-
Journal of Proteome Research Jul 2021The spindle assembly checkpoint (SAC) is critical for sensing defective microtubule-kinetochore attachments and tension across the kinetochore and functions to arrest...
The spindle assembly checkpoint (SAC) is critical for sensing defective microtubule-kinetochore attachments and tension across the kinetochore and functions to arrest cells in prometaphase to allow time to repair any errors before proceeding into anaphase. Dysregulation of the SAC leads to chromosome segregation errors that have been linked to human diseases like cancer. Although much has been learned about the composition of the SAC and the factors that regulate its activity, the proximity associations of core SAC components have not been explored in a systematic manner. Here, we have taken a BioID2-proximity-labeling proteomic approach to define the proximity protein environment for each of the five core SAC proteins BUB1, BUB3, BUBR1, MAD1L1, and MAD2L1 in mitotic-enriched populations of cells where the SAC is active. These five protein association maps were integrated to generate a SAC proximity protein network that contains multiple layers of information related to core SAC protein complexes, protein-protein interactions, and proximity associations. Our analysis validated many known SAC complexes and protein-protein interactions. Additionally, it uncovered new protein associations, including the ELYS-MAD1L1 interaction that we have validated, which lend insight into the functioning of core SAC proteins and highlight future areas of investigation to better understand the SAC.
Topics: Cell Cycle Proteins; Humans; Kinetochores; M Phase Cell Cycle Checkpoints; Protein Serine-Threonine Kinases; Proteomics; Spindle Apparatus
PubMed: 34087075
DOI: 10.1021/acs.jproteome.0c00941 -
Nature Communications Jun 2023The centromere is the chromosome region where microtubules attach during cell division. In contrast to monocentric chromosomes with one centromere, holocentric species...
The centromere is the chromosome region where microtubules attach during cell division. In contrast to monocentric chromosomes with one centromere, holocentric species usually distribute hundreds of centromere units along the entire chromatid. We assembled the chromosome-scale reference genome and analyzed the holocentromere and (epi)genome organization of the lilioid Chionographis japonica. Remarkably, each of its holocentric chromatids consists of only 7 to 11 evenly spaced megabase-sized centromere-specific histone H3-positive units. These units contain satellite arrays of 23 and 28 bp-long monomers capable of forming palindromic structures. Like monocentric species, C. japonica forms clustered centromeres in chromocenters at interphase. In addition, the large-scale eu- and heterochromatin arrangement differs between C. japonica and other known holocentric species. Finally, using polymer simulations, we model the formation of prometaphase line-like holocentromeres from interphase centromere clusters. Our findings broaden the knowledge about centromere diversity, showing that holocentricity is not restricted to species with numerous and small centromere units.
Topics: Centromere; Cell Cycle Proteins; Cell Division; Chromatids; Heterochromatin
PubMed: 37311740
DOI: 10.1038/s41467-023-38922-7 -
Stem Cell Reports Nov 2017The spindle assembly checkpoint plays a pivotal role in preventing aneuploidy and transformation. Many studies demonstrate impairment of this checkpoint in cancer cells....
The spindle assembly checkpoint plays a pivotal role in preventing aneuploidy and transformation. Many studies demonstrate impairment of this checkpoint in cancer cells. While leukemia is frequently driven by transformed hematopoietic stem and progenitor cells (HSPCs), the biology of the spindle assembly checkpoint in such primary cells is not very well understood. Here, we reveal that the checkpoint is fully functional in murine progenitor cells and, to a lesser extent, in hematopoietic stem cells. We show that HSPCs arrest at prometaphase and induce p53-dependent apoptosis upon prolonged treatment with anti-mitotic drugs. Moreover, the checkpoint can be chemically and genetically abrogated, leading to premature exit from mitosis, subsequent enforced G1 arrest, and enhanced levels of chromosomal damage. We finally demonstrate that, upon checkpoint abrogation in HSPCs, hematopoiesis is impaired, manifested by loss of differentiation potential and engraftment ability, indicating a critical role of this checkpoint in HSPCs and hematopoiesis.
Topics: Animals; Antimitotic Agents; Apoptosis; Cells, Cultured; Hematopoiesis; Hematopoietic Stem Cells; M Phase Cell Cycle Checkpoints; Mice; Mice, Inbred C57BL; Tumor Suppressor Protein p53
PubMed: 29056333
DOI: 10.1016/j.stemcr.2017.09.017 -
EMBO Reports Jun 2020The anaphase-promoting complex (APC/C) is the key E3 ubiquitin ligase which directs mitotic progression and exit by catalysing the sequential ubiquitination of specific...
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/C ) 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.
Topics: Anaphase-Promoting Complex-Cyclosome; Cdc20 Proteins; Cell Cycle Proteins; HeLa Cells; Humans; M Phase Cell Cycle Checkpoints; Mitosis; Prometaphase; Spindle Apparatus; Ubiquitination
PubMed: 32307883
DOI: 10.15252/embr.201949831 -
Journal of Cell Science Jun 2022Mitotic kinesin-like protein 2 (MKLP2; also known as KIF20A) is a motor protein with a well-established function in promoting cytokinesis. However, our results with...
Mitotic kinesin-like protein 2 (MKLP2; also known as KIF20A) is a motor protein with a well-established function in promoting cytokinesis. However, our results with siRNAs targeting MKLP2 and small-molecule inhibitors of MKLP2 (MKLP2i) suggest that it also has a function earlier in mitosis, prior to anaphase. In this study, we provide direct evidence that MKLP2 facilitates chromosome congression in prometaphase. We employed live imaging to observe HeLa cells with fluorescently tagged histones treated with MKLP2i and discovered a pronounced chromosome congression defect. We show that MKLP2 facilitates error correction, as inhibited cells have a significant increase in unstable, syntelic kinetochore-microtubule attachments. We find that the aberrant attachments are accompanied by elevated Aurora kinase (A and B) activity and phosphorylation of the downstream target HEC1 (also known as NDC80) at Ser55. Finally, we show that MKLP2 inhibition results in aneuploidy, confirming that MKLP2 safeguards cells against chromosomal instability. This article has an associated First Person interview with the first author of the paper.
Topics: Aurora Kinase B; Chromosome Segregation; Chromosomes; HeLa Cells; Humans; Kinesins; Kinetochores; Microtubules; Mitosis; Spindle Apparatus
PubMed: 35638575
DOI: 10.1242/jcs.259560 -
Proceedings of the National Academy of... Jun 2020Topoisomerase IIα (TOP2A) is a core component of mitotic chromosomes and important for establishing mitotic chromosome condensation. The primary roles of TOP2A in...
Topoisomerase IIα (TOP2A) is a core component of mitotic chromosomes and important for establishing mitotic chromosome condensation. The primary roles of TOP2A in mitosis have been difficult to decipher due to its multiple functions across the cell cycle. To more precisely understand the role of TOP2A in mitosis, we used the auxin-inducible degron (AID) system to rapidly degrade the protein at different stages of the human cell cycle. Removal of TOP2A prior to mitosis does not affect prophase timing or the initiation of chromosome condensation. Instead, it prevents chromatin condensation in prometaphase, extends the length of prometaphase, and ultimately causes cells to exit mitosis without chromosome segregation occurring. Surprisingly, we find that removal of TOP2A from cells arrested in prometaphase or metaphase cause dramatic loss of compacted mitotic chromosome structure and conclude that TOP2A is crucial for maintenance of mitotic chromosomes. Treatments with drugs used to poison/inhibit TOP2A function, such as etoposide and ICRF-193, do not phenocopy the effects on chromosome structure of TOP2A degradation by AID. Our data point to a role for TOP2A as a structural chromosome maintenance enzyme locking in condensation states once sufficient compaction is achieved.
Topics: Chromosome Segregation; Chromosome Structures; Chromosomes, Human; Cytokinesis; DNA Topoisomerases, Type II; HCT116 Cells; Heterochromatin; Humans; Metaphase; Mitosis
PubMed: 32414923
DOI: 10.1073/pnas.2001760117 -
BMC Molecular and Cell Biology Apr 2019The calmodulin-regulated spectrin-associated proteins (CAMSAPs) belong to a conserved protein family, which includes members that bind the polymerizing mcrotubule (MT)...
BACKGROUND
The calmodulin-regulated spectrin-associated proteins (CAMSAPs) belong to a conserved protein family, which includes members that bind the polymerizing mcrotubule (MT) minus ends and remain associated with the MT lattice formed by minus end polymerization. Only one of the three mammalian CAMSAPs, CAMSAP1, localizes to the mitotic spindle but its function is unclear. In Drosophila, there is only one CAMSAP, named Patronin. Previous work has shown that Patronin stabilizes the minus ends of non-mitotic MTs and is required for proper spindle elongation. However, the precise role of Patronin in mitotic spindle assembly is poorly understood.
RESULTS
Here we have explored the role of Patronin in Drosophila mitosis using S2 tissue culture cells as a model system. We show that Patronin associates with different types of MT bundles within the Drosophila mitotic spindle, and that it is required for their stability. Imaging of living cells expressing Patronin-GFP showed that Patronin displays a dynamic behavior. In prometaphase cells, Patronin accumulates on short segments of MT bundles located near the chromosomes. These Patronin "seeds" extend towards the cell poles and stop growing just before reaching the poles. Our data also suggest that Patronin localization is largely independent of proteins acting at the MT minus ends such as Asp and Klp10A.
CONCLUSION
Our results suggest a working hypothesis about the mitotic role of Patronin. We propose that Patronin binds the minus ends within MT bundles, including those generated from the walls of preexisting MTs via the augmin-mediated pathway. This would help maintaining MT association within the mitotic bundles, thereby stabilizing the spindle structure. Our data also raise the intriguing possibility that the minus ends of bundled MTs can undergo a limited polymerization.
Topics: Animals; Cell Cycle Proteins; Cell Line; Centrosome; Chromosome Segregation; Drosophila Proteins; Drosophila melanogaster; Kinesins; Microtubule-Associated Proteins; Microtubules; Mitosis; Polymerization; Protein Binding; Spindle Apparatus
PubMed: 31284878
DOI: 10.1186/s12860-019-0189-0 -
Scientific Reports Oct 2019We previously showed that curcumin, a phytopolyphenol found in turmeric (Curcuma longa), targets a series of enzymes in the ROS metabolic pathway, induces irreversible...
We previously showed that curcumin, a phytopolyphenol found in turmeric (Curcuma longa), targets a series of enzymes in the ROS metabolic pathway, induces irreversible growth arrest, and causes apoptosis. In this study, we tested Pentagamavunon-1 (PGV-1), a molecule related to curcumin, for its inhibitory activity on tumor cells in vitro and in vivo. PGV-1 exhibited 60 times lower GI compared to that of curcumin in K562 cells, and inhibited the proliferation of cell lines derived from leukemia, breast adenocarcinoma, cervical cancer, uterine cancer, and pancreatic cancer. The inhibition of growth by PGV-1 remained after its removal from the medium, which suggests that PGV-1 irreversibly prevents proliferation. PGV-1 specifically induced prometaphase arrest in the M phase of the cell cycle, and efficiently induced cell senescence and cell death by increasing intracellular ROS levels through inhibition of ROS-metabolic enzymes. In a xenograft mouse model, PGV-1 had marked anti-tumor activity with little side effects by oral administration, whereas curcumin rarely inhibited tumor formation by this administration. Therefore, PGV-1 is a potential therapeutic to induce tumor cell apoptosis with few side effects and low risk of relapse.
Topics: Administration, Oral; Alcohol Oxidoreductases; Animals; Antineoplastic Agents, Phytogenic; Carrier Proteins; Cell Death; Cell Division; Cell Movement; Cell Proliferation; Cellular Senescence; Curcumin; Gene Expression Regulation, Neoplastic; Glutathione S-Transferase pi; Glutathione Transferase; HEK293 Cells; HeLa Cells; Humans; K562 Cells; Lactoylglutathione Lyase; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; MCF-7 Cells; Mice, Nude; NAD(P)H Dehydrogenase (Quinone); Peroxiredoxins; Prometaphase; Reactive Oxygen Species; Tumor Burden; Xenograft Model Antitumor Assays
PubMed: 31619723
DOI: 10.1038/s41598-019-51244-3 -
Cytoskeleton (Hoboken, N.J.) Nov 2019Chromosome segregation is mediated by spindle microtubules that attach to the kinetochore via dynamic protein complexes, such as Ndc80, Ska, Cdt1 and ch-TOG during...
Chromosome segregation is mediated by spindle microtubules that attach to the kinetochore via dynamic protein complexes, such as Ndc80, Ska, Cdt1 and ch-TOG during mitotic metaphase. While experimental studies have previously shown that these proteins and protein complexes are all essential for maintaining a stable kinetochore-microtubule (kMT) interface, their exact roles in the mitotic metaphase remains elusive. In this study, we employed experimental and computational methods in order to characterize how these proteins can strengthen kMT attachments in both nonload-bearing and load-bearing conditions, typical of prometaphase and metaphase, respectively. Immunofluorescence staining of HeLa cells showed that the levels of Ska and Cdt1 significantly increased from prometaphase to metaphase, while levels of the Ndc80 complex remained unchanged. Our new computational model showed that by incorporating binding and unbinding of each protein complex coupled with a biased diffusion mechanism, the displacement of a possible complex formed by Ndc80-Ska-Cdt1 is significantly higher than that of Ndc80 alone or Ndc80-Ska. In addition, when we incorporate Ndc80/ch-TOG in the model, rupture force and time of attachment of the kMT interface increases. These results support the hypothesis that Ndc80-associated proteins strengthen kMT attachments, and that the interplay between kMT protein complexes in metaphase ensures stable attachments.
Topics: Cell Cycle Proteins; Chromosomal Proteins, Non-Histone; Computer Simulation; Cytoskeletal Proteins; HeLa Cells; Humans; Kinetochores; Metaphase; Microtubules; Mitosis; Protein Binding
PubMed: 31525284
DOI: 10.1002/cm.21562 -
Cell Cycle (Georgetown, Tex.) Sep 2019A single inner centromere protein (INCENP) found throughout eukaryotes modulates Aurora B kinase activity and chromosomal passenger complex (CPC) localization, which is...
A single inner centromere protein (INCENP) found throughout eukaryotes modulates Aurora B kinase activity and chromosomal passenger complex (CPC) localization, which is essential for timely mitotic progression. It has been proposed that INCENP might act as a rheostat to regulate Aurora B activity through mitosis, with successively higher activity threshold levels for chromosome alignment, the spindle checkpoint, anaphase spindle transfer and finally spindle elongation and cytokinesis. It remains mechanistically unclear how this would be achieved. Here, we reveal that the urochordate, , possesses two INCENP paralogs, which display distinct localizations and subfunctionalization in order to complete M-phase. INCENPa was localized on chromosome arms and centromeres by prometaphase, and modulated Aurora B activity to mediate H3S10/S28 phosphorylation, chromosome condensation, spindle assembly and transfer of the CPC to the central spindle. Polo-like kinase (Plk1) recruitment to CDK1 phosphorylated INCENPa was crucial for INCENPa-Aurora B enrichment on centromeres. The second paralog, INCENPb was enriched on centromeres from prometaphase, and relocated to the central spindle at anaphase onset. In the absence of INCENPa, meiotic spindles failed to form, and homologous chromosomes did not segregate. INCENPb was not required for early to mid M-phase events but became essential for the activity and localization of Aurora B on the central spindle and midbody during cytokinesis in order to allow abscission to occur. Together, our results demonstrate that INCENP paralog switching on centromeres modulates Aurora B kinase localization, thus chronologically regulating CPC functions during fast embryonic divisions in the urochordate . CCAN: constitutive centromere-associated network; CENPs: centromere proteins; cmRNA: capped messenger RNA; CPC: chromosomal passenger complex; INCENP: inner centromere protein; Plk1: polo-like kinase 1; PP1: protein phosphatase 1; PP2A: protein phosphatase 2A; SAC: spindle assembly checkpoint; SAH: single α-helix domain.
Topics: Aurora Kinase B; CDC2 Protein Kinase; Cell Cycle Proteins; Chromosomal Proteins, Non-Histone; Chromosome Segregation; Chromosomes; Cytokinesis; Humans; Kinetochores; Mitosis; Phosphorylation; Plankton; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Spindle Apparatus; Polo-Like Kinase 1
PubMed: 31306061
DOI: 10.1080/15384101.2019.1634954