Fig. 4. Removal of the CP following fertilization. Unfertilized eggs were injected with PH::GFP protein and cultured for 20 minutes to permit translocation to the PM. Following this, the eggs were injected with Fura-2-dextran and fertilized. Soon after extrusion of the first polar body pb1 (I), and once the CP was clearly visible (II) and the second series of Ca²⁺ waves had begun to originate from the CP (III), the eggs were treated with 2 µg ml^–1 cytochalasin B. Rounded-up eggs (IV) reflect the loss of the actin basket; microvilli are also lost from the CP as is the accumulation of PH::GFP (V). Even though the CP has been lost and PtdIns(4,5)P₂ is now redistributed, the Ca²⁺ waves still originate from the site where the CP previously resided (VI). To confirm that the site where the Ca²⁺ waves originate is identical to the site where the CP previously resided, we attached Nile Blue beads to the surface of the egg before fertilization [see arrow and bead (x) in I]. Following fertilization, the bead is located on the border of the CP (I); after the pharmacological removal of the CP the bead borders the site where the repetitive Ca²⁺ waves originated (IV, x). Finally, we noted the position of the sperm aster following removal of the CP (VII, marked ^*). The sperm aster is not near the site of origin of the Ca²⁺ waves (VII, arrow). Temperature 19°C, n=5 replicates, 3 animals.