Polarized actin bundles formed by human fascin‐1: their sliding and disassembly on myosin II and myosin V <i>in vitro</i>

Ishikawa, Ryoki; Sakamoto, Takeshi; Ando, Toshio; Higashi-Fujime, Sugie; Kohama, Kazuhiro

doi:10.1046/j.1471-4159.2003.02058.x

Cited by 104 publications

(102 citation statements)

References 54 publications

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“…The bundles showed transverse periodicity of 36.9 nm, roughly corresponding to the crossover distance of the actin filament (35.7 nm) (16). Our observations are generally consistent with a previous study of wild type fascin-actin bundles (17), but contrary to these authors, we found that the transverse pattern occurred at an angle of 63.0 Ϯ 6.23° (Fig. 2B and Table 2) and not perpendicular to the bundle axis as they had reported.…”

Section: Resultssupporting

confidence: 91%

“…Most likely, fascin binds at the interface between actin subunits in the filament, a location where most actin-binding proteins bind (25,26), bridging the narrow area of neighboring filaments. Another observation made here is that the transverse periodicity in wild type fascin-actin bundles occurs at an angle of ϳ63.0°, which is in agreement with a study of reconstituted bundles formed by mouse fascin (18) but disagrees with another study that found that the repeat is perpendicular to the bundle axis (17). Our results thus suggest that neighboring filaments in the bundle are either uniformly rotated or staggered by ϳ4.1 nm, which could help accommodate fascin molecules in the tight bundle.…”

Section: Discussionsupporting

confidence: 62%

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Mechanism of Actin Filament Bundling by Fascin

Jansen

Collins

Yang

et al. 2011

Journal of Biological Chemistry

163

173

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Fascin is the main actin filament bundling protein in filopodia. Because of the important role filopodia play in cell migration, fascin is emerging as a major target for cancer drug discovery. However, an understanding of the mechanism of bundle formation by fascin is critically lacking. Fascin consists of four ␤-trefoil domains. Here, we show that fascin contains two major actin-binding sites, coinciding with regions of high sequence conservation in ␤-trefoil domains 1 and 3. The site in ␤-trefoil-1 is located near the binding site of the fascin inhibitor macroketone and comprises residue Ser-39, whose phosphorylation by protein kinase C down-regulates actin bundling and formation of filopodia. The site in ␤-trefoil-3 is related by pseudo-2-fold symmetry to that in ␤-trefoil-1. The two sites are ϳ5 nm apart, resulting in a distance between actin filaments in the bundle of ϳ8.1 nm. Residue mutations in both sites disrupt bundle formation in vitro as assessed by co-sedimentation with actin and electron microscopy and severely impair formation of filopodia in cells as determined by rescue experiments in fascin-depleted cells. Mutations of other areas of the fascin surface also affect actin bundling and formation of filopodia albeit to a lesser extent, suggesting that, in addition to the two major actin-binding sites, fascin makes secondary contacts with other filaments in the bundle. In a high resolution crystal structure of fascin, molecules of glycerol and polyethylene glycol are bound in pockets located within the two major actin-binding sites. These molecules could guide the rational design of new anticancer fascin inhibitors.

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Section: Resultssupporting

confidence: 91%

Section: Discussionsupporting

confidence: 62%

Mechanism of Actin Filament Bundling by Fascin

Jansen

Collins

Yang

et al. 2011

Journal of Biological Chemistry

163

173

View full text Add to dashboard Cite

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“…It is thus reasonable to assume that no individual filament coexists with the bundles. Authors have observed that for a molar ratio [fascin]/[actin] > 0.25, which is the case here, the number of filaments in a bundle saturates to 20 (22,23). Because about 45 bundles are visible in the image of Fig.…”

Section: Significancesupporting

confidence: 63%

Power transduction of actin filaments ratcheting in vitro against a load

Démoulin

Carlier

Bibette

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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The actin cytoskeleton has the unique capability of producing pushing forces at the leading edge of motile cells without the implication of molecular motors. This phenomenon has been extensively studied theoretically, and molecular models, including the widely known Brownian ratchet, have been proposed. However, supporting experimental work is lacking, due in part to hardly accessible molecular length scales. We designed an experiment to directly probe the mechanism of force generation in a setup where a population of actin filaments grows against a load applied by magnetic microparticles. The filaments, arranged in stiff bundles by fascin, are constrained to point toward the applied load. In this protrusion-like geometry, we are able to directly measure the velocity of filament elongation and its dependence on force. Using numerical simulations, we provide evidence that our experimental data are consistent with a Brownian ratchet-based model. We further demonstrate the existence of a force regime far below stalling where the mechanical power transduced by the ratcheting filaments to the load is maximal. The actin machinery in migrating cells may tune the number of filaments at the leading edge to work in this force regime.cell motility | force generation | lamellipodium | filopodium

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“…Fascin also preferentially makes parallel bundles in vitro (Fig. 1F) (35,41,43) and localizes to cellular structures composed of parallel bundles including filopodia (60,61). Fission yeast Fim1 localizes to endocytic patches composed of branched filaments with barbed ends thought to be oriented toward the membrane as well as the contractile ring composed of antiparallel bundles of filaments and likely actin cables composed of parallel filaments (Figs.…”

Section: Discussionmentioning

confidence: 99%

“…Conversely, fascin creates bundles composed exclusively of parallel filaments (Fig. 1F, supplemental Movie 6) (35,43). Because Fim1 binds to structures composed of both parallel and antiparallel filaments in vivo, this may be an essential feature of Fim1.…”

Section: -E)mentioning

confidence: 99%

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

Skau

Courson

Bestul

et al. 2011

Journal of Biological Chemistry

118

117

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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.

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Polarized actin bundles formed by human fascin‐1: their sliding and disassembly on myosin II and myosin V in vitro

Cited by 104 publications

References 54 publications

Mechanism of Actin Filament Bundling by Fascin

Mechanism of Actin Filament Bundling by Fascin

Power transduction of actin filaments ratcheting in vitro against a load

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

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