Molecular regulation of cellular protrusions by Rac and Cdc42 subfamily GTPases
Zusammenfassung der Projektergebnisse
Cell migration requires the coordinated formation of cellular protrusions at the cell front, like lamellipodia and filopodia, which are composed of meshworks and bundles of polymerising actin filaments, respectively. Small GTPases of the Rho-family such as Rac and Cdc42 subfamily members are important stimulatory switches driving the formation of lamellipodia and filopodia, although the precise signalling pathways from a given GTPase to the respective actin polymerisation machinery are far from being elucidated. We could demonstrate that Rac1-mediated lamellipodia protrusion requires a constitutively associated protein assembly generally referred to as WAVE-complex. WAVE-complex consists of five conserved subunits, namely WAVE, Abi, Sra-1, Nap1 and Brck1. This protein complex binds to and activates the actin filament nucleating enzyme Arp2/3-complex, both of which is absolutely essential for Rac1-induced lamellipodium protrusion. Of note, the WAVE-complex component Abi was described before to be engaged in protein complexes other than WAVE-complex, namely in growth factor induced Rac activation. Consequently it co-localized with N-WASP during clathrin mediated endocytosis and EGFR distribution was altered in its absence. Unlike the other subunits, Brck1 also exists as a free un-complexed form and we have in a collaborative project established that this free form is an essential precursor in the assembly of a functional Wave complex, and solved its high resolution crystal structure. Finally, our data demonstrated that filopodia and lamellipodia formation is functionally separable. Moreover, we have studied the molecular mechanisms of filopodium protrusion downstream of small GTPases of the Cdc42- and RhoD-subclasses. In the context of GTPase driven lamellipodia and filopodia formation, we have also examined the role of the adaptor proteins IRSp53 and Eps8, that act both upstream of these GTPases and downstream directly in actin and membrane rearrangements during protrusion. Finally, one focus was to understand how the above actin driven processes impact on host pathogen interaction and how pathogens subvert host cellular regulation of actin rearrangements. To this end, we have studied the role of Arp2/3 dependent and independent actin rearrangements for Salmonella invasion, the contributions of Rac- and Cdc42-GTPases to Listeria invasion and of Eps8 to Listeria intracellular motility, or the molecular mechanisms of action of IRSp53 during EHEC pedestals and of the Shigella Rho-GEF IpgB2. Together, these studies significantly added to our molecular understanding of cellular motility processes and increased our knowledge on host pathogen interactions.
