Centralspindlin is essential for the formation of microtubule bundle structures and the equatorial recruitment of factors critical for cytokinesis. Stable accumulation of centralspindlin at the spindle midzone requires its multimerization into clusters and Aurora B kinase activity, which peaks at the central spindle during anaphase. Although Aurora B phosphorylates centralspindlin directly, how this regulates centralspindlin localization is unknown. Here we identify a novel regulatory mechanism by which Aurora B enables centralspindlin to accumulate stably at the spindle midzone. We show that 14-3-3 protein binds centralspindlin when the kinesin-6 component MKLP1 is phosphorylated at S710. 14-3-3 prevents centralspindlin from clustering in vitro, and an MKLP1 mutant that is unable to bind 14-3-3 forms aberrant clusters in vivo. Interestingly, 14-3-3 binding is inhibited by phosphorylation of S708, a known Aurora B target site that lies within the motif bound by 14-3-3. S708 phosphorylation is required for MKLP1 to stably localize to the central spindle, but it is dispensable in an MKLP1 mutant that does not bind 14-3-3. We propose that 14-3-3 serves as a global inhibitor of centralspindlin that allows Aurora B to locally activate clustering and the stable accumulation of centralspindlin between segregating chromosomes.

Opitz syndrome (OS) is a genetically heterogeneous disorder characterized by defects of the ventral midline, including hypertelorism, cleft lip and palate, heart defects, and mental retardation. We recently identified the gene responsible for X-linked OS. The ubiquitously expressed gene product, MID1, is a member of the RING finger family. These proteins are characterized by an N-terminal tripartite protein-protein interaction domain and a conserved C terminus of unknown function. Unlike other RING finger proteins for which diverse cellular functions have been proposed, the function of MID1 is as yet undefined. By using the green f luorescent protein as a tag, we show here that MID1 is a microtubule-associated protein that inf luences microtubule dynamics in MID1-overexpressing cells. We confirm this observation by demonstrating a colocalization of MID1 and tubulin in subcellular fractions and the association of endogenous MID1 with microtubules after in vitro assembly. Furthermore, overexpressed MID1 proteins harboring mutations described in OS patients lack the capability to associate with microtubules, forming cytoplasmic clumps instead. These data give an idea of the possible molecular pathomechanism underlying the OS phenotype.

Summary To take full advantage of fast-acting temperature-sensitive mutations, thermal control must be extremely rapid. We developed the Therminator, a device capable of shifting sample temperature in ~17s while simultaneously imaging cell division in vivo. Applying this technology to six key regulators of cytokinesis, we found that each has a distinct temporal requirement in the C. elegans zygote. Specifically, myosin-II is required throughout cytokinesis until contractile ring closure. In contrast, formin-mediated actin nucleation is only required during assembly and early contractile ring constriction. Centralspindlin is required to maintain division after ring closure, though its GAP activity is only required until just prior to closure. Finally, the Chromosomal Passenger Complex is required for cytokinesis only early in mitosis, but not during metaphase or cytokinesis. Together, our results provide a precise functional timeline for molecular regulators of cytokinesis using the Therminator, a powerful tool for ultra-rapid protein inactivation.

In asymmetrically dividing C. elegans embryos, the core cortical PAR proteins are required to retain septin and anillin at the anterior cortex away from the contractile ring and to promote normal F-actin levels at the contractile ring and successful cytokinesis.

Centralspindlin, a constitutive 2:2 heterotetramer of MKLP1 (a kinesin-6) and the non-motor subunit CYK4, plays important roles in cytokinesis. It is crucial for the formation of central spindle microtubule bundle structure. Its accumulation at the central antiparallel overlap zone is key for recruitment and regulation of downstream cytokinesis factors and for stable anchoring of the plasma membrane at the midbody. Both MKLP1 and CYK4 are required for efficient microtubule bundling. However, the mechanism by which CYK4 contributes to this is unclear. Here we performed structural and functional analyses of centralspindlin using high-speed atomic force microscopy, Fӧrster resonance energy transfer analysis, and in vitro reconstitution. Our data reveal that CYK4 binds to a globular mass in the atypically long MKLP1 neck domain between the catalytic core and the coiled coil and thereby reconfigures the two motor domains in the MKLP1 dimer to be suitable for antiparallel microtubule bundling. Our work provides insights into the microtubule bundling during cytokinesis and into the working mechanisms of the kinesins with non-canonical neck structures.

SUMMARYKinesin heavy chain (KHC), the force-generating component of Kinesin-1, is required for the localization of oskar mRNA and the anchoring of the nucleus in the Drosophila oocyte. These events are crucial for the establishment of the anterior-posterior and dorsal-ventral axes. KHC is also essential for the localization of Dynein and for all ooplasmic flows. Interestingly, oocytes without Kinesin light chain show no major defects in these KHC-dependent processes, suggesting that KHC binds its cargoes and is activated by a novel mechanism. Here, we shed new light on the molecular mechanism of Kinesin function in the germline. Using a combination of genetic, biochemical and motor-tracking studies, we show that PAT1, an APP-binding protein, interacts with Kinesin-1, functions in the transport of oskar mRNA and Dynein and is required for the efficient motility of KHC along microtubules. This work suggests that the role of PAT1 in cargo transport in the cell is linked to PAT1 function as a positive regulator of Kinesin motility.

Cytokinesis, the physical division of one cell into two, is powered by constriction of an actomyosin contractile ring. It has long been assumed that all animal cells divide by a similar molecular mechanism, but growing evidence suggests that cytokinetic regulation in individual cell types has more variation than previously realized. In the four-cell Caenorhabditis elegans embryo, each blastomere has a distinct cell fate, specified by conserved pathways. Using fast-acting temperature-sensitive mutants and acute drug treatment, we identified cell-type-specific variation in the cytokinetic requirement for a robust forminCYK-1-dependent filamentous-actin (F-actin) cytoskeleton. In one cell (P2), this cytokinetic variation is cell-intrinsically regulated, whereas in another cell (EMS) this variation is cell-extrinsically regulated, dependent on both SrcSRC-1 signaling and direct contact with its neighbor cell, P2. Thus, both cell-intrinsic and -extrinsic mechanisms control cytokinetic variation in individual cell types and can protect against division failure when the contractile ring is weakened.

We have investigated the potential utility of monoclonal antibodies against mini-chromosome maintenance-2 protein (Mcm2) in predicting meningioma recurrence. MCM proteins are members of the DNA-binding prereplicative complex and are essential for eukaryotic DNA replication. They are present throughout the cell cycle, but are down-regulated in quiescence and cell differentiation, making them specific markers of proliferating cells. We analysed 10 benign meningiomas that subsequently recurred within a 5-year period, together with 20 matched non-recurrent benign meningiomas. There was no significant correlation between histological subtype, mitotic count or Ki-67 labelling index and tumour recurrence. We observed that whilst the average Mcm2 labelling index (LI) of the tumour section as a whole (LI(Ave)) is not significantly different between recurrent and nonrecurrent meningiomas, the Mcm2 labelling index in the area of highest proliferative activity within the tumour section (LI(Max)) is significantly higher in recurrent meningiomas (p < 0.0001). Seven out of the 10 recurrent meningiomas displayed a Mcm2 LI((Max) greater than 30%, compared to 0 out of 20 for non-recurrent tumours. In conclusion, these results suggest that analysis of Mcm2 expression may facilitate identification of patients with a high risk of meningioma recurrence, for whom adjuvant radiotherapy may be of benefit.