bims-micesi 2023-11-19 papers

bims-micesi

Biomed News

on Mitotic cell signalling

Issue of 2023–11–19
nine papers selected by
Valentina Piano, Uniklinik Köln

Spindle architecture constrains karyotype in budding yeast.
Quantitative Super-Resolution Microscopy Reveals the Relationship between CENP-A Stoichiometry and Centromere Physical Size.
Centromere innovations within a mouse species.
Dysregulation of the endoplasmic reticulum blocks recruitment of centrosome-associated proteins resulting in mitotic failure.
Humanization reveals pervasive incompatibility of yeast and human kinetochore components.
REV7 Monomer Is Unable to Participate in Double Strand Break Repair and Translesion Synthesis but Suppresses Mitotic Errors.
A role for β-1,6- and β-1,3-Glucans in Kinetochore Function in Saccharomyces cerevisiae.
Overexpression of JNK-associated leucine zipper protein induces chromosomal instability through interaction with dynein light intermediate chain 1.
Bridging condensins mediate compaction of mitotic chromosomes.

bioRxiv. 2023 Oct 25. pii: 2023.10.25.563899. [Epub ahead of print]

Spindle architecture constrains karyotype in budding yeast.

Jana Helsen, Md Hashim Reza, Gavin Sherlock, Gautam Dey.

The eukaryotic cell division machinery must rapidly and reproducibly duplicate and partition the cell's chromosomes in a carefully coordinated process. However, chromosome number varies dramatically between genomes, even on short evolutionary timescales. We sought to understand how the mitotic machinery senses and responds to karyotypic changes by using a set of budding yeast strains in which the native chromosomes have been successively fused. Using a combination of cell biological profiling, genetic engineering, and experimental evolution, we show that chromosome fusions are well tolerated up until a critical point. However, with fewer than five centromeres, outward forces in the metaphase spindle cannot be countered by kinetochore-microtubule attachments, triggering mitotic defects. Our findings demonstrate that spindle architecture is a constraining factor for karyotype evolution.

DOI: https://doi.org/10.1101/2023.10.25.563899
Int J Mol Sci. 2023 Nov 01. pii: 15871. [Epub ahead of print]24(21):

Quantitative Super-Resolution Microscopy Reveals the Relationship between CENP-A Stoichiometry and Centromere Physical Size.

Yaqian Li, Jiabin Wang, Xuecheng Chen, Daniel M Czajkowsky, Zhifeng Shao.

  Centromeric chromatin is thought to play a critical role in ensuring the faithful segregation of chromosomes during mitosis. However, our understanding of this role is presently limited by our poor understanding of the structure and composition of this unique chromatin. The nucleosomal variant, CENP-A, localizes to narrow regions within the centromere, where it plays a major role in centromeric function, effectively serving as a platform on which the kinetochore is assembled. Previous work found that, within a given cell, the number of microtubules within kinetochores is essentially unchanged between CENP-A-localized regions of different physical sizes. However, it is unknown if the amount of CENP-A is also unchanged between these regions of different sizes, which would reflect a strict structural correspondence between these two key characteristics of the centromere/kinetochore assembly. Here, we used super-resolution optical microscopy to image and quantify the amount of CENP-A and DNA within human centromere chromatin. We found that the amount of CENP-A within CENP-A domains of different physical sizes is indeed the same. Further, our measurements suggest that the ratio of CENP-A- to H3-containing nucleosomes within these domains is between 8:1 and 11:1. Thus, our results not only identify an unexpectedly strict relationship between CENP-A and microtubules stoichiometries but also that the CENP-A centromeric domain is almost exclusively composed of CENP-A nucleosomes.

Keywords:  CENP-A; STORM; bio-macromolecules; centromere chromatin structure; stoichiometry

DOI:  https://doi.org/10.3390/ijms242115871
Sci Adv. 2023 Nov 17. 9(46): eadi5764

Centromere innovations within a mouse species.

Craig W Gambogi, Nootan Pandey, Jennine M Dawicki-McKenna, Uma P Arora, Mikhail A Liskovykh, Jun Ma, Piero Lamelza, Vladimir Larionov, Michael A Lampson, Glennis A Logsdon, Beth L Dumont, Ben E Black.

Mammalian centromeres direct faithful genetic inheritance and are typically characterized by regions of highly repetitive and rapidly evolving DNA. We focused on a mouse species, Mus pahari, that we found has evolved to house centromere-specifying centromere protein-A (CENP-A) nucleosomes at the nexus of a satellite repeat that we identified and termed π-satellite (π-sat), a small number of recruitment sites for CENP-B, and short stretches of perfect telomere repeats. One M. pahari chromosome, however, houses a radically divergent centromere harboring ~6 mega-base pairs of a homogenized π-sat-related repeat, π-satB, that contains >20,000 functional CENP-B boxes. There, CENP-B abundance promotes accumulation of microtubule-binding components of the kinetochore and a microtubule-destabilizing kinesin of the inner centromere. We propose that the balance of pro- and anti-microtubule binding by the new centromere is what permits it to segregate during cell division with high fidelity alongside the older ones whose sequence creates a markedly different molecular composition.

DOI: https://doi.org/10.1126/sciadv.adi5764
Development. 2023 Nov 15. pii: dev201917. [Epub ahead of print]150(22):

Dysregulation of the endoplasmic reticulum blocks recruitment of centrosome-associated proteins resulting in mitotic failure.

Katherine R Rollins, J Todd Blankenship.

  The endoplasmic reticulum (ER) undergoes a remarkable transition in morphology during cell division to aid in the proper portioning of the ER. However, whether changes in ER behaviors modulate mitotic events is less clear. Like many animal embryos, the early Drosophila embryo undergoes rapid cleavage cycles in a lipid-rich environment. Here, we show that mitotic spindle formation, centrosomal maturation, and ER condensation occur with similar time frames in the early syncytium. In a screen for Rab family GTPases that display dynamic function at these stages, we identified Rab1. Rab1 disruption led to an enhanced buildup of ER at the spindle poles and produced an intriguing 'mini-spindle' phenotype. ER accumulation around the mitotic space negatively correlates with spindle length/intensity. Importantly, centrosomal maturation is defective in these embryos, as mitotic recruitment of key centrosomal proteins is weakened after Rab1 disruption. Finally, division failures and ER overaccumulation is rescued by Dynein inhibition, demonstrating that Dynein is essential for ER spindle recruitment. These results reveal that ER levels must be carefully tuned during mitotic processes to ensure proper assembly of the division machinery.

Keywords:  Endoplasmic reticulum; Mitotic spindle; Rab proteins; Rab1; Syncytial divisions

DOI:  https://doi.org/10.1242/dev.201917
G3 (Bethesda). 2023 Nov 14. pii: jkad260. [Epub ahead of print]

Humanization reveals pervasive incompatibility of yeast and human kinetochore components.

Guðjón Ólafsson, Max A B Haase, Jef D Boeke.

  Kinetochores assemble on centromeres to drive chromosome segregation in eukaryotic cells. Humans and budding yeast share most of the structural subunits of the kinetochore, whereas protein sequences have diverged considerably. The conserved centromeric histone-H3 variant, CenH3 (CENP-A in humans and Cse4 in budding yeast) marks the site for kinetochore assembly in most species. A previous effort to complement Cse4 in yeast with human CENP-A was unsuccessful, however co-complementation with the human core nucleosome was not attempted. Previously, our lab successfully humanized the core nucleosome in yeast, however this severely affected cellular growth. We hypothesized that yeast Cse4 is incompatible with humanized nucleosomes and that the kinetochore represented a limiting factor for efficient histone humanization. Thus, we argued that including the human CENP-A or a Cse4-CENP-A chimera might improve histone humanization and facilitate kinetochore function in humanized yeast. The opposite was true: CENP-A expression reduced histone humanization efficiency, was toxic to yeast, and disrupted cell-cycle progression and kinetochore function in wild-type cells. Suppressors of CENP-A toxicity included gene deletions of subunits of three conserved chromatin-remodeling complexes, highlighting their role in CenH3 chromatin positioning. Finally, we attempted to complement the subunits of the NDC80 kinetochore complex, individually and in combination, without success, in contrast to a previous study indicating complementation by the human NDC80/HEC1 gene. Our results suggest that limited protein sequence similarity between yeast and human components in this very complex structure leads to failure of complementation.

Keywords:  CENP-A; centromere; histone variant; humanization; kinetochore; yeast

DOI:  https://doi.org/10.1093/g3journal/jkad260
Int J Mol Sci. 2023 Oct 31. pii: 15799. [Epub ahead of print]24(21):

REV7 Monomer Is Unable to Participate in Double Strand Break Repair and Translesion Synthesis but Suppresses Mitotic Errors.

Faye M Vassel, Daniel J Laverty, Ke Bian, Cortt G Piett, Michael T Hemann, Graham C Walker, Zachary D Nagel.

  Rev7 is a regulatory protein with roles in translesion synthesis (TLS), double strand break (DSB) repair, replication fork protection, and cell cycle regulation. Rev7 forms a homodimer in vitro using its HORMA (Hop, Rev7, Mad2) domain; however, the functional importance of Rev7 dimerization has been incompletely understood. We analyzed the functional properties of cells expressing either wild-type mouse Rev7 or Rev7K44A/R124A/A135D, a mutant that cannot dimerize. The expression of wild-type Rev7, but not the mutant, rescued the sensitivity of Rev7-/- cells to X-rays and several alkylating agents and reversed the olaparib resistance phenotype of Rev7-/- cells. Using a novel fluorescent host-cell reactivation assay, we found that Rev7K44A/R124A/A135D is unable to promote gap-filling TLS opposite an abasic site analog. The Rev7 dimerization interface is also required for shieldin function, as both Rev7-/- cells and Rev7-/- cells expressing Rev7K44A/R124A/A135D exhibit decreased proficiency in rejoining some types of double strand breaks, as well as increased homologous recombination. Interestingly, Rev7K44A/R124A/A135D retains some function in cell cycle regulation, as it maintains an interaction with Ras-related nuclear protein (Ran) and partially rescues the formation of micronuclei. The mutant Rev7 also rescues the G2/M accumulation observed in Rev7-/- cells but does not affect progression through mitosis following nocodazole release. We conclude that while Rev7 dimerization is required for its roles in TLS, DSB repair, and regulation of the anaphase promoting complex, dimerization is at least partially dispensable for promoting mitotic spindle assembly through its interaction with Ran.

Keywords:  DNA repair; DNA replication; FM-HCR; cell cycle; shieldin; translesion synthesis

DOI:  https://doi.org/10.3390/ijms242115799
Genetics. 2023 Nov 10. pii: iyad195. [Epub ahead of print]

A role for β-1,6- and β-1,3-Glucans in Kinetochore Function in Saccharomyces cerevisiae.

Rucha Kshirsagar, Arno Munhoven, Tra My Tran Nguyen, Ann E Ehrenhofer-Murray.

  Chromosome segregation is crucial for the faithful inheritance of DNA to the daughter cells after DNA replication. For this, the kinetochore, a megadalton protein complex, assembles on centromeric chromatin containing the histone H3 variant CENP-A and provides a physical connection to the microtubules. Here, we report an unanticipated role for enzymes required for β-1,6- and β-1,3 glucan biosynthesis in regulating kinetochore function in Saccharomyces cerevisiae. These carbohydrates are the major constituents of the yeast cell wall. We found that the deletion of KRE6, which encodes a glycosylhydrolase/transglycosidase required for β-1,6 glucan synthesis, suppressed the centromeric defect of mutations in components of the kinetochore, foremost the NDC80 components Spc24, Spc25, the MIND component Nsl1, and Okp1, a CCAN protein. Similarly, the absence of Fks1, a β-1,3 glucan synthase, and Kre11/Trs65, a TRAPPII component, suppressed a mutation in SPC25. Genetic analysis indicates that the reduction of intracellular β-1,6 and β -1,3 glucan, rather than the cell wall glucan content, regulates kinetochore function. Furthermore, we found a physical interaction between Kre6 and CENP-A/Cse4 in yeast, suggesting a potential function for Kre6 in glycosylating CENP-A/Cse4 or another kinetochore protein. This work shows a moonlighting function for selected cell wall synthesis proteins in regulating kinetochore assembly, which may provide a mechanism to connect the nutritional status of the cell to cell-cycle progression and chromosome segregation.

Keywords:  CENP-A; Chs1; Cse4; Fks1; Kre11/Trs65; Kre6

DOI:  https://doi.org/10.1093/genetics/iyad195
Genes Cells. 2023 Nov 14.

Overexpression of JNK-associated leucine zipper protein induces chromosomal instability through interaction with dynein light intermediate chain 1.

Ryusuke Suzuki, Masato T Kanemaki, Takeshi Suzuki, Katsuji Yoshioka.

  The c-Jun N-terminal kinase-associated leucine zipper protein (JLP), a scaffold protein of mitogen-activated protein kinase signaling pathways, is a multifunctional protein involved in a variety of cellular processes. It has been reported that JLP is overexpressed in various types of cancer and is expected to be a potential therapeutic target. However, whether and how JLP overexpression affects non-transformed cells remain unknown. Here, we aimed to investigate the effect of JLP overexpression on chromosomal stability in human non-transformed cells and the mechanisms involved. We found that aneuploidy was induced in JLP-overexpressed cells. Moreover, we established JLP-inducible cell lines and observed an increased frequency of chromosome missegregation, reduced time from nuclear envelope breakdown to anaphase onset, and decreased levels of the spindle assembly checkpoint (SAC) components at the prometaphase kinetochore in cells overexpressing the wild-type JLP. In contrast, we observed that a point mutant JLP lacking the ability to interact with dynein light intermediate chain 1 (DLIC1) failed to induce chromosomal instability. Our results suggest that overexpression of the wild-type JLP facilitates premature SAC silencing through interaction with DLIC1, leading to aneuploidy. This study provides a novel insight into the mechanism through which JLP overexpression is associated with cancer development and progression.

Keywords:  aneuploidy; chromosomal stability; dynein; mitosis; spindle assembly checkpoint

DOI:  https://doi.org/10.1111/gtc.13083
J Cell Biol. 2024 Jan 01. pii: e202209113. [Epub ahead of print]223(1):

Bridging condensins mediate compaction of mitotic chromosomes.

Giada Forte, Lora Boteva, Filippo Conforto, Nick Gilbert, Peter R Cook, Davide Marenduzzo.

Eukaryotic chromosomes compact during mitosis into elongated cylinders-and not the spherical globules expected of self-attracting long flexible polymers. This process is mainly driven by condensin-like proteins. Here, we present Brownian-dynamic simulations involving two types of such proteins with different activities. One, which we refer to as looping condensins, anchors long-lived chromatin loops to create bottlebrush structures. The second, referred to as bridging condensins, forms multivalent bridges between distant parts of these loops. We show that binding of bridging condensins leads to the formation of shorter and stiffer mitotic-like cylinders without requiring any additional energy input. These cylinders have several features matching experimental observations. For instance, the axial condensin backbone breaks up into clusters as found by microscopy, and cylinder elasticity qualitatively matches that seen in chromosome pulling experiments. Additionally, simulating global condensin depletion or local faulty condensin loading gives phenotypes seen experimentally and points to a mechanistic basis for the structure of common fragile sites in mitotic chromosomes.

DOI: https://doi.org/10.1083/jcb.202209113