III. In vitro reconstitution of diverse actin cytoskeleton networks


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. Previous studies have revealed the major ABPs associated with each network, but the molecular mechanisms by which they facilitate specific F-actin architectures and dynamics remain unclear. In the Kovar Lab we combine multi-color TIRF microscopy imaging, micropatterning and biomimetic techniques to reconstitute the assembly of complex F-actin networks with purified fluorescently labeled ABPs (movie). These approaches provide precise control and manipulation of components within the reconstituted systems, which allows characterization of ABPs with millisecond time resolution and single-molecule spatial resolution.

Legend for movie:
Two-color TIRF microscopy imaging of individual red-labeled Ena molecules processively elongating the barbed ends of two green-labeled actin filaments bundled by Fascin.