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Figure 1. Nef-DKQTLL mutant colocalizes with clathrin at the plasma membrane. HeLa cells transiently expressing the DKQTLL Nef substitution mutant (A) and DsRed-clathrin (B) were imaged by TIR-FM as in Fig. 2. Insets show higher magnification of a representative area and arrows stress colocalizing spots. Scale bar, 10 μm.

Figure 2. Distribution of the Nef-ERQPLL mutant within dynamic clathrin spot populations. Live HeLa cells expressing either wild-type (black bars) or ERQPLL (grey bars) GFP-tagged Nef fusions in combination with DsRed-clathrin were imaged by TIR-FM. The presence of Nef-GFP inside 40 CS (from more than 5 cells) from each CS population was determined using MetaMorph. Results are expressed as the percentage of clathrin spots of each population that contain Nef (see Fig. 3).

Figure 3: Expression of Nef does not increase the number of clathrin spots at the plasma membrane. HeLa cells transiently expressing wild-type (blue) or LL/AA (white) Nef GFP fusion together with dsRed-clathrin, or CD4-wt (red) or CD4- D cyt (grey) HeLa cell lines expressing Nef-GFP fusions together with DsRed-clathrin were imaged by TIR-FM. For each cell (n=4), the total number of clathrin spots present at the adherent membrane was quantified and then normalized to arbitrary surface unit.

Video 1: Time-lapse movie showing a disappearing spot imaged by TIR-FM which contained both clathrin (top) and Nef (bottom). ~ 0.3 s/frame.

Video 2: Time-lapse movie showing a laterally mobile spot imaged by TIR-FM which contained both clathrin (top) and Nef (bottom). ~ 0.3 s/frame.

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tra_525_sm_8_1cfig2.jpg208KSupporting info item
tra_525_sm_8_1cfig3.jpg413KSupporting info item
tra_525_sm_8_1cmov1.mpeg42KSupporting info item
tra_525_sm_8_1cmov2.mpeg687KSupporting info item

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