bims-micesi Biomed News
on Mitotic cell signalling
Issue of 2022‒07‒31
twelve papers selected by
Valentina Piano
Max Planck Institute of Molecular Physiology
  1. Yeast Kinesin-5 Motor Protein CIN8 Promotes Accurate Chromosome Segregation.
  2. The N-Terminal Domain of Bfa1 Coordinates Mitotic Exit Independent of GAP Activity in Saccharomyces cerevisiae.
  3. Small changes in phospho-occupancy at the kinetochore-microtubule interface drive mitotic fidelity.
  4. Three-dimensional structure of kinetochore-fibers in human mitotic spindles.
  5. Making Mitotic Chromosomes in a Test Tube.
  6. Self-organization of kinetochore-fibers in human mitotic spindles.
  7. Proteomic analysis of the actin cortex in interphase and mitosis.
  8. Genetic Control of Kinetochore-Driven Microtubule Growth in Drosophila Mitosis.
  9. Interaction with TopBP1 Is Required for Human Papillomavirus 16 E2 Plasmid Segregation/Retention Function during Mitosis.
  10. HFE promotes mitotic cell division through recruitment of cytokinetic abscission machinery in hepatocellular carcinoma.
  11. Chromosomes: A nuclear neighborhood conducive to centromere formation.
  12. Ki-67 index assessment on FNA specimens of gastrointestinal stromal tumor: Correlation with mitotic rate and potential predictive value for risk stratification.
  1. Cells. 2022 Jul 07. pii: 2144. [Epub ahead of print]11(14):
    Yeast Kinesin-5 Motor Protein CIN8 Promotes Accurate Chromosome Segregation.
    Delaney Sherwin, Abigail Huetteman, Yanchang Wang.
      Accurate chromosome segregation depends on bipolar chromosome-microtubule attachment and tension generation on chromosomes. Incorrect chromosome attachment results in chromosome missegregation, which contributes to genome instability. The kinetochore is a protein complex that localizes at the centromere region of a chromosome and mediates chromosome-microtubule interaction. Incorrect chromosome attachment leads to checkpoint activation to prevent anaphase onset. Kinetochore detachment activates the spindle assembly checkpoint (SAC), while tensionless kinetochore attachment relies on both the SAC and tension checkpoint. In budding yeast Saccharomyces cerevisiae, kinesin-5 motor proteins Cin8 and Kip1 are needed to separate spindle pole bodies for spindle assembly, and deletion of CIN8 causes lethality in the absence of SAC. To study the function of Cin8 and Kip1 in chromosome segregation, we constructed an auxin-inducible degron (AID) mutant, cin8-AID. With this conditional mutant, we first confirmed that cin8-AID kip1∆ double mutants were lethal when Cin8 is depleted in the presence of auxin. These cells arrested in metaphase with unseparated spindle pole bodies and kinetochores. We further showed that the absence of either the SAC or tension checkpoint was sufficient to abolish the cell-cycle delay in cin8-AID mutants, causing chromosome missegregation and viability loss. The tension checkpoint-dependent phenotype in cells with depleted Cin8 suggests the presence of tensionless chromosome attachment. We speculate that the failed spindle pole body separation in cin8 mutants could increase the chance of tensionless syntelic chromosome attachments, which depends on functional tension checkpoint for survival.
    Keywords:  Cin8; Kip1; kinesin-5 motor proteins; tension checkpoint; tension on chromosomes
    DOI:  https://doi.org/10.3390/cells11142144
  2. Cells. 2022 Jul 12. pii: 2179. [Epub ahead of print]11(14):
    The N-Terminal Domain of Bfa1 Coordinates Mitotic Exit Independent of GAP Activity in Saccharomyces cerevisiae.
    Yan Li, Kiwon Song.
      The spindle position checkpoint (SPOC) of budding yeast delays mitotic exit in response to misaligned spindles to ensure cell survival and the maintenance of genomic stability. The GTPase-activating protein (GAP) complex Bfa1-Bub2, a key SPOC component, inhibits the GTPase Tem1 to induce mitotic arrest in response to DNA and spindle damage, as well as spindle misorientation. However, previous results strongly suggest that Bfa1 exerts a GAP-independent function in blocking mitotic exit in response to misaligned spindles. Thus, the molecular mechanism by which Bfa1 controls mitotic exit in response to misaligned spindles remains unclear. Here, we observed that overexpression of the N-terminal domain of Bfa1 (Bfa1-D16), which lacks GAP activity and cannot localize to the spindle pole body (SPB), induced cell cycle arrest along with hyper-elongation of astral microtubules (aMTs) as Bfa1 overexpression in Δbub2. We found that Δbub2 cells overexpressing Bfa1 or Bfa1-D16 inhibited activation of Mob1, which is responsible for mitotic exit. In anaphase-arrested cells, Bfa1-D16 overexpression inhibited Tem1 binding to the SPB as well as Bfa1 overexpression. Additionally, endogenous levels of Bfa1-D16 showed minor SPOC activity that was not regulated by Kin4. These results suggested that Bfa1-D16 may block mitotic exit through inhibiting Tem1 activity outside of SPBs. Alternatively, Bfa1-D16 dispersed out of SPBs may block Tem1 binding to SPBs by physically interacting with Tem1 as previously reported. Moreover, we observed hyper-elongated aMTs in tem1-3, cdc15-2, and dbf2-2 mutants that induce anaphase arrest and cannot undergo mitotic exit at restrictive temperatures, suggesting that aMT dynamics are closely related to the regulation of mitotic exit. Altogether, these observations suggest that Bfa1 can control the SPOC independent of its GAP activity and SPB localization.
    Keywords:  Bfa1; SPOC; budding yeast; microtubule hyper-elongation; mitotic exit; spindle misorientation; spindle pole body
    DOI:  https://doi.org/10.3390/cells11142179
  3. J Cell Biol. 2022 Sep 05. pii: e202107107. [Epub ahead of print]221(9):
    Small changes in phospho-occupancy at the kinetochore-microtubule interface drive mitotic fidelity.
    Thomas J Kucharski, Rufus Hards, Sarah E Vandal, Maria Alba Abad, A Arockia Jeyaprakash, Edward Kaye, Aymen Al-Rawi, Tony Ly, Kristina M Godek, Scott A Gerber, Duane A Compton.
      Kinetochore protein phosphorylation promotes the correction of erroneous microtubule attachments to ensure faithful chromosome segregation during cell division. Determining how phosphorylation executes error correction requires an understanding of whether kinetochore substrates are completely (i.e., all-or-none) or only fractionally phosphorylated. Using quantitative mass spectrometry (MS), we measured phospho-occupancy on the conserved kinetochore protein Hec1 (NDC80) that directly binds microtubules. None of the positions measured exceeded ∼50% phospho-occupancy, and the cumulative phospho-occupancy changed by only ∼20% in response to changes in microtubule attachment status. The narrow dynamic range of phospho-occupancy is maintained, in part, by the ongoing phosphatase activity. Further, both Cdk1-Cyclin B1 and Aurora kinases phosphorylate Hec1 to enhance error correction in response to different types of microtubule attachment errors. The low inherent phospho-occupancy promotes microtubule attachment to kinetochores while the high sensitivity of kinetochore-microtubule attachments to small changes in phospho-occupancy drives error correction and ensures high mitotic fidelity.
    DOI:  https://doi.org/10.1083/jcb.202107107
  4. Elife. 2022 Jul 27. pii: e75459. [Epub ahead of print]11
    Three-dimensional structure of kinetochore-fibers in human mitotic spindles.
    Robert Kiewisz, Gunar Fabig, William Conway, Daniel Baum, Daniel J Needleman, Thomas Müller-Reichert.
      During cell division, kinetochore microtubules (KMTs) provide a physical linkage between the chromosomes and the rest of the spindle. KMTs in mammalian cells are organized into bundles, so-called kinetochore-fibers (k-fibers), but the ultrastructure of these fibers is currently not well characterized. Here we show by large-scale electron tomography that each k-fiber in HeLa cells in metaphase is composed of approximately nine KMTs, only half of which reach the spindle pole. Our comprehensive reconstructions allowed us to analyze the three-dimensional (3D) morphology of k-fibers and their surrounding MTs in detail. We found that k-fibers exhibit remarkable variation in circumference and KMT density along their length, with the pole-proximal side showing a broadening. Extending our structural analysis then to other MTs in the spindle, we further observed that the association of KMTs with non-KMTs predominantly occurs in the spindle pole regions. Our 3D reconstructions have implications for KMT growth and k-fiber self-organization models as covered in a parallel publication applying complementary live-cell imaging in combination with biophysical modeling (Conway et al., 2022). Finally, we also introduce a new visualization tool allowing an interactive display of our 3D spindle data that will serve as a resource for further structural studies on mitosis in human cells.
    Keywords:  cell biology; human; physics of living systems
    DOI:  https://doi.org/10.7554/eLife.75459
  5. Epigenomes. 2022 Jul 20. pii: 20. [Epub ahead of print]6(3):
    Making Mitotic Chromosomes in a Test Tube.
    Keishi Shintomi.
      Mitotic chromosome assembly is an essential preparatory step for accurate transmission of the genome during cell division. During the past decades, biochemical approaches have uncovered the molecular basis of mitotic chromosomes. For example, by using cell-free assays of frog egg extracts, the condensin I complex central for the chromosome assembly process was first identified, and its functions have been intensively studied. A list of chromosome-associated proteins has been almost completed, and it is now possible to reconstitute structures resembling mitotic chromosomes with a limited number of purified factors. In this review, I introduce how far we have come in understanding the mechanism of chromosome assembly using cell-free assays and reconstitution assays, and I discuss their potential applications to solve open questions.
    Keywords:  Xenopus egg extract; condensin; histone; mitotic chromosome; reconstitution; topoisomerase II
    DOI:  https://doi.org/10.3390/epigenomes6030020
  6. Elife. 2022 Jul 25. pii: e75458. [Epub ahead of print]11
    Self-organization of kinetochore-fibers in human mitotic spindles.
    William Conway, Robert Kiewisz, Gunar Fabig, Colm P Kelleher, Hai-Yin Wu, Maya Anjur-Dietrich, Thomas Müller-Reichert, Daniel J Needleman.
      During eukaryotic cell division, chromosomes are linked to microtubules (MTs) in the spindle by a macromolecular complex called the kinetochore. The bound kinetochore microtubules (KMTs) are crucial to ensuring accurate chromosome segregation. Recent reconstructions by electron tomography (Kiewisz et al. 2021) captured the positions and configurations of every MT in human mitotic spindles, revealing that roughly half the KMTs in these spindles do not reach the pole. Here, we investigate the processes that give rise to this distribution of KMTs using a combination of analysis of large-scale electron tomography, photoconversion experiments, quantitative polarized light microscopy, and biophysical modeling. Our results indicate that in metaphase, KMTs grow away from the kinetochores along well-defined trajectories, with the speed of the KMT minus ends continually decreasing as the minus ends approach the pole, implying that longer KMTs grow more slowly than shorter KMTs. The locations of KMT minus ends, and the turnover and movements of tubulin in KMTs, are consistent with models in which KMTs predominately nucleate de novo at kinetochores in metaphase and are inconsistent with substantial numbers of non-KMTs being recruited to the kinetochore in metaphase. Taken together, this work leads to a mathematical model of the self-organization of kinetochore-fibers in human mitotic spindles.
    Keywords:  cell biology; human; physics of living systems
    DOI:  https://doi.org/10.7554/eLife.75458
  7. J Cell Sci. 2022 Jul 27. pii: jcs.259993. [Epub ahead of print]
    Proteomic analysis of the actin cortex in interphase and mitosis.
    Neza Vadnjal, Sami Nourreddine, Geneviève Lavoie, Murielle Serres, Philippe P Roux, Ewa K Paluch.
      Many animal cell shape changes are driven by gradients in the contractile tension of the actomyosin cortex, a thin cytoskeletal network supporting the plasma membrane. Elucidating cortical tension control is thus essential for understanding cell morphogenesis. Increasing evidence shows that alongside myosin activity, actin network organisation and composition are key to cortex tension regulation. However, due to poor understanding of how cortex composition changes when tension changes, which cortical components are important remains unclear. In this Resource article, we compared cortices from cells with low and high cortex tensions. We purified cortex-enriched fractions from cells in interphase and mitosis, as mitosis is characterised by high cortical tension. Mass spectrometry analysis identified 922 proteins consistently represented in both interphase and mitotic cortices. Focusing on actin-related proteins narrowed down the list to 238 candidate regulators of the mitotic cortical tension increase. Among these candidates, we found a role for septins in mitotic cell rounding control. Overall, our study provides a comprehensive dataset of candidate cortex regulators, paving the way for systematic investigations of the regulation of cell surface mechanics.
    Keywords:  Actin; Cell cortex; Cell division; Cortical tension; Mass specotometry; Septin
    DOI:  https://doi.org/10.1242/jcs.259993
  8. Cells. 2022 Jul 06. pii: 2127. [Epub ahead of print]11(14):
    Genetic Control of Kinetochore-Driven Microtubule Growth in Drosophila Mitosis.
    Julia V Popova, Gera A Pavlova, Alyona V Razuvaeva, Lyubov A Yarinich, Evgeniya N Andreyeva, Alina F Anders, Yuliya A Galimova, Fioranna Renda, Maria Patrizia Somma, Alexey V Pindyurin, Maurizio Gatti.
      Centrosome-containing cells assemble their spindles exploiting three main classes of microtubules (MTs): MTs nucleated by the centrosomes, MTs generated near the chromosomes/kinetochores, and MTs nucleated within the spindle by the augmin-dependent pathway. Mammalian and Drosophila cells lacking the centrosomes generate MTs at kinetochores and eventually form functional bipolar spindles. However, the mechanisms underlying kinetochore-driven MT formation are poorly understood. One of the ways to elucidate these mechanisms is the analysis of spindle reassembly following MT depolymerization. Here, we used an RNA interference (RNAi)-based reverse genetics approach to dissect the process of kinetochore-driven MT regrowth (KDMTR) after colcemid-induced MT depolymerization. This MT depolymerization procedure allows a clear assessment of KDMTR, as colcemid disrupts centrosome-driven MT regrowth but not KDMTR. We examined KDMTR in normal Drosophila S2 cells and in S2 cells subjected to RNAi against conserved genes involved in mitotic spindle assembly: mast/orbit/chb (CLASP1), mei-38 (TPX2), mars (HURP), dgt6 (HAUS6), Eb1 (MAPRE1/EB1), Patronin (CAMSAP2), asp (ASPM), and Klp10A (KIF2A). RNAi-mediated depletion of Mast/Orbit, Mei-38, Mars, Dgt6, and Eb1 caused a significant delay in KDMTR, while loss of Patronin had a milder negative effect on this process. In contrast, Asp or Klp10A deficiency increased the rate of KDMTR. These results coupled with the analysis of GFP-tagged proteins (Mast/Orbit, Mei-38, Mars, Eb1, Patronin, and Asp) localization during KDMTR suggested a model for kinetochore-dependent spindle reassembly. We propose that kinetochores capture the plus ends of MTs nucleated in their vicinity and that these MTs elongate at kinetochores through the action of Mast/Orbit. The Asp protein binds the MT minus ends since the beginning of KDMTR, preventing excessive and disorganized MT regrowth. Mei-38, Mars, Dgt6, Eb1, and Patronin positively regulate polymerization, bundling, and stabilization of regrowing MTs until a bipolar spindle is reformed.
    Keywords:  Asp; Dgt6; Drosophila; Eb1; Klp10A; Mars; Mast/Orbit/Chb; Mei-38; Patronin; S2 cells; colcemid; kinetochores; microtubule depolymerization; microtubule regrowth; mitosis
    DOI:  https://doi.org/10.3390/cells11142127
  9. J Virol. 2022 Jul 26. e0083022
    Interaction with TopBP1 Is Required for Human Papillomavirus 16 E2 Plasmid Segregation/Retention Function during Mitosis.
    Apurva T Prabhakar, Claire D James, Dipon Das, Christian T Fontan, Raymonde Otoa, Xu Wang, Molly L Bristol, Iain M Morgan.
      Human papillomavirus 16 (HPV16) E2 is a DNA-binding protein that regulates transcription, replication and potentially, segregation of the HPV16 genome during the viral life cycle. In the segregation model, E2 simultaneously binds to viral and host chromatin, acting as a bridge to ensure that viral genomes reside in daughter nuclei following cell division. The host chromatin receptor for E2 mediating this function is unknown. Recently, we demonstrated that CK2 phosphorylation of E2 on serine 23 (S23) is required for interaction with TopBP1, and that this interaction promotes E2 and TopBP1 recruitment to mitotic chromatin. Here, we demonstrate that in U2OS cells expressing wild-type E2 and a non-TopBP1-binding mutant (S23A, serine 23 mutated to alanine), interaction with TopBP1 is essential for E2 recruitment of plasmids to mitotic chromatin. Using novel quantitative segregation assays, we demonstrate that interaction with TopBP1 is required for E2 plasmid segregation function in U2OS and N/Tert-1 cells. Small interfering RNA (siRNA) knockdown of TopBP1 or CK2 enzyme components disrupts E2 segregation/retention function. The interaction of E2 with TopBP1 promotes increased levels of E2 protein during mitosis in U2OS and N/Tert-1 cells, as well as in human foreskin keratinocytes (HFK) immortalized by the HPV16 genome. Overall, our results demonstrate that E2 has plasmid segregation activity, and that the E2-TopBP1 interaction is essential for this E2 function. IMPORTANCE HPV16 causes 3% to 4% of all human cancers. It is proposed that during the viral life cycle, the viral genome is actively segregated into daughter nuclei, ensuring viral replication in the subsequent S phase. The E2 protein potentially bridges the viral and host genomes during mitosis to mediate segregation of the circular viral plasmid. Here, we demonstrate that E2 has the ability to mediate plasmid segregation, and that this function is dependent upon interaction with the host protein TopBP1. Additionally, we demonstrate that the E2-TopBP1 interaction promotes enhanced E2 expression during mitosis, which likely promotes the plasmid segregation function of E2. Overall, our results present a mechanism of how HPV16 can segregate its viral genome during an active infection, a critical aspect of the viral life cycle.
    Keywords:  E2; TopBP1; chromosome segregation; papillomavirus; plasmid retention
    DOI:  https://doi.org/10.1128/jvi.00830-22
  10. Oncogene. 2022 Jul 26.
    HFE promotes mitotic cell division through recruitment of cytokinetic abscission machinery in hepatocellular carcinoma.
    Pingping Dong, Ziqing Cai, Bingfeng Li, Yueqin Zhu, Alice K Y Chan, Michael W L Chiang, Chun Hang Au, Wing Kin Sung, Tan To Cheung, Chung Mau Lo, Kwan Man, Nikki P Lee.
      HFE (Hemochromatosis) is a conventional iron level regulator and its loss of function due to gene mutations increases the risk of cancers including hepatocellular carcinoma (HCC). Likewise, studies focusing on HFE overexpression in cancers are all limited to linking up these events as a consequence of iron level deregulation. No study has explored any iron unrelated role of HFE in cancers. Here, we first reported HFE as an oncogene in HCC and its undescribed function on promoting abscission in cytokinesis during mitotic cell division, independent of its iron-regulating ability. Clinical analyses revealed HFE upregulation in tumors linking to large tumor size and poor prognosis. Functionally and mechanistically, HFE promoted cytokinetic abscission via facilitating ESCRT abscission machinery recruitment to the abscission site through signaling a novel HFE/ALK3/Smads/LIF/Hippo/YAP/YY1/KIF13A axis. Pharmacological blockage of HFE signaling axis impeded tumor phenotypes in vitro and in vivo. Our data on HFE-driven HCC unveiled a new mechanism utilized by cancer cells to propel rapid cell division. This study also laid the groundwork for tumor intolerable therapeutics development given the high cytokinetic dependency of cancer cells and their vulnerability to cytokinetic blockage.
    DOI:  https://doi.org/10.1038/s41388-022-02419-2
  11. Curr Biol. 2022 Jul 25. pii: S0960-9822(22)00979-4. [Epub ahead of print]32(14): R776-R778
    Chromosomes: A nuclear neighborhood conducive to centromere formation.
    Janardan N Gavade, Ben E Black.
      Centromere identity is specified by nucleosomes containing the histone variant CENP-A. A new study reveals that subnuclear location dictates the efficiency with which a new centromere forms.
    DOI:  https://doi.org/10.1016/j.cub.2022.06.020
  12. Cancer Cytopathol. 2022 Jul 25.
    Ki-67 index assessment on FNA specimens of gastrointestinal stromal tumor: Correlation with mitotic rate and potential predictive value for risk stratification.
    Xi Wang, Rita Abi-Raad, Haiming Tang, Guoping Cai.
      BACKGROUND: Risk assessment of gastrointestinal stromal tumor (GIST) is challenging on cytology specimens. This study aims to determine whether Ki-67 index evaluated on fine-needle aspiration (FNA) specimens can correlate with the mitotic rate of GIST in surgical specimens and provide further risk assessment.METHODS: Cases with cell blocks containing adequate tumor cells and surgical resections were included. Ki-67 immunostain was retrospectively performed on cell block sections, and Ki-67 index was calculated on the "hot spot" areas.
    RESULTS: This study included 50 GIST cases from stomach (n = 45; 90%), duodenum (n = 4; 8%), and distal esophagus (n = 1; 2%). The tumor size ranged from 1.5 cm to 21 cm (mean, 5.4 cm). Based on the mitotic count, 37 GISTs (74%) had low mitotic rate (LMR) and 13 GISTs (26%) had high mitotic rate (HMR). The spindle cell, epithelioid, and mixed types accounted for 60%, 14%, and 26% of GIST, respectively. Ki-67 index counted on cell block sections correlated well with mitotic count evaluated in surgical specimens (r = 0.8031). Mean Ki-67 index was higher in HMR than LMR groups (3.5% vs. 1%, p < .001). The receiver operating characteristic curve using Ki-67 index to predict mitotic rate was further analyzed, and area under the curve was 0.839. Using a cutoff of 2.5% yielded a sensitivity of 70% at 92% specificity.
    CONCLUSIONS: This study demonstrates good correlations between Ki-67 index and mitotic count or risk stratification, suggesting that Ki-67 index evaluated on cytology specimens may offer a promising approach to preoperatively predict the mitotic rate and risk of GIST.
    Keywords:  Ki-67 index; fine-needle aspiration; gastrointestinal stromal tumor; prognosis; risk stratification
    DOI:  https://doi.org/10.1002/cncy.22630
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