Pedigree diagrams of families FGBR and FGJN. Pedigree identifier and the specific mutations segregating in each family are shown.

a. Disease-segregating INF2 mutations shown aligned with wild-type INF2 protein sequence from humans, chimpanzee, mouse, rat, opossum, and zebrafish. All of these disease mutations occur in evolutionarily conserved residues within the DID.
b. Schematic showing INF2 protein domain structure and location of mutations.
c, d, and e: Model of mouse INF2 amino acids 1–330, based on the structure of mDia1 (1). Mutated residues are shown in red, and residues important for the interaction with DAD are shown in blue.
c. View of mDia1 showing the positions of A13 and R218 (red). Residues important for the direct interaction with DAD are shown in blue, including R106 (corresponding to K213 in mDia1), N110 (corresponding to N217 in mDia1), A149 (corresponding to A256 in mDia1), and I152 (corresponding to I259 in mDia1). Based on the crystal structure of the mDia1 DID/DAD complex (reference 22), the alpha helical INF2 DAD is predicted to lie in the pocket containing these residues, with its N-terminus (D974) contacting R106 and N110, and L986 contacting A149 and I152. In this model, we predict that R218 would contact residues C-terminal to L986.
d. Close-up of the portion of the INF2 region predicted to interact with the DAD.
e. 180 degree rotation of the structure shown in F1, showing L42, S186, and E220.

a, b: Kidney biopsy findings in an affected member of family FSB with an E220K INF2 mutation. This patient was 21 years of age at the time of biopsy with an estimated glomerular filtration rate (GFR) of 96 ml/min/1.73m². She had 3+ urine protein and no hematuria. She developed ESRD seven years after the biopsy was performed. a. Light micrograph (PAS) showing focal global and segmental glomerulosclerosis. b. Electron micrograph showing segmental foot process effacement in some loops (arrowheads) and focally irregular morphology of preserved foot processes. Inset: Higher magnification electron micrograph showing foot processes en face, projecting from a major process. The foot processes appear irregular and jagged, often with prominent longitudinal actin bundles. c, d: Kidney biopsy findings from a different INF2 mutant patient with an R218Q mutation, from family FGJN. This patient was 26 years old at the time of biopsy. Estimated GFR was 46 ml/min/1.73m² and urine showed 3+ protein and trace blood at the time. c: Light micrograph (PAS) showing focal and segmental glomerulosclerosis, with moderate chronic parenchymal damage. d: Electron micrograph showing segmental foot process effacement (arrowheads).

a. RNA in situ hybridization in adult human kidney with digoxigenin-labeled probe targeted against INF2 mRNA, followed by immunohistochemical staining with labeled antibody to digoxigenin. INF2 mRNA expression is apparent in podocytes as well as some tubule cells. Sense control experiment is shown on the right. b. Immunofluoresence staining of mouse kidney with DAPI (blue) and antibodies against nephrin (red) and INF2 (green). INF2 staining is observed in the glomeruli in an epithelial cell pattern. Arrows show areas of nephrin and INF2 colocalization. Arrowhead shows a cell expressing INF2 but not nephrin. d. Northern blot. Gel lanes: 1, Brain; 2, Placenta; 3, Skeletal muscle; 4, Heart; 5, Kidney; 6, Pancreas; 7, Liver; 8, Lung; 9, Spleen; 10, Colon.

Localization of INF2 variants in undifferentiated cultured human podocytes. Cells were cultured on coverslips coated with type I collagen. INF2 is visualized using a Cy3 conjugated antibody against its C-terminus 7. FITC phalloidin was used to stain F-actin, and nuclei are stained with DAPI. First column: cells expressing wild-type and three different mutant forms of INF2. Second column: INF2 channel. Third column: Phalloidin. Rows: results for WT and three mutant forms of INF2: E184K, S186P, and R218Q. E184K and R218Q mutants show diffuse localization of INF2 and F-actin compared with the perinuclear INF2 staining seen with the WT expressing cells. The S186P looked similar to WT expressing cells, but INF2 expression was more vermiform in appearance. In all three mutants, stress fibers and cortical actin appeared to be less prominent.