Going mobile: microtubule motors and chromosome segregation.

Proper chromosome segregation in eukaryotes depends upon the mitotic and meiotic spindles, which assemble at the time of cell division and then disassemble upon its completion. These spindles are composed in large part of microtubules, which either generate force by controlled polymerization and depolymerization or transduce force generated by molecular microtubule motors. In this review, we discuss recent insights into chromosome segregation mechanisms gained from the analyses of force generation during meiosis and mitosis. These analyses have demonstrated that members of the kinesin superfamily and the dynein family are essential in all organisms for proper chromosome and spindle behavior. It is also apparent that forces generated by microtubule polymerization and depolymerization are capable of generating forces sufficient for chromosome movement in vitro; whether they do so in vivo is as yet unclear. An important realization that has emerged is that some spindle activities can be accomplished by more than one motor so that functional redundancy is evident. In addition, some meiotic or mitotic movements apparently occur through the cooperative action of independent semiredundant processes. Finally, the molecular characterization of kinesin-related proteins has revealed that variations both in primary sequence and in associations with other proteins can produce motor complexes that may use a variety of mechanisms to transduce force in association with microtubules. Much remains to be learned about the regulation of these activities and the coordination of opposing and cooperative events involved in chromosome segregation; this set of problems represents one of the most important future frontiers of research.

Two genes required for the binding of an essential Saccharomyces cerevisiae kinetochore complex to DNA.

Kinetochores are DNA-protein structures that assemble on centromeric DNA and attach chromosomes to spindle microtubules. Because of their simplicity, the 125-bp centromeres of Saccharomyces cerevisiae are particularly amenable to molecular analysis. Budding yeast centromeres contain three sequence elements of which centromere DNA sequence element III (CDEIII) appears to be particularly important. cis-acting mutations in CDEIII and trans-acting mutations in genes encoding subunits of the CDEIII-binding complex (CBF3) prevent correct chromosome transmission. Using temperature-sensitive mutations in CBF3 subunits, we show a strong correlation between DNA-binding activity measured in vitro and kinetochore activity in vivo. We extend previous findings by Goh and Kilmartin [Goh, P.-Y. & Kilmartin, J.V. (1993) J. Cell Biol. 121, 503-512] to argue that DNA-bound CBF3 may be involved in the operation of a mitotic checkpoint but that functional CBF3 is not required for the assembly of a bipolar spindle.

Emergent spindle oscillations and intermittent burst firing in a thalamic model: specific neuronal mechanisms.

The rhythmogenesis of 10-Hz sleep spindles is studied in a large-scale thalamic network model with two cell populations: the excitatory thalamocortical (TC) relay neurons and the inhibitory nucleus reticularis thalami (RE) neurons. Spindle-like bursting oscillations emerge naturally from reciprocal interactions between TC and RE neurons. We find that the network oscillations can be synchronized coherently, even though the RE-TC connections are random and sparse, and even though individual neurons fire rebound bursts intermittently in time. When the fast gamma-aminobutyrate type A synaptic inhibition is blocked, synchronous slow oscillations resembling absence seizures are observed. Near-maximal network synchrony is established with even modest convergence in the RE-to-TC projection (as few as 5-10 RE inputs per TC cell suffice). The hyperpolarization-activated cation current (Ih) is found to provide a cellular basis for the intermittency of rebound bursting that is commonly observed in TC neurons during spindles. Such synchronous oscillations with intermittency can be maintained only with a significant degree of convergence for the TC-to-RE projection.

Cdc37 is required for association of the protein kinase Cdc28 with G1 and mitotic cyclins.

Studies of the temperature-sensitive cdc37-1 mutant of Saccharomyces cerevisiae suggest that Cdc37 is required for passage through the G1 phase of the cell cycle, but its precise function is not known. We have investigated the role of Cdc37 in the regulation of the cyclin-dependent protein kinase Cdc28. We find that G1 arrest in the cdc37-1 mutant is accompanied by a decrease in the Cdc28 activity associated with the G1 cyclin Cln2. This defect appears to be caused by a decrease in the binding of Cdc28 and Cln2. cdc37-1 mutants also exhibit a defect in the binding and activation of Cdc28 by the mitotic cyclin Clb2. Thus Cdc37 may be a regulator that is required for the association of Cdc28 with multiple cyclins.

Cell cycle-related shifts in subcellular localization of BCR: association with mitotic chromosomes and with heterochromatin.

The disruption of the BCR gene and its juxtaposition to and consequent activation of the ABL gene has been implicated as the critical molecular defect in Philadelphia chromosome-positive leukemias. The normal BCR protein is a multifunctional molecule with domains that suggest its participation in phosphokinase and GTP-binding pathways. Taken together with its localization to the cytoplasm of uncycled cells, it is therefore presumed to be involved in cytoplasmic signaling. By performing a double aphidicolin block for cell cycle synchronization, we currently demonstrate that the subcellular localization of BCR shifts from being largely cytoplasmic in interphase cells to being predominantly perichromosomal in mitosis. Furthermore, with the use of immunogold labeling and electron microscopy, association of BCR with DNA, in particular heterochromatin, can be demonstrated even in quiescent cells. Results were similar in cell lines of lymphoid or myeloid origin. These observations suggest a role for BCR in the phosphokinase interactions linked to condensed chromatin, a network previously implicated in cell cycle regulation.

Dual Regulation of the mitotic exit network (MEN) by PP2A-Cdc55 phosphatase.

Exit from mitosis in budding yeast is triggered by activation of the key mitotic phosphatase Cdc14. At anaphase onset, the protease separase and Zds1 promote the downregulation of PP2A(Cdc55) phosphatase, which facilitates Cdk1-dependent phosphorylation of Net1 and provides the first wave of Cdc14 activity. Once Cdk1 activity starts to decline, the mitotic exit network (MEN) is activated to achieve full Cdc14 activation. Here we describe how the PP2A(Cdc55) phosphatase could act as a functional link between FEAR and MEN due to its action on Bfa1 and Mob1. We demonstrate that PP2A(Cdc55) regulates MEN activation by facilitating Cdc5- and Cdk1-dependent phosphorylation of Bfa1 and Mob1, respectively. Downregulation of PP2A(Cdc55) initiates MEN activity up to Cdc15 by Bfa1 inactivation. Surprisingly, the premature Bfa1 inactivation observed does not entail premature MEN activation, since an additional Cdk1-Clb2 inhibitory signal acting towards Dbf2-Mob1 activity restrains MEN activity until anaphase. In conclusion, we propose a clear picture of how PP2A(Cdc55) functions affect the regulation of various MEN components, contributing to mitotic exit.

A treatise on spinning machinery : illustrated with plans of different machines made use of in that art, from the spindle and distaff of the ancients to the machines which have been invented or improved by the moderns ... /

Plates by T. Clerk.

Preliminary test on axial spindle expansion of nuber 739 Ex-Cell-O numerical tape control machine /

"U.S. Atomic Energy Commission Contract AT(29-1)-1106."

The Double-spindle Hartness flat turret lathe.

Mode of access: Internet.

Organizational maintenance manual : lathe, engine, bench mounted, solid bed type, 10 inch swing, no. 2 Morse taper center, 1-3/8 spindle hole, 110-volts, 60 cycle, single phase 3/4-horsepower, w/Army dwg. no. 7550151 bench (standard-modern tool series 2,000 model, 11 inch) (3416-517-0955).

"November 1965."

Operator's manual : grinding machine, machine tool attachment, internal and external grinding, 5" maximum external diameter wheel, 1/4" minimum internal diameter wheel, 4,600 to 42,400 rpm spindle speed, 1/2 hp, AC/DC, 115 V, 60-C, single phase (Dumore series 5) ...

"October 1967."

Transient production of recombinant proteins by chinese hamster ovary cells using polyethyleneimine/DNA complexes in combination with microtubule disrupting anti-mitotic agents

We have developed a simple and robust transient expression system utilizing the 25 kDa branched cationic polymer polyethylenimine (PEI) as a vehicle to deliver plasmid DNA into suspension-adapted Chinese hamster ovary cells synchronized in G2/M phase of the cell cycle by anti-mitotic microtubule disrupting agents. The PEI-mediated transfection process was optimized with respect to PEI nitrogen to DNA phosphate molar ratio and the plasmid DNA mass to cell ratio using a reporter construct encoding firefly luciferase. Optimal production of luciferase was observed at a PEI N to DNA P ratio of 10:1 and 5 mug DNA 10(6) cells(-1). To manipulate transgene expression at mitosis, we arrested cells in G2/M phase of the cell cycle using the microtubule depolymerizing agent nocodazole. Using secreted human alkaline phosphatase (SEAP) and enhanced green fluorescent protein (eGFP) as reporters we showed that continued inclusion of nocodazole in cell culture medium significantly increased both transfection efficiency and reporter protein production. In the presence of nocodazole, greater than 90% of cells were eGFP positive 24 h post-transfection and qSEAP was increased almost fivefold, doubling total SEAP production. Under optimal conditions for PEI-mediated transfection, transient production of a recombinant chimeric IgG(4) encoded on a single vector was enhanced twofold by nocodazole, a final yield of approximately 5 mug mL(-1) achieved at an initial viable cell density of 1 x 10(6) cells mL(-1). The glycosylation of the recombinant antibody at Asn(297) was not significantly affected by nocodazole during transient production by this method. (C) 2004 Wiley Periodicals, Inc.

Cell death mechanisms associated with G(2) radiosensitivity in patients with prostate cancer and benign prostatic hyperplasia

Cells respond to genotoxic insults such as ionizing radiation by halting in the G(2) phase of the cell cycle. Delayed cell death (mitotic death) can occur when the cell is released from G(2), and specific spindle defects form endopolyploid cells (endoreduplication/tetraploidy). Enhanced G(2) chromosomal radiosensitivity has been observed in many cancers and genomic instability syndromes, and it is manifested by radiation-induced chromatid aberrations observed in lymphocytes of patients. Here we compare the G(2) chromosomal radiosensitivity in prostate patients with benign prostatic hyperplasia (BPH) or prostate cancer with disease-free controls. We also investigated whether there is a correlation between G(2) chromosomal radiosensitivity and aneuploidy (tetraploidy and endoreduplication), which are indicative of mitotic cell death. The G(2) assay was carried out on all human blood samples. Metaphase analysis was conducted on the harvested chromosomes by counting the number of aberrations and the mitotic errors (endoreduplication/tetraploidy) separately per 100 metaphases. A total of 1/14 of the controls were radiosensitive in G(2) compared to 6/15 of the BPH patients and 15/17 of the prostate cancer patients. Radiation-induced mitotic inhibition was assessed to determine the efficacy of G(2) checkpoint control in the prostate patients. There was no significant correlation of G(2) radiosensitivity scores and mitotic inhibition in BPH patients (P = 0.057), in contrast to prostate cancer patients, who showed a small but significant positive correlation (P = 0.029). Furthermore, there was no significant correlation between G(2) radiosensitivity scores of BPH patients and endoreduplication/ tetraploidy (P = 0.136), which contrasted with an extremely significant correlation observed in prostate cancer patients (P < 0.0001). In conclusion, cells from prostate cancer patients show increased sensitivity to the induction of G(2) aberrations from ionizing radiation exposure but paradoxically show reduced mitotic indices and aneuploidy as a function of aberration frequency.

Specific morphological features predictive for the basal phenotype in grade 3 invasive ductal carcinoma of breast

Aims: Cytokeratin (CK) 14, a myoepithelial marker, is also expressed in a proportion of breast carcinomas. There is evidence that these tumours show a differing metastatic pattern and clinical outcome from other invasive ductal carcinomas (IDCs) and may need different management. Currently, they are not identified in routine practice and no morphological guidelines exist to aid their identification. The aim of this study was to analyse the histological features of CK14+ IDC. Methods and results: A detailed histological review of 453 grade 3 IDCs revealed 88 (19.4%) that expressed CK14. Assessment was made independently by two pathologists using a standardized 'tick-box' proforma covering grade, architectural and cytological features. The results were analysed using logistic regression to identify features that predicted for basal phenotype. Concordance between the two pathologists was fair to good for most parameters (kappa 0.4-0.6). On multiple logistic regression, the basal phenotype was highly significantly associated with the presence of a central scar (P = 0.005), tumour necrosis (P < 0.0001), presence of spindle cells (P = 0.006) or squamous metaplasia (P < 0.0001), high total mitotic count (> 40 per 10 high-power field) (P = 0.0002) and high nuclear-cytoplasmic ratio (P = 0.0002). Conclusions: Specific morphological features are strongly associated with basal-like breast carcinoma. These could be used in routine diagnostic practice to identify this important subset of tumours.

Inhibition of S/G(2) phase CDK4 reduces mitotic fidelity

Cyclin-dependent kinase 4 (CDK4)/cyclin D has a key role in regulating progression through late G(1) into S phase of the cell cycle. CDK4-cyclin D complexes then persist through the latter phases of the cell cycle, although little is known about their potential roles. We have developed small molecule inhibitors that are highly selective for CDK4 and have used these to define a role for CDK4-cyclin D in G(2) phase. The addition of the CDK4 inhibitor or small interfering RNA knockdown of cyclin D3, the cyclin D partner, delayed progression through G(2) phase and mitosis. The G(2) phase delay was independent of ATM/ATR and p38 MAPK but associated with elevated Wee1. The mitotic delay was because of failure of chromosomes to migrate to the metaphase plate. However, cells eventually exited mitosis, with a resultant increase in cells with multiple or micronuclei. Inhibiting CDK4 delayed the expression of the chromosomal passenger proteins survivin and borealin, although this was unlikely to account for the mitotic phenotype. These data provide evidence for a novel function for CDK4-cyclin D3 activity in S and G(2) phase that is critical for G(2)/M progression and the fidelity of mitosis.