Regulation and coordination of different actin machineries on a membrane – COORDACTIN

Cell migration is essential for processes like embryonic development, immune response, wound repair, and metastasis. It implies sequential cell protrusion (membrane deformation), formation of focal adhesions, traction and retraction of the cell rear. All these processes are induced by the rapid reorganization of the actin cytoskeleton under extracellular signalling. In vivo observation of motile cells showed the coexistence of different actin filament structures: highly dense branched network in the lamellipodium (flat extension at the cell front), parallel bundles in filopodia (finger-like extensions at the leading edge of the lamellipodium), fibers in focal adhesion complexes' How the cell manages to coordinate all these dynamics structures in space and time is a crucial issue that is still unanswered. In vivo and in vitro studies have shown that the lamellipodial and filopodial protrusions are based on spatially controlled polymerization of actin filaments at the cell membrane, induced by two different nucleation machineries (WASP-Arp2/3 generates a branched network in the lamellipodium; formins generate long unbranched filaments in filopodia). The regulation of the two systems is now partially understood, but the question still remains of how they are controlled in a concerted fashion at the membrane. The goal of our project is to combine the two actin machineries on membranes and to study how membrane properties participate to the regulation and coordination of the two arrays. Before that, we will study each system independently to understand the role of the membrane in the growth and organization of each structure on the surface. We will follow the distribution of the activators which induce actin polymerization at the membrane, and will determine what changes this distribution while the actin structure grows. Anchorage of the branched network on membranes allowed to show that the llink between the activator N-WASP and the actin filament is transient, and that it is mediated by the Arp2/3 complex. Using photobleaching (FRAP) and Fluorescence Correlation Spectroscopy (FCS) methods we will measure N-WASP diffusion on membrane surface, in order to derive the lifetime of the N-WASP-filament interaction. Our results will give insight in the kinetics of the branching reaction mechanism. For formins, we think that membrane fluidity plays an important role. In vivo, they are clustered at the filopodia tips where they nucleate long parallel actin filaments. By varying the regulatory proteins of the biochemical medium and the membrane properties, we will determine which parameters tune the formin clusterization and filament bundle growth. We will also try to define conditions in which the membrane can be invaginated by actin bundles like in filopodia. In the last part, the biochemical environment (regulatory proteins and activator densities) must be first optimized so that the two machineries generate both actin structures in parallel. Using epifluorescence microscopy, the growth of the actin filament network (the two arrays cannot be distinguished) and the activator distributions will be followed simultaneously. We will study how the two arrays reorganize and regulate mutually as function of medium composition and membrane properties. In particular, our observations should allow to verify if parallel bundles grow with the branched array simultaneously, or if they appear later because they are generated due to local rearrangement of the branched filaments, as has been proposed by some groups. Our results should lead to an integrated model of the concerted regulation/coordination of he protruding machineries by regulatory proteins that control actin assembly and membrane.