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Fig. 7. Bovine brain cytosol dissociates and exchanges clathrin on the clathrin-coated pits and active Hsc70 is required for clathrin uncoating. (A) Permeabilized GFP-clathrin cells loaded with transferrin were incubated with cytosol and nucleotide for the indicated times. GFP-clathrin dissociates (a-c), whereas there is no change in transferrin (d-f). (B) In cells expressing GFP-clathrin, clathrin uncoating by cytosol ( ${diamondsuit}$ ) was established by measuring GFP-clathrin fluorescence at 20-second intervals. To establish whether Hsc70 is responsible for clathrin uncoating in the cytosol, two Hsc70 inhibitors were used, YDJ1 ( ${bullet}$ ), and the dominant-negative mutant Hsp70(K71E) ( ${blacksquare}$ ). The permeabilized cell membranes and also the bovine brain cytosol were treated with 1 µM YDJ1, and the decrease in fluorescence was measured in 20-second intervals ( ${bullet}$ ). Bovine-brain cytosol was treated with 5 µM Hsp70(K71E) and the decrease in fluorescence was measured in 20-second intervals ( ${blacksquare}$ ). Measurements were carried out by using a confocal microscope. (C) Clathrin-coated pits uncoated by cytosol also show rebinding of clathrin. Cells were treated with bovine-brain cytosol and 1 mM ATP for 5 minutes and then fixed and immunostained. Permeabilized GFP-clathrin cells shown before (a-c) and after (d-f) cytosol treatment. Fluorescence intensity of the GFP signals was increased to clearly distinguish the puncta (inset in d). The same area is shown in the immunostained cells (inset in e) with no manipulation of fluorescence intensity. The merged image (inset in f) shows that clathrin rebinds to the same pits. Bar, 10 µm.

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