bims-micesi Biomed News
on Mitotic cell signalling
Issue of 2022–02–06
eleven papers selected by
Valentina Piano, Max Planck Institute of Molecular Physiology
  1. Non-centrosomal microtubules at kinetochores promote rapid chromosome biorientation during mitosis in human cells.
  2. Plasticity in centromere organization and kinetochore composition: Lessons from diversity.
  3. A monoastral mitotic spindle determines lineage fate and position in the mouse embryo.
  4. Cyclin B/CDK1 and Cyclin A/CDK2 phosphorylate DENR to promote mitotic protein translation and faithful cell division.
  5. Live-cell Imaging and Analysis of Germline Stem Cell Mitosis in Caenorhabditis elegans.
  6. Drosophila centrocortin is dispensable for centriole duplication but contributes to centrosome separation.
  7. TubulinTracker, a Novel In Vitro Reporter Assay to Study Intracellular Microtubule Dynamics, Cell Cycle Progression, and Aneugenicity.
  8. Overexpression of SKA Complex Is Associated With Poor Prognosis in Gliomas.
  9. Atomic force microscopy reveals distinct protofilament-scale structural dynamics in depolymerizing microtubule arrays.
  10. Lysate-based pipeline to characterize microtubule-associated proteins uncovers unique microtubule behaviours.
  11. In vitro cell cycle oscillations exhibit a robust and hysteretic response to changes in cytoplasmic density.
  1. Curr Biol. 2022 Jan 26. pii: S0960-9822(22)00024-0. [Epub ahead of print]
    Non-centrosomal microtubules at kinetochores promote rapid chromosome biorientation during mitosis in human cells.
    Fioranna Renda, Christopher Miles, Irina Tikhonenko, Rebecca Fisher, Lina Carlini, Tarun M Kapoor, Alex Mogilner, Alexey Khodjakov.
      Proper segregation of chromosomes during mitosis depends on "amphitelic attachments"-load-bearing connections of sister kinetochores to the opposite spindle poles via bundles of microtubules, termed as the "K-fibers." Current models of spindle assembly assume that K-fibers arise largely from stochastic capture of microtubules, which occurs at random times and locations and independently at sister kinetochores. We test this assumption by following the movements of all kinetochores in human cells and determine that most amphitelic attachments form synchronously at a specific stage of spindle assembly and within a spatially distinct domain. This biorientation domain is enriched in bundles of antiparallel microtubules, and perturbation of microtubule bundling changes the temporal and spatial dynamics of amphitelic attachment formation. Structural analyses indicate that interactions of kinetochores with microtubule bundles are mediated by non-centrosomal short microtubules that emanate from most kinetochores during early prometaphase. Computational analyses suggest that momentous molecular motor-driven interactions with antiparallel bundles rapidly convert these short microtubules into nascent K-fibers. Thus, load-bearing connections to the opposite spindle poles form simultaneously on sister kinetochores. In contrast to the uncoordinated sequential attachments of sister kinetochores expected in stochastic models of spindle assembly, our model envisions the formation of amphitelic attachments as a deterministic process in which the chromosomes connect with the spindle poles synchronously at a specific stage of spindle assembly and at a defined location determined by the spindle architecture. Experimental analyses of changes in the kinetochore behavior in cells with perturbed activity of molecular motors CenpE and dynein confirm the predictive power of the model.
    Keywords:  agent-based simulation; chromosome segregation; computational model; kinetochore; mitosis; spindle assembly
    DOI:  https://doi.org/10.1016/j.cub.2022.01.013
  2. Curr Opin Cell Biol. 2022 Jan 30. pii: S0955-0674(22)00001-1. [Epub ahead of print]74 47-54
    Plasticity in centromere organization and kinetochore composition: Lessons from diversity.
    Midori Ishii, Bungo Akiyoshi.
      Kinetochores are the macromolecular protein complexes that govern chromosome movement by binding spindle microtubules during mitosis and meiosis. Centromeres are the specific chromosomal regions that serve as the platform on which kinetochores assemble. Despite their essentiality for proper chromosome segregation, the size and organization of centromeres vary dramatically between species, while different compositions of kinetochores are found among eukaryotes. Here we discuss recent progress in understanding centromeres and kinetochores in non-traditional model eukaryotes. We specifically focus on select lineages (holocentric insects, early diverging fungi, and kinetoplastids) that lack CENP-A, a centromere-specific histone H3 variant that is critical for kinetochore specification and assembly in many eukaryotes. We also highlight some organisms that might have hitherto unknown types of kinetochore proteins.
    DOI:  https://doi.org/10.1016/j.ceb.2021.12.007
  3. Nat Cell Biol. 2022 Jan 31.
    A monoastral mitotic spindle determines lineage fate and position in the mouse embryo.
    Oz Pomp, Hui Yi Grace Lim, Robin M Skory, Adam A Moverley, Piotr Tetlak, Stephanie Bissiere, Nicolas Plachta.
      During mammalian development, the first asymmetric cell divisions segregate cells into inner and outer positions of the embryo to establish the pluripotent and trophectoderm lineages. Typically, polarity components differentially regulate the mitotic spindle via astral microtubule arrays to trigger asymmetric division patterns. However, early mouse embryos lack centrosomes, the microtubule-organizing centres (MTOCs) that usually generate microtubule asters. Thus, it remains unknown whether spindle organization regulates lineage segregation. Here we find that heterogeneities in cell polarity in the early 8-cell-stage mouse embryo trigger the assembly of a highly asymmetric spindle organization. This spindle arises in an unusual modular manner, forming a single microtubule aster from an apically localized, non-centrosomal MTOC, before joining it to the rest of the spindle apparatus. When fully assembled, this 'monoastral' spindle triggers spatially asymmetric division patterns to segregate cells into inner and outer positions. Moreover, the asymmetric inheritance of spindle components causes differential cell polarization to determine pluripotent versus trophectoderm lineage fate.
    DOI:  https://doi.org/10.1038/s41556-021-00826-3
  4. Nat Commun. 2022 Feb 03. 13(1): 668
    Cyclin B/CDK1 and Cyclin A/CDK2 phosphorylate DENR to promote mitotic protein translation and faithful cell division.
    Katharina Clemm von Hohenberg, Sandra Müller, Sibylle Schleich, Matthias Meister, Jonathan Bohlen, Thomas G Hofmann, Aurelio A Teleman.
      DENR and MCTS1 have been identified as oncogenes in several different tumor entities. The heterodimeric DENR·MCTS1 protein complex promotes translation of mRNAs containing upstream Open Reading Frames (uORFs). We show here that DENR is phosphorylated on Serine 73 by Cyclin B/CDK1 and Cyclin A/CDK2 at the onset of mitosis, and then dephosphorylated as cells exit mitosis. Phosphorylation of Ser73 promotes mitotic stability of DENR protein and prevents its cleavage at Asp26. This leads to enhanced translation of mRNAs involved in mitosis. Indeed, we find that roughly 40% of all mRNAs with elevated translation in mitosis are DENR targets. In the absence of DENR or of Ser73 phosphorylation, cells display elevated levels of aberrant mitoses and cell death. This provides a mechanism how the cell cycle regulates translation of a subset of mitotically relevant mRNAs during mitosis.
    DOI:  https://doi.org/10.1038/s41467-022-28265-0
  5. Bio Protoc. 2022 Jan 05. 12(1): e4272
    Live-cell Imaging and Analysis of Germline Stem Cell Mitosis in Caenorhabditis elegans.
    Réda M Zellag, Yifan Zhao, Abigail R Gerhold.
      Model organisms offer the opportunity to decipher the dynamic and complex behavior of stem cells in their native environment; however, imaging stem cells in situ remains technically challenging. C. elegans germline stem cells (GSCs) are distinctly accessible for in situ live imaging but relatively few studies have taken advantage of this potential. Here we provide our protocol for mounting and live imaging dividing C. elegans GSCs, as well as analysis tools to facilitate the processing of large datasets. While the present protocol was optimized for imaging and analyzing mitotic GSCs, it can easily be adapted to visualize dividing cells or other subcellular processes in C. elegans at multiple developmental stages. Our image analysis pipeline can also be used to analyze mitosis in other cell types and model organisms.
    Keywords:  C. elegans; Germline; Live cell and tissue imaging; Mitosis; Spindle dynamics; Stem cells
    DOI:  https://doi.org/10.21769/BioProtoc.4272
  6. G3 (Bethesda). 2021 Dec 23. pii: jkab434. [Epub ahead of print]
    Drosophila centrocortin is dispensable for centriole duplication but contributes to centrosome separation.
    Dipen S Mehta, Hala Zein-Sabatto, Pearl V Ryder, Jina Lee, Dorothy A Lerit.
      Centrosomes are microtubule-organizing centers that duplicate exactly once to organize the bipolar mitotic spindle required for error-free mitosis. Prior work indicated that Drosophila centrocortin (cen) is required for normal centrosome separation, although a role in centriole duplication was not closely examined. Through time-lapse recordings of rapid syncytial divisions, we monitored centriole duplication and the kinetics of centrosome separation in control vs cen null embryos. Our data suggest that although cen is dispensable for centriole duplication, it contributes to centrosome separation.
    Keywords:  RNA localization; centrocortin; centrosome; mitosis
    DOI:  https://doi.org/10.1093/g3journal/jkab434
  7. Toxicol Sci. 2022 Jan 30. pii: kfac008. [Epub ahead of print]
    TubulinTracker, a Novel In Vitro Reporter Assay to Study Intracellular Microtubule Dynamics, Cell Cycle Progression, and Aneugenicity.
    M E Geijer, N Moelijker, G Zhang, R Derr, T Osterlund, G Hendriks, I Brandsma.
      Aneuploidy is characterized by the presence of an abnormal number of chromosomes and is a common hallmark of cancer. However, exposure to aneugenic compounds does not necessarily lead to cancer. Aneugenic compounds are mainly identified using the in vitro micronucleus assay but this assay cannot standardly discriminate between aneugens and clastogens and cannot be used to identify the exact mode-of-action (MOA) of aneugens; tubulin stabilization, tubulin destabilization, or inhibition of mitotic kinases. To improve the classification of aneugenic substances and determine their MOA, we developed and validated the TubulinTracker assay that uses a GFP-tagged tubulin reporter cell line to study microtubule stability using flow cytometry. Combining the assay with a DNA stain also enables cell cycle analysis. Substances whose exposure resulted in an accumulation of cells in G2/M phase, combined with increased or decreased tubulin levels, were classified as tubulin poisons. All known tubulin poisons included were classified correctly. Moreover, we correctly classified compounds, including aneugens, that did not affect microtubule levels. However, the MOA of aneugens not affecting tubulin stability, such as Aurora kinase inhibitors, could not be identified. Here we show that the TubulinTracker assay can be used to classify microtubule stabilizing and destabilizing compounds in living cells. This insight into the MOA of aneugenic agents is important, for example to support a weight-of-evidence approach for risk assessment, and the classification as an aneugen as opposed to a clastogen or mutagen, has a big impact on the assessment.
    Keywords:  Aneugenicity; Genotoxicity; Genotoxicity testing; Microtubules; Tubulin
    DOI:  https://doi.org/10.1093/toxsci/kfac008
  8. Front Neurol. 2021 ;12 755681
    Overexpression of SKA Complex Is Associated With Poor Prognosis in Gliomas.
    Shoukai Yu.
      The spindle and kinetochore-associated complex is composed of three members: SKA1, SKA2, and SKA3. It is necessary for stabilizing spindle microtubules attaching to kinetochore (KT) in the middle stage of mitosis. The SKA complex is associated with poor prognosis in several human cancers. However, the role of SKA complex in rare malignant diseases, such as gliomas, has not been fully investigated. We investigated several databases, including Oncomine, UALCAN, and cBioPortal to explore the expression profile and prognostic significance of SKA complex in patients with gliomas. Gene ontology and Kyoto Encyclopedia of Genes and Genome pathways were used to analyze the potential enriched pathways. The genes co-expressed with SKA complex were identified and used for developing a protein-protein interaction (PPI) network using the STRING database. We found a significant overexpression of the mRNA levels of SKA1, SKA2, and SKA3 in patients with glioma patients. Higher expression of SKA1 and SKA3, but not SKA2, was significantly correlated with shorter overall survival of patients with glioma. In glioma, SKA complex was found to be involved in nuclear division, chromosome segregation, and DNA replication. The results of PPI network identified 10 hub genes (CCNB2, UBE2C, BUB1B, TPX2, CCNA2, CCNB1, MELK, TOP2A, PBK, and KIF11), all of which were overexpressed and negatively associated with prognosis of patients with glioma. In conclusion, our study sheds new insights into the biological role and prognostic significance of SKA complex in glioma.
    Keywords:  SKA complex; cell cycle; gliomas; prognosis; rare malignant tumor
    DOI:  https://doi.org/10.3389/fneur.2021.755681
  9. Proc Natl Acad Sci U S A. 2022 Feb 01. pii: e2115708119. [Epub ahead of print]119(5):
    Atomic force microscopy reveals distinct protofilament-scale structural dynamics in depolymerizing microtubule arrays.
    Sithara S Wijeratne, Michelle F Marchan, Jason S Tresback, Radhika Subramanian.
      The dynamic reorganization of microtubule-based cellular structures, such as the spindle and the axoneme, fundamentally depends on the dynamics of individual polymers within multimicrotubule arrays. A major class of enzymes implicated in both the complete demolition and fine size control of microtubule-based arrays are depolymerizing kinesins. How different depolymerases differently remodel microtubule arrays is poorly understood. A major technical challenge in addressing this question is that existing optical or electron-microscopy methods lack the spatial-temporal resolution to observe the dynamics of individual microtubules within larger arrays. Here, we use atomic force microscopy (AFM) to image depolymerizing arrays at single-microtubule and protofilament resolution. We discover previously unseen modes of microtubule array destabilization by conserved depolymerases. We find that the kinesin-13 MCAK mediates asynchronous protofilament depolymerization and lattice-defect propagation, whereas the kinesin-8 Kip3p promotes synchronous protofilament depolymerization. Unexpectedly, MCAK can depolymerize the highly stable axonemal doublets, but Kip3p cannot. We propose that distinct protofilament-level activities underlie the functional dichotomy of depolymerases, resulting in either large-scale destabilization or length regulation of microtubule arrays. Our work establishes AFM as a powerful strategy to visualize microtubule dynamics within arrays and reveals how nanometer-scale substrate specificity leads to differential remodeling of micron-scale cytoskeletal structures.
    Keywords:  atomic force microscopy; axoneme; cytoskeleton; kinesin; microtubule arrays
    DOI:  https://doi.org/10.1073/pnas.2115708119
  10. Nat Cell Biol. 2022 Jan 31.
    Lysate-based pipeline to characterize microtubule-associated proteins uncovers unique microtubule behaviours.
    A S Jijumon, Satish Bodakuntla, Mariya Genova, Mamata Bangera, Violet Sackett, Laetitia Besse, Fatlinda Maksut, Veronique Henriot, Maria M Magiera, Minhajuddin Sirajuddin, Carsten Janke.
      The microtubule cytoskeleton forms complex macromolecular assemblies with a range of microtubule-associated proteins (MAPs) that have fundamental roles in cell architecture, division and motility. Determining how an individual MAP modulates microtubule behaviour is an important step in understanding the physiological roles of various microtubule assemblies. To characterize how MAPs control microtubule properties and functions, we developed an approach allowing for medium-throughput analyses of MAPs in cell-free conditions using lysates of mammalian cells. Our pipeline allows for quantitative as well as ultrastructural analyses of microtubule-MAP assemblies. Analysing 45 bona fide and potential mammalian MAPs, we uncovered previously unknown activities that lead to distinct and unique microtubule behaviours such as microtubule coiling or hook formation, or liquid-liquid phase separation along the microtubule lattice that initiates microtubule branching. We have thus established a powerful tool for a thorough characterization of a wide range of MAPs and MAP variants, thus opening avenues for the determination of mechanisms underlying their physiological roles and pathological implications.
    DOI:  https://doi.org/10.1038/s41556-021-00825-4
  11. Proc Natl Acad Sci U S A. 2022 Feb 08. pii: e2109547119. [Epub ahead of print]119(6):
    In vitro cell cycle oscillations exhibit a robust and hysteretic response to changes in cytoplasmic density.
    Minjun Jin, Franco Tavella, Shiyuan Wang, Qiong Yang.
      Cells control the properties of the cytoplasm to ensure proper functioning of biochemical processes. Recent studies showed that cytoplasmic density varies in both physiological and pathological states of cells undergoing growth, division, differentiation, apoptosis, senescence, and metabolic starvation. Little is known about how cellular processes cope with these cytoplasmic variations. Here, we study how a cell cycle oscillator comprising cyclin-dependent kinase (Cdk1) responds to changes in cytoplasmic density by systematically diluting or concentrating cycling Xenopus egg extracts in cell-like microfluidic droplets. We found that the cell cycle maintains robust oscillations over a wide range of deviations from the endogenous density: as low as 0.2× to more than 1.22× relative cytoplasmic density (RCD). A further dilution or concentration from these values arrested the system in a low or high steady state of Cdk1 activity, respectively. Interestingly, diluting an arrested cytoplasm of 1.22× RCD recovers oscillations at lower than 1× RCD. Thus, the cell cycle switches reversibly between oscillatory and stable steady states at distinct thresholds depending on the direction of tuning, forming a hysteresis loop. We propose a mathematical model which recapitulates these observations and predicts that the Cdk1/Wee1/Cdc25 positive feedback loops do not contribute to the observed robustness, supported by experiments. Our system can be applied to study how cytoplasmic density affects other cellular processes.
    Keywords:  cell cycle oscillator; cytoplasmic density; hysteresis; macromolecular crowding; robustness
    DOI:  https://doi.org/10.1073/pnas.2109547119
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