In cells, filamentous actin (F-actin) polymers dynamically assemble and disassemble. Diverse sets of actin-binding proteins (ABPs) help self-organize F-actin into different types of networks including lamellipodia, filopodia and contractile rings. These networks display characteristic architectures and dynamics that are tailored to drive different fundamental cellular processes. In addition to cell biological techniques that allow us to observe and manipulate these F-actin networks in vivo, we can reconstitute actin networks in vitro using purified and fluorescently labeled proteins in order to determine the components that are necessary and sufficient for building distinct networks. We combine multi-color TIRF microscopy, micropatterning and biomimetic techniques, which provide precise control and manipulation of components within the reconstituted systems and allow for characterization of ABPs with millisecond time resolution and single-molecule spatial resolution.

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Two-color TIRF microscopy imaging of Filopodia Like Network (FLN) reconstitution from purified proteins over time. Pattern is coated with Arp2/3 complex activator pWA. Polymerization start in presence of actin (green), profilin, Arp2/3 complex, fascin and capping protein. The addition of VASP (red) triggers the formation of FLN.