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Figure 1. ISOCE during Xenopus oocyte maturation. (A) Voltage protocol used to measure ISOCE development and I–V relationship. The membrane potential was stepped to −140 mV (100 ms), and then ramped from −140 to +60 mV (2 s) from a −40 mV holding potential. The protocol was applied once every 30 s. (B and C) ISOCE development after Ca2+ store depletion with ionomycin (10 μM) in an oocyte (B) and an egg (C). Cells were injected with 7 nmol BAPTA to allow for ISOCE recording (Machaca and Haun, 2000). The current at −140 mV (A, arrow) was plotted over time. La3+ (100 μM) was added at the end of the experiment to block SOCE and obtain a measure of absolute ISOCE. (D) Representative I–V relationships obtained from the ramp voltage stimulation shown in A from an oocyte and an egg. I–V relationships were obtained by subtracting the current after La3+ addition from the current before addition. (E) Time course of progesterone-mediated GVBD. Progesterone was added to a population of at least 50 oocytes, and the occurrence of GVBD over time was recorded by following white spot appearance on the animal pole. The data shown represent the average ± SE from seven donor females. The time of GVBDi ± SD is also shown. GVBDi represents the time at which GVBD was first observed in the population. (F and G) ISOCE levels from individual oocytes at different times during oocyte maturation. Each square represents the current from a single cell. ISOCE data from oocytes are shown before progesterone addition. The data was normalized to average current in oocytes. ISOCE data from 80 cells at different times during maturation are shown in F. (G) ISOCE levels from cells incubated in progesterone without undergoing GVBD (n = 63). The average time ± SD of GVBDi is shown for reference.
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Figure 2. Time course of MPF, MAPK, and Mos activation during progesterone-induced maturation. (A) Time course of GVBD. GVBDi occurred at 5.5 h, and GVBD50 â¼7.25 h after progesterone addition. At each time point, five cells were pooled to quantify kinase activity. (BâD) MPF, P-MAPK, and Mos levels during oocyte maturation. MPF activity was measured as the histone H1 kinase activity as described in Materials and methods. The levels of P-MAPK and Mos were determined by Western blot analysis using a P-MAPKâspecific and anti-Mos antibody, respectively, as described in Materials and methods. In the case of MAPK, the same blot was reprobed with an antibody that detects total MAPK (T-MAPK) levels, showing that MAPK was present in the lysates but was not phosphorylated. To be able to compare P-MAPK and Mos levels between different experiments, we always ran a positive control lysate from eggs on every gel, and normalized P-MAPK and Mos levels to that sample. Therefore, Western data for P-MAPK and Mos throughout this manuscript are normalized to the same egg lysate (see Materials and methods for more details). The time scale is divided into two phases: after progesterone addition; and after GVBD. The 35-kD molecular mass marker is shown on the right of the gels. (E) MPF, P-MAPK, and Mos levels in cells treated with progesterone, but that did not undergo GVBD. GVBDi is indicated for reference. This time course of kinase activation is representative of five similar experiments.
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Figure 3. Correlation between ISOCE and MPF and P-MAPK levels after Î87cyclin B1 protein injection. Oocytes were either directly injected with Î87cyclin B1 protein (A and B) or preincubated in the MEK inhibitor PD98059 (50â100 μM) for 0.5â2 h before Î87cyclin B1 injection. ISOCE levels were recorded at various times after Î87cyclin B1 injection. After ISOCE recording, individual cells were lysed and assayed for MPF and P-MAPK levels. This allowed us to obtain ISOCE (squares), MPF (circles), and P-MAPK (triangles) levels from the same oocyte. The data for individual cells are connected by drop lines to help in matching ISOCE and kinase activity from the same cell. For Î87cyclin B1 and PD98059-Î87cyclin B1 injections, current and kinase data from 40 and 33 cells, respectively, are shown. ISOCE levels were normalized to the levels in untreated oocytes, and MPF and MAPK levels were normalized to the levels found in fully mature eggs, as described in Fig. 2. The time scale was normalized to the time at which GVBD initially appeared in the population (GVBDi) after Î87cyclin B1 injection. The standard deviation of GVBDi is shown in A and B (n = 7). PD98059-treated cells are plotted on a time scale normalized to GVBDi in Î87cyclin B1âinjected cells (A and B). Individual cell data were fitted with a Boltzman function to obtain a general trend of ISOCE inactivation and the activation of the different kinases.
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Figure 4. Representative individual cell data from Î87cyclin B1âinjected cells. MPF, P-MAPK, and Mos Western data are shown in conjunction with the levels of each kinase in fully mature eggs (EGG). (A) ISOCE was activated in response to store depletion with ionomycin (10 μM) in an oocyte, and MPF, P-MAPK, and Mos were not detected. (B) A representative oocyte injected with Î87cyclin B1. ISOCE was not activated in response to store depletion. MPF and P-MAPK levels were 1.6 and 0.9 times those in eggs, whereas Mos kinase was not detected in this oocyte. (C) A representative oocyte preincubated with PD98059 (100 μM) before Î87cyclin B1 injection. ISOCE was not activated in response to store depletion, MPF levels were 2.7 times higher than those found in the control egg lysate, and both P-MAPK and Mos were not detectable.
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Figure 5. Correlation between ISOCE and Mos, P-MAPK, and MPF levels after injection of GSTâMos RNA. GSTâMos is a fusion protein between GST and Xenopus Mos, which allowed the separation between endogenous Mos (39 kD) and recombinant, injected GSTâMos (64 kD) (see Materials and methods). Oocytes were either directly injected with 10 ng GSTâMos RNA (AâC) or preinjected with 10 ng of GSTâ107Wee1 (Wee) 13â16 h before Mos RNA injection. GSTâ107Wee1 is a constitutively active form of the Wee1 kinase, which blocks MPF activation (Howard et al., 1999). ISOCE levels recorded at various times after GSTâMos RNA injection. After ISOCE recording, individual cells were lysed and assayed for kinase activities. This allowed us to obtain ISOCE (squares), Mos (diamonds), P-MAPK (triangles), and MPF (circles) levels from the same oocyte. For Mos and Wee1âMos injections, current and kinase data from 40 and 27 cells, respectively, are shown. The plotted Mos data is for GSTâMos protein levels, as endogenous Mos was not detected in these experiments. The data for individual cells are connected by drop lines to help in matching ISOCE and kinase activity from the same cell. ISOCE levels were normalized to the levels in untreated oocytes, and Mos, MAPK, and MPF levels were normalized to the levels found in fully mature eggs as described in Fig. 2 and Materials and methods. The time scale was normalized to GVBDi, the standard deviation of which is shown in AâC (n = 5). Cells preinjected with GSTâ107Wee1 were plotted on the same time scale as GSTâMos-injected cells (DâF). Individual cell data were fitted with a Boltzman function to obtain a general trend of ISOCE inactivation and the activation of the different kinases.
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Figure 6. Representative individual cell data from Wee1- and Mos-injected cells. MPF, P-MAPK, and Mos Western data are shown in conjunction with the levels of each kinase from a lysate of fully mature eggs (EGG). (A) ISOCE was activated in response to store depletion with ionomycin (10 μM) in an oocyte. MPF, P-MAPK, and Mos were not detected in this cell. (B) A representative cell injected with 10 ng GSTâMos RNA. ISOCE was not activated in response to store depletion. ISOCE was recorded at the 1GVBDi time point in this cell, i.e., at about the same time that GVBD first occurred in the population. MPF and P-MAPK levels were 1.3 and 1.6 times, respectively, higher than those in the egg lysate. GSTâMos protein (arrowhead) levels were 13.1 times higher than egg levels and no endogenous Mos protein (arrow) was detected. (C) A representative cell preinjected with 10 ng GSTâ107Wee1 RNA 14 h before GSTâMos RNA (10 ng) injection. We recorded ISOCE at the 1GVBDi time point. SOCE was readily activated, in response to store depletion, to levels similar to those found in oocytes. Injection of GSTâ107Wee1 effectively blocked MPF activation, which was detected at 0.2 times egg level. In contrast, P-MAPK and GSTâMos (arrowhead) were detected at 0.76 and 8.7 times egg levels, respectively.
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Figure 7. Continuous recording of ISOCE through the GVBD transition. (A) Prolonged recording of ISOCE from a control untreated oocyte. MPF activity in this cell is also shown. (B) ISOCE was activated in a progesterone-treated oocyte before GVBD and continuously recorded until, and past, the time point at which GVBD occurred (arrow). At the end of the experiment, the cell was lysed in extraction buffer and MPF activity was measured. This cell had high MPF activity, indicating that the prolonged recording did not interfere with MPF activation at GVBD. MPF activation at GVBD is not able to block ISOCE that has been preactivated before GVBD.
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Figure 8. Pre-activated ISOCE does not inactivate in response to MPF activation. Oocytes were incubated in Ca2+-free medium (L-15 with Ca2+, buffered at 50 μM), containing thapsigargin (1 μM) to deplete intracellular Ca2+ store and activate ISOCE. Cells were voltage clamped in a Ca2+-free solution (70Mg; see Materials and methods) and then switched to a solution containing 30 mM Ca2+ (30Ca; see Materials and methods) to induce Ca2+ influx through SOCE channels. (A) ISOCE and MPF levels from a control thapsigarginâtreated oocyte. (B) Oocytes were treated with progesterone (5 μg/ml) for 1 h and then thapsigargin (1 μM) was added, and the cells were incubated until GVBD occurred. ISOCE was measured â¼15 min after GVBD occurrence. ISOCE was still present in this cell, although MPF was activated at high levels, indicating that MPF is unable to block SOCE that has been preactivated before MPF induction. ISOCE (C) and MPF (D) levels in oocytes (n = 5) and cells treated with progesterone 10â15 min (n = 8) and 15â30 min after GVBD (n = 7). ISOCE levels gradually declined after GVBD and MPF levels remained relatively stable.
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