Molecular mechanism of integrin-[beta]3 endocytosis at the podosome on viscous RGD-membranes

Abstract

The turnover of integrin-mediated adhesions through integrin endocytosis and recycling is closely associated with cell migration. Most studies to date have focused on the endocytosis of integrin-β1, whereas little is known about the endocytosis of integrin-β3. Podosomes are integrin-mediated adhesions at the cell-matrix interface. Each podosome consists of a dense F-actin core surrounded by a ring of adhesion proteins such as integrin-β3. However, integrin-β3 regulations by receptor endocytosis at the podosome remain elusive.

This study aims to investigate the functional link between podosome and integrin-β3 endocytosis. Utilizing viscous RGD (Arg-Gly-Asp)-membranes as the platform, combined with total internal reflection microscopy as well as confocal microscopy, we found the endocytosis of integrin-β3 is positively correlated with podosome formation. Integrin-β3 and RGD ligands were eliminated from the podosome ring during podosome formation through Dab2/clathrin-mediated endocytosis and were later sorted into early, late and recycling endosomes. Moreover, in the initial phase of podosome formation, plasma membrane deformation around the protrusive F-actin podosome core was observed, followed by the specific recruitment of BIN1 (also known as Amphiphysin2) through the NBAR (N-terminal amphiphathic helix Bin/amphiphysin/Rvs) domain which could sense membrane curvature. Meanwhile, the SH3 (SRC Homology 3) domain of BIN1 contributed to the dramatic increase of dynamin2 at the podosome ring. The accumulated dynamin2 then pinched off clathrin-coated pits and achieved endocytosis. Furthermore, knockdown of BIN1, the presence of BIN1-∆SH3 mutant, or introduction of dominant-negative dynamin2-K44A mutant decreased the endocytosis of RGD ligands.

In summary, these observations demonstrated the molecular mechanism of integrin-β3 endocytosis at the podosome ring. Protrusive F-actin polymerization generates plasma membrane invagination that surrounds F- actin podosome core. The deformed membrane promotes BIN1-dependent dynamin2 recruitment which then leads to the endocytosis of integrin-β3. The functional role of podosome in integrin-β3 endocytosis provides new insights into cell motility and cancer cell invasion.