Rab21 regulates caveolin-1-mediated endocytic trafficking to promote immature neurite pruning (2022)
Figure 6

Figure 6. Rab21 regulates caveolin-1 membrane localization and protein levels.

(A-B) Primary cortical neurons from E15 cerebral cortices were transfected with the indicated plasmids plus pCAG-PM-mAG1 (green) and incubated for two days in vitro. Immunocytochemical analyses with anti-caveolin-1 (red) antibody were performed. The images are obtained with high-resolution microscopy (Nikon). The graph in (B) indicates the ratio of the caveolin-1 staining signals in the plasma membrane to total fluorescence intensities of caveolin-1 in each immature neuron. Each score represents the mean of ratios with the individual points. Control: n = 20 cells, Rab21-sh115: n = 22 cells, Rab21-sh115 + wt-Rab21: n = 23 cells. Overexpression of wt-Rab21 resulted in neuronal cell death, which making it difficult to determine the accurate DNA concentration of wt-Rab21 in the rescue experiments.

(C-F) Primary cortical neurons from E15 cerebral cortices were transfected with the indicated plasmids and incubated for two days in vitro. Immunoblot analyses of cell lysates with the indicated antibodies were performed. The graphs in (D) and (F) indicate the mean ratios of immunoblot band intensities of caveolin-1/β-tubulin with the individual points.

(G) Immature neurons in the IZ of the cerebral cortices at E17, electroporated with the indicated plasmids plus pCAG-EGFP at E14. Frozen sections were immunostained with the indicated antibodies. Blue alone channels are shown in black and white images.

(H-I) Primary cortical neurons from E15 cerebral cortices were transfected with the indicated plasmids, incubated for two days in vitro, treated with Bafilomycin A1 (Baf A1) for 6 h and stained with the indicated antibodies. The images are obtained with high-resolution microscopy (Nikon) and the lower panels are high magnification images of the lysosomes, indicated by white or blue rectangles. Blue alone channels are shown in black and white images. The graph in (I) shows the Pearson's correlation coefficient between caveolin-1 and Lamp1, a lysosomal marker, in control and Rab21-sh115-transfected neurons. Each score represents the mean of ratios with the individual points. Control: n = 31 cells, Rab21-sh115: n = 22 cells.

Data information: (B) Significance was determined by one-way ANOVA with post hoc Dunnett and Tukey-Kramer. **: < the critical value at 1%. Significant difference was observed between control and Rab21-sh115, but not between control and the coexpression of Rab21-sh115 and wt-Rab21, by Dunnett and Tukey-Kramer. However, no significance was observed between Rab21-sh115 alone and coexpression of Rab21-sh115 and wt-Rab21 by Tukey-Kramer. (D and F) Significance was determined by one-way ANOVA with post hoc Tukey-Kramer (D: n = 9 biological replicates) and Mann-Whitney's U test (F: P = 0.02102, n = 4 biological replicates). n.s.: no significant differences, *: < the critical value at 5%, **: < the critical value at 1%. (I) Significance was determined by Mann-Whitney's U test (P = 0.0000006290). **: P<0.01. Scale bars: 1 μm in (A, lower panels in H), 4 μm in (G), 5 μm in (upper panels in H).