Rab21 regulates caveolin-1-mediated endocytic trafficking to promote immature neurite pruning (2022)
Figure 7

Figure 7. Rab21 regulates neuronal maturation through the maintenance of caveolin-1 protein levels.

(A) Locomoting neurons in the upper IZ of the cerebral cortices at E17, electroporated with the indicated plasmids plus pCAG-EGFP at E14.

(B) The ratio of locomoting neurons with more than three primary neurites. Each score represents the mean of ratios with the individual points. Control: n = 4 brains, Rab21-sh115: n = 5 brains, Rab21-sh115+Caveolin-1: n = 6 brains.

(C) The box-and-whisker plot shows average leading process length of the locomoting neurons in the IZ. Control: n = 115 cells, Rab21-sh115: n = 188 cells, Rab21-sh115+Caveolin-1: n = 185 cells.

(D-E) Cerebral cortices at P0, electroporated with the indicated plasmids plus pCAG-EGFP at E14. The lower graphs in (D) and the graph in (E) show the estimation of cell migration, as measured by EGFP fluorescence intensities in distinct regions of the cerebral cortices using Leica SP5 software. Each bar in the graph in (E) represents the mean percentage of relative intensities. Rab21-sh115: n = 4 brains, Rab21-sh115+CAG-wt-caveolin-1: n = 6 brains. II-IV: layers II-IV of the cortical plate, V-VI: layers V-VI of the cortical plate, IZ: intermediate zone, WM: white matter, SVZ/VZ: subventricular zone/ventricular zone.

(F) Rab5 and Rab21, members of the same subfamily, mainly regulate distinct endocytic pathways, clathrin-mediated and caveolin-1-mediated, respectively, and different steps of neuronal maturation and migration. While Rab5 is required for the locomotion mode of neuronal migration Kawauchi et al., 2010(), our present study indicates that Rab21 and caveolin-1 cooperatively regulate immature neurite pruning and leading process elongation. Rab21 promotes the membrane localization of caveolin-1 and maintains the caveolin-1 protein levels, which is required for immature neurite pruning via the enhanced internalization of N-cadherin. Rab21 also has caveolin-1-independent functions in the trafficking in early endosomes.

Data information: (B) Significance was determined by Kruskal-Wallis test (P = 0.007458) with post hoc Steel-Dwass test. *: < the critical value at 5% (compared with control), n.s.: no significant differences. (C) In the box-and-whisker plots, the central band and the upper and lower sides of the boxes indicate the median and the upper and lower quartiles. The whiskers of the depicted boxplots go from the minimum to the lower quartile and from the upper quartile to the maximum. "x" indicates the average value. Significance was determined by Kruskal-Wallis test (P = 2745E-13) with post hoc Steel-Dwass test. **: < the critical value at 1%. (E) Significance compared to Rab21-sh115 was determined by Student's t test (Rab21-sh115+CAG-wt-caveolin-1 (Layer II-IV): P = 0.003543, Rab21-sh115+CAG-wt-caveolin-1 (IZ): P = 0.003405) and one-way ANOVA with post hoc Tukey-Kramer. **: P<0.01. n.s.: no significant differences, *: < the critical value at 5%, **: < the critical value at 1%. See Fig. EV2B-C for the ratio of the number of the electroporated cells in each layer. Scale bars: 10 μm in (A), 100 μm in (D).

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