Fig. 3

The artificial stabilization of CapZ on early endosomes inhibits endosomal maturation. A Schematic depiction of the strategy to induce the stable association of CapZ with early endosomes by conjugating RAB5 with FRB and CapZβ with FKBP in the presence of rapamycin (1 mM). B, C HeLa cells were transiently transfected with FRB-RAB5 and FKBP-CapZβ, and then they were incubated with rapamycin (1 mM) for 12 h to induce an interaction between RAB5 and CapZ, followed by anti-EEA1 immunostaining and confocal imaging (B). The colocalization coefficients (MCC) of RAB5A, CapZ, or EEA1 were quantified (C). D, E HeLa cells were transiently transfected with FRB-RAB5, FKBP-CapZβ, HA-Rabex-5 (D), and/or Flag-Rabaptin-5 (E); then, they were incubated with rapamycin (1 mM) for 12 h followed by anti-HA or anti-Flag immunostaining and confocal imaging. The colocalization coefficients (MCC) of RAB5A, CapZ, Rabex-5, or Rabaptin-5 were quantified. F HeLa cells were transiently transfected with FRB-RAB5 and FKBP-CapZβ, and then they were incubated with rapamycin (1 mM) for 12 h, followed by anti-Rab7 immunostaining and confocal imaging. The colocalization coefficients (MCC) of RAB5A, CapZ, or Rab7 were quantified. G Control or FRB-RAB5A/FKBP-CapZβ-expressing HeLa cells were incubated with or without rapamycin (1 mM) for 12 h, and they were then treated with EGF for 0 h, 0.5 h or 1 h. The cell lysates were subjected to EGFR immunoblot analysis. The scale bars are 5 μm. The blots, images, and graphs represent data from at least three independent experiments. Data quantifications were analyzed using unpaired Student’s t-test (two-group comparison in C, D, E, and F) or ANOVA (multiple comparisons in G) and expressed as mean ± S.D., *P < 0.05, **P < 0.01, ***P < 0.001