Nephrin Regulates Lamellipodia Formation by Assembling a Protein Complex That Includes Ship2, Filamin and Lamellipodin

Actin dynamics has emerged at the forefront of podocyte biology. Slit diaphragm junctional adhesion protein Nephrin is necessary for development of the podocyte morphology and transduces phosphorylation-dependent signals that regulate cytoskeletal dynamics. The present study extends our understanding of Nephrin function by showing in cultured podocytes that Nephrin activation induced actin dynamics is necessary for lamellipodia formation. Upon activation Nephrin recruits and regulates a protein complex that includes Ship2 (SH2 domain containing 5′ inositol phosphatase), Filamin and Lamellipodin, proteins important in regulation of actin and focal adhesion dynamics, as well as lamellipodia formation. Using the previously described CD16-Nephrin clustering system, Nephrin ligation or activation resulted in phosphorylation of the actin crosslinking protein Filamin in a p21 activated kinase dependent manner. Nephrin activation in cell culture results in formation of lamellipodia, a process that requires specialized actin dynamics at the leading edge of the cell along with focal adhesion turnover. In the CD16-Nephrin clustering model, Nephrin ligation resulted in abnormal morphology of actin tails in human podocytes when Ship2, Filamin or Lamellipodin were individually knocked down. We also observed decreased lamellipodia formation and cell migration in these knock down cells. These data provide evidence that Nephrin not only initiates actin polymerization but also assembles a protein complex that is necessary to regulate the architecture of the generated actin filament network and focal adhesion dynamics

ER-Bound Protein Tyrosine Phosphatase PTP1B Interacts with Src at the Plasma Membrane/Substrate Interface

PTP1B is an endoplasmic reticulum (ER) anchored enzyme whose access to substrates is partly dependent on the ER distribution and dynamics. One of these substrates, the protein tyrosine kinase Src, has been found in the cytosol, endosomes, and plasma membrane. Here we analyzed where PTP1B and Src physically interact in intact cells, by bimolecular fluorescence complementation (BiFC) in combination with temporal and high resolution microscopy. We also determined the structural basis of this interaction. We found that BiFC signal is displayed as puncta scattered throughout the ER network, a feature that was enhanced when the substrate trapping mutant PTP1B-D181A was used. Time-lapse and co-localization analyses revealed that BiFC puncta did not correspond to vesicular carriers; instead they localized at the tip of dynamic ER tubules. BiFC puncta were retained in ventral membrane preparations after cell unroofing and were also detected within the evanescent field of total internal reflection fluorescent microscopy (TIRFM) associated to the ventral membranes of whole cells. Furthermore, BiFC puncta often colocalized with dark spots seen by surface reflection interference contrast (SRIC). Removal of Src myristoylation and polybasic motifs abolished BiFC. In addition, PTP1B active site and negative regulatory tyrosine 529 on Src were primary determinants of BiFC occurrence, although the SH3 binding motif on PTP1B also played a role. Our results suggest that ER-bound PTP1B dynamically interacts with the negative regulatory site at the C-terminus of Src at random puncta in the plasma membrane/substrate interface, likely leading to Src activation and recruitment to adhesion complexes. We postulate that this functional ER/plasma membrane crosstalk could apply to a wide array of protein partners, opening an exciting field of research

Chromosomal contacts connect loci associated with autism, BMI and head circumference phenotypes

Copy number variants (CNVs) are major contributors to genomic imbalance disorders. Phenotyping of 137 unrelated deletion and reciprocal duplication carriers of the distal 16p11.2 220 kb BP2-BP3 interval showed that these rearrangements are associated with autism spectrum disorders and mirror phenotypes of obesity/underweight and macrocephaly/microcephaly. Such phenotypes were previously associated with rearrangements of the non-overlapping proximal 16p11.2 600 kb BP4-BP5 interval. These two CNV-prone regions at 16p11.2 are reciprocally engaged in complex chromatin looping, as successfully confirmed by 4C-seq, fluorescence in situ hybridization and Hi-C, as well as coordinated expression and regulation of encompassed genes. We observed that genes differentially expressed in 16p11.2 BP4-BP5 CNV carriers are concomitantly modified in their chromatin interactions, suggesting that disruption of chromatin interplays could participate in the observed phenotypes. We also identified cis- and trans-acting chromatin contacts to other genomic regions previously associated with analogous phenotypes. For example, we uncovered that individuals with reciprocal rearrangements of the trans-contacted 2p15 locus similarly display mirror phenotypes on head circumference and weight. Our results indicate that chromosomal contacts’ maps could uncover functionally and clinically related genes.Molecular Psychiatry advance online publication, 31 May 2016; doi:10.1038/mp.2016.84

Podocyte-secreted angiopoietin-like-4 mediates proteinuria in glucocorticoid-sensitive nephrotic syndrome

The main manifestations of nephrotic syndrome include proteinuria, hypoalbuminemia, edema, hyperlipidemia and lipiduria. Common causes of nephrotic syndrome are diabetic nephropathy, minimal change disease (MCD), focal and segmental glomerulosclerosis (FSGS) and membranous nephropathy. Among the primary glomerular diseases, MCD is usually sensitive to glucocorticoid treatment, whereas the other diseases show variable responses(1). Despite the identification of key structural proteins in the glomerular capillary loop which may contribute to defects in ultrafiltration, many of the disease mechanisms of nephrotic syndrome remain unresolved. In this study, we show that the glomerular expression of angiopoietin-like-4 (Angptl4), a secreted glycoprotein, is glucocorticoid sensitive and is highly upregulated in the serum and in podocytes in experimental models of MCD and in the human disease. Podocyte-specific transgenic overexpression of Angptl4 (NPHS2-Angptl4) in rats induced nephrotic-range, and selective, proteinuria (over 500-fold increase in albuminuria), loss of glomerular basement membrane (GBM) charge and foot process effacement, whereas transgenic expression specifically in the adipose tissue (aP2-Angptl4) resulted in increased circulating Angptl4, but no proteinuria. Angptl4-/-mice that were injected with lipopolysaccharide (LPS) or nephritogenic antisera developed markedly less proteinuria than did control mice. Angptl4 secreted from podocytes in some forms of nephrotic syndrome lacks normal sialylation. When we fed the sialic acid precursor N-acetyl-D-mannosamine (ManNAc) to NPHS2-Angptl4 transgenic rats it increased the sialylation of Angptl4 and decreased albuminuria by more than 40%. These results suggest that podocyte-secreted Angptl4 has a key role in nephrotic syndrome

Glycoprotein hormone receptors: link between receptor homodimerization and negative cooperativity

The monomeric model of rhodopsin-like G protein-coupled receptors (GPCRs) has progressively yielded the floor to the concept of GPCRs being oligo(di)mers, but the functional correlates of dimerization remain unclear. In this report, dimers of glycoprotein hormone receptors were demonstrated in living cells, with a combination of biophysical (bioluminescence resonance energy transfer and homogenous time resolved fluorescence/fluorescence resonance energy transfer), functional and biochemical approaches. Thyrotropin (TSHr) and lutropin (LH/CGr) receptors form homo- and heterodimers, via interactions involving primarily their heptahelical domains. The large hormone-binding ectodomains were dispensable for dimerization but modulated protomer interaction. Dimerization was not affected by agonist binding. Observed functional complementation indicates that TSHr dimers may function as a single functional unit. Finally, heterologous binding-competition studies, performed with heterodimers between TSHr and LH/CG–TSHr chimeras, demonstrated the unsuspected existence of strong negative cooperativity of hormone binding. Tracer desorption experiments indicated an allosteric behavior in TSHr and, to a lesser extent, in LH/CGr and FSHr homodimers. This study is the first report of homodimerization associated with negative cooperativity in rhodopsin-like GPCRs. As such, it may warrant revisitation of allosterism in the whole GPCR family