Figure 4.

VPS4A Is Critical for Normal Erythroblast Cytokinesis

(A) Imaging flow cytometry (IFC) analysis of VPS4 localization in dividing erythroblasts cultured from normal human CD34⁺ cells showing β-tubulin (green), DNA/Hoechst (blue), and VPS4 (red).

(B) Representative images of erythroblasts produced from control and proband 1-derived iPSCs undergoing cytokinesis showing congruent staining of VPS4 (red) and tubulin (green) in the midbody of control cells (panel 1) but mislocalization of VPS4 relative to tubulin in p1-derived erythroblasts (panels 2 and 3). Post-mitotic erythroblasts with mutant VPS4A are frequently observed to remain connected with persistent cytoplasmic bridges, indicating abscission failure (panel 4).

(C) Analysis of VPS4 localization in midbodies demonstrated that VPS4 was present throughout the midbody in 8 out of 9 events observed in control cells, but only in 1 out of 7 events observed in the proband line.

(D) Confocal immunofluorescence imaging of an erythroblast generated in vitro from proband 1-derived iPSCs with a cytoplasmic bridge containing no DNA (blue). Scale bar 5 μm.

(E) A higher frequency of binucleated erythroblasts was observed in proband 1-derived iPSC cultures versus control (>300 cells analyzed by IFC out of two biologic repeats, mean ± SEM).

(F) Representative IFC images of normal nuclei in control erythroblasts and binucleated erythroblasts from ex vivo erythropoiesis cultures from control and proband 1-derived iPSCs.