Ena/VASP regulates mDia2-initiated filopodial length, dynamics, and function

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[superscript 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[superscript 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.National Institutes of Health (U.S.) (Grant GM58801)National Cancer Institute (U.S.). Integrative Cancer Biology Program (Grant 1-U54-CA112967

Ena/VASP proteins have an anti-capping independent function in filopodia formation

Author Posting. © American Society for Cell Biology, 2007. This article is posted here by permission of American Society for Cell Biology for personal use, not for redistribution. The definitive version was published in Molecular Biology of the Cell 18 (2007): 2579-2591, doi:10.1091/mbc.E06-11-0990.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.This work was supported in part by National Institutes of Health Grants GM7542201 to D.A.A., GM58801 to F.B.G., and GM62431 to G.G.B. and by Cell Migration Consortium Grants GM64346 to D.A.A and G.G.B

Struktur- und Funktionsuntersuchungen an der Phosphatidylinositol-spezifischen Phospholipase C (PI-PLC) aus Listeria monocytogenes und der Ena/VASP-Homologie 1 (EVH1)-Domäne des murinen Vesl-2

1. Studien zur Substratbindung der PI-PLC aus L. monocytogenes Um Informationen über die Bindung des Diacylglycerinphosphatanteils von PI durch die PI-PLC aus L. monocytogenes zu erhalten, wurden PI-PLC-Mutanten heterolog in E. coli produziert, gereinigt und zur Kokristallisation mit verschiedenen Substratanaloga eingesetzt. Zusätzlich wurde die Aktivität der Mutanten mit zwei unterschiedlichen Substraten untersucht. Der Austausch einzelner Aminosäuren im aktiven Zentrum der PI-PLC führte zur starken Reduktion der katalytischen Aktivität, wobei die Faltungstopologie der PI-PLC-Mutanten mit der des Wildtyp-Enzyms nahezu identisch war. Mittels Röntgenstrukturanalyse konnten die Substratanaloga jedoch in keiner der aufgeklärten PI-PLC-Strukturen zweifelsfrei nachgewiesen werden. 2. Die EVH1-Domäne von mVesl Die EVH1-Domäne des murinen Vesl-2 (mVesl), einem Mitglied der Homer/Vesl-Proteinfamilie, wurde heterolog in E. coli produziert, gereinigt und zur Kokristallisation mit synthetischen prolinreichen Peptiden eingesetzt, die von den natürlichen Interaktionspartnern abgeleitet worden waren. Die Struktur der mVesl EVH1-Domäne wurde sowohl in freier Form als auch in Komplex mit Teilen eines IP3R-Peptids bei einer Auflösung von 2.2 Å gelöst. Basierend auf diesen strukturellen Daten wurde eine Klassifizierung der bekannten EVH1-Domänen in zwei Gruppen vorgenommen. Die EVH1-Domänen der Ena/VASP-Proteinfamilie wurden der Klasse I zugeordnet, während die Homer/Vesl EVH1-Domänen die Klasse II bilden. Zusätzlich wurde mittels Oberflächen-Plasmonenresonanzspektroskopie gezeigt, dass die verwendeten prolinreichen Peptide mit mäßiger Affinität (KD: 1.5 - 2.9 mM) von mVesl EVH1 gebunden werden.1. Substrate binding studies of PI-PLC from L. monocytogenes Enzymatic activity and cocrystallization experiments of site-directed mutants using diverse substrate analogs were used to analyze the binding of the diacylglycerolphosphate moiety of PI to PI-PLC. Single-amino acid substitutions within the active site significantly decreased catalytic activity, despite marginal structural differences between mutants and wild-type enzyme. Of the crystal structures of PI-PLC determined in this work, none, however, contained a clearly defined substrate analogue in the active site. 2. The EVH1 domain of mVesl The EVH1 domain of murine Vesl-2, a member of the recently discovered Homer/Vesl protein family was cloned, purified and crystallized. The structure was subsequently solved and refined at 2.2 Å resolution. The Homer/Vesl EVH1 domain was identified as a new class II EVH1 domain. Furthermore, ligand binding by the Vesl-2 EVH1 domain was investigated using surface plasmon resonance spectroscopy

Selectivity in subunit composition of Ena/VASP tetramers

The members of the actin regulatory family of Ena/VASP proteins form stable tetramers. The vertebrate members of the Ena/VASP family, VASP, Mena and EVL, have many overlapping properties and expression patterns, but functional and regulatory differences between paralogues have been observed. The formation of mixed oligomers may serve a regulatory role to refine Ena/VASP activity. While it has been assumed that family members can form mixed oligomers, this possibility has not been investigated systematically. Using cells expressing controlled combinations of VASP, Mena and EVL, we evaluated the composition of Ena/VASP oligomers and found that VASP forms oligomers without apparent bias with itself, Mena or EVL. However, Mena and EVL showed only weak hetero-oligomerization, suggesting specificity in the association of Ena/VASP family members. Co-expression of VASP increased the ability of Mena and EVL to form mixed oligomers. Additionally, we found that the tetramerization domain (TD) at the C-termini of Ena/VASP proteins conferred the observed selectivity. Finally, we demonstrate that replacement of the TD with a synthetic tetramerizing coiled coil sequence supports homo-oligomerization and normal VASP subcellular localization.Massachusetts Institute of Technology. Ludwig Center for Molecular OncologyNational Institutes of Health (U.S.) (Grants U54- CA112967 and 1-DP5-OD019815)National Cancer Institute (U.S.) (David H. Koch Institute for Integrative Cancer Research at MIT. Grant P30-CA14051)National Institutes of Health (U.S.) ( Pre-Doctoral Training Grant T32GM007287

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

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

VASP is a CXCR2-interacting protein that regulates CXCR2-mediated polarization and chemotaxis

Chemotaxis regulates the recruitment of leukocytes, which is integral for a number of biological processes and is mediated through the interaction of chemokines with seven transmembrane G-protein-coupled receptors. Several studies have indicated that chemotactic signaling pathways might be activated via G-protein-independent mechanisms, perhaps through novel receptor-interacting proteins. CXCR2 is a major chemokine receptor expressed on neutrophils. We used a proteomics approach to identify unique ligand-dependent CXCR2-interacting proteins in differentiated neutrophil-like HL-60 cells. Using this approach, vasodilator-stimulated phosphoprotein (VASP) was identified as a CXCR2-interacting protein. The interaction between CXCR2 and VASP is direct and enhanced by CXCL8 stimulation, which triggers VASP phosphorylation via PKA- and PKCδ-mediated pathways. The interaction between CXCR2 and VASP requires free F-actin barbed ends to recruit VASP to the leading edge. Finally, knockdown of VASP in HL-60 cells results in severely impaired CXCR2-mediated chemotaxis and polarization. These data provide the first demonstration that direct interaction of VASP with CXCR2 is essential for proper CXCR2 function and demonstrate a crucial role for VASP in mediating chemotaxis in leukocytes

Actin at stereocilia tips is regulated by mechanotransduction and ADF/cofilin

Stereocilia on auditory sensory cells are actin-based protrusions that mechanotransduce sound into an electrical signal. These stereocilia are arranged into a bundle with three rows of increasing length to form a staircase-like morphology that is required for hearing. Stereocilia in the shorter rows, but not the tallest row, are mechanotransducing because they have force-sensitive channels localized at their tips. The onset of mechanotransduction during mouse postnatal development refines stereocilia length and width. However, it is unclear how actin is differentially regulated between stereocilia in the tallest row of the bundle and the shorter, mechanotransducing rows. Here, we show actin turnover is increased at the tips of mechanotransducing stereocilia during bundle maturation. Correspondingly, from birth to postnatal day 6, these stereocilia had increasing amounts of available actin barbed ends, where monomers can be added or lost readily, as compared with the non-mechanotransducing stereocilia in the tallest row. The increase in available barbed ends depended on both mechanotransduction and MYO15 or EPS8, which are required for the normal specification and elongation of the tallest row of stereocilia. We also found that loss of the F-actin-severing proteins ADF and cofilin-1 decreased barbed end availability at stereocilia tips. These proteins enriched at mechanotransducing stereocilia tips, and their localization was perturbed by the loss of mechanotransduction, MYO15, or EPS8. Finally, stereocilia lengths and widths were dysregulated in Adf and Cfl1 mutants. Together, these data show that actin is remodeled, likely by a severing mechanism, in response to mechanotransduction