Figure 2.

VPS4A Disease-Associated Variants Have a Dominant-Negative Effect on Endosomal Morphology

(A) Representative VPS4A immunoblots of fibroblast cell lysates from a proband (proband 1) with the p.Arg284Trp sequence change and her parents (control 1 and control 2), and from a proband (proband 2) with the p.Arg284Gly sequence change.

(B) Immunoblot band intensities from three such experiments were quantified, normalized to GAPDH loading control values, and plotted in the corresponding graph.

(C–I) HeLa cells were transfected with constructs expressing wild-type myc-VPS4A, myc-VPS4A containing the rationally designed ATPase-defective p.Glu228Gln mutant, or myc-VPS4A harboring the sequence changes identified in probands. Cells were fixed, labeled with anti-Myc and anti-RAB5 antibodies, and visualized with confocal microscopy. The inset panels show higher magnification views of the boxed regions; examples of large vacuolar endosomal structures are shown.

(J–L) Cultured fibroblasts from the control subjects and probands indicated were fixed, labeled with EEA1 (early endosomes) (J), CD63 (preferentially labels late endosomes) (K) and LAMP1 (predominantly labels lysosomes) (L), then visualized by widefield immunofluorescence microscopy. The percentage of cells with an endosomal organelle over a nominal cut-off size and the number of labeled organelles per cell was counted in n = 3 biological repeats for each marker (in 100 cells per experimental condition in each repeat), then quantified in the corresponding charts. Bars in all plots show mean ± SEM, p values calculated by one-way ANOVA with Tukey’s post hoc test for repeated-measures. Scale bars = 10 μm.