Arabidopsis VILLIN1 Generates Actin Filament Cables That Are Resistant to Depolymerization

Abstract

Dynamic cytoplasmic streaming, organelle positioning, and nuclear migration use 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. VILLIN1 (VLN1) 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 (K d ;1 mM) and generates bundled filament networks; both properties are independent of the free Ca 2þ 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 preexisting filaments. In kinetic assays with ADF/cofilin, 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.

The Formin Homology 1 Domain Modulates the Actin Nucleation and Bundling Activity of Arabidopsis FORMIN1

“…During the fluorescence light microscopy assays, we discovered that FH1-FH2-Cter was able to organize individual actin filaments ( Figure 9A) into long actin bundles ( Figure 9C) similar to actin filament bundles observed in the presence of Arabidopsis VILLIN1, a well-characterized plant actin filament bundler ( Figure 9B; Huang et al, 2005). No bundles were observed in absence of either VILLIN1 or FH1-FH2-Cter ( Figure 9A).…”

CASEIN KINASE1-LIKE PROTEIN2 Regulates Actin Filament Stability and Stomatal Closure via Phosphorylation of Actin Depolymerizing Factor

“…Quantification of GFP-fABD2-GFP Fluorescence Pixel Intensity, Density, and Skewness of the Actin Filaments in Guard Cells Measurement of relative fluorescence pixel intensity levels of GFP-fABD2-GFP-labeled actin filaments in guard cells and hypocotyl epidermal cells was performed as described previously (Huang et al, 2005). The average fluorescence pixel intensity associated with GFP-fABD2-GFP was measured using ImageJ software (http://rsb.info.nih.gov/ij/) to determine the relative amount of actin filaments.…”

“…High-speed cosedimentation assays were performed to examine the binding of CKL2 to actin filaments as previously described (Kovar et al, 2000;Huang et al, 2005). Direct visualization of actin filaments by epifluorescence light microscopy was performed as described previously (Okada et al, 2002).…”

14-3-3 λ protein interacts with ADF1 to regulate actin cytoskeleton dynamics in Arabidopsis

“…The high speed cosedimentation assays were performed according to Kovar et al [36] and Huang et al [37]. Actin was purified as previously published method according to Spudich and Watt [38].…”