bims-micesi 2024-10-20 papers

bims-micesi

Biomed News

on Mitotic cell signalling

Issue of 2024–10–20
twelve papers selected by
Valentina Piano, Uniklinik Köln

Aurora B/AIR-2 regulates sister centromere resolution and CENP-A/HCP-3 organization to prevent merotelic attachments.
How condensed are mitotic chromosomes?
Helical coiled nucleosome chromosome architectures during cell cycle progression.
Kinesin-5/Cut7 C-terminal tail phosphorylation is essential for microtubule sliding force and bipolar mitotic spindle assembly.
TRIM37 employs peptide motif recognition and substrate-dependent oligomerization to prevent ectopic spindle pole assembly.
Alignment of a Trivalent Chromosome on the Metaphase Plate Is Associated with Differences in Microtubule Density at Each Kinetochore.
Negative regulation of APC/C activation by MAPK-mediated attenuation of Cdc20Slp1 under stress.
ROBUST APPROACH FOR PRODUCTION OF THE HUMAN ONCOLOGY TARGET AURORA KINASE B IN COMPLEX WITH ITS BINDING PARTNER INCENP.
PIWIL1 is recruited to centrosomes during mitosis in colorectal cancer cells and is linked to cell cycle progression.
Dysfunction of Telomeric Cdc13-Stn1-Ten1 Simultaneously Activates DNA Damage and Spindle Checkpoints.
Regulation of p53 by the mitotic surveillance/stopwatch pathway: implications in neurodevelopment and cancer.
Biochemical characterization of the feedforward loop between CDK1 and FOXM1 in epidermal stem cells.

J Mol Cell Biol. 2024 Oct 16. pii: mjae045. [Epub ahead of print]

Aurora B/AIR-2 regulates sister centromere resolution and CENP-A/HCP-3 organization to prevent merotelic attachments.

Yue Wang, Charmaine Yan Yu Wong, Karen Wing Yee Yuen.

  During cell division, the accurate capture of sister kinetochores that are built on the centromeres of chromosomes by microtubules emanating from opposite spindle poles governs faithful chromosome segregation. To ensure sister chromatids separate correctly, sister centromeres undergo resolution to achieve bi-polar orientation prior to microtubule attachments. Failure of centromere resolution increases the frequency of merotelic attachments, with microtubules from opposite poles attaching to the same sister kinetochore, causing lagging chromosome, aneuploidy, and even cancer progression. The Aurora B-mediated tension-sensing machinery to correct erroneous kinetochore-microtubule attachments has been well studied. However, preventative mechanisms to avoid merotelic attachments that occur in the earlier mitotic stage are poorly understood. In this study, we found that inactivation of mitotic kinase Aurora B/AIR-2 increases merotelic attachments in Caenorhabditis elegans. On one hand, Aurora B/AIR-2-deficient cells exhibited a delay in the occurrence of centromere resolution and a disruption in targeting condensin II components to chromatin. On the other hand, loss of Aurora B/AIR-2 results in an increased localization of centromeric proteins CENP-A/HCP-3 and M18BP1/KNL-2 as well as the kinetochore protein MIS-12 on chromatin, which may generate ectopic kinetochores causing erroneous attachments. To conclude, this study elucidated that Aurora B/AIR-2 regulates sister centromere resolution and CENP-A/HCP-3 deposition to actively prevent merotely and chromosome instability in cells.

Keywords:  Aurora B/AIR-2; centromere organization; chromosome segregation; merotelic attachments; sister centromere resolution

DOI:  https://doi.org/10.1093/jmcb/mjae045
J Cell Biol. 2024 Nov 04. pii: e202409044. [Epub ahead of print]223(11):

How condensed are mitotic chromosomes?

Hide A Konishi, Hironori Funabiki.

Chromosomes undergo dramatic compaction during mitosis, but accurately measuring their volume has been challenging. Employing serial block face scanning electron microscopy, Cisneros-Soberanis et al. (https://doi.org/10.1083/jcb.202403165) report that mitotic chromosomes compact to a nucleosome concentration of ∼760 µM.

DOI: https://doi.org/10.1083/jcb.202409044
Proc Natl Acad Sci U S A. 2024 Oct 22. 121(43): e2410584121

Helical coiled nucleosome chromosome architectures during cell cycle progression.

Angus McDonald, Cornelis Murre, John W Sedat.

  Recent studies showed an interphase chromosome architecture-a specific coiled nucleosome structure-derived from cryopreserved EM tomograms, and dispersed throughout the nucleus. The images were computationally processed to fill in the missing wedges of data caused by incomplete tomographic tilts. The resulting structures increased z-resolution enabling an extension of the proposed architecture to that of mitotic chromosomes. Here, we provide additional insights into the chromosome architecture that was recently published [M. Elbaum et al., Proc. Natl. Acad. Sci. U.S.A. 119, e2119101119 (2022)]. We build on the defined chromosomes time-dependent structures in an effort to probe their dynamics. Variants of the coiled chromosome structures, possibly further defining specific regions, are discussed. We propose, based on generalized specific uncoiling of mitotic chromosomes in telophase, large-scale reorganization of interphase chromosomes. Chromosome territories, organized as micron-sized small patches, are constructed, satisfying complex volume considerations. Finally, we unveiled the structures of replicated coiled chromosomes, still attached to centromeres, as part of chromosome architecture.

Keywords:  chromosome architecture; computer modeling; nuclear structure

DOI:  https://doi.org/10.1073/pnas.2410584121
Curr Biol. 2024 Oct 15. pii: S0960-9822(24)01150-3. [Epub ahead of print]

Kinesin-5/Cut7 C-terminal tail phosphorylation is essential for microtubule sliding force and bipolar mitotic spindle assembly.

Michele H Jones, Zachary R Gergely, Daniel Steckhahn, Bojun Zhou, Meredith D Betterton.

  Kinesin-5 motors play an essential role during mitotic spindle assembly in many organisms1,2,3,4,5,6,7,8,9,10,11: they crosslink antiparallel spindle microtubules, step toward plus ends, and slide the microtubules apart.12,13,14,15,16,17 This activity separates the spindle poles and chromosomes. Kinesin-5s are not only plus-end-directed but can walk or be carried toward MT minus ends,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34 where they show enhanced localization.3,5,7,27,29,32 The kinesin-5 C-terminal tail interacts with and regulates the motor, affecting structure, motility, and sliding force of purified kinesin-535,36,37 along with motility and spindle assembly in cells.27,38,39 The tail contains phosphorylation sites, particularly in the conserved BimC box.6,7,40,41,42,43,44 Nine mitotic tail phosphorylation sites were identified in the kinesin-5 motor of the fission yeast Schizosaccharomyces pombe,45,46,47,48 suggesting that multi-site phosphorylation may regulate kinesin-5s. Here, we show that mutating all nine sites to either alanine or glutamate causes temperature-sensitive lethality due to a failure of bipolar spindle assembly. We characterize kinesin-5 localization and sliding force in the spindle based on Cut7-dependent microtubule minus-end protrusions in cells lacking kinesin-14 motors.39,49,50,51,52 Imaging and computational modeling show that Cut7p simultaneously moves toward the minus ends of protrusion MTs and the plus ends of spindle midzone MTs. Phosphorylation mutants show dramatic decreases in protrusions and sliding force. Comparison to a model of force to create protrusions suggests that tail truncation and phosphorylation mutants decrease Cut7p sliding force similarly to tail-truncated human Eg5.36 Our results show that C-terminal tail phosphorylation is required for kinesin-5/Cut7 sliding force and bipolar spindle assembly in fission yeast.

Keywords:  fission yeast; kinesin-5; microtubule; microtubule sliding; mitosis; mitotic spindle; modeling; motor protein; phosphoregulation; spindle assembly

DOI:  https://doi.org/10.1016/j.cub.2024.08.035
bioRxiv. 2024 Oct 09. pii: 2024.10.09.617493. [Epub ahead of print]

TRIM37 employs peptide motif recognition and substrate-dependent oligomerization to prevent ectopic spindle pole assembly.

Andrew Bellaart, Amanda Brambila, Jiawei Xu, Francisco Mendez Diaz, Amar Deep, John Anzola, Franz Meitinger, Midori Ohta, Kevin D Corbett, Arshad Desai, Karen Oegema.

Tightly controlled duplication of centrosomes, the major microtubule-organizing centers of animal cells, ensures bipolarity of the mitotic spindle and accurate chromosome segregation. The RBCC (RING-B-box-coiled coil) ubiquitin ligase TRIM37, whose loss is associated with elevated chromosome missegregation and the tumor-prone developmental human disorder Mulibrey nanism, prevents the formation of ectopic spindle poles that assemble around structured condensates containing the centrosomal protein centrobin. Here, we show that TRIM37's TRAF domain, unique in the extended TRIM family, engages peptide motifs in centrobin to suppress condensate formation. TRIM proteins form anti-parallel coiled-coil dimers with RING-B-box domains on each end. Oligomerization due to RING-RING interactions and conformational regulation by B-box-2-B-box-2 interfaces are critical for TRIM37 to suppress centrobin condensate formation. These results indicate that, analogous to anti-viral TRIM ligases, TRIM37 activation is linked to the detection of oligomerized substrates. Thus, TRIM37 couples peptide motif recognition and substrate-dependent oligomerization to effect ubiquitination-mediated clearance of ectopic centrosomal protein assemblies.

DOI: https://doi.org/10.1101/2024.10.09.617493
Int J Mol Sci. 2024 Oct 05. pii: 10719. [Epub ahead of print]25(19):

Alignment of a Trivalent Chromosome on the Metaphase Plate Is Associated with Differences in Microtubule Density at Each Kinetochore.

Ashley B Borseth, Hedyeh D Kianersi, Paige Galloway, Grace Gercken, Emily L Stowe, Marie Pizzorno, Leocadia V Paliulis.

  Chromosome alignment on the metaphase plate is a conserved phenomenon and is an essential function for correct chromosome segregation for many organisms. Organisms with naturally-occurring trivalent chromosomes provide a useful system for understanding how chromosome alignment is evolutionarily regulated, as they align on the spindle with one kinetochore facing one pole and two facing the opposite pole. We studied chromosome alignment in a praying mantid that has not been previously studied chromosomally, the giant shield mantis Rhombodera megaera. R. megaera has a chromosome number of 2n = 27 in males. Males have X1, X2, and Y chromosomes that combine to form a trivalent in meiosis I. Using live-cell imaging of spermatocytes in meiosis I, we document that sex trivalent Y chromosomes associate with one spindle pole and the two X chromosomes associate with the opposing spindle pole. Sex trivalents congress alongside autosomes, align with them on the metaphase I plate, and then the component chromosomes segregate alongside autosomes in anaphase I. Immunofluorescence imaging and quantification of brightness of kinetochore-microtubule bundles suggest that the X1 and X2 kinetochores are associated with fewer microtubules than the Y kinetochore, likely explaining the alignment of the sex trivalent at the spindle equator with autosomes. These observations in R. megaera support the evolutionary significance of the metaphase alignment of chromosomes and provide part of the explanation for how this alignment is achieved.

Keywords:  chromosome alignment; chromosomes; meiosis; metaphase; trivalent

DOI:  https://doi.org/10.3390/ijms251910719
Elife. 2024 Oct 16. pii: RP97896. [Epub ahead of print]13

Negative regulation of APC/C activation by MAPK-mediated attenuation of Cdc20Slp1 under stress.

Li Sun, Xuejin Chen, Chunlin Song, Wenjing Shi, Libo Liu, Shuang Bai, Xi Wang, Jiali Chen, Chengyu Jiang, Shuang-Min Wang, Zhou-Qing Luo, Ruiwen Wang, Yamei Wang, Quan-Wen Jin.

  Mitotic anaphase onset is a key cellular process tightly regulated by multiple kinases. The involvement of mitogen-activated protein kinases (MAPKs) in this process has been established in Xenopus egg extracts. However, the detailed regulatory cascade remains elusive, and it is also unknown whether the MAPK-dependent mitotic regulation is evolutionarily conserved in the single-cell eukaryotic organisms such as fission yeast (Schizosaccharomyces pombe). Here, we show that two MAPKs in S. pombe indeed act in concert to restrain anaphase-promoting complex/cyclosome (APC/C) activity upon activation of the spindle assembly checkpoint (SAC). One MAPK, Pmk1, binds to and phosphorylates Slp1Cdc20, the co-activator of APC/C. Phosphorylation of Slp1Cdc20 by Pmk1, but not by Cdk1, promotes its subsequent ubiquitylation and degradation. Intriguingly, Pmk1-mediated phosphorylation event is also required to sustain SAC under environmental stress. Thus, our study establishes a new underlying molecular mechanism of negative regulation of APC/C by MAPK upon stress stimuli, and provides a previously unappreciated framework for regulation of anaphase entry in eukaryotic cells.

Keywords:  APC/C; MAPK; S. pombe; SAC; Slp1/Cdc20; anaphase-promoting complex/cyclosome; cell biology; phosphorylation; spindle assembly checkpoint; ubiquitylation

DOI:  https://doi.org/10.7554/eLife.97896
Biochimie. 2024 Oct 16. pii: S0300-9084(24)00237-2. [Epub ahead of print]

ROBUST APPROACH FOR PRODUCTION OF THE HUMAN ONCOLOGY TARGET AURORA KINASE B IN COMPLEX WITH ITS BINDING PARTNER INCENP.

Jonna Mattsson, Per Rogne, Maréne Landström, Magnus Wolf-Watz.

  Protein kinases are key players in many eukaryotic signal transduction cascades and are as a result often linked to human disease. In humans, the mitotic protein kinase family of Aurora kinases consist of three members: Aurora A, B and C. All three members are involved in cell division with proposed implications in various human cancers. The human Aurora kinase B has in particular proven challenging to study with structural biology approaches, and this is mainly due to difficulties in producing the large quantities of active enzyme required for such studies. Here, we present a novel and E. coli-based production system that allows for production of milligram quantities of well-folded and active human Aurora B in complex with its binding partner INCENP. The complex is produced as a continuous polypeptide chain and the resulting fusion protein is cleaved with TEV protease to generate a stable and native heterodimer of the Aurora B:INCENP complex. The activity, stability and degree of phosphorylation of the protein complex was quantified by using a coupled ATPase assay, 31P NMR spectroscopy and mass spectrometry. The developed production system enables isotope labelling and we here report the first 1H-15N-HSQC of the human Aurora B:INCENP complex. Our developed production strategy paves the way for future structural and functional studies of Aurora B and can as such assist the development of novel anticancer drugs targeting this important mitotic protein kinase.

Keywords:  Aurora kinase B; Human protein kinase; INCENP; Mitotic protein kinase; Protein NMR; Protein characterization; Protein purification

DOI:  https://doi.org/10.1016/j.biochi.2024.10.011
Sci Rep. 2024 10 13. 14(1): 23928

PIWIL1 is recruited to centrosomes during mitosis in colorectal cancer cells and is linked to cell cycle progression.

Maria Rosa Garcia-Silva, Sofía Montenegro, Sofía Dacosta, Juan Pablo Tosar, Alfonso Cayota.

  PIWI proteins, traditionally associated with germline development, have recently gained attention for their expression in various cancers, including colorectal cancer. However, the molecular mechanisms underlying their reactivation and impact on cancer initiation and progression remain elusive. Here, we found that PIWIL1 is expressed at relatively high levels in CRC-derived samples and cell lines, where it undergoes a dynamic relocalization to the centrosome during mitosis. Knockdown of PIWIL1 induces G2/M arrest associated with disruption of the mitotic spindle and aberrant metaphase events, highlighting its role in cell cycle progression. We also found that the expression of PIWIL1 is lost during the differentiation of Caco-2 cells into enterocytes and that PIWIL1 is expressed in cells at the base of the intestinal crypts in normal human colon tissue, where intestinal stem cells are known to reside. Thus, it is possible that the presence of PIWIL1 in cancer cells reflects a physiological role of this protein in stem cell maintenance, which would argue in favor of the proposed stem cell origin of CRC. Supporting this view, dedifferentiation of human fibroblasts into induced pluripotent stem cells (iPSCs) involves the reactivation of PIWIL2 expression, another member of the PIWI protein family. Overall, our findings suggest a role of PIWIL1 in mediating cell cycle dynamics, both in colorectal cancer cells and possibly also in intestinal stem cells. In a broader aspect, we provide evidence supporting an involvement of PIWI proteins in somatic stem cell maintenance, thus expanding the known non-gonadal functions of this protein family.

Keywords:  Cell cycle; Colorectal cancer; PIWI

DOI:  https://doi.org/10.1038/s41598-024-75098-6
Cells. 2024 Sep 25. pii: 1605. [Epub ahead of print]13(19):

Dysfunction of Telomeric Cdc13-Stn1-Ten1 Simultaneously Activates DNA Damage and Spindle Checkpoints.

Nathalie Grandin, Michel Charbonneau.

  Telomeres, the ends of eukaryotic linear chromosomes, are composed of repeated DNA sequences and specialized proteins, with the conserved telomeric Cdc13/CTC1-Stn1-Ten1 (CST) complex providing chromosome stability via telomere end protection and the regulation of telomerase accessibility. In this study, SIZ1, coding for a SUMO E3 ligase, and TOP2 (a SUMO target for Siz1 and Siz2) were isolated as extragenic suppressors of Saccharomyces cerevisiae CST temperature-sensitive mutants. ten1-sz, stn1-sz and cdc13-sz mutants were isolated next due to being sensitive to intracellular Siz1 dosage. In parallel, strong negative genetic interactions between mutants of CST and septins were identified, with septins being noticeably sumoylated through the action of Siz1. The temperature-sensitive arrest in these new mutants of CST was dependent on the G2/M Mad2-mediated and Bub2-mediated spindle checkpoints as well as on the G2/M Mec1-mediated DNA damage checkpoint. Our data suggest the existence of yet unknown functions of the telomeric Cdc13-Stn1-Ten1 complex associated with mitotic spindle positioning and/or assembly that could be further elucidated by studying these new ten1-sz, stn1-sz and cdc13-sz mutants.

Keywords:  Bub2 and Mad2 spindle checkpoints; Cdc13-Stn1-Ten1 complex; Mec1 DNA damage checkpoint; Siz1 SUMO E3 ligase; budding yeast telomeres; cell cycle

DOI:  https://doi.org/10.3390/cells13191605
Front Cell Dev Biol. 2024 ;12 1451274

Regulation of p53 by the mitotic surveillance/stopwatch pathway: implications in neurodevelopment and cancer.

Travis H Stracker.

  The transcription factor p53 (encoded by TP53) plays diverse roles in human development and disease. While best known for its role in tumor suppression, p53 signaling also influences mammalian development by triggering cell fate decisions in response to a wide variety of stresses. After over 4 decades of study, a new pathway that triggers p53 activation in response to mitotic delays was recently identified. Termed the mitotic surveillance or mitotic stopwatch pathway, the USP28 and 53BP1 proteins activate p53 in response to delayed mitotic progression to control cell fate and promote genomic stability. In this Minireview, I discuss its identification, potential roles in neurodevelopmental disorders and cancer, as well as explore outstanding questions about its function, regulation and potential use as a biomarker for anti-mitotic therapies.

Keywords:  53BP1; USP28; apoptosis; c-MYC; cancer; microcephaly; neurodevelopment; p53

DOI:  https://doi.org/10.3389/fcell.2024.1451274
Biol Direct. 2024 Oct 13. 19(1): 91

Biochemical characterization of the feedforward loop between CDK1 and FOXM1 in epidermal stem cells.

Maria Pia Polito, Alessio Romaldini, Lorenzo Tagliazucchi, Grazia Marini, Federica Radice, Gaia Andrea Gozza, Giulia Bergamini, Maria Paola Costi, Elena Enzo.

  The complex network governing self-renewal in epidermal stem cells (EPSCs) is only partially defined. FOXM1 is one of the main players in this network, but the upstream signals regulating its activity remain to be elucidated. In this study, we identify cyclin-dependent kinase 1 (CDK1) as the principal kinase controlling FOXM1 activity in human primary keratinocytes. Mass spectrometry identified CDK1 as a key hub in a stem cell-associated protein network, showing its upregulation and interaction with essential self renewal-related markers. CDK1 phosphorylates FOXM1 at specific residues, stabilizing the protein and enhancing its nuclear localization and transcriptional activity, promoting self-renewal. Additionally, FOXM1 binds to the CDK1 promoter, inducing its expression.We identify the CDK1-FOXM1 feedforward loop as a critical axis sustaining EPSCs during in vitro cultivation. Understanding the upstream regulators of FOXM1 activity offers new insights into the biochemical mechanisms underlying self-renewal and differentiation in human primary keratinocytes.

Keywords:  CDK1; Epidermal stem cell; FOXM1; Phosphorylation

DOI:  https://doi.org/10.1186/s13062-024-00540-8