Carroll DJ, Ramarao CS, Mehlmann LM, Roche S, Terasaki M, Jaffe LA.

	Figure 2. Delay of Ca2+ release during fertilization of starfish eggs injected with PLCγSH2(N+C) protein. Eggs were co-injected with 10 μM Ca-green dextran; photomultiplier current indicating Ca-green fluorescence is shown as a function of time. Each fluorescence trace was normalized to the fluorescence of the unfertilized egg. Arrows indicate the time of sperm addition (1:5,000 dilution of the suspension from the testis). (A) An egg that was injected with 1,100 μg/ml of the GST control protein. The asterisk indicates the action potential. (B and C) Eggs that were injected with 105 (B) or 900 (C) μg/ml of the PLCγSH2 (N+C) protein. In C, the lower line shows the continuation of the same record. (D) An egg that was injected with 1,000 μg/ml of the SHP2-SH2(N+C) control protein.
	Figure 3. Ca2+ imaging during fertilization of starfish eggs injected with PLCγSH2(N+C) or GST proteins. The control egg on the left was injected with 190 μg/ml GST and 10 μM Ca-green dextran. The egg on the right was injected with 130 μg/ml PLCγSH2(N+C) and 10 μM Ca-green dextran. Fluorescence intensity (0–255) is displayed using the 32-color look-up table of NIH Image (increasing values correspond to purple, blue, green, yellow, orange, red). Images are shown at 3-s intervals; the sequence is shown reading down each column, starting at the upper left. Sperm (1:5,000 dilution of the suspension from the testis) were added to the recording chamber 14 s before the first image. In the second image in the first column, an action potential occurred in both eggs (increased fluorescence at the egg surface). In the third image in the first column, the Ca2+ wave is starting in the control egg. In the first image in the fourth column, a local Ca2+ rise occurs in the PLCγSH2(N+C)-injected egg but does not propagate across the egg. In the last image in the fourth column, another Ca2+ rise occurs in the PLCγSH2(N+C)-injected egg, and a Ca2+ wave propagates across the egg in the fifth column. The difference in fluorescence intensity between the egg cortex and interior is due at least in part to greater absorbance of light in the center of the egg where the specimen is thicker; it is also possible that there is a real difference in Ca2+ activity between the cortex and interior. A Quicktime movie sequence from this experiment is available at: http://www.uchc.edu/∼terasaki/data/sh2.html. Bar, 200 μm.
	Figure 4. Inhibition of Ca2+ release during fertilization of starfish eggs injected with PLCγSH2(N+C) protein and exposed to a low concentration of sperm (1:50,000 dilution of the suspension from the testis). Photomultiplier current as a function of time. (A) An egg that was injected with 1,000 μg/ml of the SHP2-SH2(N+C) control protein. (B) An egg that was injected with 1,000 μg/ml of the PLCγSH2(N+C) protein. Arrows indicate the time of sperm addition. The lower lines show the continuation of the same record. (C and D) Images of eggs injected with 1,000 μg/ml SHP2-SH2(N+C) and 1,000 μg/ml PLCγ-SH2(N+C) and inseminated with a 1:50,000 sperm dilution; images were taken at the time of first cleavage in uninjected eggs, using the scanning transmission mode of the BioRad confocal microscope. (C) The SHP2-SH2(N+C)-injected egg showed a Ca-green record like that in A, elevated a normal fertilization envelope, and cleaved normally. Photographed at 3.0 h after insemination. (D) The PLCγSH2(N+C)-injected egg showed a Ca-green record like that in B and did not elevate a fertilization envelope or cleave; however, the presence of two pronuclei in the cytoplasm indicated that it was fertilized. Photographed at 2.6 h after insemination. These pronuclei were similar in appearance to those described by Sluder et al. (1989) for normal starfish fertilization, although the time course of their development was not compared with that in control eggs. Asterisks indicate pronuclei; “oil” indicates the oil drop introduced during microinjection. Bar, 100 μm.
	Figure 5. Normal Ca2+ release during fertilization of starfish eggs injected with a point-mutated PLCγ SH2(N+C) protein. Photomultiplier current as a function of time. Arrows indicate the time of sperm addition (1:5,000 dilution of the suspension from the testis). (A) An egg that was injected with 260 μg/ml of the PLCγ SH2(N+C)-mut protein. (B) An egg that was injected with 220 μg/ml of the PLCγ SH2(N+C)-wt protein.
	Figure 6. Normal Ca2+ release in starfish eggs injected with the PLCγSH2(N+C) protein and treated with IP3 or serotonin. (A and B) Photomultiplier records from oocytes that were injected with Ca-green dextran and for B, 1,000 μg/ml PLCγSH2(N+C), and then matured and injected with 1% of the cell volume of 1 μM IP3 (arrows). (C and D) Prophase-stage oocytes were injected with serotonin 2c receptor RNA (30 pg/oocyte). 24–27 h later the oocytes were injected with 10 μM Ca-green dextran and for D, 1,000 μg/ml PLCγSH2(N+C), and then treated with 1-methyladenine to cause maturation to the first meiotic metaphase stage. Serotonin (1 μM) was applied (arrows), while recording Ca-green fluorescence. Photomultiplier current indicating Ca-green fluorescence is shown as a function of time.

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