Cytokinesis, the final event of the cell cycle, involves acto-myosin based constriction of the cleavage furrow to produce two daughter cells. Recent studies demonstrate that targeted membrane addition at the cleavage furrow is also essential for normal cytokinesis. This insight opens entirely new avenues of investigation exploring the sources, sites, mechanisms and timing of membrane addition during cytokinesis. These studies have a direct relevance to our understanding of cancer, because failures in membrane addition result in profound defects in cytokinesis. Failed cytokinesis produces a cell with two nuclei. These abnormal tetraploid cells are genetically unstable and are thought to be an early event of tumorigenesis. We address the role of membrane addition in driving cytokinesis by focusing on the furrows that form during the divisions immediately prior to and after cellularization in the early Drosophila embryo. During these cortical divisions, thousands of furrows form every fifteen minutes, and furrow ingression is driven almost entirely by vesicle-mediated membrane addition, making it an ideal system to study this process. In addition, these divisions are amenable to molecular genetic and biochemical experiments, and can be followed using high-resolution live imaging techniques. A major focus of this proposal is the investigation of the mechanisms driving endocytic-based vesicle trafficking to the ingressing cleavage furrow. We have discovered that vesicle-mediated addition from the re- cycling-endosome (RE) plays an important role in maintaining F-actin at the cleavage furrow, which is required for stable furrows. Insight into this mechanism comes from our discovery during the last funding period that normal RE function is required to localize RhoGEF, a potent actin remodeler, to the furrow. We propose experiments to define the role of the RE in transporting and targeting RhoGEF to the furrows. A second goal is to understand how endosome-based vesicle delivery to the cleavage furrow is coordinated with the cell cycle. We will address this question by focusing on the role of cycle-cell kinases in activating RE-based vesicle delivery to the cleavage furrow. Specifically, we will focus on Rab11, a small GTPase associated with the recycling endosome, and its cell-cycle-regulated association with the conserved effector protein Nuf/FIP3. We hypothe- size that cell-cycle-regulated, microtubule-motor-based recruitment of Nuf/FIP3 to Rab11 at the RE activates RE-based vesicle targeting to the cleavage furrow. We will take advantage of a comprehensive set of Drosophila transgenic lines that express dominant negative Rabs in order to identify additional endosomal and trafficking components required for vesicle-mediated membrane delivery during cytokinesis. Finally, using the unique genetics of the early Drosophila embryo, we can screen the entire genome for all additional trafficking components required for cytokinesis. These studies will not only shed light on the origin of cancer, but will have immediate implications for the design of new anti-cancer therapies.
Work over the past decade has made it clear the endosome-based vesicle delivery plays a key role in all stages of cytokinesis. This has opened up entirely new areas of research in the cell-cycle-regulation, which we are addressing in this grant. Defects in cytokinesis lead to tetraploidy, genomic instability, and ultimately cancer. Therefore, these studies will have direct impact on our understanding of the origins of cancer and in the design of new anti-cancer therapies.
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