Characterization of several Arabidopsis villin isovariants

Parul Khurana, Purdue University

Abstract

Actin filaments in plants form structural networks comprised of single filaments and prominent higher-order structures like cables and bundles. The latter have been implicated in several important functions including cytoplasmic streaming, organelle motility, cell division and cell morphogenesis. The molecular mechanisms that underpin bundle formation and maintenance, however, are largely unknown. At least four families of actin-binding proteins are involved in bundling; these are the fimbrins, formins, LIMs and villins. The villins belong to the villin/gelsolin/fragmin superfamily and are encoded by five genes in Arabidopsis thaliana. Multiple different villin protein isovariants are coexpressed spatially and temporally within the plant. It is not clear whether these isovariants function together and act redundantly or whether they have unique activities. VILLIN1 (VLN1) is a simple actin filament bundling protein that is not regulated by Ca²⁺, unlike other members of the villin/gelsolin/fragmin superfamily. Based on phylogenetic analyses and conservation of Ca²⁺-binding sites, we hypothesize that VLN3 is a Ca²⁺-regulated villin that has the ability to sever actin filaments and is involved in bundle formation and turnover. Detailed biochemical analyses were performed to characterize VLN3, as described in Chapter 2. Time-lapse imaging and total internal reflection fluorescence microscopy allowed the direct visualization of actin bundle formation by both VLN isovariants in vitro, which was similar to the “catch and zipper” mechanism observed in vivo. The fragmentation of actin in the presence of VLN3 was observed directly as well at physiological Ca ²⁺ concentrations and quantified to confirm that VLN3 is a bona fide severing protein. Moreover, both actin filaments and bundles are severed by VLN3 in the presence of micromolar Ca²⁺, irrespective of the presence of VLN1. The results thus demonstrate that the two Arabidopsis villin isovariants have overlapping and distinct activities important for actin bundle generation, turnover and maintenance. This is an important step in understanding how co-existing isovariants might cooperate within a cell to bring about actin cytoskeletal reorganization. Further experimental evidence for their significance in plant growth and development could be obtained from in vivo loss-of-function and gain-of-function studies. Chapter 3 focuses on the generation of plant material and resources for the reverse-genetic analyses of the Arabidopsis villin gene family. Single homozygous mutants that are knock-outs for four villin isovariants were obtained and a double mutant for vln1;vln4 was recovered. We demonstrated that an appropriate sized protein band is missing in each of the vln1 and vln4 single mutants, and two bands are missing in the homozygous double mutant plants. Villins have been previously demonstrated to localize on actin cables in pollen tubes and root hairs. Mutant studies in flies and mammals, along with loss-of-function by microinjection of antibodies in plants suggest the significance of villin in actin organization and dynamics. With the recent surge in live-cell imaging of actin cytoskeleton dynamics, in vivo rearrangements of actin have been proposed to be regulated by various actin-binding proteins, including the villins. Collectively, biochemical characterization of the different isovariants, reverse genetics and the analysis of the actin cytoskeleton in mutants, will help dissect a role for villins in actin dynamics.^

Degree

Ph.D.

Advisors

Christopher J. Staiger, Purdue University.

Subject Area

Biology, Botany|Biology, Cell

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