bims-micesi Biomed News
on Mitotic cell signalling
Issue of 2022–05–01
twelve papers selected by
Valentina Piano, Max Planck Institute of Molecular Physiology
  1. CDK1-cyclin-B1-induced kindlin degradation drives focal adhesion disassembly at mitotic entry.
  2. Endomembranes promote chromosome missegregation by ensheathing misaligned chromosomes.
  3. Synthetic antibody binders detect distinct cellular states of Chromosome Passenger Complex proteins.
  4. Spindle and Kinetochore-Associated Complex is Associated With Poor Prognosis in Adrenocortical Carcinoma.
  5. Interphase microtubule disassembly is a signaling cue that drives cell rounding at mitotic entry.
  6. Structure and function of MuvB complexes.
  7. Functional dissection of human mitotic genes using CRISPR-Cas9 tiling screens.
  8. Single-Molecule Micromanipulation and Super-Resolution Imaging Resolve Nanodomains Underlying Chromatin Folding in Mitotic Chromosomes.
  9. Photocontrolling Microtubule Dynamics with Photoswitchable Chemical Reagents.
  10. [The kiss of life: Aurora A embraces the phosphate of its cofactor Bora to trigger mitotic entry].
  11. Glial-secreted Netrins regulate Robo1/Rac1-Cdc42 signaling threshold levels during Drosophila asymmetric neural stem/progenitor cell division.
  12. A new kind of cell division.
  1. Nat Cell Biol. 2022 Apr 25.
    CDK1-cyclin-B1-induced kindlin degradation drives focal adhesion disassembly at mitotic entry.
    Nan-Peng Chen, Jonas Aretz, Reinhard Fässler.
      The disassembly of integrin-containing focal adhesions (FAs) at mitotic entry is essential for cell rounding, mitotic retraction fibre formation, bipolar spindle positioning and chromosome segregation. The mechanism that drives FA disassembly at mitotic entry is unknown. Here, we show that the CDK1-cyclin B1 complex phosphorylates the integrin activator kindlin, which results in the recruitment of the cullin 9-FBXL10 ubiquitin ligase complex that mediates kindlin ubiquitination and degradation. This molecular pathway is essential for FA disassembly and cell rounding, as phospho-inhibitory mutations of the CDK1 motif prevent kindlin degradation, FA disassembly and mitotic cell rounding. Conversely, phospho-mimetic mutations promote kindlin degradation in interphase, accelerate mitotic cell rounding and impair mitotic retraction fibre formation. Despite the opposing effects on kindlin stability, both types of mutations cause severe mitotic spindle defects, apoptosis and aneuploidy. Thus, the exquisite regulation of kindlin levels at mitotic entry is essential for cells to progress accurately through mitosis.
    DOI:  https://doi.org/10.1038/s41556-022-00886-z
  2. J Cell Biol. 2022 Jun 06. pii: e202203021. [Epub ahead of print]221(6):
    Endomembranes promote chromosome missegregation by ensheathing misaligned chromosomes.
    Nuria Ferrandiz, Laura Downie, Georgina P Starling, Stephen J Royle.
      Errors in mitosis that cause chromosome missegregation lead to aneuploidy and micronucleus formation, which are associated with cancer. Accurate segregation requires the alignment of all chromosomes by the mitotic spindle at the metaphase plate, and any misalignment must be corrected before anaphase is triggered. The spindle is situated in a membrane-free "exclusion zone"; beyond this zone, endomembranes (mainly endoplasmic reticulum) are densely packed. We investigated what happens to misaligned chromosomes localized beyond the exclusion zone. Here we show that such chromosomes become ensheathed in multiple layers of endomembranes. Chromosome ensheathing delays mitosis and increases the frequency of chromosome missegregation and micronucleus formation. We use an induced organelle relocalization strategy in live cells to show that clearance of endomembranes allows for the rescue of chromosomes that were destined for missegregation. Our findings indicate that endomembranes promote the missegregation of misaligned chromosomes that are outside the exclusion zone and therefore constitute a risk factor for aneuploidy.
    DOI:  https://doi.org/10.1083/jcb.202203021
  3. J Mol Biol. 2022 Apr 22. pii: S0022-2836(22)00182-6. [Epub ahead of print] 167602
    Synthetic antibody binders detect distinct cellular states of Chromosome Passenger Complex proteins.
    Marcin Ura, Somnath Mukherjee, Edyta Marcon, Stefan A Koestler, Anthony A Kossiakoff.
      High performance affinity reagents are essential tools to enable biologists to profile the cellular location and composition of macromolecular complexes undergoing dynamic reorganization. To support further development of such tools, we have assembled a high-throughput phage display pipeline to generate Fab-based affinity reagents that target different dynamic forms of a large macromolecular complex, using the Chromosomal Passenger Complex (CPC), as an example. The CPC is critical for the maintenance of chromosomal and cytoskeleton processes during cell division. The complex contains 4 protein components: Aurora B kinase, survivin, borealin and INCENP. The CPC acts as a node to dynamically organize other partnering subcomplexes to build multiple functional structures during mitotic progression. Using phage display mutagenesis, a cohort of synthetic antibodies (sABs) were generated against different domains of survivin, borealin and INCENP. Immunofluorescence established that a set of these sABs can discriminate between the form of the CPC complex in the midbody versus the spindle. Others localize to targets, which appear to be less organized, in the nucleus or cytoplasm. This differentiation suggests that different CPC epitopes have dynamic accessibility depending upon the mitotic state of the cell. An IP/mass spec analysis was performed using sABs that bound specifically to the CPC in either the midbody or MT spindle macromolecular assemblies. Thus, sABs can be exploited as high performance reagents to profile the accessibility of different components of the CPC within macromolecular assemblies during different stages of mitosis suggesting this high throughput approach will be applicable to other complex macromolecular systems.
    Keywords:  Borealin; INCENP; Phage display; Survivin; mitosis; synthetic antibody binders
    DOI:  https://doi.org/10.1016/j.jmb.2022.167602
  4. J Surg Res. 2022 Apr 20. pii: S0022-4804(22)00170-6. [Epub ahead of print]277 50-59
    Spindle and Kinetochore-Associated Complex is Associated With Poor Prognosis in Adrenocortical Carcinoma.
    Shoukai Yu, Jun Ma.
       INTRODUCTION: The spindle and kinetochore-associated (SKA) complex, composed of three subunits (SKA1, SKA2, and SKA3), stabilizes spindle microtubule attachment to the kinetochore (KT) in the middle stage of mitosis. High expression of this complex is associated with poor prognosis for several tumors. However, the potential role of SKA complex overexpression in rare malignant diseases, such as adrenocortical carcinoma (ACC), has not been well investigated.
    MATERIALS AND METHODS: In this study, we used several databases to explore the relationship between SKA subunit expression and prognosis in ACC patients. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genome (KEGG) databases were used to analyze enriched pathways in ACC.
    RESULTS: The results suggest that each of the three SKA subunits are overexpressed in ACC and that high expression is correlated with poor patient prognosis. Overexpression of the SKA complex is associated with the expression of organelle fission, nuclear division, and chromosome segregation pathways. Furthermore, differential expression of hub genes for proteins that interact physically or functionally with the SKA complex (CCNB2, UBE2C, BUB1B, TPX2, CCNA2, CDCA8, CCNB1, MELK, TOP2A, and KIF2C) revealed additional potential biomarkers for ACC.
    CONCLUSIONS: Our findings provide additional understanding of the mechanisms of ACC and suggest an approach for biomarker discovery using publicly available resources.
    Keywords:  Adrenocortical carcinoma; Cell cycle; Prognosis; Rare malignant tumor; SKA complex
    DOI:  https://doi.org/10.1016/j.jss.2022.03.022
  5. J Cell Biol. 2022 Jun 06. pii: e202109065. [Epub ahead of print]221(6):
    Interphase microtubule disassembly is a signaling cue that drives cell rounding at mitotic entry.
    Kévin Leguay, Barbara Decelle, Islam E Elkholi, Michel Bouvier, Jean-François Côté, Sébastien Carréno.
      At mitotic entry, reorganization of the actomyosin cortex prompts cells to round-up. Proteins of the ezrin, radixin, and moesin family (ERM) play essential roles in this process by linking actomyosin forces to the plasma membrane. Yet, the cell-cycle signal that activates ERMs at mitotic entry is unknown. By screening a compound library using newly developed biosensors, we discovered that drugs that disassemble microtubules promote ERM activation. We further demonstrated that disassembly of interphase microtubules at mitotic entry directs ERM activation and metaphase cell rounding through GEF-H1, a Rho-GEF inhibited by microtubule binding, RhoA, and its kinase effector SLK. We finally demonstrated that GEF-H1 and Ect2, another Rho-GEF previously identified to control actomyosin forces, act together to drive activation of ERMs and cell rounding in metaphase. In summary, we report microtubule disassembly as a cell-cycle signal that controls a signaling network ensuring that actomyosin forces are efficiently integrated at the plasma membrane to promote cell rounding at mitotic entry.
    DOI:  https://doi.org/10.1083/jcb.202109065
  6. Oncogene. 2022 Apr 26.
    Structure and function of MuvB complexes.
    Gerd A Müller, Anushweta Asthana, Seth M Rubin.
      Proper progression through the cell-division cycle is critical to normal development and homeostasis and is necessarily misregulated in cancer. The key to cell-cycle regulation is the control of two waves of transcription that occur at the onset of DNA replication (S phase) and mitosis (M phase). MuvB complexes play a central role in the regulation of these genes. When cells are not actively dividing, the MuvB complex DREAM represses G1/S and G2/M genes. Remarkably, MuvB also forms activator complexes together with the oncogenic transcription factors B-MYB and FOXM1 that are required for the expression of the mitotic genes in G2/M. Despite this essential role in the control of cell division and the relationship to cancer, it has been unclear how MuvB complexes inhibit and stimulate gene expression. Here we review recent discoveries of MuvB structure and molecular interactions, including with nucleosomes and other chromatin-binding proteins, which have led to the first mechanistic models for the biochemical function of MuvB complexes.
    DOI:  https://doi.org/10.1038/s41388-022-02321-x
  7. Genes Dev. 2022 Apr 01. 36(7-8): 495-510
    Functional dissection of human mitotic genes using CRISPR-Cas9 tiling screens.
    Jacob A Herman, Sonali Arora, Lucas Carter, Jun Zhu, Sue Biggins, Patrick J Paddison.
      The identity of human protein-coding genes is well known, yet our in-depth knowledge of their molecular functions and domain architecture remains limited by shortcomings in homology-based predictions and experimental approaches focused on whole-gene depletion. To bridge this knowledge gap, we developed a method that leverages CRISPR-Cas9-induced mutations across protein-coding genes for the a priori identification of functional regions at the sequence level. As a test case, we applied this method to 48 human mitotic genes, revealing hundreds of regions required for cell proliferation, including domains that were experimentally characterized, ones that were predicted based on homology, and novel ones. We validated screen outcomes for 15 regions, including amino acids 387-402 of Mad1, which were previously uncharacterized but contribute to Mad1 kinetochore localization and chromosome segregation fidelity. Altogether, we demonstrate that CRISPR-Cas9-based tiling mutagenesis identifies key functional domains in protein-coding genes de novo, which elucidates separation of function mutants and allows functional annotation across the human proteome.
    Keywords:  CRISPR–Cas9; MAD1L1; Mad1; functional genomics; human genome; human proteome; kinetochore; mitosis; spindle assembly checkpoint
    DOI:  https://doi.org/10.1101/gad.349319.121
  8. ACS Nano. 2022 Apr 29.
    Single-Molecule Micromanipulation and Super-Resolution Imaging Resolve Nanodomains Underlying Chromatin Folding in Mitotic Chromosomes.
    Jiabin Wang, Chuansheng Hu, Xuecheng Chen, Yaqian Li, Jielin Sun, Daniel M Czajkowsky, Zhifeng Shao.
      The folding of interphase chromatin into highly compact mitotic chromosomes is one of the most recognizable changes during the cell cycle. However, the structural organization underlying this drastic compaction remains elusive. Here, we combine several super resolution methods, including structured illumination microscopy (SIM), binding-activated localization microscopy (BALM), and atomic force microscopy (AFM), to examine the structural details of the DNA within the mitotic chromosome, both in the native state and after up to 30-fold extension using single-molecule micromanipulation. Images of native chromosomes reveal widespread ∼125 nm compact granules (CGs) throughout the metaphase chromosome. However, at maximal extensions, we find exclusively ∼90 nm domains (mitotic nanodomains, MNDs) that are unexpectedly resistant to extensive forces of tens of nanonewtons. The DNA content of the MNDs is estimated to be predominantly ∼80 kb, which is comparable to the size of the inner loops predicted by a recent nested loop model of the mitotic chromosome. With this DNA content, the total volume expected of the human genome assuming closely packed MNDs is nearly identical to what is observed. Thus, altogether, these results suggest that these mechanically stable MNDs, and their higher-order assembly into CGs, are the dominant higher-level structures that underlie the compaction of chromatin from interphase to metaphase.
    Keywords:  AFM; BALM; mitotic chromosome; structural domains; super-resolution
    DOI:  https://doi.org/10.1021/acsnano.2c01025
  9. Methods Mol Biol. 2022 ;2430 403-430
    Photocontrolling Microtubule Dynamics with Photoswitchable Chemical Reagents.
    Oliver Thorn-Seshold, Joyce C M Meiring.
      Microtubule dynamics can be inhibited with sub-second temporal resolution and cellular-scale spatial resolution, by using precise illuminations to optically pattern where and when photoswitchable microtubule-inhibiting chemical reagents exert their latent bioactivity. The recently available reagents (SBTub, PST, STEpo, AzTax, PHTub) now enable researchers to use light to reversibly modulate microtubule-dependent processes in eukaryotes, in 2D and 3D cell culture as well as in vivo, across a variety of model organisms: with applications in fields from cargo transport to cell migration, cell division, and embryonic development.Here we give an introduction to using these photoswitchable microtubule inhibitors in cells. We describe the theory of small molecule photoswitching, and the unique performance features, usage requirements, and limitations that photoswitchable chemical reagents have; then we summarize the major classes of photoswitchable microtubule inhibitors that are currently available, with the properties that suit them to different applications, and troubleshooting measures for avoiding common mistakes. We outline workflows to establish cellular assays where they are used to optically control microtubule dynamics in a temporally reversible fashion with spatial specificity down to a single selected cell within a field of view. The methods in this chapter also equip the reader to tackle advanced uses of photoswitchable chemical reagents, in 3D culture and in vivo.
    Keywords:  Cell division; Cell migration; Cytoskeleton; Development; Microtubule dynamics; Optical control; Optogenetics; Photocontrol; Photopharmaceutical; Photopharmacology; Photoswitch; Tubulin polymerization inhibitor
    DOI:  https://doi.org/10.1007/978-1-0716-1983-4_26
  10. Med Sci (Paris). 2022 Apr;38(4): 345-347
    [The kiss of life: Aurora A embraces the phosphate of its cofactor Bora to trigger mitotic entry].
    Anaïs Pillan, Nicolas Tavernier, Lionel Pintard.
      
    DOI:  https://doi.org/10.1051/medsci/2022033
  11. Curr Biol. 2022 Apr 18. pii: S0960-9822(22)00566-8. [Epub ahead of print]
    Glial-secreted Netrins regulate Robo1/Rac1-Cdc42 signaling threshold levels during Drosophila asymmetric neural stem/progenitor cell division.
    Ana de Torres-Jurado, Sandra Manzanero-Ortiz, Ana Carmena.
      Asymmetric stem cell division (ASCD) is a key mechanism in development, cancer, and stem cell biology. Drosophila neural stem cells, called neuroblasts (NBs), divide asymmetrically through intrinsic mechanisms. Here, we show that the extrinsic axon guidance cues Netrins, secreted by a glial niche surrounding larval brain neural stem cell lineages, regulate NB ASCD. Netrin-Frazzled/DCC signaling modulates, through Abelson kinase, Robo1 signaling threshold levels in Drosophila larval brain neural stem and progenitor cells of NBII lineages. Unbalanced Robo1 signaling levels induce ectopic NBs and progenitor cells due to failures in the ASCD process. Mechanistically, Robo1 signaling directly impinges on the intrinsic ASCD machinery, such as aPKC, Canoe/Afadin, and Numb, through the small GTPases Rac1 and Cdc42, which are required for the localization in mitotic NBs of Par-6, a Cdc42 physical partner and a core component of the Par (Par-6-aPKC-Par3/Bazooka) apical complex.
    Keywords:  Drosophila; Netrin-Frazzled/DCC-like signaling; Slit-Robo signaling; asymmetric stem cell division; neural stem cells; neuroblast; niche; small GTPases Rac1 and Cdc42
    DOI:  https://doi.org/10.1016/j.cub.2022.04.001
  12. Nature. 2022 Apr 29.
    A new kind of cell division.
    Nick Petrić Howe.
      
    Keywords:  Cell biology
    DOI:  https://doi.org/10.1038/d41586-022-01184-2
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