Cofilin cooperates with fascin to disassemble filopodial actin filaments

Abstract

SummaryCells use a large repertoire of proteins to remodel the actin cytoskeleton. Depending on the proteins involved, F-actin is organized in specialized protrusions such as lamellipodia or filopodia, which serve diverse functions in cell migration and sensing. Although factors responsible for directed filament assembly in filopodia have been extensively characterized, the mechanisms of filament disassembly in these structures are mostly unknown. We investigated how the actin-depolymerizing factor cofilin-1 affects the dynamics of fascincrosslinked actin filaments in vitro and in live cells. By multicolor total internal reflection fluorescence microscopy and fluorimetric assays, we found that cofilin-mediated severing is enhanced in fascin-crosslinked bundles compared with isolated filaments, and that fascin and cofilin act synergistically in filament severing. Immunolabeling experiments demonstrated for the first time that besides its known localization in lamellipodia and membrane ruffles, endogenous cofilin can also accumulate in the tips and shafts of filopodia. Live-cell imaging of fluorescently tagged proteins revealed that cofilin is specifically targeted to filopodia upon stalling of protrusion and during their retraction. Subsequent electron tomography established filopodial actin filament and/or bundle fragmentation to precisely correlate with cofilin accumulation. These results identify a new mechanism of filopodium disassembly involving both fascin and cofilin.

Fascin 1 is transiently expressed in mouse melanoblasts during development and promotes migration and proliferation

“…We were surprised that pseudopods generated by fascin 1 nulls had a slightly increased lifetime, but we also found that expressing fascin 1 in M4 melanocytes in culture decreased lamellipodial persistence, which also indicates that fascin 1 promotes dynamics. This agrees with studies showing increased lamellipodial dynamics of fascin 1-expressing Drosophila haemocytes (Zanet et al, 2009) and is interesting in light of a recent study showing that actin bundles containing fascin 1 are more susceptible to turnover by cofilin (Breitsprecher et al, 2011). Furthermore, fascin 1 can interact with LIMK, a cofilin regulatory kinase (Jayo et al, 2012) and thus fascin…”

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.…”

“…For better visualization of skewness, representative pictures of one ratio are presented with a custom LUT Fire. To quantify actin bundle thickness, we supposed that the Alexa Fluor 488 fluorescence signal was proportional to the amount of actin present within analyzed bundles (Breitsprecher et al, 2011;Smertenko et al, 2010). The absolute signal of each bundle was measured and normalized to the fluorescence intensity of a single actin filament to obtain the number of filaments per bundle.…”

A new role for cofilin in retinal neovascularization

“…Pak1 mediates its effects on cytoskeleton remodeling through phosphorylation and inactivation of cofilin (Delorme et al, 2007). Furthermore, many studies have also demonstrated that, although cofilin inhibition stabilizes F-actin stress fibers, its activation disassembles these F-actin filaments (Breitsprecher et al, 2011;Sumi et al, 1999). Because the Pak1-cofilin axis plays an important role in cytoskeleton remodeling, which is required for both cell migration and proliferation (Ho et al, 2013;Zipfel et al, 2006), in this study we have tested the role of Pak1 and its downstream effector cofilin in VEGFA-induced endothelial cell cytoskeleton remodeling and angiogenic signaling.…”