Clustering of VASP actively drives processive, WH2 domain-mediated actin filament elongation

Breitsprecher, Dennis; Kiesewetter, Antje K; Linkner, Joern; Urbanke, Claus; Resch, Guenter P.; Small, J. Victor; Faix, Jan

doi:10.1038/emboj.2008.211

Cited by 215 publications

(323 citation statements)

References 52 publications

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“…Comparable to formins, Ena/VASP proteins associate with growing barbed ends and accelerate filament elongation twofold to sevenfold (6,9,19,20). However, contrary to formins, they deliver actin monomers directly to filament barbed ends by virtue of their GAB domains (6,9,19,20).…”

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confidence: 99%

“…Specific protein assemblies, composed of various actin-binding proteins, operate in these processes to nucleate and elongate new actin filaments, arrange them into complex 3D arrays, and subsequently recycle them to replenish the G-actin pool (5). The only protein families known to date that actively drive the elongation of actin filaments by incorporation of actin monomers at growing barbed ends are formins and Ena/VASP proteins, albeit their modes of action are considerably different (6)(7)(8)(9).…”

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Distinct VASP tetramers synergize in the processive elongation of individual actin filaments from clustered arrays

Brühmann

Ushakov

Winterhoff

et al. 2017

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

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Ena/VASP proteins act as actin polymerases that drive the processive elongation of filament barbed ends in membrane protrusions or at the surface of bacterial pathogens. Based on previous analyses of fast and slow elongating VASP proteins by in vitro total internal reflection fluorescence microscopy (TIRFM) and kinetic and thermodynamic measurements, we established a kinetic model of Ena/VASP-mediated actin filament elongation. At steady state, it entails that tetrameric VASP uses one of its arms to processively track growing filament barbed ends while three G-actin-binding sites (GABs) on other arms are available to recruit and deliver monomers to the filament tip, suggesting that VASP operates as a single tetramer in solution or when clustered on a surface, albeit processivity and resistance toward capping protein (CP) differ dramatically between both conditions. Here, we tested the model by variation of the oligomerization state and by increase of the number of GABs on individual polypeptide chains. In excellent agreement with model predictions, we show that in solution the rates of filament elongation directly correlate with the number of free GABs. Strikingly, however, irrespective of the oligomerization state or presence of additional GABs, filament elongation on a surface invariably proceeded with the same rate as with the VASP tetramer, demonstrating that adjacent VASP molecules synergize in the elongation of a single filament. Additionally, we reveal that actin ATP hydrolysis is not required for VASP-mediated filament assembly. Finally, we show evidence for the requirement of VASP to form tetramers and provide an amended model of processive VASPmediated actin assembly in clustered arrays.actin | Ena/VASP | TIRF | cluster | formin

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Distinct VASP tetramers synergize in the processive elongation of individual actin filaments from clustered arrays

Brühmann

Ushakov

Winterhoff

et al. 2017

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

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“…The extension of filopodia likely involves the action of formins and/or VASP (9)(10)(11)(12), two actin polymerization machines that operate at the growing barbed end as processive polymerases to create the linear actin filaments that fill filopodia. Although both proteins are fairly effective at physically shielding the barbed end from CP (10,13,14), it is likely that their robustness as filopodia generators in vivo would be increased by a reduction in CP levels. Given the recent work demonstrating that formins and the Arp2/3 complex compete for G-actin in vivo (15)(16)(17), the increase in filopodia number seen upon CP knockdown may also be due in part to an increase in the amount of monomer available for formin/VASP after the reduction in Arp2/ 3-dependent nucleation caused by CP knockdown.…”

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V-1 regulates capping protein activity in vivo

Jung

Alexander

et al. 2016

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

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Capping Protein (CP) plays a central role in the creation of the Arp2/ 3-generated branched actin networks comprising lamellipodia and pseudopodia by virtue of its ability to cap the actin filament barbed end, which promotes Arp2/3-dependent filament nucleation and optimal branching. The highly conserved protein V-1/Myotrophin binds CP tightly in vitro to render it incapable of binding the barbed end. Here we addressed the physiological significance of this CP antagonist in Dictyostelium, which expresses a V-1 homolog that we show is very similar biochemically to mouse V-1. Consistent with previous studies of CP knockdown, overexpression of V-1 in Dictyostelium reduced the size of pseudopodia and the cortical content of Arp2/3 and induced the formation of filopodia. Importantly, these effects scaled positively with the degree of V-1 overexpression and were not seen with a V-1 mutant that cannot bind CP. V-1 is present in molar excess over CP, suggesting that it suppresses CP activity in the cytoplasm at steady state. Consistently, cells devoid of V-1, like cells overexpressing CP described previously, exhibited a significant decrease in cellular F-actin content. Moreover, V-1-null cells exhibited pronounced defects in macropinocytosis and chemotactic aggregation that were rescued by V-1, but not by the V-1 mutant. Together, these observations demonstrate that V-1 exerts significant influence in vivo on major actin-based processes via its ability to sequester CP. Finally, we present evidence that V-1's ability to sequester CP is regulated by phosphorylation, suggesting that cells may manipulate the level of active CP to tune their "actin phenotype."T he addition of Capping Protein (CP) to seed-initiated actin polymerization assays results in the rapid cessation of polymerization because CP binds with very high affinity to the fastgrowing barbed end of the actin filament to block further monomer addition (1). Direct extrapolation of this simple, potent biochemical property would suggest that the cell's content of F-actin should rise and fall as its content of CP is artificially forced to fall and rise, respectively. Indeed, this finding was reported many years ago in Dictyostelium amoeba (2). This simple view of CP's role in regulating actin assembly in vivo falls short of the whole story, however. The additional complexity arises from the critical relationship between CP and the Arp2/3 complex, the major actin nucleating machine that generates the branched actin networks comprising lamellipodia and pseudopodia (3). At the heart of this relationship is the fact that CP increases the rate of Arp2/3-dependent filament nucleation and promotes optimal branching by rapidly capping filaments (4). As a result, CP promotes actin-related proteins 2 and 3 (Arp2/3)-driven actin assembly and motility (4, 5). This effect was evident from early solution experiments focused on defining the function of the Arp2/3 complex (6), verified by in vitro reconstitution of the Arp2/3-dependent motility of Listeria (5), and explained mecha...

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“…This insertional polymerization mechanism, which was first proposed in a sequence of theory papers that relied strongly on kinetic and thermodynamic considerations that are well understood from a chemical engineering perspective, was followed by much validating biochemical and biophysical experimentation from various groups, ultimately culminating with direct observation of insertional polymerization with formins, 13 VASP, 14 or engineered molecules containing the essential actin-and filament tip-binding domains of the VASP's family of NPFs. 15 These latter experiments directly validated the kinetic model for assembly proposed seven years earlier by Dickinson et al 11 It is now widely accepted that insertional polymerization by NPFs is how actin filaments are assembled within cell protrusions at the leading edge of a crawling tissue cell, if not every else as well in vivo.…”

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confidence: 99%

Chemical Engineering Principles in the Field of Cell Mechanics

Dickinson

Lele

2015

Ind. Eng. Chem. Res.

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The field of cell mechanics involves problems at the interface of mechanics, molecular transport, biochemical kinetics, thermodynamics, and cell biology. We argue that chemical engineering training is uniquely suited for fruitful research in the field by discussing three examples in the research area. Cell mechanics is already well-represented in many chemical engineering departments and promises to be a vibrant subarea in the profession.

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Clustering of VASP actively drives processive, WH2 domain-mediated actin filament elongation

Cited by 215 publications

References 52 publications

Distinct VASP tetramers synergize in the processive elongation of individual actin filaments from clustered arrays

Distinct VASP tetramers synergize in the processive elongation of individual actin filaments from clustered arrays

V-1 regulates capping protein activity in vivo

Chemical Engineering Principles in the Field of Cell Mechanics

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