Cloning and Functional Characterization of a Formin-Like Protein (AtFH8) from Arabidopsis

Abstract

The actin cytoskeleton is required for many cellular processes in plant cells. The nucleation process is the rate-limiting step for actin assembly. Formins belong to a new class of conserved actin nucleator, which includes at least 2 formin homology domains, FH1 and FH2, which direct the assembly of unbranched actin filaments. The function of plant formins is quite poorly understood. Here, we provide the first biochemical study of the function of conserved domains of a formin-like protein (AtFH8) from Arabidopsis (Arabidopsis thaliana). The purified recombinant AtFH8(FH1FH2) domain has the ability to nucleate actin filaments in vitro at the barbed end and caps the barbed end of actin filaments, decreasing the rate of subunit addition and dissociation. In addition, purified AtFH8(FH1FH2) binds actin filaments and severs them into short fragments. The proline-rich domain (FH1) of the AtFH8 binds directly to profilin and is necessary for nucleation when actin monomers are profilin bound. However, profilin inhibits the nucleation mediated by AtFH8(FH1FH2) to some extent, but increases the rate of actin filament elongation in the presence of AtFH8(FH1FH2). Moreover, overexpression of the full-length AtFH8 in Arabidopsis causes a prominent change in root hair cell development and its actin organization, indicating the involvement of AtFH8 in polarized cell growth through the actin cytoskeleton.The actin cytoskeleton of plant cells is temporally and spatially regulated in response to external stimuli and plays an important role in many physiological processes, like cytoplasmic streaming, division plane coordination, and tip growth (Volkmann and Baluska, 1999;Staiger et al., 2000). A host of accessory proteins are required to regulate the equilibrium between monomeric G-actin and F-actin, to bundle and crosslink actin filaments to one another, or to promote nucleation of new actin filaments (Ayscough, 1998). The formation of actin trimers, the smallest nuclei for polymerization, is the rate-limiting step during spontaneous filament assembly. Thus, cellular activities that either block or stimulate trimer formation have a large influence on actin polymerization (Cooper et al., 1983;Winter et al., 1997). Relatively recent studies have provided important new insights into how the assembly and disassembly of the actin cytoskeleton is regulated. The Arp2/3 complex has been demonstrated as a nucleation factor in the stimulation of branched filament formation (for review, see Pollard and Beltzner, 2002). Recently, a conserved family of formin homology proteins has emerged as a new class of actin nucleator that directs assembly of straight filaments independently of the Arp2/3 complex (for review, see Zigmond, 2004).Formins are large multidomain proteins that have been found in all eukaryotes examined and are required for multiple actin-related processes, such as cytokinesis and maintenance of cell polarity, in many organisms (Wasserman, 1998;Sawin, 2002). The mouse limb deformity gene encodes the first formin identified. Subse...

A Class I ADP-Ribosylation Factor GTPase-Activating Protein Is Critical for Maintaining Directional Root Hair Growth in Arabidopsis

“…Actin-disrupting drugs inhibited or delocalized root hair growth (Bibikova et al, 1999;Ketelaar et al, 2003), consistent with reports showing that knockouts to the vegetative ACT2 gene in Arabidopsis have distorted root hair morphology (Ringli et al, 2002). Moreover, altering the expression of genes encoding proteins that affect actin turnover, such as profilin, actin-depolymering factor, formin, actininteracting protein, and cyclase-associated protein, disrupted normal root hair development (Ramachandran et al, 2000;Dong et al, 2001;Yi et al, 2005;Deeks et al, 2007;Ketelaar et al, 2007).…”

Deep Sequencing of the Medicago truncatula Root Transcriptome Reveals a Massive and Early Interaction between Nodulation Factor and Ethylene Signals

“…Root hair deformation results from cytoskeletal reorganization and cell wall deposition that redirects polar tip growth. Two genes up-regulated in the 3-to 6-h interval are homologs of Arabidopsis Formin-Like8 (AtFH8) and Abscisic Acid Insensitive-Like (ABIL), genes directly linked to actin nucleation and severing (Yi et al, 2005). ABIL functions in the SCAR/WAVE complex that regulates the actin cytoskeleton in tip-growing cells and during infection thread progression in legumes (Yokota et al, 2009;Miyahara et al, 2010;Hossain et al, 2012) and was recently observed to be up-regulated in root hairs undergoing infection (Breakspear et al, 2014).…”

Actin bundling in plants

“…The over-expression of Arabidopsis formin AtFH8 was observed to cause an increase in the overall amount of filamentous actin within root hairs, as well as the precocious extension of actin bundles to the extreme tip [Yi et al, 2005]. In addition, the morphological effects on root hairs resemble those induced on pollen tubes by the over-expression of AtFH1 [Cheung and Wu, 2004], including swelling, defects in polarization and growth arrest.…”

“…fimbrins and LIM proteins. Indeed, nucleating, capping and/or severing activities have been attributed to members of the villin and formin families [Deeks et al, 2005;Ingouff et al, 2005;Michelot et al, 2005;Yi et al, 2005] but not yet to a plant fimbrin or LIM protein. Also, additional rigorous biochemical work is needed to assert that fimbrin and LIM proteins are specifically dedicated to the bundling of AFs.…”