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1.
J Cell Sci ; 136(5)2023 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-35502670

RESUMO

The precise regulation of microtubule length during mitosis is essential to assemble and position the mitotic spindle and segregate chromosomes. The kinesin-13 Kif2C or MCAK acts as a potent microtubule depolymerase that diffuses short distances on microtubules, whereas the kinesin-8 Kif18b is a processive motor with weak depolymerase activity. However, the individual activities of these factors cannot explain the dramatic increase in microtubule dynamics in mitosis. Using in vitro reconstitution and single-molecule imaging, we demonstrate that Kif18b, MCAK and the plus-end tracking protein EB3 (also known as MAPRE3) act in an integrated manner to potently promote microtubule depolymerization at very low concentrations. We find that Kif18b can transport EB3 and MCAK and promotes their accumulation to microtubule plus ends through multivalent weak interactions. Together, our work defines the mechanistic basis for a cooperative Kif18b-MCAK-EB network at microtubule plus ends, that acts to efficiently shorten and regulate microtubules in mitosis, essential for correct chromosome segregation.


Assuntos
Cinesinas , Microtúbulos , Segregação de Cromossomos , Cinesinas/genética , Microtúbulos/metabolismo , Mitose , Fuso Acromático/metabolismo
2.
Methods Mol Biol ; 2519: 17-26, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36066706

RESUMO

Cellular division is a fundamental process of cellular growth. First, cells replicate their DNA in S phase and then undergo mitosis which, under normal conditions, leads to complete cell division. Moreover, mitotic activity correlates to cellular growth activity. The simplest and classical method to measure mitotic activity (mitotic index (MI)), is the manual counting of mitotic cells among a given cell population of interest. The latter can be accomplished via phase contrast microscope observation. However, Giemsa staining may improve accuracy and consistency. Fluorescence immunostaining targeting specific phosphorylations of proteins at critical cell cycle steps will provide further improved analysis via high-throughput capacity of flow or imaging cytometer. Finally, time lapse image analysis provides quantitative and qualitative metrics delineating the process of cellular division including timing of division, duration of mitosis, and failure to procced through or complete mitosis.


Assuntos
Mitose , Ciclo Celular , Índice Mitótico , Fosforilação , Fase S
3.
Methods Mol Biol ; 2519: 27-40, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36066707

RESUMO

Cell cycle-dependent regulation of chromosome is a dynamic event. After replication in S phase, sister chromatids show dynamic behavior including condensation, alignment, and segregation in M phase. These beautiful behaviors of chromosomes observed through the microscope have fascinated people since more than 100 years ago, and now we can sketch the dynamics of regulatory proteins and their posttranscriptional modifications through the fluorescent microscope. The purpose of this chapter is describing the basic methods of immunofluorescence analysis of mitotic cells and chromosomes. Besides, the key ideas for improving the preparation of the specimen are also described. Because the characteristic of the proteins of your interest differs one by one, modifying the method might cause the crucial improvement in the observation.


Assuntos
Cromátides , Mitose , Segregação de Cromossomos , Cromossomos , Humanos , Microscopia de Fluorescência
4.
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
5.
Mol Cell Proteomics ; 21(1): 100169, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-34742921

RESUMO

Comprehensive proteome analysis of rare cell phenotypes remains a significant challenge. We report a method for low cell number MS-based proteomics using protease digestion of mildly formaldehyde-fixed cells in cellulo, which we call the "in-cell digest." We combined this with averaged MS1 precursor library matching to quantitatively characterize proteomes from low cell numbers of human lymphoblasts. About 4500 proteins were detected from 2000 cells, and 2500 proteins were quantitated from 200 lymphoblasts. The ease of sample processing and high sensitivity makes this method exceptionally suited for the proteomic analysis of rare cell states, including immune cell subsets and cell cycle subphases. To demonstrate the method, we characterized the proteome changes across 16 cell cycle states (CCSs) isolated from an asynchronous TK6 cells, avoiding synchronization. States included late mitotic cells present at extremely low frequency. We identified 119 pseudoperiodic proteins that vary across the cell cycle. Clustering of the pseudoperiodic proteins showed abundance patterns consistent with "waves" of protein degradation in late S, at the G2&M border, midmitosis, and at mitotic exit. These clusters were distinguished by significant differences in predicted nuclear localization and interaction with the anaphase-promoting complex/cyclosome. The dataset also identifies putative anaphase-promoting complex/cyclosome substrates in mitosis and the temporal order in which they are targeted for degradation. We demonstrate that a protein signature made of these 119 high-confidence cell cycle-regulated proteins can be used to perform unbiased classification of proteomes into CCSs. We applied this signature to 296 proteomes that encompass a range of quantitation methods, cell types, and experimental conditions. The analysis confidently assigns a CCS for 49 proteomes, including correct classification for proteomes from synchronized cells. We anticipate that this robust cell cycle protein signature will be crucial for classifying cell states in single-cell proteomes.


Assuntos
Peptídeo Hidrolases , Proteômica , Contagem de Células , Ciclo Celular , Proteínas de Ciclo Celular/metabolismo , Mitose , Proteômica/métodos
6.
Proc Natl Acad Sci U S A ; 119(45): e2116167119, 2022 11 08.
Artigo em Inglês | MEDLINE | ID: mdl-36322767

RESUMO

How cells adjust their growth to the spatial and mechanical constraints of their surrounding environment is central to many aspects of biology. Here, we examined how extracellular matrix (ECM) rigidity affects cell division. We found that cells divide more rapidly when cultured on rigid substrates. While we observed no effect of ECM rigidity on rounding or postmitotic spreading duration, we found that changes in matrix stiffness impact mitosis progression. We noticed that ECM elasticity up-regulates the expression of the linker of nucleoskeleton and cytoskeleton (LINC) complex component SUN2, which in turn promotes metaphase-to-anaphase transition by acting on mitotic spindle formation, whereas when cells adhere to soft ECM, low levels of SUN2 expression perturb astral microtubule organization and delay the onset of anaphase.


Assuntos
Citoesqueleto , Matriz Nuclear , Matriz Nuclear/metabolismo , Citoesqueleto/metabolismo , Microtúbulos/metabolismo , Mitose , Matriz Extracelular , Fuso Acromático , Anáfase
7.
Elife ; 112022 11 08.
Artigo em Inglês | MEDLINE | ID: mdl-36346735

RESUMO

During cell division, the spindle generates force to move chromosomes. In mammals, microtubule bundles called kinetochore-fibers (k-fibers) attach to and segregate chromosomes. To do so, k-fibers must be robustly anchored to the dynamic spindle. We previously developed microneedle manipulation to mechanically challenge k-fiber anchorage, and observed spatially distinct response features revealing the presence of heterogeneous anchorage (Suresh et al., 2020). How anchorage is precisely spatially regulated, and what forces are necessary and sufficient to recapitulate the k-fiber's response to force remain unclear. Here, we develop a coarse-grained k-fiber model and combine with manipulation experiments to infer underlying anchorage using shape analysis. By systematically testing different anchorage schemes, we find that forces solely at k-fiber ends are sufficient to recapitulate unmanipulated k-fiber shapes, but not manipulated ones for which lateral anchorage over a 3 µm length scale near chromosomes is also essential. Such anchorage robustly preserves k-fiber orientation near chromosomes while allowing pivoting around poles. Anchorage over a shorter length scale cannot robustly restrict pivoting near chromosomes, while anchorage throughout the spindle obstructs pivoting at poles. Together, this work reveals how spatially regulated anchorage gives rise to spatially distinct mechanics in the mammalian spindle, which we propose are key for function.


Assuntos
Cinetocoros , Fuso Acromático , Animais , Fuso Acromático/fisiologia , Microtúbulos/fisiologia , Divisão Celular , Mamíferos , Mitose
8.
Results Probl Cell Differ ; 70: 191-220, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36348108

RESUMO

Chromosome organization is highly dynamic and plays an essential role during cell function. It was recently found that pairs of the homologous chromosomes are continuously separated at mitosis and display a haploid (1n) chromosome set, or "antipairing," organization in human cells. Here, we provide an introduction to the current knowledge of homologous antipairing in humans and its implications in human disease.


Assuntos
Cromossomos , Mitose , Humanos
9.
Open Biol ; 12(11): 220203, 2022 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-36321416

RESUMO

The spindle position checkpoint (SPOC) is a mitotic surveillance mechanism in Saccharomyces cerevisiae that prevents cells from completing mitosis in response to spindle misalignment, thereby contributing to genomic integrity. The kinase Kin4, one of the most downstream SPOC components, is essential to stop the mitotic exit network (MEN), a signalling pathway that promotes the exit from mitosis and cell division. Previous work, however, suggested that a Kin4-independent pathway contributes to SPOC, yet the underlying mechanisms remain elusive. Here, we established the glycogen-synthase-kinase-3 (GSK-3) homologue Mck1, as a novel component that works independently of Kin4 to engage SPOC. Our data indicate that both Kin4 and Mck1 work in parallel to counteract MEN activation by the Cdc14 early anaphase release (FEAR) network. We show that Mck1's function in SPOC is mediated by the pre-replication complex protein and mitotic cyclin-dependent kinase (M-Cdk) inhibitor, Cdc6, which is degraded in a Mck1-dependent manner prior to mitosis. Moderate overproduction of Cdc6 phenocopies MCK1 deletion and causes SPOC deficiency via its N-terminal, M-Cdk inhibitory domain. Our data uncover an unprecedented role of GSK-3 kinases in coordinating spindle orientation with cell cycle progression.


Assuntos
Proteínas de Saccharomyces cerevisiae , Saccharomycetales , Humanos , Proteínas de Saccharomyces cerevisiae/genética , Quinase 3 da Glicogênio Sintase/genética , Quinase 3 da Glicogênio Sintase/metabolismo , Fuso Acromático/metabolismo , Saccharomycetales/metabolismo , Proteínas Serina-Treonina Quinases , Fosforilação , Saccharomyces cerevisiae/genética , Mitose , Proteínas de Ciclo Celular/metabolismo , Proteínas Tirosina Fosfatases/genética , Proteínas Tirosina Fosfatases/metabolismo
10.
Biol Open ; 11(11)2022 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-36318115

RESUMO

During mitosis, spindle assembly relies on centrosomal and acentrosomal microtubule nucleation pathways that all require the γ-Tubulin Ring Complex (γ-TuRC) and its adaptor protein NEDD1. The activity of these different pathways needs to be coordinated to ensure bipolar spindle assembly ( Cavazza et al., 2016) but the underlying mechanism is still unclear. Previous studies have identified three sites in NEDD1 (S377, S405 and S411) that when phosphorylated drive MT nucleation at the centrosomes, around the chromosomes and on pre-existing MTs respectively ( Lüders et al., 2006; Pinyol et al., 2013; Sdelci et al., 2012). Here we aimed at getting additional insights into the mechanism that coordinates the different MT nucleation pathways in dividing cells using a collection of HeLa stable inducible cell lines expressing NEDD1 phospho-variants at these three sites and Xenopus egg extracts. Our results provide further support for the essential role of phosphorylation at the three residues. Moreover, we directly demonstrate that S411 phosphorylation is essential for MT branching using TIRF microscopy in Xenopus egg extracts and we show that it plays a crucial role in ensuring the balance between centrosome and chromosome-dependent MT nucleation required for bipolar spindle assembly in mitotic cells.


Assuntos
Proteínas Associadas aos Microtúbulos , Fuso Acromático , Humanos , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Mitose , Fosforilação , Fuso Acromático/metabolismo , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismo , Xenopus , Animais , Células HeLa
11.
Mol Cell Biol ; 42(11): e0021722, 2022 Nov 17.
Artigo em Inglês | MEDLINE | ID: mdl-36317925

RESUMO

Pre-mRNA splicing is an indispensable mechanism for eukaryotic gene expression. Splicing inhibition causes cell cycle arrest at the G1 and G2/M phases, and this is thought to be one of the reasons for the potent antitumor activity of splicing inhibitors. However, the molecular mechanisms underlying the cell cycle arrest have many unknown aspects. In particular, the mechanism of G2/M-phase arrest caused by splicing inhibition is completely unknown. Here, we found that lower and higher concentrations of pladienolide B caused M-phase and G2-phase arrest, respectively. We analyzed protein levels of cell cycle regulators and found that a truncated form of the p27 cyclin-dependent kinase inhibitor, named p27*, accumulated in G2-arrested cells. Overexpression of p27* caused partial G2-phase arrest. Conversely, knockdown of p27* accelerated exit from G2/M phase after washout of splicing inhibitor. These results suggest that p27* contributes to G2/M-phase arrest caused by splicing inhibition. We also found that p27* bound to and inhibited M-phase cyclins, although it is well known that p27 regulates the G1/S transition. Intriguingly, p27*, but not full-length p27, was resistant to proteasomal degradation and remained in G2/M phase. These results suggest that p27*, which is a very stable truncated protein in G2/M phase, contributes to G2-phase arrest caused by splicing inhibition.


Assuntos
Ciclinas , Precursores de RNA , Precursores de RNA/genética , Precursores de RNA/metabolismo , Inibidor de Quinase Dependente de Ciclina p27/genética , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Ciclinas/genética , Mitose , Quinases Ciclina-Dependentes/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/genética , Ciclo Celular , Proteínas de Ciclo Celular/metabolismo , Quinase 2 Dependente de Ciclina/genética
12.
Melanoma Res ; 32(6): 469-476, 2022 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-36317389

RESUMO

The 8th Edition of the American Joint Committee on Cancer (AJCC) Staging Manual removed the mitotic rate (MR) as a staging criterion for T1 melanomas, thus leading to a debate on sentinel lymph node biopsy (SLNB) in thin melanomas. This study investigates whether MR plays a role in selecting patients with T1 melanoma for SLNB. We analyzed clinical and histological data from the Florence Melanoma & Skin Cancer Unit database for 313 patients with a single thin melanoma who had undergone SLNB. We determined sentinel lymph node (SLN) positivity percentages in T1 melanomas according to the AJCC 8th Edition focusing on MR. Of the 313 T1 patients, 108 had MR = 0, 127 had MR = 1 and 78 had MR ≥2. The overall SLN positivity rate was 8.6%, (5.6% with MR = 0, 6.3% with MR = 1 and 16.7% with MR ≥2). The SLNB positivity rate in T1b melanomas was 12.1%, (8.5% with MR = 0, 5.7% with MR = 1 and 24.4% with MR ≥2), whereas in T1a melanomas it was 5.8%, (3.3% with MR = 0, 6.8% with MR = 1 and 8.1% with MR ≥2). In a logistic regression analysis, MR ≥2 had an odds ratio of almost three in comparison with MR = 0/1 also adjusting for thickness. Thus, MR ≥2 significantly predicted SLN metastases in T1 melanomas. Of those patients with positive SLN, 37% were classified as T1a according to the AJCC 8th edition. These findings underline the importance of MR ≥2 in selecting patients with T1 cutaneous melanomas for SLNB.


Assuntos
Melanoma , Neoplasias Cutâneas , Humanos , Melanoma/patologia , Biópsia de Linfonodo Sentinela , Neoplasias Cutâneas/patologia , Seleção de Pacientes , Estadiamento de Neoplasias , Mitose , Síndrome , Prognóstico , Estudos Retrospectivos
13.
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
14.
Sci Rep ; 12(1): 18184, 2022 Oct 28.
Artigo em Inglês | MEDLINE | ID: mdl-36307489

RESUMO

The quest for different natural compounds for different biomedical applications especially in the treatment of cancer is at a high pace with increasing incidence of severity. D-limonene has been portrayed as one of the effective potential candidate centered to the context of breast cancer. The anticipation of its count as an effective biomedical agent required a detailed understanding of their molecular mechanism of biocompatibility. This study elucidates the mechanistic action of D-limonene channelized by the induction of apoptosis for controlling proliferation in breast cancer cells. The possible mechanism was explored through an experimental and computational approach to estimate cell proliferation inhibition, cell cycle phase distribution, apoptosis analysis using a flow cytometry, western blotting and molecular docking. The results showed reduced dose and time-dependent viability of MCF7 cells. The study suggested the arrest of the cell cycle at G2/M phase leading to apoptosis and other discrepancies of molecular activity mediated via significant alteration in protein expression pattern of anti-apoptotic proteins like Cyclin B1 and CDK1. Computational analysis showed firm interaction of D-limonene with Cyclin B1 and CDK1 proteins influencing their structural and functional integrity indicating the mediation of mechanism. This study concluded that D-limonene suppresses the proliferation of breast cancer cells by inducing G2/M phase arrest via deregulation of Cyclin B1/CDK1.


Assuntos
Neoplasias da Mama , Proteína Quinase CDC2 , Humanos , Feminino , Ciclina B1/metabolismo , Limoneno/farmacologia , Simulação de Acoplamento Molecular , Linhagem Celular Tumoral , Proteína Quinase CDC2/metabolismo , Proliferação de Células , Apoptose , Mitose
15.
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
16.
Cancer Invest ; 40(10): 842-851, 2022 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-36200765

RESUMO

Ulceration and high mitosis are considered among the major unfavorable prognostic factors in the survival of cutaneous melanoma patients. The aim of this study was to investigate the clinical significance of these parameters and to compare them to see which one is superior to predicting prognosis across all clinical stages of melanoma. A total of 1,074 melanomas were analyzed retrospectively. Tumor ulceration was found to be limited to the local stage for predicting survival, whereas, mitosis maintained its prognostic strength for predicting survival across all clinical stages. Furthermore, no survival differences were observed between ulceration and mitosis across clinical stages.


Assuntos
Melanoma , Neoplasias Cutâneas , Humanos , Neoplasias Cutâneas/patologia , Estudos Retrospectivos , Prognóstico , Mitose
17.
BMC Biol ; 20(1): 240, 2022 10 24.
Artigo em Inglês | MEDLINE | ID: mdl-36280838

RESUMO

BACKGROUND: The centrosome is one of the most important non-membranous organelles regulating microtubule organization and progression of cell mitosis. The coiled-coil alpha-helical rod protein 1 (CCHCR1, also known as HCR) gene is considered to be a psoriasis susceptibility gene, and the protein is suggested to be localized to the P-bodies and centrosomes in mammalian cells. However, the exact cellular function of HCR and its potential regulatory role in the centrosomes remain unexplored. RESULTS: We found that HCR interacts directly with astrin, a key factor in centrosome maturation and mitosis. Immunoprecipitation assays showed that the coiled-coil region present in the C-terminus of HCR and astrin respectively mediated the interaction between them. Astrin not only recruits HCR to the centrosome, but also protects HCR from ubiquitin-proteasome-mediated degradation. In addition, depletion of either HCR or astrin significantly reduced centrosome localization of CEP72 and subsequent MCPH proteins, including CEP152, CDK5RAP2, and CEP63. The absence of HCR also caused centriole duplication defects and mitotic errors, resulting in multipolar spindle formation, genomic instability, and DNA damage. CONCLUSION: We conclude that HCR is localized and stabilized at the centrosome by directly binding to astrin. HCR are required for the centrosomal recruitment of MCPH proteins and centriolar duplication. Both HCR and astrin play key roles in keeping normal microtubule assembly and maintaining genomic stability.


Assuntos
Proteínas de Ciclo Celular , Centríolos , Animais , Centríolos/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Centrossomo/metabolismo , Mitose , Ubiquitinas/genética , Fuso Acromático/metabolismo , Mamíferos
19.
Curr Biol ; 32(20): R1025-R1027, 2022 Oct 24.
Artigo em Inglês | MEDLINE | ID: mdl-36283349

RESUMO

The dramatic cell-shape changes involved in mitosis and cell division challenge the integrity of epithelial tissues. A new study reveals a surprising role for atypical protein kinase C in keeping apical contractility in balance and thus preventing epithelial disruption.


Assuntos
Células Epiteliais , Mitose , Células Epiteliais/metabolismo , Epitélio/metabolismo , Forma Celular
20.
Genome Biol ; 23(1): 201, 2022 10 03.
Artigo em Inglês | MEDLINE | ID: mdl-36184650

RESUMO

BACKGROUND: During normal zygotic division, two haploid parental genomes replicate, unite and segregate into two biparental diploid blastomeres. RESULTS: Contrary to this fundamental biological tenet, we demonstrate here that parental genomes can segregate to distinct blastomeres during the zygotic division resulting in haploid or uniparental diploid and polyploid cells, a phenomenon coined heterogoneic division. By mapping the genomic landscape of 82 blastomeres from 25 bovine zygotes, we show that multipolar zygotic division is a tell-tale of whole-genome segregation errors. Based on the haplotypes and live-imaging of zygotic divisions, we demonstrate that various combinations of androgenetic, gynogenetic, diploid, and polyploid blastomeres arise via distinct parental genome segregation errors including the formation of additional paternal, private parental, or tripolar spindles, or by extrusion of paternal genomes. Hence, we provide evidence that private parental spindles, if failing to congress before anaphase, can lead to whole-genome segregation errors. In addition, anuclear blastomeres are common, indicating that cytokinesis can be uncoupled from karyokinesis. Dissociation of blastocyst-stage embryos further demonstrates that whole-genome segregation errors might lead to mixoploid or chimeric development in both human and cow. Yet, following multipolar zygotic division, fewer embryos reach the blastocyst stage and diploidization occurs frequently indicating that alternatively, blastomeres with genome-wide errors resulting from whole-genome segregation errors can be selected against or contribute to embryonic arrest. CONCLUSIONS: Heterogoneic zygotic division provides an overarching paradigm for the development of mixoploid and chimeric individuals and moles and can be an important cause of embryonic and fetal arrest following natural conception or IVF.


Assuntos
Blastômeros , Zigoto , Animais , Blastocisto , Bovinos , Feminino , Genoma , Humanos , Mitose
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