion is powered by actin polymerization, which is mediated by the actin-related protein 2/3 (ARP2/3)-induced nucleation of branched actin networks and the elongation of actin filaments. Recently, advances have been made in our understanding of positive and negative ARP2/3 regulators (such as the SCAR/WAVE (SCAR/WASP family verprolin-homologous protein) complex and Arpin, respectively) and of proteins that control actin branch stability (such as glial maturation factor (GMF)) or actin filament elongation (such as ENA/VASP proteins) in lamellipodium dynamics and cell migration. This Review highlights how the balance between actin filament branching and elongation, and between the positive and negative feedback loops that regulate these activities, determines Lamellipodial persistence. Importantly, directional persistence, which results from Lamellipodial persistence, emerges as a critical factor in steering cell migration. Lamellipodial protrusion is powered by actin polymerization that is mediated through the actin-related protein 2/3 (ARP2/3)-induced nucleation of branched actin networks and the elongation of actin filaments. These processes are regulated by positive and negative feedback loops centred around the GTPase RAC, and the balance between them determines Lamellipodial and directional persistence during cell migration.
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steering cell migration lamellipodium dynamics and the regulation of directional persistence
Nature Reviews Molecular Cell Biology, 2014Co-Authors: Matthias Krause, Alexis GautreauAbstract:Membrane protrusions at the leading edge of cells, known as Lamellipodia, drive cell migration in many normal and pathological situations. Lamellipodial protrusion is powered by actin polymerization, which is mediated by the actin-related protein 2/3 (ARP2/3)-induced nucleation of branched actin networks and the elongation of actin filaments. Recently, advances have been made in our understanding of positive and negative ARP2/3 regulators (such as the SCAR/WAVE (SCAR/WASP family verprolin-homologous protein) complex and Arpin, respectively) and of proteins that control actin branch stability (such as glial maturation factor (GMF)) or actin filament elongation (such as ENA/VASP proteins) in lamellipodium dynamics and cell migration. This Review highlights how the balance between actin filament branching and elongation, and between the positive and negative feedback loops that regulate these activities, determines Lamellipodial persistence. Importantly, directional persistence, which results from Lamellipodial persistence, emerges as a critical factor in steering cell migration.
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antagonism between ena vasp proteins and actin filament capping regulates fibroblast motility
Cell, 2002Co-Authors: James E Bear, Tatyana M Svitkina, Matthias Krause, Dorothy A Schafer, Joseph Loureiro, Geraldine A Strasser, Ivan V Maly, Oleg Y Chaga, John A Cooper, Gary G. BorisyAbstract:Abstract Cell motility requires Lamellipodial protrusion, a process driven by actin polymerization. Ena/VASP proteins accumulate in protruding Lamellipodia and promote the rapid actin-driven motility of the pathogen Listeria . In contrast, Ena/VASP negatively regulate cell translocation. To resolve this paradox, we analyzed the function of Ena/VASP during Lamellipodial protrusion. Ena/VASP-deficient Lamellipodia protruded slower but more persistently, consistent with their increased cell translocation rates. Actin networks in Ena/VASP-deficient Lamellipodia contained shorter, more highly branched filaments compared to controls. Lamellipodia with excess Ena/VASP contained longer, less branched filaments. In vitro, Ena/VASP promoted actin filament elongation by interacting with barbed ends, shielding them from capping protein. We conclude that Ena/VASP regulates cell motility by controlling the geometry of actin filament networks within Lamellipodia.
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zyxin is not colocalized with vasodilator stimulated phosphoprotein vasp at Lamellipodial tips and exhibits different dynamics to vinculin paxillin and vasp in focal adhesions
Molecular Biology of the Cell, 2001Co-Authors: Klemens Rottner, Matthias Krause, Victor J Small, Mario Gimona, Jurgen WehlandAbstract:Actin polymerization is accompanied by the formation of protein complexes that link extracellular signals to sites of actin assembly such as membrane ruffles and focal adhesions. One candidate recently implicated in these processes is the LIM domain protein zyxin, which can bind both Ena/vasodilator-stimulated phosphoprotein (VASP) proteins and the actin filament cross-linking protein α-actinin. To characterize the localization and dynamics of zyxin in detail, we generated both monoclonal antibodies and a green fluorescent protein (GFP)-fusion construct. The antibodies colocalized with ectopically expressed GFP-VASP at focal adhesions and along stress fibers, but failed to label Lamellipodial and filopodial tips, which also recruit Ena/VASP proteins. Likewise, neither microinjected, fluorescently labeled zyxin antibodies nor ectopically expressed GFP-zyxin were recruited to these latter sites in live cells, whereas both probes incorporated into focal adhesions and stress fibers. Comparing the dynamics of zyxin with that of the focal adhesion protein vinculin revealed that both proteins incorporated simultaneously into newly formed adhesions. However, during spontaneous or induced focal adhesion disassembly, zyxin delocalization preceded that of either vinculin or paxillin. Together, these data identify zyxin as an early target for signals leading to adhesion disassembly, but exclude its role in recruiting Ena/VASP proteins to the tips of Lamellipodia and filopodia.
Tatyana M Svitkina - One of the best experts on this subject based on the ideXlab platform.
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Myosin II and Arp2/3 crosstalk governs intracellular hydraulic pressure and Lamellipodia formation.
Molecular biology of the cell, 2021Co-Authors: Shivani Patel, Tatyana M Svitkina, Changsong Yang, Donna Mckeon, Kimheak Sao, Nicole M Naranjo, Ryan J PetrieAbstract:Human fibroblasts can switch between Lamellipodia-dependent and -independent migration mechanisms on 2D surfaces and in 3D matrices. RhoA GTPase activity governs the switch from low-pressure Lamellipodia to high-pressure lobopodia in response to the physical structure of the 3D matrix. Inhibiting actomyosin contractility in these cells reduces intracellular pressure and reverts lobopodia to Lamellipodial protrusions via an unknown mechanism. To test the hypothesis that high pressure physically prevents Lamellipodia formation, we manipulated pressure by activating RhoA or changing the osmolarity of the extracellular environment and imaged cell protrusions. We find RhoA activity inhibits Rac1-mediated Lamellipodia formation through two distinct pathways. First, RhoA boosts intracellular pressure by increasing actomyosin contractility and water influx but acts upstream of Rac1 to inhibit Lamellipodia formation. Increasing osmotic pressure revealed a second RhoA pathway which acts through non-muscle myosin II (NMII) to disrupt Lamellipodia downstream of Rac1 and elevate pressure. Interestingly, Arp2/3 inhibition triggered a NMII-dependent increase in intracellular pressure, along with Lamellipodia disruption. Together, these results suggest that actomyosin contractility and water influx are coordinated to increase intracellular pressure, and RhoA signaling can inhibit Lamellipodia formation via two distinct pathways in high-pressure cells. [Media: see text] [Media: see text] [Media: see text].
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antagonism between ena vasp proteins and actin filament capping regulates fibroblast motility
Cell, 2002Co-Authors: James E Bear, Tatyana M Svitkina, Matthias Krause, Dorothy A Schafer, Joseph Loureiro, Geraldine A Strasser, Ivan V Maly, Oleg Y Chaga, John A Cooper, Gary G. BorisyAbstract:Abstract Cell motility requires Lamellipodial protrusion, a process driven by actin polymerization. Ena/VASP proteins accumulate in protruding Lamellipodia and promote the rapid actin-driven motility of the pathogen Listeria . In contrast, Ena/VASP negatively regulate cell translocation. To resolve this paradox, we analyzed the function of Ena/VASP during Lamellipodial protrusion. Ena/VASP-deficient Lamellipodia protruded slower but more persistently, consistent with their increased cell translocation rates. Actin networks in Ena/VASP-deficient Lamellipodia contained shorter, more highly branched filaments compared to controls. Lamellipodia with excess Ena/VASP contained longer, less branched filaments. In vitro, Ena/VASP promoted actin filament elongation by interacting with barbed ends, shielding them from capping protein. We conclude that Ena/VASP regulates cell motility by controlling the geometry of actin filament networks within Lamellipodia.
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arp2 3 complex and actin depolymerizing factor cofilin in dendritic organization and treadmilling of actin filament array in Lamellipodia
Journal of Cell Biology, 1999Co-Authors: Tatyana M Svitkina, Gary G. BorisyAbstract:The leading edge (∼1 μm) of Lamellipodia in Xenopus laevis keratocytes and fibroblasts was shown to have an extensively branched organization of actin filaments, which we term the dendritic brush. Pointed ends of individual filaments were located at Y-junctions, where the Arp2/3 complex was also localized, suggesting a role of the Arp2/3 complex in branch formation. Differential depolymerization experiments suggested that the Arp2/3 complex also provided protection of pointed ends from depolymerization. Actin depolymerizing factor (ADF)/cofilin was excluded from the distal 0.4 μm of the Lamellipodial network of keratocytes and in fibroblasts it was located within the depolymerization-resistant zone. These results suggest that ADF/cofilin, per se, is not sufficient for actin brush depolymerization and a regulatory step is required. Our evidence supports a dendritic nucleation model (Mullins, R.D., J.A. Heuser, and T.D. Pollard. 1998. Proc. Natl. Acad. Sci. USA. 95:6181–6186) for Lamellipodial protrusion, which involves treadmilling of a branched actin array instead of treadmilling of individual filaments. In this model, Arp2/3 complex and ADF/cofilin have antagonistic activities. Arp2/3 complex is responsible for integration of nascent actin filaments into the actin network at the cell front and stabilizing pointed ends from depolymerization, while ADF/cofilin promotes filament disassembly at the rear of the brush, presumably by pointed end depolymerization after dissociation of the Arp2/3 complex.
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Arp2/3 Complex and Actin Depolymerizing Factor/Cofilin in Dendritic Organization and Treadmilling of Actin Filament Array in Lamellipodia
Journal of Cell Biology, 1999Co-Authors: Tatyana M Svitkina, Gary G. BorisyAbstract:The leading edge (∼1 μm) of Lamellipodia in Xenopus laevis keratocytes and fibroblasts was shown to have an extensively branched organization of actin filaments, which we term the dendritic brush. Pointed ends of individual filaments were located at Y-junctions, where the Arp2/3 complex was also localized, suggesting a role of the Arp2/3 complex in branch formation. Differential depolymerization experiments suggested that the Arp2/3 complex also provided protection of pointed ends from depolymerization. Actin depolymerizing factor (ADF)/cofilin was excluded from the distal 0.4 μm of the Lamellipodial network of keratocytes and in fibroblasts it was located within the depolymerization-resistant zone. These results suggest that ADF/cofilin, per se, is not sufficient for actin brush depolymerization and a regulatory step is required. Our evidence supports a dendritic nucleation model (Mullins, R.D., J.A. Heuser, and T.D. Pollard. 1998. Proc. Natl. Acad. Sci. USA. 95:6181–6186) for Lamellipodial protrusion, which involves treadmilling of a branched actin array instead of treadmilling of individual filaments. In this model, Arp2/3 complex and ADF/cofilin have antagonistic activities. Arp2/3 complex is responsible for integration of nascent actin filaments into the actin network at the cell front and stabilizing pointed ends from depolymerization, while ADF/cofilin promotes filament disassembly at the rear of the brush, presumably by pointed end depolymerization after dissociation of the Arp2/3 complex.
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analysis of the actin myosin ii system in fish epidermal keratocytes mechanism of cell body translocation
Journal of Cell Biology, 1997Co-Authors: Tatyana M Svitkina, Alexande Verkhovsky, Kyle M Mcquade, Gary G OrisyAbstract:While the protrusive event of cell locomotion is thought to be driven by actin polymerization, the mechanism of forward translocation of the cell body is unclear. To elucidate the mechanism of cell body translocation, we analyzed the supramolecular organization of the actin–myosin II system and the dynamics of myosin II in fish epidermal keratocytes. In Lamellipodia, long actin filaments formed dense networks with numerous free ends in a brushlike manner near the leading edge. Shorter actin filaments often formed T junctions with longer filaments in the brushlike area, suggesting that new filaments could be nucleated at sides of preexisting filaments or linked to them immediately after nucleation. The polarity of actin filaments was almost uniform, with barbed ends forward throughout most of the Lamellipodia but mixed in arc-shaped filament bundles at the Lamellipodial/cell body boundary. Myosin II formed discrete clusters of bipolar minifilaments in Lamellipodia that increased in size and density towards the cell body boundary and colocalized with actin in boundary bundles. Time-lapse observation demonstrated that myosin clusters appeared in the Lamellipodia and remained stationary with respect to the substratum in locomoting cells, but they exhibited retrograde flow in cells tethered in epithelioid colonies. Consequently, both in locomoting and stationary cells, myosin clusters approached the cell body boundary, where they became compressed and aligned, resulting in the formation of boundary bundles. In locomoting cells, the compression was associated with forward displacement of myosin features. These data are not consistent with either sarcomeric or polarized transport mechanisms of cell body translocation. We propose that the forward translocation of the cell body and retrograde flow in the Lamellipodia are both driven by contraction of an actin–myosin network in the Lamellipodial/cell body transition zone.
