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Files in this Data Supplement:
Fig. S1. (A) HSB-2 cells stained for the endogenous clathrin heavy chain (red), one of them expressing GFP-ClLC (green). The straight-line distribution of green and red fluorescence indicates a high statistical correlation of intensity values in the entire cell at a mid-confocal section. DAPI staining (blue), and ClHC merged with DIC images are shown. (B) HSB-2 cells expressing GFP-ClLC (green) and stained for Golgin95 (red). As expected, intracellular clathrin partially colocalizes with TGN at the uropod. (C) A375 melanoma cell transfected with GFP-ClLC (green). Numerous differently sized CS can be observed at the basal membrane (adhesion plane). The DIC image is shown, and the enlarged area of CS is outlined in blue. (D) High magnification of an isolated small CS colocalizing with one Tfn-Tx spot (red) at the plasma membrane (arrowhead). The measured length for the GFP-ClLC spot is ∼212 nm, which most probably corresponds to a single CCP. This cell is the same as that analyzed in Fig. 1A,D and Movie 1. The location of the enlarged area is shown in blue. (E) T cell transfected with GFP-ClLC (green) and labeled for CD45 membrane protein (red). A mid-uropod section is shown, where the enlarged areas appear outlined in blue. Note how differently sized CS are located adjacent to the membrane (lengths are indicated). Bars, 5 μm.
Fig. S2. (A) Co-immunoprecipitation assays to check the effects of Y-27632 and blebbistatin on the self-assembly of clathrin and AP-2, and its association. Abs against the α-adaptin and the clathrin heavy chain (X22 and AP6 Abs, respectively) were used for IP. The relative precipitated amounts of β2-adaptin and clathrin heavy and light chains were estimated by immunoblot densitometry from three independent experiments, showing no significant differences between them or to control untreated cells. (B) Effects of short-time treatments with cytochalasin D (1 hour) on CS distribution in GFP-ClLC-transfected cells plated on PLL. F-actin disruption resulted in a drastic redistribution of CS out of the trailing tail (arrowhead) and all over the cell surface, especially at the basal membrane (quantified in Fig. 6B). Lateral views, and several enlarged areas from the basal membrane of both the control and actin-disrupted cells (blue rectangles) are shown. (C) Colchicine-treated cells were allowed to take up Tfn-A488 as described for Fig. 5, fixed and surface-stained for TfnR (red). (D) Colchicine-treated cells stained for AP-2. (E) Distribution of endogenous AP-2 in blebbistatin pre-treated cells. Note that the usual polarized distribution at the uropod is lost and numerous CS labeled with α-adaptin are located at the basal membrane. Bars, 5 μm.
Fig. S3. (A,B) Tfn-uptake assay quantification; (A) control and (B) Y-27632-treated representative cells. To clearly differentiate between internal and external pools of transferrin, cells were after the Tfn (green) uptake assay, fixed and surface stained for TfnR (red). Internalized Tfn was analyzed in nonsaturated dual-channel plots as the single green pixels above the threshold (white upper left quadrant and corresponding white mask). The mean and the total intensities (au) corresponding to the white-mask pixels are indicated. Statistical analysis of data presented in Fig. 5 was performed for the gated single green events. This analysis excludes double-positive events, which correspond to surface Tfn (blue upper left quadrant and corresponding blue mask).
Movie 1. Confocal sectioning of a GFP-ClLC transfected HSB-2 cell. In vivo confocal sectioning of a T cell transfected with GFP-ClLC and pulsed with Tfn-Tx for 4 minutes. Nineteen confocal sections taken every 300 nm are shown.
Movie 2. In vivo imaging of CME during T cell migration. T cells incubated with Tfn-A488 and imaged for random migration during a 12-minute period, at intervals of 21 seconds. Follow the simultaneous migration and CME in a polarized T cell from the upper part of the image.
Movie 3. Tfn-A488 recovery after rear-membrane photo-bleaching. Post-bleaching Movie that corresponds to Fig. 4D. Surface Tfn-A488 was photo-bleached at the rear pole of the T lymphoblast (see the small uropod in the DIC image of Fig. 4D), and the post-bleaching sequence recorded at 30-second intervals over a period of 300 seconds, see Material and Methods. The first frame shown is the last pre-bleaching image, and the second frame corresponds to the first post-bleaching point (exactly 1.3 seconds after bleaching). Note the rapid recovery on the rear membrane, how the leading pseudopods begin to move at the front pole when temperature rises and, most importantly, note the scarce accumulation of fluorescent Tfn at the rear-bleached cell compared with neighboring cells. Photo-bleaching does not damage CME (see Fig. 4C). An additional distant cell has been included in Fig. 4D.
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