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1.
Proc Natl Acad Sci U S A ; 121(16): e2309621121, 2024 Apr 16.
Artigo em Inglês | MEDLINE | ID: mdl-38588415

RESUMO

Chromosomal instability (CIN) is the persistent reshuffling of cancer karyotypes via chromosome mis-segregation during cell division. In cancer, CIN exists at varying levels that have differential effects on tumor progression. However, mis-segregation rates remain challenging to assess in human cancer despite an array of available measures. We evaluated measures of CIN by comparing quantitative methods using specific, inducible phenotypic CIN models of chromosome bridges, pseudobipolar spindles, multipolar spindles, and polar chromosomes. For each, we measured CIN fixed and timelapse fluorescence microscopy, chromosome spreads, six-centromere FISH, bulk transcriptomics, and single-cell DNA sequencing (scDNAseq). As expected, microscopy of tumor cells in live and fixed samples significantly correlated (R = 0.72; P < 0.001) and sensitively detect CIN. Cytogenetics approaches include chromosome spreads and 6-centromere FISH, which also significantly correlate (R = 0.76; P < 0.001) but had limited sensitivity for lower rates of CIN. Bulk genomic DNA signatures and bulk transcriptomic scores, CIN70 and HET70, did not detect CIN. By contrast, scDNAseq detects CIN with high sensitivity, and significantly correlates with imaging methods (R = 0.82; P < 0.001). In summary, single-cell methods such as imaging, cytogenetics, and scDNAseq can measure CIN, with the latter being the most comprehensive method accessible to clinical samples. To facilitate the comparison of CIN rates between phenotypes and methods, we propose a standardized unit of CIN: Mis-segregations per Diploid Division. This systematic analysis of common CIN measures highlights the superiority of single-cell methods and provides guidance for measuring CIN in the clinical setting.


Assuntos
Instabilidade Cromossômica , Neoplasias , Humanos , Linhagem Celular Tumoral , Instabilidade Cromossômica/genética , Centrômero , Cariotipagem , Perfilação da Expressão Gênica , Segregação de Cromossomos , Aneuploidia
2.
Life Sci Alliance ; 7(6)2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38575358

RESUMO

For establishing sister chromatid cohesion and proper chromosome segregation in mitosis in fission yeast, the acetyltransferase Eso1 plays a key role. Eso1 acetylates cohesin complexes, at two conserved lysine residues K105 and K106 of the cohesin subunit Psm3. Although Eso1 also contributes to reductional chromosome segregation in meiosis, the underlying molecular mechanisms have remained elusive. Here, we purified meiosis-specific Rec8 cohesin complexes localized at centromeres and identified a new acetylation at Psm3-K1013, which largely depends on the meiotic kinetochore factor meikin (Moa1). Our molecular genetic analyses indicate that Psm3-K1013 acetylation cooperates with canonical acetylation at Psm3-K105 and K106, and plays a crucial role in establishing reductional chromosome segregation in meiosis.


Assuntos
Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces , Segregação de Cromossomos/genética , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Acetilação , Meiose/genética , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo
3.
Parasit Vectors ; 17(1): 180, 2024 Apr 05.
Artigo em Inglês | MEDLINE | ID: mdl-38581071

RESUMO

BACKGROUND: Toxoplasma gondii is an apicomplexan intracellular obligate parasite and the etiological agent of toxoplasmosis in humans, domestic animals and wildlife, causing miscarriages and negatively impacting offspring. During its intracellular development, it relies on nutrients from the host cell, controlling several pathways and the cytoskeleton. T. gondii has been proven to control the host cell cycle, mitosis and cytokinesis, depending on the time of infection and the origin of the host cell. However, no data from parallel infection studies have been collected. Given that T. gondii can infect virtually any nucleated cell, including those of humans and animals, understanding the mechanism by which it infects or develops inside the host cell is essential for disease prevention. Therefore, we aimed here to reveal whether this modulation is dependent on a specific cell type or host cell species. METHODS: We used only primary cells from humans and bovines at a maximum of four passages to ensure that all cells were counted with appropriate cell cycle checkpoint control. The cell cycle progression was analysed using fluorescence-activated cell sorting (FACS)-based DNA quantification, and its regulation was followed by the quantification of cyclin B1 (mitosis checkpoint protein). The results demonstrated that all studied host cells except bovine colonic epithelial cells (BCEC) were arrested in the S-phase, and none of them were affected in cyclin B1 expression. Additionally, we used an immunofluorescence assay to track mitosis and cytokinesis in uninfected and T. gondii-infected cells. RESULTS: The results demonstrated that all studied host cell except bovine colonic epithelial cells (BCEC) were arrested in the S-phase, and none of them were affected in cyclin B1 expression. Our findings showed that the analysed cells developed chromosome segregation problems and failed to complete cytokinesis. Also, the number of centrosomes per mitotic pole was increased after infection in all cell types. Therefore, our data suggest that T. gondii modulates the host cell cycle, chromosome segregation and cytokinesis during infection or development regardless of the host cell origin or type.


Assuntos
Toxoplasma , Toxoplasmose , Humanos , Animais , Bovinos , Toxoplasma/fisiologia , Citocinese , Ciclina B1/genética , Ciclina B1/metabolismo , Segregação de Cromossomos , Toxoplasmose/parasitologia
4.
Curr Biol ; 34(5): R211-R213, 2024 Mar 11.
Artigo em Inglês | MEDLINE | ID: mdl-38471453

RESUMO

In most eukaryotes, balanced chromosome segregation at meiosis requires crossovers, but female Bombyx mori lack these structures. Instead, the synaptonemal complex is repurposed to compensate for this absence of crossovers, a remarkable example of exaptation.


Assuntos
Bombyx , Elefantes , Animais , Feminino , Elefantes/genética , Bombyx/genética , Meiose , Complexo Sinaptonêmico , Eucariotos/genética , Segregação de Cromossomos
5.
Curr Biol ; 34(6): 1295-1308.e5, 2024 Mar 25.
Artigo em Inglês | MEDLINE | ID: mdl-38452759

RESUMO

Lysine acetylation of non-histone proteins plays crucial roles in many cellular processes. In this study, we examine the role of lysine acetylation during sister chromatid separation in mitosis. We investigate the acetylation of securin at K21 by cell-cycle-dependent acetylome analysis and uncover its role in separase-triggered chromosome segregation during mitosis. Prior to the onset of anaphase, the acetylated securin via TIP60 prevents its degradation by the APC/CCDC20-mediated ubiquitin-proteasome system. This, in turn, restrains precocious activation of separase and premature separation of sister chromatids. Additionally, the acetylation-dependent stability of securin is also enhanced by its dephosphorylation. As anaphase approaches, HDAC1-mediated deacetylation of securin promotes its degradation, allowing released separase to cleave centromeric cohesin. Blocking securin deacetylation leads to longer anaphase duration and errors in chromosome segregation. Thus, this study illustrates the emerging role of securin acetylation dynamics in mitotic progression and genetic stability.


Assuntos
Cromátides , Lisina , Separase/metabolismo , Securina/genética , Securina/metabolismo , Cromátides/metabolismo , Acetilação , Lisina/genética , Lisina/metabolismo , Proteínas de Ciclo Celular/metabolismo , Anáfase , Endopeptidases , Segregação de Cromossomos
6.
Nat Commun ; 15(1): 2737, 2024 Mar 28.
Artigo em Inglês | MEDLINE | ID: mdl-38548820

RESUMO

Bacterial chromosomes are folded into tightly regulated three-dimensional structures to ensure proper transcription, replication, and segregation of the genetic information. Direct visualization of chromosomal shape within bacterial cells is hampered by cell-wall confinement and the optical diffraction limit. Here, we combine cell-shape manipulation strategies, high-resolution fluorescence microscopy techniques, and genetic engineering to visualize the shape of unconfined bacterial chromosome in real-time in live Bacillus subtilis cells that are expanded in volume. We show that the chromosomes predominantly exhibit crescent shapes with a non-uniform DNA density that is increased near the origin of replication (oriC). Additionally, we localized ParB and BsSMC proteins - the key drivers of chromosomal organization - along the contour of the crescent chromosome, showing the highest density near oriC. Opening of the BsSMC ring complex disrupted the crescent chromosome shape and instead yielded a torus shape. These findings help to understand the threedimensional organization of the chromosome and the main protein complexes that underlie its structure.


Assuntos
Bacillus subtilis , Segregação de Cromossomos , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Segregação de Cromossomos/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Complexo de Reconhecimento de Origem/metabolismo , Replicação do DNA/genética , Cromossomos Bacterianos/genética , Cromossomos Bacterianos/metabolismo , DNA Bacteriano/metabolismo , Origem de Replicação
7.
Cell Rep ; 43(3): 113901, 2024 Mar 26.
Artigo em Inglês | MEDLINE | ID: mdl-38446663

RESUMO

Condensin shapes mitotic chromosomes by folding chromatin into loops, but whether it does so by DNA-loop extrusion remains speculative. Although loop-extruding cohesin is stalled by transcription, the impact of transcription on condensin, which is enriched at highly expressed genes in many species, remains unclear. Using degrons of Rpb1 or the torpedo nuclease Dhp1XRN2 to either deplete or displace RNAPII on chromatin in fission yeast metaphase cells, we show that RNAPII does not load condensin on DNA. Instead, RNAPII retains condensin in cis and hinders its ability to fold mitotic chromatin and to support chromosome segregation, consistent with the stalling of a loop extruder. Transcription termination by Dhp1 limits such a hindrance. Our results shed light on the integrated functioning of condensin, and we argue that a tight control of transcription underlies mitotic chromosome assembly by loop-extruding condensin.


Assuntos
Adenosina Trifosfatases , Segregação de Cromossomos , Complexos Multiproteicos , Schizosaccharomyces , Proteínas de Ligação a DNA/genética , Cromatina , Cromossomos , DNA , Schizosaccharomyces/genética , RNA Polimerase II/genética , Mitose , Proteínas de Ciclo Celular/genética
8.
PLoS Genet ; 20(3): e1011185, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38489251

RESUMO

The segregation of homologous chromosomes during meiosis typically requires tight end-to-end chromosome pairing. However, in Drosophila spermatogenesis, male flies segregate their chromosomes without classic synaptonemal complex formation and without recombination, instead compartmentalizing homologs into subnuclear domains known as chromosome territories (CTs). How homologs find each other in the nucleus and are separated into CTs has been one of the biggest riddles in chromosome biology. Here, we discuss our current understanding of pairing and CT formation in flies and review recent data on how homologs are linked and partitioned during meiosis in male flies.


Assuntos
Recombinação Genética , Complexo Sinaptonêmico , Animais , Masculino , Complexo Sinaptonêmico/genética , Meiose/genética , Pareamento Cromossômico/genética , Drosophila/genética , Segregação de Cromossomos/genética
9.
Elife ; 132024 Mar 08.
Artigo em Inglês | MEDLINE | ID: mdl-38456462

RESUMO

The physical basis of phase separation is thought to consist of the same types of bonds that specify conventional macromolecular interactions yet is unsatisfyingly often referred to as 'fuzzy'. Gaining clarity on the biogenesis of membraneless cellular compartments is one of the most demanding challenges in biology. Here, we focus on the chromosome passenger complex (CPC), that forms a chromatin body that regulates chromosome segregation in mitosis. Within the three regulatory subunits of the CPC implicated in phase separation - a heterotrimer of INCENP, Survivin, and Borealin - we identify the contact regions formed upon droplet formation using hydrogen/deuterium exchange mass spectrometry (HXMS). These contact regions correspond to some of the interfaces seen between individual heterotrimers within the crystal lattice they form. A major contribution comes from specific electrostatic interactions that can be broken and reversed through initial and compensatory mutagenesis, respectively. Our findings reveal structural insight for interactions driving liquid-liquid demixing of the CPC. Moreover, we establish HXMS as an approach to define the structural basis for phase separation.


Assuntos
Proteínas de Ciclo Celular , Proteínas de Ciclo Celular/genética , Cromossomos , Mitose , Citoesqueleto , Segregação de Cromossomos , Aurora Quinase B/genética
10.
Biochim Biophys Acta Mol Basis Dis ; 1870(4): 167116, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38447882

RESUMO

The Aurora-A kinase (AurkA) and its major regulator TPX2 (Targeting Protein for Xklp2) are key mitotic players frequently co-overexpressed in human cancers, and the link between deregulation of the AurkA/TPX2 complex and tumourigenesis is actively investigated. Chromosomal instability, one of the hallmarks of cancer related to the development of intra-tumour heterogeneity, metastasis and chemo-resistance, has been frequently associated with TPX2-overexpressing tumours. In this study we aimed to investigate the actual contribution to chromosomal instability of deregulating the AurkA/TPX2 complex, by overexpressing it in nontransformed hTERT RPE-1 cells. Our results show that overexpression of both AurkA and TPX2 results in increased AurkA activation and severe mitotic defects, compared to AurkA overexpression alone. We also show that AurkA/TPX2 co-overexpression yields increased aneuploidy in daughter cells and the generation of micronucleated cells. Interestingly, the p53/p21 axis response is impaired in AurkA/TPX2 overexpressing cells subjected to different stimuli; consistently, cells acquire increased ability to proliferate after independent induction of mitotic errors, i.e. following nocodazole treatment. Based on our observation that increased levels of the AurkA/TPX2 complex affect chromosome segregation fidelity and interfere with the activation of a pivotal surveillance mechanism in response to altered cell division, we propose that co-overexpression of AurkA and TPX2 per se represents a condition promoting the generation of a genetically unstable context in nontransformed human cells.


Assuntos
Aurora Quinase A , Proteínas de Ciclo Celular , Humanos , Aurora Quinase A/genética , Aurora Quinase A/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteína Supressora de Tumor p53/genética , Segregação de Cromossomos/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Instabilidade Genômica , Instabilidade Cromossômica/genética , Cromossomos/metabolismo
11.
J Cell Sci ; 137(6)2024 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-38372383

RESUMO

Male meiotic division exhibits two consecutive chromosome separation events without apparent pausing. Several studies have shown that spermatocyte divisions are not stringently regulated as in mitotic cells. In this study, we investigated the role of the canonical spindle assembly (SAC) pathway in Caenorhabditis elegans spermatogenesis. We found the intensity of chromosome-associated outer kinetochore protein BUB-1 and SAC effector MDF-1 oscillates between the two divisions. However, the SAC target securin is degraded during the first division and remains undetectable for the second division. Inhibition of proteasome-dependent protein degradation did not affect the progression of the second division but stopped the first division at metaphase. Perturbation of spindle integrity did not affect the duration of meiosis II, and only slightly lengthened meiosis I. Our results demonstrate that male meiosis II is independent of SAC regulation, and male meiosis I exhibits only weak checkpoint response.


Assuntos
Caenorhabditis elegans , Fuso Acromático , Animais , Masculino , Caenorhabditis elegans/metabolismo , Fuso Acromático/metabolismo , Espermatócitos/metabolismo , Meiose , Cinetocoros/metabolismo , Segregação de Cromossomos , Espermatogênese , Oócitos/metabolismo , Proteínas de Ciclo Celular/metabolismo
12.
Chromosome Res ; 32(1): 3, 2024 02 26.
Artigo em Inglês | MEDLINE | ID: mdl-38403686

RESUMO

Centromere is the chromosomal site of kinetochore assembly and microtubule attachment for chromosome segregation. Given its importance, markers that allow specific labeling of centromeric chromatin throughout the cell cycle and across all chromosome types are sought for facilitating various centromere studies. Antibodies against the N-terminal region of CENH3 are commonly used for this purpose, since CENH3 is the near-universal marker of functional centromeres. However, because the N-terminal region of CENH3 is highly variable among plant species, antibodies directed against this region usually function only in a small group of closely related species. As a more versatile alternative, we present here antibodies targeted to the conserved domains of two outer kinetochore proteins, KNL1 and NDC80. Sequence comparison of these domains across more than 350 plant species revealed a high degree of conservation, particularly within a six amino acid motif, FFGPVS in KNL1, suggesting that both antibodies would function in a wide range of plant species. This assumption was confirmed by immunolabeling experiments in angiosperm (monocot and dicot) and gymnosperm species, including those with mono-, holo-, and meta-polycentric chromosomes. In addition to centromere labeling on condensed chromosomes during cell division, both antibodies detected the corresponding regions in the interphase nuclei of most species tested. These results demonstrated that KNL1 and NDC80 are better suited for immunolabeling centromeres than CENH3, because antibodies against these proteins offer incomparably greater versatility across different plant species which is particularly convenient for studying the organization and function of the centromere in non-model species.


Assuntos
Centrômero , Cinetocoros , Sequência de Aminoácidos , Cromatina , Segregação de Cromossomos
13.
Int J Mol Sci ; 25(4)2024 Feb 09.
Artigo em Inglês | MEDLINE | ID: mdl-38396778

RESUMO

Chromosome segregation in female germ cells and early embryonic blastomeres is known to be highly prone to errors. The resulting aneuploidy is therefore the most frequent cause of termination of early development and embryo loss in mammals. And in specific cases, when the aneuploidy is actually compatible with embryonic and fetal development, it leads to severe developmental disorders. The main surveillance mechanism, which is essential for the fidelity of chromosome segregation, is the Spindle Assembly Checkpoint (SAC). And although all eukaryotic cells carry genes required for SAC, it is not clear whether this pathway is active in all cell types, including blastomeres of early embryos. In this review, we will summarize and discuss the recent progress in our understanding of the mechanisms controlling chromosome segregation and how they might work in embryos and mammalian embryos in particular. Our conclusion from the current literature is that the early mammalian embryos show limited capabilities to react to chromosome segregation defects, which might, at least partially, explain the widespread problem of aneuploidy during the early development in mammals.


Assuntos
Segregação de Cromossomos , Desenvolvimento Embrionário , Animais , Feminino , Humanos , Desenvolvimento Embrionário/genética , Aneuploidia , Mamíferos/genética , Tamanho Celular , Cromossomos
14.
Cell Mol Life Sci ; 81(1): 100, 2024 Feb 22.
Artigo em Inglês | MEDLINE | ID: mdl-38388697

RESUMO

Cell division is a crucial process, and one of its essential steps involves copying the genetic material, which is organized into structures called chromosomes. Before a cell can divide into two, it needs to ensure that each newly copied chromosome is paired tightly with its identical twin. This pairing is maintained by a protein complex known as cohesin, which is conserved in various organisms, from single-celled ones to humans. Cohesin essentially encircles the DNA, creating a ring-like structure to handcuff, to keep the newly synthesized sister chromosomes together in pairs. Therefore, chromosomal cohesion and separation are fundamental processes governing the attachment and segregation of sister chromatids during cell division. Metaphase-to-anaphase transition requires dissolution of cohesins by the enzyme Separase. The tight regulation of these processes is vital for safeguarding genomic stability. Dysregulation in chromosomal cohesion and separation resulting in aneuploidy, a condition characterized by an abnormal chromosome count in a cell, is strongly associated with cancer. Aneuploidy is a recurring hallmark in many cancer types, and abnormalities in chromosomal cohesion and separation have been identified as significant contributors to various cancers, such as acute myeloid leukemia, myelodysplastic syndrome, colorectal, bladder, and other solid cancers. Mutations within the cohesin complex have been associated with these cancers, as they interfere with chromosomal segregation, genome organization, and gene expression, promoting aneuploidy and contributing to the initiation of malignancy. In summary, chromosomal cohesion and separation processes play a pivotal role in preserving genomic stability, and aberrations in these mechanisms can lead to aneuploidy and cancer. Gaining a deeper understanding of the molecular intricacies of chromosomal cohesion and separation offers promising prospects for the development of innovative therapeutic approaches in the battle against cancer.


Assuntos
Proteínas de Ciclo Celular , Neoplasias , Humanos , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Cromátides/genética , Cromátides/metabolismo , Carcinogênese/genética , Transformação Celular Neoplásica , Neoplasias/genética , Segregação de Cromossomos , Aneuploidia , Instabilidade Genômica
15.
Methods Mol Biol ; 2740: 275-293, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38393482

RESUMO

In this chapter, we describe a software called MAARS (Mitotic Analysis And Recording System) that enables automatic and quantitative analysis of mitotic progression on an open-source platform. This computer-assisted analysis of cell division allows the unbiased acquisition of multiple parameters such as cell shape or size, metaphase or anaphase delays, as well as various mitotic abnormalities. This chapter describes the power of such an expert system to highlight the complexity of the mechanisms required to prevent mitotic chromosome segregation errors, leading to aneuploidy.


Assuntos
Mitose , Fuso Acromático , Metáfase , Anáfase , Segregação de Cromossomos , Software
16.
Elife ; 122024 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-38315099

RESUMO

Structural maintenance of chromosomes (SMC) complexes share conserved structures and serve a common role in maintaining chromosome architecture. In the bacterium Escherichia coli, the SMC complex MukBEF is necessary for rapid growth and the accurate segregation and positioning of the chromosome, although the specific molecular mechanisms involved are still unknown. Here, we used a number of in vivo assays to reveal how MukBEF controls chromosome conformation and how the MatP/matS system prevents MukBEF activity. Our results indicate that the loading of MukBEF occurs preferentially on newly replicated DNA, at multiple loci on the chromosome where it can promote long-range contacts in cis even though MukBEF can promote long-range contacts in the absence of replication. Using Hi-C and ChIP-seq analyses in strains with rearranged chromosomes, the prevention of MukBEF activity increases with the number of matS sites and this effect likely results from the unloading of MukBEF by MatP. Altogether, our results reveal how MukBEF operates to control chromosome folding and segregation in E. coli.


Assuntos
Proteínas de Escherichia coli , Escherichia coli , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas Repressoras/genética , Cromossomos Bacterianos/genética , Origem de Replicação , Proteínas Cromossômicas não Histona/genética , Cromossomos , Segregação de Cromossomos
17.
Nat Commun ; 15(1): 981, 2024 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-38302485

RESUMO

Despite drastic cellular changes during cleavage, a mitotic spindle assembles in each blastomere to accurately segregate duplicated chromosomes. Mechanisms of mitotic spindle assembly have been extensively studied using small somatic cells. However, mechanisms of spindle assembly in large vertebrate embryos remain little understood. Here, we establish functional assay systems in medaka (Oryzias latipes) embryos by combining CRISPR knock-in with auxin-inducible degron technology. Live imaging reveals several unexpected features of microtubule organization and centrosome positioning that achieve rapid, accurate cleavage. Importantly, Ran-GTP assembles a dense microtubule network at the metaphase spindle center that is essential for chromosome segregation in early embryos. This unique spindle structure is remodeled into a typical short, somatic-like spindle after blastula stages, when Ran-GTP becomes dispensable for chromosome segregation. We propose that despite the presence of centrosomes, the chromosome-derived Ran-GTP pathway has essential roles in functional spindle assembly in large, rapidly dividing vertebrate early embryos, similar to acentrosomal spindle assembly in oocytes.


Assuntos
Oryzias , Animais , Oryzias/genética , Segregação de Cromossomos , Centrossomo/metabolismo , Fuso Acromático/metabolismo , Microtúbulos/metabolismo , Vertebrados , Guanosina Trifosfato/metabolismo , Mitose
18.
Proc Natl Acad Sci U S A ; 121(9): e2318782121, 2024 Feb 27.
Artigo em Inglês | MEDLINE | ID: mdl-38381793

RESUMO

Regulation of microtubule dynamics by microtubule-associated proteins (MAPs) is essential for mitotic spindle assembly and chromosome segregation. Altered microtubule dynamics, particularly increased microtubule growth rates, were found to be a contributing factor for the development of chromosomal instability, which potentiates tumorigenesis. The MAP XMAP215/CKAP5 is the only known microtubule growth factor, and whether other MAPs regulate microtubule growth in cells is unclear. Our recent in vitro reconstitution experiments have demonstrated that Cytoskeleton-Associated Protein 2 (CKAP2) increases microtubule nucleation and growth rates, and here, we find that CKAP2 is also an essential microtubule growth factor in cells. By applying CRISPR-Cas9 knock-in and knock-out (KO) as well as microtubule plus-end tracking live cell imaging, we show that CKAP2 is a mitotic spindle protein that ensures faithful chromosome segregation by regulating microtubule growth. Live cell imaging of endogenously labeled CKAP2 showed that it localizes to the spindle during mitosis and rapidly shifts its localization to the chromatin upon mitotic exit before being degraded. Cells lacking CKAP2 display reduced microtubule growth rates and an increased proportion of chromosome segregation errors and aneuploidy that may be a result of an accumulation of kinetochore-microtubule misattachments. Microtubule growth rates and chromosome segregation fidelity can be rescued upon ectopic CKAP2 expression in KO cells, revealing a direct link between CKAP2 expression and microtubule dynamics. Our results unveil a role of CKAP2 in regulating microtubule growth in cells and provide a mechanistic explanation for the oncogenic potential of CKAP2 misregulation.


Assuntos
Segregação de Cromossomos , Microtúbulos , Microtúbulos/metabolismo , Fuso Acromático/metabolismo , Proteínas do Citoesqueleto/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo
19.
J Cell Sci ; 137(3)2024 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-38319136

RESUMO

The kinetochore is an essential structure for chromosome segregation. Although the kinetochore is usually formed on a centromere locus, it can be artificially formed at a non-centromere locus by protein tethering. An artificial kinetochore can be formed by tethering of CENP-C or CENP-I, members of the constitutive centromere-associated network (CCAN). However, how CENP-C or CENP-I recruit the centromere-specific histone CENP-A to form an artificial kinetochore remains unclear. In this study, we analyzed this issue using the tethering assay combined with an auxin-inducible degron (AID)-based knockout method in chicken DT40 cells. We found that tethering of CENP-C or CENP-I induced CENP-A incorporation at the non-centromeric locus in the absence of Knl2 (or MIS18BP1), a component of the Mis18 complex, and that Knl2 tethering recruited CENP-A in the absence of CENP-C. We also showed that CENP-C coimmunoprecipitated with HJURP, independently of Knl2. Considering these results, we propose that CENP-C recruits CENP-A by HJURP binding to form an artificial kinetochore. Our results suggest that CENP-C or CENP-I exert CENP-A recruitment activity, independently of Knl2, for artificial kinetochore formation in chicken DT40 cells. This gives us a new insight into mechanisms for CENP-A incorporation.


Assuntos
Proteína Centromérica A , Centrômero , Cinetocoros , Proteína Centromérica A/metabolismo , Segregação de Cromossomos , Animais , Galinhas
20.
Biochem Soc Trans ; 52(1): 29-39, 2024 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-38305688

RESUMO

Accurate chromosome segregation in mitosis relies on sister kinetochores forming stable attachments to microtubules (MTs) extending from opposite spindle poles and establishing biorientation. To achieve this, erroneous kinetochore-MT interactions must be resolved through a process called error correction, which dissolves improper kinetochore-MT attachment and allows new interactions until biorientation is achieved. The Aurora B kinase plays key roles in driving error correction by phosphorylating Dam1 and Ndc80 complexes, while Mps1 kinase, Stu2 MT polymerase and phosphatases also regulate this process. Once biorientation is formed, tension is applied to kinetochore-MT interaction, stabilizing it. In this review article, we discuss the mechanisms of kinetochore-MT interaction, error correction and biorientation. We focus mainly on recent insights from budding yeast, where the attachment of a single MT to a single kinetochore during biorientation simplifies the analysis of error correction mechanisms.


Assuntos
Proteínas de Saccharomyces cerevisiae , Saccharomycetales , Saccharomyces cerevisiae/genética , Cinetocoros , Microtúbulos/genética , Mitose , Segregação de Cromossomos , Proteínas de Saccharomyces cerevisiae/genética
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