The human nephrin Y1139RSL motif is essential for podocyte foot process organization and slit diaphragm formation during glomerular development

Abstract

Nephrin is an immunoglobulin-type cell-adhesion molecule with a key role in the glomerular interpodocyte slit diaphragm. Mutations in the nephrin gene are associated with defects in the slit diaphragm, leading to early-onset nephrotic syndrome, typically resistant to treatment. Although the endocytic trafficking of nephrin is essential for the assembly of the slit diaphragm, nephrin's specific endocytic motifs remain unknown. To search for endocytic motifs, here we performed a multisequence alignment of nephrin and identified a canonical YXXØ-type motif, Y1139RSL, in the nephrin cytoplasmic tail, expressed only in primates. Using site-directed mutagenesis, various biochemical methods, single-plane illumination microscopy, a human podocyte line, and a human nephrin-expressing zebrafish model, we found that Y1139RSL is a novel endocytic motif and a structural element for clathrin-mediated nephrin endocytosis that functions as a phosphorylation-sensitive signal. We observed that Y1139RSL motif–mediated endocytosis helps to localize nephrin to specialized plasma membrane domains in podocytes and is essential for normal foot process organization into a functional slit diaphragm between neighboring foot processes in zebrafish. The importance of nephrin Y1139RSL for healthy podocyte development was supported by population-level analyses of genetic variations at this motif, revealing that such variations are very rare, suggesting that mutations in this motif have autosomal-recessive negative effects on kidney health. These findings expand our understanding of the mechanism underlying nephrin endocytosis and may lead to improved diagnostic tools or therapeutic strategies for managing early-onset, treatment-resistant nephrotic syndrome.

Multivalent nephrin/Nck interactions define a threshold for clustering and tyrosine-dependent nephrin endocytosis

“…The association of nephrin phosphorylation and clustering with actin assembly has been established; however, their connection with endocytosis is less well understood (Swiatecka-Urban, 2017). Several studies have shown that nephrin phosphorylation regulates its trafficking (Espiritu et al, 2019;Martin et al, 2018;Qin et al, 2009;Quack et al, 2006), and that dynamin is required for both clathrin-dependent and raft-mediated nephrin endocytosis (Espiritu et al, 2019;Qin et al, 2009). Consistent with these findings, we show that Fyn induces removal of nephrin from the cell surface, and that this effect is blocked with both the nephrin-Y3F mutant and the dynamin K44A mutant.…”

“…By contrast, clathrin-mediated endocytosis is more rapid and may instead regulate steady-state recycling of nephrin (Espiritu et al, 2019;Satoh et al, 2014;Soda et al, 2012). We demonstrate that clathrin associates with nephrin clusters, and a recent study highlights a putative interaction between nephrin and clathrin (Espiritu et al, 2019). Altogether, our results support the cooperation of nephrin phosphorylation and endocytosis in slit diaphragm remodeling, and suggest that Nck functions at the interface of these signaling pathways.…”

“…Nephrin can be tethered by podocin into raft-like membrane clusters (Schwarz et al, 2001;Simons et al, 2001), and raft-mediated endocytosis has been proposed as a mechanism of slow internalization of phosphorylated nephrin following signal activation (Lahdenperä et al, 2003;Qin et al, 2009). By contrast, clathrin-mediated endocytosis is more rapid and may instead regulate steady-state recycling of nephrin (Espiritu et al, 2019;Satoh et al, 2014;Soda et al, 2012). We demonstrate that clathrin associates with nephrin clusters, and a recent study highlights a putative interaction between nephrin and clathrin (Espiritu et al, 2019).…”

Podocyte Endocytosis in Regulating the Glomerular Filtration Barrier

“…Using site-directed mutagenesis, various biochemical methods, single-plane illumination microscopy, and a human nephrin-expressing zebrafish model, canonical Y XX Ø-type motif, Y 1139 RSL, was demonstrated as an endocytic motif for clathrin-mediated nephrin endocytosis. Y 1139 RSL motif–mediated endocytosis facilitates nephrin delivery leading to foot process organization and a functional slit diaphragm in a human nephrin-expressing zebrafish model ( 112 ). Phosphorylation (activation) and dephosphorylation (deactivation) of nephrin have been identified as an essential posttranslational event that maintains proper podocyte structure and function.…”