FIG. 8.

The GPPX3Y motif of TEX14 inhibits the CEP55-ALIX and CEP55-TSG101 interactions and the entry of ALIX to the midbody, resulting in formation of stable intercellular bridges. (A to C) pcDNA3 vectors lacking an insert (Empty) or containing the truncated TEX14 (TX), ALIX (ALIX), TSG101(TSG), and TEX14 mutant (AAAX3A) were cotransfected into HEK293T cells along with VP16-AD-X, GAL4-BD-Y, and GAL4.31-mCherry vectors indicated at the bottom of each panel (see Fig. 6B and Table 2 for additional details). The relative ratios of the interactions of protein X and protein Y are shown. (D) Transfection of the full-length TEX14 vectors into HeLa cells. Immunofluorescence using goat anti-TEX14 and guinea pig anti-MKLP1 antibodies was performed: red, TEX14; green, MKLP1; blue, DAPI; and yellow, merged. (E and F) Cotransfection of pcDNA-YFP-full-length ALIX overexpression vector with pcDNA-mCherry-truncated GPPX3Y TEX14 (TX) (E) and TEX14 mutant AAAX3A (F) overexpression vectors into HeLa cells. The localization patterns of ALIX were microscopically examined for yellow fluorescence within a background of cells expressing TX or AAAX3A (red fluorescence). Arrow, midbody; arrowhead, ALIX. (G) Quantification of the experiment in panels E and F. The graphs were made by analyzing 1,000 double-positive cells with RFP and YFP from 11 to 13 separate experiments. The graphs show the percentage of the number of bridge containing cells/the number of double-positive RFP and YFP cells (left) and the number of ALIX localized in midbody/the number of RFP positive bridges in double-positive RFP and YFP cells (right).