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Figure 3


Fig. 3. PM PtdIns(4,5)P2 hydrolysis significantly suppresses LDL receptor endocytosis and weakly suppresses PM localization of epsin and CALM adaptors. (A,B) Following treatment with either DMSO or iRap, cells expressing Lyn-FRB and CF-Inp were simultaneously assayed for internalization of DiI-labeled LDL and Alexa-Fluor-488-conjugated transferrin. (A) Representative image of CF-Inp-expressing cells treated with iRap and fixed after transferrin and LDL uptake, showing suppression of the internalization of both receptors in the same cells. Dashed area indicates the location of Lyn-FRB- and CF-Inp-expressing cells. Scale bars: 10 µm. (B) Quantification of transferrin and LDL uptake. For each tracer, the intracellular fluorescence intensity was normalized to that measured in cells expressing Lyn-FRB and CF-Inp(D281A) treated with iRap. Error bars represent s.e.m. (C) Antibody against CALM (top row) or epsin (bottom row) reveals PM-associated punctate localization in untransfected control cells but a more cytosolic distribution in PtdIns(4,5)P2-depleted cells. 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 lines indicate the location of Lyn-FRB- and CF-Inp-expressing cells. Scale bars: 10 µm.





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