NANO-MEMEC is structured along three main objectives: Objective 1: to dissect mechanical and biochemical coupling of mechanosensing at the cell membrane ascertaining how forces alter membrane physical properties (tension, fluidity), the cortical actin cytoskeleton and the glycocalyx matrix, affecting the nanoscale organization and molecular conformation of integrin receptors. Objective 2: to visualize and quantify the coordinated spatiotemporal recruitment of integrin-associated signaling proteins in response to force and associated to mechanotransduction, dissecting how these events couple back to remodel the cell membrane. Objective 3: to determine how changes in spatiotemporal remodeling of integrin receptor nanoplatforms and interactions with their ligands propagate through the intracellular machinery to impact on cell response, from adhesion to migration of immune cells. In this reporting period we have mainly concentrated on objectives 1 and objectives 3 and started to perform preliminary experiments along objective 2. In particular, we have developed a new high-density single molecule-based approach to map how receptors at the cell surface dynamically explore the 2D space over multiple spatiotemporal scales. By using the prototypical receptor CD44, known to directly interact with the cortical actin cytoskeleton and the extracellular matrix, we have been able to discriminate the role of actin and the glycocalyx matrix orchestrating the organization of many cell surface receptors (Mol. Bio. Cell 2020). Our results indicate that the glycocalyx matrix strengthen interactions between receptors, while the cortical cytoskeleton imposes dynamic fences that constrain and relax the diffusion and interaction between multiple partners of the cell membrane. Interestingly, actin remodeling occurs at multiple temporal scales and our work conciliates the different dynamical scales that have been observed in separate experiments by us and other groups around the world. In addition, as part of objective 1, we have exploited our photonic antennas configuration to investigate the role of extra-cellular glycans on the patterning of the lipid bilayer composing the cell surface. Our work reveals that glycans have a profound impact modulating the physico-chemical properties of the lipid bilayer itself, synergizing with cholesterol to alter the local fluidity and mechanical properties of the membrane at the nanoscale (J. Phys. Chem. Lett. 2021). In relation to objective 3, we have provided evidence that prolonged physiological shear-forces promote the formation of ICAM-1 nanoclusters on endothelial cells and cause actin-dependent polarization of ICAM-1 against the flow direction. Importantly, we determined for the first time, to our knowledge, that this shear-force induced ICAM-1 nanoclustering is sufficient to alter the migration of T cells under flow, inducing a more migratory T cell profile and accelerating their migration (Biophys. J. 2021). Regarding objective 2, we are currently investigating the nanoscale organization of different integrin receptors and their main molecular adaptors (paxillin, talin and vinculin) both on fibroblasts and T cells. So far, our unpublished data reveals that: a) integrins and their molecular adaptors organize as segregated nanoclusters inside adhesion structures forming dynamic nano-hubs of activity; b) interactions between integrins and their molecular adaptors (in particular talin and vinculin) are highly dynamic so that at a given moment of time, only a few integrins are actively engaged with their partners; c) integrin receptors are highly sensitive to mechanical force, with a modest shear stress application being sufficient to induce conformation changes of integrins that promote their activation.