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International Journal of Biological... 2024Cysteine-rich angiogenic inducer 61 (CYR61), also called CCN1, has long been characterized as a secretory protein. Nevertheless, the intracellular function of CYR61...
Cysteine-rich angiogenic inducer 61 (CYR61), also called CCN1, has long been characterized as a secretory protein. Nevertheless, the intracellular function of CYR61 remains unclear. Here, we found that CYR61 is important for proper cell cycle progression. Specifically, CYR61 interacts with microtubules and promotes microtubule polymerization to ensure mitotic entry. Moreover, CYR61 interacts with PLK1 and accumulates during the mitotic process, followed by degradation as mitosis concludes. The proteolysis of CYR61 requires the PLK1 kinase activity, which directly phosphorylates two conserved motifs on CYR61, enhancing its interaction with the SCF E3 complex subunit FBW7 and mediating its degradation by the proteasome. Mutations of phosphorylation sites of Ser167 and Ser188 greatly increase CYR61's stability, while deletion of CYR61 extends prophase and metaphase and delays anaphase onset. In summary, our findings highlight the precise control of the intracellular CYR61 by the PLK1-FBW7 pathway, accentuating its significance as a microtubule-associated protein during mitotic progression.
Topics: Protein Serine-Threonine Kinases; Humans; Polo-Like Kinase 1; Mitosis; Cell Cycle Proteins; Proto-Oncogene Proteins; Cysteine-Rich Protein 61; Microtubules; F-Box-WD Repeat-Containing Protein 7; HeLa Cells; Phosphorylation; Ubiquitin-Protein Ligases; Microtubule-Associated Proteins
PubMed: 38904029
DOI: 10.7150/ijbs.93335 -
BioRxiv : the Preprint Server For... Jan 2024Accurate chromosome segregation during mitosis requires that all chromosomes establish stable bi-oriented attachments with the spindle apparatus. Kinetochores form the...
Accurate chromosome segregation during mitosis requires that all chromosomes establish stable bi-oriented attachments with the spindle apparatus. Kinetochores form the interface between chromosomes and spindle microtubules and as such are under tight control by complex regulatory circuitry. As part of the chromosomal passenger complex (CPC), the Aurora B kinase plays a central role within this circuitry by destabilizing improper kinetochore-microtubule attachments and relaying the attachment status to the spindle assembly checkpoint, a feedback control system that delays the onset of anaphase by inhibiting the anaphase-promoting complex/cyclosome. Intriguingly, Aurora B is conserved even in kinetoplastids, an evolutionarily divergent group of eukaryotes, whose kinetochores are composed of a unique set of structural and regulatory proteins. Kinetoplastids do not have a canonical spindle checkpoint and it remains unclear how their kinetochores are regulated to ensure the fidelity and timing of chromosome segregation. Here, we show in , the kinetoplastid parasite that causes African sleeping sickness, that inhibition of Aurora B using an analogue-sensitive approach arrests cells in metaphase, with a reduction in properly bi-oriented kinetochores. Aurora B phosphorylates several kinetochore proteins , including the N-terminal region of the divergent Bub1-like protein KKT14. Depletion of KKT14 partially overrides the cell cycle arrest caused by Aurora B inhibition, while overexpression of a non-phosphorylatable KKT14 protein results in a prominent delay in the metaphase-to-anaphase transition. Finally, we demonstrate using a nanobody-based system that re-targeting the catalytic module of the CPC to the outer kinetochore is sufficient to promote mitotic exit but causes massive chromosome mis-segregation in anaphase. Our results indicate that the CPC and KKT14 are involved in an unconventional pathway controlling mitotic exit and error-free chromosome segregation in trypanosomes.
PubMed: 38293145
DOI: 10.1101/2024.01.20.576407 -
Journal of Cell Science Feb 2024The mammalian cell cycle alternates between two phases - S-G2-M with high levels of A- and B-type cyclins (CycA and CycB, respectively) bound to cyclin-dependent kinases...
The mammalian cell cycle alternates between two phases - S-G2-M with high levels of A- and B-type cyclins (CycA and CycB, respectively) bound to cyclin-dependent kinases (CDKs), and G1 with persistent degradation of CycA and CycB by an activated anaphase promoting complex/cyclosome (APC/C) bound to Cdh1 (also known as FZR1 in mammals; denoted APC/C:Cdh1). Because CDKs phosphorylate and inactivate Cdh1, these two phases are mutually exclusive. This 'toggle switch' is flipped from G1 to S by cyclin-E bound to a CDK (CycE:CDK), which is not degraded by APC/C:Cdh1, and from M to G1 by Cdc20-bound APC/C (APC/C:Cdc20), which is not inactivated by CycA:CDK or CycB:CDK. After flipping the switch, cyclin E is degraded and APC/C:Cdc20 is inactivated. Combining mathematical modelling with single-cell timelapse imaging, we show that dysregulation of CycB:CDK disrupts strict alternation of the G1-S and M-G1 switches. Inhibition of CycB:CDK results in Cdc20-independent Cdh1 'endocycles', and sustained activity of CycB:CDK drives Cdh1-independent Cdc20 endocycles. Our model provides a mechanistic explanation for how whole-genome doubling can arise, a common event in tumorigenesis that can drive tumour evolution.
Topics: Animals; Cell Cycle; Anaphase-Promoting Complex-Cyclosome; Cell Cycle Proteins; Cyclins; Cyclin-Dependent Kinases; Mitosis; Cdc20 Proteins; Mammals
PubMed: 38206091
DOI: 10.1242/jcs.261364 -
EMBO Reports May 2024ELYS is a nucleoporin that localizes to the nuclear side of the nuclear pore complex (NPC) in interphase cells. In mitosis, it serves as an assembly platform that...
ELYS is a nucleoporin that localizes to the nuclear side of the nuclear pore complex (NPC) in interphase cells. In mitosis, it serves as an assembly platform that interacts with chromatin and then with nucleoporin subcomplexes to initiate post-mitotic NPC assembly. Here we identify ELYS as a major binding partner of the membrane protein VAPB during mitosis. In mitosis, ELYS becomes phosphorylated at many sites, including a predicted FFAT (two phenylalanines in an acidic tract) motif, which mediates interaction with the MSP (major sperm protein)-domain of VAPB. Binding assays using recombinant proteins or cell lysates and co-immunoprecipitation experiments show that VAPB binds the FFAT motif of ELYS in a phosphorylation-dependent manner. In anaphase, the two proteins co-localize to the non-core region of the newly forming nuclear envelope. Depletion of VAPB results in prolonged mitosis, slow progression from meta- to anaphase and in chromosome segregation defects. Together, our results suggest a role of VAPB in mitosis upon recruitment to or release from ELYS at the non-core region of the chromatin in a phosphorylation-dependent manner.
Topics: Mitosis; Humans; Phosphorylation; Protein Binding; HeLa Cells; Chromatin; Transcription Factors; Chromosome Segregation; Nuclear Pore Complex Proteins; Nuclear Envelope; Membrane Proteins; Anaphase
PubMed: 38605278
DOI: 10.1038/s44319-024-00125-6 -
Qualitative rather than quantitative phosphoregulation shapes the end of meiosis I in budding yeast.The EMBO Journal Apr 2024Exit from mitosis is brought about by dramatic changes in the phosphoproteome landscape. A drop in Cyclin-dependent kinase (Cdk) activity, the master regulatory kinase,...
Exit from mitosis is brought about by dramatic changes in the phosphoproteome landscape. A drop in Cyclin-dependent kinase (Cdk) activity, the master regulatory kinase, and activation of counteracting phosphatases such as Cdc14 in budding yeast, results in ordered substrate dephosphorylation, allowing entry into a new cell cycle and replication licensing. In meiosis however, two cell divisions have to be executed without intermediate DNA replication, implying that global phosphorylation and dephosphorylation have to be adapted to the challenges of meiosis. Using a global time-resolved phosphoproteomics approach in budding yeast, we compared the phosphoproteome landscape between mitotic exit and the transition from meiosis I to meiosis II. We found that unlike exit from mitosis, Cdk phosphomotifs remain mostly stably phosphorylated at the end of meiosis I, whereas a majority of Cdk-unrelated motifs are reset by dephosphorylation. However, inducing an artificial drop of Cdk at metaphase of meiosis I leads to ordered substrate dephosphorylation, comparable to mitosis, indicating that phosphoregulation of substrates at the end of meiosis I is thus mainly qualitatively rather than quantitatively ordered.
Topics: Cell Cycle Proteins; Saccharomycetales; Saccharomyces cerevisiae Proteins; Protein Tyrosine Phosphatases; Mitosis; Phosphorylation; Meiosis
PubMed: 38321267
DOI: 10.1038/s44318-024-00032-5 -
The Korean Journal of Physiology &... Sep 2023Mitotic arrest deficient 2 like 2 (Mad2L2, also known as Mad2B), the human homologue of the yeast Rev7 protein, is a regulatory subunit of DNA polymerase ζ that shares...
Mitotic arrest deficient 2 like 2 (Mad2L2, also known as Mad2B), the human homologue of the yeast Rev7 protein, is a regulatory subunit of DNA polymerase ζ that shares high sequence homology with Mad2, the mitotic checkpoint protein. Previously, we demonstrated the involvement of Mad2B in the cisplatin-induced DNA damage response. In this study, we extend our findings to show that Mad2B is recruited to sites of DNA damage in human cancer cells in response to cisplatin treatment. We found that in undamaged cells, Mad2B exists in a complex with Polζ-Rev1 and the APC/C subunit Cdc27. Following cisplatin-induced DNA damage, we observed an increase in the recruitment of Mad2B and Cdc20 (the activators of the APC/C), to the complex. The involvement of Mad2B-Cdc20-APC/C during DNA damage has not been reported before and suggests that the APC/C is activated following cisplatin-induced DNA damage. Using an in vitro ubiquitination assay, our data confirmed Mad2B-dependent activation of APC/C in cisplatin-treated cells. Mad2B may act as an accelerator for APC/C activation during DNA damage response. Our data strongly suggest a role for Mad2B-APC/C-Cdc20 in the ubiquitination of proteins involved in the DNA damage response.
PubMed: 37641805
DOI: 10.4196/kjpp.2023.27.5.427 -
Biochimie Jun 2024Alterations in cell cycle regulation contribute to Zika virus (ZIKV)-associated pathogenesis and may have implications for the development of therapeutic avenues. As a...
Alterations in cell cycle regulation contribute to Zika virus (ZIKV)-associated pathogenesis and may have implications for the development of therapeutic avenues. As a matter of fact, ZIKV alters cell cycle progression at multiple stages, including G1, S, G2, and M phases. During a cell cycle, the progression of mitosis is particularly controlled to avoid any abnormalities in cell division. In this regard, the critical metaphase-anaphase transition is triggered by the activation of anaphase-promoting complex/cyclosome (APC/C) by its E3 ubiquitin ligase subunit Cdc20. Cdc20 recognizes substrates by interacting with a destruction box motif (D-box). Recently, the ZIKV nonstructural protein 5 (NS5), one of the most highly conserved flavivirus proteins, has been shown to localize to the centrosome in each pole and to spindle fibers during mitosis. Inducible expression of NS5 reveals an interaction of this viral factor with centrosomal proteins leading to an increase in the time required to complete mitosis. By analyzing the NS5 sequence, we discovered the presence of a D-box. Taken together, these data support the idea that, in addition to its role in viral replication, NS5 plays a critical role in the control of the cell cycle of infected cells and, more specifically, in the regulation of the mitotic spindle. Here we propose that the NS5 protein may interfere with the metaphase-anaphase progression, and thus cause the observed delay in mitosis via the regulation of APC/C.
Topics: Humans; Anaphase-Promoting Complex-Cyclosome; Cdc20 Proteins; Cell Cycle; Centrosome; Mitosis; Viral Nonstructural Proteins; Virus Replication; Zika Virus; Zika Virus Infection
PubMed: 38307244
DOI: 10.1016/j.biochi.2024.01.016 -
BioRxiv : the Preprint Server For... Aug 2023The cytoplasmic dynein-1 (dynein) motor organizes cells by shaping microtubule networks and moving a large variety of cargoes along them. However, dynein's diverse roles...
The cytoplasmic dynein-1 (dynein) motor organizes cells by shaping microtubule networks and moving a large variety of cargoes along them. However, dynein's diverse roles complicate studies of its functions significantly. To address this issue, we have used gene editing to generate a series of missense mutations in Dynein heavy chain (Dhc). We find that mutations associated with human neurological disease cause a range of defects in larval and adult flies, including impaired cargo trafficking in neurons. We also describe a novel mutation in the microtubule-binding domain (MTBD) of Dhc that, remarkably, causes metaphase arrest of mitotic spindles in the embryo but does not impair other dynein-dependent processes. We demonstrate that the mitotic arrest is independent of dynein's well-established roles in silencing the spindle assembly checkpoint. reconstitution and optical trapping assays reveal that the mutation only impairs the performance of dynein under load. all-atom molecular dynamics simulations show that this effect correlates with increased flexibility of the MTBD, as well as an altered orientation of the stalk domain, with respect to the microtubule. Collectively, our data point to a novel role of dynein in anaphase progression that depends on the motor operating in a specific load regime. More broadly, our work illustrates how cytoskeletal transport processes can be dissected by manipulating mechanical properties of motors.
PubMed: 37577480
DOI: 10.1101/2023.08.03.551815 -
G3 (Bethesda, Md.) Jun 2024Meiosis is a complex variant of the mitotic cell cycle, and as such relies on many of the same proteins involved in mitosis, but utilizes these in novel ways. As in...
Meiosis is a complex variant of the mitotic cell cycle, and as such relies on many of the same proteins involved in mitosis, but utilizes these in novel ways. As in mitosis, Cdk1 and its cyclin partners, Cyclin A, B, and B3 are required at multiple steps in meiosis. Here, we study the effect of stabilized forms of the three mitotic cyclins to study the consequences of failure to degrade the cyclins in meiosis. We find that stabilized Cyclin B3 promotes ectopic microtubule polymerization throughout the egg, dependent on APC/C activity and apparently due to the consequent destruction of Cyclin A and Cyclin B. We present data that suggests CycB, and possibly CycA, can also promote APC/C activity at specific stages of meiosis. We also present evidence that in meiosis APC/CCort and APC/CFzy are able to target Cyclin B via a novel degron. Overall, our findings highlight the distinct functions of the three mitotic Cdk-cyclin complexes in meiosis.
Topics: Animals; Meiosis; Drosophila Proteins; Cyclin B; Mitosis; Cyclins; Cyclin A; Drosophila; Microtubules; Anaphase-Promoting Complex-Cyclosome; Drosophila melanogaster
PubMed: 38551147
DOI: 10.1093/g3journal/jkae066 -
International Journal of Molecular... Oct 2023Rev7 is a regulatory protein with roles in translesion synthesis (TLS), double strand break (DSB) repair, replication fork protection, and cell cycle regulation. Rev7...
Rev7 is a regulatory protein with roles in translesion synthesis (TLS), double strand break (DSB) repair, replication fork protection, and cell cycle regulation. Rev7 forms a homodimer in vitro using its HORMA (Hop, Rev7, Mad2) domain; however, the functional importance of Rev7 dimerization has been incompletely understood. We analyzed the functional properties of cells expressing either wild-type mouse Rev7 or Rev7, a mutant that cannot dimerize. The expression of wild-type Rev7, but not the mutant, rescued the sensitivity of Rev7 cells to X-rays and several alkylating agents and reversed the olaparib resistance phenotype of Rev7 cells. Using a novel fluorescent host-cell reactivation assay, we found that Rev7 is unable to promote gap-filling TLS opposite an abasic site analog. The Rev7 dimerization interface is also required for shieldin function, as both Rev7 cells and Rev7 cells expressing Rev7 exhibit decreased proficiency in rejoining some types of double strand breaks, as well as increased homologous recombination. Interestingly, Rev7 retains some function in cell cycle regulation, as it maintains an interaction with Ras-related nuclear protein (Ran) and partially rescues the formation of micronuclei. The mutant Rev7 also rescues the G2/M accumulation observed in Rev7 cells but does not affect progression through mitosis following nocodazole release. We conclude that while Rev7 dimerization is required for its roles in TLS, DSB repair, and regulation of the anaphase promoting complex, dimerization is at least partially dispensable for promoting mitotic spindle assembly through its interaction with Ran.
Topics: Animals; Mice; Anaphase-Promoting Complex-Cyclosome; DNA Repair; DNA Replication; Mad2 Proteins; Mitosis
PubMed: 37958783
DOI: 10.3390/ijms242115799