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PMID: 11093254 Published · ppublish English Journal Article Research Support, U.S. Gov't, P.H.S.

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

Cell motility and the cytoskeleton ·Vol. 47 ·No. 4 ·2000-12-00 ·Pages 351-64

Bearer EL, Prakash JM, Manchester RD, Allen PG

Abstract

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) approximately 0.07 microM) and its ability to promote polymerization (1:20 VASP actin ratio) were greater with Ca(++)-actin than with Mg(++)-actin (K(d) approximately 1 microM 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.

MeSH Terms
Actins/metabolism Blood Platelets/metabolism Calcium/metabolism Cell Adhesion Molecules/metabolism,physiology Cell Nucleus/metabolism Cytoskeleton/metabolism Dose-Response Relationship, Drug Electrophoresis, Polyacrylamide Gel Gelsolin/isolation & purification,metabolism Humans Kinetics Listeria/metabolism Magnesium/metabolism Microfilament Proteins Microscopy, Fluorescence Models, Biological Peptides/metabolism Phosphoproteins/metabolism,physiology Pseudopodia/metabolism Time Factors
Chemicals
Actins Cell Adhesion Molecules Gelsolin Microfilament Proteins Peptides Phosphoproteins vasodilator-stimulated phosphoprotein polyproline Magnesium Calcium
Authors & Affiliations
4 authors, click to expand affiliations / ORCID
Bearer E L
Department of Pathology and Laboratory Medicine, Brown University, Providence, Rhode Island 02912, USA. Elaine_Bearer@Brown.edu
Prakash J M
Manchester R D
Allen P G
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Article Info
Journal
Cell motility and the cytoskeleton
Abbr.
Cell Motil Cytoskeleton
ISSN
0886-1544
DOI
10.1002/1097-0169(200012)47:4<351::AID-CM8>3.0.CO;2-8
Email
elaine_bearer@brown.edu
Published
2000-12-00
Pages
351-64
Language
English
Region
United States
NLM ID
8605339
PMCID
PMC3376085
Subset
IM
Grants
NIGMS NIH HHS · R01 GM047368 · United States
NIGMS NIH HHS · R01 GM047368-05 · United States
NIGMS NIH HHS · GM57256 · United States
NIGMS NIH HHS · GM47368 · United States
NIGMS NIH HHS · R01 GM057256 · United States
External Links
PubMed source DOI: 10.1002/1097-0169(200012)47:4<351::AID-CM8>3.0.CO;2-8 PMC Full Text View journal details
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