The primary cilium is an antenna-like organelle that is dynamically regulated during the cell cycle. Ciliogenesis is initiated as cells enter quiescence, while cilium resorption precedes mitosis. The mechanisms coordinating ciliogenesis with the cell cycle are unknown. Here we identify the centrosomal protein, Nde1, as a negative regulator of ciliary length. Nde1 is expressed at high levels in mitosis, low levels in quiescence and localizes at the mother centriole, which nucleates the primary cilium. Cells depleted of Nde1 show longer cilia and a delay in cell cycle re-entry that correlates with ciliary length. Knockdown of Nde1 in zebrafish embryos results in increased ciliary length, suppression of cell division, reduction of the number of cells forming the Kupffer’s vesicle, and left-right patterning defects. These data suggest that Nde1 is an integral component of a network coordinating ciliary length with cell cycle progression and have implications in the transition from quiescence to a proliferative state.

Timely dissolution of sister-chromatid cohesion in mitosis ensures accurate chromosome segregation to guard against aneuploidy and tumorigenesis. The complex of shugoshin and protein phosphatase 2A (Sgo1–PP2A) protects cohesin at centromeres from premature removal by mitotic kinases and Wapl in prophase. Here we address the regulation and mechanism of human Sgo1 in centromeric cohesion protection, and show that cyclin-dependent kinase (Cdk)-mediated, mitosis-specific phosphorylation of Sgo1 activates its cohesion-protection function and enables its direct binding to cohesin. The phospho-Sgo1-bound cohesin complex contains PP2A, Pds5, and hypophosphorylated sororin, but lacks Wapl. Expression of non-phosphorylatable sororin bypasses the requirement for Sgo1–PP2A in centromeric cohesion. Thus, mitotic phosphorylation of Sgo1 targets Sgo1–PP2A to cohesin, promotes dephosphorylation of Pds5-bound sororin, and protects centromeric cohesin from Wapl. PP2A-orchestrated, selective removal of a specific subset of phosphorylation from cohesin and its regulators underlies centromeric cohesion protection.

We have identified a regulator of sister chromatid cohesion in a screen for cell cycle-controlled proteins. This 35 kDa protein is degraded through anaphase-promoting complex (APC)-dependent ubiquitination in G1. The protein is nuclear in interphase cells, dispersed from the chromatin in mitosis, and interacts with the cohesin complex. In Xenopus embryos, overexpression of the protein causes failure to resolve and segregate sister chromatids in mitosis and an increase in the level of cohesin associated with metaphase chromosomes. In cultured cells, depletion of the protein causes mitotic arrest and complete failure of sister chromatid cohesion. This protein is thus an essential, cell cycle-dependent mediator of sister chromatid cohesion. Based on sequence analysis, this protein has no apparent orthologs outside of the vertebrates. We speculate that the protein, which we have named sororin, regulates the ability of the cohesin complex to mediate sister chromatid cohesion, perhaps by altering the nature of the interaction of cohesin with the chromosomes.

Summary Background Chromosome instability is thought to be a major contributor to cancer malignancy and birth defects. For balanced chromosome segregation in mitosis, kinetochores on sister chromatids bind and pull on microtubules emanating from opposite spindle poles. This tension contributes to the correction of improper kinetochore attachments and is opposed by the cohesin complex that holds the sister chromatids together. Normally, within minutes of alignment at the metaphase plate, chromatid cohesion is released, allowing each cohort of chromatids to move synchronously to opposite poles in anaphase, an event closely coordinated with mitotic exit. Results Here we show that during experimentally induced metaphase delay spindle pulling forces can cause asynchronous chromatid separation, a phenomenon we term “cohesion fatigue.” Cohesion fatigue is not blocked by inhibition of Plk1, a kinase essential for the “prophase pathway” of cohesin release from chromosomes or by depletion of separase, the protease that normally drives chromatid separation at anaphase. Cohesion fatigue is inhibited by drug-induced depolymerization of mitotic spindle microtubules and by experimentally increasing the levels of cohesin on mitotic chromosomes. In cells undergoing cohesion fatigue, cohesin proteins remain associated with the separated chromatids. Conclusion In cells arrested at metaphase, pulling forces originating from kinetochore-microtubule interactions can, with time, rupture normal sister chromatid cohesion. This cohesion fatigue, resulting in unscheduled chromatid separation in cells delayed at metaphase, constitutes a previously overlooked source for chromosome instability in mitosis and meiosis.

Enteropathogenic Escherichia coli (EPEC) translocates effector proteins into mammalian cells to promote reorganization of the cytoskeleton into filamentous actin pedestals. One effector, Tir, is a transmembrane receptor for the bacterial surface adhesin intimin, and intimin binding by the extracellular domain of Tir is required for actin assembly. The cytoplasmic NH2 terminus of Tir interacts with focal adhesion proteins, and its tyrosine-phosphorylated COOH terminus binds Nck, a host adaptor protein critical for pedestal formation. To define the minimal requirements for EPEC-mediated actin assembly, Tir derivatives were expressed in mammalian cells in the absence of all other EPEC components. Replacement of the NH2 terminus of Tir with a viral membrane-targeting sequence promoted efficient surface expression of a COOH-terminal Tir fragment. Artificial clustering of this fusion protein revealed that the COOH terminus of Tir, by itself, is sufficient to initiate a complete signaling cascade leading to pedestal formation. Consistent with this finding, clustering of Nck by a 12-residue Tir phosphopeptide triggered actin tail formation in Xenopus egg extracts.

Sister chromatids are held together, from the time they are made during S phase until they are pulled apart just before cell division, by a protein complex called cohesin. The mechanistic details by which sister chromatid cohesion is established and maintained have remained elusive, particularly in vertebrate systems. Sororin, a protein that interacts with the cohesin complex, is essential for cohesion in vertebrates, but how it participates in the process is unknown. Here we demonstrate that sororin recruitment depends on active DNA replication and that sororin loading onto chromosomes depends upon another essential cohesion factor, the acetyltransferase Eco2. We find that Eco2, like sororin, is a substrate of the anaphasepromoting complex (APC), which ensures that protein levels remain low before S phase. These findings demonstrate that sororin and Eco2 work together to form a unique regulatory module that limits cohesion to cells with replicated chromatin and support a model in which cohesion in vertebrates is not fully established until the G2 phase of the cell cycle.anaphase-promoting complex | cohesion establishment | acetylation T he events that result in sister chromatid cohesion are conceptually divisible into three discrete steps: cohesin loading, cohesion establishment, and cohesion maintenance.Cohesion establishment, thought of as the conversion of DNAbound cohesin complexes to a state in which they hold two sister chromatids together, is poorly understood at the molecular level. Several lines of evidence suggest that establishment occurs during DNA replication (1). An emerging model is that cohesin loads on the chromatin before replication (2, 3) and the Smc3 subunit of the complex is then modified by members of the Eco family of acetyltransferase proteins (Eco1/Ctf7 in yeast) (4-7). Eco-dependent acetylation makes cohesin refractory to the activity of the Wapl protein, which destabilizes cohesin/chromatin interactions in G2 (5, 8). Eco1/Ctf7 interacts directly with replication fork components, perhaps explaining how cohesion establishment is integrated with DNA replication (9-11). In the absence of both Eco1 and Wapl/Rad61, cohesion is still established (5). This suggests that cohesion establishment is in fact a default state that in the absence of Wapl/Rad61 need not be integrated with DNA replication by Eco1.It is not clear whether cohesion maintenance is an active process, or simply the result of stable associations that develop during DNA replication. In support of the latter hypothesis, in vertebrates a pool of cohesin that associates stably with the chromatin develops during DNA replication and persists into G2 (12). It has been proposed that this stable pool of cohesin represents complexes that are actively engaged in cohesion. Just what this stability represents at a molecular level is not known. To date, the only factors exclusively involved in maintenance have their most obvious impact in metaphase where they influence the ability to resist removal of cohesin from the chromosomes in mitos...

Entry into mitosis requires the activation of cdk1/cyclin B, while mitotic exit is achieved when the same kinase activity decreases, as cyclin B is degraded. Cyclin B proteolysis is mediated by the anaphase promoting complex, or APC, an E3 ligase that is active at anaphase in mitosis through G1. We have identified a G1 substrate of the APC that we have termed Tome-1, for trigger of mitotic entry. Tome-1 is a cytosolic protein required for proper activation of cdk1/cyclin B and mitotic entry. Tome-1 associates with Skp-1 and is required for degradation of the cdk1 inhibitory tyrosine kinase wee1; Tome-1 therefore appears to be acting as part of an SCF-type E3 for wee1. Degradation of Tome-1 during G1 allows for wee 1 accumulation during interphase, thereby providing a critical link between the APC and SCF pathways in regulation of cdk1/cyclin B activity and thus mitotic entry and exit.

Sister chromatids are tethered together by the cohesin complex from the time they are made until their separation at anaphase. The ability of cohesin to tether sister chromatids together depends on acetylation of its Smc3 subunit by members of the Eco1 family of cohesin acetyltransferases. Vertebrates express two orthologs of Eco1, called Esco1 and Esco2, both of which are capable of modifying Smc3, but their relative contributions to sister chromatid cohesion are unknown. We therefore set out to determine the precise contributions of Esco1 and Esco2 to cohesion in vertebrate cells. Here we show that cohesion establishment is critically dependent upon Esco2. Although most Smc3 acetylation is Esco1 dependent, inactivation of the gene has little effect on mitotic cohesion. The unique ability of Esco2 to promote cohesion is mediated by sequences in the N terminus of the protein. We propose that Esco1-dependent modification of Smc3 regulates almost exclusively the noncohesive activities of cohesin, such as DNA repair, transcriptional control, chromosome loop formation, and/or stabilization. Collectively, our data indicate that Esco1 and Esco2 contribute to distinct and separable activities of cohesin in vertebrate cells.