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.

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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.

Upon wounding, syndecan-4 detects the appearance of fibronectin in the wound bed and mediates regulation of the small GTPases, Rac1, RhoA and RhoG. Cohesive regulation of these molecules results in cycles of membrane protrusion and cytoskeletal contraction, and triggers the endocytosis of α5β1-integrin, which collectively lead to immigration of fibroblasts into the wound bed. In this manuscript we identify the regulation of a fourth GTPase, Arf6 that is responsible for α5β1-integrin recycling and thereby completes the cycle of syndecan-4-regulated integrin trafficking. We demonstrate that each of the GTPase signals can be regulated by syndecan-4, but that they are independent of one another. By doing so we identify syndecan-4 as the coordinating center of pro-migratory signals.

We describe a mutation in human erythrocyte band 3 (anion exchanger 1; SLC4A1) causing both hereditary spherocytosis and distal renal tubular acidosis. The proband developed a transfusion-dependent, hemolytic anemia following birth. Immunoblotting showed band 3 was reduced to approximately 35% of wildtype; other proteins of the band 3/Rh macrocomplex were also reduced. DNA sequence analysis revealed a novel homozygous mutation, c.2000C>T, leading to the amino acid substitution Ser667Phe. The parents were heterozygous for the same mutation. Sulfate influx in the patient's erythrocytes was approximately 40% wild type. The mutant band 3 produced very little chloride influx when expressed in Xenopus oocytes. Influx was partially rescued by coexpression of glycophorin A and also rescued by coexpression of wild-type band 3. At 2 years of age, an ammonium chloride challenge showed the child has incomplete distal renal tubular acidosis (dRTA). Stable expression of mutant kidney band 3 in both nonpolarized and polarized Madin-Darby canine kidney cells showed that most of the mutant protein was retained in the endoplasmic reticulum. Overall our results suggest that the Ser667Phe does not affect the anion transport function of band 3, but causes a trafficking defect in both erythrocytes and kidney cells.

Key points• A mutant electrogenic sodium bicarbonate cotransporter NBCe1 (A799V) is associated with an inability of the kidney to regulate blood pH as well as weakness of muscles.• In the present study we employ biotinylation and electrophysiology on Xenopus oocytes as well as confocal microscopy on non-polarized MDCK cells. We study A799V, plus three laboratory-generated mutants A799G, A799I and A799S.• A799V and A799I show increased intracellular retention in MDCK cells. All four mutants exhibit a reduced per-molecule Na + /HCO 3 − cotransport activity in oocytes. These observations probably underlie the inability of A799V to regulate blood pH.• A799V and A799I exhibit a novel, DIDS-stimulated, HCO 3 − -independent conductance -the first example in an electrogenic NBC. This observation could underlie the contribution of A799V towards muscle weakness.• A799G and A799S exhibit unusual outward rectification. A799G is unusually insensitive to DIDS and tenidap. Thus Alanine-799 is a critical determinant of correct NBCe1 function. AbstractThe renal electrogenic Na + /HCO 3 − cotransporter (NBCe1-A) contributes to the basolateral step of transepithelial HCO 3 − reabsorption in proximal tubule epithelia, contributing to the buffering of blood pH. Elsewhere in the body (e.g. muscle cells) NBCe1 variants contribute to, amongst other processes, maintenance of intracellular pH. Others have described a homozygous mutation in NBCe1 (NBCe1-A p.Ala799Val) in an individual with severe proximal renal tubular acidosis (pRTA; usually associated with defective HCO 3 − reabsorption in proximal tubule cells) and hypokalaemic periodic paralysis (hypoPP; usually associated with leaky cation channels in muscle cells). Using biotinylation and two-electrode voltage-clamp on Xenopus oocytes expressing NBCe1, we demonstrate that the mutant NBCe1-A (A A799V ) exhibits a per-molecule transport defect that probably contributes towards the observed pRTA. Furthermore, we find that A A799V expression is associated with an unusual HCO 3 − -independent conductance that, if associated with mutant NBCe1 in muscle cells, could contribute towards the appearance of hypokalaemic paralysis in the affected individual. We also study three novel lab mutants of NBCe1-A: p.Ala799Ile, p.Ala799Gly and p.Ala799Ser. All three exhibit a per-molecule transport defect, but only A A799I exhibits an A A799V -like ion conductance. A A799G and A A799S exhibit unusual outward rectification in their HCO 3 − -dependent conductance and A A799G exhibits reduced sensitivity to both DIDS and tenidap. A799G is the first mutation shown to affect the apparent tenidap affinity of NBCe1. Finally we show that A A799V and A A799I , which accumulate poorly in the plasma membrane of oocytes, exhibit signs of abnormal intracellular accumulation in a non-polarized renal cell-line. Abbreviations A A799V , mutant NBCe1-A p.Ala799Val with a C-terminal EGFP tag; A WT , wild-type NBCe1-A with a C-terminal EGFP tag; B A843V , mutant NBCe1-B p.Ala843Val with a C-terminal EGFP tag; BIC, HCO 3 − ...