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Fig. 1. PM PtdIns(4,5)P₂ hydrolysis disrupts endocytosis of the transferrin receptor, but not fluid-phase endocytosis. (A,B) iRap induces rapid translocation of CFP-FKBP-Inp (CF-Inp) from the cytosol to the PM and dissociation of PtdIns(4,5)P₂ biosensor YFP-PLC-PH from the PM. Sample image (A), and corresponding time course of CF-Inp and YFP-PLC-PH translocation (B). Scale bars: 10 µm. (C,D) Rapid PtdIns(4,5)P₂ hydrolysis inhibits transferrin uptake. HeLa cells expressing Lyn-FRB and either CF-Inp or CF-Inp(D281A) were treated with iRap or DMSO for 1 minute before addition of Alexa-Fluor-594-conjugated transferrin. (C) Sample image of CF-Inp-expressing cells treated with iRap and fixed after 15 minutes of Alexa-Fluor-594-conjugated-transferrin uptake. Dashed area indicates the location of Lyn-FRB- and CF-Inp-expressing cells. Scale bars: 50 µm. (D) Time-course of Alexa-Fluor-594-conjugated-transferrin uptake (significance of difference P<9x10^–46 at the 15-minute time point). Error bars represent s.e.m. (E,F) Rapid PtdIns(4,5)P₂ hydrolysis does not affect dextran uptake. Following treatment with either DMSO or iRap, cells expressing Lyn-FRB and CF-Inp were assayed for internalization of Alexa-Fluor-594-conjugated transferrin and fluorescein-dextran simultaneously. (E) Representative image of CF-Inp-expressing cells treated with iRap and fixed after Alexa-Fluor-594-conjugated-transferrin and fluorescein-dextran uptake. Dashed area indicates the location of Lyn-FRB- and CF-Inp-expressing cells. Scale bars: 10 µm. (F) Quantification of transferrin and dextran uptake. For each tracer, the intracellular fluorescence intensity was normalized to that measured in cells expressing Lyn-FRB and CF-Inp(D281A) that were treated with iRap. Error bars represent s.e.m.

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