bims-micesi 2021-11-28 papers

bims-micesi

Biomed News

on Mitotic cell signalling

Issue of 2021–11–28
ten papers selected by
Valentina Piano, Max Planck Institute of Molecular Physiology

Multisite phosphorylation by Cdk1 initiates delayed negative feedback to control mitotic transcription.
Highway to hell-thy meiotic divisions: Chromosome passenger complex functions driven by microtubules: CPC interactions with both the chromosomes and microtubules are important for spindle assembly and function.
Ccp1-Ndc80 switch at the N terminus of CENP-T regulates kinetochore assembly.
Conserved Mitotic Phosphorylation of a Proteasome Subunit Regulates Cell Proliferation.
The phosphatase CTDSPL2 is phosphorylated in mitosis and a target for restraining tumor growth and motility in pancreatic cancer.
The Multifaceted Regulation of Mitochondrial Dynamics During Mitosis.
Chromatin bridges, not micronuclei, activate cGAS after drug-induced mitotic errors in human cells.
Mitotic Errors Promote Genomic Instability and Leukemia in a Novel Mouse Model of Fanconi Anemia.
Mitotic Centromere-Associated Kinesin (MCAK/KIF2C) Regulates Cell Migration and Invasion by Modulating Microtubule Dynamics and Focal Adhesion Turnover.
Diverse mechanisms of centromere specification.

Curr Biol. 2021 Nov 16. pii: S0960-9822(21)01522-0. [Epub ahead of print]

Multisite phosphorylation by Cdk1 initiates delayed negative feedback to control mitotic transcription.

Jonathan B Asfaha, Mihkel Örd, Christopher R Carlson, Ilona Faustova, Mart Loog, David O Morgan.

  Cell-cycle progression is driven by the phosphorylation of cyclin-dependent kinase (Cdk) substrates.1-3 The order of substrate phosphorylation depends in part on the general rise in Cdk activity during the cell cycle,4-7 together with variations in substrate docking to sites on associated cyclin and Cks subunits.3,6,8-10 Many substrates are modified at multiple sites to provide more complex regulation.10-14 Here, we describe an elegant regulatory circuit based on multisite phosphorylation of Ndd1, a transcriptional co-activator of budding yeast genes required for mitotic progression.11,12 As cells enter mitosis, Ndd1 phosphorylation by Cdk1 is known to promote mitotic cyclin (CLB2) gene transcription, resulting in positive feedback.13-16 Consistent with these findings, we show that low Cdk1 activity promotes CLB2 expression at mitotic entry. We also find, however, that when high Cdk1 activity accumulates in a mitotic arrest, CLB2 expression is inhibited. Inhibition is accompanied by Ndd1 degradation, and we present evidence that degradation is triggered by multisite Ndd1 phosphorylation by high mitotic Cdk1-Clb2 activity. Complete Ndd1 phosphorylation by Clb2-Cdk1-Cks1 requires the phosphothreonine-binding site of Cks1, as well as a recently identified phosphate-binding pocket on the cyclin Clb2.17 We therefore propose that initial phosphorylation by Cdk1 primes Ndd1 for delayed secondary phosphorylation at suboptimal sites that promote degradation. Together, our results suggest that rising levels of mitotic Cdk1 activity act at multiple phosphorylation sites on Ndd1, first triggering rapid positive feedback and then promoting delayed negative feedback, resulting in a pulse of mitotic gene expression.

Keywords:  Cdk; Cks1; Clb2; Ndd1; cell cycle; cyclin gene expression; multisite phosphorylation; phosphodegron

DOI:  https://doi.org/10.1016/j.cub.2021.11.001
Bioessays. 2021 Nov 25. e2100202

Highway to hell-thy meiotic divisions: Chromosome passenger complex functions driven by microtubules: CPC interactions with both the chromosomes and microtubules are important for spindle assembly and function.

Kim S McKim.

  The chromosome passenger complex (CPC) localizes to chromosomes and microtubules, sometimes simultaneously. The CPC also has multiple domains for interacting with chromatin and microtubules. Interactions between the CPC and both the chromatin and microtubules is important for spindle assembly and error correction. Such dual chromatin-microtubule interactions may increase the concentration of the CPC necessary for efficient kinase activity while also making it responsive to specific conditions or structures in the cell. CPC-microtubule dependent functions are considered in the context of the first meiotic division. Acentrosomal spindle assembly is a process that depends on transfer of the CPC from the chromosomes to the microtubules. Furthermore, transfer to the microtubules is not only to position the CPC for a later role in cytokinesis; metaphase I error correction and subsequent bi-orientation of bivalents may depend on microtubule associated CPC interacting with the kinetochores.

Keywords:  central spindle; chromosome passenger complex; chromosome segregation; kinetochore; meiosis; oocyte

DOI:  https://doi.org/10.1002/bies.202100202
Proc Natl Acad Sci U S A. 2021 Nov 30. pii: e2104459118. [Epub ahead of print]118(48):

Ccp1-Ndc80 switch at the N terminus of CENP-T regulates kinetochore assembly.

Qianhua Dong, Xue-Lei Liu, Xiao-Hui Wang, Yu Zhao, Yu-Hang Chen, Fei Li.

  Kinetochores, a protein complex assembled on centromeres, mediate chromosome segregation. In most eukaryotes, centromeres are epigenetically specified by the histone H3 variant CENP-A. CENP-T, an inner kinetochore protein, serves as a platform for the assembly of the outer kinetochore Ndc80 complex during mitosis. How CENP-T is regulated through the cell cycle remains unclear. Ccp1 (counteracter of CENP-A loading protein 1) associates with centromeres during interphase but delocalizes from centromeres during mitosis. Here, we demonstrated that Ccp1 directly interacts with CENP-T. CENP-T is important for the association of Ccp1 with centromeres, whereas CENP-T centromeric localization depends on Mis16, a homolog of human RbAp48/46. We identified a Ccp1-interaction motif (CIM) at the N terminus of CENP-T, which is adjacent to the Ndc80 receptor motif. The CIM domain is required for Ccp1 centromeric localization, and the CIM domain-deleted mutant phenocopies ccp1Δ. The CIM domain can be phosphorylated by CDK1 (cyclin-dependent kinase 1). Phosphorylation of CIM weakens its interaction with Ccp1. Consistent with this, Ccp1 dissociates from centromeres through all stages of the cell cycle in the phosphomimetic mutant of the CIM domain, whereas in the phospho-null mutant of the domain, Ccp1 associates with centromeres during mitosis. We further show that the phospho-null mutant disrupts the positioning of the Ndc80 complex during mitosis, resulting in chromosome missegregation. This work suggests that competitive exclusion between Ccp1 and Ndc80 at the N terminus of CENP-T via phosphorylation ensures precise kinetochore assembly during mitosis and uncovers a previously unrecognized mechanism underlying kinetochore assembly through the cell cycle.

Keywords:  CENP-A; CENP-T; centromere; kinetochore; phosphorylation

DOI:  https://doi.org/10.1073/pnas.2104459118
Cells. 2021 Nov 08. pii: 3075. [Epub ahead of print]10(11):

Conserved Mitotic Phosphorylation of a Proteasome Subunit Regulates Cell Proliferation.

Jinyuan Duan, Wenzhu Li, Xin Shu, Bing Yang, Xiangwei He, Xing Guo.

  Reversible phosphorylation has emerged as an important mechanism for regulating proteasome function in various physiological processes. Essentially all proteasome phosphorylations characterized thus far occur on proteasome holoenzyme or subcomplexes to regulate substrate degradation. Here, we report a highly conserved phosphorylation that only exists on the unassembled α5 subunit of the proteasome. The modified residue, α5-Ser16, is within a SP motif typically recognized by cyclin-dependent kinases (CDKs). Using a phospho-specific antibody generated against this site, we found that α5-S16 phosphorylation is mitosis-specific in both yeast and mammalian cells. Blocking this site with a S16A mutation caused growth defect and G2/M arrest of the cell cycle. α5-S16 phosphorylation depends on CDK1 activity and is highly abundant in some but not all mitotic cells. Immunoprecipitation and mass spectrometry (IP-MS) studies identified numerous proteins that could interact with phosphorylated α5, including PLK1, a key regulator of mitosis. α5-PLK1 interaction increased upon mitosis and could be facilitated by S16 phosphorylation. CDK1 activation downstream of PLK1 activity was delayed in S16A mutant cells, suggesting an important role of α5-S16 phosphorylation in regulating PLK1 and mitosis. These data have revealed an unappreciated function of "exo-proteasome" phosphorylation of a proteasome subunit and may bring new insights to our understanding of cell cycle control.

Keywords:  PSMA5/α5; cell cycle; mitosis; phosphorylation; proteasome

DOI:  https://doi.org/10.3390/cells10113075
Cancer Lett. 2021 Nov 20. pii: S0304-3835(21)00585-1. [Epub ahead of print]526 53-65

The phosphatase CTDSPL2 is phosphorylated in mitosis and a target for restraining tumor growth and motility in pancreatic cancer.

Yi Xiao, Yuanhong Chen, Aimin Peng, Jixin Dong.

  Carboxy-terminal domain (CTD) small phosphatase like 2 (CTDSPL2), also known as SCP4 or HSPC129, is a new member of the small CTD phosphatase (SCP) family and its role in cancers remains unclear. Here, we used a Phos-tag technique to screen a series of phosphatases and identified CTDSPL2 as a mitotic regulator. We demonstrated that CTDSPL2 was phosphorylated at T86, S104, and S134 by cyclin-dependent kinase 1 (CDK1) in mitosis. Depletion of CTDSPL2 led to mitotic defects and prolonged mitosis. Resultantly, CTDSPL2 deletion restrained proliferation, migration, and invasion in pancreatic cancer cells. We further confirmed the dominant negative effects of a phosphorylation-deficient mutant form of CTDSPL2, implying the biological significance of CTDSPL2 mitotic phosphorylation. Moreover, RT2 cell cycle array analysis revealed p21 and p27 as downstream regulators of CTDSPL2, and inhibition of p21 and/or p27 partially rescued the phenotype in CTDSPL2-deficient cell lines. Importantly, both CTDSPL2 depletion and phosphorylation-deficient mutant CTDSPL2 hindered tumor growth in xenograft models. Together, our findings for the first time highlight the novel role of CTDSPL2 in regulating cell mitosis, proliferation and motility in pancreatic cancer and point out the implications of CTDSPL2 in regulating two critical cell cycle participants (p21 and p27), providing an alternative molecular target for pancreatic cancer treatment.

Keywords:  CDK1; CTDSPL2; Mitosis; Pancreatic cancer; Phosphorylation; p21; p27

DOI:  https://doi.org/10.1016/j.canlet.2021.11.018
Front Cell Dev Biol. 2021 ;9 767221

The Multifaceted Regulation of Mitochondrial Dynamics During Mitosis.

Evanthia Pangou, Izabela Sumara.

  Mitosis ensures genome integrity by mediating precise segregation of the duplicated genetic material. Segregation of subcellular organelles during mitosis also needs to be tightly coordinated in order to warrant their proper inheritance and cellular homeostasis. The inheritance of mitochondria, a powerhouse of the cell, is tightly regulated in order to meet the high energy demand to fuel the mitotic machinery. Mitochondria are highly dynamic organelles, which undergo events of fission, fusion and transport during different cell cycle stages. Importantly, during mitosis several kinases phosphorylate the key mitochondrial factors and drive fragmentation of mitochondria to allow for their efficient distribution and inheritance to two daughter cells. Recent evidence suggests that mitochondrial fission can also actively contribute to the regulation of mitotic progression. This review aims at summarizing established and emerging concepts about the complex regulatory networks which couple crucial mitotic factors and events to mitochondrial dynamics and which could be implicated in human disease.

Keywords:  disease; fission; fusion; mitochondria; mitosis; transport

DOI:  https://doi.org/10.3389/fcell.2021.767221
Proc Natl Acad Sci U S A. 2021 Nov 30. pii: e2103585118. [Epub ahead of print]118(48):

Chromatin bridges, not micronuclei, activate cGAS after drug-induced mitotic errors in human cells.

Patrick J Flynn, Peter D Koch, Timothy J Mitchison.

  Mitotic errors can activate cyclic GMP-AMP synthase (cGAS) and induce type I interferon (IFN) signaling. Current models propose that chromosome segregation errors generate micronuclei whose rupture activates cGAS. We used a panel of antimitotic drugs to perturb mitosis in human fibroblasts and measured abnormal nuclear morphologies, cGAS localization, and IFN signaling in the subsequent interphase. Micronuclei consistently recruited cGAS without activating it. Instead, IFN signaling correlated with formation of cGAS-coated chromatin bridges that were selectively generated by microtubule stabilizers and MPS1 inhibitors. cGAS activation by chromatin bridges was suppressed by drugs that prevented cytokinesis. We confirmed cGAS activation by chromatin bridges in cancer lines that are unable to secrete IFN by measuring paracrine transfer of 2'3'-cGAMP to fibroblasts, and in mouse cells. We propose that cGAS is selectively activated by self-chromatin when it is stretched in chromatin bridges. Immunosurveillance of cells that fail mitosis, and antitumor actions of taxanes and MPS1 inhibitors, may depend on this effect.

Keywords:  cGAS; cancer; chemotherapy; interferon; mitosis

DOI:  https://doi.org/10.1073/pnas.2103585118
Front Oncol. 2021 ;11 752933

Mitotic Errors Promote Genomic Instability and Leukemia in a Novel Mouse Model of Fanconi Anemia.

Donna M Edwards, Dana K Mitchell, Zahi Abdul-Sater, Ka-Kui Chan, Zejin Sun, Aditya Sheth, Ying He, Li Jiang, Jin Yuan, Richa Sharma, Magdalena Czader, Pei-Ju Chin, Yie Liu, Guillermo de Cárcer, Grzegorz Nalepa, Hal E Broxmeyer, D Wade Clapp, Elizabeth A Sierra Potchanant.

  Fanconi anemia (FA) is a disease of genomic instability and cancer. In addition to DNA damage repair, FA pathway proteins are now known to be critical for maintaining faithful chromosome segregation during mitosis. While impaired DNA damage repair has been studied extensively in FA-associated carcinogenesis in vivo, the oncogenic contribution of mitotic abnormalities secondary to FA pathway deficiency remains incompletely understood. To examine the role of mitotic dysregulation in FA pathway deficient malignancies, we genetically exacerbated the baseline mitotic defect in Fancc-/- mice by introducing heterozygosity of the key spindle assembly checkpoint regulator Mad2. Fancc-/-;Mad2+/- mice were viable, but died from acute myeloid leukemia (AML), thus recapitulating the high risk of myeloid malignancies in FA patients better than Fancc-/-mice. We utilized hematopoietic stem cell transplantation to propagate Fancc-/-; Mad2+/- AML in irradiated healthy mice to model FANCC-deficient AMLs arising in the non-FA population. Compared to cells from Fancc-/- mice, those from Fancc-/-;Mad2+/- mice demonstrated an increase in mitotic errors but equivalent DNA cross-linker hypersensitivity, indicating that the cancer phenotype of Fancc-/-;Mad2+/- mice results from error-prone cell division and not exacerbation of the DNA damage repair defect. We found that FANCC enhances targeting of endogenous MAD2 to prometaphase kinetochores, suggesting a mechanism for how FANCC-dependent regulation of the spindle assembly checkpoint prevents chromosome mis-segregation. Whole-exome sequencing revealed similarities between human FA-associated myelodysplastic syndrome (MDS)/AML and the AML that developed in Fancc-/-; Mad2+/- mice. Together, these data illuminate the role of mitotic dysregulation in FA-pathway deficient malignancies in vivo, show how FANCC adjusts the spindle assembly checkpoint rheostat by regulating MAD2 kinetochore targeting in cell cycle-dependent manner, and establish two new mouse models for preclinical studies of AML.

Keywords:  FANCC; Fanconi anemia; genomic instability; leukemia; spindle assembly checkpoint

DOI:  https://doi.org/10.3389/fonc.2021.752933
Cancers (Basel). 2021 Nov 12. pii: 5673. [Epub ahead of print]13(22):

Mitotic Centromere-Associated Kinesin (MCAK/KIF2C) Regulates Cell Migration and Invasion by Modulating Microtubule Dynamics and Focal Adhesion Turnover.

Ha Hyung Moon, Nina-Naomi Kreis, Alexandra Friemel, Susanne Roth, Dorothea Schulte, Christine Solbach, Frank Louwen, Juping Yuan, Andreas Ritter.

  The microtubule (MT) cytoskeleton is crucial for cell motility and migration by regulating multiple cellular activities such as transport and endocytosis of key components of focal adhesions (FA). The kinesin-13 family is important in the regulation of MT dynamics and the best characterized member of this family is the mitotic centromere-associated kinesin (MCAK/KIF2C). Interestingly, its overexpression has been reported to be related to increased metastasis in various tumor entities. Moreover, MCAK is involved in the migration and invasion behavior of various cell types. However, the precise molecular mechanisms were not completely clarified. To address these issues, we generated CRISPR/dCas9 HeLa and retinal pigment epithelium (RPE) cell lines overexpressing or downregulating MCAK. Both up- or downregulation of MCAK led to reduced cell motility and poor migration in malignant as well as benign cells. Specifically, it's up- or downregulation impaired FA protein composition and phosphorylation status, interfered with a proper spindle and chromosome segregation, disturbed the assembly and disassembly rate of FA, delayed cell adhesion, and compromised the plus-tip dynamics of MTs. In conclusion, our data suggest MCAK act as an important regulator for cell motility and migration by affecting the actin-MT cytoskeleton dynamics and the FA turnover, providing molecular mechanisms by which deregulated MCAK could promote malignant progression and metastasis of tumor cells.

Keywords:  CRISPR/dCas9; MCAK; actin cytoskeleton; depolymerization; focal adhesion; invasion; microtubule dynamics; migration; motility; plus-tip

DOI:  https://doi.org/10.3390/cancers13225673
Curr Biol. 2021 Nov 22. pii: S0960-9822(21)01351-8. [Epub ahead of print]31(22): R1491-R1504

Diverse mechanisms of centromere specification.

Barbara G Mellone, Daniele Fachinetti.

The centromere performs a universally conserved function, to accurately partition genetic information upon cell division. Yet, centromeres are among the most rapidly evolving regions of the genome and are bound by a varying assortment of centromere-binding factors that are themselves highly divergent at the protein-sequence level. A common thread in most species is the dependence on the centromere-specific histone variant CENP-A for the specification of the centromere site. However, CENP-A is not universally required in all species or cell types, making the identification of a general mechanism for centromere specification challenging. In this review, we examine our current understanding of the mechanisms of centromere specification in CENP-A-dependent and independent systems, focusing primarily on recent work.

DOI: https://doi.org/10.1016/j.cub.2021.09.083