Fig. 5

Effect of changes in intracellular calcium on PYK2 activation state in the zebrafish oocyte. In order to establish whether oocyte PYK2 could be activated by artificially increasing intracellular calcium levels Oocytes were injected with injection buffer only as a control (A) or with injection buffer containing 25 μM IP3 (B) to artificially drive an increase in intracellular free calcium levels. The control and IP3-injected oocytes were maintained in Hanks-BSA for 4.5 min then fixed. In order to establish whether prevention of the fertilization-induced calcium transient would block PYK2 activation in response to fertilization, oocytes were injected with calcium clamping buffer (C) then fertilized by a mixture of sperm and aquarium water, followed by fixation at 4.5 m.p.i. Oocytes were then prepared for immunofluorescence detection of activated PYK2 with anti-PYK2-PY⁵⁷⁹ and images representative of three experiments performed with five oocytes in each group are presented with green fluorescence representing the distribution of PYK2-PY⁵⁷⁹ labeling. Arrows in panel C point out the position of the micropyle. In separate experiments designed to confirm that the above treatments had the desired effect on intracellular calcium signaling (bottom panels), oocytes were first preloaded with 50 μM calcium-green dextran, and allowed to recover for 30 min. The injection pipet was replaced, calcium induced fluorescence recording was begun, and a second injection with injection buffer (Panel A, bottom), injection buffer with 25 μM IP3 (Panel B, bottom), or with calcium clamping buffer followed by in vitro fertilization (panel C, bottom) was performed after a baseline was established. The relative change in calcium green fluorescence over time is depicted graphically in the bottom panels which show patterns representative of the five oocytes from each group that were recorded. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)