Figure 7.
Figure 7. Down-regulation of NEEP21 leads to missorting of endosomal NgCAM to the somatodendritic surface and to lysosomes. (A) The A/D PI was determined for NgCAMY33A at steady state (left) and for NgCAM for endosomal recycling to the plasma membrane (t = 2.5 h; right) in cells coexpressing GFP as controls (black) or AS-NEEP21 (gray). Error bars indicate SEM; **, statistical significance from GFP controls at P < 0.001. n = 4 independent experiments scoring 12–17 cells per experiment for each condition. (B) The extent of anti-NgCAM antibody loading at t = 0 into somatic endosomes was quantified in control cells (black) or AS-NEEP-GFP coexpressing neurons (gray). Values were normalized to GFP-expressing control cells (Ab loading). (C) The presence of endocytosed NgCAM in somatic endosomes (soma retention) was quantitated at t = 0 and t = 40 min for cells coexpressing either GFP as control (black) or antisense NEEP21-GFP (AS-NEEP21; gray). Values were normalized to the t = 0 levels for each condition. n = 4 independent experiments, scoring 12–17 cells per experiment for each condition. Error bars indicate SEM. *, statistical significance from controls at P < 0.01; #, statistical significance from controls at P = 0.055 (Mann Whitney U test). (D) The extent of colocalization between the lysosomal lgp120 (blue) and endocytosed NgCAM (90-min chase; red) was visualized for cells coexpressing NgCAM and GFP (top) or NgCAM and AS-NEEP21-GFP (bottom). Single channel and merged images of the soma region are shown separately on the right. In these panels, lgp120 is displayed in aqua. Overlap with the red channel appears white. (E) Quantification of the number of colocalizing puncta from the experiment in D. n = 3 independent experiments scoring 12–17 cells per experiment and condition. Error bars indicate SEM; **, statistical significance from GFP controls at P < 0.001. (F) Localization of internalized Tf (aqua) and endocytosed NgCAM (red; 20-min load at t = 0) in cells coexpressing NgCAM and AS-NEEP21-GFP. The right panel shows the single channel for Tf. Although endocytosed NgCAM can be detected in endosomes along axons (red; left, arrows), no missorting of Tf (aqua) to the axon (arrows) is observed.

Down-regulation of NEEP21 leads to missorting of endosomal NgCAM to the somatodendritic surface and to lysosomes. (A) The A/D PI was determined for NgCAMY33A at steady state (left) and for NgCAM for endosomal recycling to the plasma membrane (t = 2.5 h; right) in cells coexpressing GFP as controls (black) or AS-NEEP21 (gray). Error bars indicate SEM; **, statistical significance from GFP controls at P < 0.001. n = 4 independent experiments scoring 12–17 cells per experiment for each condition. (B) The extent of anti-NgCAM antibody loading at t = 0 into somatic endosomes was quantified in control cells (black) or AS-NEEP-GFP coexpressing neurons (gray). Values were normalized to GFP-expressing control cells (Ab loading). (C) The presence of endocytosed NgCAM in somatic endosomes (soma retention) was quantitated at t = 0 and t = 40 min for cells coexpressing either GFP as control (black) or antisense NEEP21-GFP (AS-NEEP21; gray). Values were normalized to the t = 0 levels for each condition. n = 4 independent experiments, scoring 12–17 cells per experiment for each condition. Error bars indicate SEM. *, statistical significance from controls at P < 0.01; #, statistical significance from controls at P = 0.055 (Mann Whitney U test). (D) The extent of colocalization between the lysosomal lgp120 (blue) and endocytosed NgCAM (90-min chase; red) was visualized for cells coexpressing NgCAM and GFP (top) or NgCAM and AS-NEEP21-GFP (bottom). Single channel and merged images of the soma region are shown separately on the right. In these panels, lgp120 is displayed in aqua. Overlap with the red channel appears white. (E) Quantification of the number of colocalizing puncta from the experiment in D. n = 3 independent experiments scoring 12–17 cells per experiment and condition. Error bars indicate SEM; **, statistical significance from GFP controls at P < 0.001. (F) Localization of internalized Tf (aqua) and endocytosed NgCAM (red; 20-min load at t = 0) in cells coexpressing NgCAM and AS-NEEP21-GFP. The right panel shows the single channel for Tf. Although endocytosed NgCAM can be detected in endosomes along axons (red; left, arrows), no missorting of Tf (aqua) to the axon (arrows) is observed.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal