bims-micesi 2024-02-04 papers

bims-micesi

Biomed News

on Mitotic cell signalling

Issue of 2024–02–04
thirteen papers selected by
Valentina Piano, Uniklinik Köln

An unconventional regulatory circuitry involving Aurora B controls anaphase onset and error-free chromosome segregation in trypanosomes.
Kinetochore-microtubule error correction for biorientation: lessons from yeast.
Fission yeast spindle dynamics and chromosome segregation fidelity show distinct thermosensitivity.
Ran-GTP assembles a specialized spindle structure for accurate chromosome segregation in medaka early embryos.
Cell cycle and mitosis progression during ZIKA virus infection: The viral non-structural protein NS5 as a master regulator of the APC/cyclosome?
Calreticulin and JAK2V617F driver mutations induce distinct mitotic defects in myeloproliferative neoplasms.
A centromere-derived retroelement RNA localizes in cis and is a core element of the transcriptional landscape of Drosophila centromeres.
Combinatorial regulation by ERK1/2 and CK1δ protein kinases leads to HIF-1α association with microtubules and facilitates its symmetrical distribution during mitosis.
A human papillomavirus 16 E2-TopBP1 dependent SIRT1-p300 acetylation switch regulates mitotic viral and human protein levels.
Computational Studies Reveal How Passive Cross-Linkers Regulate Anaphase Spindle Elongation.
A missense SNP in the tumor suppressor SETD2 reduces H3K36me3 and mitotic spindle integrity in Drosophila.
Multiscale biophysical analysis of nucleolus disassembly during mitosis.
Environmental physical factors produce mitotic aberration and multicellularity in the ovarian cancer cell line SKOV3.

bioRxiv. 2024 Jan 20. pii: 2024.01.20.576407. [Epub ahead of print]

An unconventional regulatory circuitry involving Aurora B controls anaphase onset and error-free chromosome segregation in trypanosomes.

Daniel Ballmer, Hua Jane Lou, Midori Ishii, Benjamin E Turk, Bungo Akiyoshi.

Accurate chromosome segregation during mitosis requires that all chromosomes establish stable bi-oriented attachments with the spindle apparatus. Kinetochores form the interface between chromosomes and spindle microtubules and as such are under tight control by complex regulatory circuitry. As part of the chromosomal passenger complex (CPC), the Aurora B kinase plays a central role within this circuitry by destabilizing improper kinetochore-microtubule attachments and relaying the attachment status to the spindle assembly checkpoint, a feedback control system that delays the onset of anaphase by inhibiting the anaphase-promoting complex/cyclosome. Intriguingly, Aurora B is conserved even in kinetoplastids, an evolutionarily divergent group of eukaryotes, whose kinetochores are composed of a unique set of structural and regulatory proteins. Kinetoplastids do not have a canonical spindle checkpoint and it remains unclear how their kinetochores are regulated to ensure the fidelity and timing of chromosome segregation. Here, we show in Trypanosoma brucei , the kinetoplastid parasite that causes African sleeping sickness, that inhibition of Aurora B using an analogue-sensitive approach arrests cells in metaphase, with a reduction in properly bi-oriented kinetochores. Aurora B phosphorylates several kinetochore proteins in vitro , including the N-terminal region of the divergent Bub1-like protein KKT14. Depletion of KKT14 partially overrides the cell cycle arrest caused by Aurora B inhibition, while overexpression of a non-phosphorylatable KKT14 protein results in a prominent delay in the metaphase-to-anaphase transition. Finally, we demonstrate using a nanobody-based system that re-targeting the catalytic module of the CPC to the outer kinetochore is sufficient to promote mitotic exit but causes massive chromosome mis-segregation in anaphase. Our results indicate that the CPC and KKT14 are involved in an unconventional pathway controlling mitotic exit and error-free chromosome segregation in trypanosomes.

DOI: https://doi.org/10.1101/2024.01.20.576407
Biochem Soc Trans. 2024 Feb 02. pii: BST20221261. [Epub ahead of print]

Kinetochore-microtubule error correction for biorientation: lessons from yeast.

Shuyu Li, Taciana Kasciukovic, Tomoyuki U Tanaka.

  Accurate chromosome segregation in mitosis relies on sister kinetochores forming stable attachments to microtubules (MTs) extending from opposite spindle poles and establishing biorientation. To achieve this, erroneous kinetochore-MT interactions must be resolved through a process called error correction, which dissolves improper kinetochore-MT attachment and allows new interactions until biorientation is achieved. The Aurora B kinase plays key roles in driving error correction by phosphorylating Dam1 and Ndc80 complexes, while Mps1 kinase, Stu2 MT polymerase and phosphatases also regulate this process. Once biorientation is formed, tension is applied to kinetochore-MT interaction, stabilizing it. In this review article, we discuss the mechanisms of kinetochore-MT interaction, error correction and biorientation. We focus mainly on recent insights from budding yeast, where the attachment of a single MT to a single kinetochore during biorientation simplifies the analysis of error correction mechanisms.

Keywords:  budding yeast; chromosome biorientation; chromosome segregation; error correction; kinetochore; microtubule

DOI:  https://doi.org/10.1042/BST20221261
MicroPubl Biol. 2024 ;2024

Fission yeast spindle dynamics and chromosome segregation fidelity show distinct thermosensitivity.

Zachary Chaba, Ishutesh Jain, Phong T Tran.

Cellular processes rely on proteins with temperature-dependent stability and activity. While thermosensitivity in biological networks is well-explored, the effect of temperature on complex mechanochemical assemblies, like the spindle, is rarely studied. We examined fission yeast spindle dynamics and chromosome segregation from 15⁰C to 40⁰C. Our findings reveal that these parameters follow U-shaped temperature-dependent curves but reach their minima at different temperatures. Specifically, spindle dynamics peak around 35⁰C, whereas chromosome segregation defects are minimized at 25⁰C. This suggests a scenario in which mitotic errors are tolerated to expedite rapid cell cycle progression.

DOI: https://doi.org/10.17912/micropub.biology.001048
Nat Commun. 2024 Feb 01. 15(1): 981

Ran-GTP assembles a specialized spindle structure for accurate chromosome segregation in medaka early embryos.

Ai Kiyomitsu, Toshiya Nishimura, Shiang Jyi Hwang, Satoshi Ansai, Masato T Kanemaki, Minoru Tanaka, Tomomi Kiyomitsu.

Despite drastic cellular changes during cleavage, a mitotic spindle assembles in each blastomere to accurately segregate duplicated chromosomes. Mechanisms of mitotic spindle assembly have been extensively studied using small somatic cells. However, mechanisms of spindle assembly in large vertebrate embryos remain little understood. Here, we establish functional assay systems in medaka (Oryzias latipes) embryos by combining CRISPR knock-in with auxin-inducible degron technology. Live imaging reveals several unexpected features of microtubule organization and centrosome positioning that achieve rapid, accurate cleavage. Importantly, Ran-GTP assembles a dense microtubule network at the metaphase spindle center that is essential for chromosome segregation in early embryos. This unique spindle structure is remodeled into a typical short, somatic-like spindle after blastula stages, when Ran-GTP becomes dispensable for chromosome segregation. We propose that despite the presence of centrosomes, the chromosome-derived Ran-GTP pathway has essential roles in functional spindle assembly in large, rapidly dividing vertebrate early embryos, similar to acentrosomal spindle assembly in oocytes.

DOI: https://doi.org/10.1038/s41467-024-45251-w
Biochimie. 2024 Jan 31. pii: S0300-9084(24)00034-8. [Epub ahead of print]

Cell cycle and mitosis progression during ZIKA virus infection: The viral non-structural protein NS5 as a master regulator of the APC/cyclosome?

Grégorie Lebeau, Mathilde Hoareau, Sébastien Rivière, Daed El Safadi, Christine Robert Da Silva, Pascale Krejbich-Trotot, Wildriss Viranaicken.

  Alterations in cell cycle regulation contribute to Zika virus (ZIKV)-associated pathogenesis and may have implications for the development of therapeutic avenues. As a matter of fact, ZIKV alters cell cycle progression at multiple stages, including G1, S, G2, and M phases. During a cell cycle, the progression of mitosis is particularly controlled to avoid any abnormalities in cell division. In this regard, the critical metaphase-anaphase transition is triggered by the activation of anaphase-promoting complex/cyclosome (APC/C) by its E3 ubiquitin ligase subunit Cdc20. Cdc20 recognizes substrates by interacting with a destruction box motif (D-box). Recently, the ZIKV nonstructural protein 5 (NS5), one of the most highly conserved flavivirus proteins, has been shown to localize to the centrosome in each pole and to spindle fibers during mitosis. Inducible expression of NS5 reveals an interaction of this viral factor with centrosomal proteins leading to an increase in the time required to complete mitosis. By analyzing the NS5 sequence, we discovered the presence of a D-box. Taken together, these data support the idea that, in addition to its role in viral replication, NS5 plays a critical role in the control of the cell cycle of infected cells and, more specifically, in the regulation of the mitotic spindle. Here we propose that the NS5 protein may interfere with the metaphase-anaphase progression, and thus cause the observed delay in mitosis via the regulation of APC/C.

Keywords:  Anaphase promoting complex; Cell cycle; Cyclosome; Flavivirus; NS5; Zika virus

DOI:  https://doi.org/10.1016/j.biochi.2024.01.016
Sci Rep. 2024 Feb 02. 14(1): 2810

Calreticulin and JAK2V617F driver mutations induce distinct mitotic defects in myeloproliferative neoplasms.

Kristin Holl, Nicolas Chatain, Susanne Krapp, Julian Baumeister, Tiago Maié, Sarah Schmitz, Anja Scheufen, Nathalie Brock, Steffen Koschmieder, Daniel Moreno-Andrés.

Myeloproliferative neoplasms (MPNs) encompass a diverse group of hematologic disorders driven by mutations in JAK2, CALR, or MPL. The prevailing working model explaining how these driver mutations induce different disease phenotypes is based on the decisive influence of the cellular microenvironment and the acquisition of additional mutations. Here, we report increased levels of chromatin segregation errors in hematopoietic cells stably expressing CALRdel52 or JAK2V617F mutations. Our investigations employing murine 32DMPL and human erythroleukemic TF-1MPL cells demonstrate a link between CALRdel52 or JAK2V617F expression and a compromised spindle assembly checkpoint (SAC), a phenomenon contributing to error-prone mitosis. This defective SAC is associated with imbalances in the recruitment of SAC factors to mitotic kinetochores upon CALRdel52 or JAK2V617F expression. We show that JAK2 mutant CD34 + MPN patient-derived cells exhibit reduced expression of the master mitotic regulators PLK1, aurora kinase B, and PP2A catalytic subunit. Furthermore, the expression profile of mitotic regulators in CD34 + patient-derived cells allows to faithfully distinguish patients from healthy controls, as well as to differentiate primary and secondary myelofibrosis from essential thrombocythemia and polycythemia vera. Altogether, our data suggest alterations in mitotic regulation as a potential driver in the pathogenesis in MPN.

DOI: https://doi.org/10.1038/s41598-024-53240-8
bioRxiv. 2024 Jan 16. pii: 2024.01.14.574223. [Epub ahead of print]

A centromere-derived retroelement RNA localizes in cis and is a core element of the transcriptional landscape of Drosophila centromeres.

B Santinello, R Sun, A Amjad, S J Hoyt, L Ouyang, C Courret, R Drennan, L Leo, A M Larracuente, L M Core, R J O'Neill, B G Mellone.

Centromeres are essential chromosomal landmarks that dictate the point of attachment between chromosomes and spindle microtubules during cell division. The stable transmission of the centromere site through generations is ensured by a unique chromatin containing the histone H3 variant CENP-A. Previous studies have highlighted the impact of transcription on promoting CENP-A deposition. However, the specific sequences undergoing this transcription and their contribution to centromere function in metazoan systems remain elusive. In this study, we unveil the centromeric transcriptional landscape and explore its correlation with CENP-A in D. melanogaster , currently the only in vivo model with assembled centromeres. We find that the centromere-enriched retroelement G2/Jockey-3 (hereafter referred to as Jockey-3 ) is a major driver of centromere transcription, producing RNAs that localize to all mitotic centromeres, with the Y centromere showing the most transcription. Taking advantage of the polymorphism of Jockey-3 , we show that these RNAs remain associated with their cognate DNA sequences in cis . Using a LacI/lacO system to generate de novo centromeres, we find that Jockey-3 transcripts do not localize to ectopic sites, suggesting they are unlikely to function as non-coding RNAs with a structural role at centromeres. At de novo centromeres on the lacO array, the presence of CENP-A augments the detection of exogenous lacO-derived transcripts specifically in metaphase. We propose that Jockey-3 contributes to the epigenetic maintenance of the centromere by promoting chromatin transcription, while inserting in a region that permits its continuous transmission. Given the conservation of retroelements as centromere components across taxa, our findings have broad implications in understanding this widespread association.

DOI: https://doi.org/10.1101/2024.01.14.574223
Cell Mol Life Sci. 2024 Feb 01. 81(1): 72

Combinatorial regulation by ERK1/2 and CK1δ protein kinases leads to HIF-1α association with microtubules and facilitates its symmetrical distribution during mitosis.

Christina Arseni, Martina Samiotaki, George Panayotou, George Simos, Ilias Mylonis.

  Hypoxia-inducible factor-1 (HIF-1) is the key transcriptional mediator of the cellular response to hypoxia and is also involved in cancer progression. Regulation of its oxygen-sensitive HIF-1α subunit involves post-translational modifications that control its stability, subcellular localization, and activity. We have previously reported that phosphorylation of the HIF-1α C-terminal domain by ERK1/2 promotes HIF-1α nuclear accumulation and stimulates HIF-1 activity while lack of this modification triggers HIF-1α nuclear export and its association with mitochondria. On the other hand, modification of the N-terminal domain of HIF-1α by CK1δ impairs HIF-1 activity by obstructing the formation of a HIF-1α/ARNT heterodimer. Investigation of these two antagonistic events by expressing double phospho-site mutants in HIF1A-/- cells under hypoxia revealed independent and additive phosphorylation effects that can create a gradient of HIF-1α subcellular localization and transcriptional activity. Furthermore, modification by CK1δ caused mitochondrial release of the non-nuclear HIF-1α form and binding to microtubules via its N-terminal domain. In agreement, endogenous HIF-1α could be shown to co-localize with mitotic spindle microtubules and interact with tubulin, both of which were inhibited by CK1δ silencing or inhibition. Moreover, CK1δ expression was necessary for equal partitioning of mother cell-produced HIF-1α to the daughter cell nuclei at the end of mitosis. Overall, our results suggest that phosphorylation by CK1δ stimulates the association of non-nuclear HIF-1α with microtubules, which may serve as a means to establish a symmetric distribution of HIF-1α during cell division under low oxygen conditions.

Keywords:  Casein kinase 1δ; ERK1/2; HIF-1α; Hypoxia; Microtubules; Mitosis

DOI:  https://doi.org/10.1007/s00018-024-05120-7
bioRxiv. 2024 Jan 16. pii: 2024.01.15.575713. [Epub ahead of print]

A human papillomavirus 16 E2-TopBP1 dependent SIRT1-p300 acetylation switch regulates mitotic viral and human protein levels.

Apurva T Prabhakar, Claire D James, Aya H Youssef, Reafa A Hossain, Ronald D Hill, Molly L Bristol, Xu Wang, Aanchal Dubey, Iain M Morgan.

An interaction between human papillomavirus 16 (HPV16) E2 and the cellular proteins TopBP1 and BRD4 is required for E2 plasmid segregation function. The E2-TopBP1 interaction promotes increased mitotic E2 protein levels in U2OS and N/Tert-1 cells, as well as in human foreskin keratinocytes immortalized by HPV16 (HFK+HPV16). SIRT1 deacetylation reduces E2 protein stability and here we demonstrate that increased E2 acetylation occurs during mitosis in a TopBP1 interacting dependent manner, promoting E2 mitotic stabilization. p300 mediates E2 acetylation and acetylation is increased due to E2 switching off SIRT1 function during mitosis in a TopBP1 interacting dependent manner, confirmed by increased p53 stability and acetylation on lysine 382, a known target for SIRT1 deacetylation. SIRT1 can complex with E2 in growing cells but is unable to do so during mitosis due to the E2-TopBP1 interaction; SIRT1 is also unable to complex with p53 in mitotic E2 wild type cells but can complex with p53 outside of mitosis. E2 lysines 111 and 112 are highly conserved residues across all E2 proteins and we demonstrate that K111 hyper-acetylation occurs during mitosis, promoting E2 interaction with Topoisomerase 1 (Top1). We also demonstrate that K112 ubiquitination promotes E2 proteasomal degradation during mitosis. The results present a model in which the E2-TopBP1 complex inactivates SIRT1 during mitosis and E2 acetylation on K111 by p300 increases, promoting interaction with Top1 that protects K112 from ubiquitination and therefore E2 proteasomal degradation.
Importance: Human papillomaviruses are causative agents in around 5% of all human cancers. While there are prophylactic vaccines that will significantly alleviate HPV disease burden on future generations, there are currently no anti-viral strategies available for the treatment of HPV cancers. To generate such reagents, we must understand more about the HPV life cycle, and in particular about viral-host interactions. Here we describe a novel mitotic complex generated by the HPV16 E2 protein interacting with the host protein TopBP1 that controls the function of the deacetylase SIRT1. The E2-TopBP1 interaction disrupts SIRT1 function during mitosis in order to enhance acetylation and stability of viral and host proteins. This novel complex is essential for the HPV16 life cycle and represents a novel anti-viral therapeutic target.

DOI: https://doi.org/10.1101/2024.01.15.575713
J Phys Chem B. 2024 Jan 30.

Computational Studies Reveal How Passive Cross-Linkers Regulate Anaphase Spindle Elongation.

Yao Wang, Yu-Ru Liu, Peng-Ye Wang, Ping Xie.

In eukaryotic cell division, a series of events are organized to produce two daughter cells. The spindle elongation in anaphase B is essential for providing enough space to maintain cell size and distribute sister chromatids properly, which is associated with microtubules and microtubule-associated proteins such as kinesin-5 Eg5 and the Ase1-related protein, PRC1. The available experimental data indicated that after the start of anaphase B more PRC1 proteins can bind to the antiparallel microtubule pairs in the spindle but the excess amount of PRC1 proteins can lead to the failure of cell division, indicating that PRC1 proteins can regulate the spindle elongation in a concentration-dependent manner. However, the underlying mechanism of the PRC1 proteins regulating the spindle elongation has not been explained up to now. Here, we use a simplified model, where only the two important participants (kinesin-5 Eg5 motors and PRC1 proteins) are considered, to study the spindle elongation during anaphase B. We first show that only in the appropriate range of the PRC1 concentration can the spindle elongation complete properly. Furthermore, we explore the underlying mechanism of PRC1 as a regulator for spindle elongation.

DOI: https://doi.org/10.1021/acs.jpcb.3c07655
Genetics. 2024 Jan 30. pii: iyae015. [Epub ahead of print]

A missense SNP in the tumor suppressor SETD2 reduces H3K36me3 and mitotic spindle integrity in Drosophila.

Jovan S Brockett, Tad Manalo, Hala Zein-Sabatto, Jina Lee, Junnan Fang, Philip Chu, Harry Feng, Dattatraya Patil, Priscilla Davidson, Kenneth Ogan, Viraj A Master, John G Pattaras, David L Roberts, Sharon H Bergquist, Matthew A Reyna, John A Petros, Dorothy A Lerit, Rebecca S Arnold.

  Mutations in SETD2 are among the most prevalent drivers of renal cell carcinoma (RCC). We identified a novel single nucleotide polymorphism (SNP) in SETD2, E902Q, within a subset of RCC patients, which manifests as both an inherited or tumor-associated somatic mutation. To determine if the SNP is biologically functional, we used CRISPR-based genome editing to generate the orthologous mutation within the Drosophila melanogaster Set2 gene. In Drosophila, the homologous amino acid substitution, E741Q, reduces H3K36me3 levels comparable to Set2 knockdown, and this loss is rescued by reintroduction of a wild-type Set2 transgene. We similarly uncovered significant defects in spindle morphogenesis, consistent with the established role of SETD2 in methylating α-Tubulin during mitosis to regulate microtubule dynamics and maintain genome stability. These data indicate the Set2 E741Q SNP affects both histone methylation and spindle integrity. Moreover, this work further suggests the SETD2 E902Q SNP may hold clinical relevance.

Keywords:   Drosophila ; H3K36me3; Renal cell carcinoma; SETD2; SNP; models of human disease

DOI:  https://doi.org/10.1093/genetics/iyae015
Proc Natl Acad Sci U S A. 2024 Feb 06. 121(6): e2312250121

Multiscale biophysical analysis of nucleolus disassembly during mitosis.

An T Pham, Madhav Mani, Xiaozhong Wang, Reza Vafabakhsh.

  During cell division, precise and regulated distribution of cellular material between daughter cells is a critical step and is governed by complex biochemical and biophysical mechanisms. To achieve this, membraneless organelles and condensates often require complete disassembly during mitosis. The biophysical principles governing the disassembly of condensates remain poorly understood. Here, we used a physical biology approach to study how physical and material properties of the nucleolus, a prominent nuclear membraneless organelle in eukaryotic cells, change during mitosis and across different scales. We found that nucleolus disassembly proceeds continuously through two distinct phases with a slow and reversible preparatory phase followed by a rapid irreversible phase that was concurrent with the nuclear envelope breakdown. We measured microscopic properties of nucleolar material including effective diffusion rates and binding affinities as well as key macroscopic properties of surface tension and bending rigidity. By incorporating these measurements into the framework of critical phenomena, we found evidence that near mitosis surface tension displays a power-law behavior as a function of biochemically modulated interaction strength. This two-step disassembly mechanism maintains structural and functional stability of nucleolus while enabling its rapid and efficient disassembly in response to cell cycle cues.

Keywords:  critical phenomena; membraneless organelles; nucleolus disassembly

DOI:  https://doi.org/10.1073/pnas.2312250121
Exp Mol Pathol. 2024 Jan 27. pii: S0014-4800(24)00003-0. [Epub ahead of print] 104884

Environmental physical factors produce mitotic aberration and multicellularity in the ovarian cancer cell line SKOV3.

N Lujea, F Chiola, M De Leon Rodriguez, C Acosta, P Kunda.

  The spread of ovarian cancer (OC) to the coelomic cavity triggers the secretion and accumulation of ascitic fluid (AF). Although its biochemical composition has been well studied, less is known about the implications of physical factors such as the pH and the mechanical properties of the AF for the malignancy of tumor cells. In this work, we investigated the effect of pH and the mechanical properties of AF on cell proliferation and mitotic morphology. We employed biopsies from patients with OC and the SKOV3 cell line as an in vitro model of OC with HeLa cells as controls. Sections of each tumor were stained with HE, analyzed, and related to clinical data. AF from patients with OC exhibited an alkaline pH (ranging from 7.3 to 7.8). Compared to control conditions, the 3 AFs significantly enhanced the proliferation of SKOV3 and HeLa cells. These effects were more pronounced at a more alkaline pH. In addition, we found that AFs have different densities that correlated with a significant increase in multinucleated tumor cells and severe morphological defects in cells undergoing mitosis. In agreement with these data, we found that higher concentrations of soft agar provoked significantly higher numbers of multinucleated and morphologically abnormal SKOV3 cells with no effect on HeLa cells. We conclude that an alkaline pH and greater rigidity could enhance the metastatic potential of OC cells. We propose that these two physical factors could be parameters of clinical importance as predictors of malignancy.

Keywords:  Ascitic fluid; Mechanical factors; Ovarian cancer; Proliferation; SKOV3

DOI:  https://doi.org/10.1016/j.yexmp.2024.104884