Dissection of the actin assembly machinery at the lamellipodium tip

• Applicant: Professor Dr. Klemens Rottner
• Subject Area: Cell Biology
• Term: from 2006 to 2013
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 14023866

Arp2/3-catalysed actin filament assembly is implicated in various cellular motility processes. The Arp2/3-complex is activated by nucleation promoting factors (NPFs), for instance WASP and WAVE family proteins, which drive actin reorganizations downstream of the Rho- GTPases Cdc42 and Rad. A macromolecular unit comprising ubiquitous WAVE2 and constitutively associated factors (WAVE-complex) is translocated to the cell periphery upon Rac1 activation and essential for Arp2/3-complex activation and induction of lamellipodia protrusion. However, the molecular details of how this occurs e.g. concerning the potential role of plasma membrane lipids in this process or regarding the lifetime of individual WAVE-complex units in lamellipodia are elusive. In this proposal, we set out to address these questions by (1) developing an in vivo reconstitution assay of WAVE-complex mediated actin filament assembly, and by (2) assessing the turnover of both WAVE- and Arp2/3-complexes during lamellipodia protrusion e.g. by FRAP (fluorescence recovery after photobleaching) experiments. These approaches will help not only to draw a more comprehensive picture of the requirements for WAVE-complex activation, but also to develop and evaluate new assays for unravelling the molecular features of actin polymerisation complexes in vivo.

• DFG Programme: Research Units
• Subproject of: FOR 629: Molecular Mechanisms of Cell Motility