Fig. 6

VAMP-2 vesicle fusion and PAE formation is inhibited by the calpain inhibitor, PD 150606. Embryos were treated with either the general protease inhibitor, leupeptin (675 μM) as a control (see panel A) or the calpain inhibitor, PD 150606 (at 250 to 500 μM; panel B). Brightfield images were captured at the beginning of the second cleavage (panels Ai and Bi) and completion of the third cleavage (panels Aii and Bii). Selected embryos were fixed for either VAMP-2 immunolabeling during the first cleavage (see panels Aiii and Biii), and at the completion of the second cleavage (see panels Aiv and Biv), or for F-actin labeling using rhodamine–phalloidin during the first cleavage (see panels Av and Bv). VAMP-2 labeling is indicated by white arrowheads, while the contractile bands and PAEs are indicated by white arrows and brackets, respectively. Panels Avi and Bvi are actin staining intensity line-scans (taken at the positions indicated by the dashed lines in panels Av and Bv). Panels Aiii to Av and Biii to Bv are 2-D images reconstructed from stacks of images captured from an animal pole view. (C) The degree of VAMP-2 vesicle fusion was quantified during late deepening of the 1st cleavage and presented as averaged fusion area ± S.E.M. (n ≥ 5 for each treatment). One-way ANOVA was performed for statistical analysis. The Neuman–Keuls multiple comparison test was performed as a post-hoc analysis. * indicates significant differences compared to the leupeptin-treated controls (at p < 0.001). Embryos treated with brefeldin A (190 μM), an inhibitor of membrane trafficking from the Golgi, were included as a control for the inhibition of vesicle fusion.