bims-micesi 2022-08-07 papers

bims-micesi

Biomed News

on Mitotic cell signalling

Issue of 2022–08–07
nine papers selected by
Valentina Piano, Max Planck Institute of Molecular Physiology

Uncoupling of Mitosis and Cytokinesis Upon a Prolonged Arrest in Metaphase Is Influenced by Protein Phosphatases and Mitotic Transcription in Fission Yeast.
Transcription factor Sp1 regulates mitotic chromosome assembly and segregation.
Length-dependent poleward flux of sister kinetochore fibers promotes chromosome alignment.
The E3 ubiquitin ligase HECTD1 contributes to cell proliferation through an effect on mitosis.
Modest increase of KIF11 exposes fragilities in the mitotic spindle causing chromosomal instability.
Dual role of deubiquitinating enzyme USP19 regulates mitotic progression and tumorigenesis by stabilizing survivin.
Moonlighting at the Poles: Non-Canonical Functions of Centrosomes.
A mitotic chromatin phase transition prevents perforation by microtubules.
Germline Missense Variants in CDC20 Result in Aberrant Mitotic Progression and Familial Cancer.

Front Cell Dev Biol. 2022 ;10 876810

Uncoupling of Mitosis and Cytokinesis Upon a Prolonged Arrest in Metaphase Is Influenced by Protein Phosphatases and Mitotic Transcription in Fission Yeast.

Nathalia Chica, Marina Portantier, Mari Nyquist-Andersen, Silvia Espada-Burriel, Sandra Lopez-Aviles.

  Depletion of the Anaphase-Promoting Complex/Cyclosome (APC/C) activator Cdc20 arrests cells in metaphase with high levels of the mitotic cyclin (Cyclin B) and the Separase inhibitor Securin. In mammalian cells this arrest has been exploited for the treatment of cancer with drugs that engage the spindle assembly checkpoint and, recently, with chemical inhibitors of the APC/C. While most cells arrested in mitosis for prolonged periods undergo apoptosis, others skip cytokinesis and enter G1 with unsegregated chromosomes. This process, known as mitotic slippage, generates aneuploidy and increases genomic instability in the cancer cell. Here, we analyze the behavior of fission yeast cells arrested in mitosis through the transcriptional silencing of the Cdc20 homolog slp1. While depletion of slp1 readily halts cells in metaphase, this arrest is only transient and a majority of cells eventually undergo cytokinesis and show steady mitotic dephosphorylation. Notably, this occurs in the absence of Cyclin B (Cdc13) degradation. We investigate the involvement of phosphatase activity in these events and demonstrate that PP2A-B55Pab1 is required to prevent septation and, during the arrest, its CDK-mediated inhibition facilitates the induction of cytokinesis. In contrast, deletion of PP2A-B56Par1 completely abrogates septation. We show that this effect is partly due to this mutant entering mitosis with reduced CDK activity. Interestingly, both PP2A-B55Pab1 and PP2A-B56Par1, as well as Clp1 (the homolog of the budding yeast mitotic phosphatase Cdc14) are required for the dephosphorylation of mitotic substrates during the escape. Finally, we show that the mitotic transcriptional wave controlled by the RFX transcription factor Sak1 facilitates the induction of cytokinesis and also requires the activity of PP2A-B56Par1 in a mechanism independent of CDK.

Keywords:  APC/CSlp1; CDK; PP2A-B55; PP2A-B56; cytokinesis; fission yeast; metaphase arrest; phosphatases

DOI:  https://doi.org/10.3389/fcell.2022.876810
Chromosoma. 2022 Aug 02.

Transcription factor Sp1 regulates mitotic chromosome assembly and segregation.

Samuel Flashner, Michelle Swift, Aislinn Sowash, Alexander N Fahmy, Jane Azizkhan-Clifford.

  Aneuploidy is a pervasive feature of cancer cells that results from chromosome missegregation. Several transcription factors have been associated with aneuploidy; however, no studies to date have demonstrated that mammalian transcription factors directly regulate chromosome segregation during mitosis. Here, we demonstrate that the ubiquitously expressed transcription factor specificity protein 1 (Sp1), which we have previously linked to aneuploidy, has a mitosis-specific role regulating chromosome segregation. We find that Sp1 localizes to mitotic centromeres and auxin-induced rapid Sp1 degradation at mitotic onset results in chromosome segregation errors and aberrant mitotic progression. Furthermore, rapid Sp1 degradation results in anomalous mitotic chromosome assembly characterized by loss of condensin complex I localization to mitotic chromosomes and chromosome condensation defects. Consistent with these defects, Sp1 degradation results in reduced chromosome passenger complex activity and histone H3 serine 10 phosphorylation during mitosis, which is essential for condensin complex I recruitment and chromosome condensation. Together, these data provide the first evidence of a mammalian transcription factor acting specifically during mitosis to regulate chromosome segregation.

Keywords:  Chromosome passenger complex; Chromosome segregation; Condensin complex I; Mitosis; Sp1; Transcription factor

DOI:  https://doi.org/10.1007/s00412-022-00778-z
Cell Rep. 2022 Aug 02. pii: S2211-1247(22)00982-2. [Epub ahead of print]40(5): 111169

Length-dependent poleward flux of sister kinetochore fibers promotes chromosome alignment.

Patrik Risteski, Domagoj Božan, Mihaela Jagrić, Agneza Bosilj, Nenad Pavin, Iva M Tolić.

  Chromosome alignment at the spindle equator promotes proper chromosome segregation and depends on pulling forces exerted at kinetochore fiber tips together with polar ejection forces. However, kinetochore fibers are also subjected to forces driving their poleward flux. Here we introduce a flux-driven centering model that relies on flux generated by forces within the overlaps of bridging and kinetochore fibers. This centering mechanism works so that the longer kinetochore fiber fluxes faster than the shorter one, moving the kinetochores toward the center. We develop speckle microscopy in human spindles and confirm the key prediction that kinetochore fiber flux is length dependent. Kinetochores are better centered when overlaps are shorter and the kinetochore fiber flux slower than the bridging fiber flux. We identify Kif18A and Kif4A as overlap and flux regulators and NuMA as a fiber coupler. Thus, length-dependent sliding forces exerted by the bridging fiber onto kinetochore fibers support chromosome alignment.

Keywords:  CP: Cell biology; chromosome alignment; kinetochore; mathematical model; microtubule poleward flux; mitotic spindle; speckle microscopy

DOI:  https://doi.org/10.1016/j.celrep.2022.111169
Sci Rep. 2022 Aug 01. 12(1): 13160

The E3 ubiquitin ligase HECTD1 contributes to cell proliferation through an effect on mitosis.

Natalie Vaughan, Nico Scholz, Catherine Lindon, Julien D F Licchesi.

The cell cycle is tightly regulated by protein phosphorylation and ubiquitylation events. During mitosis, the multi-subunit cullin-RING E3 ubiquitin ligase APC/c functions as a molecular switch which signals for one cell to divide into two daughter cells, through the ubiquitylation and proteasomal degradation of mitotic cyclins. The contributions of other E3 ligase families during cell cycle progression remain less well understood. Similarly, the roles of ubiquitin chain types beyond homotypic K48 chains in S-phase or branched K11/K48 chains during mitosis, also remain to be fully determined. Our recent findings that HECTD1 ubiquitin ligase activity assembles branched K29/K48 ubiquitin linkages prompted us to evaluate HECTD1 function during the cell cycle. We used transient knockdown and genetic knockout to show that HECTD1 depletion in HEK293T and HeLa cells decreases cell number and we established that this is mediated through loss of ubiquitin ligase activity. Interestingly, we found that HECTD1 depletion increases the proportion of cells with aligned chromosomes (Prometa/Metaphase) and we confirmed this molecularly using phospho-Histone H3 (Ser28) as a marker of mitosis. Time-lapse microscopy of NEBD to anaphase onset established that HECTD1-depleted cells take on average longer to go through mitosis. In line with this data, HECTD1 depletion reduced the activity of the Spindle Assembly Checkpoint, and BUB3, a component of the Mitosis Checkpoint Complex, was identified as novel HECTD1 interactor. BUB3, BUBR1 or MAD2 protein levels remained unchanged in HECTD1-depleted cells. Overall, this study reveals a novel putative role for HECTD1 during mitosis and warrants further work to elucidate the mechanisms involved.

DOI: https://doi.org/10.1038/s41598-022-16965-y
J Cell Sci. 2022 Aug 05. pii: jcs.260031. [Epub ahead of print]

Modest increase of KIF11 exposes fragilities in the mitotic spindle causing chromosomal instability.

Katie L Dale, Jonathan W Armond, Robert E Hynds, Elina Vladimirou.

  Chromosomal instability (CIN), the process of increased chromosomal alterations, compromises genomic integrity and has profound consequences on human health. Yet, our understanding of the molecular and mechanistic basis of CIN initiation remains limited. We developed a high-throughput, single-cell image-based pipeline employing deep learning and spot counting models to detect CIN by automatically counting chromosomes and micronuclei. To identify CIN-initiating conditions, we used CRISPR activation in human diploid cells to upregulate, at physiologically-relevant levels, 14 genes that are functionally important in cancer. We found that upregulation of CCND1, FOXA1, and NEK2 resulted in pronounced changes in chromosome counts and KIF11 upregulation resulted in micronuclei formation. We identified KIF11-dependent fragilities within the mitotic spindle; increased KIF11 causes centrosome fragmentation, higher microtubule stability, lagging chromosomes or mitotic catastrophe. Our findings demonstrate that even modest average single gene expression changes in a karyotypically stable background are sufficient for initiating CIN by exposing fragilities of the mitotic spindle which can lead to a genomically-diverse cell population.

Keywords:  CRISPR activation; Chromosomal instability; Confocal imaging; Deep learning; KIF11; Mitosis

DOI:  https://doi.org/10.1242/jcs.260031
Mol Ther. 2022 Aug 01. pii: S1525-0016(22)00440-3. [Epub ahead of print]

Dual role of deubiquitinating enzyme USP19 regulates mitotic progression and tumorigenesis by stabilizing survivin.

Arun Pandian Chandrasekaran, Apoorvi Tyagi, Naresh Poondla, Neha Sarodaya, Janardhan Keshav Karapurkar, Kamini Kaushal, Chang-Hwan Park, Seok-Ho Hong, Kye-Seong Kim, Suresh Ramakrishna.

  Survivin is a component of the chromosomal passenger complex, which includes Aurora B, INCENP and Borealin, and is required for chromosome segregation and cytokinesis. We performed a genome-wide screen of deubiquitinating enzymes for survivin. For the first time, we report that USP19 has a dual role in the modulation of mitosis and tumorigenesis by regulating survivin expression. Our results found that USP19 stabilizes and interacts with survivin in HCT116 cells. USP19 deubiquitinates survivin protein and extends its half-life. We also found that USP19 functions as a mitotic regulator by controlling the downstream signaling of survivin protein. Targeted genome knockout verified that USP19 depletion leads to several mitotic defects, including cytokinesis failure. Additionally, USP19 depletion results in significant enrichment of apoptosis and reduces the growth of tumors in the mouse xenograft. We envision that simultaneous targeting of USP19 and survivin in oncologic drug development would increase therapeutic value and minimize redundancy.

Keywords:  USP19; apoptosis; chromosome; deubiquitinating enzyme; mitosis; survivin

DOI:  https://doi.org/10.1016/j.ymthe.2022.07.019
Front Cell Dev Biol. 2022 ;10 930355

Moonlighting at the Poles: Non-Canonical Functions of Centrosomes.

Laurence Langlois-Lemay, Damien D'Amours.

  Centrosomes are best known as the microtubule organizing centers (MTOCs) of eukaryotic cells. In addition to their classic role in chromosome segregation, centrosomes play diverse roles unrelated to their MTOC activity during cell proliferation and quiescence. Metazoan centrosomes and their functional doppelgängers from lower eukaryotes, the spindle pole bodies (SPBs), act as important structural platforms that orchestrate signaling events essential for cell cycle progression, cellular responses to DNA damage, sensory reception and cell homeostasis. Here, we provide a critical overview of the unconventional and often overlooked roles of centrosomes/SPBs in the life cycle of eukaryotic cells.

Keywords:  Cdc5; MTOCs; PLK1; cell cycle; centrosomes; spindle pole bodies

DOI:  https://doi.org/10.3389/fcell.2022.930355
Nature. 2022 Aug 03.

A mitotic chromatin phase transition prevents perforation by microtubules.

Maximilian W G Schneider, Bryan A Gibson, Shotaro Otsuka, Maximilian F D Spicer, Mina Petrovic, Claudia Blaukopf, Christoph C H Langer, Paul Batty, Thejaswi Nagaraju, Lynda K Doolittle, Michael K Rosen, Daniel W Gerlich.

Dividing eukaryotic cells package extremely long chromosomal DNA molecules into discrete bodies to enable microtubule-mediated transport of one genome copy to each of the newly forming daughter cells1-3. Assembly of mitotic chromosomes involves DNA looping by condensin4-8 and chromatin compaction by global histone deacetylation9-13. Although condensin confers mechanical resistance to spindle pulling forces14-16, it is not known how histone deacetylation affects material properties and, as a consequence, segregation mechanics of mitotic chromosomes. Here we show how global histone deacetylation at the onset of mitosis induces a chromatin-intrinsic phase transition that endows chromosomes with the physical characteristics necessary for their precise movement during cell division. Deacetylation-mediated compaction of chromatin forms a structure dense in negative charge and allows mitotic chromosomes to resist perforation by microtubules as they are pushed to the metaphase plate. By contrast, hyperacetylated mitotic chromosomes lack a defined surface boundary, are frequently perforated by microtubules and are prone to missegregation. Our study highlights the different contributions of DNA loop formation and chromatin phase separation to genome segregation in dividing cells.

DOI: https://doi.org/10.1038/s41586-022-05027-y
Cancer Res. 2022 Aug 01. pii: CAN-21-3956. [Epub ahead of print]

Germline Missense Variants in CDC20 Result in Aberrant Mitotic Progression and Familial Cancer.

Owen J Chen, Ester Castellsagué, Mohamed Moustafa-Kamal, Javad Nadaf, Barbara Rivera, Somayyeh Fahiminiya, Yilin Wang, Isabelle Gamache, Caterina Pacifico, Lai Jiang, Jian Carrot-Zhang, Leora Witkowski, Albert M Berghuis, Stefan Schoenberger, Dominik Schneider, Morten Hillmer, Susanne Bens, Reiner Siebert, Colin J R Stewart, Ziguo Zhang, William C H Chao, Celia M T Greenwood, David Barford, Marc Tischkowitz, Jacek Majewski, William D Foulkes, Jose G Teodoro.

CDC20 is a co-activator of the anaphase promoting complex/cyclosome (APC/C) and is essential for mitotic progression. APC/CCDC20 is inhibited by the spindle assembly checkpoint (SAC), which prevents premature separation of sister chromatids and aneuploidy in daughter cells. Although overexpression of CDC20 is common in many cancers, oncogenic mutations have never been identified in humans. Using whole exome sequencing, we identified heterozygous missense CDC20 variants (L151R and N331K) that segregate with ovarian germ cell tumors in two families. Functional characterization showed these mutants retain APC/C activation activity but have impaired binding to BUBR1, a component of the SAC. Expression of L151R and N331K variants promoted mitotic slippage in HeLa cells and primary skin fibroblasts derived from carriers. Generation of mice carrying the N331K variant using CRISPR-Cas9 showed that, while homozygous N331K mice were non-viable, heterozygotes displayed accelerated oncogenicity of Myc-driven cancers. These findings highlight an unappreciated role for CDC20 variants as tumor-promoting genes.

DOI: https://doi.org/10.1158/0008-5472.CAN-21-3956