bims-micesi Biomed News
on Mitotic cell signalling
Issue of 2023–02–05
twelve papers selected by
Valentina Piano, Uniklinik Köln
  1. Meiotic Cells Escape Prolonged Spindle Checkpoint Activity Through Premature Silencing and Slippage.
  2. CTCF is essential for proper mitotic spindle structure and anaphase segregation.
  3. Multi-site phosphorylation of yeast Mif2/CENP-C promotes inner kinetochore assembly.
  4. Nuclear-enriched protein phosphatase 4 ensures outer kinetochore assembly prior to nuclear dissolution.
  5. Microtubule nucleation from the fibrous corona by LIC1-pericentrin promotes chromosome congression.
  6. Mitotic events depend on regulation of PLK-1 levels by the mitochondrial protein SPD-3.
  7. Single molecule visualization of native centromeric nucleosome formation reveals coordinated deposition by kinetochore proteins and centromere DNA sequence.
  8. Polyploid yeast are dependent on elevated levels of Mps1 for successful chromosome segregation.
  9. Nuclear architecture and the structural basis of mitotic memory.
  10. Cyclin A-CDK1 suppresses the expression of the CDK1 activator CDC25A to safeguard timely mitotic entry.
  11. Repurposing a Cyclin-Dependent Kinase 1 (CDK1) Mitotic Regulatory Network to Complete Terminal Differentiation in Lens Fiber Cells.
  12. Kinase PLK1 regulates the disassembly of the lateral elements and the assembly of the inner centromere during the diakinesis/metaphase I transition in male mouse meiosis.
  1. bioRxiv. 2023 Jan 02. pii: 2023.01.02.522494. [Epub ahead of print]
    Meiotic Cells Escape Prolonged Spindle Checkpoint Activity Through Premature Silencing and Slippage.
    Anne MacKenzie, Victoria Vicory, Soni Lacefield.
      To prevent chromosome mis-segregation, a surveillance mechanism known as the spindle checkpoint delays the cell cycle if kinetochores are not attached to spindle microtubules, allowing the cell additional time to correct improper attachments. During spindle checkpoint activation, checkpoint proteins bind the unattached kinetochore and send a diffusible signal to inhibit the anaphase promoting complex/cyclosome (APC/C). Previous work has shown that mitotic cells with depolymerized microtubules can escape prolonged spindle checkpoint activation in a process called mitotic slippage. During slippage, spindle checkpoint proteins bind unattached kinetochores, but the cells cannot maintain the checkpoint arrest. We asked if meiotic cells had as robust of a spindle checkpoint response as mitotic cells and whether they also undergo slippage after prolonged spindle checkpoint activity. We performed a direct comparison between mitotic and meiotic budding yeast cells that signal the spindle checkpoint due to a lack of either kinetochore-microtubule attachments or due to a loss of tension-bearing attachments. We find that the spindle checkpoint is not as robust in meiosis I or meiosis II compared to mitosis, overcoming a checkpoint arrest approximately 150 minutes earlier in meiosis. In addition, cells in meiosis I escape spindle checkpoint signaling using two mechanisms, silencing the checkpoint at the kinetochore and through slippage. We propose that meiotic cells undertake developmentally-regulated mechanisms to prevent persistent spindle checkpoint activity to ensure the production of gametes.
    AUTHOR SUMMARY: Mitosis and meiosis are the two major types of cell divisions. Mitosis gives rise to genetically identical daughter cells, while meiosis is a reductional division that gives rise to gametes. Cell cycle checkpoints are highly regulated surveillance mechanisms that prevent cell cycle progression when circumstances are unfavorable. The spindle checkpoint promotes faithful chromosome segregation to safeguard against aneuploidy, in which cells have too many or too few chromosomes. The spindle checkpoint is activated at the kinetochore and then diffuses to inhibit cell cycle progression. Although the checkpoint is active in both mitosis and meiosis, most studies involving checkpoint regulation have been performed in mitosis. By activating the spindle checkpoint in both mitosis and meiosis in budding yeast, we show that cells in meiosis elicit a less persistent checkpoint signal compared to cells in mitosis. Further, we show that cells use distinct mechanisms to escape the checkpoint in mitosis and meiosis I. While cells in mitosis and meiosis II undergo anaphase onset while retaining checkpoint proteins at the kinetochore, cells in meiosis I prematurely lose checkpoint protein localization at the kinetochore. If the mechanism to remove the checkpoint components from the kinetochore is disrupted, meiosis I cells can still escape checkpoint activity. Together, these results highlight that cell cycle checkpoints are differentially regulated during meiosis to avoid long delays and to allow gametogenesis.
    DOI:  https://doi.org/10.1101/2023.01.02.522494
  2. bioRxiv. 2023 Jan 10. pii: 2023.01.09.523293. [Epub ahead of print]
    CTCF is essential for proper mitotic spindle structure and anaphase segregation.
    Katherine Chiu, Yasmin Berrada, Nebiyat Eskndir, Dasol Song, Claire Fong, Sarah Naughton, Tina Chen, Savanna Moy, Sarah Gyurmey, Liam James, Chimere Ezeiruaku, Caroline Capistran, Daniel Lowey, Vedang Diwanji, Samantha Peterson, Harshini Parakh, Ayanna R Burgess, Cassandra Probert, Annie Zhu, Bryn Anderson, Nehora Levi, Gabi Gerlitz, Mary C Packard, Katherine A Dorfman, Michael Seifu Bahiru, Andrew D Stephens.
      Mitosis is an essential process in which the duplicated genome is segregated equally into two daughter cells. CTCF has been reported to be present in mitosis but its importance for mitotic fidelity remains to be determined. To evaluate the importance of CTCF in mitosis, we tracked mitotic behaviors in wild type and two different CTCF CRISPR-based genetic knockdowns. We find that knockdown of CTCF results in prolonged mitoses and failed anaphase segregation via time lapse imaging of SiR-DNA. CTCF knockdown did not alter cell cycling or the mitotic checkpoint, which was activated upon nocodazole treatment. Immunofluorescence imaging of the mitotic spindle in CTCF knockdowns revealed disorganization via tri/tetrapolar spindles and chromosomes behind the spindle pole. Imaging of interphase nuclei showed that nuclear size increased drastically, consistent with failure to divide the duplicated genome in anaphase. Population measurements of nuclear shape in CTCF knockdowns do not display decreased circularity or increased nuclear blebbing relative to wild type. However, failed mitoses do display abnormal nuclear morphologies relative to successful mitoses, suggesting population images do not capture individual behaviors. Thus, CTCF is important for both proper metaphase organization and anaphase segregation which impacts the size and shape of the interphase nucleus.
    DOI:  https://doi.org/10.1101/2023.01.09.523293
  3. Curr Biol. 2023 Jan 30. pii: S0960-9822(23)00012-X. [Epub ahead of print]
    Multi-site phosphorylation of yeast Mif2/CENP-C promotes inner kinetochore assembly.
    Stephen M Hinshaw, Yun Quan, Jiaxi Cai, Ann L Zhou, Huilin Zhou.
      Kinetochores control eukaryotic chromosome segregation by connecting chromosomal centromeres to spindle microtubules. Duplication of centromeric DNA necessitates kinetochore disassembly and subsequent reassembly on nascent sisters. To search for a regulatory mechanism that controls the earliest steps of this process, we studied Mif2/CENP-C, an essential basal component of the kinetochore. We found that phosphorylation of a central region of Mif2 (Mif2-PEST) enhances inner kinetochore assembly. Eliminating Mif2-PEST phosphorylation sites progressively impairs cellular fitness. The most severe Mif2-PEST mutations are lethal in cells lacking otherwise non-essential inner kinetochore factors. These data show that multi-site phosphorylation of Mif2/CENP-C controls inner kinetochore assembly.
    Keywords:  cell cycle; centromere; chromosome segregation; kinase; kinetochore; mitosis; phosphorylation
    DOI:  https://doi.org/10.1016/j.cub.2023.01.012
  4. J Cell Biol. 2023 Mar 06. pii: e202208154. [Epub ahead of print]222(3):
    Nuclear-enriched protein phosphatase 4 ensures outer kinetochore assembly prior to nuclear dissolution.
    Helder Rocha, Patrícia A Simões, Jacqueline Budrewicz, Pablo Lara-Gonzalez, Ana Xavier Carvalho, Julien Dumont, Arshad Desai, Reto Gassmann.
      A landmark event in the transition from interphase to mitosis in metazoans is nuclear envelope breakdown (NEBD). Important mitotic events occur prior to NEBD, including condensation of replicated chromosomes and assembly of kinetochores to rapidly engage spindle microtubules. Here, we show that nuclear-enriched protein phosphatase 4 (PP4) ensures robust assembly of the microtubule-coupling outer kinetochore prior to NEBD. In the absence of PP4, chromosomes exhibit extended monopolar orientation after NEBD and subsequently mis-segregate. A secondary consequence of diminished outer kinetochore assembly is defective sister chromatid resolution. After NEBD, a cytoplasmic activity compensates for PP4 loss, leading to outer kinetochore assembly and recovery of chromosomes from monopolar orientation to significant bi-orientation. The Ndc80-Ska microtubule-binding module of the outer kinetochore is required for this recovery. PP4 associates with the inner kinetochore protein CENP-C; however, disrupting the PP4-CENP-C interaction does not perturb chromosome segregation. These results establish that PP4-dependent outer kinetochore assembly prior to NEBD is critical for timely and proper engagement of chromosomes with spindle microtubules.
    DOI:  https://doi.org/10.1083/jcb.202208154
  5. Curr Biol. 2023 Jan 24. pii: S0960-9822(23)00010-6. [Epub ahead of print]
    Microtubule nucleation from the fibrous corona by LIC1-pericentrin promotes chromosome congression.
    Jingchao Wu, Ainhoa Larreategui-Aparicio, Maaike L A Lambers, Dani L Bodor, Sjoerd J Klaasen, Eveline Tollenaar, Marta de Ruijter-Villani, Geert J P L Kops.
      Error-free chromosome segregation in mitosis and meiosis relies on the assembly of a microtubule-based spindle that interacts with kinetochores to guide chromosomes to the cell equator before segregation in anaphase. Microtubules sprout from nucleation sites such as centrosomes, but kinetochores can also promote microtubule formation. It is unclear, however, how kinetochore-derived microtubules are generated and what their role is in chromosome segregation. Here, we show that the transient outer-kinetochore meshwork known as the fibrous corona serves as an autonomous microtubule nucleation platform. The fibrous corona is essential for the nucleation of kinetochore-derived microtubules, and when dissociated from the core kinetochore, it retains microtubule nucleation capacity. Nucleation relies on a fibrous-corona-bound pool of the LIC1 subunit of the dynein motor complex, which interacts with the γ-tubulin-tethering protein pericentrin (PCNT). PCNT is essential for microtubule nucleation from fibrous coronas, and in centrosome-depleted cells, where nearly all mitotic nucleation occurs at fibrous coronas, chromosome congression is fully dependent on PCNT. We further show that chromosomes in bovine oocytes, which naturally lack centrosomes, have highly expanded fibrous coronas that drive chromosome-derived microtubule nucleation. Preventing fibrous corona expansion in these cells impairs chromosome congression and causes spindle assembly defects. Our results show that fibrous coronas are autonomous microtubule-organizing centers that are important for spindle assembly, which may be especially relevant in acentrosomal cells such as oocytes.
    Keywords:  cell division; chromosome; fibrous corona; kinetochore; meiosis; microtubule; microtubule nucleation; mitosis; oocyte; pericentrin
    DOI:  https://doi.org/10.1016/j.cub.2023.01.010
  6. bioRxiv. 2023 Jan 12. pii: 2023.01.11.523633. [Epub ahead of print]
    Mitotic events depend on regulation of PLK-1 levels by the mitochondrial protein SPD-3.
    Yu-Zen Chen, Vitaly Zimyanin, Stefanie Redemann.
      In metazoans, Polo Kinase (Plk1) controls several mitotic events including nuclear envelope breakdown, centrosome maturation and kinetochore assembly. Here we show that mitotic events regulated by Polo Like Kinase (PLK-1) in early C. elegans embryos depend on the mitochondrial-localized protein SPD-3. spd-3 mutant one-cell embryos contain abnormally positioned mitotic chromosomes and prematurely and asymmetrically disassemble the nuclear lamina. Nuclear envelope breakdown (NEBD) in C. elegans requires direct dephosphorylation of lamin by PLK-1. In spd-3 mutants PLK-1 levels are ~6X higher in comparison to control embryos and PLK-1::GFP was highly accumulated at centrosomes, the nuclear envelope, nucleoplasm, and chromosomes prior to NEBD. Partial depletion of plk-1 in spd-3 mutant embryos rescued mitotic chromosome and spindle positioning defects indicating that these phenotypes result from higher PLK-1 levels and thus activity. Our data suggests that the mitochondrial SPD-3 protein controls NEBD and chromosome positioning by regulating the endogenous levels of PLK-1 during early embryogenesis in C. elegans . This finding suggests a novel link between mitochondria and mitotic events by controlling the amount of a key mitotic regulator, PLK-1 and thus may have further implications in the context of cancers or age-related diseases and infertility as it provides a novel link between mitochondria and mitosis.
    DOI:  https://doi.org/10.1101/2023.01.11.523633
  7. bioRxiv. 2023 Jan 21. pii: 2023.01.20.524981. [Epub ahead of print]
    Single molecule visualization of native centromeric nucleosome formation reveals coordinated deposition by kinetochore proteins and centromere DNA sequence.
    Andrew R Popchock, Joshua D Larson, Julien Dubrulle, Charles L Asbury, Sue Biggins.
      Eukaryotic chromosome segregation requires the kinetochore, a conserved megadalton-sized machine that forms on specialized centromeric chromatin containing CENP-A, a histone H3 variant. CENP-A deposition requires a conserved chaperone protein HJURP that targets it to the centromere, but it has remained unclear whether HJURP has additional functions beyond CENP-A targeting and why high AT DNA content, which disfavors nucleosome assembly, is widely conserved at centromeres. To overcome the difficulties of studying nucleosome formation in vivo, we developed a microscopy assay that enables direct observation of de novo centromeric nucleosome recruitment and maintenance at single molecule resolution. Using this assay, we discover that CENP-A can arrive at centromeres without its chaperone, but stable incorporation depends on HJURP and on DNA-binding proteins of the inner kinetochore. We also show that homopolymer AT runs in the yeast centromeres are essential for efficient CENP-A deposition. Together, our findings reveal requirements for stable nucleosome formation and provide a foundation for further studies of the assembly and dynamics of native kinetochore complexes.
    DOI:  https://doi.org/10.1101/2023.01.20.524981
  8. bioRxiv. 2023 Jan 10. pii: 2023.01.09.523325. [Epub ahead of print]
    Polyploid yeast are dependent on elevated levels of Mps1 for successful chromosome segregation.
    Régis E Meyer, Ashlea Sartin, Madeline Gish, Jillian Harsha, Emily Wilkie, Dawson Haworth, Rebecca LaVictoire, Isabel Alberola, Hoa H Chuong, Gary J Gorbsky, Dean S Dawson.
      Tumor cell lines with elevated chromosome numbers frequently have correlated elevations of Mps1 expression and these tumors are more dependent on Mps1 activity for their survival than control cell lines. Mps1 is a conserved kinase involved in controlling aspects of chromosome segregation in mitosis and meiosis. The mechanistic explanation for the Mps1-addiction of aneuploid cells is unknown. To address this question, we explored Mps1-dependence in yeast cells with increased sets of chromosomes. These experiments revealed that in yeast, increasing ploidy leads to delays and failures in orienting chromosomes on the mitotic spindle. Yeast cells with elevated numbers of chromosomes proved vulnerable to reductions of Mps1 activity. Cells with reduced Mps1 activity exhibit an extended prometaphase with longer spindles and delays in orienting the chromosomes. One known role of Mps1 is in recruiting Bub1 to the kinetochore in meiosis. We found that the Mps1-addiction of polyploid yeast cells is due in part to its role in Bub1 recruitment. Together, the experiments presented here demonstrate that increased ploidy renders cells more dependent on Mps1 for orienting chromosomes on the spindle. The phenomenon described here may be relevant in understanding why hyper-diploid cancer cells exhibit elevated reliance on Mps1 expression for successful chromosome segregation.
    Author summary: Losing or gaining chromosomes during cell division leads to aneuploidy (an abnormal number of chromosomes) and can contribute to cancer and other diseases. Indeed, most cells in solid tumors carry abnormally elevated numbers of chromosomes. Mps1 is a regulator of the machinery that distributes chromosomes to daughter cells. In tumors with elevated chromosome numbers the expression of Mps1 is often also elevated. In some aneuploid tumor cell lines these elevated Mps1 levels have been shown to be critical for survival tumor survival. To determine how cells with higher ploidy become dependent on Mps1, we explored Mps1-dependence in yeast cells with increased numbers of chromosomes. We report that yeast cells with elevated chromosome number are sensitive to reductions Mps1 expression. In cells with high ploidy and reduced levels of Mps1, the progression of the cell cycle is delayed and the ability of the cells to properly orient and segregate their chromosomes on the spindle is greatly reduced.
    DOI:  https://doi.org/10.1101/2023.01.09.523325
  9. Chromosome Res. 2023 Feb 02. 31(1): 8
    Nuclear architecture and the structural basis of mitotic memory.
    Mamilla Soujanya, Ashish Bihani, Nikhil Hajirnis, Rashmi U Pathak, Rakesh K Mishra.
      The nucleus is a complex organelle that hosts the genome and is essential for vital processes like DNA replication, DNA repair, transcription, and splicing. The genome is non-randomly organized in the three-dimensional space of the nucleus. This functional sub-compartmentalization was thought to be organized on the framework of nuclear matrix (NuMat), a non-chromatin scaffold that functions as a substratum for various molecular processes of the nucleus. More recently, nuclear bodies or membrane-less subcompartments of the nucleus are thought to arise due to phase separation of chromatin, RNA, and proteins. The nuclear architecture is an amalgamation of the relative organization of chromatin, epigenetic landscape, the nuclear bodies, and the nucleoskeleton in the three-dimensional space of the nucleus. During mitosis, the nucleus undergoes drastic changes in morphology to the degree that it ceases to exist as such; various nuclear components, including the envelope that defines the nucleus, disintegrate, and the chromatin acquires mitosis-specific epigenetic marks and condenses to form chromosome. Upon mitotic exit, chromosomes are decondensed, re-establish hierarchical genome organization, and regain epigenetic and transcriptional status similar to that of the mother cell. How this mitotic memory is inherited during cell division remains a puzzle. NuMat components that are a part of the mitotic chromosome in the form of mitotic chromosome scaffold (MiCS) could potentially be the seeds that guide the relative re-establishment of the epigenome, chromosome territories, and the nuclear bodies. Here, we synthesize the advances towards understanding cellular memory of nuclear architecture across mitosis and propose a hypothesis that a subset of NuMat proteome essential for nucleation of various nuclear bodies are retained in MiCS to serve as seeds of mitotic memory, thus ensuring the daughter cells re-establish the complex status of nuclear architecture similar to that of the mother cells, thereby maintaining the pre-mitotic transcriptional status.
    Keywords:  Mitotic bookmarking; Mitotic chromosome scaffold (MiCS); Mitotic memory; Nuclear architecture; Nuclear bodies; Nuclear matrix (NuMat); Phase separation; Ribonucleoprotein network; Transcriptional memory
    DOI:  https://doi.org/10.1007/s10577-023-09714-y
  10. J Biol Chem. 2023 Jan 27. pii: S0021-9258(23)00089-3. [Epub ahead of print] 102957
    Cyclin A-CDK1 suppresses the expression of the CDK1 activator CDC25A to safeguard timely mitotic entry.
    Lau Yan Ng, Hoi Tang, Randy Y C Poon.
      Cyclin A and CDC25A are both activators of cyclin-dependent kinases (CDKs): cyclin A acts as an activating subunit of CDKs and CDC25A a phosphatase of the inhibitory phosphorylation sites of the CDKs. In this study, we uncovered an inverse relationship between the two CDK activators. As cyclin A is an essential gene, we generated a conditional silencing cell line using a combination of CRISPR-Cas9 and degron-tagged cyclin A. Destruction of cyclin A promoted an acute accumulation of CDC25A. The increase of CDC25A after cyclin A depletion occurred throughout the cell cycle and was independent on cell cycle delay caused by cyclin A deficiency. Moreover, we determined that the inverse relationship with cyclin A was specific for CDC25A and not for other CDC25 family members or kinases that regulate the same sites in CDKs. Unexpectedly, the upregulation of CDC25A was mainly caused by an increase in transcriptional activity instead of a change in the stability of the protein. Reversing the accumulation of CDC25A severely delayed G2-M in cyclin A-depleted cells. Taken togther, these data provide evidence of a compensatory mechanism involving CDC25A that ensures timely mitotic entry at different levels of cyclin A.
    Keywords:  cell cycle; cyclin-dependent kinases; mitosis; phosphatase
    DOI:  https://doi.org/10.1016/j.jbc.2023.102957
  11. Invest Ophthalmol Vis Sci. 2023 Feb 01. 64(2): 6
    Repurposing a Cyclin-Dependent Kinase 1 (CDK1) Mitotic Regulatory Network to Complete Terminal Differentiation in Lens Fiber Cells.
    Allen Taylor, Yumei Gu, Min-Lee Chang, Wenxin Yang, Sarah Francisco, Sheldon Rowan, Eloy Bejarano, Steven Pruitt, Liang Zhu, Grant Weiss, Lisa Brennan, Marc Kantorow, Elizabeth A Whitcomb.
       Purpose: During lens fiber cell differentiation, organelles are removed in an ordered manner to ensure lens clarity. A critical step in this process is removal of the cell nucleus, but the mechanisms by which this occurs are unclear. In this study, we investigate the role of a cyclin-dependent kinase 1 (CDK1) regulatory loop in controlling lens fiber cell denucleation (LFCD).
    Methods: We examined lens differentiation histologically in two different vertebrate models. An embryonic chick lens culture system was used to test the role of CDK1, cell division cycle 25 (CDC25), WEE1, and PP2A in LFCD. Additionally, we used three mouse models that express high levels of the CDK inhibitor p27 to test whether increased p27 levels affect LFCD.
    Results: Using chick lens organ cultures, small-molecule inhibitors of CDK1 and CDC25 inhibit LFCD, while inhibiting the CDK1 inhibitory kinase WEE1 potentiates LFCD. Additionally, treatment with an inhibitor of PP2A, which indirectly inhibits CDK1 activity, also increased LFCD. Three different mouse models that express increased levels of p27 through different mechanisms show impaired LFCD.
    Conclusions: Here we define a conserved nonmitotic role for CDK1 and its upstream regulators in controlling LFCD. We find that CDK1 functionally interacts with WEE1, a nuclear kinase that inhibits CDK1 activity, and CDC25 activating phosphatases in cells where CDK1 activity must be exquisitely regulated to allow for LFCD. We also provide genetic evidence in multiple in vivo models that p27, a CDK1 inhibitor, inhibits lens growth and LFCD.
    DOI:  https://doi.org/10.1167/iovs.64.2.6
  12. Front Cell Dev Biol. 2022 ;10 1069946
    Kinase PLK1 regulates the disassembly of the lateral elements and the assembly of the inner centromere during the diakinesis/metaphase I transition in male mouse meiosis.
    Rocío Gómez, Alberto Viera, Tania Moreno-Mármol, Inés Berenguer, Andrea Guajardo-Grence, Attila Tóth, María Teresa Parra, José A Suja.
      PLK1 is a serine/threonine kinase with crucial roles during mitosis. However, its involvement during mammalian male meiosis remains largely unexplored. By inhibiting the kinase activity of PLK1 using BI 2536 on organotypic cultures of seminiferous tubules, we found that the disassembly of SYCP3 and HORMAD1 from the lateral elements of the synaptonemal complex during diakinesis is impeded. We also found that the normal recruitment of SYCP3 and HORMAD1 to the inner centromere in prometaphase I spermatocytes did not occur. Additionally, we analyzed the participation of PLK1 in the assembly of the inner centromere by studying its implication in the Bub1-H2AT120ph-dependent recruitment of shugoshin SGO2, and the Haspin-H3T3ph-dependent recruitment of Aurora B/C and Borealin. Our results indicated that both pathways are regulated by PLK1. Altogether, our results demonstrate that PLK1 is a master regulator of the late prophase I/metaphase I transition in mouse spermatocytes.
    Keywords:  H2AT120ph; H3T3ph; PLK1; inner centromere; lateral elements; meiosis; mouse
    DOI:  https://doi.org/10.3389/fcell.2022.1069946
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