Open AccessVASP protects actin filaments from gelsolin: An in vitro study with implications for platelet actin reorganizations
Author(s) -
Bearer E.L.,
Prakash J.M.,
Manchester R.D.,
Allen P.G.
Publication year - 2000
Publication title -
cell motility and the cytoskeleton
Language(s) - English
Resource type - Journals
eISSN - 1097-0169
pISSN - 0886-1544
DOI - 10.1002/1097-0169(200012)47:4<351::aid-cm8>3.0.co;2-8
Subject(s) - gelsolin , biology , actin , in vitro , microbiology and biotechnology , actin remodeling , mdia1 , microfilament , actin cytoskeleton , cytoskeleton , biochemistry , cell
An initial step in platelet shape change is disassembly of actin filaments, which are then reorganized into new actin structures, including filopodia and lamellipodia. This disassembly is thought to be mediated primarily by gelsolin, an abundant actin filament‐severing protein in platelets. Shape change is inhibited by VASP, another abundant actin‐binding protein. Paradoxically, in vitro VASP enhances formation of actin filaments and bundles them, activities that would be expected to increase shape change, not inhibit it. We hypothesized that VASP might inhibit shape change by stabilizing filaments and preventing their disassembly by gelsolin. Such activity would explain VASP's known physiological role. Here, we test this hypothesis in vitro using either purified recombinant or endogenous platelet VASP by fluorescence microscopy and biochemical assays. VASP inhibited gelsolin's ability to disassemble actin filaments in a dose‐dependent fashion. Inhibition was detectable at the low VASP:actin ratio found inside the platelet (1:40 VASP:actin). Gelsolin bound to VASP‐actin filaments at least as well as to actin alone. VASP inhibited gelsolin‐induced nucleation at higher concentrations (1:5 VASP:actin ratios). VASP's affinity for actin (K d ∼0.07 μM) and its ability to promote polymerization (1:20 VASP actin ratio) were greater with Ca ++ ‐actin than with Mg ++ ‐actin (K d ∼1 μM and 1:1 VASP), regardless of the presence of gelsolin. By immunofluorescence, VASP and gelsolin co‐localized in the filopodia and lamellipodia of platelets spreading on glass, suggesting that these in vitro interactions could take place within the cell as well. We conclude that VASP stabilizes actin filaments to the severing effects of gelsolin but does not inhibit gelsolin from binding to the filaments. These results suggest a new concept for actin dynamics inside cells: that bundling proteins protect the actin superstructure from disassembly by severing, thereby preserving the integrity of the cytoskeleton. Cell Motil. Cytoskeleton 47:351–364, 2000. © 2000 Wiley‐Liss, Inc.