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Figure 5


Fig. 5. The effect of PtdIns(4,5)P2 conversion on the location of PLCβ1a. (A) HeLa cells coexpressing CFP-pm-FRB, RFP-5-phosphatase-FKBP12, YFP-PLCβ1a and B2R were monitored upon addition of rapamycin. A region of interest was selected in the cytosol. The red trace depicts the mean normalized fluorescence intensity of RFP-5-phosphatase-FKBP12; the black trace depicts the mean normalized fluorescence intensity of YFP-PLCβ1a (n=11, error bars depict standard error). The decrease in the fluorescence intensity of RFP before addition of rapamycin is due to photobleaching. Subsequently, bradykinin (BK, 1 µM) was added to completely dissociate YFP-PLCβ1a from the plasma membrane. (B) HeLa cells coexpressing CFP-pm-FRB, RFP-5-phosphatase-FKBP12, YFP-PH{delta}1 and B2R were monitored upon addition of rapamycin. A region of interest was selected in the cytosol. The red trace depicts the mean normalized fluorescence intensity of RFP-5-phosphatase-FKBP12; the black trace depicts the mean normalized fluorescence intensity of YFP-PH{delta}1 (n=3, error bars depict standard error). Subsequently, bradykinin (BK, 1 µM) was added to completely dissociate YFP-PH{delta}1 from the plasma membrane. (C) HeLa cells coexpressing CFP-pm-FRB, RFP-5-phosphatase-FKBP12 and YFP-C2{gamma} were monitored upon addition of rapamycin. The red trace depicts the mean normalized fluorescence intensity of RFP-5-phosphatase-FKBP12; the black trace depicts the mean normalized fluorescence intensity of YFP-C2{gamma} (n=5, error bars depict standard error). Subsequently, ionomycin (5 µM) was added to translocate the YFP-C2{gamma} domain to the plasma membrane. (D) HeLa cells coexpressing CFP-pm-FRB and YFP-PLCβ1a were monitored upon addition of rapamycin. The black trace depicts the mean normalized fluorescence intensity of YFP-PLCβ1a (n=4, error bars depict standard error). (E,F) Models of the situation in the cell before (E) and after (F) the addition of rapamycin. Note that PLCβ1a moves to the cytosol, whereas 5-phosphatase moves to the plasma membrane through heterodimerization of the plasma membrane-localized FRB domain and the FKBP12 domain fused to 5-phosphatase. In E and F, red circles in plasma membrane indicate PtdIns(4,5)P2, blue circles indicate PtdIns(4)P; the encircled R in panel F indicates rapamycin.





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