Mechanosensing during directed cell migration requires dynamic actin polymerization at focal adhesions.
ABSTRACT: The mechanical properties of a cell's microenvironment influence many aspects of cellular behavior, including cell migration. Durotaxis, the migration toward increasing matrix stiffness, has been implicated in processes ranging from development to cancer. During durotaxis, mechanical stimulation by matrix rigidity leads to directed migration. Studies suggest that cells sense mechanical stimuli, or mechanosense, through the acto-myosin cytoskeleton at focal adhesions (FAs); however, FA actin cytoskeletal remodeling and its role in mechanosensing are not fully understood. Here, we show that the Ena/VASP family member, Ena/VASP-like (EVL), polymerizes actin at FAs, which promotes cell-matrix adhesion and mechanosensing. Importantly, we show that EVL regulates mechanically directed motility, and that suppression of EVL expression impedes 3D durotactic invasion. We propose a model in which EVL-mediated actin polymerization at FAs promotes mechanosensing and durotaxis by maturing, and thus reinforcing, FAs. These findings establish dynamic FA actin polymerization as a central aspect of mechanosensing and identify EVL as a crucial regulator of this process.
Project description:Shigella spp. are intracellular bacterial pathogens that cause diarrhoeal disease in humans. Shigella utilize the host actin cytoskeleton to enter cells, move through the cytoplasm of cells and pass into adjacent cells. Ena/VASP family proteins are highly conserved proteins that participate in actin-dependent dynamic cellular processes. We tested whether Ena/VASP family members VASP (vasodilator-stimulated phosphoprotein), Mena (mammalian-enabled) or EVL (Ena-VASP-like) contribute to Shigella flexneri spread through cell monolayers. VASP and EVL restricted cell-to-cell spread without significantly altering actin-based motility, whereas Mena had no effect on these processes. Phosphorylation of VASP on Ser153, Ser235 and Thr274 regulated its subcellular distribution and function. VASP derivatives that lack the Ena/VASP homology 1 (EVH1) domain or contain a phosphoablative mutation of Ser153 were defective in restricting S. flexneri spread, indicating that the EVH1 domain and phosphorylation on Ser153 are required for this process. The EVH1 domain and Ser153 of VASP were required for VASP localization to focal adhesions, and localization of VASP to focal adhesions and/or the leading edge was required for restriction of spread. The contribution of the EVH1 domain was from both the donor and the recipient cell, whereas the contribution of Ser153 phosphorylation was only from the donor cell. Thus, unlike host proteins characterized in Shigella pathogenesis that promote bacterial spread, VASP and EVL function to limit it. The ability of VASP and EVL to limit spread highlights the critical role of focal adhesion complexes and/or the leading edge in bacterial passage between cells.
Project description:Proteins of the Enabled/vasodilator-stimulated phosphoprotein (Ena/VASP) family link signal transduction pathways to actin cytoskeleton dynamics. VASP is substrate of cAMP-dependent, cGMP-dependent and AMP-activated protein kinases that primarily phosphorylate the sites S157, S239 and T278, respectively. Here, we systematically analyzed functions of VASP phosphorylation patterns for actin assembly and subcellular targeting in vivo and compared the phosphorylation effects of Ena/VASP family members. Methods used were the reconstitution of VASP-null cells with ;locked' phosphomimetic VASP mutants, actin polymerization of VASP mutants in vitro and in living cells, site-specific kinase-mediated VASP phosphorylation, and analysis of the endogenous protein with phosphorylation-status-specific antibodies. Phosphorylation at S157 influenced VASP localization, but had a minor impact on F-actin assembly. Phosphorylation of the S157-equivalent site in the Ena/VASP family members Mena and EVL had no effect on the ratio of cellular F-actin to G-actin. By contrast, VASP phosphorylation at S239 (and the equivalent site in Mena) or T278 impaired VASP-driven actin filament formation. The data show that VASP functions are precisely regulated by differential phosphorylation and provide new insights into cytoskeletal control by serine/threonine kinase-dependent signaling pathways.
Project description:Dynamic rearrangements of the actin cytoskeleton power cell motility in contexts ranging from intracellular microbial pathogenesis to axon guidance. The Ena/VASP family proteins-Mena, VASP, and Evl-are believed to control cell motility by serving as a direct link between signaling events and the actin cytoskeleton. It has previously been reported that a novel miniature protein, pGolemi, binds with high affinity to the EVH1 domain of Mena (Mena1-112) but not to those of VASP (VASP1-115) or Evl (Evl1-115) and also causes an unusual defect in actin-driven Listeria monocytogenes motility. Here, scanning mutagenesis was used to examine the effects of single amino acid changes within pGolemi on EVH1 domain affinity and specificity, miniature protein secondary structure, and L. monocytogenes motility. The data suggest that pGolemi contains the expected aPP-like fold and binds Mena1-112 in a manner highly analogous to the proline-rich repeat region of L. monocytogenes ActA protein. Residues throughout pGolemi contribute to both EVH1 domain affinity and paralog specificity. Moreover, the affinities of pGolemi variants for Mena1-112 correlate with selectivity against the EVH1 domains of VASP and Evl. In L. monocytogenes motility assays, speed and speed variability correlate strongly with EVH1 paralog specificity, suggesting that the Ena/VASP paralogs do not play equivalent roles in the process of L. monocytogenes actin tail maturation.
Project description:Tight regulation of cell motility is essential for many physiological processes, such as formation of a functional nervous system and wound healing. Drosophila Abl negatively regulates the actin cytoskeleton effector protein Ena during neuronal development in flies, and it has been postulated that this may occur through an unknown intermediary. Lamellipodin (Lpd) regulates cell motility and recruits Ena/VASP proteins (Ena, Mena, VASP, EVL) to the leading edge of cells. However, the regulation of this recruitment has remained unsolved.Here we show that Lpd is a substrate of Abl kinases and binds to the Abl SH2 domain. Phosphorylation of Lpd positively regulates the interaction between Lpd and Ena/VASP proteins. Consistently, efficient recruitment of Mena and EVL to Lpd at the leading edge requires Abl kinases. Furthermore, transient Lpd phosphorylation by Abl kinases upon netrin-1 stimulation of primary cortical neurons positively correlates with an increase in Lpd-Mena coprecipitation. Lpd is also transiently phosphorylated by Abl kinases upon platelet-derived growth factor (PDGF) stimulation, regulates PDGF-induced dorsal ruffling of fibroblasts and axonal morphogenesis, and cooperates with c-Abl in an Ena/VASP-dependent manner.Our findings suggest that Abl kinases positively regulate Lpd-Ena/VASP interaction, Ena/VASP recruitment to Lpd at the leading edge, and Lpd-Ena/VASP function in axonal morphogenesis and in PDGF-induced dorsal ruffling. Our data do not support the suggested negative regulatory role of Abl for Ena. Instead, we propose that Lpd is the hitherto unknown intermediary between Abl and Ena/VASP proteins.
Project description:Vasodilator-stimulated phosphoprotein (VASP) and Ena-VASP-like (EVL) are cytoskeletal effector proteins implicated in regulating cell morphology, adhesion, and migration in various cell types. However, the role of these proteins in T-cell motility, adhesion, and in vivo trafficking remains poorly understood. This study identifies a specific role for EVL and VASP in T-cell diapedesis and trafficking. We demonstrate that EVL and VASP are selectively required for activated T-cell trafficking but are not required for normal T-cell development or for naïve T-cell trafficking to lymph nodes and spleen. Using a model of multiple sclerosis, we show an impairment in trafficking of EVL/VASP-deficient activated T cells to the inflamed central nervous system of mice with experimental autoimmune encephalomyelitis. Additionally, we found a defect in trafficking of EVL/VASP double-knockout (dKO) T cells to the inflamed skin and secondary lymphoid organs. Deletion of EVL and VASP resulted in the impairment in ?4 integrin (CD49d) expression and function. Unexpectedly, EVL/VASP dKO T cells did not exhibit alterations in shear-resistant adhesion to, or in crawling on, primary endothelial cells under physiologic shear forces. Instead, deletion of EVL and VASP impaired T-cell diapedesis. Furthermore, T-cell diapedesis became equivalent between control and EVL/VASP dKO T cells upon ?4 integrin blockade. Overall, EVL and VASP selectively mediate activated T-cell trafficking by promoting the diapedesis step of transendothelial migration in a ?4 integrin-dependent manner.
Project description:Cells can control actin polymerization by nucleating new filaments or elongating existing ones. We recently identified CRMP-1 as a factor that stimulates the formation of <i>Listeria monocytogenes</i> actin comet tails, thereby implicating it in actin assembly. We now show that CRMP-1 is a major contributor to actin assembly in epithelial cells, where it works with the Ena/VASP family member EVL to assemble the actin cytoskeleton in the apical cortex and in protruding lamellipodia. CRMP-1 and EVL bind to one another and together accelerate actin filament barbed-end elongation. CRMP-1 also stimulates actin assembly in the presence of VASP and Mena in vitro, but CRMP-1-dependent actin assembly in MDCK cells is EVL specific. Our results identify CRMP-1 as a novel regulator of actin filament elongation and reveal a surprisingly important role for CRMP-1, EVL, and actin polymerization in maintaining the structural integrity of epithelial sheets.
Project description:Cells within the tumor microenvironment influence tumor growth through multiple mechanisms. Pericytes such as hepatic stellate cells are an important cell within the tumor microenvironment; their transformation into highly motile myofibroblasts leads to angiogenesis, stromal cell recruitment, matrix deposition, and ensuing tumor growth. Thus, a better understanding of mechanisms that regulate motility of pericytes is required. Focal adhesions (FAs) form a physical link between the extracellular environment and the actin cytoskeleton, a requisite step for cell motility. FAs contain a collection of proteins including the Ena/VASP family member, vasodilator-stimulated phosphoprotein (VASP); however, a role for VASP in FA development has been elusive. Using a comprehensive siRNA knockdown approach and a variety of VASP mutants coupled with complementary cell imaging methodologies, we demonstrate a requirement of VASP for optimal development of FAs and cell spreading in LX2 liver myofibroblasts, which express high levels of endogenous VASP. Rac1, a binding partner of VASP, acts in tandem with VASP to regulate FAs. In vivo, perturbation of Ena/VASP function in tumor myofibroblast precursor cells significantly reduces pericyte recruitment to tumor vasculature, myofibroblastic transformation, tumor angiogenesis, and tumor growth, providing in vivo pathobiologic relevance to these findings. Taken together, our results identify Ena/VASP as a significant modifier of tumor growth through regulation of FA dynamics and ensuing pericyte/myofibroblast function within the tumor microenvironment.
Project description:T cell receptor (TCR)-driven activation of helper T cells induces a rapid polarization of their cytoskeleton towards bound antigen presenting cells (APCs). We have identified the Fyn- and SLP-76-associated protein Fyb/SLAP as a new ligand for Ena/ vasodilator-stimulated phosphoprotein (VASP) homology 1 (EVH1) domains. Upon TCR engagement, Fyb/SLAP localizes at the interface between T cells and anti-CD3-coated beads, where Evl, a member of the Ena/VASP family, Wiskott-Aldrich syndrome protein (WASP) and the Arp2/3 complex are also found. In addition, Fyb/SLAP is restricted to lamellipodia of spreading platelets. In activated T cells, Fyb/SLAP associates with Ena/VASP family proteins and is present within biochemical complexes containing WASP, Nck, and SLP-76. Inhibition of binding between Fyb/SLAP and Ena/VASP proteins or WASP and the Arp2/3 complex impairs TCR-dependent actin rearrangement, suggesting that these interactions play a key role in linking T cell signaling to remodeling of the actin cytoskeleton.
Project description:Dynamic actin cytoskeletal reorganization is integral to cell motility. Profilins are well-characterized regulators of actin polymerization; however, functional differences among coexpressed profilin isoforms are not well defined. Here, we demonstrate that profilin-1 and profilin-2 differentially regulate membrane protrusion, motility, and invasion; these processes are promoted by profilin-1 and suppressed by profilin-2. Compared to profilin-1, profilin-2 preferentially drives actin polymerization by the Ena/VASP protein, EVL. Profilin-2 and EVL suppress protrusive activity and cell motility by an actomyosin contractility-dependent mechanism. Importantly, EVL or profilin-2 downregulation enhances invasion in vitro and in vivo. In human breast cancer, lower EVL expression correlates with high invasiveness and poor patient outcome. We propose that profilin-2/EVL-mediated actin polymerization enhances actin bundling and suppresses breast cancer cell invasion.
Project description:Cancer invasion is a hallmark of metastasis. The mesenchymal mode of cancer cell invasion is mediated by elongated membrane protrusions driven by the assembly of branched F-actin networks. How deregulation of actin regulators promotes cancer cell invasion is still enigmatic. We report that increased expression and membrane localization of the actin regulator Lamellipodin correlate with reduced metastasis-free survival and poor prognosis in breast cancer patients. In agreement, we find that Lamellipodin depletion reduced lung metastasis in an orthotopic mouse breast cancer model. Invasive 3D cancer cell migration as well as invadopodia formation and matrix degradation was impaired upon Lamellipodin depletion. Mechanistically, we show that Lamellipodin promotes invasive 3D cancer cell migration via both actin-elongating Ena/VASP proteins and the Scar/WAVE complex, which stimulates actin branching. In contrast, Lamellipodin interaction with Scar/WAVE but not with Ena/VASP is required for random 2D cell migration. We identified a phosphorylation-dependent mechanism that regulates selective recruitment of these effectors to Lamellipodin: Abl-mediated Lamellipodin phosphorylation promotes its association with both Scar/WAVE and Ena/VASP, whereas Src-dependent phosphorylation enhances binding to Scar/WAVE but not to Ena/VASP. Through these selective, regulated interactions Lamellipodin mediates directional sensing of epidermal growth factor (EGF) gradients and invasive 3D migration of breast cancer cells. Our findings imply that increased Lamellipodin levels enhance Ena/VASP and Scar/WAVE activities at the plasma membrane to promote 3D invasion and metastasis.