Browsing by Subject "Mitosis"

Now showing 1 - 9 of 9
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    Analysis of Aurora B Regulation and Signaling
    (2006-05-16) Oncel, Dilhan; Yu, Hongtao
    Aurora B is a serine/threonine kinase that functions in a complex with two other chromosomal passenger proteins called INCENP and Survivin. Its function is implicated in a variety of processes related to mitosis, such as chromosome condensation, regulation of arm cohesion, spindle assembly, chromosome bi-orientation and cytokinesis. During the cell cycle, the level of this protein is tightly controlled and its deregulated abundance is suspected to contribute to aneuploidy. The cell cycle profile for Aurora B is reminiscent of those for substrates of the anaphase-promoting complex/cyclosome (APC/C), an ubiquitin ligase essential for mitotic progression. Here, we showed that Aurora B is a substrate of APC/C both in vitro and in vivo. Aurora B is efficiently ubiquitinated iv in an in vitro reconstituted system by APC/C that had been activated by Cdh1. The recognition of Aurora B by APC/CCdh1 is specific as it requires the presence of a conserved KEN-box motif at the amino terminus of Aurora B. Degradation of Aurora B at the end of mitosis requires Cdh1 in vivo as the reduction of Cdh1 level by RNA interference stabilizes Aurora B protein. We conclude that, as a key mitotic regulator, Aurora B is degraded by APC/CCdh1 in late mitosis. Aurora B lies at the heart of the cellular mechanism that resolves synthelic and merotelic attachments. A failure to eliminate such events results in gain or loss of chromosomes. Therefore, identifying the physiological substrates of Aurora B is of pivotal importance for research. We screened Aurora B substrates using an in vitro expression cloning system. However, the methodology we employed didn't lead to candidate substrates to be further validated by more rigorous in vivo approaches. The use of high concentrations of misfolded recombinant Aurora B was partially responsible for the loss of specificity. Therefore, purifying active recombinant Aurora B has become a primary goal for future biochemical and structural work. Two molecular chaperones Hsp90 and Cdc37 assist the folding of a variety of kinases in vivo, among which Aurora B is also a candidate. This gave us the final idea of expressing Aurora B-INCENP complexes in bacteria via the coexpression of Hsp90-Cdc37 molecular chaperones.
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    CDK5RAP2 Regulates Centriole Licensing to Restrict Centriole Duplication in Mice
    (2009-09-04) Barrera, Jose Anselmo; Megraw, Timothy
    Cells division is a highly coordinated series of events that must occur with extreme precision. Defects during segregation of genetic material (DNA) can have adverse effects on the health of the cell, surrounding tissue, organ, and the organism as a whole. Accurate assembly of the bipolar mitotic spindle apparatus is crucial for precise chromosome segregation. Centrosomes play a crucial role in establishment of the mitotic spindle and therefore are vital to the maintenance of genetic stability. Centrosomes are composed of two centrioles that arrange a specialized conglomerate of proteins into a pericentriolar matrix. Centrosomes are highly regulated throughout the cell cycle, and duplicate only once per cell cycle ensuring that each cell inherits one centrosome after mitotic exit, and contains only two centrosomes at the following mitosis. Truncating mutations in the Cyclin-Dependent Kinase 5 Regulatory Associated Protein 2 gene (CDK5RAP2), which encodes a centrosomal protein, result in autosomal recessive primary microcephaly (MCPH, [MIM 251200]) in humans. The major phenotypic manifestation of this rare genetic disorder is a small head. Affected individuals have head circumferences at least 4 standard deviations below sex- and age-matched individuals and suffer mental retardation. In order to investigate how mutations in CDK5RAP2 affect centrosome structure and regulation, and how this leads to MCPH, we derived two distinct mouse mutant lines with truncating mutations within the CDK5RAP2 locus similar to those found in affected humans. We show that centriole engagement and cohesion, two distinct centriole-binding processes, are disrupted in CDK5RAP2 mutant cells. Partial disruption of CDK5RAP2 affected centriole cohesion, whereas complete CDK5RAP2 disruption deregulated the centriole duplication cycle leading to centriole/centrosome amplification. During mitosis amplified centrosomes in CDK5RAP2 mutant cells were potent microtubule organizing centers that drove formation of multipolar spindles. Furthermore, cells formed multiple primary cilia from multiple centrioles inherited from the previous cell cycle. Together these results define a role for CDK5RAP2 in the regulation of centriole duplication and also provide a basis for the development of MCPH.
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    Centrosomin Self-Assembly and Centrosomal Protein Recruitment
    (2005-08-11) Bauer, Ruth Anne; Megraw, Timothy
    Centrosomes, the major microtubule organizing centers in animal cells, are important for mitotic spindle formation. Normally, each cell has two centrosomes which migrate to opposite sides of the nuclear envelope prior to entry into mitosis. Centrosomin (Cnn) is a major centrosomal protein that is important for nucleation and organization of bipolar spindle microtubules at mitosis. Cnn protein localizes to the pericentriolar matrix and from there other centrosomal proteins 'load' onto the centrosome, including gamma-tubulin. Centrosomes are non-functional without the addition of Cnn since it is responsible for recruitment of other centrosomal proteins. There are two conserved motifs in Cnn protein, currently of unknown function. One of these motifs is most likely responsible for interaction with gamma-tubulin and other centrosomal proteins which make the centrosome capable for microtubule nucleation. Cnn full-length and half proteins were expressed in E. coli and purified in vitro. The properties of these Cnn proteins show self-assembly and recruitment of centrosomal proteins. These activities of Cnn in vitro are novel and will help further the investigation of Cnn protein function in the context of biological systems. Cnn fusion proteins show characteristics similar to centrosomal 'satellite' or 'flare' particles described in animal cells. It is likely that these satellites communicate with the actin cytoskeleton in syncytial Drosophila embryos.
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    From poles to equator: functional analysis of DdAurora during mitosis and cytokinesis in Dictyostelium discoideum
    (2007) Li, Hui, 1976-; De Lozanne, Arturo
    The Aurora kinases are highly conserved serine/threonine kinases that play essential roles throughout mitosis. In metazoans, these functions are mediated by Aurora A and B at the spindle poles and the equatorial region respectively. I show here that Dictyostelium contains a single Aurora kinase, DdAurora that displays characteristics of both Aurora A and B. Like Aurora A, DdAurora has an extended N-terminal domain with an A-box and localizes to the spindle poles during early mitosis. Like Aurora B, DdAurora localizes to centromeres in metaphase, the central spindle during anaphase and the cleavage furrow at the end of cytokinesis. In addition to these known features of Aurora A and B, I found that DdAurora remains associated with centromeres during anaphase and telophase which has not been shown in any other organisms. INCENP is known to be an important binding partner of Aurora B. In Dictyostelium the conserved C-terminal IN-box domain of DdINCENP is essential for its interaction with DdAurora and for the localization of DdAurora to the central spindle. In contrast, the centromeric and spindle pole localization of DdAurora does not require an interaction with DdINCENP. Surprisingly, a truncated DdINCENP protein lacking the IN-box domain can still localize on centromeres and the central spindle even though it does not bind to DdAurora. I also found that the localization of DdAurora to the central spindle requires Kif12, a protein similar to mitotic kinesin like protein 2 (MKLP2). However, this requirement is suppressed by the overexpression of GFP-DdINCENP. GFP-DdINCENP can localize to the central spindle in the absence of Kif12 and it probably recruits DdAurora to the same location through their strong interaction. Finally, I demonstrated that Myosin II heavy chain is important for the proper localization of the DdAurora/DdINCENP complex to the cleavage furrow during late cytokinesis. With the exception of DdINCENP, no other binding partner or substrate of DdAurora has been identified in Dictyostelium. By performing large-scale immunoprecipitation in wild-type cells, I identified several potential binding partners/substrates of DdAurora, including topoisomerase B and HspA. Future esearch on these proteins may help to elucidate DdAurora function in different stages of M phase.
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    Function And Recruitment Of Centromeric Heterochromatin Protein 1
    (2011-02-01T19:34:12Z) Chaudhary, Jaideep; Yu, Hongtao
    During early mitosis, the sister chromatids are held together by Cohesin, a protein complex composed of Smc3, Smc1, Scc1/Rad21 and Scc3. Cohesin is first released from the arms of chromosomes, leaving it intact at the centromere. At the metaphase – anaphase transition, centromeric cohesin is cleaved, allowing the chromatids to segregate to two daughter cells. Shugoshin (Sgo-1) is a known protector of cohesin at the centromere. It prevents phosphorylation of cohesin complex by Plk1 before the metaphase – anaphase transition, which would otherwise lead to cohesin release, causing the two chromatids to separate untimely. In this study we show that Sgo1 localizes on centromeres through HP1 during interphase in human cells. Also, Sgo1 binds all three forms of HP1 (i.e. alpha, beta, gamma) through its chromoshadow domain. We have determined the dissociation constant of this interaction to be in the sub-micromolar range. We have shown conclusively that Sgo1 binds to HP1 chromoshadow domain via one PxVxL motif. We have further shown that, in mitosis, HP1 is recruited to centromeres by Incenp, a subunit of the chromosomal passenger complex via the chromoshadow domain of HP1. This interaction is most likely at the HP1 CSD dimer interface, where PxVxL motifsbind. Hence, it seems that Incenp may provide competition to Sgo1 for HP1 binding.
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    Functional analysis of DdINCENP, a chromosomal passenger protein, in Dictyostelium
    (2006-08) Chen, Qian, 1975-; De Lozanne, Arturo
    Dictyostelium DdINCENP is a chromosomal passenger protein associated with centromeres, the spindle midzone and poles during mitosis and the cleavage furrow during cytokinesis. Disruption of the single DdINCENP gene revealed important roles for this protein in mitosis and cytokinesis. DdINCENP null cells lack a robust spindle midzone and are hypersensitive to microtubule depolymerizing drugs suggesting that their spindles may not be stable. Furthermore DdCP224, a protein homologous to the microtubule-stabilizing protein TOGp/XMAP215, was absent from the spindle midzone of DdINCENP null cells. Overexpression of DdCP224 rescued the weak spindle midzone defect of DdINCENP null cells. While not required for the localization of the myosin II contractile ring and subsequent formation of a cleavage furrow, DdINCENP is important for the abscission of daughter cells at the end of cytokinesis. The localization of DdINCENP at the cleavage furrow is modulated by myosin II. Loss of myosin II restricted the localization of DdINCENP to a narrow zone at the cleavage furrow. Kif12, a homolog of mitotic kinesin like protein (MKLP), was essential for relocalization of DdINCENP from the central spindle to the cleavage furrow. Furthermore, Kif12 was also localized at the cortex of the cleavage furrow and its localization during cytokinesis closely resembled that of DdINCENP, suggesting a possible interaction between them. The correct localization of DdINCENP during cytokinesis also required its N-terminal sequence. DdINCENP1-500 was found at the cleavage furrow and interacted with the actin cytoskeleton. Domain analysis of DdINCENP also revealed that its DdINCENP1-500 was sufficient to rescue the weak spindle defect of DdINCENP null cells.
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    Generating the Spindle Assembly Checkpoint Signal at the Kinetochore
    (2004-08-19) Bharadwaj, Rajnish; Yu, Hongtao
    To avoid missegregation of chromosomes during mitosis cells employ a surveillance mechanism termed Spindle assembly checkpoint that senses the lack of tension/attachment on the kinetochores and consequently blocks anaphase onset by inhibiting an E3 ubiquitin ligase called anaphase-promoting complex. The roles of two kinases- BubR1 and Mps1, implicated in spindle assembly checkpoint were investigated. A checkpoint complex containing BubR1 and Bub3 has been purified from mitotic human cells. BubR1 directly interacts with Cdc20 and inhibits the activity of APC in vitro,much more efficiently than Mad2. Surprisingly, the kinase activity of BubR1 or association with Bub3 is not required for the inhibition of APCCdc20. Furthermore, BubR1 restores the mitotic arrest in Cdc20-overexpressing cells treated with nocodazole. Mps1 is a dual specificity kinase that localizes to kinetochores in mitosis. Depletion of Mps1 by RNAi leads to the abrogation of spindle assembly checkpoint. The kinetochore proteins involved in the recruitment of checkpoint proteins and the generation of wait-anaphase signal have not been identified. Kinetochores also provide the attachment sites for spindle microtubules and are required for the alignment of chromosomes at the metaphase plate (chromosome congression). Components of the conserved Ndc80 complex have been implicated in both these function. To better understand the function of the Ndc80 complex, we have identified two novel subunits of the human Ndc80 complex, termed human Spc25 (hSpc25) and human Spc24 (hSpc24), using an immuno-affinity approach. Human Spc25 interacts with Hec1 (human Ndc80) throughout the cell cycle and localizes to kinetochores during mitosis. RNAi-mediated depletion of hSpc25 in HeLa cells causes aberrant mitosis followed by cell death, a phenotype similar to that of cells depleted for Hec1. Loss of hSpc25 also causes multiple spindle aberrations, including elongated, multipolar, and fractured spindles. In the absence of hSpc25, Mad1 and Hec1 fail to localize to kinetochores during mitosis whereas the kinetochore localization of Bub1 and BubR1 is largely unaffected. Interestingly, the kinetochore localization of Mad1 in cells with a compromised Ndc80 function is restored upon microtubule depolymerization. Thus, hSpc25 is an essential kinetochore component that plays a significant role in proper execution of mitotic events.
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    The Mitotic Spindle Mediates Inheritance of the Golgi Ribbon Structures
    (2010-05-14) Wei, Jen-Hsuan; Seemann, Joachim
    The mammalian Golgi ribbon disassembles during mitosis and reforms in both daughter cells after division. Mitotic Golgi membranes concentrate around the spindle poles, suggesting that the spindle may control Golgi partitioning. To test this, cells were induced to divide asymmetrically with the entire spindle segregated into only one daughter cell. A ribbon reforms in the nucleated karyoplasts, whereas the Golgi stacks in the cytoplasts are scattered. However, the scattered Golgi stacks are polarized and transport cargo. Microinjection of Golgi extract together with tubulin or incorporation of spindle materials rescues Golgi ribbon formation. Therefore, the factors required for postmitotic Golgi ribbon assembly are transferred by the spindle, but the constituents of functional stacks are partitioned independently, suggesting that Golgi inheritance is regulated by two distinct mechanisms.
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    The role of Nek2 and TRF1 in mitotic perturbations: Potential implications for breast cancer treatment
    (2013-05) Lee, Jaehyung; Gollahon, Lauren; Rodgers, Brenda E.; Butler, Boyd; Thomas, Jeffrey; Zhang, Kai
    Although the anti-cancer drugs paclitaxel and doxorubicin are commonly used to treat many solid tumors, their effectiveness is highly variable due to tumor cell resistance. Therefore, it is important to find mechanisms that can be targeted to increase the sensitivity of cancer cells to current chemotherapy agents. NIMA related kinase 2 (Nek2), a serine/threonine kinase, is emerging as an important oncogene because of its regulatory role in multiple mitosis-related events including timing of mitotic entry, chromatin condensation, spindle organization and cytokinesis. Thus, regulation of the Nek2 expression levels may prove important as a target for cancer treatment. In order to determine the potential of Nek2 as a viable anti-cancer target that increases drug sensitivity, the triple negative breast cancer cell lines MDA-MB-231 and MDA-MB-468 were used. The approach design utilized was to pretreat the cells with small interfering RNA (siRNA) and antisense oligonucleotides (ASOs) against Nek2, and then expose them to various concentrations of paclitaxel and doxorubicin. Results demonstrated that drug susceptibility in these pre-treated cells was dramatically increased compared with either agent alone. FACS results showed that apoptosis was induced in siRNA or ASO pretreated. Furthermore, Nek2 knockdown worked synergistically with paclitaxel and doxorubicin by inhibition of cell proliferation. Hence, results suggest that these drugs in combination with Nek2 depletion may improve the sensitivity of breast cancer cells to chemotherapy treatments. To understand how Nek2 affects cancer development caused by chromosomal instability, the relationship between Nek2 and telomeric repeat binding factor 1 (TRF1) was investigated. TRF1 not only regulates telomere length, but is also associated with cell cycle regulation. Nek2 depleted cells lead to centrosome separation failure while Nek2 overexpression results in premature centrosome separation. While it has been shown in mice that TRF1 interacts directly with Nek2, the interactions and correlations between Nek2 and TRF1 in human cells are far from clear. In this study, the results showed that mitotic aberrations through Nek2 overexpression require TRF1. Kinase assay results demonstrate that Nek2 directly binds to and phosphorylates TRF1 in vitro and in vivo at multiple sites. Nek2 overexpression MCF 7 and MDA-MB-231 breast cancer cells resulted in increased numbers of centrosomes and multinucleated cells, ultimately leading to cytokinetic failure and aneuploidization. Furthermore, TRF1 depletion by siRNA prevented Nek2-induced unaligned chromosomes during metaphase. Concurrent Nek2 overexpression and TRF1 depletion showed that cells with  2 centrosomes restored cytokinetic failure and chromosome instability similar to controls. Therefore, I propose that TRF1 is required for overexpressed Nek2 to trigger abnormal mitosis and chromosomal instability. Taken together, overexpression of the Nek2 in breast cancer suggests that inhibition of Nek2 beneficially interferes with the cancer proliferation. Furthermore, combinational treatments involving Nek2 depletion in conjunction with anti-cancer drugs could potentially be developed as a cancer inhibitor. Therefore, Nek2’s contribution to cancer development may serve as a new starting point to exploit as a therapeutic target.