ABSTRACT
Error-free chromosome segregation requires stable attachment of sister kinetochores to the opposite spindle poles (amphitelic attachment). Exactly how amphitelic attachments are achieved during spindle assembly remains elusive. We employed photoactivatable GFP and high-resolution live-cell confocal microscopy to visualize complete 3D movements of individual kinetochores throughout mitosis in nontransformed human cells. Combined with electron microscopy, molecular perturbations, and immunofluorescence analyses, this approach reveals unexpected details of chromosome behavior. Our data demonstrate that unstable lateral interactions between kinetochores and microtubules dominate during early prometaphase. These transient interactions lead to the reproducible arrangement of chromosomes in an equatorial ring on the surface of the nascent spindle. A computational model predicts that this toroidal distribution of chromosomes exposes kinetochores to a high density of microtubules which facilitates subsequent formation of amphitelic attachments. Thus, spindle formation involves a previously overlooked stage of chromosome prepositioning which promotes formation of amphitelic attachments.
Subject(s)
Chromosomes/metabolism , Prometaphase , Spindle Apparatus/metabolism , Animals , Cell Line , Centromere/metabolism , Humans , Kinetochores/metabolism , Mice , Microtubules/metabolism , Models, MolecularABSTRACT
The establishment of a metaphase plate in which all chromosomes are attached to mitotic spindle microtubules and aligned at the cell equator is required for faithful chromosome segregation in metazoans. The achievement of this configuration relies on the precise coordination between several concurrent mechanisms that start upon nuclear envelope breakdown, mediate chromosome capture at their kinetochores during mitotic spindle assembly and culminate with the congression of all chromosomes to the spindle equator. This period is called 'prometaphase'. Because the nature of chromosome capture by mitotic spindle microtubules is error prone, the cell is provided of error correction mechanisms that sense and correct most erroneous kinetochore-microtubule attachments before committing to separate sister chromatids in anaphase. In this review, aimed for newcomers in the field, more than providing an exhaustive mechanistic coverage of each and every concurrent mechanism taking place during prometaphase, we provide an integrative overview of these processes that ultimately promote the subsequent faithful segregation of chromosomes during mitosis.
Subject(s)
Mitosis/physiology , Prometaphase/physiology , Humans , Spindle Apparatus/metabolismABSTRACT
The early stages of development involve complex sequences of morphological changes that are both reproducible from embryo to embryo and often robust to environmental variability. To investigate the relationship between reproducibility and robustness we examined cell cycle progression in early Drosophila embryos at different temperatures. Our experiments show that while the subdivision of cell cycle steps is conserved across a wide range of temperatures (5-35 â°C), the relative duration of individual steps varies with temperature. We find that the transition into prometaphase is delayed at lower temperatures relative to other cell cycle events, arguing that it has a different mechanism of regulation. Using an in vivo biosensor, we quantified the ratio of activities of the major mitotic kinase, Cdk1 and one of the major mitotic phosphatases PP1. Comparing activation profile with cell cycle transition times at different temperatures indicates that in early fly embryos activation of Cdk1 drives entry into prometaphase but is not required for earlier cell cycle events. In fact, chromosome condensation can still occur when Cdk1 activity is inhibited pharmacologically. These results demonstrate that different kinases are rate-limiting for different steps of mitosis, arguing that robust inter-regulation may be needed for rapid and ordered mitosis.
Subject(s)
CDC2 Protein Kinase/metabolism , Cell Cycle Checkpoints , Cell Cycle , Drosophila Proteins/metabolism , Embryo, Nonmammalian/cytology , Mitosis , Animals , CDC2 Protein Kinase/antagonists & inhibitors , Cyclin B/metabolism , Drosophila Proteins/antagonists & inhibitors , Drosophila melanogaster/embryology , Embryo, Nonmammalian/enzymology , Enzyme Activation , Metaphase , Prometaphase , Prophase , Protein Phosphatase 1/metabolism , TemperatureABSTRACT
Human retinal pigment epithelial-1 (RPE-1) cells are increasingly being used as a model to study mitosis because they represent a non-transformed alternative to cancer cell lines, such as HeLa cervical adenocarcinoma cells. However, the lack of an efficient method to synchronize RPE-1 cells in mitosis precludes their application for large-scale biochemical and proteomics assays. Here, we report a protocol to synchronize RPE-1 cells based on sequential treatments with the Cdk4 and Cdk6 inhibitor PD 0332991 (palbociclib) and the microtubule-depolymerizing drug nocodazole. With this method, the vast majority (80-90%) of RPE-1 cells arrested at prometaphase and exited mitosis synchronously after release from nocodazole. Moreover, the cells fully recovered and re-entered the cell cycle after the palbociclib-nocodazole block. Finally, we show that this protocol could be successfully employed for the characterization of the protein-protein interaction network of the kinetochore protein Ndc80 by immunoprecipitation coupled with mass spectrometry. This synchronization method significantly expands the versatility and applicability of RPE-1 cells to the study of cell division and might be applied to other cell lines that do not respond to treatments with DNA synthesis inhibitors.
Subject(s)
Kinetochores , Mitosis , Humans , Nocodazole/pharmacology , Prometaphase , Retinal PigmentsABSTRACT
CCCTC-binding factor (CTCF) plays a key role in the formation of topologically associating domains (TADs) and loops in interphase. During mitosis TADs are absent, but how TAD formation is dynamically controlled during the cell cycle is not known. Several contradicting observations have been made regarding CTCF binding to mitotic chromatin using both genomics- and microscopy-based techniques. Here, we have used four different assays to address this debate. First, using 5C, we confirmed that TADs and CTCF loops are readily detected in interphase, but absent during prometaphase. Second, ATAC-seq analysis showed that CTCF sites display greatly reduced accessibility and lose the CTCF footprint in prometaphase, suggesting loss of CTCF binding and rearrangement of the nucleosomal array around the binding motif. In contrast, transcription start sites remain accessible in prometaphase, although adjacent nucleosomes can also become repositioned and occupy at least a subset of start sites during mitosis. Third, loss of site-specific CTCF binding was directly demonstrated using CUT&RUN. Histone modifications and histone variants are maintained in mitosis, suggesting a role in bookmarking of active CTCF sites. Finally, live-cell imaging, fluorescence recovery after photobleaching, and single molecule tracking showed that almost all CTCF chromatin binding is lost in prometaphase. Combined, our results demonstrate loss of CTCF binding to CTCF sites during prometaphase and rearrangement of the chromatin landscape around CTCF motifs. This, combined with loss of cohesin, would contribute to the observed loss of TADs and CTCF loops during mitosis and reveals that CTCF sites, key architectural cis-elements, display cell cycle stage-dependent dynamics in factor binding and nucleosome positioning.
Subject(s)
CCCTC-Binding Factor/metabolism , Cell Cycle/genetics , Nucleosomes/physiology , Binding Sites , Cells, Cultured , Chromatin/chemistry , HeLa Cells , Histone Code , Humans , Interphase/genetics , Mitosis/genetics , Nucleotide Motifs , Prometaphase/genetics , Transcription Initiation SiteABSTRACT
Cell division depends on the timely degradation of numerous proteins by the anaphase-promoting complex/cyclosome (APC/C). The APC/C is a large E3 ubiquitin ligase that in complex with Cdc20 recognises degrons in its substrates. The ability of APC/C-Cdc20 to bind degrons is prevented by the binding of the mitotic checkpoint complex (MCC) which constitutes the "wait anaphase" signal. Curiously, the mitotic kinase Nek2A is insensitive to the presence of the MCC. How Nek2A avoids MCC inhibition has been unclear but now work from Alfieri and colleagues published in this issue of EMBO reports provides an explanation [1]. It shows that Nek2A is able to bind a specific open conformation of the APC/C-MCC complex that allows Nek2A ubiquitination. A dimer of Nek2A binds two distinct binding pockets on the APC/C through C-terminal MR motifs and thus independently of degrons. One of the MR binding pockets is only available for interaction in the open form of APC/C-MCC explaining Nek2A selectivity for this conformation. Whether other substrates bind the APC/C directly without using canonical degrons will be important to determine.
Subject(s)
Cell Cycle Proteins , Prometaphase , Anaphase-Promoting Complex-Cyclosome/genetics , Anaphase-Promoting Complex-Cyclosome/metabolism , Cdc20 Proteins/metabolism , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , UbiquitinationABSTRACT
The anaphase-promoting complex (APC/C) is the key E3 ubiquitin ligase which directs mitotic progression and exit by catalysing the sequential ubiquitination of specific substrates. The activity of the APC/C in mitosis is restrained by the spindle assembly checkpoint (SAC), which coordinates chromosome segregation with the assembly of the mitotic spindle. The SAC effector is the mitotic checkpoint complex (MCC), which binds and inhibits the APC/C. It is incompletely understood how the APC/C switches substrate specificity in a cell cycle-specific manner. For instance, it is unclear how in prometaphase, when APC/C activity towards cyclin B and securin is repressed by the MCC, the kinase Nek2A is ubiquitinated. Here, we combine biochemical and structural analysis with functional studies in cells to show that Nek2A is a conformational-specific binder of the APC/C-MCC complex (APC/CMCC ) and that, in contrast to cyclin A, Nek2A can be ubiquitinated efficiently by the APC/C in conjunction with both the E2 enzymes UbcH10 and UbcH5. We propose that these special features of Nek2A allow its prometaphase-specific ubiquitination.
Subject(s)
M Phase Cell Cycle Checkpoints , Prometaphase , Anaphase-Promoting Complex-Cyclosome/genetics , Anaphase-Promoting Complex-Cyclosome/metabolism , Cdc20 Proteins/metabolism , Cell Cycle Proteins/metabolism , HeLa Cells , Humans , Mitosis , Spindle Apparatus/metabolism , UbiquitinationABSTRACT
BACKGROUND: The World Health Organization (WHO) proposed COVID-19 vaccination as an emergent and important method to end the COVID-19 pandemic. Since China started vaccination programs in December 2020, vaccination has spread to provinces and municipalities nationwide. Previous research has focused on people's vaccination willingness and its influencing factors but has not examined vaccination behavior. We examine the effectiveness of psychosocial factors in predicting vaccination behavior. METHODS: A cross-sectional online survey was performed among Chinese adults on 8 May and 4 June 2021. The statistical analysis of the data included univariate analysis, receiver operator characteristics (ROC) analysis and ordinal multiclassification logistic regression model analysis. RESULTS: Of the 1300 respondents, 761 (58.5%) were vaccinated. Univariate analysis showed that a high education level and good subjective health status were protective factors for vaccination behavior, while suffering from chronic diseases was a risk factor. ROC analysis showed that subjective health status (AUC = 0.625, 95% CI: 0.594-0.656, P < 0.001) was the best predictor of vaccination behavior. Logistic regression analysis with subjective health status as a dependent variable indicated that older age, female sex, depression, neurasthenia, obsession, hypochondriasis and chronic disease were significant risk factors, while positive coping tendencies were a significant protective factor. CONCLUSION: Our study found a simple and effective marker, subjective health status, that can predict vaccination behavior. This finding can guide future epidemic prevention work.
Subject(s)
COVID-19 , Diagnostic Self Evaluation , Adult , COVID-19 Vaccines , China/epidemiology , Cross-Sectional Studies , Female , Humans , Pandemics/prevention & control , Prometaphase , Vaccination/psychologyABSTRACT
Microtubules are highly strategic targets of cancer therapies. Small molecule antimitotic agents are so far the best chemotherapeutic medication in cancer treatment. However, the high rate of neuropathy and drug resistance limit their clinical usage. Inspired by the multicomponent-targeting feature of molecular self-assembly (MSA) overcoming drug resistance, we synthesized peptide-based rotor molecules that self-assemble in response to the surrounding environment to target the microtubule array. The MSAs self-adjust morphologically in response to the pH change and viscosity variations during Golgi-endosome trafficking, escape trafficking cargos, and eventually bind to the microtubule array physically in a nonspecific manner. Such unrefined nano-bio interactions suppress regional tubulin polymerization triggering atypical prometaphase--metaphase oscillations to inhibit various cancer cells proliferating without inducing obvious neurotoxicity. The MSA also exerts potent antiproliferative effects in the subcutaneous cervix cancer xenograft tumor model equivalent to Cisplatin, better than the classic antimitotic drug Taxol.
Subject(s)
Neoplasms , Prometaphase , Female , Humans , Metaphase , Microtubules , TubulinABSTRACT
ARHGAP19 is a hematopoietic-specific Rho GTPase-activating protein (RhoGAP) that acts through the RhoA/ROCK pathway to critically regulate cell elongation and cytokinesis during lymphocyte mitosis. We report here that, during mitosis progression, ARHGAP19 is sequentially phosphorylated by the RhoA-activated kinases ROCK1 and ROCK2 (hereafter ROCK) on serine residue 422, and by CDK1 on threonine residues 404 and 476. The phosphorylation of ARHGAP19 by ROCK occurs before mitosis onset and generates a binding site for 14-3-3 family proteins. ARHGAP19 is then phosphorylated by CDK1 in prometaphase. The docking of 14-3-3 proteins to phosphorylated S422 protects ARHGAP19 from dephosphorylation of the threonine sites and prevents ARHGAP19 from relocating to the plasma membrane during prophase and metaphase, thus allowing RhoA to become activated. Disruption of these phosphorylation sites results in premature localization of ARHGAP19 at the cell membrane and in its enrichment to the equatorial cortex in anaphase leading to cytokinesis failure and cell multinucleation.
Subject(s)
Cytokinesis/genetics , GTPase-Activating Proteins/genetics , Mitosis/genetics , rhoA GTP-Binding Protein/genetics , 14-3-3 Proteins/genetics , CDC2 Protein Kinase/genetics , Humans , Jurkat Cells , Phosphorylation/genetics , Prometaphase/genetics , Serine/genetics , rho-Associated Kinases/geneticsABSTRACT
During the prometaphase stage of mitosis, the cell builds a bipolar spindle of microtubules that mechanically segregates sister chromatids between two daughter cells in anaphase. The spindle assembly checkpoint (SAC) is a quality control mechanism that monitors proper attachment of microtubules to chromosome kinetochores during prometaphase. Segregation occurs only when each chromosome is bi-oriented with each kinetochore pair attached to microtubules emanating from opposite spindle poles. Overexpression of the protein kinase Aurora A is a feature of various cancers and is thought to enable tumour cells to bypass the SAC, leading to aneuploidy. Here, we took advantage of a chemical and chemical-genetic approach to specifically inhibit Aurora A kinase activity in late prometaphase. We observed that a loss of Aurora A activity directly affects SAC function, that Aurora A is essential for maintaining the checkpoint protein Mad2 on unattached kinetochores and that inhibition of Aurora A leads to loss of the SAC, even in the presence of nocodazole or Taxol. This is a new finding that should affect the way Aurora A inhibitors are used in cancer treatments.This article has an associated First Person interview with the first authors of the paper.
Subject(s)
Aurora Kinase A/genetics , M Phase Cell Cycle Checkpoints/genetics , Mad2 Proteins/genetics , Prometaphase/genetics , Anaphase/genetics , Aurora Kinase A/antagonists & inhibitors , Azepines/pharmacology , Cell Line, Tumor , Chromatids/genetics , Chromosome Segregation/drug effects , Gene Expression Regulation, Enzymologic/genetics , Humans , Kinetochores/drug effects , Microtubules/drug effects , Mitosis/drug effects , Mitosis/genetics , Nocodazole/pharmacology , Paclitaxel/pharmacology , Prometaphase/drug effects , Pyrimidines/pharmacology , Spindle Apparatus/geneticsABSTRACT
Kinetochore fibers (K-fibers) are microtubule bundles attached to chromosomes. Efficient K-fiber formation is required for chromosome congression, crucial for faithful chromosome segregation in cells. However, the mechanisms underlying K-fiber formation before chromosome biorientation remain unclear. Depletion of hepatoma up-regulated protein (HURP), a RanGTP-dependent microtubule-associated protein localized on K-fibers, has been shown to result in low-efficiency K-fiber formation. Therefore, here we sought to identify critical interaction partners of HURP that may modulate this function. Using co-immunoprecipitation and bimolecular fluorescence complementation assays, we determined that HURP interacts directly with the centrosomal protein transforming acidic coiled coil-containing protein 3 (TACC3), a centrosomal protein, both in vivo and in vitro through the HURP1-625 region. We found that HURP is important for TACC3 function during kinetochore microtubule assembly at the chromosome region in prometaphase. Moreover, HURP regulates stable lateral kinetochore attachment and chromosome congression in early mitosis by modulation of TACC3. These findings provide new insight into the coordinated regulation of K-fiber formation and chromosome congression in prometaphase by microtubule-associated proteins.
Subject(s)
Chromosome Positioning , Microtubule-Associated Proteins/genetics , Microtubules/metabolism , Neoplasm Proteins/genetics , Prometaphase , Amino Acid Sequence , Cell Cycle Proteins/antagonists & inhibitors , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Chromosome Segregation , Gene Expression Regulation , HEK293 Cells , HeLa Cells , Humans , Kinetochores/metabolism , Kinetochores/ultrastructure , Microtubule-Associated Proteins/antagonists & inhibitors , Microtubule-Associated Proteins/metabolism , Microtubules/ultrastructure , Neoplasm Proteins/metabolism , Protein Transport , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Spindle Apparatus/metabolism , Spindle Apparatus/ultrastructure , Time-Lapse ImagingABSTRACT
Formation of stable kinetochore-microtubule attachments is essential for accurate chromosome segregation in human cells and depends on the NDC80 complex. We recently showed that Chmp4c, an endosomal sorting complex required for transport protein involved in membrane remodelling, localises to prometaphase kinetochores and promotes cold-stable kinetochore microtubules, faithful chromosome alignment and segregation. In the present study, we show that Chmp4c associates with the NDC80 components Hec1 and Nuf2 and is required for optimal NDC80 stability and Hec1-Nuf2 localisation to kinetochores in prometaphase. However, Chmp4c-depletion does not cause a gross disassembly of outer or inner kinetochore complexes. Conversely, Nuf2 is required for Chmp4c kinetochore targeting. Constitutive Chmp4c kinetochore tethering partially rescues cold-stable microtubule polymers in cells depleted of the endogenous Nuf2, showing that Chmp4c also contributes to kinetochore-microtubule stability independently of regulating Hec1 and Nuf2 localisation. Chmp4c interacts with tubulin in cell extracts, and binds and bundles microtubules in vitro through its highly basic N-terminal region (amino acids 1-77). Furthermore, the N-terminal region of Chmp4c is required for cold-stable kinetochore microtubules and efficient chromosome alignment. We propose that Chmp4c promotes stable kinetochore-microtubule attachments by regulating Hec1-Nuf2 localisation to kinetochores in prometaphase and by binding to spindle microtubules. These results identify Chmp4c as a novel protein that regulates kinetochore-microtubule interactions to promote accurate chromosome segregation in human cells.
Subject(s)
Endosomal Sorting Complexes Required for Transport/metabolism , Kinetochores/metabolism , Microtubules/metabolism , Cell Cycle Proteins/metabolism , Chromosome Segregation , Cytoskeletal Proteins , Endosomal Sorting Complexes Required for Transport/genetics , HeLa Cells , Humans , Nuclear Proteins/metabolism , Prometaphase/physiologyABSTRACT
The spindle assembly checkpoint (SAC) restricts mitotic exit to cells that have completed chromosome-microtubule attachment. Cdc20 is a bifunctional protein. In complex with SAC proteins Mad2, BubR1, and Bub3, Cdc20 forms the mitotic checkpoint complex (MCC), which binds the anaphase-promoting complex (APC/C) and inhibits its mitotic exit-promoting activity. When devoid of SAC proteins, Cdc20 serves as an APC/C coactivator and promotes mitotic exit. During mitotic arrest, Cdc20 is continuously degraded via ubiquitin-dependent proteolysis and resynthesized. It is believed that this cycle keeps the levels of Cdc20 below a threshold above which Cdc20 would promote mitotic exit. We report that p31(comet), a checkpoint antagonist, is necessary for mitotic destabilization of Cdc20. p31(comet) depletion stabilizes the MCC, super-inhibits the APC/C, and delays mitotic exit, indicating that Cdc20 proteolysis in prometaphase opposes the checkpoint. Our studies reveal a homeostatic network in which checkpoint-sustaining and -repressing forces oppose each other during mitotic arrest and suggest ways for enhancing the sensitivity of cancer cells to antitubulin chemotherapeutics.
Subject(s)
Homeostasis , Mitosis , Adaptor Proteins, Signal Transducing/metabolism , Anaphase-Promoting Complex-Cyclosome , Cdc20 Proteins , Cell Cycle Proteins/metabolism , HeLa Cells , Homeostasis/drug effects , Humans , M Phase Cell Cycle Checkpoints/drug effects , Mitosis/drug effects , Nocodazole/pharmacology , Nuclear Proteins/metabolism , Prometaphase/drug effects , Ubiquitin-Protein Ligase Complexes/metabolismABSTRACT
The aim of investigation was to learn cytogenetic karyotype characteristics of children with asthma, with varying degrees of control in order to find markers of disease severity. The study involved 82 children aged 6 to 18 years, patients with asthma who were treated in Allergic Department of Regional Pediatric Hospital of Ivano-Frankivsk. Regarding the level of controlled asthma as a result of the application of test control asthma (GINA, 2011) children were distributed as follows: - 22 (30.6%) with controlled (CBA), 24 (33.3%) - is partly controlled (PCBA) 26 (36.1%) - with uncontrolled asthma (NCBA). The control group children were selected by random sampling, living in different parts of the Ivano-Frankivsk region (10 persons). Research of cytogenetic features of the karyotype in children with bronchial asthma was conducted by studying specimens of prometaphase chromosomes of peripheral blood lymphocytes. We analyzed associations of acrocentral number of chromosomes, karyotype features. The study involved 82 children aged 6 to 18 years with asthma of varying degrees of its control over the outcome of asthma control test. Children with uncontrolled asthma received significantly higher rate of chromosome acrocentral associations 13-15 (DD), 21-22 (GG) and 13-15 - 21-22 (DG). In patients with asthma we observed a lower mitotic activity compared with that in healthy ones (PN <0.05), and with a lower degree of controllability it decreased. Recognition of the genetic mechanisms of asthma leads to a new understanding of the genesis of the disease and can move forward in developing methods of treatment and prevention.
Subject(s)
Anti-Asthmatic Agents/therapeutic use , Asthma/drug therapy , Chromosomal Instability , Adolescent , Asthma/genetics , Asthma/physiopathology , Case-Control Studies , Child , Genetic Markers , Humans , Karyotyping , PrometaphaseABSTRACT
The Anaphase Promoting Complex/Cyclosome (APC/C) in complex with its co-activator Cdc20 is responsible for targeting proteins for ubiquitin-mediated degradation during mitosis. The activity of APC/C-Cdc20 is inhibited during prometaphase by the Spindle Assembly Checkpoint (SAC) yet certain substrates escape this inhibition. Nek2A degradation during prometaphase depends on direct binding of Nek2A to the APC/C via a C-terminal MR dipeptide but whether this motif alone is sufficient is not clear. Here, we identify Kif18A as a novel APC/C-Cdc20 substrate and show that Kif18A degradation depends on a C-terminal LR motif. However in contrast to Nek2A, Kif18A is not degraded until anaphase showing that additional mechanisms contribute to Nek2A degradation. We find that dimerization via the leucine zipper, in combination with the MR motif, is required for stable Nek2A binding to and ubiquitination by the APC/C. Nek2A and the mitotic checkpoint complex (MCC) have an overlap in APC/C subunit requirements for binding and we propose that Nek2A binds with high affinity to apo-APC/C and is degraded by the pool of Cdc20 that avoids inhibition by the SAC.
Subject(s)
Cell Cycle Proteins/physiology , Kinesins/metabolism , Protein Serine-Threonine Kinases/metabolism , Proteolysis , Ubiquitin-Protein Ligase Complexes/physiology , Anaphase-Promoting Complex-Cyclosome , Apc8 Subunit, Anaphase-Promoting Complex-Cyclosome , Cdc20 Proteins , Cell Cycle Proteins/metabolism , HeLa Cells , Humans , M Phase Cell Cycle Checkpoints/physiology , NIMA-Related Kinases , Prometaphase/physiology , Protein Binding , Protein Multimerization , Time Factors , Tumor Cells, Cultured , Ubiquitin-Protein Ligase Complexes/metabolismABSTRACT
Nek2 isoform A (Nek2A) is a presumed substrate of the anaphase-promoting complex/cyclosome containing Cdc20 (APC/C(Cdc20)). Nek2A, like cyclin A, is degraded in mitosis while the spindle checkpoint is active. Cyclin A prevents spindle checkpoint proteins from binding to Cdc20 and is recruited to the APC/C in prometaphase. We found that Nek2A and cyclin A avoid being stabilized by the spindle checkpoint in different ways. First, enhancing mitotic checkpoint complex (MCC) formation by nocodazole treatment inhibited the degradation of geminin and cyclin A, whereas Nek2A disappeared at a normal rate. Second, depleting Cdc20 effectively stabilized cyclin A but not Nek2A. Nevertheless, Nek2A destruction crucially depended on Cdc20 binding to the APC/C. Third, in contrast to cyclin A, Nek2A was recruited to the APC/C before the start of mitosis. Interestingly, the spindle checkpoint very effectively stabilized an APC/C-binding mutant of Nek2A, which required the Nek2A KEN box. Apparently, in cells, the spindle checkpoint primarily prevents Cdc20 from binding destruction motifs. Nek2A disappearance marks the prophase-to-prometaphase transition, when Cdc20, regardless of the spindle checkpoint, activates the APC/C. However, Mad2 depletion accelerated Nek2A destruction, showing that spindle checkpoint release further increases APC/C(Cdc20) catalytic activity.
Subject(s)
Cdc20 Proteins/metabolism , Geminin/metabolism , M Phase Cell Cycle Checkpoints , Prometaphase , Prophase , Protein Serine-Threonine Kinases/metabolism , Anaphase-Promoting Complex-Cyclosome/metabolism , Cell Line, Tumor , Cyclin A/metabolism , Humans , NIMA-Related Kinases , Nocodazole/chemistryABSTRACT
In the course of our continuing studies on the 2-(benzo[b]thiophene-3'-yl)-6,8,8-triethyldesmosdumotin B (TEDB-TB) series, we designed and synthesized nine amino-TEDB-TB derivatives to improve pharmaceutical properties, identify structure activity relationships, and discover novel antitubulin agents. Among all newly synthesized amino-TEDB-TBs, the 5'- and 6'-amino derivatives, 6 and 7, exhibited significant antiproliferative activity against five human tumor cell lines, including an MDR subline overexpressing P-gp. The IC50 values of 0.50-1.01µM were 3-6 times better than those of previously reported hydroxy-TEDB-TBs. Compounds 6 and 7 inhibited tubulin polymerization, induced both depolymerization of interphase microtubules and multiple spindle formations, and caused cell arrest at prometaphase. Among all compounds, compound 7 scored best pharmaceutically with LogP 2.11 and biologically with greater antiproliferative activity and induction of cell cycle arrest at prometaphase.
Subject(s)
Antineoplastic Agents/pharmacology , Chromones/pharmacology , Flavonoids/pharmacology , Thiophenes/pharmacology , Tubulin/metabolism , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/chemistry , Cell Cycle Checkpoints/drug effects , Cell Line, Tumor , Cell Proliferation/drug effects , Chromones/chemical synthesis , Chromones/chemistry , Dose-Response Relationship, Drug , Drug Screening Assays, Antitumor , Flavonoids/chemical synthesis , Flavonoids/chemistry , Humans , Molecular Structure , Polymerization/drug effects , Prometaphase/drug effects , Structure-Activity Relationship , Thiophenes/chemical synthesis , Thiophenes/chemistry , Tubulin ModulatorsABSTRACT
In budding yeast, meiotic commitment is the irreversible continuation of the developmental path of meiosis. After reaching meiotic commitment, cells finish meiosis and gametogenesis, even in the absence of the meiosis-inducing signal. In contrast, if the meiosis-inducing signal is removed and the mitosis-inducing signal is provided prior to reaching meiotic commitment, cells exit meiosis and return to mitosis. Previous work has shown that cells commit to meiosis after prophase I but before entering the meiotic divisions. Since the Ndt80 transcription factor induces expression of middle meiosis genes necessary for the meiotic divisions, we examined the role of the NDT80 transcriptional network in meiotic commitment. Using a microfluidic approach to analyze single cells, we found that cells commit to meiosis in prometaphase I, after the induction of the Ndt80-dependent genes. Our results showed that high-level expression of NDT80 is important for the timing and irreversibility of meiotic commitment. A modest reduction in NDT80 levels delayed meiotic commitment based on meiotic stages, although the timing of each meiotic stage was similar to that of wildtype cells. A further reduction of NDT80 resulted in the surprising finding of inappropriately uncommitted cells: withdrawal of the meiosis-inducing signal and addition of the mitosis-inducing signal to cells at stages beyond metaphase I caused return to mitosis, leading to multi-nucleate cells. Since Ndt80 enhances its own transcription through positive feedback, we tested whether positive feedback ensured the irreversibility of meiotic commitment. Ablating positive feedback in NDT80 expression resulted in a complete loss of meiotic commitment. These findings suggest that irreversibility of meiotic commitment is a consequence of the NDT80 transcriptional positive feedback loop, which provides the high-level of Ndt80 required for the developmental switch of meiotic commitment. These results also illustrate the importance of irreversible meiotic commitment for maintaining genome integrity by preventing formation of multi-nucleate cells.
Subject(s)
DNA-Binding Proteins/genetics , Meiosis/genetics , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae/genetics , Transcription Factors/genetics , Cell Cycle Proteins/biosynthesis , DNA, Fungal/biosynthesis , DNA, Fungal/genetics , DNA-Binding Proteins/biosynthesis , Gametogenesis/genetics , Microfluidics/methods , Prometaphase/genetics , Promoter Regions, Genetic/genetics , Protein Serine-Threonine Kinases/biosynthesis , Saccharomyces cerevisiae Proteins/biosynthesis , Spindle Apparatus/metabolism , Transcription Factors/biosynthesisABSTRACT
The Bub1-Bub3 and BubR1-Bub3 checkpoint complexes, or the Bubs, contribute to the accurate segregation of chromosomes during mitosis by promoting chromosome bi-orientation and halting exit from mitosis if this fails. The complexes associate with kinetochores during mitosis, which is required for proper chromosome segregation. The outer kinetochore protein KNL1 (also known as CASC5, Blinkin and AF15Q14) is the receptor for Bub proteins, but the exact nature of the functional binding sites on KNL1 are yet to be determined. Here, we show that KNL1 contains multiple binding sites for the Bub proteins, with the Mps1-phosphorylated MELT repeats constituting individual functional docking sites for direct binding of Bub3. Surprisingly, chromosome congression and the spindle assembly checkpoint (SAC) are still functional when KNL1 is deleted of all but four of its twelve MELT repeats. Systematically reducing the number of MELT repeats to less than four reduced KNL1 functionality. Furthermore, we show that protein phosphatase 1 (PP1) binding to KNL1 during prometaphase reduces the levels of Bub proteins at kinetochores to approximately the level recruited by four active MELT repeats.