bims-micesi Biomed News
on Mitotic cell signalling
Issue of 2022‒08‒14
eleven papers selected by
Valentina Piano
Max Planck Institute of Molecular Physiology
  1. The Roles of Histone Post-Translational Modifications in the Formation and Function of a Mitotic Chromosome.
  2. Biochemical, biophysical, and functional characterisation of the E3 ubiquitin ligase APC/C regulator CDC20 from Arabidopsis thaliana.
  3. Proximity Labeling Reveals Spatial Regulation of the Anaphase-Promoting Complex/Cyclosome by a Microtubule Adaptor.
  4. Banp regulates DNA damage response and chromosome segregation during the cell cycle in zebrafish retina.
  5. Evidence for a HURP/EB free mixed-nucleotide zone in kinetochore-microtubules.
  6. Duplication and Segregation of Centrosomes during Cell Division.
  7. Timely Schwann cell division drives peripheral myelination in vivo via Laminin/cAMP pathway.
  8. Antibody for Serine 65 Phosphorylated Ubiquitin Identifies PLK1-Mediated Phosphorylation of Mitotic Proteins and APC1.
  9. Damage-induced regeneration of the intestinal stem cell pool through enteroblast mitosis in the Drosophila midgut.
  10. CDC20-Mediated hnRNPU Ubiquitination Regulates Chromatin Condensation and Anti-Cancer Drug Response.
  11. The vacuole shapes the nucleus and the ribosomal DNA loop during mitotic delays.
  1. Int J Mol Sci. 2022 Aug 05. pii: 8704. [Epub ahead of print]23(15):
    The Roles of Histone Post-Translational Modifications in the Formation and Function of a Mitotic Chromosome.
    Marco A Andonegui-Elguera, Rodrigo E Cáceres-Gutiérrez, Alejandro López-Saavedra, Fernanda Cisneros-Soberanis, Montserrat Justo-Garrido, José Díaz-Chávez, Luis A Herrera.
      During mitosis, many cellular structures are organized to segregate the replicated genome to the daughter cells. Chromatin is condensed to shape a mitotic chromosome. A multiprotein complex known as kinetochore is organized on a specific region of each chromosome, the centromere, which is defined by the presence of a histone H3 variant called CENP-A. The cytoskeleton is re-arranged to give rise to the mitotic spindle that binds to kinetochores and leads to the movement of chromosomes. How chromatin regulates different activities during mitosis is not well known. The role of histone post-translational modifications (HPTMs) in mitosis has been recently revealed. Specific HPTMs participate in local compaction during chromosome condensation. On the other hand, HPTMs are involved in CENP-A incorporation in the centromere region, an essential activity to maintain centromere identity. HPTMs also participate in the formation of regulatory protein complexes, such as the chromosomal passenger complex (CPC) and the spindle assembly checkpoint (SAC). Finally, we discuss how HPTMs can be modified by environmental factors and the possible consequences on chromosome segregation and genome stability.
    Keywords:  arsenic; centromere; chromosome condensation; histones; kinetochore; mitosis; nickel
    DOI:  https://doi.org/10.3390/ijms23158704
  2. Front Physiol. 2022 ;13 938688
    Biochemical, biophysical, and functional characterisation of the E3 ubiquitin ligase APC/C regulator CDC20 from Arabidopsis thaliana.
    Maria-Alexa Cosma, Natalie L Curtis, Charlotte Pain, Verena Kriechbaumer, Victor M Bolanos-Garcia.
      The Anaphase Promoting Complex (APC/C), a large cullin-RING E3-type ubiquitin ligase, constitutes the ultimate target of the Spindle Assembly Checkpoint (SAC), an intricate regulatory circuit that ensures the high fidelity of chromosome segregation in eukaryotic organisms by delaying the onset of anaphase until each chromosome is properly bi-oriented on the mitotic spindle. Cell-division cycle protein 20 homologue (CDC20) is a key regulator of APC/C function in mitosis. The formation of the APC/CCDC20 complex is required for the ubiquitination and degradation of select substrates, which is necessary to maintain the mitotic state. In contrast to the roles of CDC20 in animal species, little is known about CDC20 roles in the regulation of chromosome segregation in plants. Here we address this gap in knowledge and report the expression in insect cells; the biochemical and biophysical characterisation of Arabidopsis thaliana (AtCDC20) WD40 domain; and the nuclear and cytoplasmic distribution of full-length AtCDC20 when transiently expressed in tobacco plants. We also show that most AtCDC20 degrons share a high sequence similarity to other eukaryotes, arguing in favour of conserved degron functions in AtCDC20. However, important exceptions were noted such as the lack of a canonical MAD1 binding motif; a fully conserved RRY-box in all six AtCDC20 isoforms instead of a CRY-box motif, and low conservation of key residues known to be phosphorylated by BUB1 and PLK1 in other species to ensure a robust SAC response. Taken together, our studies provide insights into AtCDC20 structure and function and the evolution of SAC signalling in plants.
    Keywords:  APC/C regulation; Arabidopsis thaliana; CDC20; Spindle Assembly Checkpoint (SAC); cell division; chromosome segregation; mitosis
    DOI:  https://doi.org/10.3389/fphys.2022.938688
  3. ACS Chem Biol. 2022 Aug 11.
    Proximity Labeling Reveals Spatial Regulation of the Anaphase-Promoting Complex/Cyclosome by a Microtubule Adaptor.
    Xiaofu Cao, Adnan Shami Shah, Ethan J Sanford, Marcus B Smolka, Jeremy M Baskin.
      The anaphase-promoting complex/cyclosome (APC/C) coordinates advancement through mitosis via temporally controlled polyubiquitination events. Despite the long-appreciated spatial organization of key events in mitosis mediated largely by cytoskeletal networks, the spatial regulation of APC/C, the major mitotic E3 ligase, is poorly understood. We describe a microtubule-resident protein, PLEKHA5, as an interactor of APC/C and spatial regulator of its activity in mitosis. Microtubule-localized proximity biotinylation tools revealed that PLEKHA5 depletion decreased APC/C association with the microtubule cytoskeleton, which prevented efficient loading of APC/C with its coactivator CDC20 and led to reduced APC/C E3 ligase activity. PLEKHA5 knockdown delayed mitotic progression, causing accumulation of APC/C substrates dependent upon the PLEKHA5-APC/C interaction in microtubules. We propose that PLEKHA5 functions as an adaptor of APC/C that promotes its subcellular localization to microtubules and facilitates its activation by CDC20, thus ensuring the timely turnover of key mitotic APC/C substrates and proper progression through mitosis.
    DOI:  https://doi.org/10.1021/acschembio.2c00527
  4. Elife. 2022 Aug 09. pii: e74611. [Epub ahead of print]11
    Banp regulates DNA damage response and chromosome segregation during the cell cycle in zebrafish retina.
    Swathy Babu, Yuki Takeuchi, Ichiro Masai.
      Btg3-associated nuclear protein (Banp) was originally identified as a nuclear matrix-associated region (MAR)-binding protein and it functions as a tumor suppressor. At the molecular level, Banp regulates transcription of metabolic genes via a CGCG-containing motif called the Banp motif. However, its physiological roles in embryonic development are unknown. Here, we report that Banp is indispensable for the DNA damage response and chromosome segregation during mitosis. Zebrafish banp mutants show mitotic cell accumulation and apoptosis in developing retina. We found that DNA replication stress and tp53-dependent DNA damage responses were activated to induce apoptosis in banp mutants, suggesting that Banp is required for regulation of DNA replication and DNA damage repair. Furthermore, consistent with mitotic cell accumulation, chromosome segregation was not smoothly processed from prometaphase to anaphase in banp morphants, leading to a prolonged M-phase. Our RNA- and ATAC-sequencing identified 31 candidates for direct Banp target genes that carry the Banp motif. Interestingly, a DNA replication fork regulator, wrnip1, and two chromosome segregation regulators, cenpt and ncapg, are included in this list. Thus, Banp directly regulates transcription of wrnip1 for recovery from DNA replication stress, and cenpt and ncapg for chromosome segregation during mitosis. Our findings provide the first in vivo evidence that Banp is required for cell-cycle progression and cell survival by regulating DNA damage responses and chromosome segregation during mitosis.
    Keywords:  Banp; DNA damage; cell biology; cenpt; chromosome segregation; developmental biology; ncapg; tp53; zebrafish
    DOI:  https://doi.org/10.7554/eLife.74611
  5. Nat Commun. 2022 Aug 10. 13(1): 4704
    Evidence for a HURP/EB free mixed-nucleotide zone in kinetochore-microtubules.
    Cédric Castrogiovanni, Alessio V Inchingolo, Jonathan U Harrison, Damian Dudka, Onur Sen, Nigel J Burroughs, Andrew D McAinsh, Patrick Meraldi.
      Current models infer that the microtubule-based mitotic spindle is built from GDP-tubulin with small GTP caps at microtubule plus-ends, including those that attach to kinetochores, forming the kinetochore-fibres. Here we reveal that kinetochore-fibres additionally contain a dynamic mixed-nucleotide zone that reaches several microns in length. This zone becomes visible in cells expressing fluorescently labelled end-binding proteins, a known marker for GTP-tubulin, and endogenously-labelled HURP - a protein which we show to preferentially bind the GDP microtubule lattice in vitro and in vivo. We find that in mitotic cells HURP accumulates on the kinetochore-proximal region of depolymerising kinetochore-fibres, whilst avoiding recruitment to nascent polymerising K-fibres, giving rise to a growing "HURP-gap". The absence of end-binding proteins in the HURP-gaps leads us to postulate that they reflect a mixed-nucleotide zone. We generate a minimal quantitative model based on the preferential binding of HURP to GDP-tubulin to show that such a mixed-nucleotide zone is sufficient to recapitulate the observed in vivo dynamics of HURP-gaps.
    DOI:  https://doi.org/10.1038/s41467-022-32421-x
  6. Cells. 2022 Aug 07. pii: 2445. [Epub ahead of print]11(15):
    Duplication and Segregation of Centrosomes during Cell Division.
    Claude Prigent, Rustem Uzbekov.
      During its division the cell must ensure the equal distribution of its genetic material in the two newly created cells, but it must also distribute organelles such as the Golgi apparatus, the mitochondria and the centrosome. DNA, the carrier of heredity, located in the nucleus of the cell, has made it possible to define the main principles that regulate the progression of the cell cycle. The cell cycle, which includes interphase and mitosis, is essentially a nuclear cycle, or a DNA cycle, since the interphase stages names (G1, S, G2) phases are based on processes that occur exclusively with DNA. However, centrosome duplication and segregation are two equally important events for the two new cells that must inherit a single centrosome. The centrosome, long considered the center of the cell, is made up of two small cylinders, the centrioles, made up of microtubules modified to acquire a very high stability. It is the main nucleation center of microtubules in the cell. Apart from a few exceptions, each cell in G1 phase has only one centrosome, consisting in of two centrioles and pericentriolar materials (PCM), which must be duplicated before the cell divides so that the two new cells formed inherit a single centrosome. The centriole is also the origin of the primary cilia, motile cilia and flagella of some cells.
    Keywords:  cell cycle; centriole; centrosome; checkpoint; regulation
    DOI:  https://doi.org/10.3390/cells11152445
  7. Development. 2022 Aug 08. pii: dev.200640. [Epub ahead of print]
    Timely Schwann cell division drives peripheral myelination in vivo via Laminin/cAMP pathway.
    Aya Mikdache, Marie-José Boueid, Emilie Lesport, Brigitte Delespierre, Julien Loisel-Duwattez, Cindy Degerny, Marcel Tawk.
      Schwann cells (SC) migrate along peripheral axons and divide intensively to generate the right number of cells prior to axonal ensheathment; however, little is known regarding the temporal and molecular control of their division and its impact on myelination. We report that Sil, a spindle pole protein associated with autosomal recessive primary microcephaly (MCPH), is required for temporal mitotic exit of SC. In sil-deficient cassiopeia (csp-/-) mutants, SC fail to radially sort and myelinate peripheral axons. Elevation of cAMP, but not Rac1 activity in csp-/- restores myelin ensheathment. Most importantly, we show a significant decrease in Laminin expression within csp-/- posterior lateral line nerve and that forcing Laminin2 expression in csp-/- fully restores SC ability to myelinate. Thus, we unravel a novel and essential role for timely SC division in mediating Laminin expression to orchestrate radial sorting and peripheral myelination in vivo.
    Keywords:  Laminin/cAMP; MCPH; Mitotic spindle; Myelin; Schwann cells; Zebrafish
    DOI:  https://doi.org/10.1242/dev.200640
  8. Molecules. 2022 Jul 29. pii: 4867. [Epub ahead of print]27(15):
    Antibody for Serine 65 Phosphorylated Ubiquitin Identifies PLK1-Mediated Phosphorylation of Mitotic Proteins and APC1.
    Guy Mann, Prasad Sulkshane, Pradeep Sadhu, Tamar Ziv, Michael H Glickman, Ashraf Brik.
      Deciphering the protein posttranslational modification (PTM) code is one of the greatest biochemical challenges of our time. Phosphorylation and ubiquitylation are key PTMs that dictate protein function, recognition, sub-cellular localization, stability, turnover and fate. Hence, failures in their regulation leads to various disease. Chemical protein synthesis allows preparation of ubiquitinated and phosphorylated proteins to study their biochemical properties in great detail. However, monitoring these modifications in intact cells or in cell extracts mostly depends on antibodies, which often have off-target binding. Here, we report that the most widely used antibody for ubiquitin (Ub) phosphorylated at serine 65 (pUb) has significant off-targets that appear during mitosis. These off-targets are connected to polo-like kinase 1 (PLK1) mediated phosphorylation of cell cycle-related proteins and the anaphase promoting complex subunit 1 (APC1).
    Keywords:  antibody off-targets; cell cycle; posttranslational modifications
    DOI:  https://doi.org/10.3390/molecules27154867
  9. EMBO J. 2022 Aug 11. e110834
    Damage-induced regeneration of the intestinal stem cell pool through enteroblast mitosis in the Drosophila midgut.
    Aiguo Tian, Virginia Morejon, Sarah Kohoutek, Yi-Chun Huang, Wu-Min Deng, Jin Jiang.
      Many adult tissues and organs including the intestine rely on resident stem cells to maintain homeostasis and regeneration. In mammals, the progenies of intestinal stem cells (ISCs) can dedifferentiate to generate ISCs upon ablation of resident stem cells. However, whether and how mature tissue cells generate ISCs under physiological conditions remains unknown. Here, we show that infection of the Drosophila melanogaster intestine with pathogenic bacteria induces entry of enteroblasts (EBs), which are ISC progenies, into the mitotic cycle through upregulation of epidermal growth factor receptor (EGFR)-Ras signaling. We also show that ectopic activation of EGFR-Ras signaling in EBs is sufficient to drive enteroblast mitosis cell autonomously. Furthermore, we find that the dividing enteroblasts do not gain ISC identity as a prerequisite to divide, and the regenerative ISCs are produced through EB mitosis. Taken together, our work uncovers a new role for EGFR-Ras signaling in driving EB mitosis and replenishing the ISC pool during fly intestinal regeneration, which may have important implications for tissue homeostasis and tumorigenesis in vertebrates.
    Keywords:  Drosophila midgut; dedifferentiation; enteroblasts; regeneration; stem cells
    DOI:  https://doi.org/10.15252/embj.2022110834
  10. Cancers (Basel). 2022 Jul 31. pii: 3732. [Epub ahead of print]14(15):
    CDC20-Mediated hnRNPU Ubiquitination Regulates Chromatin Condensation and Anti-Cancer Drug Response.
    Cindy Wavelet-Vermuse, Olena Odnokoz, Yifan Xue, Xinghua Lu, Massimo Cristofanilli, Yong Wan.
      Cell division cycle 20 (CDC20) functions as a critical cell cycle regulator. It plays an important role in cancer development and drug resistance. However, the molecular mechanisms by which CDC20 regulates cellular drug response remain poorly understood. Chromatin-associated CDC20 interactome in breast cancer cells was analyzed by using affinity purification coupled with mass spectrometry. hnRNPU as a CDC20 binding partner was validated by co-immunoprecipitation and immunostaining. The molecular domain, comprising amino acid residues 461-653, on hnRNPU required for its interaction with CDC20 was identified by mapping of interactions. Co-immunoprecipitation showed that CDC20-mediated hnRNPU ubiquitination promotes its interaction with the CTCF and cohesin complex. The effects of CDC20-hnRNPU on nuclear size and chromatin condensation were investigated by analyzing DAPI and H2B-mCherry staining, respectively. The role of CDC20-hnRNPU in tumor progression and drug resistance was examined by CCK-8 cell survival and clonogenic assays. Our study indicates that CDC20-mediated ubiquitination of hnRNPU modulates chromatin condensation by regulating the interaction between hnRNPU and the CTCF-cohesin complex. Dysregulation of the CDC20-hnRNPU axis contributes to tumor progression and drug resistance.
    Keywords:  CDC20; chromatin condensation; cohesin complex; drug resistance; hnRNPU; mitotic slippage
    DOI:  https://doi.org/10.3390/cancers14153732
  11. Life Sci Alliance. 2022 Oct;pii: e202101161. [Epub ahead of print]5(10):
    The vacuole shapes the nucleus and the ribosomal DNA loop during mitotic delays.
    Emiliano Matos-Perdomo, Silvia Santana-Sosa, Jessel Ayra-Plasencia, Sara Medina-Suárez, Félix Machín.
      The ribosomal DNA (rDNA) array of Saccharomyces cerevisiae has served as a model to address chromosome organization. In cells arrested before anaphase (mid-M), the rDNA acquires a highly structured chromosomal organization referred to as the rDNA loop, whose length can double the cell diameter. Previous works established that complexes such as condensin and cohesin are essential to attain this structure. Here, we report that the rDNA loop adopts distinct presentations that arise as spatial adaptations to changes in the nuclear morphology triggered during mid-M arrests. Interestingly, the formation of the rDNA loop results in the appearance of a space under the loop (SUL) which is devoid of nuclear components yet colocalizes with the vacuole. We show that the rDNA-associated nuclear envelope (NE) often reshapes into a ladle to accommodate the vacuole in the SUL, with the nucleus becoming bilobed and doughnut-shaped. Finally, we demonstrate that the formation of the rDNA loop and the SUL require TORC1, membrane synthesis and functional vacuoles, yet is independent of nucleus-vacuole junctions and rDNA-NE tethering.
    DOI:  https://doi.org/10.26508/lsa.202101161
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