bims-micesi 2022-10-30 papers

bims-micesi

Biomed News

on Mitotic cell signalling

Issue of 2022‒10‒30
thirteen papers selected by
Valentina Piano
Uniklinik Köln

Juxtaposition of Bub1 and Cdc20 on phosphorylated Mad1 during catalytic mitotic checkpoint complex assembly.
Lis1-dynein drives corona compaction and limits erroneous microtubule attachment at kinetochores.
Preprint Highlight: Mitotic clustering of pulverized chromosomes from micronuclei.
Morphological growth dynamics, mechanical stability, and active microtubule mechanics underlying spindle self-organization.
GRANT Motif Regulates CENP-A Incorporation and Restricts RNA Polymerase II Accessibility at Centromere.
DNA damage checkpoint execution and the rules of its disengagement.
Cohesin is required for meiotic spindle assembly independent of its role in cohesion in C. elegans.
Elongator stabilizes microtubules to control central spindle asymmetry and polarized trafficking of cell fate determinants.
Design and Synthesis of Aminopyrimidinyl Pyrazole Analogs as PLK1 Inhibitors Using Hybrid 3D-QSAR and Molecular Docking.
CCHCR1-astrin interaction promotes centriole duplication through recruitment of CEP72.
SCFFBXW7 regulates G2-M progression through control of CCNL1 ubiquitination.
Cytokinetic Abscission Regulation in Neural Stem Cells and Tissue Development.
FtsK, a DNA Motor Protein, Coordinates the Genome Segregation and Early Cell Division Processes in Deinococcus radiodurans.

Nat Commun. 2022 Oct 26. 13(1): 6381

Juxtaposition of Bub1 and Cdc20 on phosphorylated Mad1 during catalytic mitotic checkpoint complex assembly.

Elyse S Fischer, Conny W H Yu, Johannes F Hevler, Stephen H McLaughlin, Sarah L Maslen, Albert J R Heck, Stefan M V Freund, David Barford.

In response to improper kinetochore-microtubule attachments in mitosis, the spindle assembly checkpoint (SAC) assembles the mitotic checkpoint complex (MCC) to inhibit the anaphase-promoting complex/cyclosome, thereby delaying entry into anaphase. The MCC comprises Mad2:Cdc20:BubR1:Bub3. Its assembly is catalysed by unattached kinetochores on a Mad1:Mad2 platform. Mad1-bound closed-Mad2 (C-Mad2) recruits open-Mad2 (O-Mad2) through self-dimerization. This interaction, combined with Mps1 kinase-mediated phosphorylation of Bub1 and Mad1, accelerates MCC assembly, in a process that requires O-Mad2 to C-Mad2 conversion and concomitant binding of Cdc20. How Mad1 phosphorylation catalyses MCC assembly is poorly understood. Here, we characterized Mps1 phosphorylation of Mad1 and obtained structural insights into a phosphorylation-specific Mad1:Cdc20 interaction. This interaction, together with the Mps1-phosphorylation dependent association of Bub1 and Mad1, generates a tripartite assembly of Bub1 and Cdc20 onto the C-terminal domain of Mad1 (Mad1CTD). We additionally identify flexibility of Mad1:Mad2 that suggests how the Cdc20:Mad1CTD interaction brings the Mad2-interacting motif (MIM) of Cdc20 near O-Mad2. Thus, Mps1-dependent formation of the MCC-assembly scaffold functions to position and orient Cdc20 MIM near O-Mad2, thereby catalysing formation of C-Mad2:Cdc20.

DOI: https://doi.org/10.1038/s41467-022-34058-2
J Cell Sci. 2022 Oct 24. pii: jcs.260226. [Epub ahead of print]

Lis1-dynein drives corona compaction and limits erroneous microtubule attachment at kinetochores.

Olivera Mitevska, Pak Wing Lam, Lydia Daly, Philip Auckland.

  Mitotic cell division requires that kinetochores form microtubule attachments that can segregate chromosomes and control mitotic progression via the spindle assembly checkpoint. During prometaphase, kinetochores shed a domain called the fibrous corona as microtubule attachments form. This shedding is mediated, in part, by the minus-end directed motor dynein, which 'strips' cargos along K-fibre microtubules. Despite its essentiality, little is known about how dynein stripping is regulated and how it responds to attachment maturation. Lis1 is a conserved dynein regulator that is mutated in the neurodevelopmental disease lissencephaly. Here, we have combined loss-of-function studies, high-resolution imaging and separation-of-function mutants to define how Lis1 contributes to dynein-mediated corona stripping. Cells depleted of Lis1 fail to disassemble the corona and delay in metaphase as a result of persistent checkpoint activation. Furthermore, we find that while kinetochore-tethered Lis1-dynein is required for error-free microtubule attachment, the contribution of Lis1 to corona disassembly can be mediated by a cytoplasmic pool. These findings support the idea that Lis1 drives dynein function at kinetochores to ensure corona disassembly and prevent chromosome mis-segregation.

Keywords:  Dynein; Kinetochore; Lis1; Microtubule; Mitosis

DOI:  https://doi.org/10.1242/jcs.260226
Mol Biol Cell. 2022 Nov 01. 33(13): mbcP22091002

Preprint Highlight: Mitotic clustering of pulverized chromosomes from micronuclei.

Huaiying Zhang.

DOI: https://doi.org/10.1091/mbc.P22-09-1002
Proc Natl Acad Sci U S A. 2022 Nov;119(44): e2209053119

Morphological growth dynamics, mechanical stability, and active microtubule mechanics underlying spindle self-organization.

Tatsuya Fukuyama, Lucan Yan, Masahito Tanaka, Megumi Yamaoka, Kei Saito, Shih-Chieh Ti, Chung-Chi Liao, Kuo-Chiang Hsia, Yusuke T Maeda, Yuta Shimamoto.

  The spindle is a dynamic intracellular structure self-organized from microtubules and microtubule-associated proteins. The spindle's bipolar morphology is essential for the faithful segregation of chromosomes during cell division, and it is robustly maintained by multifaceted mechanisms. However, abnormally shaped spindles, such as multipolar spindles, can stochastically arise in a cell population and cause chromosome segregation errors. The physical basis of how microtubules fail in bipolarization and occasionally favor nonbipolar assembly is poorly understood. Here, using live fluorescence imaging and quantitative shape analysis in Xenopus egg extracts, we find that spindles of varied shape morphologies emerge through nonrandom, bistable self-organization paths, one leading to a bipolar and the other leading to a multipolar phenotype. The bistability defines the spindle's unique morphological growth dynamics linked to each shape phenotype and can be promoted by a locally distorted microtubule flow that arises within premature structures. We also find that bipolar and multipolar spindles are stable at the steady-state in bulk but can infrequently switch between the two phenotypes. Our microneedle-based physical manipulation further demonstrates that a transient force perturbation applied near the assembled pole can trigger the phenotypic switching, revealing the mechanical plasticity of the spindle. Together with molecular perturbation of kinesin-5 and augmin, our data propose the physical and molecular bases underlying the emergence of spindle-shape variation, which influences chromosome segregation fidelity during cell division.

Keywords:  microtubule; mitosis; self-organization

DOI:  https://doi.org/10.1073/pnas.2209053119
Genes (Basel). 2022 Sep 22. pii: 1697. [Epub ahead of print]13(10):

GRANT Motif Regulates CENP-A Incorporation and Restricts RNA Polymerase II Accessibility at Centromere.

Hwei Ling Tan, Ee Sin Chen.

  Precise chromosome segregation is essential for maintaining genomic stability, and its proper execution centers on the centromere, a chromosomal locus that mounts the kinetochore complex to mediate attachment of chromosomes to the spindle microtubules. The location of the centromere is epigenetically determined by a centromere-specific histone H3 variant, CENP-A. Many human cancers exhibit overexpression of CENP-A, which correlates with occurrence of aneuploidy in these malignancies. Centromeric targeting of CENP-A depends on its histone fold, but recent studies showed that the N-terminal tail domain (NTD) also plays essential roles. Here, we investigated implications of NTD in conferring aneuploidy formation when CENP-A is overexpressed in fission yeast. A series of mutant genes progressively lacking one amino acid of the NTD have been constructed for overexpression in wild-type cells using the intermediate strength nmt41 promoter. Constructs hosting disrupted GRANT (Genomic stability-Regulating site within CENP-A N-Terminus) motif in NTD results in growth retardation, aneuploidy, increased localization to the centromere, upregulated RNA polymerase II accessibility and transcriptional derepression of the repressive centromeric chromatin, suggesting that GRANT residues fine-tune centromeric CENP-A incorporation and restrict RNA polymerase II accessibility. This work highlighted the importance of CENP-A NTD, particularly the GRANT motif, in aneuploidy formation of overexpressed CENP-A in fission yeast.

Keywords:  CENP-A; N-terminus; RNA polymerase II; centromere; chromatin; epigenetic; histone

DOI:  https://doi.org/10.3390/genes13101697
Front Cell Dev Biol. 2022 ;10 1020643

DNA damage checkpoint execution and the rules of its disengagement.

Candice Qiu Xia Yam, Hong Hwa Lim, Uttam Surana.

  Chromosomes are susceptible to damage during their duplication and segregation or when exposed to genotoxic stresses. Left uncorrected, these lesions can result in genomic instability, leading to cells' diminished fitness, unbridled proliferation or death. To prevent such fates, checkpoint controls transiently halt cell cycle progression to allow time for the implementation of corrective measures. Prominent among these is the DNA damage checkpoint which operates at G2/M transition to ensure that cells with damaged chromosomes do not enter the mitotic phase. The execution and maintenance of cell cycle arrest are essential aspects of G2/M checkpoint and have been studied in detail. Equally critical is cells' ability to switch-off the checkpoint controls after a successful completion of corrective actions and to recommence cell cycle progression. Interestingly, when corrective measures fail, cells can mount an unusual cellular response, termed adaptation, where they escape checkpoint arrest and resume cell cycle progression with damaged chromosomes at the cost of genome instability or even death. Here, we discuss the DNA damage checkpoint, the mitotic networks it inhibits to prevent segregation of damaged chromosomes and the strategies cells employ to quench the checkpoint controls to override the G2/M arrest.

Keywords:  DNA damage; adaptation to chromosome damage; cell division; checkpoint; mitosis

DOI:  https://doi.org/10.3389/fcell.2022.1020643
PLoS Genet. 2022 Oct 24. 18(10): e1010136

Cohesin is required for meiotic spindle assembly independent of its role in cohesion in C. elegans.

Karen P McNally, Elizabeth A Beath, Brennan M Danlasky, Consuelo Barroso, Ting Gong, Wenzhe Li, Enrique Martinez-Perez, Francis J McNally.

Accurate chromosome segregation requires a cohesin-mediated physical attachment between chromosomes that are to be segregated apart, and a bipolar spindle with microtubule plus ends emanating from exactly two poles toward the paired chromosomes. We asked whether the striking bipolar structure of C. elegans meiotic chromosomes is required for bipolarity of acentriolar female meiotic spindles by time-lapse imaging of mutants that lack cohesion between chromosomes. Both a spo-11 rec-8 coh-4 coh-3 quadruple mutant and a spo-11 rec-8 double mutant entered M phase with separated sister chromatids lacking any cohesion. However, the quadruple mutant formed an apolar spindle whereas the double mutant formed a bipolar spindle that segregated chromatids into two roughly equal masses. Residual non-cohesive COH-3/4-dependent cohesin on separated sister chromatids of the double mutant was sufficient to recruit haspin-dependent Aurora B kinase, which mediated bipolar spindle assembly in the apparent absence of chromosomal bipolarity. We hypothesized that cohesin-dependent Aurora B might activate or inhibit spindle assembly factors in a manner that would affect their localization on chromosomes and found that the chromosomal localization patterns of KLP-7 and CLS-2 correlated with Aurora B loading on chromosomes. These results demonstrate that cohesin is essential for spindle assembly and chromosome segregation independent of its role in sister chromatid cohesion.

DOI: https://doi.org/10.1371/journal.pgen.1010136
Nat Cell Biol. 2022 Oct 27.

Elongator stabilizes microtubules to control central spindle asymmetry and polarized trafficking of cell fate determinants.

Vicente Jose Planelles-Herrero, Alice Bittleston, Carole Seum, Marcos Gonzalez Gaitan, Emmanuel Derivery.

Asymmetric cell division gives rise to two daughter cells that inherit different determinants, thereby acquiring different fates. Polarized trafficking of endosomes containing fate determinants recently emerged as an evolutionarily conserved feature of asymmetric cell division to enhance the robustness of asymmetric cell fate determination in flies, fish and mammals. In particular, polarized sorting of signalling endosomes by an asymmetric central spindle contributes to asymmetric cell division in Drosophila melanogaster. However, how central spindle asymmetry arises remains elusive. Here we identify a moonlighting function of the Elongator complex-an established protein acetylase and tRNA methylase involved in the fidelity of protein translation-as a key factor for central spindle asymmetry. Elongator controls spindle asymmetry by stabilizing microtubules differentially on the anterior side of the central spindle. Accordingly, lowering the activity of Elongator on the anterior side using nanobodies mistargets endosomes to the wrong cell. Molecularly, Elongator regulates microtubule dynamics independently of its acetylation and methylation enzymatic activities. Instead, Elongator directly binds to microtubules and increases their polymerization speed while decreasing their catastrophe frequency. Our data establish a non-canonical role of Elongator at the core of cytoskeleton polarity and asymmetric signalling.

DOI: https://doi.org/10.1038/s41556-022-01020-9
Pharmaceuticals (Basel). 2022 Sep 21. pii: 1170. [Epub ahead of print]15(10):

Design and Synthesis of Aminopyrimidinyl Pyrazole Analogs as PLK1 Inhibitors Using Hybrid 3D-QSAR and Molecular Docking.

Swapnil P Bhujbal, Hyejin Kim, Hyunah Bae, Jung-Mi Hah.

  Cancer continues to be one of the world's most severe public health issues. Polo-like kinase 1 (PLK1) is one of the most studied members of the polo-like kinase subfamily of serine/threonine protein kinases. PLK1 is a key mitotic regulator responsible for cell cycle processes, such as mitosis initiation, bipolar mitotic spindle formation, centrosome maturation, the metaphase to anaphase transition, and mitotic exit, whose overexpression is often associated with oncogenesis. Moreover, it is also involved in DNA damage response, autophagy, cytokine signaling, and apoptosis. Due to its fundamental role in cell cycle regulation, PLK1 has been linked to various types of cancer onset and progression, such as lung, colon, prostate, ovary, breast cancer, melanoma, and AML. Hence, PLK1 is recognized as a critical therapeutic target in the treatment of various proliferative diseases. PLK1 inhibitors developed in recent years have been researched and studied through clinical trials; however, most of them have failed because of their toxicity and poor therapeutic response. To design more potent and selective PLK1 inhibitors, we performed a receptor-based hybrid 3D-QSAR study of two datasets, possessing similar common scaffolds. The developed hybrid CoMFA (q2 = 0.628, r2 = 0.905) and CoMSIA (q2 = 0.580, r2 = 0.895) models showed admissible statistical results. Comprehensive, molecular docking of one of the most active compounds from the dataset and hybrid 3D-QSAR studies revealed important active site residues of PLK1 and requisite structural characteristics of ligand to design potent PLK1 inhibitors. Based on this information, we have proposed approximately 38 PLK1 inhibitors. The newly designed PLK1 inhibitors showed higher activity (predicted pIC50) than the most active compounds of all the derivatives selected for this study. We selected and synthesized two compounds, which were ultimately found to possess good IC50 values. Our design strategy provides insight into development of potent and selective PLK1 inhibitors.

Keywords:  3D-QSAR; PLK1; cancer; inhibitors; kinase; molecular docking

DOI:  https://doi.org/10.3390/ph15101170
BMC Biol. 2022 Oct 24. 20(1): 240

CCHCR1-astrin interaction promotes centriole duplication through recruitment of CEP72.

Zhenguang Ying, Kaifang Wang, Junfeng Wu, Mingyu Wang, Jing Yang, Xia Wang, Guowei Zhou, Haibin Chen, Hongwu Xu, Stephen Cho Wing Sze, Feng Gao, Chunman Li, Ou Sha.

  BACKGROUND: The centrosome is one of the most important non-membranous organelles regulating microtubule organization and progression of cell mitosis. The coiled-coil alpha-helical rod protein 1 (CCHCR1, also known as HCR) gene is considered to be a psoriasis susceptibility gene, and the protein is suggested to be localized to the P-bodies and centrosomes in mammalian cells. However, the exact cellular function of HCR and its potential regulatory role in the centrosomes remain unexplored.RESULTS: We found that HCR interacts directly with astrin, a key factor in centrosome maturation and mitosis. Immunoprecipitation assays showed that the coiled-coil region present in the C-terminus of HCR and astrin respectively mediated the interaction between them. Astrin not only recruits HCR to the centrosome, but also protects HCR from ubiquitin-proteasome-mediated degradation. In addition, depletion of either HCR or astrin significantly reduced centrosome localization of CEP72 and subsequent MCPH proteins, including CEP152, CDK5RAP2, and CEP63. The absence of HCR also caused centriole duplication defects and mitotic errors, resulting in multipolar spindle formation, genomic instability, and DNA damage.
CONCLUSION: We conclude that HCR is localized and stabilized at the centrosome by directly binding to astrin. HCR are required for the centrosomal recruitment of MCPH proteins and centriolar duplication. Both HCR and astrin play key roles in keeping normal microtubule assembly and maintaining genomic stability.

Keywords:  Astrin; CCHCR1; CEP72; Centrosome; Microtubule organization; Mitosis

DOI:  https://doi.org/10.1186/s12915-022-01437-6
EMBO Rep. 2022 Oct 24. e55044

SCFFBXW7 regulates G2-M progression through control of CCNL1 ubiquitination.

Siobhan O'Brien, Susan Kelso, Zachary Steinhart, Stephen Orlicky, Monika Mis, Yunhye Kim, Sichun Lin, Frank Sicheri, Stephane Angers.

  FBXW7, which encodes a substrate-specific receptor of an SCF E3 ligase complex, is a frequently mutated human tumor suppressor gene known to regulate the post-translational stability of various proteins involved in cellular proliferation. Here, using genome-wide CRISPR screens, we report a novel synthetic lethal genetic interaction between FBXW7 and CCNL1 and describe CCNL1 as a new substrate of the SCF-FBXW7 E3 ligase. Further analysis showed that the CCNL1-CDK11 complex is critical at the G2-M phase of the cell cycle since defective CCNL1 accumulation, resulting from FBXW7 mutation, leads to shorter mitotic time. Cells harboring FBXW7 loss-of-function mutations are hypersensitive to treatment with a CDK11 inhibitor, highlighting a genetic vulnerability that could be leveraged for cancer treatment.

Keywords:  CDK11; FBXW7; cell cycle; cyclin L1; mitosis

DOI:  https://doi.org/10.15252/embr.202255044
Curr Stem Cell Rep. 2021 Dec;7(4): 161-173

Cytokinetic Abscission Regulation in Neural Stem Cells and Tissue Development.

Katrina C McNeely, Noelle D Dwyer.

  Purpose of Review: How stem cells balance proliferation with differentiation, giving rise to specific daughter cells during development to build an embryo or tissue, remains an open question. Here, we discuss recent evidence that cytokinetic abscission regulation in stem cells, particularly neural stem cells (NSCs), is part of the answer. Abscission is a multi-step process mediated by the midbody, a microtubule-based structure formed in the intercellular bridge between daughter cells after mitosis.Recent Findings: Human mutations and mouse knockouts in abscission genes reveal that subtle disruptions of NSC abscission can cause brain malformations. Experiments in several epithelial systems have shown that midbodies serve as scaffolds for apical junction proteins and are positioned near apical membrane fate determinants. Abscission timing is tightly controlled and developmentally regulated in stem cells, with delayed abscission in early embryos and faster abscission later. Midbody remnants (MBRs) contain over 400 proteins and may influence polarity, fate, and ciliogenesis.
Summary: As NSCs and other stem cells build tissues, they tightly regulate three aspects of abscission: midbody positioning, duration, and MBR handling. Midbody positioning and remnants establish or maintain cell polarity. MBRs are deposited on the apical membranes of epithelia, can be released or internalized by surrounding cells, and may sequester fate determinants or transfer information between cells. Work in cell lines and simpler systems has shown multiple roles for abscission regulation influencing stem cell polarity, potency, and daughter fates during development. Elucidating how the abscission process influences cell fate and tissue growth is important for our continued understanding of brain development and stem cell biology.

Keywords:  Abscission; Cell fate; Cytokinesis; Microcephaly; Midbody; Neural stem cells

DOI:  https://doi.org/10.1007/s40778-021-00193-7
mBio. 2022 Oct 27. e0174222

FtsK, a DNA Motor Protein, Coordinates the Genome Segregation and Early Cell Division Processes in Deinococcus radiodurans.

Shruti Mishra, Hari S Misra, Swathi Kota.

  Filament temperature-sensitive mutant K (FtsK)/SpoIIIE family proteins are DNA translocases known as the fastest DNA motor proteins that use ATP for their movement on DNA. Most of the studies in single chromosome-containing bacteria have established the role of FtsK in chromosome dimer resolution (CDR), connecting the bacterial chromosome segregation process with cell division. Only limited reports, however, are available on the interdependent regulation of genome segregation and cell division in multipartite genome harboring (MGH) bacteria. In this study, for the first time, we report the characterization of FtsK from the radioresistant MGH bacterium Deinococcus radiodurans R1 (drFtsK). drFtsK shows the activity characteristics of a typical FtsK/SpoIIIE/Tra family. It stimulates the site-specific recombination catalyzed by Escherichia coli tyrosine recombinases. drFtsK interacts with various cell division and genome segregation proteins of D. radiodurans. Microscopic examination of different domain deletion mutants of this protein reveals alterations in cellular membrane architecture and nucleoid morphology. In vivo localization studies of drFtsK-RFP show that it forms multiple foci on nucleoid as well as on the membrane with maximum density on the septum. drFtsK coordinates its movement with nucleoid separation. The alignment of its foci shifts from old to new septum indicating its cellular dynamics with the FtsZ ring during the cell division process. Nearly, similar positional dynamicity of FtsK was observed in cells recovering from gamma radiation exposure. These results suggest that FtsK forms a part of chromosome segregation, cell envelope, and cell division machinery in D. radiodurans. IMPORTANCE Deinococcus radiodurans show extraordinary resistance to gamma radiation. It is polyploid and harbors a multipartite genome comprised of 2 chromosomes and 2 plasmids, packaged in a doughnut-shaped toroidal nucleoid. Very little is known about how the tightly packed genome is accurately segregated and the next divisional plane is determined. Filament temperature-sensitive mutant K (FtsK), a multifunctional protein, helps in pumping the septum-trapped DNA in several bacteria. Here, we characterized FtsK of D. radiodurans R1 (drFtsK) for the first time and showed it to be an active protein. The absence of drFtsK causes many defects in morphology at both cellular and nucleoid levels. The compact packaging of the deinococcal genome and cell membrane formation is hindered in ftsK mutants. In vivo drFtsK is dynamic, forms foci on both nucleoid and septum, and coordinates with FtsZ for the next cell division. Thus, drFtsK role in maintaining the normal genome phenotype and cell division in D. radiodurans is suggested.

Keywords:  DNA translocation; Deinococcus radiodurans; FtsK; bacterial cell division

DOI:  https://doi.org/10.1128/mbio.01742-22