Actin Bundling Via LIM Domains

Article Addendum Actin bundling via LIM domains

Clément Thomas,* Monika Dieterle, Sabrina Gatti, Céline Hoffmann, Flora Moreau, Jessica Papuga and André Steinmetz

Centre de Recherche Public‑Santé; Val Fleuri 84; L‑1526; Luxembourg Key words: Actin‑binding proteins, actin‑bundling, cysteine‑rich proteins, cytoskeleton, LIM domain.

The LIM domain is defined as a protein‑protein interaction module involved in the regulation of diverse cellular processes including gene expression and cytoskeleton organization. We distribute. have recently shown that the tobacco WLIM1, a two LIM domain‑containing protein, is able to bind to, stabilize and bundle actin filaments, suggesting that it participates to the regulation of actin cytoskeleton structure and dynamics. In the December issue of the Journal of Biological Chemistry we report a domain analysis not that specifically ascribes the actin‑related activities of WLIM1 to its two LIM domains. Results suggest that LIM domains func‑ Figure 1. Domain maps for wild‑type WLIM1 (A) and GFP‑fused chimeric tion synergistically in the full‑length protein to achieve optimal 3xWLIM1 (B). A. WLIM1 basically comprises a short N‑terminal domain (Nt), two LIM domains (LIM1Do and LIM2), an interLIM spacer (IL) and a C‑terminal activities. Here we briefly summarize relevant data regarding the domain (Ct). B. 3xWLIM1 consists of three tandem WLIM1 copies. This chi‑ actin‑related properties/functions of two LIM domain‑containing meric protein has been fused in C‑terminus to GFP and transiently expressed proteins in plants and animals. In addition, we provide further in tobacco BY2 cells. evidence of cooperative effects between LIM domains by transiently expressing a chimeric multicopy WLIM1 protein in BY2 cells. two proteins were shown to arrange AF into cables both in vitro and in vivo and thus join the list of actin bundlers. The LIM domain is a ≈55 amino acid peptide domain that was To identify the peptide domains of WLIM1 responsible for its first identified in 1990 as a common cystein‑rich sequence found in actin‑related properties/activities, we generated domain‑deleted and the three homeodomain proteins LIN‑11, Isl1 and MEC‑3. It has single domain variants and submitted them to a series of in vivo and since been found in a wide variety of eukaryotic proteins of diverse in vitro assays.4 Localization experiments established that both LIM functions. Animals possess several families of LIM proteins, with domains are required to efficiently target the actin cytoskeleton in members containing 1‑5 LIM domains occasionally linked to other tobacco BY2 cells. High‑speed (200,000 g) cosedimentation data catalytic or protein‑binding domains such as homeodomain, kinase confirmed that the actin‑binding activity of WLIM1 relies on its and SH3 domains. In contrast, plants only possessBioscience. two distinct sets LIM domains. Indeed, the deletion of either the first or the second of LIM proteins. One is plant‑specific and has not been functionally LIM domain respectively resulted in a 5‑fold and 10‑fold decrease characterized yet. The other one comprises proteins that exhibit the of the protein affinity for AF. Importantly, each single LIM domain same overall structure as the animal cystein rich proteins (CRPs), i.e., was found able to interact with AF in an autonomous manner, two very similar LIM domains separated by a ≈50 amino acid‑long although with a reduced affinity compared to the wild‑type WLIM1. interLIM domain and a relatively short and variable C‑terminal Low‑speed (12,500 g) cosedimentation data and electron microscopy domain (Fig. 1A). The mouse CRP2 protein was the first CRP observations revealed that the actin bundling activity of WLIM1 is reported to interact directly with actin filaments (AF) and to stabilize also triggered by its LIM domains. Surprisingly each single LIM the latter.1 IdenticalLandes observations were subsequently described for the domain was able to bundle AF in an autonomous manner, suggesting chicken CRP1 and tobacco WLIM1 proteins.2,3 In addition, these that WLIM1 has two discrete actin‑bundling sites. However, the bundles induced by the variants containing only one LIM domain, *Correspondence to: Clément Thomas; Centre de Recherche Public‑Santé; Val Fleuri i.e., LIM domain‑deleted mutants and single LIM domains, differed 84; L‑1526 Luxembourg; Email: clement.thomas@crp‑sante.lu from those induced by the full‑length WLIM1. They appeared more wavy and loosely packed and formed only at relatively high Submitted: 11/14/07; Accepted: 11/19/07 protein:actin ratios. Together these data suggest that LIM domains Previously published online as a Plant Signaling & Behavior E-publication: are autonomous actin‑binding and ‑bundling modules that function ©2008www.landesbioscience.com/journals/psb/article/5310 in synergy in wild‑type WLIM1 to achieve optimal activities. Addendum to: Thomas C, Moreau F, Dieterle M, Hoffmann C, Gatti S, Hofmann C, To further assess the mechanism of cooperation between the LIM Van Troys M, Ampe C, Steinmetz A. The LIM domains of WLIM1 define a new class domains of plant CRP‑related proteins, we generated a chimeric of actin bundling modules. J Biol Chem 2007; 282:33599–608; PMID: 17827159; protein composed of three WLIM1 copies in tandem (3 x WLIM1, doi: 10.1074/jbc.M703691200.

320 Plant Signaling & Behavior 2008; Vol. 3 Issue 5 Plant actin cytoskeleton remodeling

Figure 2. Typical actin cytoskeleton patterns in tobacco BY2 cells that have been transiently transformed, using a particle gun, with GFP‑fABD2 (A), WLIM1‑GFP (B), and 3xWLIM1‑GFP (C and D). For each construct, more than 60 cells were analyzed by confocal microscopy. In the case of 3xWLIM1‑GFP, two prevalent patterns have been observed (C and D). Bars = 20 mm.

(Fig. 2C) that, at times (≈30 %), form atypical long looped struc‑ tures (Fig. 2D). The appearance of such structures may result from the increase of cable stability and thickness induced by the 3xWLIM1‑GFP protein, as these parameters are likely to determine, at least partially, the maximal length of actin bundles. Together the present observations support earlier data showing that LIM domains work in concert in LIM proteins to regulate actin bundling in plant cells. Strikingly, vertebrate and plant CRPs invariably contain two LIM domains. The lack, in these organisms, of CRP‑related proteins combining more than two LIM domainsdistribute. may be explained by the fact that very thick cables, such as those induced by the artificial 3xWLIM1, may be too stable structures incompatible with the neces‑ sary high degree of actin cytoskeleton plasticity. As an exception, a muscle CRP‑related protein with five LIM domains (Mlp84B) has been identified in Drosophila.not6 However, rather than decorating actin filaments in an homogenous manner, this protein has been found to concentrate in a specialized region of the Z‑discs where it stabilizes, in concertDo with D‑titin, muscle sarcomeres.7 The relatively well conserved spacer length (≈50 amino acids) that separates the two LIM domains in vertebrate CRPs and related plant LIM proteins remains an intriguing feature the importance of which in actin cable organization remains to be established. Using electron microscopy we are currently evaluating the effects of the modifica‑ tion of the interLIM domain length on the structural properties of actin cables. References 1. Grubinger M, Gimona M. CRP2 is an autonomous actin‑binding protein. FEBS Lett 2004; 55:88‑92. 2. Tran TC, Singleton C, Fraley TS, Greenwood JA. Cysteine‑rich protein 1 (CRP1) regulates actin filament bundling. BMC Cell Biol 2005; 6:45. 3. Thomas C, Hoffmann C, Dieterle M, Van Troys M, Ampe C, Steinmetz A. Tobacco WLIM1 is a novel F‑actin binding protein involved in actin cytoskeleton remodeling. Plant Bioscience.Cell 2006; 18:2194‑206. 4. Thomas C, Moreau F, Dieterle M, Hoffmann C, Gatti S, Hofmann C, Van Troys M, Ampe C, Steinmetz A. The LIM Domains of WLIM1 define a new class of actin bundling mod‑ ules. J Biol Chem 2007; 282:33599‑608. 5. Sheahan MB, Staiger CJ, Rose RJ, McCurdy DW. A green fluorescent protein fusion to Fig. 1B), and transiently expressed it as a GFP‑fusion in tobacco actin‑binding domain 2 of Arabidopsis fimbrin highlights new features of a dynamic actin cytoskeleton in live plant cells. Plant Physiol 2004; 136:3968‑78. BY2 cells. We anticipated that such a six LIM domain‑containing 6. Stronach BE, Siegrist SE, Beckerle MC. Two muscle‑specific LIM proteins in Drosophila. J protein displays an even higher actin‑bundling activity. (Fig. 2A) Cell Biol 1996; 134:1179‑95. shows the typical actin cytoskeleton pattern in an expanding BY2 7. Clark KA, Bland JM, Beckerle MC. The Drosophila muscle LIM protein, Mlp84B, cooper‑ ates with D‑titin to maintain muscle structural integrity. J Cell Sci 2007; 120:2066‑77. cell as visualized using the actin marker GFP‑fABD2.5 As previously reported by SheahanLandes et al.,5 GFP‑fABD2 decorated dense, trans‑ versely oriented, cortical networks as well as transvacuolar strands connecting the subcortical‑perinuclear region to the cortex. Ectopic expression of WLIM1‑GFP (BY2 cells normally do not express the WLIM1 gene) induced moderate but perceptible modifications of the actin cytoskeleton structure (Fig. 2B). Most AF are arranged in bundles thicker than those observed in GFP‑fABD2 expressing ©2008cells and fine AF arrays are less frequently observed. As expected, this phenotype was significantly enhanced in cells transformed with the 3xWLIM1‑GFP protein (Fig. 2C). Indeed, cells were almost devoided of fine AF arrays and exhibited very thick actin cables