Fig. 8

Recruitment of VAMP-2 vesicles to the cleavage furrow requires kif23. Transient expression of kif23-EGFP (green) and VAMP-2-HT (red) in live embryos. (A) Time-series showing the temporal and spatial relationship between the recruitment of both kif23-EGFP and VAMP-2 to the furrow region in a representative zebrafish embryo undergoing cleavage at the 64- to 128-cell stage. The recruitment of kif23-EGFP to the cleavage furrow plane is indicated by yellow arrowheads (see panels Aii to Av) while the appearance of VAMP-2 labeling in the new furrow membrane is indicated by white arrowheads (see panels Aiv and Av). (B and C) Embryos were injected at the single cell stage with mRNA encoding either functional kif23-EGFP or a dominant-negative form of kif23 (kif23-trnc-EGFP), and were then fixed at the 8-cell stage for subsequent VAMP-2 immunofluorescence analysis. Panel Bi clearly indicates that kif23-EGFP is recruited to the cleavage furrow during the onset of furrow deepening (see yellow arrowhead in panel Bi and yellow bracket in panel Bi′). This coincides with the recruitment of VAMP-2 vesicles, which are concentrated at the furrow as shown in panel Bii (see white arrowheads in panels Bii and Bii′). Panel Biii indicates a normal blastoderm with mononucleate cells and characteristic kif23-EGFP spots between daughter cells that have successfully completed cytokinesis. Panel Ci on the other hand clearly shows that kif23-trnc-EGFP fails to be recruited to the cleavage furrow during furrow deepening and has a dominant-negative effect on VAMP-2 vesicle recruitment to the deepening furrow, as shown in panel Cii. This leads to a failure of normal cytokinesis during the late cleavage period as shown by the presence of multiple syncitial nuclei and an abnormal distribution of kif23-trnc-EGFP (see panel Ciii). Images shown in panels B and C are reconstructed 2-D images from stacks of images captured from the top view of blastomeres undergoing 3rd cleavage. Panels Bi′ and Ci′ are the y–z plane images of panels Bi and Ci, respectively, while panels Bii′ and Cii′ are the x–z plane images of panels Bii and Cii, respectively.