bims-micesi Biomed News
on Mitotic cell signalling
Issue of 2024–09–29
thirteen papers selected by
Valentina Piano, Uniklinik Köln
  1. CEP192 localises mitotic Aurora-A activity by priming its interaction with TPX2.
  2. Conditional Localization Pharmacology Manipulates the Cell Cycle with Spatiotemporal Precision.
  3. Dynamic Mitotic Localization of the Centrosomal Kinases CDK1, Plk, AurK, and Nek2 in Dictyostelium amoebae.
  4. Long-range repulsion between chromosomes in mammalian oocyte spindles.
  5. "Mitotic" kinesin-5 is a dynamic brake for axonal growth.
  6. Spatiotemporal regulation of organelle transport by spindle position checkpoint kinase Kin4.
  7. Septin Organization and Dynamics for Budding Yeast Cytokinesis.
  8. Central Role of the Actomyosin Ring in Coordinating Cytokinesis Steps in Budding Yeast.
  9. Optimized expansion microscopy reveals species-specific spindle microtubule organization in Xenopus egg extracts.
  10. Molecular and mechanical mechanisms of animal cell abscission.
  11. Binding Modalities and Phase-Specific Regulation of Cyclin/Cyclin-Dependent Kinase Complexes in the Cell Cycle.
  12. The Myosin-V Myo51 and Alpha-Actinin Ain1p Cooperate during Contractile Ring Assembly and Disassembly in Fission Yeast Cytokinesis.
  13. Lamin chromatin binding is modulated by interactions of different LAP2α domains with lamins and chromatin.
  1. EMBO J. 2024 Sep 26.
    CEP192 localises mitotic Aurora-A activity by priming its interaction with TPX2.
    James Holder, Jennifer A Miles, Matthew Batchelor, Harrison Popple, Martin Walko, Wayland Yeung, Natarajan Kannan, Andrew J Wilson, Richard Bayliss, Fanni Gergely.
      Aurora-A is an essential cell-cycle kinase with critical roles in mitotic entry and spindle dynamics. These functions require binding partners such as CEP192 and TPX2, which modulate both kinase activity and localisation of Aurora-A. Here we investigate the structure and role of the centrosomal Aurora-A:CEP192 complex in the wider molecular network. We find that CEP192 wraps around Aurora-A, occupies the binding sites for mitotic spindle-associated partners, and thus competes with them. Comparison of two different Aurora-A conformations reveals how CEP192 modifies kinase activity through the site used for TPX2-mediated activation. Deleting the Aurora-A-binding interface in CEP192 prevents centrosomal accumulation of Aurora-A, curtails its activation-loop phosphorylation, and reduces spindle-bound TPX2:Aurora-A complexes, resulting in error-prone mitosis. Thus, by supplying the pool of phosphorylated Aurora-A necessary for TPX2 binding, CEP192:Aurora-A complexes regulate spindle function. We propose an evolutionarily conserved spatial hierarchy, which protects genome integrity through fine-tuning and correctly localising Aurora-A activity.
    Keywords:  Aurora-A; Centrosome; Kinase; Mitosis; Mitotic Spindle
    DOI:  https://doi.org/10.1038/s44318-024-00240-z
  2. bioRxiv. 2024 Sep 15. pii: 2024.09.12.612697. [Epub ahead of print]
    Conditional Localization Pharmacology Manipulates the Cell Cycle with Spatiotemporal Precision.
    Changfeng Deng, Yung-Chi Lan, Geng-Yuan Chen, Chigozie S Ekeabu, Megan Chung, Micheal A Lampson, David M Chenoweth.
      Traditional pharmacology has limited control of drug activity and localization in space and time. Herein, we described an approach for kinase regulation using conditional localization pharmacology (CLP), where an inactive caged inhibitor is localized to a site of interest in a dormant state using intracellular protein tethering. The activity of the inhibitor can be regulated with spatial and temporal precision in a live cellular environment using light. As a proof of concept, a photocaged MPS1 kinase inhibitor (reversine) bearing a Halo-tag ligand tether was designed to manipulate the cell cycle. We demonstrate that this new caged reversine halo probe (CRH) strategy is capable of efficient localization and exceptional spatiotemporal control over spindle assembly checkpoint (SAC) silencing and mitotic exit.
    DOI:  https://doi.org/10.1101/2024.09.12.612697
  3. Cells. 2024 Sep 10. pii: 1513. [Epub ahead of print]13(18):
    Dynamic Mitotic Localization of the Centrosomal Kinases CDK1, Plk, AurK, and Nek2 in Dictyostelium amoebae.
    Stefan Krüger, Nathalie Pfaff, Ralph Gräf, Irene Meyer.
      The centrosome of the amoebozoan model Dictyostelium discoideum provides the best-established model for an acentriolar centrosome outside the Opisthokonta. Dictyostelium exhibits an unusual centrosome cycle, in which duplication is initiated only at the G2/M transition and occurs entirely during the M phase. Little is known about the role of conserved centrosomal kinases in this process. Therefore, we have generated knock-in strains for Aurora (AurK), CDK1, cyclin B, Nek2, and Plk, replacing the endogenous genes with constructs expressing the respective green fluorescent Neon fusion proteins, driven by the endogenous promoters, and studied their behavior in living cells. Our results show that CDK1 and cyclin B arrive at the centrosome first, already during G2, followed by Plk, Nek2, and AurK. Furthermore, CDK1/cyclin B and AurK were dynamically localized at kinetochores, and AurK in addition at nucleoli. The putative roles of all four kinases in centrosome duplication, mitosis, cytokinesis, and nucleolar dynamics are discussed.
    Keywords:  Aurora; CDK1; Dictyostelium; Nek2; Plk; centrosome; cyclin; kinase; mitosis
    DOI:  https://doi.org/10.3390/cells13181513
  4. Sci Adv. 2024 Sep 27. 10(39): eadq7540
    Long-range repulsion between chromosomes in mammalian oocyte spindles.
    Colm P Kelleher, Yash P Rana, Daniel J Needleman.
      During eukaryotic cell division, a microtubule-based structure called the spindle exerts forces on chromosomes. The best-studied spindle forces, including those responsible for the separation of sister chromatids, are directed parallel to the spindle's long axis. By contrast, little is known about forces perpendicular to the spindle axis, which determine the metaphase plate configuration and thus the location of chromosomes in the subsequent nucleus. Using live-cell microscopy, we find that metaphase chromosomes are spatially anti-correlated in mouse oocyte spindles, evidence of previously unknown long-range forces acting perpendicular to the spindle axis. We explain this observation by showing that the spindle's microtubule network behaves as a nematic liquid crystal and that deformation of the nematic field around embedded chromosomes causes long-range repulsion between them.
    DOI:  https://doi.org/10.1126/sciadv.adq7540
  5. bioRxiv. 2024 Sep 15. pii: 2024.09.12.612721. [Epub ahead of print]
    "Mitotic" kinesin-5 is a dynamic brake for axonal growth.
    Wen Lu, Brad S Lee, Helen Xue Ying Deng, Margot Lakonishok, Enrique Martin-Blanco, Vladimir I Gelfand.
      During neuronal development, neurons undergo significant microtubule reorganization to shape axons and dendrites, establishing the framework for efficient wiring of the nervous system. Previous studies from our laboratory demonstrated the key role of kinesin-1 in driving microtubule-microtubule sliding, which provides the mechanical forces necessary for early axon outgrowth and regeneration in Drosophila melanogaster . In this study, we reveal the critical role of kinesin-5, a mitotic motor, in modulating the development of postmitotic neurons. Kinesin-5, a conserved homotetrameric motor, typically functions in mitosis by sliding antiparallel microtubules apart in the spindle. Here, we demonstrate that the Drosophila kinesin-5 homolog, Klp61F, is expressed in larval brain neurons, with high levels in ventral nerve cord (VNC) neurons. Knockdown of Klp61F using a pan-neuronal driver leads to severe locomotion defects and complete lethality in adult flies, mainly due to the absence of kinesin-5 in VNC motor neurons during early larval development. Klp61F depletion results in significant axon growth defects, both in cultured and in vivo neurons. By imaging individual microtubules, we observe a significant increase in microtubule motility, and excessive penetration of microtubules into the axon growth cone in Klp61F -depleted neurons. Adult lethality and axon growth defects are fully rescued by a chimeric human- Drosophila kinesin-5 motor, which accumulates at the axon tips, suggesting a conserved role of kinesin-5 in neuronal development. Altogether, our findings show that at the growth cone, kinesin-5 acts as a brake on kinesin-1-driven microtubule sliding, preventing premature microtubule entry into the growth cone. This regulatory role of kinesin-5 is essential for precise axon pathfinding during nervous system development.
    DOI:  https://doi.org/10.1101/2024.09.12.612721
  6. J Cell Sci. 2024 Sep 25. pii: jcs.261948. [Epub ahead of print]
    Spatiotemporal regulation of organelle transport by spindle position checkpoint kinase Kin4.
    Lakhan Ekal, Abdulaziz M S Alqahtani, Kathryn R Ayscough, Ewald H Hettema.
      Asymmetric cell division in Saccharomyces cerevisiae involves Class V myosin-dependent transport of organelles along the polarised actin cytoskeleton to the emerging bud. Vac17 is the vacuole/lysosome-specific myosin receptor. Its timely breakdown terminates transport and results in the proper positioning of vacuoles in the bud. Vac17 breakdown is controlled by the bud-concentrated p21-activated kinase, Cla4, and the E3-Ubiquitin ligase, Dma1. We found that the spindle position checkpoint kinase, Kin4, and to a lesser extent its paralog Frk1, contribute to successful vacuole transport by preventing the premature breakdown of Vac17 by Cla4 and Dma1. Furthermore, Kin4 and Cla4 contribute to the regulation of peroxisome transport. We conclude that Kin4 acts antagonistically to the Cla4-/Dma1-pathway to coordinate spatiotemporal regulation of organelle transport.
    Keywords:  Organelle inheritance; P21-activated kinase (PAK); Peroxisome; Spindle position checkpoint (SPOC); Vacuole
    DOI:  https://doi.org/10.1242/jcs.261948
  7. J Fungi (Basel). 2024 Sep 09. pii: 642. [Epub ahead of print]10(9):
    Septin Organization and Dynamics for Budding Yeast Cytokinesis.
    Maritzaida Varela Salgado, Simonetta Piatti.
      Cytokinesis, the process by which the cytoplasm divides to generate two daughter cells after mitosis, is a crucial stage of the cell cycle. Successful cytokinesis must be coordinated with chromosome segregation and requires the fine orchestration of several processes, such as constriction of the actomyosin ring, membrane reorganization, and, in fungi, cell wall deposition. In Saccharomyces cerevisiae, commonly known as budding yeast, septins play a pivotal role in the control of cytokinesis by assisting the assembly of the cytokinetic machinery at the division site and controlling its activity. Yeast septins form a collar at the division site that undergoes major dynamic transitions during the cell cycle. This review discusses the functions of septins in yeast cytokinesis, their regulation and the implications of their dynamic remodelling for cell division.
    Keywords:  S. cerevisiae; cytokinesis; septins
    DOI:  https://doi.org/10.3390/jof10090642
  8. J Fungi (Basel). 2024 Sep 21. pii: 662. [Epub ahead of print]10(9):
    Central Role of the Actomyosin Ring in Coordinating Cytokinesis Steps in Budding Yeast.
    Magdalena Foltman, Alberto Sanchez-Diaz.
      Eukaryotic cells must accurately transfer their genetic material and cellular components to their daughter cells. Initially, cells duplicate their chromosomes and subsequently segregate them toward the poles. The actomyosin ring, a crucial molecular machinery normally located in the middle of the cells and underneath the plasma membrane, then physically divides the cytoplasm and all components into two daughter cells, each ready to start a new cell cycle. This process, known as cytokinesis, is conserved throughout evolution. Defects in cytokinesis can lead to the generation of genetically unstable tetraploid cells, potentially initiating uncontrolled proliferation and cancer. This review focuses on the molecular mechanisms by which budding yeast cells build the actomyosin ring and the preceding steps involved in forming a scaffolding structure that supports the challenging structural changes throughout cytokinesis. Additionally, we describe how cells coordinate actomyosin ring contraction, plasma membrane ingression, and extracellular matrix deposition to successfully complete cytokinesis. Furthermore, the review discusses the regulatory roles of Cyclin-Dependent Kinase (Cdk1) and the Mitotic Exit Network (MEN) in ensuring the precise timing and execution of cytokinesis. Understanding these processes in yeast provides insights into the fundamental aspects of cell division and its implications for human health.
    Keywords:  actomyosin ring; budding yeast; cell division; cytokinesis; extracellular matrix remodeling; ingression; ingression progression complexes (IPCs); septin; septum formation
    DOI:  https://doi.org/10.3390/jof10090662
  9. bioRxiv. 2024 Sep 11. pii: 2024.09.11.612005. [Epub ahead of print]
    Optimized expansion microscopy reveals species-specific spindle microtubule organization in Xenopus egg extracts.
    Gabriel Guilloux, Maiko Kitaoka, Karel Mocaer, Claire Heichette, Laurence Duchesne, Rebecca Heald, Thierry Pécot, Romain Gibeaux.
      The spindle is a key structure in cell division as it orchestrates the accurate segregation of genetic material. While its assembly and function are well-studied, the mechanisms regulating spindle architecture remain elusive. In this study, we focus on the differences in spindle organization between Xenopus laevis and Xenopus tropicalis , leveraging expansion microscopy (ExM) to overcome the limitations of conventional imaging techniques. We optimized an ExM protocol tailored for Xenopus egg extract spindles, improving upon fixation, denaturation and gelation methods to achieve higher resolution imaging of spindles. Our protocol preserves spindle integrity and allows effective pre-expansion immunofluorescence. This method enabled detailed analysis of the differences in microtubule organization between the two species. X. laevis spindles overall exhibit a broader range of bundle sizes, while X. tropicalis spindles are more limited to smaller bundles. Moreover, while both species favor larger bundle sizes near and at the spindle center, X. tropicalis spindles otherwise prefer very small bundles, and X. laevis spindles medium-sized bundles. By enhancing resolution and minimizing distortions and fixation artifacts, our optimized ExM approach offers new insights into spindle morphology and provides a robust tool for studying the structural intricacies of these large cellular assemblies. This work advances our understanding of spindle architecture and opens up new avenues for exploring spindle-related questions.
    SIGNIFICANCE STATEMENT: Correct spindle morphology is key to its function; however, traditional microscopy methods limit our view of spindle architecture. This study addresses the gap in resolving detailed spindle microtubule organization by using advanced imaging. The research utilizes Expansion Microscopy (ExM) to reveal previously unobservable details of spindle morphology in egg extracts of two Xenopus species ( X. laevis and X. tropicalis ). This approach provides unprecedented clarity on microtubule arrangement and variations in spindle architecture. This work establishes a new protocol for high-resolution imaging of spindle structures, offering insights into how spindle architecture is adapted in differently-sized spindles to ensure proper function.
    DOI:  https://doi.org/10.1101/2024.09.11.612005
  10. FEBS Lett. 2024 Sep 26.
    Molecular and mechanical mechanisms of animal cell abscission.
    Amber Öztop, Agathe Chaigne.
      Cytokinesis leads to the distribution of segregated chromosomes, membrane, and cytoplasmic material in the two daughter cells, and ultimately concludes with abscission, their physical separation. In this Graphical Review, we outline the key events that lead to abscission and discuss mechanisms of delayed abscisison.
    Keywords:  Aurora B; abscission; cytokinesis; midbody; mitosis
    DOI:  https://doi.org/10.1002/1873-3468.15015
  11. J Phys Chem B. 2024 Sep 24.
    Binding Modalities and Phase-Specific Regulation of Cyclin/Cyclin-Dependent Kinase Complexes in the Cell Cycle.
    Michael T Bergman, Wengang Zhang, Yonglan Liu, Hyunbum Jang, Ruth Nussinov.
      Cyclin-dependent kinases (CDKs) are activated upon cyclin-binding to enable progression through the cell cycle. Dominant CDKs and cyclins in mammalian cells include CDK1, CDK2, CDK4, and CDK6 and corresponding cyclins A, B, D, and E. While only certain, "typical" cyclin/CDK complexes are primarily responsible for cell cycle progression, "atypical" cyclin/CDK complexes can form and sometimes perform the same roles as typical complexes. We asked what structural features of cyclins and CDKs favor the formation of typical complexes, a vital yet not fully explored question. We use computational docking and biophysical analyses to exhaustively evaluate the structure and stability of all CDK and cyclin complexes listed above. We find that binding of the complexes is generally stronger for typical than for atypical complexes, especially when the CDK is in an active conformation. Typical complexes have denser clusters, indicating that they have more defined cyclin-binding sites than atypical complexes. Our results help explain three notable features of cyclin/CDK function in the cell cycle: (i) why CDK4 and cyclin-D have exceptionally high specificity for each other; (ii) why both cyclin-A and cyclin-B strongly activate CDK1, whereas CDK2 is only strongly activated by cyclin-A; and (iii) why cyclin-E normally activates CDK2 but not CDK1. Overall, this work reveals the binding modalities of cyclin/CDK complexes, how the modalities lead to the preference for typical complexes versus atypical complexes, and how binding modalities differ between typical complexes. Our observations suggest targeting CDK catalytic actions through destabilizing their native differential cyclin interfaces.
    DOI:  https://doi.org/10.1021/acs.jpcb.4c03243
  12. J Fungi (Basel). 2024 Sep 12. pii: 647. [Epub ahead of print]10(9):
    The Myosin-V Myo51 and Alpha-Actinin Ain1p Cooperate during Contractile Ring Assembly and Disassembly in Fission Yeast Cytokinesis.
    Zoe L Tyree, Kimberly Bellingham-Johnstun, Jessica Martinez-Baird, Caroline Laplante.
      Cytokinesis is driven in part by the constriction of a ring of actin filaments, myosin motors and other proteins. In fission yeast, three myosins contribute to cytokinesis including a Myosin-V Myo51. As Myosin-Vs typically carry cargo along actin filaments, the role of Myo51 in cytokinesis remains unclear. The previous work suggests that Myo51 may crosslink actin filaments. We hypothesized that if Myo51 crosslinks actin filaments, cells carrying double deletions of ain1, which encodes the crosslinker alpha-actinin, and myo51 (∆ain1 ∆myo51 cells) will exhibit more severe cytokinesis phenotypes than cells with the single ∆ain1 mutation. Contrary to our expectations, we found that the loss of Myo51 in ∆ain1 cells partially rescued the severity of the node clumping phenotype measured in ∆ain1 cells. Furthermore, we describe a normal process of contractile ring "shedding", the appearance of fragments of ring material extending away from the contractile ring along the ingressing septum that occurs in the second half of constriction. We measured that ∆ain1 ∆myo51 cells exhibit premature and exaggerated shedding. Our work suggests that Myo51 is not a simple actin filament crosslinker. Instead, a role in effective node motion better recapitulates its function during ring assembly and disassembly.
    Keywords:  Myosin-V; alpha-actinin; cytokinesis
    DOI:  https://doi.org/10.3390/jof10090647
  13. iScience. 2024 Oct 18. 27(10): 110869
    Lamin chromatin binding is modulated by interactions of different LAP2α domains with lamins and chromatin.
    Daria Filipczak, Anna Souchet, Konstantina Georgiou, Roland Foisner, Nana Naetar.
      Lamins A and C are components of the lamina at the nuclear periphery and associate with heterochromatin. A distinct, relatively mobile pool of lamin A/C in the nuclear interior associates with euchromatic regions and with lamin-associated polypeptide 2α (LAP2α). Here we show that phosphorylation-dependent impairment of lamin assembly had no effect on its chromatin association, while LAP2α depletion was sufficient to increase chromatin association of lamins. This suggests that complex interactions between LAP2α, chromatin, and lamins regulate lamin chromatin binding. Both the C terminus of LAP2α and its N-terminal LAP2-Emerin-MAN1 (LEM) domain, mediating interaction with lamin A/C indirectly via barrier-to-autointegration factor (BAF), are required for binding to lamins. The N-terminal LEM-like domain of LAP2α, but not its LEM domain, mediates chromatin association of LAP2α and requires LAP2α dimerization via its C terminus. Our data suggest that formation of several LAP2α-, lamin A/C-, and BAF-containing complexes in the nucleoplasm and on chromatin affects lamin chromatin association.
    Keywords:  Cell biology; Chromosome organization; Molecular interaction; Nucleic acids
    DOI:  https://doi.org/10.1016/j.isci.2024.110869
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