Multiple actin binding domains of Ena/VASP proteins determine actin network stiffening

Abstract

Vasodilator-stimulated phosphoprotein (Ena/VASP) is an actin binding protein, important for actin dynamics in motile cells and developing organisms. Though VASP's main activity is the promotion of barbed end growth, it has an F-actin binding site and can form tetramers, and so could additionally play a role in actin crosslinking and bundling in the cell. To test this activity, we performed rheology of reconstituted actin networks in the presence of wild-type VASP or mutants lacking the ability to tetramerize or to bind G-actin and/or F-actin. We show that increasing amounts of wild-type VASP increase network stiffness up to a certain point, beyond which stiffness actually decreases with increasing VASP concentration. The maximum stiffness is 10-fold higher than for pure actin networks. Confocal microscopy shows that VASP forms clustered actin filament bundles, explaining the reduction in network elasticity at high VASP concentration. Removal of the tetramerization site results in significantly reduced bundling and bundle clustering, indicating that VASP's flexible tetrameric structure causes clustering. Removing either the F-actin or the G-actin binding site diminishes VASP's effect on elasticity, but does not eliminate it. Mutating the F-actin and G-actin binding site together, or mutating the F-actin binding site and saturating the G-actin binding site with monomeric actin, eliminates VASP's ability to increase network stiffness. We propose that, in the cell, VASP crosslinking confers only moderate increases in linear network elasticity, and unlike other crosslinkers, VASP's network stiffening activity may be tuned by the local concentration of monomeric actin.

Actin Dynamics, Architecture, and Mechanics in Cell Motility

“…Indeed, Ena/VASP is found to magnify the effect of fascin on network rigidity, perhaps explaining the presence of these two proteins in filopodia (301). However, on their own, Ena/VASP proteins only very modestly increase the rigidity of actin networks, possibly due to the flexibility the Ena/VASP tetramer (101,302).…”

Septins promote F-actin ring formation by crosslinking actin filaments into curved bundles

“…Fluorescently labelled actin was purified from skeletal rabbit muscle and labelled with Alexa Fluor-488 or -594 (Invitrogen) 70 . Recombinant GSTtagged mouse Fascin (the plasmid was a gift from S. Hansen and R. D. Mullins, University of California San Francisco, USA) was produced in E. coli and purified using standard procedures.…”

Cytochalasin B Modulates Macrophage-Mediated Inflammatory Responses

“…However, interestingly, Cyto B strongly suppressed these phosphorylation events, indicating that actin cytoskeleton disruption itself or actin cytoskeleton disruption-linked molecular events may autologously participate in regulating upstream events for actin polymerization. Indeed, it has been reported that VASP and HSP27 are actin-binding proteins (Wang and Bitar, 1998; Gentry et al ., 2012). Thus, the EVH2 domain is known as an important part of VASP for actin binding capacity (Gentry et al ., 2012), indicating direct regulation between actin cytoskeleton and the activation of VASP and HSP27.…”

“…Indeed, it has been reported that VASP and HSP27 are actin-binding proteins (Wang and Bitar, 1998; Gentry et al ., 2012). Thus, the EVH2 domain is known as an important part of VASP for actin binding capacity (Gentry et al ., 2012), indicating direct regulation between actin cytoskeleton and the activation of VASP and HSP27. Mean-while, considering that Src is a critical enzyme linked to actin cytoskeleton (Kim et al ., 2010), a possibility that actin cytoskeleton-controlled Src is able to modulate the phosphorylation of VASP and HSP27 could be hypothesed.…”