SummaryCell migration during wound healing requires adhesion receptor turnover to enable the formation and disassembly of cell-extracellular matrix contacts. Although recent advances have improved our understanding of integrin trafficking pathways, it is not known how extracellular ligand engagement controls receptor dynamics. Using atomic force microscopy, we have measured cell avidity for fibronectin and defined a mechanism for the outside-in regulation of α5β1-integrin. Surprisingly, adhesive strength was attenuated by the syndecan-4-binding domain of fibronectin due to a rapid triggering of α5β1-integrin endocytosis. Association of syndecan-4 with PKCα was found to trigger RhoG activation and subsequent dynamin- and caveolin-dependent integrin uptake. Like disruption of syndecan-4 or caveolin, gene disruption of RhoG in mice was found to retard closure of dermal wounds due to a migration defect of the fibroblasts and keratinocytes of RhoG null mice. Thus, this syndecan-4-regulated integrin endocytic pathway appears to play a key role in tissue repair.

No abstract

Sustained forward migration through a fibrillar extracellular matrix requires localization of protrusive signals. Contact with fibronectin at the tip of a cell protrusion activates Rac1, and for linear migration it is necessary to dampen Rac1 activity in off-axial positions and redistribute Rac1 from non-protrusive membrane to the leading edge. Here, we identify interactions between coronin-1C (Coro1C), RCC2 and Rac1 that focus active Rac1 to a single protrusion. Coro1C mediates release of inactive Rac1 from non-protrusive membrane and is necessary for Rac1 redistribution to a protrusive tip and fibronectin-dependent Rac1 activation. The second component, RCC2, attenuates Rac1 activation outside the protrusive tip by binding to the Rac1 switch regions and competitively inhibiting GEF action, thus preventing off-axial protrusion. Depletion of Coro1C or RCC2 by RNA interference causes loss of cell polarity that results in shunting migration in 1D or 3D culture systems. Furthermore, morpholinos against Coro1C or RCC2, or mutation of any of the binding sites in the Rac1–RCC2–Coro1C complex delays the arrival of neural crest derivatives at the correct location in developing zebrafish, demonstrating the crucial role in migration guidance in vivo.

Highlights► Syndecans are cleared from adhesion sites to allow integrin engagement. ► Literature-curated interactomes differ between syndecan-1 and syndecan-4. ► Overlap between interactomes of syndecan or integrin describe adhesion formation. ► Switching of syndecan signalling by phosphorylation.

An important question in renal physiology is how the α-intercalated cells of the kidney regulate the distribution of the basolateral kidney anion exchanger 1 (kAE1) according to systemic acid-base status. Previous work using a MDCKI model system demonstrated that kAE1 basolateral targeting requires an N-terminal determinant and a critical C-terminal tyrosine (Y904). Here, we show that the N-terminal determinant is residue Y359, because a Y359A substitution mutant was mistargeted to the apical membrane. Further determinants might exist because a range of N-terminal kAE1 truncations that contained Y359 were incorrectly targeted to the TGN. Y359 and Y904 in kAE1 are phosphorylated upon pervanadate treatment and this phosphorylation is sensitive to specific Src kinase family inhibitors. We tested a range of stimuli on this model system and only the application of high nonphysiological concentrations of extracellular bicarbonate, and to a lesser extent hypertonicity or hyperosmolarity, induced tyrosine phosphorylation of kAE1. Treatment with pervanadate caused internalisation of kAE1 from the plasma membrane, but treatment with high concentrations of bicarbonate did not, because of the hypertonicity of the solution. We propose that α-intercalated cells control the distribution of kAE1 by reversible phosphorylation of tyrosine residues Y359 and Y904.