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Fig. 1. CME components are polarized toward the uropod. (A) In vivo confocal sectioning of a representative T cell transfected with GFP-clathrin light chain (ClLC) and pulsed with Tfn-Tx (10 µg/ml). The z projection of 19 confocal sections taken each 300 nm, the lateral reconstruction, and the basal and mid-uropod planes, are shown. The selected middle section avoids most of the cytoplasmic TGN-labeling, and several CSs located at the cell periphery are enlarged below. A number of Tfn spots are observed in some CSs (arrows), probably corresponding to CCPs or budding-CCV. The double labeling can be viewed section by section in Movie 1 (see supplementary material). (B) Polarized distribution of endogenous AP-2 (
-adaptin) in a T lymphoblast. The z projection, a confocal section and the DIC image are shown. (C) AP-2 (-adaptin, red) and GFP-ClLC (green) double labeling. Several co-localizing large CSs distributed at the tip and the uropod neck (arrows) are observed in the mid-uropod confocal section. The single AP-2 labeling and the merged image are shown; the enlarged area appears outlined in blue. (D) Intensity profile of clathrin staining along the cell contour (yellow line). This is the same cell shown in A. (E) Schematic representation of membrane CS distribution (green circles) in a polarized T cell. Cells were virtually divided in uropod, uropod-neck and front. However, generally, both the uropod and its neck are overall similarly rich in CSs/µm, in contrast to the front cell pole. (F) Histograms summarizing the frequency of membrane CSs/µm at the uropod, uropod-neck and the front part of the cell (check all three domains in E). Over 1000 µm of membrane were carefully checked in highly magnified images for 200-800 µm CSs co-localizing with Tfn or AP-2. Bars, 5 µm, or as indicated.
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