Figure 4.

ANKHD1 Regulates Rab5-Positive Organelles and Recruitment at the Scission Site

(A) Confocal microscopic images of overexpressed EGFP-Rab5 in HeLa cells treated with control siRNA or ANKHD1 siRNA. Scale bars, 10 μm.

(B and C) Quantification of the number (B) and average area (C) of EGFP-Rab5 puncta per cell in (A). Data represent the mean of 21 and 18 cells from three independent experiments for control siRNA and ANKHD1 siRNA, respectively.

(D) Confocal microscopic images of overexpressed EGFP-Rab5Q79L in HeLa cells treated with control siRNA or ANKHD1 siRNA. Scale bars, 10 μm.

(E and F) Quantification of the number (E) and the average area (F) of EGFP-Rab5Q79L puncta per cell in (D). Data represent the mean of 30 and 29 cells from three independent experiments for control siRNA and ANKHD1 siRNA, respectively.

(G) Live imaging of HeLa cells co-transfected with ANKHD1 siRNA, ANKHD1-Venus×3, and mCherry-Rab5. Arrows indicate early endosome scission. Scale bar, 1 μm.

(H) Live imaging of HeLa cells co-transfected with ANKHD1 siRNA, ANKHD1-Venus×3, and mCherry-Rab5Q79L. Arrows indicate early endosome scission. Scale bar, 1 μm.

(I) Proposed function of ANKHD1. ANKHD1 is recruited to Rab5-positive early endosomes for membrane vesiculation.

All error bars represent SE. *p < 0.05 and ***p < 0.001. Statistical significance was determined with the Student's t test. ns, not significant.