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United States Department of Agriculture

Agricultural Research Service

Title: Arabidopsis Villin1 Generates Actin Filament Cables That Are Resistant to Depolymerization

Authors
item Huang, Shanjin - PURDUE UNIVERSITY
item Robinson, Robert - UPPSALA UNIVERSITY
item Gao, Lisa - PURDUE UNIVERSITY
item Matsumoto Brower, Tracie
item Brunet, Arnaud - CELLULAIRE VEGETALE
item Blanchion, Laurent - CELLULAIRE VEGETALE
item Staiger, Christopher - PURDUE UNIVERSITY

Submitted to: Plant Cell and Environment
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: December 2, 2004
Publication Date: February 20, 2004
Citation: Huang, S., R.C. Robinson, L.Y. Gao, T. Matsumoto, A. Brunet, L. Blanchoin and C.J. Staiger. 2005. Arabidopsis VILLIN1 generates actin filament cables that are resistant to depolymerization. Plant Cell 17: 486-501.

Interpretive Summary: The cell cytoskeleton is composed of a dynamic network of actin microfilaments and microtubules which facilitate the movement and positioning of nutrients, metabolites and genetic molecules throughout the cell. Accessory proteins associated with cytoskeleton are assumed to be cellular sensors that respond to different environmental or cellular signals. Villin, which was originally identified from intestinal epithelial cell microvilli, is a major actin cytoskeleton regulator. Villin belongs to a superfamily of actin-binding proteins called the villin/gelsolin family. In the presence of calcium, most, but not all, villins nucleate actin filament formation, cap filament barbed ends and sever actin filaments. Here we describe the characterization of one of the villin-like protein (VLN1) which is a member of multi-gene family in Arabidopsis. Unlike human gelsolins, VLN1 from Arabidopsis does not nucleate the formation of actin filaments or cap the barbed ends of actin filaments. Instead VLN1 binds F-actin with a high affinity and bundles actin filaments independently of calcium and calmodulin. VLN1 decorated actin filaments are protected against Actin Depolymerizing Factor (ADF)-mediated depolymerization. These data suggest that VLN1 is a major regulator of the actin cytoskeleton and provides stability to the cable network in the presence of stimuli that result in depolymerization of other actin arrays.

Technical Abstract: Dynamic cytoplasmic streaming, organelle positioning and nuclear migration utilize molecular tracks generated from actin filaments arrayed into higher-order structures like actin cables and bundles. How these arrays are formed and stabilized against cellular depolymerizing forces remains an open question. Villin and fimbrin are the best characterized actin-filament bundling or cross-linking proteins in plants and each is encoded by a multigene family of five members in Arabidopsis thaliana. The related villins and gelsolins are conserved proteins that are constructed from a core of six homologous gelsolin domains. Gelsolin is a calcium-regulated actin filament severing, nucleating and barbed end capping factor. Villin has a seventh domain at its C-terminus, the villin headpiece, which can bind to an actin filament, conferring the ability to crosslink or bundle actin filaments. Many, but not all, villins retain the ability to sever, nucleate and cap filaments. Here we have identified a putative calcium-insensitive villin isoform, through comparison of sequence alignments between human gelsolin and plant villins with x-ray crystallography data for vertebrate gelsolin. AtVLN1 has the least well-conserved type 1 and type 2 calcium-binding sites among the Arabidopsis VILLIN isoforms. Recombinant VLN1 binds to actin filaments with high affinity (Kd ~ 1 uM) and generates bundled filament networks; both properties are independent of the free Ca2+ concentration. Unlike human plasma gelsolin, VLN1 does not nucleate the assembly of filaments from monomer, does not block the polymerization of profilin-actin onto barbed ends, and does not stimulate depolymerization or sever pre-existing filaments. In kinetic assays with ADF/cofilin, the villin appears to bind first to growing filaments and protects filaments against ADF-mediated depolymerization. We propose that VLN1 is a major regulator of the formation and stability of actin filament bundles in plant cells and that it functions to maintain the cable network even in the presence of stimuli that result in depolymerization of other actin arrays.

Last Modified: 12/21/2014
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