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First published online May 28, 2005
doi: 10.1242/10.1242/jcs.02356

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Exchange of clathrin, AP2 and epsin on clathrin-coated pits in permeabilized tissue culture cells

Laboratory of Cell Biology, NHLBI, National Institutes of Heath, Bethesda, MD 20892-0301, USA

View larger version (127K): [in a new window]   Fig. 1. Effects of permeabilization on clathrin-coated pits. (A-D) Clathrin-coated pits were fixed before (a,b) and after (c,d) permeabilization. Cells were imaged for both GFP (a,c) or Alexa 546 (b,d) fluorescence. Immunostained cells were imaged for clathrin in (A, b,d), for AP2 (B, b,d), and for epsin (C, b,d). In D, cells were loaded with Alexa 546 transferrin and imaged for transferrin (b,d). Notice that, all fluorescence settings used in comparing nonpermeabilized and permeabilized cells were identical. Bars, 10 µm.

View larger version (123K): [in a new window]   Fig. 2. Uncoating of clathrin-coated pits requires Hsc70, auxilin and ATP. Factors known to affect the uncoating of CVs were incubated with the permeabilized cells for 5 minutes, in an attempt to uncoat permeabilized cells. Conditions were buffer A plus ATP, 1 mM; Hsc70, 2 µM; auxilin, 0.2 µM as indicated. Bars, 10 µm.

View larger version (67K): [in a new window]   Fig. 3. Kinetics of uncoating of clathrin-coated pits by Hsc70-auxilin-ATP mixture. (A) Uncoating mixture composed of Hsc70 (2 µM), auxilin (0.2 µM) and ATP (1 mM) was added to the permeabilized cells and imaged at 30-second intervals. (B) The decrease in fluorescence intensity of three different regions of interest (ROI) (circles, numbered 1-3) for the time course in A is plotted against time. Data were normalized to the maximal initial fluorescence in each plot. Bar, 10 µm.

View larger version (36K): [in a new window]   Fig. 4. Time course of uncoating of clathrin puncta by Hsc70-auxilin-ATP mixture. (A,B) Fluorescence image (A) showing the circled GFP-clathrin puncta (1-5) used to obtain the time course of uncoating (B). Bar, 5 µm. (C) Distribution in uncoating time obtained from 200 puncta (five cells) uncoated by Hsc70, auxilin and ATP using the same concentrations as in Fig. 2. Puncta measured were no larger than 0.3 µm. Cells were imaged using the 63x, 1.4 NA objective.

View larger version (34K): [in a new window]   Fig. 5. Dependency of auxilin and Hsc70 concentration on the uncoating of clathrin-coated pits. (A) Different concentrations of auxilin (between 2x105 µM and 2 µM) were added to permeabilized cells in the presence of 1 mM ATP and 2 µM Hsc70. (B) Different concentrations of Hsc70 (between 2x104 µM and 10 µM) were added to permeabilized cells in the presence of 1 mM ATP and 0.2 µM auxilin. Cells were imaged at 30-second intervals for 5 minutes. Cells of similar fluorescence intensity were analyzed. The decrease in relative fluorescence intensity was measured as a function of time. Data were normalized to the maximal initial fluorescence in each plot.

View larger version (98K): [in a new window]   Fig. 6. Hsc70-auxilin-ATP mixture does not dissociate adaptors or transferrin from clathrin-coated pits. GFP-clathrin cells were uncoated with Hsc70, auxilin, and ATP for 10 min, at which time they were fixed. Cells were immunostained for clathrin (a,b), AP2 (c,d) and epsin (e,f) both before (a,c,e) and after uncoating (b,d,f). The GFP-clathrin cells were preloaded with transferrin, permeabilized, and imaged before (g) and after uncoating (h). Bars, 10 µm.

View larger version (61K): [in a new window]   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 () 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 (), and the dominant-negative mutant Hsp70(K71E) (). 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 (). Bovine-brain cytosol was treated with 5 µM Hsp70(K71E) and the decrease in fluorescence was measured in 20-second intervals (). 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.

View larger version (47K): [in a new window]   Fig. 8. Cytosol induces clathrin to exchange gradually on the coated-pits. (A) permeabilized GFP-clathrin cells (a,c,e,g) were incubated with cytosol (2 mg/ml) for the indicated times, fixed and then stained with X22, the anti-clathrin antibody (b,d,f,h). Notice that, all fluorescence settings were held constant when comparing samples. Bars, 5 µm. (B) Distribution of uncoating time obtained from 200 puncta (five cells) uncoated by cytosol. Measured puncta were no larger than 0.3 µm.

View larger version (75K): [in a new window]   Fig. 9. AP2 and epsin rebind to clathrin-coated pits that were uncoated by cytosol. Cells were treated with bovine-brain cytosol (10 mg/ml) and ATP (1 mM) for 5 minutes. (A) GFP-AP2 (a-c) and GFP-epsin (d-f) dissociate from puncta in the presence of cytosol and ATP. (B) Cytosol-induced release of AP2 (,) and epsin (,) was measured at 20-second intervals in the presence (, ) and absence (, ) of YDJ1. The dashed line represents the release of clathrin, obtained under conditions identical to the ones described above. (C) AP2 and epsin release by cytosol are accompanied by their rebinding. Cells were treated with bovine-brain cytosol and 1 mM ATP for 5 minutes, then fixed and immunostained. Permeabilized cells with GFP-AP2 (a,b,e,f) and GFP-epsin (c,d,g,h) are shown before (a-d) and after (e-h) treatment with cytosol. GFP-fluorescence (a,c,e,g) and immunostaining (b,d,f,h) are shown.

View larger version (78K): [in a new window]   Fig. 10. When cytosol is present, clathrin and AP2 rebind to the identical clathrin-coated pit. Permeabilized GFP-clathrin-cells (a-h) were uncoated and fixed, and simultaneously stained with anti-clathrin and anti-AP2 antibodies. Clathrin (b,f) was stained with CHC5.9 antibody and AP2 (c,g) was stained with AP.6 antibody. For double-staining, CHC5.9 and AP.6 were visualized with cy5-conjugated anti-mouse IgM antibody and rhodamine-conjugated anti-mouse IgG1, respectively.

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