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Figure 2


Fig. 2. PM PtdIns(4,5)P2 hydrolysis partially inhibits transferrin receptor recycling to the surface while fully suppressing AP-2{alpha} PM localization and targeting of transferrin receptor (TfR) to endocytic sites. (A,B) Rapid PtdIns(4,5)P2 hydrolysis causes partial inhibition of the reinsertion of internalized transferrin receptors back to the cell surface. Cells expressing Lyn-FRB and either CF-Inp or CF-Inp(D281A) were treated with Alexa-Fluor-594-conjugated transferrin for 20 minutes, then kept in Alexa-Fluor-594-conjugated-transferrin-free media for an additional 5, 15 or 30 minutes with iRap or DMSO. (A) Internal Alexa-Fluor-594-conjugated transferrin remaining in CF-Inp-translocated cells after 15 minutes of recycling. Arrows point to Lyn-FRB- and CF-Inp-expressing cells. Scale bars: 10 µm. (B) Time course of internal transferrin loss. Intracellular fluorescence intensity was normalized to that measured in cells expressing Lyn-FRB and CF-Inp(D281A) that were fixed immediately after Alexa-Fluor-594-conjugated-transferrin uptake without recycling (significance of difference P<7x10–11 at the 15-minute time point). Error bars represent s.e.m. (C) Rapid PtdIns(4,5)P2 hydrolysis increases the amount of transferrin receptors expressed at the PM, suggesting that PtdIns(4,5)P2 regulates endocytosis significantly more than it does recycling. Cells expressing Lyn-FRB and CF-Inp were treated with iRap or DMSO for 15 or 30 minutes, then stained with transferrin receptor antibody to label surface transferrin receptors. Receptor expression is normalized to the fluorescence intensity measured in cells without iRap or DMSO treatment. Error bars represent s.e.m. (D) Antibody against AP-2{alpha} shows that PtdIns(4,5)P2 depletion causes a dissociation of AP-2{alpha} puncta from the PM. Cells expressing Lyn-FRB and CF-Inp were treated with Alexa-Fluor-594-conjugated transferrin for 5 minutes following a 1-minute treatment with iRap. Dashed area indicates the location of Lyn-FRB- and CF-Inp-expressing cells. Scale bars: 10 µm. (E) Quantitative analysis of the PM dissociation of AP-2{alpha} using ratio imaging of the fluorescence intensity near the PM versus that of the midsection. Error bars represent s.e.m. (F) TIRF imaging of AP-2-µ2–YFP fluorescence at the PM better resolves the rapid loss of AP-2-µ2-YFP from the PM upon iRap-induced translocation of CF-Inp. Scale bars: 5 µm. (G-I) The AP-2-adaptor-mediated clustering of transferrin receptors is also lost upon PtdIns(4,5)P2 hydrolysis. Cells expressing Lyn-FRB and either CF-Inp or CF-Inp(D281A) were treated with either DMSO or iRap for 5 minutes, then incubated with Alexa-Fluor-594-conjugated transferrin at 4°C to label transferrin receptors at the surface. Representative images are shown in G. Arrows point to Lyn-FRB- and CF-Inp-expressing cells. Scale bar: 10 µm. (H) Linescan of transferrin intensity from broken lines drawn in G reveals the loss of sharp peaks of transferrin intensity upon PtdIns(4,5)P2 hydrolysis. (I) Mean of the standard deviations of transferrin intensity calculated from each linescan collected. Error bars represent s.e.m.





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