VASP protects actin filaments from gelsolin: An in vitro study with implications for platelet actin reorganizations

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 g elsolin, an abundant actin filament-severing protein in platelets. Shape ch ange is inhibited by VASP, another abundant actin-binding protein. Paradoxi cally, in vitro VASP enhances formation of actin filaments and bundles them , activities that would be expected to increase shape change, not inhibit i t. We hypothesized that VASP might inhibit shape change by stabilizing fila ments and preventing their disassembly by gelsolin. Such activity would exp lain VASP's known physiological role. Here, we test this hypothesis in vitr o using either purified recombinant or endogenous platelet VASP by fluoresc ence microscopy and biochemical assays. VASP inhibited gelsolin's ability t o disassemble actin filaments in a dose-dependent fashion. Inhibition was d etectable 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 similar to0.07 muM ) and its ability to promote polymerization (1:20 VASP actin ratio) were gr eater with Ca++-actin than with Mg++-actin (K-d similar to1 muM and 1:1 VAS P), regardless of the presence of gelsolin. By immunofluorescence, VASP and gelsolin co-localized in the filopodia and lamellipodia of platelets sprea ding 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 bind ing to the filaments. These results suggest a new concept for actin dynamic s inside cells: that bundling proteins protect the actin superstructure fro m disassembly by severing, thereby preserving the integrity of the cytoskel eton. Cell Motil. Cytoskeleton 47:351-364, 2000. (C) 2000 Wiley-Liss, Inc.