CD16/7-nephrin(WT)^IC phosphorylation and Nck recruitment increases over time following antibody-induced clustering. (A) CD16/7-nephrin(WT)^IC was cotransfected with Flag-Nck1 into Nck-null MEFs. Anti-CD16 antibodies, followed by goat anti-mouse Alexa Fluor 488-conjugated antibody, were added to the cell growth medium to induce clustering for the indicated amounts of time (shown in minutes). Localization of nephrin (green), Nck (anti-Flag [blue]) and F-actin (phalloidin [red]) is shown. Scale bars = 10 μm. (B) GFP-tagged CD16/7-nephrin(WT)^IC was immunoprecipitated (IP) from cells clustered with anti-CD16 for the indicated time periods. Immunoprecipitates were immunoblotted (IB) with antibodies to GFP to identify CD16/7-nephrin(WT)^IC (top panel), anti-pTyr to identify tyrosine phosphorylated CD16/7-nephrin(WT)^IC (second panel), and anti-Flag to identify CD16/7-nephrin(WT)^IC-associated Nck1 (third panel). The bottom panel shows Nck1 levels in whole-cell lysates. The time of incubation with clustering antibodies is indicated above the panels.

Nephrin-induced actin polymerization is dependent on the SH2, SH3-2, and SH3-3 domains of Nck. (A) The SH2 and SH3 domains of Nck1 were rendered nonfunctional by specific amino acid substitutions. The various constructs used are depicted, with the mutations indicated in red. (B) Nck-null MEFs were cotransfected with myc-tagged CD16/7-nephrin(WT)^IC, GFP-tagged N-WASP, and either WT or mutant forms of Flag-Nck1, as indicated at the top of the panels. GFP-N-WASP was immunoprecipitated (IP) from cells expressing CD16/7-nephrin(WT)^IC-myc, either unclustered (−) or clustered with anti-CD16 antibodies for 10 min (+). Immunoprecipitates were immunoblotted (IB) with antibodies to N-WASP to identify GFP-N-WASP (top panel) and Flag to identify GFP-N-WASP-associated Nck1 proteins (middle panels). The middle panels show two different exposures of the same blot. The bottom panel shows Nck1 levels in whole-cell lysates. (C) Nck-null MEFs were cotransfected with GFP-tagged CD16/7-nephrin(WT)^IC and mutant forms of Flag-Nck1. Cells were incubated with anti-CD16 antibody for 30 min, followed by a 30-min incubation with goat anti-mouse Alexa Fluor 488 antibodies. The resulting localization of CD16/7-nephrin(WT)^IC (green), Flag-Nck (anti-Flag [blue]), and F-actin (phalloidin [red]) is shown. Scale bars = 10 μm. (D) Cells coexpressing GFP-tagged CD16/7-nephrin(WT)^IC with various Flag-Nck1 mutants were analyzed for the presence of actin polymerization at clusters. The numbers of cells that showed actin polymerization at CD16/7-nephrin(WT)^IC clusters were scored and are presented as percentages of total cells analyzed. Numbers above each bar indicate the total number of cells counted. Error bars represent standard deviations calculated from at least four independent experiments. Asterisks denote differences with statistical significance (P < 0.005) relative to the WT, as determined by the Student t test.

Combining nephrin and Nck mutants suppresses localized actin polymerization. Cells coexpressing Flag-Nck1 and CD16/7-nephrin^IC were analyzed for the presence of actin polymerization at clusters. The various combinations of nephrin and Nck1 mutants are indicated. The numbers of cells that showed actin polymerization at CD16/7-nephrin^IC clusters were scored and are presented as percentages of total cells counted. Numbers above bars indicate the total numbers of cells counted. Error bars represent standard deviations calculated from at least four independent experiments. Asterisks denote differences with statistical significances (P < 0.005) relative to the WT, as determined by the Student t test.

(A) Nephrin is a transmembrane protein consisting of eight extracellular immunoglobulin (Ig)-like domains, a fibronectin type three (FNIII) domain, a transmembrane region, and an intracellular region. Nephrin is located at the slit diaphragm, where it interacts with nephrin molecules from adjacent foot processes of podocyte cells. Upon tyrosine phosphorylation by Fyn, nephrin recruits the adaptor protein Nck, which is required for proper foot process formation. (B) An alignment of the intracellular regions of human and mouse nephrin. The tyrosine residues located in the three YDXV motifs are shown (Y is shown in red). The sequences of the mouse peptides used for binding studies are underlined. Numbers to the left of the sequence denote amino acid residues. (C) A schematic of the CD16/7-nephrin^IC clustering system is shown. CD16/7-nephrin^IC fusion protein localizes to the plasma membrane in cells. Upon the addition of anti-CD16 antibodies to the cellular growth medium, the fusion protein clusters, causing nephrin^IC to become phosphorylated and recruit Nck, leading to localized polymerization of actin.

Nephrin clusters are internalized. CD16/7-nephrin(WT)^IC was cotransfected with Flag-Nck1 into Nck-null MEFs. Anti-CD16 antibodies, followed by goat anti-mouse Alexa Fluor 488-conjugated antibodies (green), were added to the cell growth medium to induce clustering for the indicated amounts of time (shown in minutes). Cells were then fixed and stained with donkey anti-goat Texas Red-conjugated antibodies, in the absence of membrane permeabilization, to visualize extracellular clusters (shown in red). Yellow clusters represent nephrin present at the cellular membrane, while green clusters represent internalized nephrin. Scale bars = 10 μm.

A single YDXV motif on nephrin is sufficient to recruit Nck and induce actin polymerization. (A) Nck-null MEFs were transfected with either GFP-tagged CD16/7-nephrin(WT)^IC or mutants with two or three nephrin YDXV tyrosine residues mutated to phenylalanine as indicated, together with WT Flag-Nck1. Cells were incubated with anti-CD16 antibodies for 10 min (+). GFP-tagged CD16/7-nephrin(WT)^IC was immunoprecipitated (IP), and immunoprecipitates were immunoblotted (IB) with antibodies to GFP to identify CD16/7-nephrin(WT)^IC (top panel) and Nck to identify CD16/7-nephrin^IC-associated Flag-Nck1 (middle panel). The bottom panel shows Flag-Nck1 levels in whole-cell lysates. (B) Cells were transfected as for panel A, and GFP-tagged CD16/7-nephrin^IC mutants were clustered with anti-CD16 antibodies for 30 min, followed by clustering with goat anti-mouse Alexa Fluor 488-conjugated antibodies for 30 min. Immunofluorescence localization of GFP-tagged CD16/7-nephrin^IC mutants (green), Flag-Nck (anti-Flag [blue]), and F-actin (phalloidin [red]) is shown. Scale bars = 10 μm. (C) Cells expressing both Flag-Nck1 and GFP-tagged CD16/7-nephrin^IC (WT or mutants) were analyzed for the presence of actin polymerization at clusters. The numbers of cells that showed actin polymerization at CD16/7-nephrin^IC clusters were scored and are presented as percentages of total cells analyzed. Numbers above bars indicate the total numbers of cells counted. Error bars represent standard deviations calculated from at least four independent experiments. Asterisks denote differences with statistical significance (P < 0.005) relative to the WT, as determined by the Student t test.

The YDXV motifs of the EPEC Tir protein and nephrin are functionally interchangeable. (A) An alignment of the sequences encompassing the YDXV motifs (boxed) of nephrin and EPEC Tir is shown. The phosphorylated Tyr is colored red. Numbers to the left of the sequence indicate the amino acid numbers of the indicated Tyr residue. (B) Nck-null MEFs were cotransfected with GFP-tagged CD16/7-nephrin(Y3F)^IC-Tir and Flag-Nck1 and incubated with anti-CD16 antibodies for 30 min, followed by a 30-min incubation with goat anti-mouse Alexa Fluor 488-conjugated antibodies. Immunofluorescence localization of CD16/7-nephrin(Y3F)^IC-Tir (green), Flag-Nck1 (anti-Flag [blue]), and F-actin (phalloidin [red]) is shown. (C) HeLa cells were infected with various strains of EPEC, either without Tir (ΔTir) or with ΔTir reconstituted with WT Tir, Tir^Y474F, Tir^Y474F-nephrin Y1217, or Tir^Y474F-nephrin Y1217F. The EPEC bacteria were visualized with anti-EPEC (blue), sites of tyrosine phosphorylation were visualized with anti-pTyr antibodies (green), and polymerized actin was visualized with phalloidin (red). Bacteria lacking Tir (ΔTir) are impaired in their abilities to adhere to host cells and, thus, show reduced levels of staining for EPEC. Scale bars = 10 μm.