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1.
J Cell Sci ; 136(5)2023 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-36274587

RESUMO

Mitotic cell division requires that kinetochores form microtubule attachments that can segregate chromosomes and control mitotic progression via the spindle assembly checkpoint. During prometaphase, kinetochores shed a domain called the fibrous corona as microtubule attachments form. This shedding is mediated, in part, by the minus-end directed motor dynein, which 'strips' cargoes along K-fibre microtubules. Despite its essentiality, little is known about how dynein stripping is regulated and how it responds to attachment maturation. Lis1 (also known as PAFAH1B1) is a conserved dynein regulator that is mutated in the neurodevelopmental disease lissencephaly. Here, we have combined loss-of-function studies, high-resolution imaging and separation-of-function mutants to define how Lis1 contributes to dynein-mediated corona stripping in HeLa cells. Cells depleted of Lis1 fail to disassemble the corona and show a delay in metaphase as a result of persistent checkpoint activation. Furthermore, we find that although kinetochore-tethered Lis1-dynein is required for error-free microtubule attachment, the contribution of Lis1 to corona disassembly can be mediated by a cytoplasmic pool. These findings support the idea that Lis1 drives dynein function at kinetochores to ensure corona disassembly and prevent chromosome mis-segregation.


Assuntos
1-Alquil-2-acetilglicerofosfocolina Esterase , Dineínas , Cinetocoros , Proteínas Associadas aos Microtúbulos , Humanos , Dineínas/metabolismo , Células HeLa , Cinetocoros/metabolismo , Pontos de Checagem da Fase M do Ciclo Celular , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Mitose , 1-Alquil-2-acetilglicerofosfocolina Esterase/genética , 1-Alquil-2-acetilglicerofosfocolina Esterase/metabolismo
2.
Science ; 378(6621): eabq7361, 2022 11 18.
Artigo em Inglês | MEDLINE | ID: mdl-36395215

RESUMO

Meiotic spindle assembly ensures proper chromosome segregation in oocytes. However, the mechanisms behind spindle assembly in human oocytes remain largely unknown. We used three-dimensional high-resolution imaging of more than 2000 human oocytes to identify a structure that we named the human oocyte microtubule organizing center (huoMTOC). The proteins TACC3, CCP110, CKAP5, and DISC1 were found to be essential components of the huoMTOC. The huoMTOC arises beneath the oocyte cortex and migrates adjacent to the nuclear envelope before nuclear envelope breakdown (NEBD). After NEBD, the huoMTOC fragments and relocates on the kinetochores to initiate microtubule nucleation and spindle assembly. Disrupting the huoMTOC led to spindle assembly defects and oocyte maturation arrest. These results reveal a physiological mechanism of huoMTOC-regulated spindle assembly in human oocytes.


Assuntos
Centro Organizador dos Microtúbulos , Oócitos , Fuso Acromático , Humanos , Segregação de Cromossomos , Cinetocoros/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Centro Organizador dos Microtúbulos/metabolismo , Oócitos/metabolismo , Fuso Acromático/metabolismo , Células Cultivadas
3.
Mol Cell ; 82(21): 4018-4032.e9, 2022 Nov 03.
Artigo em Inglês | MEDLINE | ID: mdl-36332605

RESUMO

Kinetochore assembly on centromeres is central for chromosome segregation, and defects in this process cause mitotic errors and aneuploidy. Besides the well-established protein network, emerging evidence suggests the involvement of regulatory RNA in kinetochore assembly; however, it has remained elusive about the identity of such RNA, let alone its mechanism of action in this critical process. Here, we report CCTT, a previously uncharacterized long non-coding RNA (lncRNA) transcribed from the arm of human chromosome 17, which plays a vital role in kinetochore assembly. We show that CCTT highly localizes to all centromeres via the formation of RNA-DNA triplex and specifically interacts with CENP-C to help engage this blueprint protein in centromeres, and consequently, CCTT loss triggers extensive mitotic errors and aneuploidy. These findings uncover a non-centromere-derived lncRNA that recruits CENP-C to centromeres and shed critical lights on the function of centromeric DNA sequences as anchor points for kinetochore assembly.


Assuntos
RNA Longo não Codificante , Humanos , Aneuploidia , Proteína Centromérica A/metabolismo , DNA , Cinetocoros/metabolismo , RNA Longo não Codificante/genética , Centrômero
4.
Int J Mol Sci ; 23(22)2022 Nov 17.
Artigo em Inglês | MEDLINE | ID: mdl-36430712

RESUMO

The protein kinase Mps1 (monopolar spindle 1) is an important regulator of the Spindle Assembly Checkpoint (SAC), the evolutionary conserved checkpoint system of higher organisms that monitors the proper bipolar attachment of all chromosomes to the mitotic spindle during cell division. Defects in the catalytic activity and the transcription regulation of Mps1 are associated with genome instability, aneuploidy, and cancer. Moreover, multiple Mps1 missense and frameshift mutations have been reported in a wide range of types of cancer of different tissue origin. Due to these features, Mps1 arises as one promising drug target for cancer therapy. In this contribution, we developed a computational biology approach to study the dynamics of human Mps1 kinase interaction with isoflavones, a class of natural flavonoids, and compared their predicted mode of binding with that observed in the crystal structure of Mps1 in complex with reversine, a small-sized inhibitor of Mps1 and Aurora B kinases. We concluded that isoflavones define a chemical scaffold that can be used to develop new Mps1 inhibitors for the treatment of cancer associated with Mps1 amplification and aberrant chromosome segregation. In a broader context, the present report illustrates how modern chemoinformatics approaches can accelerate drug development in oncology.


Assuntos
Isoflavonas , Neoplasias , Humanos , Cinetocoros/metabolismo , Proteínas Tirosina Quinases/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas Serina-Treonina Quinases , Mitose , Biologia Computacional , Isoflavonas/farmacologia , Isoflavonas/metabolismo , Microtúbulos/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo
5.
J Cell Biol ; 221(11)2022 11 07.
Artigo em Inglês | MEDLINE | ID: mdl-36200976

RESUMO

Barbosa et al. discuss work by Mussachio and colleagues (2022. J. Cell Biol.https://doi.org/10.1083/jcb.202206131) finding that conformational changes in the DYNEIN adaptor SPINDLY can precisely control DYNEIN activation at kinetochores.


Assuntos
Proteínas de Ciclo Celular , Divisão Celular , Dineínas , Fuso Acromático , Proteínas de Ciclo Celular/metabolismo , Dineínas/metabolismo , Cinetocoros/metabolismo , Fuso Acromático/metabolismo
6.
J Vis Exp ; (187)2022 09 13.
Artigo em Inglês | MEDLINE | ID: mdl-36190266

RESUMO

Aneuploidy is the leading genetic abnormality causing early miscarriage and pregnancy failure in humans. Most errors in chromosome segregation that give rise to aneuploidy occur during meiosis in oocytes, but why oocyte meiosis is error-prone is still not fully understood. During cell division, cells prevent errors in chromosome segregation by activating the spindle assembly checkpoint (SAC). This control mechanism relies on detecting kinetochore (KT)-microtubule (MT) attachments and sensing tension generated by spindle fibers. When KTs are unattached, the SAC is activated and prevents cell-cycle progression. The SAC is activated first by MPS1 kinase, which triggers the recruitment and formation of the mitotic checkpoint complex (MCC), composed of MAD1, MAD2, BUB3, and BUBR1. Then, the MCC diffuses into the cytoplasm and sequesters CDC20, an anaphase-promoting complex/cyclosome (APC/C) activator. Once KTs become attached to microtubules and chromosomes are aligned at the metaphase plate, the SAC is silenced, CDC20 is released, and the APC/C is activated, triggering the degradation of Cyclin B and Securin, thereby allowing anaphase onset. Compared to somatic cells, the SAC in oocytes is not as effective because cells can undergo anaphase despite having unattached KTs. Understanding why the SAC is more permissive and if this permissiveness is one of the causes of chromosome segregation errors in oocytes still needs further investigation. The present protocol describes the three techniques to comprehensively evaluate SAC integrity in mouse oocytes. These techniques include using nocodazole to depolymerize MTs to evaluate the SAC response, tracking SAC silencing by following the kinetics of Securin destruction, and evaluating the recruitment of MAD2 to KTs by immunofluorescence. Together these techniques probe mechanisms needed to produce healthy eggs by providing a complete evaluation of SAC integrity.


Assuntos
Pontos de Checagem da Fase M do Ciclo Celular , Fuso Acromático , Ciclossomo-Complexo Promotor de Anáfase/genética , Ciclossomo-Complexo Promotor de Anáfase/metabolismo , Aneuploidia , Animais , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Cinetocoros/metabolismo , Camundongos , Nocodazol , Oócitos , Securina/genética , Securina/metabolismo , Fuso Acromático/metabolismo
7.
J Phys Chem B ; 126(43): 8720-8734, 2022 Nov 03.
Artigo em Inglês | MEDLINE | ID: mdl-36269085

RESUMO

Kinesin-14s constitute a subfamily of the large superfamily of adenosine triphosphate-dependent microtubule-based motor proteins. Kinesin-14s have the motor domain at the C-terminal end of the peptide, playing key roles during spindle assembly and maintenance. Some of them are nonprocessive motors, whereas others can move processively on microtubules. Here, we take budding yeast Cik1-Kar3 and human HSET as examples to study theoretically the dynamics of the processive kinesin-14 motor moving on the single microtubule under load, the dynamics of the motor coupled with an Ndc80 protein moving on the single microtubule, the dynamics of the motor moving in microtubule arrays, and so on. The dynamics of the nonprocessive Drosophila Ncd motor is also discussed. The studies explain well the available experimental data and, moreover, provide predicted results. We show that the processive kinesin-14 motors can move efficiently in microtubule arrays toward the minus ends, and after reaching the minus ends, they can stay there stably, thus performing the function of organizing the microtubules in the bipolar spindle into polar arrays at the spindle poles.


Assuntos
Cinesinas , Proteínas de Saccharomyces cerevisiae , Animais , Humanos , Microtúbulos/química , Proteínas dos Microtúbulos/análise , Proteínas dos Microtúbulos/metabolismo , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/metabolismo , Drosophila/metabolismo , Proteínas Nucleares/análise , Proteínas Nucleares/metabolismo , Cinetocoros/metabolismo
8.
Nat Commun ; 13(1): 6381, 2022 Oct 26.
Artigo em Inglês | MEDLINE | ID: mdl-36289199

RESUMO

In response to improper kinetochore-microtubule attachments in mitosis, the spindle assembly checkpoint (SAC) assembles the mitotic checkpoint complex (MCC) to inhibit the anaphase-promoting complex/cyclosome, thereby delaying entry into anaphase. The MCC comprises Mad2:Cdc20:BubR1:Bub3. Its assembly is catalysed by unattached kinetochores on a Mad1:Mad2 platform. Mad1-bound closed-Mad2 (C-Mad2) recruits open-Mad2 (O-Mad2) through self-dimerization. This interaction, combined with Mps1 kinase-mediated phosphorylation of Bub1 and Mad1, accelerates MCC assembly, in a process that requires O-Mad2 to C-Mad2 conversion and concomitant binding of Cdc20. How Mad1 phosphorylation catalyses MCC assembly is poorly understood. Here, we characterized Mps1 phosphorylation of Mad1 and obtained structural insights into a phosphorylation-specific Mad1:Cdc20 interaction. This interaction, together with the Mps1-phosphorylation dependent association of Bub1 and Mad1, generates a tripartite assembly of Bub1 and Cdc20 onto the C-terminal domain of Mad1 (Mad1CTD). We additionally identify flexibility of Mad1:Mad2 that suggests how the Cdc20:Mad1CTD interaction brings the Mad2-interacting motif (MIM) of Cdc20 near O-Mad2. Thus, Mps1-dependent formation of the MCC-assembly scaffold functions to position and orient Cdc20 MIM near O-Mad2, thereby catalysing formation of C-Mad2:Cdc20.


Assuntos
Proteínas de Ciclo Celular , Pontos de Checagem da Fase M do Ciclo Celular , Proteínas de Ciclo Celular/metabolismo , Cinetocoros/metabolismo , Mitose , Catálise , Proteínas Mad2/metabolismo , Fuso Acromático/metabolismo , Proteínas Cdc20/metabolismo
9.
Proc Natl Acad Sci U S A ; 119(41): e2208255119, 2022 10 11.
Artigo em Inglês | MEDLINE | ID: mdl-36191188

RESUMO

Aneuploidy, the incorrect number of whole chromosomes, is a common feature of tumors that contributes to their initiation and evolution. Preventing aneuploidy requires properly functioning kinetochores, which are large protein complexes assembled on centromeric DNA that link mitotic chromosomes to dynamic spindle microtubules and facilitate chromosome segregation. The kinetochore leverages at least two mechanisms to prevent aneuploidy: error correction and the spindle assembly checkpoint (SAC). BubR1, a factor involved in both processes, was identified as a cancer dependency and therapeutic target in multiple tumor types; however, it remains unclear what specific oncogenic pressures drive this enhanced dependency on BubR1 and whether it arises from BubR1's regulation of the SAC or error-correction pathways. Here, we use a genetically controlled transformation model and glioblastoma tumor isolates to show that constitutive signaling by RAS or MAPK is necessary for cancer-specific BubR1 vulnerability. The MAPK pathway enzymatically hyperstimulates a network of kinetochore kinases that compromises chromosome segregation, rendering cells more dependent on two BubR1 activities: counteracting excessive kinetochore-microtubule turnover for error correction and maintaining the SAC. This work expands our understanding of how chromosome segregation adapts to different cellular states and reveals an oncogenic trigger of a cancer-specific defect.


Assuntos
Neoplasias , Proteínas Serina-Treonina Quinases , Aneuploidia , Carcinogênese/metabolismo , Proteínas de Ciclo Celular/metabolismo , Segregação de Cromossomos , Humanos , Cinetocoros/metabolismo , Microtúbulos/metabolismo , Mitose/genética , Neoplasias/metabolismo , Proteínas Serina-Treonina Quinases/genética , Fuso Acromático/metabolismo
10.
Nucleic Acids Res ; 50(19): 10914-10928, 2022 10 28.
Artigo em Inglês | MEDLINE | ID: mdl-36200823

RESUMO

Centromeres of most eukaryotes consist of two distinct chromatin domains: a kinetochore domain, identified by the histone H3 variant, CENP-A, and a heterochromatic domain. How these two domains are separated is unclear. Here, we show that, in Schizosaccharomyces pombe, mutation of the chromatin remodeler RSC induced CENP-ACnp1 misloading at pericentromeric heterochromatin, resulting in the mis-assembly of kinetochore proteins and a defect in chromosome segregation. We find that RSC functions at the kinetochore boundary to prevent CENP-ACnp1 from spreading into neighbouring heterochromatin, where deacetylated histones provide an ideal environment for the spread of CENP-ACnp1. In addition, we show that RSC decompacts the chromatin structure at this boundary, and propose that this RSC-directed chromatin decompaction prevents mis-propagation of CENP-ACnp1 into pericentromeric heterochromatin. Our study provides an insight into how the distribution of distinct chromatin domains is established and maintained.


Assuntos
Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces , Heterocromatina/metabolismo , Cromatina/metabolismo , Proteína Centromérica A/metabolismo , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Centrômero/metabolismo , Cinetocoros/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Histonas/metabolismo
11.
J Vis Exp ; (188)2022 10 11.
Artigo em Inglês | MEDLINE | ID: mdl-36314815

RESUMO

Time-lapse fluorescence microscopy has revolutionized the understanding of meiotic cell-cycle events by providing temporal and spatial data that is often not seen by imaging fixed cells. Budding yeast has proved to be an important model organism to study meiotic chromosome segregation because many meiotic genes are highly conserved. Time-lapse microscopy of meiosis in budding yeast allows the monitoring of different meiotic mutants to show how the mutation disrupts meiotic processes. However, many proteins function at multiple points in meiosis. The use of loss-of-function or meiotic null mutants can therefore disrupt an early process, blocking or disturbing the later process and making it difficult to determine the phenotypes associated with each individual role. To circumvent this challenge, this protocol describes how the proteins can be conditionally depleted from the nucleus at specific stages of meiosis while monitoring meiotic events using time-lapse microscopy. Specifically, this protocol describes how the cells are synchronized in prophase I, how the anchor away technique is used to deplete proteins from the nucleus at specific meiotic stages, and how time-lapse imaging is used to monitor meiotic chromosome segregation. As an example of the usefulness of the technique, the kinetochore protein Ctf19 was depleted from the nucleus at different time points during meiosis, and the number of chromatin masses was analyzed at the end of meiosis II. Overall, this protocol can be adapted to deplete different nuclear proteins from the nucleus while monitoring the meiotic divisions.


Assuntos
Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Imagem com Lapso de Tempo , Microscopia , Meiose , Cinetocoros/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Ciclo Celular/metabolismo
12.
Biophys J ; 121(21): 4048-4062, 2022 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-36199251

RESUMO

In the mitotic spindle, microtubules attach to chromosomes via kinetochores. The microtubule-binding Ndc80 complex is an integral part of kinetochores, and is essential for kinetochores to attach to microtubules and to transmit forces from dynamic microtubule ends to the chromosomes. The Ndc80 complex has a rod-like appearance with globular domains at its ends that are separated by a long coiled coil. Its mechanical properties are considered important for the dynamic interaction between kinetochores and microtubules. Here, we present a novel method that allows us to time trace the effective stiffness of Ndc80 complexes following shortening microtubule ends against applied force in optical trap experiments. Applying this method to wild-type Ndc80 and three variants (calponin homology (CH) domains mutated or Hec1 tail unphosphorylated, phosphorylated, or truncated), we reveal that each variant exhibits strain stiffening; i.e., the effective stiffness increases under tension that is built up by a depolymerizing microtubule. The strain stiffening relation is roughly linear and independent of the state of the microtubule. We introduce structure-based models that show that the strain stiffening can be traced back to the specific architecture of the Ndc80 complex with a characteristic flexible kink, to thermal fluctuations of the microtubule, and to the bending elasticity of flaring protofilaments, which exert force to move the Ndc80 complexes. Our model accounts for changes in the amount of load-bearing attachments at various force levels and reproduces the roughly linear strain stiffening behavior, highlighting the importance of force-dependent binding affinity.


Assuntos
Cinetocoros , Proteínas Nucleares , Cinetocoros/metabolismo , Proteínas Nucleares/metabolismo , Microtúbulos/metabolismo , Fuso Acromático/metabolismo , Segregação de Cromossomos
13.
PLoS Genet ; 18(9): e1010397, 2022 09.
Artigo em Inglês | MEDLINE | ID: mdl-36108046

RESUMO

The activated spindle assembly checkpoint (SAC) potently inhibits the anaphase-promoting complex/cyclosome (APC/C) to ensure accurate chromosome segregation at anaphase. Early studies have recognized that the SAC should be silenced within minutes to enable rapid APC/C activation and synchronous segregation of chromosomes once all kinetochores are properly attached, but the underlying silencers are still being elucidated. Here, we report that the timely silencing of SAC in fission yeast requires dnt1+, which causes severe thiabendazole (TBZ) sensitivity and increased rate of lagging chromosomes when deleted. The absence of Dnt1 results in prolonged inhibitory binding of mitotic checkpoint complex (MCC) to APC/C and attenuated protein levels of Slp1Cdc20, consequently slows the degradation of cyclin B and securin, and eventually delays anaphase entry in cells released from SAC activation. Interestingly, Dnt1 physically associates with APC/C upon SAC activation. We propose that this association may fend off excessive and prolonged MCC binding to APC/C and help to maintain Slp1Cdc20 stability. This may allow a subset of APC/C to retain activity, which ensures rapid anaphase onset and mitotic exit once SAC is inactivated. Therefore, our study uncovered a new player in dictating the timing and efficacy of APC/C activation, which is actively required for maintaining cell viability upon recovery from the inhibition of APC/C by spindle checkpoint.


Assuntos
Proteínas de Ciclo Celular , Tiabendazol , Ciclossomo-Complexo Promotor de Anáfase/genética , Ciclossomo-Complexo Promotor de Anáfase/metabolismo , Proteínas Cdc20/genética , Proteínas Cdc20/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Cinetocoros/metabolismo , Pontos de Checagem da Fase M do Ciclo Celular/genética , Securina/genética , Fuso Acromático/genética , Fuso Acromático/metabolismo , Tiabendazol/metabolismo
14.
J Cell Biol ; 221(11)2022 11 07.
Artigo em Inglês | MEDLINE | ID: mdl-36107127

RESUMO

Cytoplasmic Dynein 1, or Dynein, is a microtubule minus end-directed motor. Dynein motility requires Dynactin and a family of activating adaptors that stabilize the Dynein-Dynactin complex and promote regulated interactions with cargo in space and time. How activating adaptors limit Dynein activation to specialized subcellular locales is unclear. Here, we reveal that Spindly, a mitotic Dynein adaptor at the kinetochore corona, exists natively in a closed conformation that occludes binding of Dynein-Dynactin to its CC1 box and Spindly motif. A structure-based analysis identified various mutations promoting an open conformation of Spindly that binds Dynein-Dynactin. A region of Spindly downstream from the Spindly motif and not required for cargo binding faces the CC1 box and stabilizes the intramolecular closed conformation. This region is also required for robust kinetochore localization of Spindly, suggesting that kinetochores promote Spindly activation to recruit Dynein. Thus, our work illustrates how specific Dynein activation at a defined cellular locale may require multiple factors.


Assuntos
Proteínas de Ciclo Celular , Dineínas do Citoplasma , Complexo Dinactina , Proteínas de Ciclo Celular/metabolismo , Dineínas do Citoplasma/metabolismo , Complexo Dinactina/metabolismo , Cinetocoros/metabolismo , Conformação Proteica
15.
Methods Mol Biol ; 2478: 609-650, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36063336

RESUMO

Optical trapping has been instrumental for deciphering translocation mechanisms of the force-generating cytoskeletal proteins. However, studies of the dynamic interactions between microtubules (MTs) and MT-associated proteins (MAPs) with no motor activity are lagging. Investigating the motility of MAPs that can diffuse along MT walls is a particular challenge for optical-trapping assays because thermally driven motions rely on weak and highly transient interactions. Three-bead, ultrafast force-clamp (UFFC) spectroscopy has the potential to resolve static and diffusive translocations of different MAPs with sub-millisecond temporal resolution and sub-nanometer spatial precision. In this report, we present detailed procedures for implementing UFFC, including setup of the optical instrument and feedback control, immobilization and functionalization of pedestal beads, and preparation of MT dumbbells. Example results for strong static interactions were generated using the Kinesin-7 motor CENP-E in the presence of AMP-PNP. Time resolution for MAP-MT interactions in the UFFC assay is limited by the MT dumbbell relaxation time, which is significantly longer than reported for analogous experiments using actin filaments. UFFC, however, provides a unique opportunity for quantitative studies on MAPs that glide along MTs under a dragging force, as illustrated using the kinetochore-associated Ska complex.


Assuntos
Proteínas de Transporte , Proteínas dos Microtúbulos , Proteínas de Transporte/metabolismo , Cinetocoros/metabolismo , Proteínas dos Microtúbulos/análise , Proteínas dos Microtúbulos/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Análise Espectral
16.
Artigo em Inglês | MEDLINE | ID: mdl-36123287

RESUMO

OBJECTIVE: This study elucidated the clinical significance, functions, and mechanism of action of spindle and kinetochore-associated complex 3 (SKA3) in oral squamous cell carcinoma (OSCC). STUDY DESIGN: The SKA3 levels within the patients with OSCC were determined using the The cancer genome atlas (TCGA) database and clinical samples. The functions of SKA3 in OSCC cells were evaluated by cell counting Kit-8 (Beyotime Biotechnology, Haimen, China), 5-ethynyl-2'-deoxyuridine, wound healing, transwell invasion, flow cytometry, and xenograft nude mice model assays. A quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and Western blot were performed to assess mRNA and protein expression levels in specimens and cells, respectively. RESULTS: The SKA3 was highly expressed in OSCC tissues, and its knockdown suppressed OSCC cell proliferation, migration, and invasion, and promoted their apoptosis. Mechanistically, SKA3 was shown to modulate OSCC cell proliferation and apoptosis via the PI3K/AKT/GSK3ß and PI3K/AKT/FOXO1 pathways. CONCLUSIONS: Biologically, SKA3 has a potential carcinogenic role in OSCC progression and is a promising prognostic biomarker and therapeutic target.


Assuntos
Carcinoma de Células Escamosas , Neoplasias de Cabeça e Pescoço , Neoplasias Bucais , Camundongos , Animais , Humanos , Neoplasias Bucais/patologia , Carcinoma de Células Escamosas/patologia , Carcinoma de Células Escamosas de Cabeça e Pescoço , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Glicogênio Sintase Quinase 3 beta/genética , Cinetocoros/metabolismo , Cinetocoros/patologia , Camundongos Nus , Prognóstico , Movimento Celular/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Biomarcadores
17.
Mol Biol Cell ; 33(14): ar143, 2022 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-36129769

RESUMO

Chromosome segregation requires assembly of the macromolecular kinetochore complex onto centromeric DNA. While most eukaryotes have canonical kinetochore proteins that are widely conserved among eukaryotes, evolutionarily divergent kinetoplastids have a unique set of kinetochore proteins. Little is known about the mechanism of kinetochore assembly in kinetoplastids. Here we characterize two homologous kinetoplastid kinetochore proteins, KKT2 and KKT3, that constitutively localize at centromeres. They have three domains that are highly conserved among kinetoplastids: an N-terminal kinase domain of unknown function, the centromere localization domain in the middle, and the C-terminal domain that has weak similarity to polo boxes of Polo-like kinases. We show that the kinase activity of KKT2 is essential for accurate chromosome segregation, while that of KKT3 is dispensable for cell growth in Trypanosoma brucei. Crystal structures of their divergent polo boxes reveal differences between KKT2 and KKT3. We also show that the divergent polo boxes of KKT3 are sufficient to recruit KKT2 in trypanosomes. Furthermore, we demonstrate that the divergent polo boxes of KKT2 interact directly with KKT1 and that KKT1 interacts with KKT6. These results show that the divergent polo boxes of KKT2 and KKT3 are protein-protein interaction domains that initiate kinetochore assembly in T. brucei.


Assuntos
Trypanosoma brucei brucei , Trypanosoma brucei brucei/metabolismo , Cinetocoros/metabolismo , Proteínas de Protozoários/metabolismo , Segregação de Cromossomos , Proteínas de Ciclo Celular/metabolismo , Centrômero/metabolismo
18.
Commun Biol ; 5(1): 818, 2022 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-35970865

RESUMO

Centromeres are established by nucleosomes containing the histone H3 variant CENP-A. CENP-A is recruited to centromeres by the Mis18-HJURP machinery. During mitosis, CENP-A recruitment ceases, implying the necessity of CENP-A maintenance at centromeres, although the exact underlying mechanism remains elusive. Herein, we show that the inner kinetochore protein Mis6 (CENP-I) and Mis15 (CENP-N) retain CENP-A during mitosis in fission yeast. Eliminating Mis6 or Mis15 during mitosis caused immediate loss of pre-existing CENP-A at centromeres. CENP-A loss occurred due to the transcriptional upregulation of non-coding RNAs at the central core region of centromeres, as confirmed by the observation RNA polymerase II inhibition preventing CENP-A loss from centromeres in the mis6 mutant. Thus, we concluded that the inner kinetochore complex containing Mis6-Mis15 blocks the indiscriminate transcription of non-coding RNAs at the core centromere, thereby retaining the epigenetic inheritance of CENP-A during mitosis.


Assuntos
Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/metabolismo , Centrômero/metabolismo , Proteína Centromérica A/genética , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Cinetocoros/metabolismo , Mitose , Nucleossomos/genética , Nucleossomos/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/genética
19.
Int J Mol Sci ; 23(15)2022 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-35955838

RESUMO

During mitosis, many cellular structures are organized to segregate the replicated genome to the daughter cells. Chromatin is condensed to shape a mitotic chromosome. A multiprotein complex known as kinetochore is organized on a specific region of each chromosome, the centromere, which is defined by the presence of a histone H3 variant called CENP-A. The cytoskeleton is re-arranged to give rise to the mitotic spindle that binds to kinetochores and leads to the movement of chromosomes. How chromatin regulates different activities during mitosis is not well known. The role of histone post-translational modifications (HPTMs) in mitosis has been recently revealed. Specific HPTMs participate in local compaction during chromosome condensation. On the other hand, HPTMs are involved in CENP-A incorporation in the centromere region, an essential activity to maintain centromere identity. HPTMs also participate in the formation of regulatory protein complexes, such as the chromosomal passenger complex (CPC) and the spindle assembly checkpoint (SAC). Finally, we discuss how HPTMs can be modified by environmental factors and the possible consequences on chromosome segregation and genome stability.


Assuntos
Proteínas Cromossômicas não Histona , Histonas , Centrômero/genética , Centrômero/metabolismo , Proteína Centromérica A/metabolismo , Cromatina/genética , Proteínas Cromossômicas não Histona/metabolismo , Segregação de Cromossomos , Histonas/metabolismo , Cinetocoros/metabolismo , Mitose/genética , Processamento de Proteína Pós-Traducional
20.
Mol Biol Rep ; 49(11): 10875-10883, 2022 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-35931874

RESUMO

The process of cell division plays a vital role in cancer progression. Cell proliferation and error-free chromosomes segregation during mitosis are central events in life cycle. Mistakes during cell division generate changes in chromosome content and alter the balances of chromosomes number. Any defects in expression of TIF1 family proteins, SAC proteins network, mitotic checkpoint proteins involved in chromosome mis-segregation and cancer development. Here we discuss the function of organelles deal with the chromosome segregation machinery, proteins and correction mechanisms involved in the accurate chromosome segregation during mitosis.


Assuntos
Segregação de Cromossomos , Neoplasias , Humanos , Mitose/genética , Ciclo Celular/genética , Pontos de Checagem da Fase M do Ciclo Celular , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Neoplasias/genética , Neoplasias/terapia , Cinetocoros/metabolismo
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