It has previously been shown that four Ca
2+ transients, generated by release from intracellular stores, are associated with cytokinesis during the early cell division cycles of zebrafish embryos (Webb et al., 1997; Chang and Lu, 2000; Lee et al., 2003). These have been termed the furrow positioning, propagation, deepening and apposition transients. Here, I clearly demonstrate that the furrow positioning Ca
2+ transient precedes the appearance of the cleavage furrow on the surface of the blastodisc, suggesting, therefore, that it is a key component of the furrow positioning signal. Through the introduction of the Ca
2+ shuttle buffer, dibromo-BAPTA, at specific times to challenge the individual transients, I demonstrate the requirement of the four Ca
2+ transients in the furrow positioning,...[
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It has previously been shown that four Ca
2+ transients, generated by release from intracellular stores, are associated with cytokinesis during the early cell division cycles of zebrafish embryos (Webb et al., 1997; Chang and Lu, 2000; Lee et al., 2003). These have been termed the furrow positioning, propagation, deepening and apposition transients. Here, I clearly demonstrate that the furrow positioning Ca
2+ transient precedes the appearance of the cleavage furrow on the surface of the blastodisc, suggesting, therefore, that it is a key component of the furrow positioning signal. Through the introduction of the Ca
2+ shuttle buffer, dibromo-BAPTA, at specific times to challenge the individual transients, I demonstrate the requirement of the four Ca
2+ transients in the furrow positioning, propagation, deepening and apposition processes. Pharmacological studies show that all four transients are generated by Ca
2+ released via inositol 1,4,5-trisphosphate (IP
3) receptors and not via ryanodine receptors or NAADP-sensitive channels. In addition, microinjection of IP
3 results in the release of Ca
2+ via IP
3-sensitive stores. Furthermore, by labeling the endoplasmic reticulum and IP
3 receptors by immunocytochemistry, I demonstrate that they are initially both localized in the blastodisc cortex at the specific position of the future cleavage furrow; i.e., they appear prior to the physical appearance of the furrow at the blastodisc surface. As the furrow ingresses, they are then localized on either side of the deepening furrow. These new data thus provide additional evidence that the ER and IP
3 receptors are the intracellular Ca
2+-store and release mechanisms, respectively, for generating all the cytokinetic transients.
I also report evidence to suggest that during the first few cell division cycles in zebrafish embryos, a dynamic population of central-spindle microtubules serve a major function in positioning the cleavage furrow at the blastoderm surface. Originating from the midzone of the mitotic spindle, they develop into a mid-spindle "pre-furrowing microtubule array" (pf-MTA) that expands upward and outward from the spindle midzone to the blastodisc surface. I suggest that this pf-MTA transmits positional information from the mitotic spindle to the blastodisc cortex that results in the correctly positioned assembly of the cytokinetic contractile apparatus. It is proposed that the pf-MTA is also involved in localizing the corticular ER to the cleavage plane, thus providing a mechanistic link between the mitotic spindle and the series of cytokinetic Ca
2+ transients. I also suggest that the pf-MTA then develops into a furrow-ingression microtubule array that helps to direct and assemble the deepening furrow as it cuts its way through the blastodisc. It then also plays a role in transporting key components to facilitate furrow apposition.
Finally, I provide evidence for some of the possible downstream targets of the cytokinetic Ca
2+ transients. I show that the furrow propagation Ca
2+ transient co-localizes with a region of actin, which suggests that this transient may be involved in the assembly of the actomyosin contractile arc. In addition, embryos treated with a myosin II ATPase inhibitor or inhibitors of exocytosis all exhibited normal cytokinetic Ca
2+ transients but abnormal cytokinesis with respect to the shape and apposition of the daughter blastomeres. These results suggest that the propagation and deepening Ca
2+ transients are upstream in the cytokinetic sequence of events from actomyosin contractile band assembly/contraction and vesicle exocytosis, respectively.
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