Actin Filament Bundling by Fimbrin Is Important for Endocytosis, Cytokinesis, and Polarization in Fission Yeast

Abstract

Through the coordinated action of diverse actin-binding proteins, cells simultaneously assemble actin filaments with distinct architectures and dynamics to drive different processes. Actin filament cross-linking proteins organize filaments into higher order networks, although the requirement of cross-linking activity in cells has largely been assumed rather than directly tested. Fission yeast Schizosaccharomyces pombe assembles actin into three discrete structures: endocytic actin patches, polarizing actin cables, and the cytokinetic contractile ring. The fission yeast filament cross-linker fimbrin Fim1 primarily localizes to Arp2/3 complex-nucleated branched filaments of the actin patch and by a lesser amount to bundles of linear antiparallel filaments in the contractile ring. It is unclear whether Fim1 associates with bundles of parallel filaments in actin cables. We previously discovered that a principal role of Fim1 is to control localization of tropomyosin Cdc8, thereby facilitating cofilinmediated filament turnover. Therefore, we hypothesized that the bundling ability of Fim1 is dispensable for actin patches but is important for the contractile ring and possibly actin cables. By directly visualizing actin filament assembly using total internal reflection fluorescence microscopy, we determined that Fim1 bundles filaments in both parallel and antiparallel orientations and efficiently bundles Arp2/3 complex-branched filaments in the absence but not the presence of actin capping protein. Examination of cells exclusively expressing a truncated version of Fim1 that can bind but not bundle actin filaments revealed that bundling activity of Fim1 is in fact important for all three actin structures. Therefore, fimbrin Fim1 has diverse roles as both a filament "gatekeeper" and as a filament cross-linker.

Live cell imaging approaches reveal actin cytoskeleton-induced self-association of the actin-bundling protein WLIM1

“…By contrast, control experiments with the human actin-bundling protein fascin yielded almost exclusively unipolar bundles (90%, n520) (Fig. 4A, right), a result consistent with the high selectivity previously reported for fascin (Breitsprecher et al, 2011;Courson and Rock, 2010;Ishikawa et al, 2003;Skau et al, 2011).…”

“…Here, we provide evidence that NtWLIM1 promotes local interaction between adjacent actin filaments and their subsequent zippering into tight bundles. A similar two-step bundling process was recently described for the Arabidopsis villin proteins VLN1 and VLN3 , fission yeast fimbrin Fim1 (Skau et al, 2011), mouse TRIOBP (Kitajiri et al, 2010) and human fascin (Breitsprecher et al, 2011), suggesting that it defines a basic mechanism 'employed' by structurally unrelated actinbundling proteins. In addition, in vitro reconstituted assays and live cell analyses conducted with NtWLIM1 and AtWLIM1, respectively, show that both proteins can effectively bind to individual unbundled actin filaments, suggesting that WLIM1 binding chronologically precedes formation of actin bundles.…”

Actin Dynamics, Architecture, and Mechanics in Cell Motility

“…Antiparallel actin structures with myosin-induced contraction are necessary for cytokinesis and for stress fiber function during the establishment of cell/cell and cell-matrix adhesions (45,126,170,281,289,311,315). As for parallel actin bundles, antiparallel organizations are stabilized by crosslinking proteins that favor this specific configuration (FIGURE 3D and Refs.…”

“…An identifying property of each crosslinking protein is the distance by which it bridges two actin filaments (FIGURE 3, B AND C); crosslink distances range from 10 nm for fimbrin to 160 nm for filamin (153,282,297). Crosslinkers that impose small crosslinking distances, such as fimbrin or fascin, tightly pack actin filaments into bundles with actin filaments oriented in a parallel, antiparallel, or mixed polarity fashion depending on the crosslinker (178,289). Larger crosslinkers, such as filamin or ␣-actinin, are present in either bundles or networks depending on their concentrations (69,148,199,277,329,330).…”

The plant LIM proteins: unlocking the hidden attractions

“…NtWLIM1 interacts locally with adjoining actin filaments and triggered them to zipper into tight bundles. Similar mechanism was also attributed by other proteins such as Arabidopsis VLN1, VLN3 (Khurana et al 2010) and fission yeast Fim1 (Skau et al 2011). The NtWLIM1 also exhibits possible dimerization and oligomerization in the cytosol, but in the absence of F-actin these conformations are not stable.…”