bims-micesi 2023-01-15 papers

bims-micesi

Biomed News

on Mitotic cell signalling

Issue of 2023–01–15
ten papers selected by
Valentina Piano, Uniklinik Köln

NuMA deficiency causes micronuclei via checkpoint-insensitive k-fiber minus-end detachment from mitotic spindle poles.
Multiple Roles of PLK1 in Mitosis and Meiosis.
EMC3 regulates mesenchymal cell survival via control of the mitotic spindle assembly.
A pluripotent developmental state confers a low fidelity of chromosome segregation.
Polo-like kinase 1 (PLK1) O-GlcNAcylation is essential for dividing mammalian cells and inhibits uterine carcinoma.
A non-catalytic N-terminus domain of WRN prevents mitotic telomere deprotection.
Inhibition of acetyl-CoA carboxylase impaired tubulin palmitoylation and induced spindle abnormalities.
Annexin A1 is a polarity cue that directs mitotic spindle orientation during mammalian epithelial morphogenesis.
The Drosophila mitotic spindle orientation machinery requires activation, not just localization.
ReCasNet: Improving consistency within the two-stage mitosis detection framework.

Curr Biol. 2023 Jan 04. pii: S0960-9822(22)01917-0. [Epub ahead of print]

NuMA deficiency causes micronuclei via checkpoint-insensitive k-fiber minus-end detachment from mitotic spindle poles.

Marvin van Toorn, Amy Gooch, Susan Boerner, Tomomi Kiyomitsu.

  Micronuclei resulting from improper chromosome segregation foster chromosome rearrangements.1,2 To prevent micronuclei formation in mitosis, the dynamic plus ends of bundled kinetochore microtubules (k-fibers) must establish bipolar attachment with all sister kinetochores on chromosomes,3 whereas k-fiber minus ends must be clustered at the two opposing spindle poles, which are normally connected with centrosomes.4 The establishment of chromosome biorientation via k-fiber plus ends is carefully monitored by the spindle assembly checkpoint (SAC).5 However, how k-fiber minus-end clustering near centrosomes is maintained and monitored remains poorly understood. Here, we show that degradation of NuMA by auxin-inducible degron technologies results in micronuclei formation through k-fiber minus-end detachment from spindle poles during metaphase in HCT116 colon cancer cells. Importantly, k-fiber minus-end detachment from one pole creates misaligned chromosomes that maintain chromosome biorientation and satisfy the SAC, resulting in abnormal chromosome segregation. NuMA depletion also causes minus-end clustering defects in non-transformed Rpe1 cells, but it additionally induces centrosome detachment from partially focused poles, resulting in highly disorganized anaphase. Moreover, we find that NuMA depletion causes centrosome clustering defects in tetraploid-like cells, leading to an increased frequency of multipolar divisions. Together, our data indicate that NuMA is required for faithful chromosome segregation in human mitotic cells, generally by maintaining k-fiber minus-end clustering but also by promoting spindle pole-centrosome or centrosome-centrosome connection in specific cell types or contexts. Similar to erroneous merotelic kinetochore attachments,6 detachment of k-fiber minus ends from spindle poles evades spindle checkpoint surveillance and may therefore be a source of genomic instability in dividing cells.

Keywords:  NuMA; aneuploidy; auxin-inducible degron; centrosome clustering; chromosome mis-segregation; k-fiber minus-end clustering; micronuclei; spindle pole-centrosome connection

DOI:  https://doi.org/10.1016/j.cub.2022.12.017
Cells. 2023 Jan 02. pii: 187. [Epub ahead of print]12(1):

Multiple Roles of PLK1 in Mitosis and Meiosis.

Jaroslav Kalous, Daria Aleshkina.

  Cells are equipped with a diverse network of signaling and regulatory proteins that function as cell cycle regulators and checkpoint proteins to ensure the proper progression of cell division. A key regulator of cell division is polo-like kinase 1 (PLK1), a member of the serine/threonine kinase family that plays an important role in regulating the mitotic and meiotic cell cycle. The phosphorylation of specific substrates mediated by PLK1 controls nuclear envelope breakdown (NEBD), centrosome maturation, proper spindle assembly, chromosome segregation, and cytokinesis. In mammalian oogenesis, PLK1 is essential for resuming meiosis before ovulation and for establishing the meiotic spindle. Among other potential roles, PLK1 regulates the localized translation of spindle-enriched mRNAs by phosphorylating and thereby inhibiting the translational repressor 4E-BP1, a downstream target of the mTOR (mammalian target of rapamycin) pathway. In this review, we summarize the functions of PLK1 in mitosis, meiosis, and cytokinesis and focus on the role of PLK1 in regulating mRNA translation. However, knowledge of the role of PLK1 in the regulation of meiosis remains limited.

Keywords:  PLK1; mRNA translation; meiosis; mitosis; oocytes; polo-like kinase 1; spindle

DOI:  https://doi.org/10.3390/cells12010187
iScience. 2023 Jan 20. 26(1): 105667

EMC3 regulates mesenchymal cell survival via control of the mitotic spindle assembly.

Xiaofang Tang, Wei Wei, John M Snowball, Ernesto S Nakayasu, Sheila M Bell, Charles Ansong, Xinhua Lin, Jeffrey A Whitsett.

  Eukaryotic cells transit through the cell cycle to produce two daughter cells. Dysregulation of the cell cycle leads to cell death or tumorigenesis. Herein, we found a subunit of the ER membrane complex, EMC3, as a key regulator of cell cycle. Conditional deletion of Emc3 in mouse embryonic mesoderm led to reduced size and patterning defects of multiple organs. Emc3 deficiency impaired cell proliferation, causing spindle assembly defects, chromosome mis-segregation, cell cycle arrest at G2/M, and apoptosis. Upon entry into mitosis, mesenchymal cells upregulate EMC3 protein levels and localize EMC3 to the mitotic centrosomes. Further analysis indicated that EMC3 works together with VCP to tightly regulate the levels and activity of Aurora A, an essential factor for centrosome function and mitotic spindle assembly: while overexpression of EMC3 or VCP degraded Aurora A, their loss led to increased Aurora A stability but reduced Aurora A phosphorylation in mitosis.

Keywords:  Biological sciences; Cell biology; Molecular biology

DOI:  https://doi.org/10.1016/j.isci.2022.105667
Stem Cell Reports. 2022 Dec 24. pii: S2213-6711(22)00597-5. [Epub ahead of print]

A pluripotent developmental state confers a low fidelity of chromosome segregation.

Chenhui Deng, Amanda Ya, Duane A Compton, Kristina M Godek.

  During in vitro propagation, human pluripotent stem cells (hPSCs) frequently become aneuploid with incorrect chromosome numbers due to mitotic chromosome segregation errors. Yet, it is not understood why hPSCs exhibit a low mitotic fidelity. Here, we investigate the mechanisms responsible for mitotic errors in hPSCs and show that the primary cause is lagging chromosomes in anaphase with improper merotelic microtubule attachments. Accordingly, short-term treatment (<24 h) with small molecules that prolong mitotic duration or destabilize chromosome microtubule attachments reduces merotelic errors and lagging chromosome rates, although hPSCs adapt and lagging chromosome rates rebound upon long-term (>24 h) microtubule destabilization. Strikingly, we also demonstrate that mitotic error rates correlate with developmental potential decreasing or increasing upon loss or gain of pluripotency, respectively. Thus, a low mitotic fidelity is an inherent and conserved phenotype of hPSCs. Moreover, chromosome segregation fidelity depends on developmental state in normal human cells.

Keywords:  aneuploidy; chromosome segregation; developmental potential; human pluripotent stem cells; mitosis

DOI:  https://doi.org/10.1016/j.stemcr.2022.12.008
J Biol Chem. 2023 Jan 07. pii: S0021-9258(23)00019-4. [Epub ahead of print] 102887

Polo-like kinase 1 (PLK1) O-GlcNAcylation is essential for dividing mammalian cells and inhibits uterine carcinoma.

Sheng Yan, Bin Peng, Shifeng Kan, Guangcan Shao, Zhikai Xiahou, Xiangyan Tang, Yong-Xiang Chen, Meng-Qiu Dong, Xiao Liu, Xingzhi Xu, Jing Li.

  The O-linked N-acetylglucosamine (O-GlcNAc) transferase (OGT) mediates intracellular O-GlcNAcylation modification. O-GlcNAcylation occurs on Ser/Thr residues and is important for numerous physiological processes. OGT is essential for dividing mammalian cells, and is involved in many human diseases; however, many of its fundamental substrates during cell division remain unknown. Here we focus on the effect of OGT on polo-like kinase 1 (PLK1), a mitotic master kinase that governs DNA replication, mitotic entry, chromosome segregation, and mitotic exit. We show that PLK1 interacts with OGT and is O-GlcNAcylated. By utilizing stepped collisional energy/higher-energy collisional dissociation (sceHCD) mass spectrometry (MS), we found a peptide fragment of PLK1 that is modified by O-GlcNAc. Further mutation analysis of PLK1 shows that the T291A mutant decreases O-GlcNAcylation. Interestingly, T291N is a uterine carcinoma mutant in the TCGA database. Our biochemical assays demonstrate that T291A and T291N both increase PLK1 stability. Using stable H2B-GFP cells, we found that PLK1-T291A and -T291N mutants display chromosome segregation defects, and result in misaligned and lagging chromosomes. In mouse xenograft models, we demonstrate that the O-GlcNAc-deficient PLK1-T291A and -T291N mutants enhance uterine carcinoma in animals. Hence, we propose that OGT partially exerts its mitotic function through O-GlcNAcylation of PLK1, which might be one mechanism by which elevated levels of O-GlcNAc promote tumorigenesis.

Keywords:  O-GlcNAc; PLK1; mitosis; ubiquitination; uterine carcinoma

DOI:  https://doi.org/10.1016/j.jbc.2023.102887
Sci Rep. 2023 Jan 12. 13(1): 645

A non-catalytic N-terminus domain of WRN prevents mitotic telomere deprotection.

Diana Romero-Zamora, Makoto T Hayashi.

Telomeric ends form a loop structure (T-loop) necessary for the repression of ATM kinase activation throughout the normal cell cycle. However, cells undergoing a prolonged mitotic arrest are prone to lose the T-loop, resulting in Aurora B kinase-dependent mitotic telomere deprotection, which was proposed as an anti-tumor mechanism that eliminates precancerous cells from the population. The mechanism of mitotic telomere deprotection has not been elucidated. Here, we show that WRN, a RECQ helicase family member, can suppress mitotic telomere deprotection independently of its exonuclease and helicase activities. Truncation of WRN revealed that N-terminus amino acids 168-333, a region that contains a coiled-coil motif, is sufficient to suppress mitotic telomere deprotection without affecting both mitotic Aurora B-dependent spindle checkpoint and ATM kinase activity. The suppressive activity of the WRN168-333 fragment is diminished in cells partially depleted of TRF2, while WRN is required for complete suppression of mitotic telomere deprotection by TRF2 overexpression. Finally, we found that phosphomimetic but not alanine mutations of putative Aurora B target sites in the WRN168-333 fragment abolished its suppressive effect. Our findings reveal a non-enzymatic function of WRN, which may be regulated by phosphorylation in cells undergoing mitotic arrest. We propose that WRN enhances the protective function of TRF2 to counteract the hypothetical pathway that resolves the mitotic T-loop.

DOI: https://doi.org/10.1038/s41598-023-27598-0
Cell Death Discov. 2023 Jan 09. 9(1): 4

Inhibition of acetyl-CoA carboxylase impaired tubulin palmitoylation and induced spindle abnormalities.

Chieh-Ting Fang, Hsiao-Hui Kuo, Oyundari Amartuvshin, Hwei-Jan Hsu, Sih-Long Liu, Jhong-Syuan Yao, Ling-Huei Yih.

Tubulin s-palmitoylation involves the thioesterification of a cysteine residue in tubulin with palmitate. The palmitate moiety is produced by the fatty acid synthesis pathway, which is rate-limited by acetyl-CoA carboxylase (ACC). While it is known that ACC is phosphorylated at serine 79 (pSer79) by AMPK and accumulates at the spindle pole (SP) during mitosis, a functional role for tubulin palmitoylation during mitosis has not been identified. In this study, we found that modulating pSer79-ACC level at the SP using AMPK agonist and inhibitor induced spindle defects. Loss of ACC function induced spindle abnormalities in cell lines and in germ cells of the Drosophila germarium, and palmitic acid (PA) rescued the spindle defects in the cell line treated transiently with the ACC inhibitor, TOFA. Furthermore, inhibition of protein palmitoylating or depalmitoylating enzymes also induced spindle defects. Together, these data suggested that precisely regulated cellular palmitate level and protein palmitoylation may be required for accurate spindle assembly. We then showed that tubulin was largely palmitoylated in interphase cells but less palmitoylated in mitotic cells. TOFA treatment diminished tubulin palmitoylation at doses that disrupt microtubule (MT) instability and cause spindle defects. Moreover, spindle MTs comprised of α-tubulins mutated at the reported palmitoylation site exhibited disrupted dynamic instability. We also found that TOFA enhanced the MT-targeting drug-induced spindle abnormalities and cytotoxicity. Thus, our study reveals that precise regulation of ACC during mitosis impacts tubulin palmitoylation to delicately control MT dynamic instability and spindle assembly, thereby safeguarding nuclear and cell division.

DOI: https://doi.org/10.1038/s41420-023-01301-8
Nat Commun. 2023 Jan 11. 14(1): 151

Annexin A1 is a polarity cue that directs mitotic spindle orientation during mammalian epithelial morphogenesis.

Maria Fankhaenel, Farahnaz S Golestan Hashemi, Larissa Mourao, Emily Lucas, Manal M Hosawi, Paul Skipp, Xavier Morin, Colinda L G J Scheele, Salah Elias.

Oriented cell divisions are critical for the formation and maintenance of structured epithelia. Proper mitotic spindle orientation relies on polarised anchoring of force generators to the cell cortex by the evolutionarily conserved protein complex formed by the Gαi subunit of heterotrimeric G proteins, the Leucine-Glycine-Asparagine repeat protein (LGN) and the nuclear mitotic apparatus protein. However, the polarity cues that control cortical patterning of this ternary complex remain largely unknown in mammalian epithelia. Here we identify the membrane-associated protein Annexin A1 (ANXA1) as an interactor of LGN in mammary epithelial cells. Annexin A1 acts independently of Gαi to instruct the accumulation of LGN and nuclear mitotic apparatus protein at the lateral cortex to ensure cortical anchoring of Dynein-Dynactin and astral microtubules and thereby planar alignment of the mitotic spindle. Loss of Annexin A1 randomises mitotic spindle orientation, which in turn disrupts epithelial architecture and luminogenesis in three-dimensional cultures of primary mammary epithelial cells. Our findings establish Annexin A1 as an upstream cortical cue that regulates LGN to direct planar cell divisions during mammalian epithelial morphogenesis.

DOI: https://doi.org/10.1038/s41467-023-35881-x
EMBO Rep. 2023 Jan 11. e56074

The Drosophila mitotic spindle orientation machinery requires activation, not just localization.

Kathryn E Neville, Tara M Finegan, Nicholas Lowe, Philip M Bellomio, Daxiang Na, Dan T Bergstralh.

  The orientation of the mitotic spindle at metaphase determines the placement of the daughter cells. Spindle orientation in animals typically relies on an evolutionarily conserved biological machine comprised of at least four proteins - called Pins, Gαi, Mud, and Dynein in flies - that exerts a pulling force on astral microtubules and reels the spindle into alignment. The canonical model for spindle orientation holds that the direction of pulling is determined by asymmetric placement of this machinery at the cell cortex. In most cell types, this placement is thought to be mediated by Pins, and a substantial body of literature is therefore devoted to identifying polarized cues that govern localized cortical enrichment of Pins. In this study we revisit the canonical model and find that it is incomplete. Spindle orientation in the Drosophila follicular epithelium and embryonic ectoderm requires not only Pins localization but also direct interaction between Pins and the multifunctional protein Discs large. This requirement can be over-ridden by interaction with another Pins interacting protein, Inscuteable.

Keywords:  Drosophila; discs large; pins; spindle orientation

DOI:  https://doi.org/10.15252/embr.202256074
Artif Intell Med. 2023 Jan;pii: S0933-3657(22)00214-7. [Epub ahead of print]135 102462

ReCasNet: Improving consistency within the two-stage mitosis detection framework.

Chawan Piansaddhayanaon, Sakun Santisukwongchote, Shanop Shuangshoti, Qingyi Tao, Sira Sriswasdi, Ekapol Chuangsuwanich.

  Mitotic count (MC) is an important histological parameter for cancer diagnosis and grading, but the manual process for obtaining MC from whole-slide histopathological images is very time-consuming and prone to error. Therefore, deep learning models have been proposed to facilitate this process. Existing approaches utilize a two-stage pipeline: the detection stage for identifying the locations of potential mitotic cells and the classification stage for refining prediction confidences. However, this pipeline formulation can lead to inconsistencies in the classification stage due to the poor prediction quality of the detection stage and the mismatches in training data distributions between the two stages. In this study, we propose a Refine Cascade Network (ReCasNet), an enhanced deep learning pipeline that mitigates the aforementioned problems with three improvements. First, window relocation was used to reduce the number of poor quality false positives generated during the detection stage. Second, object re-cropping was performed with another deep learning model to adjust poorly centered objects. Third, improved data selection strategies were introduced during the classification stage to reduce the mismatches in training data distributions. ReCasNet was evaluated on two large-scale mitotic figure recognition datasets, canine cutaneous mast cell tumor (CCMCT) and canine mammary carcinoma (CMC), which resulted in up to 4.8% percentage point improvements in the F1 scores for mitotic cell detection and 44.1% reductions in mean absolute percentage error (MAPE) for MC prediction. Techniques that underlie ReCasNet can be generalized to other two-stage object detection pipeline and should contribute to improving the performances of deep learning models in broad digital pathology applications.

Keywords:  Image classification; Mitotic count; Multi-stage deep learning; Object detection; Whole slide image

DOI:  https://doi.org/10.1016/j.artmed.2022.102462