Keary, S. | bioRxiv Science

bioRxiv2023

Differential spatial regulation and activation of integrin nanoclusters inside focal adhesions

5{beta}1 and v{beta}3 integrins are core components of focal adhesions (FAs) involved in cell attachment, migration, and mechanobiology-dependent processes. Recent works indicate that both integrins organize in nanoclusters inside FAs, with sub-populations of active and inactive {beta}1 nanoclusters. However, whether both integrins work in concert or their activities are spatially regulated is not fully understood. Using dual-color super-resolution microscopy (STORM, STED and DNA-PAINT) we show that integrins 5{beta}1 and v{beta}3 and their main adaptor proteins exhibit similar functional nanoscale segregation across all adhesion proteins examined, independent of FA maturation state. Notably, both integrins never mix at the nanoscale indicating that their functions might be spatially regulated. We find a nearly 1:1 relationship between active integrin and adaptor nanoclusters suggesting that coordinated integrin activation occurs via the concurrent engagement of adaptor nanoclusters. Interestingly, 5{beta}1 nanoclusters preferentially localize at the FA periphery near adaptor nanoclusters, establishing regions of multi-nanocluster enrichment, whereas v{beta}3 nanoclusters uniformly distribute throughout FAs. Overall, our results show that adhesion proteins are arranged as modular nanoscale units that distinctively organize inside FAs to spatially regulate integrin activation and function.

cell biology↗

bioRxiv2020

Shear flow-driven actin re-organization induces ICAM-1 nanoclustering on endothelial cells that impact T-cell migration

The leukocyte specific {beta}2-integrin LFA-1, and its ligand ICAM-1 expressed on endothelial cells (ECs), are involved in the arrest, adhesion and transendothelial migration of leukocytes. Although the role of mechanical forces on LFA-1 activation is well established, the impact of forces on its major ligand ICAM-1, has received less attention. Using a parallel-plate flow chamber combined with confocal and super-resolution microscopy, we show that prolonged shear-flow induces a global translocation of ICAM-1 on ECs upstream of flow direction. Interestingly, shear-forces promoted ICAM-1 nanoclustering prior to LFA-1 engagement. This spatial nanoscale organization was driven by actin cytoskeleton re-arrangements induced by shear-force. We further assessed the impact of prolonged shear-stress EC stimulation on T cell migration. T cells adhered to mechanically pre-stimulated ECs developed a more pro-migratory phenotype, migrated faster and exhibited shorter EC interactions than when adhered to non-mechanically stimulated ECs. Together, our results indicate that shear-forces increase the number of ICAM-1/LFA-1 bonds due to ICAM-1 nanoclustering, strengthening adhesion and thereby reducing actin retrograde flow of T-cells, leading to their increased migration speed. Our data also underscores the importance of mechanical forces regulating the spatial organization of cell membrane receptors and their contribution to adhesion regulation, regardless of integrin activation. Summary statementWe show that shear forces promote ICAM-1 spatial re-arrangement and actin-dependent nanoclustering on ECs prior to integrin engagement. This mechanism might be important for firm leukocyte adhesion and migration during the immune response.

cell biology↗