Fig 1: CapZ controls the actin density around early endosomes.(A, B) HeLa cells were stably transfected with EGFP/mCherry or CapZa1-EGFP/CapZß-mCherry constructs. These cells were either immunostained with phalloidin (A), or with an anti-RAB5A antibody (B). The number and size of the early endosomes in these cells were quantified (B). (C) Control or CapZß-knockout HeLa cells were immunostained with antibodies against actin and RAB5A. The single plane whole-cell confocal images were then captured with a resolution of 2048×2048 pixels (left panels). The selected area in the cells was further scanned with a resolution of 4096×4096 pixels (right panels). The percentage of RAB5 puncta that are actin positive against total RAB5 puncta (MCC) in control or CapZß-knockout cells was quantified. (D, E) The control or CapZß-knockout HeLa cells were stained with antibodies against RAB5 and p16-Arc, followed by confocal imaging and colocalization coefficients (MCC or PCC) analysis of RAB5/p16-Arc (D) or N-SIM S Super-resolution imaging (E). (F) Control or CapZß-knockout HeLa cells were transiently transfected with RFP-RAB5A, and cells were then imaged without delay for 3 min by time-lapse Nikon high-speed confocal microscopy. 2D track analysis was performed using NIS-Elements AR software. The images and graphs represent data from at least three independent experiments. The data are presented as mean ± SD. The difference between the two groups was analyzed using two-tailed Student’s t-test. Differences were considered statistically significant when p<0.05. * p<0.05, ** p<0.01, and *** p<0. 001. Scale bar, 5 µm. MCC, Manders colocalization coefficient; PCC, Pearson’s correlation coefficient. Figure 3—source data 1.Data for 3A–3D, 3F.