bims-micesi Biomed News
on Mitotic cell signalling
Issue of 2024–02–25
twelve papers selected by
Valentina Piano, Uniklinik Köln
  1. The spindle protein CKAP2 regulates microtubule dynamics and ensures faithful chromosome segregation.
  2. MAARS Software for Automatic and Quantitative Analysis of Mitotic Progression.
  3. Identification of a motif in TPX2 that regulates spindle architecture in Xenopus egg extracts.
  4. Measuring Mitotic Spindle and Microtubule Dynamics in Marine Embryos and Non-model Organisms.
  5. Phospho-KNL-1 recognition by a TPR domain targets the BUB-1-BUB-3 complex to C. elegans kinetochores.
  6. Increases in cyclin A/Cdk activity and in PP2A-B55 inhibition by FAM122A are key mitosis-inducing events.
  7. Dissecting the Multiple Functions of the Polo-Like Kinase 1 in the C. elegans Zygote.
  8. C. elegans spermatocyte divisions show weak spindle checkpoint response.
  9. Chromosome Division in Early Embryos-Is Everything under Control? And Is the Cell Size Important?
  10. Fluorescence complementation-based FRET imaging reveals centromere assembly dynamics.
  11. Transcriptional repression across mitosis: mechanisms and functions.
  12. Cell cycle perturbation uncouples mitotic progression and invasive behavior in a post-mitotic cell.
  1. Proc Natl Acad Sci U S A. 2024 Feb 27. 121(9): e2318782121
    The spindle protein CKAP2 regulates microtubule dynamics and ensures faithful chromosome segregation.
    Lia Mara Gomes Paim, Azriel Abraham Lopez-Jauregui, Thomas S McAlear, Susanne Bechstedt.
      Regulation of microtubule dynamics by microtubule-associated proteins (MAPs) is essential for mitotic spindle assembly and chromosome segregation. Altered microtubule dynamics, particularly increased microtubule growth rates, were found to be a contributing factor for the development of chromosomal instability, which potentiates tumorigenesis. The MAP XMAP215/CKAP5 is the only known microtubule growth factor, and whether other MAPs regulate microtubule growth in cells is unclear. Our recent in vitro reconstitution experiments have demonstrated that Cytoskeleton-Associated Protein 2 (CKAP2) increases microtubule nucleation and growth rates, and here, we find that CKAP2 is also an essential microtubule growth factor in cells. By applying CRISPR-Cas9 knock-in and knock-out (KO) as well as microtubule plus-end tracking live cell imaging, we show that CKAP2 is a mitotic spindle protein that ensures faithful chromosome segregation by regulating microtubule growth. Live cell imaging of endogenously labeled CKAP2 showed that it localizes to the spindle during mitosis and rapidly shifts its localization to the chromatin upon mitotic exit before being degraded. Cells lacking CKAP2 display reduced microtubule growth rates and an increased proportion of chromosome segregation errors and aneuploidy that may be a result of an accumulation of kinetochore-microtubule misattachments. Microtubule growth rates and chromosome segregation fidelity can be rescued upon ectopic CKAP2 expression in KO cells, revealing a direct link between CKAP2 expression and microtubule dynamics. Our results unveil a role of CKAP2 in regulating microtubule growth in cells and provide a mechanistic explanation for the oncogenic potential of CKAP2 misregulation.
    Keywords:  chromosome segregation; kinetochore; microtubule dynamics; mitosis; spindle
    DOI:  https://doi.org/10.1073/pnas.2318782121
  2. Methods Mol Biol. 2024 ;2740 275-293
    MAARS Software for Automatic and Quantitative Analysis of Mitotic Progression.
    Tong Li, Yannick Gachet, Sylvie Tournier.
      In this chapter, we describe a software called MAARS (Mitotic Analysis And Recording System) that enables automatic and quantitative analysis of mitotic progression on an open-source platform. This computer-assisted analysis of cell division allows the unbiased acquisition of multiple parameters such as cell shape or size, metaphase or anaphase delays, as well as various mitotic abnormalities. This chapter describes the power of such an expert system to highlight the complexity of the mechanisms required to prevent mitotic chromosome segregation errors, leading to aneuploidy.
    Keywords:  Fission yeast; High-content analysis; Mitosis; Neuploidy
    DOI:  https://doi.org/10.1007/978-1-0716-3557-5_17
  3. bioRxiv. 2024 Feb 11. pii: 2024.02.10.579770. [Epub ahead of print]
    Identification of a motif in TPX2 that regulates spindle architecture in Xenopus egg extracts.
    Guadalupe E Pena, Xiao Zhou, Lauren Slevin, Christopher Brownlee, Rebecca Heald.
      A bipolar spindle composed of microtubules and many associated proteins functions to segregate chromosomes during cell division in all eukaryotes, yet spindle size and architecture varies dramatically across different species and cell types. Targeting protein for Xklp2 (TPX2) is one candidate factor for modulating spindle microtubule organization through its roles in branching microtubule nucleation, activation of the mitotic kinase Aurora A, and association with the kinesin-5 (Eg5) motor. Here we identify a conserved nuclear localization sequence (NLS) motif, 123 KKLK 126 in X. laevis TPX2, which regulates astral microtubule formation and spindle pole morphology in Xenopus egg extracts. Addition of recombinant TPX2 with this sequence mutated to AALA dramatically increased spontaneous formation of microtubule asters and recruitment of phosphorylated Aurora A, pericentrin, and Eg5 to meiotic spindle poles. We propose that TPX2 is a linchpin spindle assembly factor whose regulation contributes to the recruitment and activation of multiple microtubule polymerizing and organizing proteins, generating distinct spindle architectures.
    DOI:  https://doi.org/10.1101/2024.02.10.579770
  4. Methods Mol Biol. 2024 ;2740 187-210
    Measuring Mitotic Spindle and Microtubule Dynamics in Marine Embryos and Non-model Organisms.
    Janet Chenevert, Morgane L V Robert, Jérémy Sallé, Sébastien Cacchia, Thierry Lorca, Anna Castro, Alex McDougall, Nicolas Minc, Stefania Castagnetti, Julien Dumont, Benjamin Lacroix.
      During eukaryotic cell division a microtubule-based structure, the mitotic spindle, aligns and segregates chromosomes between daughter cells. Understanding how this cellular structure is assembled and coordinated in space and in time requires measuring microtubule dynamics and visualizing spindle assembly with high temporal and spatial resolution. Visualization is often achieved by the introduction and the detection of molecular probes and fluorescence microscopy. Microtubules and mitotic spindles are highly conserved across eukaryotes; however, several technical limitations have restricted these investigations to only a few species. The ability to monitor microtubule and chromosome choreography in a wide range of species is fundamental to reveal conserved mechanisms or unravel unconventional strategies that certain forms of life have developed to ensure faithful partitioning of chromosomes during cell division. Here, we describe a technique based on injection of purified proteins that enables the visualization of microtubules and chromosomes with a high contrast in several divergent marine embryos. We also provide analysis methods and tools to extract microtubule dynamics and monitor spindle assembly. These techniques can be adapted to a wide variety of species in order to measure microtubule dynamics and spindle assembly kinetics when genetic tools are not available or in parallel to the development of such techniques in non-model organisms.
    Keywords:  Confocal live imaging; Histone purification; Marine embryos; Microtubule dynamics; Mitotic spindle; Non-model organisms; Tubulin labelling
    DOI:  https://doi.org/10.1007/978-1-0716-3557-5_12
  5. bioRxiv. 2024 Feb 11. pii: 2024.02.09.579536. [Epub ahead of print]
    Phospho-KNL-1 recognition by a TPR domain targets the BUB-1-BUB-3 complex to C. elegans kinetochores.
    Jack Houston, Clémence Vissotsky, Amar Deep, Hiro Hakozaki, Enice Crews, Karen Oegema, Kevin D Corbett, Pablo Lara-Gonzalez, Taekyung Kim, Arshad Desai.
      During mitosis, the Bub1-Bub3 complex concentrates at kinetochores, the microtubule-coupling interfaces on chromosomes, where it contributes to spindle checkpoint activation, kinetochore-spindle microtubule interactions, and protection of centromeric cohesion. Bub1 has a conserved N-terminal tetratricopeptide (TPR) domain followed by a binding motif for its conserved interactor Bub3. The current model for Bub1-Bub3 localization to kinetochores is that Bub3, along with its bound motif from Bub1, recognizes phosphorylated "MELT" motifs in the kinetochore scaffold protein Knl1. Motivated by the greater phenotypic severity of BUB-1 versus BUB-3 loss in C. elegans , we show that the BUB-1 TPR domain directly recognizes a distinct class of phosphorylated motifs in KNL-1 and that this interaction is essential for BUB-1-BUB-3 localization and function. BUB-3 recognition of phospho-MELT motifs additively contributes to drive super-stoichiometric accumulation of BUB-1-BUB-3 on its KNL-1 scaffold during mitotic entry. Bub1's TPR domain interacts with Knl1 in other species, suggesting that collaboration of TPR-dependent and Bub3-dependent interfaces in Bub1-Bub3 localization and functions may be conserved.
    DOI:  https://doi.org/10.1101/2024.02.09.579536
  6. EMBO J. 2024 Feb 20.
    Increases in cyclin A/Cdk activity and in PP2A-B55 inhibition by FAM122A are key mitosis-inducing events.
    Benjamin Lacroix, Suzanne Vigneron, Jean Claude Labbé, Lionel Pintard, Corinne Lionne, Gilles Labesse, Anna Castro, Thierry Lorca.
      Entry into mitosis has been classically attributed to the activation of a cyclin B/Cdk1 amplification loop via a partial pool of this kinase becoming active at the end of G2 phase. However, how this initial pool is activated is still unknown. Here we discovered a new role of the recently identified PP2A-B55 inhibitor FAM122A in triggering mitotic entry. Accordingly, depletion of the orthologue of FAM122A in C. elegans prevents entry into mitosis in germline stem cells. Moreover, data from Xenopus egg extracts strongly suggest that FAM122A-dependent inhibition of PP2A-B55 could be the initial event promoting mitotic entry. Inhibition of this phosphatase allows subsequent phosphorylation of early mitotic substrates by cyclin A/Cdk, resulting in full cyclin B/Cdk1 and Greatwall (Gwl) kinase activation. Subsequent to Greatwall activation, Arpp19/ENSA become phosphorylated and now compete with FAM122A, promoting its dissociation from PP2A-B55 and taking over its phosphatase inhibition role until the end of mitosis.
    Keywords:  Arpp19; Cyclin A.; FAM122A; Mitosis; PP2A-B55
    DOI:  https://doi.org/10.1038/s44318-024-00054-z
  7. Methods Mol Biol. 2024 ;2740 63-88
    Dissecting the Multiple Functions of the Polo-Like Kinase 1 in the C. elegans Zygote.
    Griselda Velez-Aguilera, Batool Ossareh-Nazari, Lionel Pintard.
      Plk1 (polo-like kinase 1) is an evolutionarily conserved serine/threonine kinase instrumental for mitotic entry and progression. Beyond these canonical functions, Plk1 also regulates cell polarization and cell fate during asymmetric cell divisions in C. elegans and D. melanogaster. Plk1 contains a specialized phosphoserine-threonine binding domain, the polo-box domain (PBD), which localizes and concentrates the kinase at its various sites of action within the cell in space and time. Here we present protocols to express and purify the C. elegans Plk1 kinase along with biochemical and phosphoproteomic approaches to interrogate the PBD interactome and to dissect Plk1 substrate interactions. These protocols are most suitable for the identification of Plk1 targets in C. elegans embryos but can be easily adapted to identify and study Plk1 substrates from any source."
    Keywords:  C. elegans embryos; Phosphoproteomics; Polo-box domain; Polo-like kinase 1
    DOI:  https://doi.org/10.1007/978-1-0716-3557-5_4
  8. J Cell Sci. 2024 Feb 19. pii: jcs.257675. [Epub ahead of print]
    C. elegans spermatocyte divisions show weak spindle checkpoint response.
    Shang-Yang Chen, Pu-Wei Cheng, Hsiao-Fang Peng, Jui-Ching Wu.
      Male meiotic division exhibits two consecutive chromosome separation events without apparent pausing. Several studies showed spermatocyte divisions are not stringently regulated as in mitotic cells. In this study, we investigated the role of the canonical spindle assembly (SAC) pathway in C. elegans spermatogenesis. We found the intensity of chromosome-associated outer kinetochore protein BUB-1 and SAC effector MDF-1 oscillate between two divisions. However, SAC target securin is degraded during the first division and remains undetectable for the second division. Inhibition of proteasome-dependent protein degradation did not affect the progression of the second division but stopped the first division at metaphase. Perturbation of spindle integrity did not affect the duration of meiosis II, and only slightly lengthened meiosis I. Our results demonstrate that male meiosis II is independent of SAC regulation, and male meiosis I exhibits only weak checkpoint response.
    Keywords:  Chromosome segregation; Male meiotic divisions; Proteasome; Securin; Spindle assembly checkpoint
    DOI:  https://doi.org/10.1242/jcs.257675
  9. Int J Mol Sci. 2024 Feb 09. pii: 2101. [Epub ahead of print]25(4):
    Chromosome Division in Early Embryos-Is Everything under Control? And Is the Cell Size Important?
    Adela Horakova, Marketa Konecna, Martin Anger.
      Chromosome segregation in female germ cells and early embryonic blastomeres is known to be highly prone to errors. The resulting aneuploidy is therefore the most frequent cause of termination of early development and embryo loss in mammals. And in specific cases, when the aneuploidy is actually compatible with embryonic and fetal development, it leads to severe developmental disorders. The main surveillance mechanism, which is essential for the fidelity of chromosome segregation, is the Spindle Assembly Checkpoint (SAC). And although all eukaryotic cells carry genes required for SAC, it is not clear whether this pathway is active in all cell types, including blastomeres of early embryos. In this review, we will summarize and discuss the recent progress in our understanding of the mechanisms controlling chromosome segregation and how they might work in embryos and mammalian embryos in particular. Our conclusion from the current literature is that the early mammalian embryos show limited capabilities to react to chromosome segregation defects, which might, at least partially, explain the widespread problem of aneuploidy during the early development in mammals.
    Keywords:  CDK1; aneuploidy; cell size; chromosome division; embryo; segregation errors; spindle; spindle assembly checkpoint
    DOI:  https://doi.org/10.3390/ijms25042101
  10. Mol Biol Cell. 2024 Feb 21. mbcE23090379
    Fluorescence complementation-based FRET imaging reveals centromere assembly dynamics.
    Zhen Dou, Ran Liu, Ping Gui, Chuanhai Fu, Jennifer Lippincott-Schwartz, Xuebiao Yao, Xing Liu.
      Visualization of specific molecules and their assembly in real time and space is essential to delineate how cellular dynamics and signaling circuit are orchestrated during cell division cycle. Our recent studies reveal structural insights into human centromere-kinetochore core CCAN complex. Here we introduce a method for optically imaging trimeric and tetrameric protein interactions at nanometer spatial resolution in live cells using fluorescence complementation-based Förster resonance energy transfer (FC-FRET). Complementary fluorescent protein molecules were first used to visualize dimerization followed by FRET measurements. Using FC- FRET, we visualized centromere CENP-SXTW tetramer assembly dynamics in live cells, and dimeric interactions between CENP-TW dimer and kinetochore protein Spc24/25 dimer in dividing cells. We further delineated the interactions of monomeric CENP-T with Spc24/25 dimer in dividing cells. Surprisingly, our analyses revealed critical role of CDK1 kinase activity in the initial recruitment of Spc24/25 by CENP-T. However, interactions between CENP-T and Spc24/25 during chromosome segregation is independent of CDK1. Thus, FC-FRET provides a unique approach to delineate spatiotemporal dynamics of trimerized and tetramerized proteins at nanometer scale and establishes a platform to report the precise regulation of multimeric protein interactions in space and time in live cells.
    DOI:  https://doi.org/10.1091/mbc.E23-09-0379
  11. Biochem Soc Trans. 2024 Feb 19. pii: BST20231071. [Epub ahead of print]
    Transcriptional repression across mitosis: mechanisms and functions.
    A Contreras, C Perea-Resa.
      Transcription represents a central aspect of gene expression with RNA polymerase machineries (RNA Pol) driving the synthesis of RNA from DNA template molecules. In eukaryotes, a total of three RNA Pol enzymes generate the plethora of RNA species and RNA Pol II is the one transcribing all protein-coding genes. A high number of cis- and trans-acting factors orchestrates RNA Pol II-mediated transcription by influencing the chromatin recruitment, activation, elongation, and/or termination steps. The levels of DNA accessibility, defining open-euchromatin versus close-heterochromatin, delimits RNA Pol II activity as well as the encounter with other factors acting on chromatin such as the DNA replication or DNA repair machineries. The stage of the cell cycle highly influences RNA Pol II activity with mitosis representing the major challenge. In fact, there is a massive inhibition of transcription during the mitotic entry coupled with chromatin dissociation of most of the components of the transcriptional machinery. Mitosis, as a consequence, highly compromises the transcriptional memory and the perpetuation of cellular identity. Once mitosis ends, transcription levels immediately recover to define the cell fate and to safeguard the proper progression of daughter cells through the cell cycle. In this review, we evaluate our current understanding of the transcriptional repression associated with mitosis with a special focus on the molecular mechanisms involved, on the potential function behind the general repression, and on the transmission of the transcriptional machinery into the daughter cells. We finally discuss the contribution that errors in the inheritance of the transcriptional machinery across mitosis might play in stem cell aging.
    Keywords:  RNA Pol II; bookmarking; centromere; gene expression; mitosis; transcription
    DOI:  https://doi.org/10.1042/BST20231071
  12. bioRxiv. 2024 Feb 07. pii: 2023.03.16.533034. [Epub ahead of print]
    Cell cycle perturbation uncouples mitotic progression and invasive behavior in a post-mitotic cell.
    Michael A Q Martinez, Chris Z Zhao, Frances E Q Moore, Callista Yee, Wan Zhang, Kang Shen, Benjamin L Martin, David Q Matus.
      The acquisition of the post-mitotic state is crucial for the execution of many terminally differentiated cell behaviors during organismal development. However, the mechanisms that maintain the post-mitotic state in this context remain poorly understood. To gain insight into these mechanisms, we used the genetically and visually accessible model of C. elegans anchor cell (AC) invasion into the vulval epithelium. The AC is a terminally differentiated uterine cell that normally exits the cell cycle and enters a post-mitotic state, initiating contact between the uterus and vulva through a cell invasion event. Here, we set out to identify the set of negative cell cycle regulators that maintain the AC in this post-mitotic, invasive state. Our findings revealed a critical role for CKI-1 (p21CIP1/p27KIP1) in redundantly maintaining the post-mitotic state of the AC, as loss of CKI-1 in combination with other negative cell cycle regulators-including CKI-2 (p21CIP1/p27KIP1), LIN-35 (pRb/p107/p130), FZR-1 (Cdh1/Hct1), and LIN-23 (β-TrCP)-resulted in proliferating ACs. Remarkably, time-lapse imaging revealed that these ACs retain their ability to invade. Upon examination of a node in the gene regulatory network controlling AC invasion, we determined that proliferating, invasive ACs do so by maintaining aspects of pro-invasive gene expression. We therefore report that the requirement for a post-mitotic state for invasive cell behavior can be bypassed following direct cell cycle perturbation.
    Keywords:  C. elegans; CKI-1; dichotomy; invasion; proliferation
    DOI:  https://doi.org/10.1101/2023.03.16.533034
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