Arp2/3 complex-mediated actin assembly at cell membranes drives the formation of protrusions or endocytic vesicles. To identify the mechanism by which different membrane deformations can be achieved, we reconstitute the basic membrane deformation modes of inward and outward bending in a confined geometry by encapsulating a minimal set of cytoskeletal proteins into giant unilamellar vesicles. Formation of membrane protrusions is favoured at low capping protein (CP) concentrations, whereas the formation of negatively bent domains is promoted at high CP concentrations. Addition of non-muscle myosin II results in full fission events in the vesicle system. The different deformation modes are rationalized by simulations of the underlying transient nature of the reaction kinetics. The relevance of the regulatory mechanism is supported by CP overexpression in mouse melanoma B16-F1 cells and therefore demonstrates the importance of the quantitative understanding of microscopic kinetic balances to address the diverse functionality of the cytoskeleton.

the dynamics of actin networks is modulated by a machinery consisting of actin binding proteins that control the turnover of filaments in space and time. To study this complex orchestration, in vitro reconstitution approaches strive to project actin dynamics in ideal, minimal systems. To this extent we reconstitute a self-supplying, dense network of globally treadmilling filaments. In this system we analyze growth and intrinsic turnover by means of fRAp measurements and thereby demonstrate how the depletion of monomers and actin binding partners modulate the dynamics in active actin networks. The described effects occur only in dense networks, as single filament dynamics are unable to produce depletion effects to this extent. Furthermore, we demonstrate a synergistic relationship between the nucleators formin and Arp2/3 when branched networks and formin-induced networks are colocalized. As a result, the formin-enhanced filament turnover depletes cofilin at the surface and thus protects the dense, Arp2/3 polymerized network from debranching. Ultimately, these results may be key for understanding the maintenance of the two contradicting requirements of network stability and dynamics in cells.

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Contrary to conventional actin cross-linkers, we show that vasodilator-stimulated phosphoprotein (VASP) bound to lipid bilayers in vitro facilitates the formation of actin bundles through alignment by polymerization. The mobility enables VASP to phase separate and track growing barbed ends while accumulating on the actin bundles. This in turn results in a reorganization of the bilayer.