Rosalind C. Williamson

A syndecan-4 hair trigger initiates wound healing through caveolin- and RhoG-regulated integrin endocytosis.

Summary Cell 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.

Band 3 multiprotein complexes in the red cell membrane; of mice and men.

The bicarbonate/chloride exchanger band 3 (Anion Exchanger 1, AE1) is the most abundant protein in the erythrocyte membrane, it has an important role in gas exchange and functions as a point of attachment for the cytoskeletons maintaining the mechanistic and osmotic properties of the erythrocyte. Band 3 is found in three distinct protein complexes within the erythrocyte membrane: an ankyrin-dependent tetrameric band 3 complex, a dimeric band 3 complex bound to the protein 4.1-GPC junctional complex and as freely diffusing dimeric band 3 complexes. Much if not all of our present knowledge of these protein complexes is derived from mouse knockout model systems and human variant blood samples. This review will explore what is known about the band 3 complexes of mice and humans, focussing on the observed species differences and their potential functional consequences.

Glycophorin A Band 3 aid

Band 3 (B3) is a major site of cytoskeletal attachment to the erythrocyte membrane and is important for gas exchange. A truncated isoform of B3 (kB3) is expressed in the alpha-intercalated cells of the kidney and its functional activity and basolateral localization are essential for acid secretion. B3 mutations generally lead to red blood cell (RBC) specific disease (hereditary spherocytosis (HS), Southeast Asian Ovalocytosis or hereditary stomatocytosis) or kidney disease (distal Renal Tubular Acidosis--dRTA). It is rare for both the RBC and kidney disease phenotypes to co-exist, but this does occur in knockout mice, and also in humans (B3 Coimbra and B3 Courcouronne) or cattle with homozygous HS mutations. This is because RBCs express a B3 chaperone-like molecule in the form of Glycophorin A that can rescue the majority of B3 mutations that cause dRTA but probably not the majority of HS mutations. The study of naturally occurring B3 variant blood and expression of B3 or kB3 mutants in heterologous expression systems has provided valuable information concerning B3 trafficking and interactions in the RBC and kidney. This article will review these studies and comment on our current understanding of the interaction between GPA with B3 and also on the proposed B3 centred macrocomplex.

Band 3 Courcouronnes (Ser667Phe): a trafficking mutant differentially rescued by wild type band 3 and glycophorin A

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 patients 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.

Syndecan and integrin interactomes: large complexes in small spaces

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.

Human kidney anion exchanger 1 localisation in MDCK cells is controlled by the phosphorylation status of two critical tyrosines

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.

Anion Exchanger 1 Interacts with Nephrin in Podocytes

The central role of the multifunctional protein nephrin within the macromolecular complex forming the glomerular slit diaphragm is well established, but the mechanisms linking the slit diaphragm to the cytoskeleton and to the signaling pathways involved in maintaining the integrity of the glomerular filter remain incompletely understood. Here, we report that nephrin interacts with the bicarbonate/chloride transporter kidney anion exchanger 1 (kAE1), detected by yeast two-hybrid assay and confirmed by immunoprecipitation and co-localization studies. We confirmed low-level glomerular expression of kAE1 in human and mouse kidneys by immunoblotting and immunofluorescence microscopy. We observed less kAE1 in human glomeruli homozygous for the NPHS1(FinMaj) nephrin mutation, whereas kAE1 expression remained unchanged in the collecting duct. We could not detect endogenous kAE1 expression in NPHS1(FinMaj) podocytes in primary culture, but heterologous re-introduction of wild-type nephrin into these podocytes rescued kAE1 expression. In kidneys of Ae1(-/-) mice, nephrin abundance was normal but its distribution was altered along with the reported kAE1-binding protein integrin-linked kinase (ILK). Ae1(-/-) mice had increased albuminuria with glomerular enlargement, mesangial expansion, mesangiosclerosis, and expansion of the glomerular basement membrane. Glomeruli with ILK-deficient podocytes also demonstrated altered AE1 and nephrin expression, further supporting the functional interdependence of these proteins. These data suggest that the podocyte protein kAE1 interacts with nephrin and ILK to maintain the structure and function of the glomerular basement membrane.

Syndecan-4 independently regulates multiple small GTPases to promote fibroblast migration during wound healing

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.

HCO3−‐independent conductance with a mutant Na+/HCO3− cotransporter (SLC4A4) in a case of proximal renal tubular acidosis with hypokalaemic paralysis

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+/HCO3− cotransport activity in oocytes. These observations probably underlie the inability of A799V to regulate blood pH. •  A799V and A799I exhibit a novel, DIDS‐stimulated, HCO3−‐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.

Coronin-1C and RCC2 guide mesenchymal migration by trafficking Rac1 and controlling GEF exposure

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.