Fig 2: The NPC subunit NUP188 binds to Nesprin-2 and promotes SV40 nuclear entry.(A) CV-1 cells were infected with SV40 (MOI ~1) and treated with either a dimethyl sulfoxide (DMSO) control or importazole. At 24 hpi, cells were fixed and stained for large TAg. Data were normalized to the DMSO control. (B) Unique peptides corresponding to SV40-interacting host proteins identified by IP mass spectrometry [32]. (C) CV-1 cells were transfected with scrambled control siRNA, 50 nM siRNA against NUP35, NUP88, NUP93, NUP98, and NUP133, or 10 nM siRNA against NUP188 for 48 h. Protein levels were assessed by immunoblotting. Hsp90 was used as a loading control. (D) CV-1 cells were transfected as in (C) and infected with SV40 (MOI ~1) for 24 h. Cells were fixed and stained for large TAg. Data were normalized to the scrambled control. (E) CV-1 cells were infected with SV40 (MOI ~25) for 20 h. Endogenous NUP188 was immunoprecipitated (IP) from whole-cell extracts and the eluted samples subjected to SDS-PAGE followed by immunoblotting for VP1. (F) CV-1 cells were infected with SV40 (MOI ~25) for 20 h. Endogenous Nesprin-2 was immunoprecipitated (IP) from whole-cell extracts and eluted samples subjected to SDS-PAGE followed by immunoblotting for NUP188 and BICD2. (G) CV-1 cells were transfected with either the scrambled control siRNA or siRNA against Nesprin-2 for 48 h. Cells were then infected with SV40 (MOI ~25) for 20 h and endogenous NUP188 immunoprecipitated from whole-cell extracts. The samples were subjected to SDS-PAGE followed by immunoblotting for VP1. (H) As in (G), except samples were immunoblotted for NUP93. (I) Confocal analysis of CV-1 cells that were transfected with either scramble control siRNA or siRNA against NUP188 for 48 h and then infected with SV40 (MOI ~25) for 20 h in the presence of 1 μM actinomycin D (ActD). Cells were stained with anti-VP2/3 (green) and anti-MAB414 that stains the NPC (red). Cells were counterstained with DAPI (blue). White arrow = VP2/3 at nuclear membrane, orange arrow = VP3 inside the nucleus. (J) Quantification of the percentage of cells in (I) with VP2/3 at the nuclear membrane normalized to the scrambled control. Actual values are shown in parenthesis. (K) As in J, except the percentage of cells with VP3 inside of the nucleus is quantified. Actual values are shown in parenthesis. Values are averages of the means (n = 3) ± SD. A standard Student’s t test was used to determine statistical significance. **, P ≤ 0.005; ***, P ≤ 0.0005; ****, P ≤ 0.0001. Scale bars, 10 μm.

Fig 3: Knockout of NUP133 impairs nuclear pore complex formation in podocytes. (a) Schematic of the nuclear pore complex (NPC) and nuclear pore proteins (NUPs), with known causative mutations for steroid-resistant nephrotic syndrome (SRNS). (b) Schematic illustrating the generation of two independent monoclonal NUP133 knockout (KO) podocyte cell lines by CRISPR/Cas9 genome editing. (c) Western blot analysis confirmed the loss of NUP133 in knockout cell lines and demonstrates the partial degradation of the Y-complex nuclear pore protein NUP107. In contrast, mAb414 (recognizing NUP358, NUP214, NUP153, and NUP62) [48,49] detected no altered protein levels. Vinculin (VCL) and alpha-tubulin (TUBA) served as loading controls. (d) Immunofluorescence analysis of mature NPCs in the nuclear envelope, by NUP133 and mAb414, confirmed loss of NUP133 expression and strongly reduced formation of mAb414 positive NPCs in the nuclear envelope. Concomitant increase of mAb414 positive vesicles in the cytoplasm was observed. Nuclei (dsDNA) were co-stained by Hoechst 33342 and whole cells by beta-Actin (ACTB). Dotted lines indicate the nuclear circumference; maximum intensity projections of z-stacks from whole nuclei are shown.

Fig 4: NUP133 variants do not significantly alter transcriptome composition. (a-1,a-2,a-3) Western blot analysis of NUP133 KO, WT, and mutant cell lines for PPME1 and COL18A1. Alpha-tubulin (TUBA) was used as loading control and three independent replicates were analyzed (scatterplots; n. s.—non-significant). (b) Schematic of the experimental setup for transcriptome analysis of NUP133 KO-1 podocytes, expressing either WT or mutation carrying NUP133 cDNAs. Three replicates, per genotype and clone, were sequenced, and mutant variants were compared to NUP133 WT. (c-1,c-2) Volcano plot of differential expression analysis for NUP133 Mutant-1 or Mutant-2 versus NUP133 WT (rescue) podocytes. Red dots indicate 45 differential expressed gene transcripts for Mutant-1 (c-1), with 37 for Mutant-2 ((c-2); p-value < 0.01). (d) Heat map showing Z-scores for these 77 by at least one mutant variant regulated transcripts, as well as unsupervised grouping of analyzed samples.

Fig 5: Loss of NUP133 disturbs dynamic cytoskeleton regulation in podocytes. (a–c) Cell spreading analysis of NUP133 WT, KO, and mutant cell lines showed reduced spreading capacity of KO and mutant podocytes (F-actin was stained by phalloidin and nuclei by Hoechst 33342). Mean cell areas, from at least three independent experiments per genotype and clone, are shown (scatterplots indicate mean values of independent experiments, at least 100 cells per replicate were analyzed; n. s.—non-significant). (d) Representative GSEA plots of enriched gene ontology (GO) gene sets, correlating with cell adhesion, protrusion, and polarization. (e–g-2) Volcano plots of differential expression analysis of NUP133 KO-1&2 versus WT-1&2 clones. Red dots indicate significantly regulated transcripts (adjusted p-value < 0.05). Analysis for consensus adhesome proteins, RhoGAP&RhoGEFs, and podocyte-specific small RhoGTPase affinity interactors demonstrated slight regulation of adhesome genes, but marked regulation of small RhoGTPase signaling pathways. (g-2) Heat map of altered RhoGTPase affinity interactors identified in (g-1), as well as the corresponding RhoGTPases demonstrates regulation of RhoA, Rac1, and Cdc42 signaling pathways.