nanomotility

Cell migration is a process having a hierarchical spatial and temporal organization. At the cellular, sub-cellular, and molecular levels, migration proceeds through cycles lasting from minutes to seconds. The robustness of these cycles relies on the precise regulation of motions and transient interactions of protein complexes involved in actin nucleation/polymerization and adhesion. During cell migration the Arp2/3 complex must be activated by the Wave complex at the tip of cellular protrusions for sustained site-directed actin polymerization. Force generation on the substrate is triggered by integrins recruitment at nascent adhesion sites. We have recently developed single molecule techniques and demonstrated their invaluable power for understanding the key role of motions and localized interactions during trafficking between different compartments of the plasma membrane. Because, current available techniques suffer major limitations such as short life times and large size of probes, the transition from the plasma membrane to confined environments such as crowded adhesion sites and intracellular actin networks is in the initial stages. In this project we want to understand the cyclic molecular mechanisms leading to actin nucleation/polymerization and initiation of early integrin-dependent adhesion sites during cell migration. For this purpose we need to elucidate the mechanisms by which the nano-dynamics of integrins, Arp2/3 and Wave2 complexes control molecular motile cycles. To reach this goal we will combine nanotechnology, cell biology, and biochemistry approaches to overcome current technological limitations and allow for the continuous and long term single molecule tracking of intracellular actin nucleation/polymerization protein complexes and adhesion proteins as they traffic in and between the different crowded macromolecular structures involved in cell motility. Specifically, by combining the expertise of our groups in cell biology of migration and protein dynamics (Giannone), nano-photonics (Lounis) and biochemistry of actin nucleation/polymerization protein complexes (Gautreau) we will: 1) transfer new and established imaging methods for the long term tracking of single proteins in intracellular and confined environments; 2) develop versatile and specific strategies of labeling with small and photostable probes, to label integrins on the extracellular side, and to label purified Arp2/3 and Wave2 complexes; 3) resolve the long term dynamics of Arp2/3 complex and Wave2 protein at the single molecule level in in vitro systems; 4) resolve the nano-dynamics of actin nucleation/polymerization complexes and integrins at dense macromolecular structures in live motile cells: adhesion sites and branched actin networks.