Roger Karess

The early Drosophila embryo undergoes two distinct membrane invagination events believed to be mechanistically related to cytokinesis: metaphase furrow formation and cellularization. Both involve actin cytoskeleton rearrangements, and both have myosin II at or near the forming furrow. Actin and myosin are thought to provide the force driving membrane invagination; however, membrane addition is also important. We have examined the role of myosin during these events in living embryos, with a fully functional myosin regulatory light-chain-GFP chimera. We find that furrow invagination during metaphase and cellularization occurs even when myosin activity has been experimentally perturbed. In contrast, the basal closure of the cellularization furrows and the first cytokinesis after cellularization are highly dependent on myosin. Strikingly, when ingression of the cellularization furrow is experimentally inhibited by colchicine treatment, basal closure still occurs at the appropriate time, suggesting that it is regulated independently of earlier cellularization events. We have also identified a previously unrecognized reservoir of particulate myosin that is recruited basally into the invaginating furrow in a microfilament-independent and microtubule-dependent manner. We suggest that cellularization can be divided into two distinct processes: furrow ingression, driven by microtubule mediated vesicle delivery, and basal closure, which is mediated by actin/myosin based constriction.

The nuclei of early syncytial Drosophila embryos migrate dramatically toward the poles. The cellular mechanisms driving this process, called axial expansion, are unclear, but myosin II activity is required. By following regulatory myosin light chain (RLC)–green fluorescent protein dynamics in living embryos, we observed cycles of myosin recruitment to the cortex synchronized with mitotic cycles. Cortical myosin is first seen in a patch at the anterocentral part of the embryo at cycle 4. With each succeeding cycle, the patch expands poleward, dispersing at the beginning of each mitosis and reassembling at the end of telophase. Each cycle of actin and myosin recruitment is accompanied by a cortical contraction. The cortical myosin cycle does not require microtubules but correlates inversely with Cdc2/cyclinB (mitosis-promoting factor) activity. A mutant RLC lacking inhibitory phosphorylation sites was fully functional with no effect on the cortical myosin cycle, indicating that Cdc2 must be modulating myosin activity by some other mechanism. An inhibitor of Rho kinase blocks the cortical myosin recruitment cycles and provokes a concomitant failure of axial expansion. These studies suggest a model in which cycles of myosin-mediated contraction and relaxation, tightly linked to Cdc2 and Rho kinase activity, are directly responsible for the axial expansion of the syncytial nuclei.

The zeste-white 10 (zw10) and rough deal (rod) genes of Drosophila both encode kinetochore components, and mutations in either gene greatly increase the missegregation of sister chromatids during mitosis. Here, we present genetic, cytological and biochemical evidence for a close, evolutionarily conserved relationship between the ROD and ZW10 proteins. We show that the phenotypes caused by disruption of either gene’s function are similar in Drosophila and in C. elegans. No additive effects are observed in zw10; rod double null mutants. In flies, the two proteins always colocalize and, moreover, require each other for their recruitment to the mitotic apparatus. The human ROD and ZW10 homologs also colocalize on HeLa cell kinetochores or kinetochore microtubules throughout most but not all of mitosis. Finally, we show that in both Drosophila and human cells, ROD and ZW10 are in fact physically associated, and in Drosophila these proteins are together constituents of a large (700-900 kDa), soluble macromolecular complex.

The metaphase–anaphase transition during mitosis is carefully regulated in order to assure high-fidelity transmission of genetic information to the daughter cells. A surveillance mechanism known as the metaphase checkpoint (or spindle-assembly checkpoint) monitors the attachment of kinetochores to the spindle microtubules, and inhibits anaphase onset until all chromosomes have achieved a proper bipolar orientation on the spindle. Defects in this checkpoint lead to premature anaphase onset, and consequently to greatly increased rates of aneuploidy. Here we show that the Drosophila kinetochore components Rough deal (Rod) and Zeste-White 10 (Zw10) are required for the proper functioning of the metaphase checkpoint in flies. Drosophila cells lacking either ROD or Zw10 exhibit a phenotype that is similar to that of bub1 mutants — they do not arrest in metaphase in response to spindle damage, but instead separate sister chromatids, degrade cyclin B and exit mitosis. These are the first checkpoint components to be identified that do not have obvious homologues in budding yeast.