bims-micesi Biomed News
on Mitotic cell signalling
Issue of 2022–06–05
eleven papers selected by
Valentina Piano, Max Planck Institute of Molecular Physiology
  1. Zombies Never Die: The Double Life Bub1 Lives in Mitosis.
  2. SIRT1 regulates mitotic catastrophe via autophagy and BubR1 signaling.
  3. Function and regulation of transcription factors during mitosis-to-G1 transition.
  4. Directly Measuring Forces within Reconstituted Active Microtubule Bundles.
  5. MKLP2 functions in early mitosis to ensure proper chromosome congression.
  6. Ribosomal RNA regulates chromosome clustering during mitosis.
  7. EML4-ALK Variant 3 Promotes Mitotic Errors and Spindle Assembly Checkpoint Deficiency Leading to Increased Microtubule Poison Sensitivity.
  8. Upregulated mitosis-associated genes CENPE, CENPF, and DLGAP5 predict poor prognosis and chemotherapy resistance of Acute Myeloid Leukemia.
  9. Tip60 acetylation of histone H3K4 temporally controls Chromosome Passenger Complex localization.
  10. Examining the mechanistic relationship of APC/CCDH1 and its interphase inhibitor EMI1.
  11. AURKA and PLK1 inhibition selectively and synergistically block cell cycle progression in diffuse midline glioma.
  1. Front Cell Dev Biol. 2022 ;10 870745
    Zombies Never Die: The Double Life Bub1 Lives in Mitosis.
    Yuqing Zhang, Chunlin Song, Lei Wang, Hongfei Jiang, Yujing Zhai, Ying Wang, Jing Fang, Gang Zhang.
      When eukaryotic cells enter mitosis, dispersed chromosomes move to the cell center along microtubules to form a metaphase plate which facilitates the accurate chromosome segregation. Meanwhile, kinetochores not stably attached by microtubules activate the spindle assembly checkpoint and generate a wait signal to delay the initiation of anaphase. These events are highly coordinated. Disruption of the coordination will cause severe problems like chromosome gain or loss. Bub1, a conserved serine/threonine kinase, plays important roles in mitosis. After extensive studies in the last three decades, the role of Bub1 on checkpoint has achieved a comprehensive understanding; its role on chromosome alignment also starts to emerge. In this review, we summarize the latest development of Bub1 on supporting the two mitotic events. The essentiality of Bub1 in higher eukaryotic cells is also discussed. At the end, some undissolved questions are raised for future study.
    Keywords:  Bub1; chromosome alignment; kinetochore; mitosis; spindle assembly checkpoint
    DOI:  https://doi.org/10.3389/fcell.2022.870745
  2. Mol Cell Biochem. 2022 May 31.
    SIRT1 regulates mitotic catastrophe via autophagy and BubR1 signaling.
    Weiwei Zhao, Qing Wang, Le Li, Chengshen Xie, Yequn Wu, Mayank Gautam, Lijia Li.
      Mitotic catastrophe (MC) is a suppressive mechanism that mediates the elimination of mitosis-deficient cells through apoptosis, necrosis or senescence after M phase block. SIRT1 is involved in the regulation of several cellular processes, including autophagy. However, the relationship between SIRT1 and MC has been largely obscure. Our study highlights that SIRT1 might be involved in the regulation of MC. We have shown that degradation of the SIRT1 protein via proteasome and lysosomal pathway was accompanied by MC induced via BMH-21. Overexpression of SIRT1 alleviated MC by decreasing the proportion of apoptotic and multinuclear cells induced by G2/M block and triggered autophagy whereas knockdown of SIRT1 aggravated MC and repressed autophagy. Furthermore, we found that serum starvation triggered autophagy evidently generated lower MC whereas siRNA of ATG5/7 suppressed autophagy leading to higher MC. ChIP analysis revealed that SIRT1 could bind to the promoter of BubR1, a component of spindle assembly checkpoint (SAC), to upregulate its expression. Overexpression of BubR1 decreased MC whereas knockdown of BubR1 increased it. These results reveal that SIRT1 regulates MC through autophagy and BubR1 signaling, and provide evidence for SIRT1, autophagy and BubR1 being the potential cancer therapeutic targets.
    Keywords:  Autophagy; BubR1; Cell cycle; Mitotic catastrophe; SIRT1
    DOI:  https://doi.org/10.1007/s11010-022-04470-9
  3. Open Biol. 2022 Jun;12(6): 220062
    Function and regulation of transcription factors during mitosis-to-G1 transition.
    Mário A F Soares, Raquel A Oliveira, Diogo S Castro.
      During cell division, drastic cellular changes characteristic of mitosis result in the inactivation of the transcriptional machinery, and global downregulation of transcription. Sequence-specific transcription factors (TFs) have thus been considered mere bystanders, devoid of any regulatory function during mitosis. This view changed significantly in recent years, upon the conclusion that many TFs associate with condensed chromosomes during cell division, even occupying a fraction of their genomic target sites in mitotic chromatin. This finding was at the origin of the concept of mitotic bookmarking by TFs, proposed as a mechanism to propagate gene regulatory information across cell divisions, by facilitating the reactivation of specific bookmarked genes. While the underlying mechanisms and biological significance of this model remain elusive, recent developments in this fast-moving field have cast new light into TF activity during mitosis, beyond a bookmarking role. Here, we start by reviewing the most recent findings on the complex nature of TF-chromatin interactions during mitosis, and on mechanisms that may regulate them. Next, and in light of recent reports describing how transcription is reinitiated in temporally distinct waves during mitosis-to-G1 transition, we explore how TFs may contribute to defining this hierarchical gene expression process. Finally, we discuss how TF activity during mitotic exit may impact the acquisition of cell identity upon cell division, and propose a model that integrates dynamic changes in TF-chromatin interactions during this cell-cycle period, with the execution of cell-fate decisions.
    Keywords:  chromatin; electrostatic interactions; mitosis-to-G1 transition; mitotic bookmarking; sequence-specific binding; transcription factor
    DOI:  https://doi.org/10.1098/rsob.220062
  4. J Vis Exp. 2022 May 10.
    Directly Measuring Forces within Reconstituted Active Microtubule Bundles.
    Jacob Palumbo, Ellinor Tai, Scott Forth.
      Microtubule networks are employed in cells to accomplish a wide range of tasks, ranging from acting as tracks for vesicle transport to working as specialized arrays during mitosis to regulate chromosome segregation. Proteins that interact with microtubules include motors such as kinesins and dynein, which can generate active forces and directional motion, as well as non-motor proteins that crosslink filaments into higher-order networks or regulate filament dynamics. To date, biophysical studies of microtubule-associated proteins have overwhelmingly focused on the role of single motor proteins needed for vesicle transport, and significant progress has been made in elucidating the force-generating properties and mechanochemical regulation of kinesins and dyneins. However, for processes in which microtubules act both as cargo and track, such as during filament sliding within the mitotic spindle, much less is understood about the biophysical regulation of ensembles of the crosslinking proteins involved. Here, we detail our methodology for directly probing force generation and response within crosslinked microtubule minimal networks reconstituted from purified microtubules and mitotic proteins. Microtubule pairs are crosslinked by proteins of interest, one microtubule is immobilized to a microscope coverslip, and the second microtubule is manipulated by an optical trap. Simultaneous total internal reflection fluorescence microscopy allows for multichannel visualization of all the components of this microtubule network as the filaments slide apart to generate force. We also demonstrate how these techniques can be used to probe pushing forces exerted by kinesin-5 ensembles and how viscous braking forces arise between sliding microtubule pairs crosslinked by the mitotic MAP PRC1. These assays provide insights into the mechanisms of spindle assembly and function and can be more broadly adapted to study dense microtubule network mechanics in diverse contexts, such as the axon and dendrites of neurons and polar epithelial cells.
    DOI:  https://doi.org/10.3791/63819
  5. J Cell Sci. 2022 May 31. pii: jcs.259560. [Epub ahead of print]
    MKLP2 functions in early mitosis to ensure proper chromosome congression.
    Morgan S Schrock, Luke Scarberry, Benjamin R Stromberg, Claire Sears, Adrian E Torres, David Tallman, Lucas Krupinski, Arnab Chakravarti, Matthew K Summers.
      Mitotic kinesin-like protein 2 (MKLP2) is a motor protein with a well-established function in promoting cytokinesis. However, our results with siRNAs targeting MKLP2 and small molecule inhibitors of MKLP2 (MKLP2i) suggested a function earlier in mitosis, prior to anaphase. In this study we provide direct evidence that MKLP2 facilitates chromosome congression in prometaphase. We employed live imaging to observe HeLa cells with fluorescently tagged histones treated with MKLP2i and discovered a pronounced chromosome congression defect. We show that MKLP2 facilitates error correction as inhibited cells had a significant increase in unstable, syntelic kinetochore-microtubule attachments. We find that the aberrant attachments are accompanied by elevated Aurora Kinase (A/B) activity and phosphorylation of the downstream target, pHEC1 (Ser 55). Lastly, we show that MKLP2 inhibition results in aneuploidy, confirming that MKLP2 safeguards cells against chromosomal instability.
    Keywords:  Aurora Kinase; Chromosome congression; KIF20A; MKLP2; Motor kinesin
    DOI:  https://doi.org/10.1242/jcs.259560
  6. Cell Discov. 2022 May 31. 8(1): 51
    Ribosomal RNA regulates chromosome clustering during mitosis.
    Kai Ma, Man Luo, Guanglei Xie, Xi Wang, Qilin Li, Lei Gao, Hongtao Yu, Xiaochun Yu.
      Noncoding RNAs are known to associate with mitotic chromosomes, but the identities and functions of chromosome-associated RNAs in mitosis remain elusive. Here, we show that rRNA species associate with condensed chromosomes during mitosis. In particular, pre-rRNAs such as 45S, 32S, and 30S are highly enriched on mitotic chromosomes. Immediately following nucleolus disassembly in mitotic prophase, rRNAs are released and associate with and coat each condensed chromosome at prometaphase. Using unbiased mass spectrometry analysis, we further demonstrate that chromosome-bound rRNAs are associated with Ki-67. Moreover, the FHA domain and the repeat region of Ki-67 recognize and anchor rRNAs to chromosomes. Finally, suppression of chromosome-bound rRNAs by RNA polymerase I inhibition or by using rRNA-binding-deficient Ki-67 mutants impair mitotic chromosome dispersion during prometaphase. Our study thus reveals an important role of rRNAs in preventing chromosome clustering during mitosis.
    DOI:  https://doi.org/10.1038/s41421-022-00400-7
  7. Mol Cancer Res. 2022 Jun 03. 20(6): 854-866
    EML4-ALK Variant 3 Promotes Mitotic Errors and Spindle Assembly Checkpoint Deficiency Leading to Increased Microtubule Poison Sensitivity.
    Kellie Lucken, Laura O'Regan, Jene Choi, Josephina Sampson, Sarah L Pashley, Richard Bayliss, Sam Khan, Andrew M Fry.
      EML4-ALK is an oncogenic fusion protein present in approximately 5% of non-small cell lung cancers (NSCLC). Alternative breakpoints in the gene encoding EML4 result in distinct variants that are linked to markedly different patient outcomes. Patients with EML4-ALK variant 3 (V3) respond poorly to ALK inhibitors and have lower survival rates compared with patients with other common variants, such as V1. Here, we use isogenic Beas-2B bronchial epithelial cell lines expressing EML4-ALK V1 or V3, as well as ALK-positive NSCLC patient cells that express V1 (H3122 cells) or V3 (H2228 cells), to show that EML4-ALK V3 but not V1 leads to hyperstabilized K-fibers in mitosis, as well as errors in chromosome congression and segregation. This is consistent with our observation that EML4-ALK V3 but not V1 localizes to spindle microtubules and that wild-type EML4 is a microtubule stabilizing protein. In addition, cells expressing EML4-ALK V3 exhibit loss of spindle assembly checkpoint control that is at least in part dependent on ALK catalytic activity. Finally, we demonstrate that cells expressing EML4-ALK V3 have increased sensitivity to microtubule poisons that interfere with mitotic spindle assembly, whereas combination treatment with paclitaxel and clinically approved ALK inhibitors leads to a synergistic response in terms of reduced survival of H2228 cells.
    IMPLICATIONS: This study suggests that combining the microtubule poison, paclitaxel, with targeted ALK inhibitors may provide an effective new treatment option for patients with NSCLC with tumors that express the EML4-ALK V3 oncogenic fusion.
    DOI:  https://doi.org/10.1158/1541-7786.MCR-21-1010
  8. Cancer Biomark. 2022 May 20.
    Upregulated mitosis-associated genes CENPE, CENPF, and DLGAP5 predict poor prognosis and chemotherapy resistance of Acute Myeloid Leukemia.
    Mingyue Shi, Honggang Guo, Yanliang Bai, Junwei Niu, Xiaona Niu, Kai Sun, Yuqing Chen.
       BACKGROUND: Mitosis-associated genes are dysregulated in many types of cancers and play important roles in disease progression and chemotherapy resistance. However, their expression and functions in chemotherapy-resistant Acute Myeloid Leukemia (AML) are still largely undetermined.
    OBJECTIVE: This study aims to explore the roles of spindle assembly checkpoint (SAC) genes CENPE, CENPF, and DLGAP5 in chemotherapy-resistant AML.
    METHODS: RNA-sequencing (RNA-seq) was performed in patients with chemotherapy-resistant AML and chemotherapy-sensitive AML. AML mRNA data from 151 patients with recurrence were downloaded from TCGA. Integrated analysis of the differentially expressed genes (DEGs), GO and KEGG pathways. CENPE, CENPF, or DLGAP5 knockdown cell lines were used to analyse proliferation, apoptosis and cell cycle alterations.
    RESULTS: A total of 87 DEGs (48 upregulated and 39 downregulated) were obtained through gene analysis of R/R-AML (Fig. 1A) and a total of 329 DEGs (202 upregulated and 127 downregulated) were obtained in refractory S-AML. Upregulated DEGs were mainly enriched in cell cycle (GO: 0007049, hsa04110) and mitotic cell cycle (GO: 0000278) processes and pathway. Venn diagram analysis identified the most upregulated DEGs (including CENPE, CENPF, and DLGAP5) in chemoresistant AML. The expression of CENPE, CENPF and DLGAP5 in R-AML (TCGA) was significantly higher than that of primary AML (GEO). The proliferation of K562 cells after CENPE and DLGAP5 knockdown was significantly decreased (P= 0.0001 and P= 0.0006). In THP-1 cells, the CCK-8 values after CENPE, CENPF and DLGAP5 knockdown were significantly decreased (P= 0.01, P= 0.0395 and P= 0.0362). Knockdown of CENPE, CENPF and DLGAP5 significantly increased cell apoptosis by regulating Caspase-9, BAX, TP-53 and bcl-2, and induced cell cycle arrested by regulating CDK1, CDK2, CDKN1A, and CyclinD1.
    CONCLUSIONS: In conclusion, the mitotic cell cycle-associated genes CENPE, CENPF, and DLGAP5 were upregulated in chemotherapy-resistant AML patients and might be useful for predicting poor prognosis.
    Keywords:  AML; CENPE; CENPF; DLGAP5; Relapsed; prognosis; refractory
    DOI:  https://doi.org/10.3233/CBM-203170
  9. Mol Biol Cell. 2022 Jun 02. mbcE21060283
    Tip60 acetylation of histone H3K4 temporally controls Chromosome Passenger Complex localization.
    Ewa Niedzialkowska, Limin Liu, Cem Kuscu, Zachary Mayo, Wladek Minor, Brian D Strahl, Mazhar Adli, P Todd Stukenberg.
      The Chromosome Passenger Complex (CPC) generates chromosome autonomous signals that regulate mitotic events critical for genome stability. Tip60 is a lysine acetyltransferase that is a tumor suppressor and is targeted for proteasomal degradation by oncogenic papilloma viruses. Mitotic regulation requires the localization of the CPC to inner centromeres, which is driven by the Haspin kinase phosphorylating histone H3 on threonine 3 (H3T3ph). Here we describe how Tip60 acetylates histone H3 at lysine 4 (H3K4ac) to block both the H3T3ph writer and reader to ensure that this mitotic signaling cannot begin before prophase. Specifically, H3K4ac inhibits Haspin phosphorylation of H3T3 and prevents binding of the Survivin subunit to H3T3ph. Tip60 acetylates H3K4 during S/G2 at centromeres. Inhibition of Tip60 allows the CPC to bind centromeres in G2 cells, and targeting of Tip60 to centromeres prevents CPC localization in mitosis. The H3K4ac mark is removed in prophase by HDAC3 to initiate the CPC localization cascade. Together, our results suggest that Tip60 and HDAC3 temporally control H3K4 acetylation to precisely time the targeting of the CPC to inner centromeres.
    DOI:  https://doi.org/10.1091/mbc.E21-06-0283
  10. Protein Sci. 2022 Jun;31(6): e4324
    Examining the mechanistic relationship of APC/CCDH1 and its interphase inhibitor EMI1.
    Derek L Bolhuis, Raquel C Martinez-Chacin, Kaeli A Welsh, Tatyana Bodrug, Liying Cui, Michael J Emanuele, Nicholas G Brown.
      Proper protein destruction by the ubiquitin (Ub)-proteasome system is vital for a faithful cell cycle. Hence, the activity of Ub ligases is tightly controlled. The Anaphase-Promoting Complex/Cyclosome (APC/C) is a 1.2 MDa Ub ligase responsible for mitotic progression and G1 maintenance. At the G1/S transition, the APC/C is inhibited by EMI1 to prevent APC/C-dependent polyubiquitination of cell cycle effectors. EMI1 uses several interaction motifs to block the recruitment of APC/C substrates as well as the APC/C-associated E2s, UBE2C, and UBE2S. Paradoxically, EMI1 is also an APC/C substrate during G1. Using a comprehensive set of enzyme assays, we determined the context-dependent involvement of the EMI1 motifs in APC/C-dependent ubiquitination of EMI1 and other substrates. Furthermore, we demonstrated that an isolated C-terminal peptide fragment of EMI1 activates APC/C-dependent substrate priming by UBE2C. Together, these findings reveal the multiple roles of the EMI1 C-terminus for G1 maintenance and the G1/S transition.
    Keywords:  anaphase-promoting complex/cyclosome; cell cycle; early mitotic inhibitor 1; ubiquitin conjugating enzyme E2 C; ubiquitin conjugating enzyme E2 S; ubiquitin ligase
    DOI:  https://doi.org/10.1002/pro.4324
  11. iScience. 2022 Jun 17. 25(6): 104398
    AURKA and PLK1 inhibition selectively and synergistically block cell cycle progression in diffuse midline glioma.
    Dennis S Metselaar, Aimée du Chatinier, Michaël H Meel, Giovanna Ter Huizen, Piotr Waranecki, Joshua R Goulding, Marianna Bugiani, Jan Koster, Gertjan J L Kaspers, Esther Hulleman.
      Diffuse midline gliomas (DMG) are highly malignant incurable pediatric brain tumors. In this study, we show that Aurora kinase A (AURKA) is overexpressed in DMG and can be used as a therapeutic target. Additionally, AURKA inhibition combined with CRISPR/Cas9 screening in DMG cells, revealed polo-like kinase 1 (PLK1) as a synergistic target with AURKA. Using a panel of patient-derived DMG culture models, we demonstrate that treatment with volasertib, a clinically relevant and selective PLK1 inhibitor, synergizes with different AURKA inhibitors, supporting the CRISPR screen results. Mechanistically, our results show that combined loss of PLK1 and AURKA causes a G2/M cell cycle arrest which blocks vital parts of DNA-damage repair and induces apoptosis, solely in DMG cells. Altogether, our findings highlight the importance of AURKA and PLK1 for DMG propagation and demonstrate the potential of concurrently targeting these proteins as a therapeutic strategy for these devastating pediatric brain tumors.
    Keywords:  Biological sciences; Cancer; Cell biology; Molecular biology
    DOI:  https://doi.org/10.1016/j.isci.2022.104398
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