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First published online 28 October 2008
doi: 10.1242/jcs.029785

Journal of Cell Science 121, 3770-3777 (2008)
Published by The Company of Biologists 2008
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Regulation of PLCβ1a membrane anchoring by its substrate phosphatidylinositol (4,5)-bisphosphate

Merel J. W. Adjobo-Hermans, Joachim Goedhart and Theodorus W. J. Gadella, Jr*

Swammerdam Institute for Life Sciences, Section of Molecular Cytology, Centre for Advanced Microscopy, University of Amsterdam, Kruislaan 316, NL-1098 SM, Amsterdam, The Netherlands


Figure 1
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  		Fig. 1. Subcellular location of PLCβ1a. (A-D) Confocal images of (A) HeLa cells expressing GFP-PLCβ1a, (B) MEF cells deficient in G{alpha}q/11 expressing GFP-PLCβ1a, (C) wild-type MEF cells expressing GFP-PLCβ1a and (D) HeLa cells expressing YFP-PLCβ1a without its CT domain. (E) TIRF image of a HeLa cell expressing GFP-PLCβ1a. (F) Fluorescence of GFP-PLCβ1a detected in TIRF mode upon stimulation with histamine (100 µM, arrow). Scale bars: 10 µm.

 

Figure 2
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  		Fig. 2. PLCβ1a and intracellular Ca2+. HeLa cells coexpressing YFP-C2{gamma} (A-C, green) and RFP-PLCβ1a (D-F, white, for higher contrast). Confocal images are shown at three different timepoints as indicated by the vertical lines in the graphs beneath (G-I). Addition of histamine (100 µM) is indicated by the arrow in G. Regions of interest were selected in the cytosol of three different cells. Graphs G, H and I show the normalized fluorescence intensity quantified from cells 1, 2 and 3 (as depicted in A), respectively. The green lines indicate the intensity of YFP-C2{gamma} in the regions of interest; the red lines indicate the intensity of RFP-PLCβ1a. Scale bar: 10 µm.

 

Figure 3
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  		Fig. 3. PLCβ1a and PtdIns(4,5)P2. (A) HeLa cells were stimulated with histamine (100 µM, arrow) after overnight incubation with 1 µM PMA. The red trace depicts the mean normalized fluorescence intensity of GFP-PLCβ1a; the black trace depicts the mean normalized fluorescence intensity of the Fura Red dye in a region of interest in the cytosol (n=7; the error bars depict standard error). The Fura Red dye was excited at 488 nm and detected with a long-pass 650-nm filter. Therefore, an increase in intracellular Ca2+ correlates with a decrease in fluorescence intensity. For the exact settings used, see Materials and Methods. (B) HeLa cells expressing RFP-PH{delta}1. Scale bar: 10 µm. (C) HeLa cells coexpressing PLCβ1a (red trace), PH{delta}1 (black trace) and bradykinin type 2 receptor (B2R) were first stimulated with histamine (100 µM; at t=15 seconds, first arrow) and then with bradykinin (1 µM; at t=115 seconds, second arrow).

 

Figure 4
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  		Fig. 4. The effect of PIP5K and 5-phosphatase on the location of PLCβ1a. (A,B) HeLa cells coexpressing GFP-PIP5K (imaged separately in A) and RFP-PLCβ1a (imaged separately in B). (C) The fluorescence intensity ratio of PLC to PIP5K was calculated for cells in which RFP-PLCβ1a does [TL (translocation)] and does not (no TL) dissociate from the plasma membrane upon stimulation with 100 µM histamine. A two-tailed Student's t-test (confidence level of 95%) indicates that the cells in which PLCβ1a does not translocate to the cytosol express significantly more PIP5K than those in which PLCβ1a diffuses into the cytosol. Error bars depict standard error. (D,E,G,H) HeLa cells coexpressing untagged 5-phosphatase IV, YFP-PLCβ1a (D,G) and CFP-PH{delta}1 (E,H). (F) The ratio between plasma membrane fluorescence and cytosolic fluorescence of YFP-PLCβ1a was calculated for cells expressing PLCβ1a alone (PLC), in the presence of PIP5K (PLC+PIP5K) and in the presence of 5-phosphatase (PLC+5ptase). A two-tailed Student's t-test (confidence level of 95%) indicates that significantly more PLCβ1a localizes at the plasma membrane in the presence of PIP5K, and that significantly less localizes at the plasma membrane in the presence of 5-phosphatase. Error bars depict standard error.

 

Figure 5
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  		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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