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Fig. 5. Echinoid is associated with the cis-endocytosis of Dl in S2 cells. Immunohistochemical detection and confocal microscopy of N, Dl, Ed-Myc and Ed-FLAG in transiently transfected S2 cells. (A) Ed-Myc-expressing cell (green) contacting an Ed-FLAG-expressing cell (red). Ed is visible at the region of contact between the cells and in cis-endocytic vesicles (arrows). (B) Single N- (N^ICD, green) and Dl (red)-expressing cell. (C) N- (N^ICD, green), Dl- (red) and Ed-FLAG (blue)-expressing cell contacting an Ed-Myc-expressing cell. Arrows indicate cis-endocytic vesicles containing all three proteins. The anti-FLAG antibody also shows some non-specific staining of cell nuclei. (D) Single Dl- (red) and Ed-FLAG (blue)-expressing cell. Dl and Ed are frequently located in the same vesicle (arrows). (E) N- (N^ECD, green) and Ed-Myc-expressing cell (not detected) contacting a Dl- (red) and Ed-FLAG (blue)-expressing cell. Ed is cis-endocytosed with Dl as N is trans-endocytosed (arrows). Trans-endocytosis of N or Dl alone is not associated with Ed (arrowheads). (F) N (N^ECD, green) and Ed-FLAG (blue)-expressing cell contacting a Dl- (red) and Ed-Myc (not visible)-expressing cell. Ed is not trans-endocytosed with N into the signalling cell. Trans-endocytosis of N or Dl alone is not associated with Ed (arrowheads). (G) Cartoon summary of S2 cell experiments. (G') In isolated S2 cells Ed is associated with the cisendocytosis of Dl/N together (1) and Dl alone (2), but not with N alone (3). (G'') In contacting S2 cells Ed is associated with the cisendocytosis of Dl (1), which is necessary for trans-endocytosis of N^ECD during N activation (Parks et al., 2000). It is not associated with other trans-endocytosis events seen in S2 cells that are unrelated to N activation (2, 3).

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