Functional Validation of an Alpha-Actinin-4 Mutation as a Potential Cause of an Aggressive Presentation of Adolescent Focal Segmental Glomerulosclerosis: Implications for Genetic Testing - Fig 2

<p>(A), pedigree of the patient and nuclear family. Partial ACTN4 genomic sequence from the patient (red circle) shows a heterozygous T793C substitution that results in a Y265H ACTN4 amino acid substitution. The patient’s mother (white circle) does not carry this mutation. The genotype of the father (white square) is unknown. (B), mutation prediction software results for Y265H ACTN4 mutation and sequence alignment showing sequence conservation of ACTN4 Y265 across difference species. (C), representative image of coomassie blue stained SDS-PAGE gel showing an F-actin bundling experiment. The distribution of ACTN4 and F-actin in supernatant (S) and pellet (P) after centrifugation is shown. Arrows point out the bundled F-actin in the pellet. 55% of F-actin (42kDa) is bundled when crosslinked by K255E ACTN4 and Y265H ACTN4 proteins while only 32% of F-actin is bundled when crosslinked by WT ACTN4. (D), representative immunofluorescence staining images. Rhodamine phalloidin stains actin (red). GFP tagged ACTN4 is green. Hoechst 33342 stains the nuclei (blue). K255E ACTN4 and Y265H ACTN4 form aggregates within the cells. Scale bar is 5μm shown in WT merged image. 2E, representative contractile force maps for podocyte expressing the different forms of ACTN4. Different colors correlate with different levels of force, with red being the largest degree of force. Arrows are force vectors. Bright field image of each cell is included in top left of each force map. Root mean square value (RMST, an index of contractile force) is plotted. Data are from one of the three independent experiments, and are expressed as mean ± SEM. *P<0.05 indicate significant difference from WT.