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

Podocyte dysfunction : an approach towards glomerular diseases with emphasis on nephrin

Podocytes are terminally differentiated and structurally unique cells having interdigitating foot processes which form filtration slits. These slits are bridged by specialised podocyte cell junctions called slit diaphragms. Nephrin is an essential structural and signalling molecule within the slit diaphragm that plays a pivotal role in actin dynamics and podocyte junctional remodelling. Most glomerular diseases are caused by podocyte dysfunction, and glomerular disease progression directly correlates with podocyte numbers. Towards facilitating the quantitation of podocyte numbers, a convenient and robust methodology was developed applicable for high throughput usage. Progression was studied using hDTR and 4-EBP1 transgenic rat models. Progression non-invasively monitored through Nephrin / Podocin mRNA ratios correlated well with podocyte morphometrics. Nephrin mRNA down-regulated after acute injury was thought to facilitate podocyte detachment and contribute towards glomerular progression. An inducible Nephrin-KO mouse was generated to study the time-dependent development of FSGS, progression and podocyte loss. Molecular studies were conducted to understand Nephrin's role in foot process maintenance related to progression mechanisms. Podocyte remodelling involves actin dynamics regulated by Fyn phosphorylation and Nck adapter proteins that bind to Nephrin and recruit other downstream molecular players. In cell culture models, Nephrin actin dynamics involved lamellipodia regulation through ship2, filamin and lamellopodin. Integrin activation phosphorylated Nephrin through an inside signalling pathway. Shp2 deletion prevented Nephrin phosphorylation protecting mice from foot processes effacement. Endocytic trafficking of Nephrin was studied with Vps34 and PIKfyve KO models. Nephrin was trapped within Vps34-KO vesicles and mistargeted from its slit diaphragm position. Together, these studies have helped better understand glomerular disease progression and Nephrin's role in foot processes maintenance and progression. Future challenges and avenues studying Nephrin involve transcriptional regulation and molecular trafficking of Nephrin to the membrane

Integrin Ligation Results in Nephrin Tyrosine Phosphorylation In Vitro.

Nephrin is expressed at the basolateral aspect of podocytes and is an important signaling protein at the glomerular slit diaphragm. In vitro studies have demonstrated that Nephrin phosphorylation-dependent signaling is able to assemble a protein complex that is able to polymerize actin. However, proximal signaling events that result in nephrin tyrosine phosphorylation are not well understood. Nephrin deletion in mice and human nephrin mutations result in developmental failure of the podocyte intercellular junction resutling in proteinuria. This has been presumed to be due to a failure to respond to an external polarized cue in the absence of nephrin or a failure to transduce an outside-in signal in patients with nephrin mutations. The nephrin extracellular domain binds to itself or neph1 across the foot process intercellular junction. Nephrin is tyrosine phosphorylation-silent in healthy glomeruli when presumably the nephrin extracellular domain is in an engaged state. These observations raise the possibility of an alternate proximal signaling mechanism that might be responsible for nephrin tyrosine phosphorylation. Here we present data showing that integrin engagement at the basal aspect of cultured podocytes results in nephrin tyrosine phosphorylation. This is abrogated by incubating podocytes with an antibody that prevents integrin β1 ligation and activation in response to binding to extracellular matrix. Furthermore, nephrin tyrosine phosphorylation was observed in podocytes expressing a membrane-targeted nephrin construct that lacks the extracellular domain. We propose, integrin-activation based signaling might be responsible for nephrin phosphorylation rather than engagment of the nephrin extracellular domain by a ligand

Nephrin is tyrosine phosphorylated when mouse podocytes are plated on laminin and fibronectin coated surface.

<p>Mouse podocytes were plated on culture dishes coated with laminin (A) and fibronectin (B) for the indicated time points. At time 0 cells were maintained in suspension (Su) on a rocker prior to plating. Cells were lysed at various time points and lysates were resolved using SDS-PAGE. Membranes were probed with the indicated antibodies. FAK Y397 phosphorylation was used as a surrogate for integrin activation. (C and D) Bands densitometry using ImageJ software for 4 separate experiments showing increase in nephrin tyrosine phosphorylation as well as FAK Y397 phosphorylaton at different time points. Data are mean values ± SEM. *P<0.001.</p

Schematic showing existing paradigm as well as proposed signaling that results in nephrin phosphorylation via integrin activation during development and following injury.

<p>Schematic showing existing paradigm as well as proposed signaling that results in nephrin phosphorylation via integrin activation during development and following injury.</p

Src Kinase Fyn is necessary for nephrin phosphorylation.

<p>(A) SYF cells that lack Src, Fyn and Yes were transfected with full-length nephrin. Cells were plated on laminin-coated surface for indicated time points. Nephrin phosphorylation as well as β1 integrin activation was observed only after introduction of Fyn in SYF cells. (B) Band densitometry using ImageJ software for 4 separate experiments showing increase in nephrin tyrosine phosphorylation in the presence and absence of Fyn at different time points. Data are mean values ± SEM. *P<0.001, using two-tailed <i>t</i> test.</p

Nephrin tyrosine phosphorylation on laminin-coated surface is abrogated by pre-incubation of podocytes with β1 blocking antibody.

<p>(A). Mouse podocytes were incubated with β1 blocking mab and mouse IgG (control) prior to plating on laminin coated surface. Time 0 denotes cell in suspension (Su). Cells were lysed at various time points and lysates were resolved using SDS-PAGE. Membranes were probed with the indicated antibodies. FAK Y397 phosphorylation was used as a surrogate for integrin activation. (B) Bands densitometry using ImageJ software for 4 separate experiments showing increase in nephrin tyrosine phosphorylation as well as FAK Y397 phosphorylaton at different time points. Data are mean values ± SEM. *P<0.001, using two-tailed <i>t</i> test.</p

Src activation is abrogated in the presence of β1 blocking mab.

<p>(A) Cultured mouse podocyte lysates show decrease in Src Y416 phosphorylation in the presence of β1 blocking mab suggesting decreased activation of Src kinases when integrin β1 activation is prevented. Mouse IgG was used as control. (B) Bands densitometry using ImageJ software for 4 separate experiments showing increase in nephrin and Src Y416 tyrosine phosphorylation, as well as activated β1 integrin at different time points. Data are mean values ± SEM. *P<0.001, using two-tailed <i>t</i> test.</p