Rab21 regulates caveolin-1-mediated endocytic trafficking to promote immature neurite pruning (2022)
Figure 5

Figure 5. Rab21 promotes the internalization and early endosomal trafficking of N-cadherin.

(A) Primary cortical neurons from E15 cerebral cortices transfected with CMV=EGFP-Rab21, incubated for two days in vitro and stained with the indicated antibodies. Blue alone channels are shown in black and white images. Insets are high magnification images indicated by white or blue rectangles.

(B) Immature neurons in the IZ of the cerebral cortices at E17, electroporated with pCAG-EGFP at E14. Frozen sections were immunostained with the indicated antibodies. The images are obtained with high-resolution microscopy (Nikon). Blue alone channels are shown in black and white images. The lower panels are high magnification images.

(C) The box-and-whisker plot shows the ratio of cell surface to total N-cadherin in primary cortical neurons (2 days in vitro). Control: n = 85 cells from 3 biological replicates, Rab21-sh115: n = 143 cells from 3 biological replicates.

(D-F) Primary cortical neurons from E15 cerebral cortices were transfected with pCAG-PM-mAG1, incubated for two days in vitro and stained with the indicated antibodies. Blue alone channels are shown in black and white images. Arrowheads indicate accumulation of N-cadherin at the plasma membrane. Lower panels in (D) are high magnification images indicated by white or blue rectangles. The images are obtained with high-resolution microscopy (Nikon). The graphs show the ratios of the N-cadherin staining signals in the plasma membrane (E) and the APPL1-positive early endosomes (F) to total fluorescence intensities of N-cadherin in each primary cortical neuron. Each score represents the mean of ratios with the individual points. Control: n = 20 cells (E) or 33 cells (F), Rab21-sh115: n = 19 cells (E) or 30 cells (F).

(G-H) Immature neurons in the IZ of the cerebral cortices at E17, electroporated with the indicated plasmids plus pCAG-PM-mAG1 and pCAG-HA-N-cadherin at E14. Frozen sections were immunostained with anti-mAG1 and anti-HA antibodies. Arrow and arrowheads indicate the accumulation of HA-N-cadherin at the perinuclear regions or the plasma membrane, respectively. The box-and-whisker plot shows the ratio of the HA-N-cadherin staining signals in the plasma membrane to total fluorescence intensities of HA-N-cadherin in each immature neuron in vivo. Control: n = 77 cells from 3 biological replicates, Rab21-sh115: n = 108 cells from 3 biological replicates.

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

(J) The ratio of locomoting neurons with more than three primary neurites in the IZ. Each score represents the mean of ratios with the individual points. Control: n = 5 brains, Rab21-sh115: n = 7 brains, Rab21-sh115+Ncad-sh1023: n = 5 brains.

Data information: (C, H) 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. In (C) significance compared to control was determined by Welch's t test (P = 0.0003106). **: P<0.01. In (H) significance compared to control was determined by Student's t test (P = 3.951E-28). **: P<0.01. (E-F) Significance compared to control was determined by Student's t test (E: P = 0.0005588, F: P = 0.00003682). **: P<0.01. (J) Significance was determined by Kruskal-Wallis test with post hoc Steel-Dwass test. *: < the critical value at 5%. Scale bars: 10 μm in (A), 4 μm in (upper panels in B), 0.2 μm in (lower panels in B), 5 μm in (D), 1 μm in (lower panels in D), 4 μm in (G), 10 μm in (I).