Rough deal and Zw10 are required for the metaphase checkpoint in Drosophila

The metaphase-anaphase transition during mitosis is carefully regulated in order to assure high-fidelity transmission of genetic information to the daughter cells. A surveillance mechanism known as the metaphase checkpoint (or spindle-assembly checkpoint) monitors the attachment of kinetochores to the spindle microtubules, and inhibits anaphase onset until all chromosomes have achieved a proper bipolar orientation on the spindle. Defects in this checkpoint lead to premature anaphase onset, and consequently to greatly increased rates of aneuploidy. Here we show that the Drosophila kinetochore components Rough deal (Rod) and Zeste-White 10 (Zw10) are required for the proper functioning of the metaphase checkpoint in flies. Drosophila cells lacking either ROD or Zw10 exhibit a phenotype that is similar to that of bub1 mutants - they do not arrest in metaphase in response to spindle damage, but instead separate sister chromatids, degrade cyclin B and exit mitosis. These are the first checkpoint components to be identified that do not have obvious homologues in budding yeast.     

Anaphase-promoting complex/cyclosome-dependent proteolysis of human cyclin A starts at the beginning of mitosis and is not subject to the spindle assembly checkpoint

Cyclin A is a stable protein in S and G2 phases, but is destabilized when cells enter mitosis and is almost completely degraded before the metaphase to anaphase transition. Microinjection of antibodies against subunits of the anaphase-promoting complex/cyclosome (APC/C) or against human Cdc20 (fizzy) arrested cells at metaphase and stabilized both cyclins A and B1. Cyclin A was efficiently polyubiquitylated by Cdc20 or Cdh1-activated APC/C in vitro, but in contrast to cyclin B1, the proteolysis of cyclin A was not delayed by the spindle assembly checkpoint. The degradation of cyclin B1 was accelerated by inhibition of the spindle assembly checkpoint. These data suggest that the APC/C is activated as cells enter mitosis and immediately targets cyclin A for degradation, whereas the spindle assembly checkpoint delays the degradation of cyclin B1 until the metaphase to anaphase transition. The "destruction box" (D-box) of cyclin A is 10-20 residues longer than that of cyclin B. Overexpression of wild-type cyclin A delayed the metaphase to anaphase transition, whereas expression of cyclin A mutants lacking a D-box arrested cells in anaphase.     

Protein phosphatase 2A regulates MPF activity and sister chromatid cohesion in budding yeast

Background Mitosis is regulated by MPF (maturation promoting factor), the active form of Cdc2/28–cyclin B complexes. Increasing levels of cyclin B abundance and the loss of inhibitory phosphates from Cdc2/28 drives cells into mitosis, whereas cyclin B destruction inactivates MPF and drives cells out of mitosis. Cells with defective spindles are arrested in mitosis by the spindle-assembly checkpoint, which prevents the destruction of mitotic cyclins and the inactivation of MPF. We have investigated the relationship between the spindle-assembly checkpoint, cyclin destruction, inhibitory phosphorylation of Cdc2/28, and exit from mitosis.Results The previously characterized budding yeast mad mutants lack the spindle-assembly checkpoint. Spindle depolymerization does not arrest them in mitosis because they cannot stabilize cyclin B. In contrast, a newly isolated mutant in the budding yeast CDC55 gene, which encodes a protein phosphatase 2A (PP2A) regulatory subunit, shows a different checkpoint defect. In the presence of a defective spindle, these cells separate their sister chromatids and leave mitosis without inducing cyclin B destruction. Despite the persistence of B-type cyclins, cdc55 mutant cells inactivate MPF. Two experiments show that this inactivation is due to inhibitory phosphorylation on Cdc28: phosphotyrosine accumulates on Cdc28 in cdc55Δ cells whose spindles have been depolymerized, and a cdc28 mutant that lacks inhibitory phosphorylation sites on Cdc28 allows spindle defects to arrest cdc55 mutants in mitosis with active MPF and unseparated sister chromatids.Conclusions We conclude that perturbations of protein phosphatase activity allow MPF to be inactivated by inhibitory phosphorylation instead of by cyclin destruction. Under these conditions, sister chromatid separation appears to be regulated by MPF activity rather than by protein degradation. We discuss the role of PP2A and Cdc28 phosphorylation in cell-cycle control, and the possibility that the novel mitotic exit pathway plays a role in adaptation to prolonged activation of the spindle-assembly checkpoint.     

Cell-cycle-dependent subcellular localization of cyclin B1, phosphorylated cyclin B1 and p34cdc2 during oocyte meiotic maturation and fertilization in mouse

M phase or maturation promoting factor (MPF), a kinase complex composed of the regulatory cyclin B and the catalytic p34cdc2 kinase, plays important roles in meiosis and mitosis. This study was designed to detect and compare the subcellular localization of cyclin B1, phosphorylated cyclin B1 and p34cdc2 during oocyte meiotic maturation and fertilization in mouse. We found that all these proteins were concentrated in the germinal vesicle of oocytes. Shortly after germinal vesicle breakdown, all these proteins were accumulated around the condensed chromosomes. With spindle formation at metaphase I, cyclin B1 and phosphorylated cyclin B1 were localized around the condensed chromosomes and concentrated at the spindle poles, while p34cdc2 was localized in the spindle region. At the anaphase/telophase transition, phosphorylated cyclin B1 was accumulated in the midbody between the separating chromosomes/chromatids, while p34cdc2 was accumulated in the entire spindle except for the midbody region. At metaphase II, both cyclin B1 and p34cdc2 were horizontally localized in the region with the aligned chromosomes and the two poles of the spindle, while phosphorylated cyclin B1 was localized in the two poles of spindle and the chromosomes. We could not detect a particular distribution of cyclin B1 in fertilized eggs when the pronuclei were initially formed, but in late pronuclei cyclin B1 was accumulated in the pronuclei. p34cdc2 and phosphorylated cyclin B1 were always concentrated in one pronucleus after parthenogenetic activation or in two pronuclei after fertilization. At metaphase of 1-cell embryos, cyclin B1 was accumulated around the condensed chromosomes. Cyclin B1 was accumulated in the nucleus of late 2-cell embryos but not in early 2-cell embryos. Furthermore, we also detected the accumulation of p34cdc2 in the nucleus of 2- and 4-cell embryos. All these results show that cyclin B1, phosphorylated cyclin B1 and p34cdc2 have similar distributions at some stages but different localizations at other stages during oocyte meiotic maturation and fertilization, suggesting that they may play a common role in some events but different roles in other events during oocyte maturation and fertilization.     

Constitutive Mad1 targeting to kinetochores uncouples checkpoint signalling from chromosome biorientation

Accurate chromosome segregation depends on biorientation, whereby sister chromatids attach to microtubules from opposite spindle poles. The spindle-assembly checkpoint is a surveillance mechanism in eukaryotes that inhibits anaphase until all chromosomes have bioriented. In present models, the recruitment of the spindle-assembly checkpoint protein Mad2, through Mad1, to non-bioriented kinetochores is needed to stop cell-cycle progression. However, it is unknown whether Mad1-Mad2 targeting to kinetochores is sufficient to block anaphase. Furthermore, it is unclear whether regulators of biorientation (for example, Aurora kinases) have checkpoint functions downstream of Mad1-Mad2 recruitment or whether they act upstream to quench the primary error signal. Here, we engineered a Mad1 construct that localizes to bioriented kinetochores. We show that the kinetochore localization of Mad1 is sufficient for a metaphase arrest that depends on Mad1-Mad2 binding. By uncoupling the checkpoint from its primary error signal, we show that Aurora, Mps1 and BubR1 kinases, but not Polo-like kinase, are needed to maintain checkpoint arrest when Mad1 is present on kinetochores. Together, our data suggest a model in which the biorientation errors, which recruit Mad1-Mad2 to kinetochores, may be signalled not only through Mad2 template dynamics, but also through the activity of widely conserved kinases, to ensure the fidelity of cell division.     

Studying Proteolysis of Cyclin B at the Single Cell Level in Whole Cell Populations

Equal distribution of chromosomes between the two daughter cells during cell division is a prerequisite for guaranteeing genetic stability ¹. Inaccuracies during chromosome separation are a hallmark of malignancy and associated with progressive disease ^2-4. The spindle assembly checkpoint (SAC) is a mitotic surveillance mechanism that holds back cells at metaphase until every single chromosome has established a stable bipolar attachment to the mitotic spindle¹. The SAC exerts its function by interference with the activating APC/C subunit Cdc20 to block proteolysis of securin and cyclin B and thus chromosome separation and mitotic exit. Improper attachment of chromosomes prevents silencing of SAC signaling and causes continued inhibition of APC/C^Cdc20 until the problem is solved to avoid chromosome missegregation, aneuploidy and malignant growths¹.Most studies that addressed the influence of improper chromosomal attachment on APC/C-dependent proteolysis took advantage of spindle disruption using depolymerizing or microtubule-stabilizing drugs to interfere with chromosomal attachment to microtubules. Since interference with microtubule kinetics can affect the transport and localization of critical regulators, these procedures bear a risk of inducing artificial effects ⁵.To study how the SAC interferes with APC/C-dependent proteolysis of cyclin B during mitosis in unperturbed cell populations, we established a histone H2-GFP-based system which allowed the simultaneous monitoring of metaphase alignment of mitotic chromosomes and proteolysis of cyclin B ⁶.To depict proteolytic profiles, we generated a chimeric cyclin B reporter molecule with a C-terminal SNAP moiety ⁶ (Figure 1). In a self-labeling reaction, the SNAP-moiety is able to form covalent bonds with alkylguanine-carriers (SNAP substrate) ^7,8 (Figure 1). SNAP substrate molecules are readily available and carry a broad spectrum of different fluorochromes. Chimeric cyclin B-SNAP molecules become labeled upon addition of the membrane-permeable SNAP substrate to the growth medium ⁷ (Figure 1). Following the labeling reaction, the cyclin B-SNAP fluorescence intensity drops in a pulse-chase reaction-like manner and fluorescence intensities reflect levels of cyclin B degradation ⁶ (Figure 1). Our system facilitates the monitoring of mitotic APC/C-dependent proteolysis in large numbers of cells (or several cell populations) in parallel. Thereby, the system may be a valuable tool to identify agents/small molecules that are able to interfere with proteolytic activity at the metaphase to anaphase transition. Moreover, as synthesis of cyclin B during mitosis has recently been suggested as an important mechanism in fostering a mitotic block in mice and humans by keeping cyclin B expression levels stable ^9,10, this system enabled us to analyze cyclin B proteolysis as one element of a balanced equilibrium ⁶.     

DNA damage during the spindle-assembly checkpoint degrades CDC25A, inhibits cyclin-CDC2 complexes, and reverses cells to interphase

Cell cycle checkpoints that monitor DNA damage and spindle assembly are essential for the maintenance of genetic integrity, and drugs that target these checkpoints are important chemotherapeutic agents. We have examined how cells respond to DNA damage while the spindle-assembly checkpoint is activated. Single cell electrophoresis and phosphorylation of histone H2AX indicated that several chemotherapeutic agents could induce DNA damage during mitotic block. DNA damage during mitotic block triggered CDC2 inactivation, histone H3 dephosphorylation, and chromosome decondensation. Cells did not progress into G1 but seemed to retract to a G2-like state containing 4N DNA content, with stabilized cyclin A and cyclin B1 binding to Thr14/Tyr15-phosphorylated CDC2. The loss of mitotic cells was not due to cell death because there was no discernible effect on caspase-3 activation, DNA fragmentation, or viability. Extensive DNA damage during mitotic block inactivated cyclin B1-CDC2 and prevented G1 entry when the block was removed. The mitotic DNA damage responses were independent of p53 and pRb, but they were dependent on ATM. CDC25A that accumulated during mitosis was rapidly destroyed after DNA damage in an ATM-dependent manner. Ectopic expression of CDC25A or nonphosphorylatable CDC2 effectively inhibited the dephosphorylation of histone H3 after DNA damage. Hence, although spindle disruption and DNA damage provide conflicting signals to regulate CDC2, the negative regulation by the DNA damage checkpoint could overcome the positive regulation by the spindle-assembly checkpoint.     

BRCA1 is required for meiotic spindle assembly and spindle assembly checkpoint activation in mouse oocytes

BRCA1 as a tumor suppressor has been widely investigated in mitosis, but its functions in meiosis are unclear. In the present study, we examined the expression, localization, and function of BRCA1 during mouse oocyte meiotic maturation. We found that expression level of BRCA1 was increased progressively from germinal vesicle to metaphase I stage, and then remained stable until metaphase II stage. Immunofluorescent analysis showed that BRCA1 was localized to the spindle poles at metaphase I and metaphase II stages, colocalizing with centrosomal protein gamma-tubulin. Taxol treatment resulted in the presence of BRCA1 onto the spindle microtubule fibers, whereas nocodazole treatment induced the localization of BRCA1 onto the chromosomes. Depletion of BRCA1 by both antibody injection and siRNA injection caused severely impaired spindles and misaligned chromosomes. Furthermore, BRCA1-depleted oocytes could not arrest at the metaphase I in the presence of low-dose nocodazole, suggesting that the spindle checkpoint is defective. Also, in BRCA1-depleted oocytes, gamma-tubulin dissociated from spindle poles and MAD2L1 failed to rebind to the kinetochores when exposed to nocodazole at metaphase I stage. Collectively, these data indicate that BRCA1 regulates not only meiotic spindle assembly, but also spindle assembly checkpoint, implying a link between BRCA1 deficiency and aneuploid embryos.     

Cyclin A is destroyed in prometaphase and can delay chromosome alignment and anaphase

Mitosis is controlled by the specific and timely degradation of key regulatory proteins, notably the mitotic cyclins that bind and activate the cyclin-dependent kinases (Cdks). In animal cells, cyclin A is always degraded before cyclin B, but the exact timing and the mechanism underlying this are not known. Here we use live cell imaging to show that cyclin A begins to be degraded just after nuclear envelope breakdown. This degradation requires the 26S proteasome, but is not affected by the spindle checkpoint. Neither deletion of its destruction box nor disrupting Cdk binding prevents cyclin A proteolysis, but Cdk binding is necessary for degradation at the correct time. We also show that increasing the levels of cyclin A delays chromosome alignment and sister chromatid segregation. This delay depends on the proteolysis of cyclin A and is not caused by a lag in the bipolar attachment of chromosomes to the mitotic spindle, nor is it mediated via the spindle checkpoint. Thus, proteolysis that is not under the control of the spindle checkpoint is required for chromosome alignment and anaphase.     

Cytoplasmic dynein/dynactin drives kinetochore protein transport to the spindle poles and has a role in mitotic spindle checkpoint inactivation.

We discovered that many proteins located in the kinetochore outer domain, but not the inner core, are depleted from kinetochores and accumulate at spindle poles when ATP production is suppressed in PtK1 cells, and that microtubule depolymerization inhibits this process. These proteins include the microtubule motors CENP-E and cytoplasmic dynein, and proteins involved with the mitotic spindle checkpoint, Mad2, Bub1R, and the 3F3/2 phosphoantigen. Depletion of these components did not disrupt kinetochore outer domain structure or alter metaphase kinetochore microtubule number. Inhibition of dynein/dynactin activity by microinjection in prometaphase with purified p50 "dynamitin" protein or concentrated 70.1 anti-dynein antibody blocked outer domain protein transport to the spindle poles, prevented Mad2 depletion from kinetochores despite normal kinetochore microtubule numbers, reduced metaphase kinetochore tension by 40%, and induced a mitotic block at metaphase. Dynein/dynactin inhibition did not block chromosome congression to the spindle equator in prometaphase, or segregation to the poles in anaphase when the spindle checkpoint was inactivated by microinjection with Mad2 antibodies. Thus, a major function of dynein/dynactin in mitosis is in a kinetochore disassembly pathway that contributes to inactivation of the spindle checkpoint.     

The disappearance of cyclin B at the end of mitosis is regulated spatially in Drosophila cells

We have followed the behaviour of a cyclin B-green fluorescent protein (GFP) fusion protein in living Drosophila embryos in order to study how the localization and destruction of cyclin B is regulated in space and time. We show that the fusion protein accumulates at centrosomes in interphase, in the nucleus in prophase, on the mitotic spindle in prometaphase and on the microtubules that overlap in the middle of the spindle in metaphase. In cellularized embryos, toward the end of metaphase, the spindle-associated cyclin B-GFP disappears from the spindle in a wave that starts at the spindle poles and spreads to the spindle equator; when the cyclin B-GFP on the spindle is almost undetectable, the chromosomes enter anaphase, and any remaining cytoplasmic cyclin B-GFP then disappears over the next few minutes. The endogenous cyclin B protein appears to behave in a similar manner. These findings suggest that the inactivation of cyclin B is regulated spatially in Drosophila cells. We show that the anaphase-promoting complex/cyclosome (APC/C) specifically interacts with microtubules in embryo extracts, but it is not confined to the spindle in mitosis, suggesting that the spatially regulated disappearance of cyclin B may reflect the spatially regulated activation of the APC/C.     

Regulation of Intracellular MEK1/2 Translocation in Mouse Oocytes: Cytoplasmic Dynein/Dynactin-Mediated Poleward Transport and Cyclin B Degradation-Dependent Release from Spindle Poles

We recently reported that MEK1/2 plays an important role in microtubule organization and spindle pole tethering in mouse oocytes, but how the intracellular transport of this protein is regulated remains unknown. In the present study, we investigated the mechanisms of poleward MEK1/2 transport during the prometaphase I/metaphase I transition and MEK1/2 release from the spindle poles during the metaphase I/anaphase I transition in mouse oocytes. Firstly, we found that p-MEK1/2 was colocalized with dynactin at the spindle poles. Inhibition of the cytoplasmic dynein/dynactin complex by antibody microinjection blocked polar accumulation of p-MEK1/2 and caused obvious spindle abnormalities. Moreover, coimmunoprecipitation of p-MEK1/2 and dynein or dynactin from mouse oocyte extracts confirmed their association at metaphase I. Secondly, disruption of microtubules by nocodazole resulted in the failure of poleward p-MEK1/2 transport. Whereas, when the nocodazole-treated oocytes were recovered in fresh culture medium, the spindle reformed and p-MEK1/2 relocalized to the spindle poles. Finally, we examined the mechanism of p-MEK1/2 release from the spindle poles. In control oocytes, polar p-MEK1/2 was gradually released during metaphase I/anaphase I transition. By contrast, in the presence of nondegradable cyclin B (△90), p-MEK1/2 still remained at the spindle poles at anaphase I. Our results indicate that poleward MEK1/2 transport is a cytoplasmic dynein/dynactin-mediated and spindle microtubule-dependent intracellular movement, and that its subsequent anaphase release from spindle poles is dependent on cyclin B degradation.     

Flies without a spindle checkpoint

Mad2 has a key role in the spindle-assembly checkpoint (SAC) - the mechanism delaying anaphase onset until all chromosomes correctly attach to the spindle. Here, we show that unlike every other reported case of SAC inactivation in metazoans, mad2-null Drosophila are viable and fertile, and their cells almost always divide correctly despite having no SAC and an accelerated 'clock', which is caused by premature degradation of cyclin B. Mitosis in Drosophila does not need the SAC because correct chromosome attachment is achieved very rapidly, before even the cell lacking Mad2 can initiate anaphase. Experimentally reducing spindle-assembly efficiency renders the cells Mad2-dependent. In fact, the robustness of the SAC may generally mask minor mitotic defects of mutations affecting spindle function. The reported lethality of other Drosophila SAC mutations may be explained by their multifunctionality, and thus the 'checkpoint' phenotypes previously ascribed to these mutations should be considered the consequence of eliminating both the checkpoint and a second mitotic function.     

The Xenopus chromokinesin Xkid is essential for metaphase chromosome alignment and must be degraded to allow anaphase chromosome movement

At anaphase, the linkage betweeh sister chromatids is dissolved and the separated sisters move toward opposite poles of the spindle. We developed a method to purify metaphase and anaphase chromosomes from frog egg extracts and identified proteins that leave chromosomes at anaphase using a new form of expression screening. This approach identified Xkid, a Xenopus homolog of human Kid (kinesin-like DNA binding protein) as a protein that is degraded in anaphase by ubiquitin-mediated proteolysis. Immunodepleting Xkid from egg extracts prevented normal chromosome alignment on the metaphase spindle. Adding a mild excess of wild-type or nondegradable Xkid to egg extracts prevented the separated chromosomes from moving toward the poles. We propose that Xkid provides the metaphase force that pushes chromosome arms toward the equator of the spindle and that its destruction is needed for anaphase chromosome movement.     

The spindle checkpoint in the dinoflagellate Crypthecodinium cohnii

Dinoflagellates are a major group of organisms with an extranuclear spindle. As the purpose of the spindle checkpoint is to ensure proper alignment of the chromosomes on the spindle, dinoflagellate cell cycle control may be compromised to accomodate the extranuclear spindle. In the present study, we demonstrated that nocodazole reversibly prolonged the G2 + M phase of the dinoflagellate cell cycle, in both metaphase and anaphase. The regulation of the spindle checkpoint involves the activation and inhibition of the anaphase promoting complex (APC), which in turn degrades specific cell cycle regulators in the metaphase to anaphase transition. In Crypthecodinium cohnii, nocodazole was also able to induce a prolongation of the degradation of mitotic cyclins and a delay in the inactivation of p13(suc1)-associated histone kinase activities. In addition, cell extracts prepared from C. cohnii in G1 phase and G2/M phase (or nocodazole treated) were able to activate and inhibit, respectively, the degradation of exogenous human cyclin B1 in vitro. The present study thus demonstrated the presence of the spindle checkpoint and APC-mediated cyclin degradation in dinoflagellates. This is discussed in relation to a possible role of the nuclear membrane in mitosis in dinoflagellates.     

Chromator is required for proper microtubule spindle formation and mitosis in Drosophila

The chromodomain protein, Chromator, has been shown to have multiple functions that include regulation of chromatin structure as well as coordination of muscle remodeling during metamorphosis depending on the developmental context. In this study we show that mitotic neuroblasts from brain squash preparations from larvae heteroallelic for the two Chromator loss-of-function alleles Chro⁷¹ and Chro⁶¹² have severe microtubule spindle and chromosome segregation defects that were associated with a reduction in brain size. The microtubule spindles formed were incomplete, unfocused, and/or without clear spindle poles and at anaphase chromosomes were lagging and scattered. Time-lapse analysis of mitosis in S2 cells depleted of Chromator by RNAi treatment suggested that the lagging and scattered chromosome phenotypes were caused by incomplete alignment of chromosomes at the metaphase plate, possibly due to a defective spindle-assembly checkpoint, as well as of frayed and unstable microtubule spindles during anaphase. Expression of full-length Chromator transgenes under endogenous promoter control restored both microtubule spindle morphology as well as brain size strongly indicating that the observed mutant defects were directly attributable to lack of Chromator function.     

Bub1 Prevents Chromosome Misalignment and Precocious Anaphase during Mouse Oocyte Meiosis

In mitosis the checkpoint proteins ensure faithful chromosome segregation by delaying onset of anaphase until all sister chromatids align at the metaphase plate of the bipolar spindle correctly. In the present study we blocked the function of Bub1 during meiosis by microinjecting anti-Bub1 specific antibody into cytoplasm of mouse oocytes, and found that depletion of Bub1 induced evident cyclin B degradation and precocious anaphase onset. Bub1 suppression also overrode the checkpoint-dependent cell cycle arrest provoked by a low dosage of nocodazole. Furthermore, Bub1 depletion induced a significantly higher percentage of oocytes with misaligned chromosomes. In addition, we depicted the localization dynamics of Bub1 in response to spindle damage and its relationship with microtubules and chromosomes, providing further evidence for Bub1’s role as a spindle checkpoint protein. Our data suggest that Bub1 is a critical spindle checkpoint protein that regulates accurate chromosome alignment and homolog disjunction in mammalian oocyte meiosis.     

Cyclin B1 is localized to unattached kinetochores and contributes to efficient microtubule attachment and proper chromosome alignment during mitosis

Cyclin B1 is a key regulatory protein controlling cell cycle progression in vertebrates. Cyclin B1 binds CDK1, a cy-clin-dependent kinase catalytic subunit, forming a complex that orchestrates mitosis through phosphorylation of key proteins. Cyclin B1 regulates both the activation of CDK1 and its subcellular localization, which may be critical for substrate selection. Here, we demonstrate that cyclin B1 is concentrated on the outer plate of the kinetochore during prometaphase. This localization requires the cyclin box region of the protein. Cyclin B1 is displaced from individual kinetochores to the spindle poles by microtubule attachment to the kinetochores, and this displacement is dependent on the dynein/dynactin complex. Depletion of cyclin B1 by vector-based siRNA causes inefficient attachment between kinetochores and microtubules, and chromosome alignment defects, and delays the onset of anaphase. We conclude that cyclin B1 accumulates at kinetochores during prometaphase, where it contributes to the correct attachment of mi- crotubules to kinetochores and efficient alignment of the chromosomes, most likely through localized phosphorylation of specific substrates by cyclin B1-CDK1. Cyclin B1 is then transported from each kinetochore as microtubule attachment is completed, and this relocalization may redirect the activity of cyclin B1-CDK1 and contribute to inactivation of the spindle assembly checkpoint.     

Dynein/Dynactin-mediated transport of kinetochore components off kinetochores and onto spindle poles induced by nordihydroguaiaretic acid

The mitotic checkpoint functions to ensure accurate chromosome segregation by regulating the progression from metaphase to anaphase. Once the checkpoint has been satisfied, it is inactivated in order to allow the cell to proceed into anaphase and complete the cell cycle. The minus end-directed microtubule motor dynein/dynactin has been implicated in the silencing of the mitotic checkpoint by "stripping" checkpoint proteins off kinetochores. A recent study suggested that Nordihydroguaiaretic acid (NDGA) stimulates dynein/dynactin-mediated transport of its cargo including ZW10 (Zeste White 10). We analyzed the effects of NDGA on dynein/dynactin dependent transport of the RZZ (Zeste White 10, Roughdeal, Zwilch) complex as well as other kinetochore components from kinetochores to spindle poles. Through this approach we have catalogued several kinetochore and centromere components as dynein/dynactin cargo. These include hZW10, hZwilch, hROD, hSpindly, hMad1, hMad2, hCENP-E, hCdc27, cyclin-B and hMps1. Furthermore, we found that treatment with NDGA induced a robust accumulation and complete stabilization of hZW10 at spindle poles. This finding suggests that NDGA may not induce dynein/dynactin transport but rather interfere with cargo release. Lastly, we determined that NDGA induced accumulation of checkpoint proteins at the poles requires dynein/dynactin-mediated transport, hZW10 kinetochore localization and kinetochore-microtubule attachments but not tension or Aurora B kinase activity.