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The dynamics of plasma membrane PtdIns(4,5)P2 at fertilization of mouse eggs

Guillaume Halet1, Richard Tunwell1,2, Tamas Balla3, Karl Swann2 and John Carroll1,*

1 Department of Physiology, University College London, Gower Street, London WC1E 6BT, UK
2 Department of Anatomy and Developmental Biology, University College London, Gower Street, London WC1E 6BT, UK
3 Endocrinology and Reproduction Research Branch, National Institutes of Health, Bethesda, MD 20892, USA



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  		Fig. 1. PH-GFP binds to plasma membrane PtdIns(4,5)P2 in mouse oocytes. Confocal images of GFP fluorescence in the equatorial plane of mouse MII oocytes, acquired 2-3 hours after injection with the cRNA encoding PH-GFP (A,B) or the mutant PH(R40L)-GFP, which does not bind to PtdIns(4,5)P2 (C). Corresponding line intensity profiles are displayed on the right, with GFP fluorescence expressed as relative fluorescence units (F) and bright field images shown on the left. (A) MII oocyte with the animal pole out of focus (fluorescence from the chromosomes, stained with Hoechst 33342, was captured in a plane above and superimposed). (B) MII oocyte with the animal pole in the focal plane. Note the higher fluorescence intensity associated with the plasma membrane in the vegetal pole. (C) MII oocyte expressing PH(R40L)-GFP. For each condition, similar patterns of staining were obtained in at least 20 oocytes. Bar, 10 µm.

 


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  		Fig. 2. Plasma membrane PtdIns(4,5)P2 increases at fertilization. MII oocytes were fertilized on the stage of the microscope and GFP fluorescence (in arbitrary units; PM, plasma membrane; C, cytoplasm) and Fura-red fluorescence ({Delta}F/F) were continuously recorded at a rate of 1 frame every 7 seconds (A) or 10 seconds (B). Note the translocation of PH-GFP from the cytoplasm to the plasma membrane. The increase in PM/C ratio indicates an increase in plasma membrane PtdIns(4,5)P2. The white and black arrowheads indicate the position for measurement of the peak change in PM/C and value of PM/C at the end of the first Ca2+ transient, respectively. This data is displayed in Table 1 as the {Delta}PM/C(peak) and {Delta}PM/C(end) and used to compare the different properties of the increase in PtdIns(4,5)P2.

 


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  		Fig. 3. The increase in PtdIns(4,5)P2 is polarized to the vegetal pole. (A) Changes in [Ca2+]i and PH-GFP distribution at fertilization in an egg in which the confocal slice was taken through both the animal and vegetal poles. Drawing on right illustrates the regions selected for measurements of PM and C fluorescence in the animal (dark/light blue) and vegetal (dark/light green) poles. Changes in PM/C in the vegetal and animal poles are illustrated by a red or orange trace, respectively. For abbreviations, see Fig. 2 legend. (B) GFP images corresponding to selective time points (arrowheads numbered 1-3 in A) for the experiment described in A. The lower region of the egg (red box) was expanded and displayed in pseudocolor to show more clearly the changes in fluorescence intensity.

 


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  		Fig. 4. PtdIns(4,5)P2 synthesis at fertilization is Ca2+-dependent. (A) Changes in [Ca2+]i and PH-GFP distribution recorded at fertilization in an egg pretreated with 1 µM BAPTA-AM. Similar results were obtained in 15 eggs. (B) Upper panel, changes in [Ca2+]i and PH-GFP distribution elicited by uncaging Ins(1,4,5)P3 using a low UV power (0.5% excitation-indicated by the horizontal bar). Similar results were obtained in eight eggs. Lower panel, changes in [Ca2+]i and PH-GFP distribution elicited by uncaging Ins(1,4,5)P3 using a tenfold higher UV power (5% excitation-indicated by the horizontal bar). Similar results were obtained in five eggs. For abbreviations, see Fig. 2 legend.

 


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  		Fig. 5. PtdIns(4,5)P2 synthesis is a consequence of exocytosis. (A) Changes in [Ca2+]i and PH-GFP distribution at fertilization in an egg treated with 100 nM jasplakinolide. Similar results were obtained in six eggs. (B) Changes in [Ca2+]i and PH-GFP distribution at fertilization in an egg injected with 5 µM BoNT/A-LC. Similar results were obtained in nine eggs. (C) To illustrate the effect of jasplakinolide and BoNT/A on the kinetics of the rise in PtdIns(4,5)P2, the traces obtained in the control experiment shown in Fig. 1C (red trace), and in the two experiments described above (green, jasplakinolide; blue, BoNT/A-LC) were aligned according to the first peak in [Ca2+]i and superimposed. For abbreviations, see Fig. 2 legend. (D) FITC-LCA labelling on (i) unfertilized MII oocyte; (ii) control fertilized egg; and (iii) jasplakinolide (100 nM)-treated fertilized egg. For each condition, the equatorial plane (left) and another focal plane close to the surface (right) of the same cell are displayed. Note the incomplete ring of fluorescence in the equatorial planes, illustrating the cortical granule-free domain in the animal pole. Exocytosis is indicated by an increased fluorescence in the equatorial plane and by a punctate fluorescence in the section close to the surface. Similar patterns of staining were observed in at least 20 cells, for each condition.

 


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  		Fig. 6. Fertilization of GV-stage oocytes does not trigger the increase in PtdIns(4,5)P2. (A) Confocal images of PH-GFP labeling in a GV-stage oocyte. DNA is stained with Hoechst 33342. The corresponding bright field image is displayed on the left. (B) Changes in [Ca2+]i and PH-GFP distribution at fertilization of a GV stage oocyte. Similar results were obtained in 4 oocytes. For abbreviations, see Fig. 2 legend.

 


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  		Fig. 7. Micromolar wortmannin inhibits the PtdIns(4,5)P2 increase but not exocytosis. (A) Fertilization-induced changes in [Ca2+]i and PH-GFP distribution recorded in an egg pre-treated with 30 µM wortmannin for 15 minutes. Similar results were obtained in eight eggs. For abbreviations, see Fig. 2 legend. (B) FITC-LCA labeling on (i) unfertilized MII oocyte; (ii) control fertilized egg; and (iii) wortmannin (30 µM, 15 minutes)-treated fertilized egg. See Fig. 5D legend for details on FITC-LCA labeling. Similar patterns of staining were observed in at least 12 cells, for each condition.

 

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