bims-micesi 2022-08-21 papers

bims-micesi

Biomed News

on Mitotic cell signalling

Issue of 2022–08–21
eight papers selected by
Valentina Piano, Max Planck Institute of Molecular Physiology

Control of division in Chlamydomonas by cyclin B/CDKB1 and the anaphase-promoting complex.
The Mis6 inner kinetochore subcomplex maintains CENP-A nucleosomes against centromeric non-coding transcription during mitosis.
Two Kinesin-14A Motors Oligomerize to Drive Poleward Microtubule Convergence for Acentrosomal Spindle Morphogenesis in Arabidopsis thaliana.
Phosphorylation of XPD drives its mitotic role independently of its DNA repair and transcription functions.
Mitotic H3K9ac is controlled by phase-specific activity of HDAC2, HDAC3, and SIRT1.
Genome characterization and CRISPR-Cas9 editing of a human neocentromere.
Revisiting the multisite phosphorylation that produces the M-phase supershift of key mitotic regulators.
Combined TRIP13 and Aurora kinase inhibition induces apoptosis in human papillomavirus-driven cancers.

PLoS Genet. 2022 Aug 18. 18(8): e1009997

Control of division in Chlamydomonas by cyclin B/CDKB1 and the anaphase-promoting complex.

Kresti Pecani, Kristi Lieberman, Natsumi Tajima-Shirasaki, Masayuki Onishi, Frederick R Cross.

In yeast and animals, cyclin B binds and activates the cyclin-dependent kinase ('CDK') CDK1 to drive entry into mitosis. We show that CYCB1, the sole cyclin B in Chlamydomonas, activates the plant-specific CDKB1 rather than the CDK1 ortholog CDKA1, confirming and extending previous results. Time-lapse microscopy shows that CYCB1 is synthesized before each division in the multiple fission cycle, then is rapidly degraded 3-5 minutes before division occurs. CYCB1 degradation is dependent on the anaphase-promoting complex (APC). Like CYCB1, CDKB1 is not synthesized until late G1; however, CDKB1 is not degraded with each division within the multiple fission cycle, but is degraded after all divisions have ceased. The microtubule plus-end-binding protein EB1 labeled with mNeonGreen allowed detection of mitotic events in live cells. The earliest detectable step in mitosis, splitting of polar EB1 signal into two foci, likely associated with future spindle poles, was dependent on CYCB1. CYCB1-GFP localized close to these foci immediately before spindle formation. Spindle breakdown, cleavage furrow formation and accumulation of EB1 in the furrow were dependent on the APC. In interphase, rapidly growing microtubules are marked by 'comets' of EB1; comets are absent in the absence of APC function. Thus CYCB1/CDKB1 and the APC modulate microtubule function and assembly while regulating mitotic progression. Genetic results suggest an independent additional role for the APC in regulating sister chromatid cohesion; this role is likely conserved across eukaryotes.

DOI: https://doi.org/10.1371/journal.pgen.1009997
Commun Biol. 2022 Aug 15. 5(1): 818

The Mis6 inner kinetochore subcomplex maintains CENP-A nucleosomes against centromeric non-coding transcription during mitosis.

Hayato Hirai, Yuki Shogaki, Masamitsu Sato.

Centromeres are established by nucleosomes containing the histone H3 variant CENP-A. CENP-A is recruited to centromeres by the Mis18-HJURP machinery. During mitosis, CENP-A recruitment ceases, implying the necessity of CENP-A maintenance at centromeres, although the exact underlying mechanism remains elusive. Herein, we show that the inner kinetochore protein Mis6 (CENP-I) and Mis15 (CENP-N) retain CENP-A during mitosis in fission yeast. Eliminating Mis6 or Mis15 during mitosis caused immediate loss of pre-existing CENP-A at centromeres. CENP-A loss occurred due to the transcriptional upregulation of non-coding RNAs at the central core region of centromeres, as confirmed by the observation RNA polymerase II inhibition preventing CENP-A loss from centromeres in the mis6 mutant. Thus, we concluded that the inner kinetochore complex containing Mis6-Mis15 blocks the indiscriminate transcription of non-coding RNAs at the core centromere, thereby retaining the epigenetic inheritance of CENP-A during mitosis.

DOI: https://doi.org/10.1038/s42003-022-03786-y
Front Cell Dev Biol. 2022 ;10 949345

Two Kinesin-14A Motors Oligomerize to Drive Poleward Microtubule Convergence for Acentrosomal Spindle Morphogenesis in Arabidopsis thaliana.

Takashi Hotta, Yuh-Ru Julie Lee, Takumi Higaki, Takashi Hashimoto, Bo Liu.

  Plant cells form acentrosomal spindles with microtubules (MTs) converged toward two structurally undefined poles by employing MT minus end-directed Kinesin-14 motors. To date, it is unclear whether the convergent bipolar MT array assumes unified poles in plant spindles, and if so, how such a goal is achieved. Among six classes of Kinesin-14 motors in Arabidopsis thaliana, the Kinesin-14A motors ATK1 (KatA) and ATK5 share the essential function in spindle morphogenesis. To understand how the two functionally redundant Kinesin-14A motors contributed to the spindle assembly, we had ATK1-GFP and ATK5-GFP fusion proteins expressed in their corresponding null mutants and found that they were functionally comparable to their native forms. Although ATK1 was a nuclear protein and ATK5 cytoplasmic prior to nuclear envelop breakdown, at later mitotic stages, the two motors shared similar localization patterns of uniform association with both spindle and phragmoplast MTs. We found that ATK1 and ATK5 were rapidly concentrated toward unified polar foci when cells were under hyperosmotic conditions. Concomitantly, spindle poles became perfectly focused as if there were centrosome-like MT-organizing centers where ATK1 and ATK5 were highly enriched and at which kinetochore fibers pointed. The separation of ATK1/ATK5-highlighted MTs from those of kinetochore fibers suggested that the motors translocated interpolar MTs. Our protein purification and live-cell imaging results showed that ATK1 and ATK5 are associated with each other in vivo. The stress-induced spindle pole convergence was also accompanied by poleward accumulation of the MT nucleator γ-tubulin. These results led to the conclusion that the two Kinesin-14A motors formed oligomeric motor complexes that drove MT translocation toward the spindle pole to establish acentrosomal spindles with convergent poles.

Keywords:  Arabidopsis; interpolar microtubules; kinesin-14; kinetochore fibers; microtubule convergence; mitotic spindle; spindle poles

DOI:  https://doi.org/10.3389/fcell.2022.949345
Sci Adv. 2022 Aug 19. 8(33): eabp9457

Phosphorylation of XPD drives its mitotic role independently of its DNA repair and transcription functions.

Emmanuel Compe, Evanthia Pangou, Nicolas Le May, Clémence Elly, Cathy Braun, Ji-Hyun Hwang, Frédéric Coin, Izabela Sumara, Kwang-Wook Choi, Jean-Marc Egly.

The helicase XPD is known as a key subunit of the DNA repair/transcription factor TFIIH. However, here, we report that XPD, independently to other TFIIH subunits, can localize with the motor kinesin Eg5 to mitotic spindles and the midbodies of human cells. The XPD/Eg5 partnership is promoted upon phosphorylation of Eg5/T926 by the kinase CDK1, and conversely, it is reduced once Eg5/S1033 is phosphorylated by NEK6, a mitotic kinase that also targets XPD at T425. The phosphorylation of XPD does not affect its DNA repair and transcription functions, but it is required for Eg5 localization, checkpoint activation, and chromosome segregation in mitosis. In XPD-mutated cells derived from a patient with xeroderma pigmentosum, the phosphomimetic form XPD/T425D or even the nonphosphorylatable form Eg5/S1033A specifically restores mitotic chromosome segregation errors. These results thus highlight the phospho-dependent mitotic function of XPD and reveal how mitotic defects might contribute to XPD-related disorders.

DOI: https://doi.org/10.1126/sciadv.abp9457
Life Sci Alliance. 2022 Oct;pii: e202201433. [Epub ahead of print]5(10):

Mitotic H3K9ac is controlled by phase-specific activity of HDAC2, HDAC3, and SIRT1.

Shashi Gandhi, Raizy Mitterhoff, Rachel Rapoport, Marganit Farago, Avraham Greenberg, Lauren Hodge, Sharon Eden, Christopher Benner, Alon Goren, Itamar Simon.

Histone acetylation levels are reduced during mitosis. To study the mitotic regulation of H3K9ac, we used an array of inhibitors targeting specific histone deacetylases. We evaluated the involvement of the targeted enzymes in regulating H3K9ac during all mitotic stages by immunofluorescence and immunoblots. We identified HDAC2, HDAC3, and SIRT1 as modulators of H3K9ac mitotic levels. HDAC2 inhibition increased H3K9ac levels in prophase, whereas HDAC3 or SIRT1 inhibition increased H3K9ac levels in metaphase. Next, we performed ChIP-seq on mitotic-arrested cells following targeted inhibition of these histone deacetylases. We found that both HDAC2 and HDAC3 have a similar impact on H3K9ac, and inhibiting either of these two HDACs substantially increases the levels of this histone acetylation in promoters, enhancers, and insulators. Altogether, our results support a model in which H3K9 deacetylation is a stepwise process-at prophase, HDAC2 modulates most transcription-associated H3K9ac-marked loci, and at metaphase, HDAC3 maintains the reduced acetylation, whereas SIRT1 potentially regulates H3K9ac by impacting HAT activity.

DOI: https://doi.org/10.26508/lsa.202201433
Chromosoma. 2022 Aug 17.

Genome characterization and CRISPR-Cas9 editing of a human neocentromere.

Antonio Palazzo, Ilaria Piccolo, Crescenzio Francesco Minervini, Stefania Purgato, Oronzo Capozzi, Pietro D'Addabbo, Cosimo Cumbo, Francesco Albano, Mariano Rocchi, Claudia Rita Catacchio.

  The maintenance of genome integrity is ensured by proper chromosome inheritance during mitotic and meiotic cell divisions. The chromosomal counterpart responsible for chromosome segregation to daughter cells is the centromere, at which the spindle apparatus attaches through the kinetochore. Although all mammalian centromeres are primarily composed of megabase-long repetitive sequences, satellite-free human neocentromeres have been described. Neocentromeres and evolutionary new centromeres have revolutionized traditional knowledge about centromeres. Over the past 20 years, insights have been gained into their organization, but in spite of these advancements, the mechanisms underlying their formation and evolution are still unclear. Today, through modern and increasingly accessible genome editing and long-read sequencing techniques, research in this area is undergoing a sudden acceleration. In this article, we describe the primary sequence of a previously described human chromosome 3 neocentromere and observe its possible evolution and repair results after a chromosome breakage induced through CRISPR-Cas9 technologies. Our data represent an exciting advancement in the field of centromere/neocentromere evolution and chromosome stability.

Keywords:  CRISPR-Cas9; Isochromosome; Long-read sequencing; Neocentromere

DOI:  https://doi.org/10.1007/s00412-022-00779-y
Mol Biol Cell. 2022 Aug 17. mbcE22040118

Revisiting the multisite phosphorylation that produces the M-phase supershift of key mitotic regulators.

Tan Tan, Chuanfen Wu, Boye Liu, Bih-Fang Pan, David H Hawke, Zehao Su, Shuaishuai Liu, Wei Zhang, Ruoning Wang, Sue-Hwa Lin, Jian Kuang.

The term M-phase supershift denotes the phosphorylation-dependent substantial increase in the apparent molecular weight of numerous proteins of varied biological functions during M-phase induction. Although the M-phase supershift of multiple key mitotic regulators has been attributed to the multisite phosphorylation catalyzed by the Cdk1/cyclin B/Cks complex, this view is challenged by multiple lines of paradoxical observations. To solve this problem, we reconstituted the M-phase supershift of Xenopus Cdc25C, Myt1, Wee1A, APC3 and Greatwall in Xenopus egg extracts and characterized the supershift-producing phosphorylations. Our results demonstrate that their M-phase supershifts are each due to simultaneous phosphorylation of a considerable portion of S/T/Y residues in a long intrinsically disordered region that is enriched in both S/T residues and S/TP motifs. Although the major mitotic kinases in Xenopus egg extracts, Cdk1, MAPK, Plx1 and RSK2, are able to phosphorylate the five mitotic regulators, they are neither sufficient nor required to produce the M-phase supershift. Accordingly, inhibition of the four major mitotic kinase activities in Xenopus oocytes did not inhibit the M-phase supershift in okadaic acid-induced oocyte maturation. These findings indicate that the M-phase supershift is produced by a previously unrecognized category of mitotic phosphorylation that likely plays important roles in M-phase induction.

DOI: https://doi.org/10.1091/mbc.E22-04-0118
Clin Cancer Res. 2022 Aug 16. pii: CCR-22-1627. [Epub ahead of print]

Combined TRIP13 and Aurora kinase inhibition induces apoptosis in human papillomavirus-driven cancers.

Soma Ghosh, Tuhina Mazumdar, Wei Xu, Reid T Powell, Clifford Stephan, Li Shen, Pooja A Shah, Curtis R Pickering, Jeffrey N Myers, Jing Wang, Mitchell J Frederick, Faye M Johnson.

PURPOSE: Human papillomavirus (HPV) causes >5% of cancers but no therapies uniquely target HPV-driven cancers.
EXPERIMENTAL DESIGN: We tested the cytotoxic effect of 864 drugs in 16 HPV-positive and 17 HPV-negative human squamous cancer cell lines. We confirmed apoptosis in vitro and in vivo using patient derived xenografts. Mitotic pathway components were manipulated with drugs, knockdown, and overexpression.
RESULTS: Aurora kinase inhibitors were more effective in vitro and in vivo in HPV-positive than in HPV-negative models. We hypothesized that the mechanism of sensitivity involves retinoblastoma (RB) expression because the viral oncoprotein E7 leads to RB protein degradation, and basal RB protein expression correlates with Aurora inhibition-induced apoptosis. Manipulating RB directly, or by inducing E7 expression, altered cells' sensitivity to Aurora kinase inhibitors. RB affects expression of the mitotic checkpoint genes MAD2L1 and BUB1B which we found to be highly expressed in HPV-positive patient tumors. Knockdown of MAD2L1 or BUB1B reduced Aurora kinase inhibition-induced apoptosis, whereas depletion of the MAD2L1 regulator TRIP13 enhanced it. TRIP13 is a potentially druggable AAA-ATPase. Combining Aurora kinase inhibition with TRIP13 depletion led to extensive apoptosis in HPV-positive cancer cells but not in HPV-negative cancer cells.
CONCLUSIONS: Our data support a model in which HPV-positive cancer cells maintain a balance of MAD2L1 and TRIP13 to allow mitotic exit and survival in the absence of RB. Because it does not affect cells with intact RB function, this novel combination may have a wide therapeutic window, enabling the effective treatment of RB-deficient cancers.

DOI: https://doi.org/10.1158/1078-0432.CCR-22-1627