Giant Rab7 vesicular structures exhibit CD63 colocalization and CHQ-sensitive intraluminal CD63 bodies, suggesting an endolysosomal origin. (A) HEK293A cells expressing endogenous TDP-43 (WT) or TDP-43-GFP (pseudocolored white in merge) were immunostained for Rab7 (pseudocolored red) and CD63 (pseudocolored green). Percentages in zoom panels indicate Rab7 foci that overlap with CD63 foci. (B) TDP-43-GFP (pseudocolored white) expressing cells were immunostained for Rab7 (pseudocolored red in merge) and CD63 (pseudocolored green in merge) in the presence or absence of CHQ. Inclusion of multiple rows reflects diverse phenotypes observed. Green arrowheads indicate CD63 foci on the intraluminal Rab7 vesicular membrane (top row) or fully within Rab7 vesicles (second and fourth row). Red arrowheads indicate Rab7 puncta fully within Rab 7 vesicles (second row). White arrowheads indicate TDP-43 puncta within Rab7 vesicles (second and fourth row; bottom right in latter case), on the external Rab7 vesicular membrane (fifth row), or in distinct cytoplasmic puncta (fourth and sixth row). Scale bar, 10 µm. (C) Quantification of CD63 signal (average and by bins) within the lumen of Rab7 vesicles. *, P < 0.05 by Mann–Whitney U test. (D) Average Rab7 vesicle lumen area ± CHQ. ***, P < 0.001 by Mann–Whitney U test. (E) Percentage of TDP-43 localization phenotypes relative to Rab7 and CD63, binned by category. Error bars in all graphical panels represent standard error of the mean.