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During cell division, genome inheritance is orchestrated by microtubule attachments formed at kinetochores of mitotic chromosomes. The primary microtubule coupler at the kinetochore, the Ndc80 complex, is regulated by Aurora kinase phosphorylation of its N-terminal tail. Dephosphorylation is proposed to stabilize kinetochore-microtubule attachments by strengthening electrostatic interactions of the tail with the microtubule lattice. Here, we show that removal of the Ndc80 tail, which compromises in vitro microtubule binding, has no effect on kinetochore-microtubule attachments in the Caenorhabditis elegans embryo. Despite this, preventing Aurora phosphorylation of the tail results in prematurely stable attachments that restrain spindle elongation. This premature stabilization requires the conserved microtubule-binding Ska complex, which enriches at attachment sites prior to anaphase onset to dampen chromosome motion. We propose that Ndc80-tail dephosphorylation promotes stabilization of kinetochore-microtubule attachments via the Ska complex and that this mechanism ensures accurate segregation by constraining chromosome motion following biorientation on the spindle.

Abscission completes cytokinesis to form the two daughter cells. Although abscission could be organized from the inside out by the microtubule-based midbody or from the outside in by the contractile ring-derived midbody ring, it is assumed that midbody microtubules scaffold the abscission machinery. In this paper, we assess the contribution of midbody microtubules versus the midbody ring in the Caenorhabditis elegans embryo. We show that abscission occurs in two stages. First, the cytoplasm in the daughter cells becomes isolated, coincident with formation of the intercellular bridge; proper progression through this stage required the septins (a midbody ring component) but not the membrane-remodeling endosomal sorting complex required for transport (ESCRT) machinery. Second, the midbody and midbody ring are released into a specific daughter cell during the subsequent cell division; this stage required the septins and the ESCRT machinery. Surprisingly, midbody microtubules were dispensable for both stages. These results delineate distinct steps during abscission and highlight the central role of the midbody ring, rather than midbody microtubules, in their execution.

The chromosomal passenger complex (CPC) and centralspindlin are conserved cytokinesis regulators that localize to the spindle midzone, which forms between the separating chromosomes. Previous work placed the CPC and centralspindlin in a linear pathway that governs midzone formation. Using Caenorhabditis elegans embryos, we test whether there is a similar linear relationship between centralspindlin and the CPC in contractile ring constriction during cytokinesis. We show that simultaneous inhibition of the CPC kinase Aurora B(AIR-2) and the centralspindlin component MKLP1(ZEN-4) causes an additive constriction defect. Consistent with distinct roles for the proteins, inhibition of filamentous septin guanosine triphosphatases alleviates constriction defects in Aurora B(AIR-2)-inhibited embryos, whereas inhibition of Rac does so in MKLP1(ZEN-4)-inhibited embryos. Centralspindlin and the CPC are not required to enrich ring proteins at the cell equator but instead regulate formation of a compact mature ring. Therefore, in contrast to the linear midzone assembly pathway, centralspindlin and the CPC make independent contributions to control transformation of the sheet-like equatorial band into a ribbon-like contractile ring at the furrow tip.

The size of subcellular structures must be tightly controlled to maintain normal cell function. Despite its importance, few studies have determined how the size of organelles or other structures is maintained during development, when cells are growing, dividing, and rearranging. The developing egg chamber is a powerful model in which to study the relative growth rates of subcellular structures. The egg chamber contains a cluster of sixteen germline cells, which are connected through intercellular bridges called ring canals. As the egg chamber grows, the germline cells and the ring canals that connect them increase in size. Here, we demonstrate that ring canal size scaling is related to lineage; the largest, "first born" ring canals increase in size at a relatively slower rate than ring canals derived from subsequent mitotic divisions. This lineage-based scaling relationship is maintained even if directed transport is reduced, ring canal size is altered, or in egg chambers with twice as many germline cells. Analysis of lines that produce larger or smaller mature eggs reveals different strategies could be used to alter final egg size., Summary Statement: Using the fruit fly egg chamber as a model, this study demonstrates that the size and scaling of germline intercellular bridges vary based on lineage.

Drosophila egg chamber development depends on a number of dynamic cellular processes that contribute to the final shape and function of the egg. We can gain insight into the mechanisms underlying these events by combining the power of Drosophila genetics and ex vivo live imaging. During developmental stages 1-8, egg chambers rotate around their anterior-posterior axes due to collective migration of the follicular epithelium. This motion is required for the proper elongation of the egg chamber. Here, we describe how to prepare stage 1-8 egg chambers for live imaging. We provide alternate protocols for the use of inverted or upright microscopes and describe ways to stabilize egg chambers to reduce drift during imaging. We discuss the advantages and limitations of these methods to assist the researcher in choosing an appropriate method based on experimental need and available resources.