Actin Cross-link Assembly and Disassembly Mechanics for α-Actinin and Fascin

Actin filaments constitute one of the main components of cell cytoskeleton. Assembled into bundles in filopodia or in stress fibres, they play a pivotal role in eukaryotes during cell morphogenesis, adhesion and motility. The bundle emergence has been extensively related to specific actin regulators 1-3 in vivo [4][5][6][7] . Such dynamic modulation was also highlighted by biochemical reconstitution of the actin-network assembly, in bulk solution or with biomimetic devices [8][9][10][11][12][13][14][15][16][17][18] . However, the question of how geometrical boundaries, such as those encountered in cells, affect the dynamic formation of highly ordered actin structures remains poorly studied 14,19 . Here we demonstrate that the nucleation geometry in itself can be the principal determinant of actin-network architecture. We developed a micropatterning method that enables the spatial control of actin nucleation sites for in vitro assays. Shape, orientation and distance between nucleation regions control filament orientation and length, filament-filament interactions and filopodium-like bundle formation. Modelling of filament growth and interactions demonstrates that basic mechanical and probabilistic laws govern actin assembly in higher-order structures.In cells, actin nucleation occurs at various locations at the plasma membrane, and bundles of parallel actin filaments are initiated at focal adhesion sites 1 or result from the rearrangement of the dynamic branched actin network of the lamellipodium 6 . Here, we modulate the positioning of nucleation sites at scales corresponding to cellular dimensions. As a first step and to precisely regulate the position of actin nucleation sites in vitro, we used a recently developed ultraviolet-based micropatterning approach 20 to create a template for the localization of the nucleation promoting factor pWA (Fig. 1a). pWA comprises the C-terminal domains from the WASP/Scar proteins, a ubiquitous family of proteins that initiate actin polymerization on a pre-existing actin filament in the presence of the Arp2/3 complex and an actin monomer [21][22][23] . A small volume of solution made of a minimal set of purified proteins ensuring actin polymerization 8,10 was placed between the pWA-coated micropatterned slide and a glass support. Functionalized micropatterns specifically initiate actin filament nucleation on their surface and promote two-dimensional growth of actin filaments (Fig. 1b). Real-time visualization of actin-filament nucleation and growth highlighted the autocatalytic process of network formation (see Supplementary Fig. S1). These networks consist of filaments growing from the pWA-coated regions, with their fast-growing, barbed end oriented outwards ( Fig. 1c and Supplementary Fig. S2 and Videos S1-S3). In agreement with actin-filament growth on glass rods 15 , as the nucleation waves propagate, dense and interconnected

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“…S6 and Video S5). This confirms that bundle emergence is tightly controlled by mechanical properties of the actin filament 24 .…”

Section: Nature Materialssupporting

confidence: 75%

Nucleation geometry governs ordered actin networks structures

et al. 2010

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“…Intersectin-1 is a cytoplasmic membrane-associated protein that coordinates endocytic membrane traffic with the actin assembly machinery, and regulates the formation of clathrin-coated vesicles (32). Fascin is an actin cross- linking protein involved in the assembly of actin filaments into bundles (33). Finally, calreticulin is a multifunctional protein that serves as a cell-surface receptor for the C1q component of complement and mediates phagocytosis upon binding to major plasma proteins (34,35).…”

Section: Expression Of Cell Remodeling Genes Is Ompu-dependent In Lgp32mentioning

confidence: 99%

Use of OmpU porins for attachment and invasion of Crassostrea gigas immune cells by the oyster pathogen Vibrio splendidus

Duperthuy

Schmitt

Garzón

et al. 2011

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

155

168

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OmpU porins are increasingly recognized as key determinants of pathogenic host Vibrio interactions. Although mechanisms remain incompletely understood, various species, including the human pathogen Vibrio cholera, require OmpU for host colonization and virulence. We have shown previously that OmpU is essential for virulence in the oyster pathogen Vibrio splendidus LGP32. Here, we showed that V. splendidus LGP32 invades the oyster immune cells, the hemocytes, through subversion of host-cell actin cytoskeleton. In this process, OmpU serves as an adhesin/invasin required for β-integrin recognition and host cell invasion. Furthermore, the major protein of oyster plasma, the extracellular superoxide dismutase CgEcSOD, is used as an opsonin mediating the OmpU-promoted phagocytosis through its RGD sequence. Finally, the endocytosed bacteria were found to survive intracellularly, evading the host defense by preventing acidic vacuole formation and limiting reactive oxygen species production. We conclude that (i) V. splendidus is a facultative intracellular pathogen that manipulates host defense mechanisms to enter and survive in host immune cells, and (ii) that OmpU is a major determinant of host cell invasion in Vibrio species, used by V. splendidus LGP32 to attach and invade oyster hemocytes through opsonisation by the oyster plasma Cg-EcSOD.T he oyster pathogen, Vibrio splendidus strain LGP32 was isolated from massive mortality events in the production of Crassostrea gigas oysters (1). However, up to now, little has been known about the route of infection and pathogenic processes of LGP32 (2, 3). A metalloprotease has been associated with toxicity (4, 5) and the outer membrane protein (OMP) OmpU was shown to be a major determinant of LGP32 virulence (6).As bacterial surface components, OMPs are both used by hosts for pathogen recognition and by pathogens for interaction with and invasion of host cells, serving as adhesion proteins (adhesins)

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“…Branching rates were measured as total branches that appeared over a period of 10 min and normalized to total amount of polymer. Measuring Off-rates by TIRF-The method was modified from Courson and Rock (35). Bundles were prepared by incubating 10 M actin filaments with 3 M TMR-Fimbrin at 4°C for 2 h in assay buffer (AB: 25 mM imidazole, pH 7.5, 25 mM KCl, 1 mM EGTA, 4 mM MgCl 2 , and 10 mM DTT).…”

Section: Methodsmentioning

confidence: 99%

“…Native fission yeast Arp2/3 com-plex was purified from a protease-deficient strain (TP150) by binding to GST-N-WASP-VCA as previously described (3). Human fascin was purified as previously described (35). Recombinant fission yeast cofilin Adf1 was expressed from pMW-SpCofilin in Escherichia coli BL21-Codon Plus(DE3)-RP and purified by ammonium sulfate precipitation, S-200 gel filtration, and ion-exchange chromatography as described (12,31).…”

Section: Methodsmentioning

confidence: 99%

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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

Self Cite

115

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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Actin Cross-link Assembly and Disassembly Mechanics for α-Actinin and Fascin

Cited by 106 publications

References 53 publications

Nucleation geometry governs ordered actin networks structures

Nucleation geometry governs ordered actin networks structures

Use of OmpU porins for attachment and invasion of Crassostrea gigas immune cells by the oyster pathogen Vibrio splendidus

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

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