Biomedical subjects
Publications and source records attributed to Tim J Yen.
In this issue, Baumann et al. (2007) identify a helicase PICH that localizes to "threads" that remain connected between sister kinetochores after they have separated in anaphase. These threads are thought to be catenated centromeric DNA. PICH contributes to the mitotic checkpoint by recruiting Mad2 to kinetochores and is proposed to regulate checkpoint signaling by monitoring tension at centromeres.
We report the interactions amongst 20 proteins that specify their assembly to the centromere-kinetochore complex in human cells. Centromere protein (CENP)-A is at the top of a hierarchy that directs three major pathways, which are specified by CENP-C, -I, and Aurora B. Each pathway consists of branches that intersect to form nodes that may coordinate the assembly process. Complementary EM studies found that the formation of kinetochore trilaminar plates depends on the CENP-I/NUF2 branch, whereas CENP-C and Aurora B affect the size, shape, and structural integrity of the plates. We found that hMis12 is not constitutively localized at kinetochores, and that it is not essential for recruiting CENP-I. Our studies also revealed that kinetochores in HeLa cells contain an excess of CENP-A, of which approximately 10% is sufficient to promote the assembly of normal levels of kinetochore proteins. We elaborate on a previous model that suggested kinetochores are assembled from repetitive modules (Zinkowski, R.P., J. Meyne, and B.R. Brinkley. 1991. J. Cell Biol. 113:1091-110).
The vertebrate kinetochore is a complex structure that specifies the attachments between the chromosomes and microtubules of the spindle and is thus essential for accurate chromosome segregation. Kinetochores are assembled on centromeric chromatin through complex pathways that are coordinated with the cell cycle. In the light of recent discoveries on how proteins assemble onto kinetochores and interact with each other, we review these findings in this article (which is part of the Chromosome Segregation and Aneuploidy series), and discuss their implications for the current mitotic checkpoint models - the template model and the two-step model. The template model proposes that Mad1-Mad2 at kinetochores acts as a template to change the conformation of another binding molecule of Mad2. This templated change in conformation is postulated as a mechanism for the amplification of the 'anaphase wait' signal. The two-step model proposes that the mitotic checkpoint complex (MCC) is the kinetochore-independent anaphase inhibitor, and the role of the unaligned kinetochore is to sensitize the anaphase-promoting complex/cyclosome (APC/C) to MCC-mediated inhibition.
The kinetochore, a multi-protein complex assembled on centromeric chromatin in mitosis, is essential for sister chromosome segregation. We show here that inhibition of histone deacetylation blocks mitotic progression at prometaphase in two human tumor cell lines by interfering with kinetochore assembly. Decreased amounts of hBUB1, CENP-F and the motor protein CENP-E were present on kinetochores of treated cells. These kinetochores failed to nucleate and inefficiently captured microtubules, resulting in activation of the mitotic checkpoint. Addition of histone deacetylase inhibitors prior to the end of S-phase resulted in decreased HP1-beta on pericentromeric heterochromatin in S-phase and G(2), decreased pericentromeric targeting of Aurora B kinase, resulting in decreased premitotic phosphorylation of pericentromeric histone H3(S10) in G(2), followed by assembly of deficient kinetochores in M-phase. HP1-beta, Aurora B and the affected kinetochore proteins all were present at normal levels in treated cells; thus, effects of the inhibitors on mitotic progression do not seem to reflect changes in gene expression. In vitro kinase activity of Aurora B isolated from treated cells was unaffected. We propose that the increased presence in pericentromeric heterochromatin of histone H3 acetylated at K9 is responsible for the mitotic defects resulting from inhibition of histone deacetylation.
Two splice variants of Nek2 kinase, a member of the NIMA-related family, have been identified as Nek2A and Nek2B. Nek2A regulates centrosome disjunction, spindle formation checkpoint signaling, and faithful chromosome segregation. A specific role for Nek2B has not yet been identified. Here, we have examined the distinct roles of Nek2A and Nek2B using timelapse video microscopy to follow the fate of cells progressing through the cell cycle in the absence of either Nek2A or Nek2B. We show that the down-regulation of Nek2B leads to a mitotic delay in the majority of cells. Upon exiting mitosis, cells exhibit mitotic defects such as the formation of multinucleated cells. Such phenotypes are not observed in cells that exit mitosis in the absence of Nek2A. These observations suggest that Nek2B may be required for the execution of mitotic exit.
Anti-centromere antibody (ACA) has been reported to be associated with Sjogren's syndrome (SS) and the clinical significance of anti-CENP-H antibody remains unknown. To determine the clinical significance of anti-CENP-H and anti-centromere antibodies in primary SS, sera from 62 patients with primary SS and 40 normal controls were examined for anti-SS-A/SS-B antibodies, ACA and anti-CENP-H antibodies, by enzyme-linked immunosorbent assay and indirect immunofluorescence (IIF), respectively. Of the 62 serum samples with primary SS, 17 were positive with ACA and anti-CENP-H antibodies. Sera from SS patients with anti-CENP-H and ACA antibodies do not contain anti-SS-A/Ro and/or anti-SS-B/La antibodies. No anti-CENP-H antibody was found in sera of normal controls. An increased frequency of ACA and anti-CENP-H antibodies was found for the first time in patients with SS. Anti-CENP-H antibodies and anti-SS-A/Ro or anti-SS-B/La antibodies are present mutually exclusive. Patients with anti-CENP-H antibodies had a lower frequency of rheumatoid factor (RF). SS can be subdivided serologically into two groups; group one with anti-SS-A/Ro and/or anti-SS-B/La antibody, group two with ACA and/or anti-CENP-H antibodies. We recommend that ACA or anti-CENP-H antibodies should be considered as one of the serological markers for SS.
The temporal and spatial regulation of cytokinesis requires an interaction between the anaphase mitotic spindle and the cell cortex. However, the relative roles of the spindle asters or the central spindle bundle are not clear in mammalian cells. The central spindle normally serves as a platform to localize key regulators of cell cleavage, including passenger proteins. Using time-lapse and immunofluorescence analysis, we have addressed the consequences of eliminating the central spindle by ablation of PRC1, a microtubule bundling protein that is critical to the formation of the central spindle. Without a central spindle, the asters guide the equatorial cortical accumulation of anillin and actin, and of the passenger proteins, which organize into a subcortical ring in anaphase. Furrowing goes to completion, but abscission to create two daughter cells fails. We conclude the central spindle bundle is required for abscission but not for furrowing in mammalian cells.
Histone deacetylase 6 (HDAC6) contains tandem catalytic domains and a ubiquitin-binding zinc finger and displays deacetylase activity toward acetylated microtubules. Here we show that unlike its orthologs from Caenorhabditis elegans, Drosophila, and mouse, human HDAC6 possesses a tetradecapeptide repeat domain located between the second deacetylase domain and the C-terminal ubiquitin-binding motif. Related to this structural difference, the cytoplasmic localization of human, but not murine, HDAC6 is resistant to treatment with leptomycin B (LMB). Although it is dispensable for the deacetylase and ubiquitin binding activities of human HDAC6, the tetradecapeptide repeat domain displays acetyl-microtubule targeting ability. Moreover, it forms a unique structure and is required for the LMB-resistant cytoplasmic localization of human HDAC6. Besides the tetradecapeptide repeat domain, human HDAC6 possesses two LMB-sensitive nuclear export signals and a nuclear localization signal. These results thus indicate that the cytoplasmic localization for murine and human HDAC6 proteins is differentially regulated and suggest that the tetradecapeptide repeat domain serves as an important sequence element to stably retain human HDAC6 in the cytoplasm.
RanGAP1 is the activating protein for the Ran GTPase. Vertebrate RanGAP1 is conjugated to a small ubiquitin-like protein, SUMO-1. This modification promotes association of RanGAP1 with the interphase nuclear pore complex (NPC) through binding to the nucleoporin RanBP2, also known as Nup358. During mitosis, RanGAP1 is concentrated at kinetochores in a microtubule- (MT) and SUMO-1-dependent fashion. RanBP2 is also abundantly found on kinetochores in mitosis. Here we show that ablation of proteins required for MT-kinetochore attachment (Hec1/Ndc80, Nuf2 ) disrupts RanGAP1 and RanBP2 targeting to kinetochores. No similar disruption was observed after ablation of proteins nonessential for MT-kinetochore interactions (CENP-I, Bub1, CENP-E ). Acquisition of RanGAP1 and RanBP2 by kinetochores is temporally correlated in untreated cells with MT attachment. These patterns of accumulation suggest a loading mechanism wherein the RanGAP1-RanBP2 complex may be transferred along the MT onto the kinetochore. Depletion of RanBP2 caused mislocalization of RanGAP1, Mad1, Mad2, CENP-E, and CENP-F, as well as loss of cold-stable kinetochore-MT interactions and accumulation of mitotic cells with multipolar spindles and unaligned chromosomes. Taken together, our observations indicate that RanBP2 and RanGAP1 are targeted as a single complex that is both regulated by and essential for stable kinetochore-MT association.
The mitotic checkpoint prevents cells with unaligned chromosomes from prematurely exiting mitosis by inhibiting the anaphase-promoting complex/cyclosome (APC/C) from targeting key proteins for ubiquitin-mediated proteolysis. Great efforts have been made recently to identify and characterize the factors involved in this checkpoint pathway. These studies will ultimately lead to a better understanding of cell cycle defects and chromosome instability. We report here a protocol for purification of an inhibitor of the APC/C from HeLa cells, called mitotic checkpoint complex (MCC). Our procedure is based on biochemical purification and characterization of the APC/C inhibitory activity from extracts of HeLa cells.
Caffeine is an efficient inhibitor of cellular DNA repair, likely through its effects on ATM (ataxia telangiectasia mutated) and ATR (ATM and Rad3-related) kinases. Here, we show that caffeine treatment causes a dose-dependent reduction in the total amount of HIV-1 and avian sarcoma virus retroviral vector DNA that is joined to host DNA in the population of infected cells and also in the number of transduced cells. These changes were observed at caffeine concentrations that had little or no effect on overall cell growth, synthesis, and nuclear import of the viral DNA, or the activities of the viral integrase in vitro. Substantial reductions in the amount of host-viral-joined DNA in the infected population, and in the number of transductants, were also observed in the presence of a dominant-negative form of the ATR protein, ATRkd. After infection, a significant fraction of these cells undergoes cell death. In contrast, retroviral transduction is not impeded in ATM-deficient cells, and addition of caffeine leads to the same reduction that was observed in ATM-proficient cells. These results suggest that activity of the ATR kinase, but not the ATM kinase, is required for successful completion of the viral DNA integration process and/or survival of transduced cells. Components of the cellular DNA damage repair response may represent potential targets for antiretroviral drug development.
Anumber of proteins are recruited to nuclear foci upon exposure to double-strand DNA damage, including 53BP1 and Rad51, but the precise role of these DNA damage-induced foci remain unclear. Here we show in a variety of human cell lines that histone deacetylase (HDAC) 4 is recruited to foci with kinetics similar to, and colocalizes with, 53BP1 after exposure to agents causing double-stranded DNA breaks. HDAC4 foci gradually disappeared in repair-proficient cells but persisted in repair-deficient cell lines or cells irradiated with a lethal dose, suggesting that resolution of HDAC4 foci is linked to repair. Silencing of HDAC4 via RNA interference surprisingly also decreased levels of 53BP1 protein, abrogated the DNA damage-induced G2 delay, and radiosensitized HeLa cells. Our combined results suggest that HDAC4 is a critical component of the DNA damage response pathway that acts through 53BP1 and perhaps contributes in maintaining the G2 cell cycle checkpoint.
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The kinetochore, a macromolecular complex located at the centromere of chromosomes, provides essential functions for accurate chromosome segregation. Kinetochores contain checkpoint proteins that monitor attachments between the kinetochore and microtubules to ensure that cells do not exit mitosis in the presence of unaligned chromosomes. Here we report that human CENP-I, a constitutive protein of the kinetochore that shares limited similarity with Mis6 of Schizosaccharomyces pombe, is required for the localization of CENP-F and the checkpoint proteins MAD1 and MAD2 to kinetochores. Depletion of CENP-I from kinetochores causes the cell cycle to delay in G2. Although monopolar chromosomes in CENP-I-depleted cells fail to establish bipolar connections, the cells are unable to arrest in mitosis. These cells are transiently delayed in mitosis in a MAD2-dependent manner, even though their kinetochores are depleted of MAD2. The delay is extended considerably when the number of unattached kinetochores is increased. This suggests that no single unattached kinetochore in CENP-I-depleted cells can arrest mitosis. The collective output from many unattached kinetochores is required to reach a threshold signal of 'wait for anaphase' to sustain a prolonged mitotic arrest.
We have determined that the previously identified dual-specificity protein kinase TTK is the human orthologue of the yeast MPS1 kinase. Yeast MPS1 (monopolar spindle) is required for spindle pole duplication and the spindle checkpoint. Consistent with the recently identified vertebrate MPS1 homologues, we found that hMPS1 is localized to centrosomes and kinetochores. In addition, hMPS1 is part of a growing list of kinetochore proteins that are localized to nuclear pores. hMPS1 is required by cells to arrest in mitosis in response to spindle defects and kinetochore defects resulting from the loss of the kinesin-like protein, CENP-E. The pattern of kinetochore localization of hMPS1 in CENP-E defective cells suggests that their interaction with the kinetochore is sensitive to microtubule occupancy rather than kinetochore tension. hMPS1 is required for MAD1, MAD2 but not hBUB1, hBUBR1 and hROD to bind to kinetochores. We localized the kinetochore targeting domain in hMPS1 and found that it can abrogate the mitotic checkpoint in a dominant negative manner. Last, hMPS1 was found to associate with the anaphase promoting complex, thus raising the possibility that its checkpoint functions extend beyond the kinetochore.
The Zeste-White 10 (ZW10) and Rough Deal (ROD) proteins are part of a complex necessary for accurate chromosome segregation. This complex recruits cytoplasmic dynein to the kinetochore and participates in the spindle checkpoint. We used immunoaffinity chromatography and mass spectroscopy to identify the Drosophila proteins in this complex. We found that the complex contains an additional protein we name Zwilch. Zwilch localizes to kinetochores and kinetochore microtubules in a manner identical to ZW10 and ROD. We have also isolated a zwilch mutant, which exhibits the same mitotic phenotypes associated with zw10 and rod mutations: lagging chromosomes at anaphase and precocious sister chromatid separation upon activation of the spindle checkpoint. Zwilch's role within the context of this complex is evolutionarily conserved. The human Zwilch protein (hZwilch) coimmunoprecipitates with hZW10 and hROD from HeLa cell extracts and localizes to the kinetochores at prometaphase. Finally, we discuss immunoaffinity chromatography results that suggest the existence of a weak interaction between the ZW10/ROD/Zwilch complex and the kinesin-like kinetochore component CENP-meta.
G(2) is defined as the time in the cell cycle after DNA synthesis is complete but before the initiation of mitosis. However, as the molecular events of the cell cycle are described, G(2) can be seen to be a sequence of events rather than a static phase. For example, CENP-F increases in amount in early G(2), after DNA synthesis is complete, but localizes to the nuclear rim and then to the kinetochores before mitosis commences. After DNA damage cells may arrest in G(2) through TP53-dependent and independent mechanisms, raising the question of the precise position of the arrest within G(2). HeLa cells lack functional TP53; this allowed us to examine the TP53-independent mechanism of G(2) arrest. Here we showed that the DNA damage-induced G(2) arrest in HeLa cells is positioned in early G(2), before redistribution of CENP-F to the nuclear envelope and kinetochores, and before chromosome condensation commences.
The mitotic checkpoint is a failsafe mechanism for the cell to ensure accurate chromosome segregation during mitosis. Mutations in genes encoding essential checkpoint proteins lead to chromosome instability and promote carcinogenesis. The BUB and MAD genes are essential components of the mitotic checkpoint pathway. BUB and MAD inhibit the ubiquitin ligase activity of the Anaphase Promoting Complex/Cyclosome (APC/C) during mitosis to ensure cells with unaligned chromosomes do not prematurely enter anaphase. Two models explain how the APC/C is inhibited by the checkpoint. The Sequestration Model postulates that Mad2 and BubR1 bind and sequester Cdc20, an APC/C activator, away from APC/C so substrates whose destruction drives mitotic exit are no longer ubiquitinated. In this model, the unattached kinetochore is postulated to catalytically convert Mad2 to a form that binds Cdc20. In the Direct Inhibition Model, the Mitotic Checkpoint Complex (MCC) consisting of BubR1, Bub3, Mad2 and Cdc20 binds and inhibits the APC/C independently of the kinetochore. However, the "wait anaphase" signal generated by unattached kinetochores sensitizes the APC/C to prolonged inhibition by the MCC. A single unattached kinetochore is proposed to amplify the "wait anaphase" signal through a kinase cascade involving checkpoint kinases such as hBubR1, hBub1 and Mps1.