Extracting isolated Drosophila centrosomes with 2 M KI generates salt-resistant scaffolds that lack the centrosomal proteins CP190, CP60, centrosomin, and gamma-tubulin. To clarify the role of these proteins in microtubule nucleation by centrosomes and to identify additional centrosome components required for nucleation, we have developed an in vitro complementation assay for centrosome function. Centrosome aster formation is reconstituted when these inactive, salt-stripped centrosome scaffolds are supplemented with a soluble fraction of a Drosophila embryo extract. The CP60 and CP190 can be removed from this extract without effect, whereas removing the gamma-tubulin destroys the complementing activity. Consistent with these results, we find no evidence that these three proteins form a complex together. Instead, gamma-tubulin is found in two distinct protein complexes of 240,000 and approximately 3,000,000 D. The larger complex, which is analogous to the Xenopus gamma-tubulin ring complex (gammaTuRC) (Zheng, Y., M.L. Wong, B. Alberts, and T. Mitchison. 1995. Nature. 378:578-583), is necessary but not sufficient for complementation. An additional factor found in the extract is required. These results provide the first evidence that the gammaTuRC is required for microtubule nucleation at the centrosome.

Centrioles are surrounded by pericentriolar material (PCM), which is proposed to promote new centriole assembly by concentrating gamma-tubulin. Here, we quantitatively monitor new centriole assembly in living Caenorhabditis elegans embryos, focusing on the conserved components SAS-4 and SAS-6. We show that SAS-4 and SAS-6 are coordinately recruited to the site of new centriole assembly and reach their maximum levels during S phase. Centriolar SAS-6 is subsequently reduced by a mechanism intrinsic to the early assembly pathway that does not require progression into mitosis. Centriolar SAS-4 remains in dynamic equilibrium with the cytoplasmic pool until late prophase, when it is stably incorporated in a step that requires gamma-tubulin and microtubule assembly. These results indicate that gamma-tubulin in the PCM stabilizes the nascent daughter centriole by promoting microtubule addition to its outer wall. Such a mechanism may help restrict new centriole assembly to the vicinity of preexisting parent centrioles that recruit PCM.

During cell division, kinetochores couple chromosomes to spindle microtubules. To protect against chromosome gain or loss, kinetochores lacking microtubule attachment locally catalyze association of the checkpoint proteins Cdc20 and Mad2, which is the key event in the formation of a diffusible checkpoint complex that prevents mitotic exit. We elucidated the mechanism of kinetochore-catalyzed Mad2-Cdc20 assembly with a probe that specifically monitors this assembly reaction at kinetochores in living cells. We found that catalysis occurs through a tripartite mechanism that includes localized delivery of Mad2 and Cdc20 substrates and two phosphorylation-dependent interactions that geometrically constrain their positions and prime Cdc20 for interaction with Mad2. These results reveal how unattached kinetochores create a signal that ensures genome integrity during cell division.

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.

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.

Centromeres are defined epigenetically in the majority of eukaryotes by the presence of chromatin containing the centromeric histone H3 variant CENP-A. Most species have a single gene encoding a centromeric histone variant whereas C. elegans has two: HCP-3 (also known as CeCENP-A) and CPAR-1. Prior RNAi replacement experiments showed that HCP-3 is the functionally dominant isoform, consistent with CPAR-1 not being detectable in embryos. GFP::CPAR-1 is loaded onto meiotic chromosomes in diakinesis and is enriched on bivalents until meiosis I. Here we show that GFP::CPAR-1 signal loss from chromosomes precisely coincides with homolog segregation during anaphase I. This loss of GFP::CPAR-1 signal reflects proteolytic cleavage between GFP and the histone fold of CPAR-1, as CPAR-1::GFP, in which GFP is fused to the C-terminus of CPAR-1, does not exhibit any loss of GFP signal. A focused candidate screen implicated separase, the protease that initiates anaphase by cleaving the kleisin subunit of cohesin, in this cleavage reaction. Examination of the N-terminal tail sequence of CPAR-1 revealed a putative separase cleavage site and mutation of the signature residues in this site eliminated the cleavage reaction, as visualized by retention of GFP::CPAR-1 signal on separating homologous chromosomes at the metaphase-anaphase transition of meiosis I. Neither cleaved nor uncleavable CPAR-1 were centromere-localized in mitosis and instead localized throughout chromatin, indicating that centromere activity has not been retained in CPAR-1. Although the functions of CPAR-1 and of its separase-dependent cleavage remain to be elucidated, this effort reveals a new substrate of separase and provides an in vivo biosensor to monitor separase activity at the onset of meiosis I anaphase.

Precise regulation of microtubule (MT) dynamics is increasingly recognized as a critical determinant of axon regeneration. In contrast to developing neurons, mature axons exhibit noncentrosomal microtubule nucleation. The factors regulating noncentrosomal MT architecture in axon regeneration remain poorly understood. We report that PTRN-1, the C. elegans member of the Patronin/Nezha/calmodulin- and spectrin-associated protein (CAMSAP) family of microtubule minus-end-binding proteins, is critical for efficient axon regeneration in vivo. ptrn-1-null mutants display generally normal developmental axon outgrowth but significantly impaired regenerative regrowth after laser axotomy. Unexpectedly, mature axons in ptrn-1 mutants display elevated numbers of dynamic axonal MTs before and after injury, suggesting that PTRN-1 inhibits MT dynamics. The CKK domain of PTRN-1 is necessary and sufficient for its functions in axon regeneration and MT dynamics and appears to stabilize MTs independent of minus-end localization. Whereas in developing neurons, PTRN-1 inhibits activity of the DLK-1 mitogen-activated protein kinase (MAPK) cascade, we find that, in regeneration, PTRN-1 and DLK-1 function together to promote axonal regrowth.