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1.
Nat Commun ; 13(1): 835, 2022 02 11.
Artigo em Inglês | MEDLINE | ID: mdl-35149709

RESUMO

The majority of high-grade serous ovarian cancers (HGSCs) are deficient in homologous recombination (HR) DNA repair, most commonly due to mutations or hypermethylation of the BRCA1/2 genes. We aimed to discover how BRCA1/2 mutations shape the cellular phenotypes and spatial interactions of the tumor microenvironment. Using a highly multiplex immunofluorescence and image analysis we generate spatial proteomic data for 21 markers in 124,623 single cells from 112 tumor cores originating from 31 tumors with BRCA1/2 mutation (BRCA1/2mut), and from 13 tumors without alterations in HR genes. We identify a phenotypically distinct tumor microenvironment in the BRCA1/2mut tumors with evidence of increased immunosurveillance. Importantly, we report a prognostic role of a proliferative tumor-cell subpopulation, which associates with enhanced spatial tumor-immune interactions by CD8+ and CD4 + T-cells in the BRCA1/2mut tumors. The single-cell spatial landscapes indicate distinct patterns of spatial immunosurveillance with the potential to improve immunotherapeutic strategies and patient stratification in HGSC.


Assuntos
Proteína BRCA1/metabolismo , Proteína BRCA2/metabolismo , Carcinoma Epitelial do Ovário/imunologia , Cistadenocarcinoma Seroso/imunologia , Neoplasias Ovarianas/imunologia , Microambiente Tumoral/imunologia , Proteína BRCA1/genética , Proteína BRCA2/genética , Linfócitos T CD4-Positivos , Linfócitos T CD8-Positivos , Carcinoma Epitelial do Ovário/genética , Cistadenocarcinoma Seroso/genética , Feminino , Genes BRCA1 , Genes BRCA2 , Genótipo , Recombinação Homóloga , Humanos , Mutação , Neoplasias Ovarianas/genética , Prognóstico , Proteômica
2.
Anal Chem ; 85(9): 4666-74, 2013 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-23607489

RESUMO

Protein kinases play critical roles in many biological and pathological processes, making them important targets for therapeutic drugs. Here, we desired to increase the throughput for kinome-wide profiling. A new workflow coupling ActivX ATP probe (AAP) affinity reagents with isotopic labeling to quantify the relative levels and modification states of kinases in cell lysates is described. We compared the new workflow to a classical proteomics approach in which fractionation was used to identify low-abundance kinases. We find that AAPs enriched approximately 90 kinases in a single analysis involving six cell lines or states in a single run, an 8-fold improvement in throughput relative to the classical approach. In general, AAPs cross-linked to both the active and inactive states of kinases but performing phosphopeptide enrichment made it possible to measure the phospho sites of regulatory residues lying in the kinase activation loops, providing information on activation state. When we compared the kinome across the six cell lines, representative of different breast cancer clinical subtypes, we observed that many kinases, particularly receptor tyrosine kinases, varied widely in abundance, perhaps explaining the differential sensitivities to kinase inhibitor drugs. The improved kinome profiling methods described here represent an effective means to perform systematic analysis of kinases involved in cell signaling and oncogenic transformation and for analyzing the effect of different inhibitory drugs.


Assuntos
Trifosfato de Adenosina/química , Sondas Moleculares/química , Proteínas Quinases/análise , Linhagem Celular Tumoral , Humanos , Células MCF-7 , Espectrometria de Massas , Proteínas Quinases/metabolismo
3.
Clin Pharmacol Ther ; 88(1): 34-8, 2010 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-20520606

RESUMO

Variability in patient responses to even the most potent and targeted therapeutics is now the primary challenge facing drug discovery and patient care, particularly in oncology and immune therapy. Variability with respect to mechanisms of induced resistance is observed both in drug-naive patients and among those who are initially responsive. Genomics has developed powerful tools for systematic interrogation of disease genotype and transcriptional states (particularly in cancer) and for correlation of these measures with parameters of disease such as histological diagnosis and outcome. In contrast, mechanistic preclinical studies remain relatively narrowly focused, leading to many apparent contradictions and poor understanding of the determinants of response. We describe the emergence of a systems pharmacology approach that is mechanistic, quantitative, probabilistic, and postgenomic and promises to do for mechanistic pharmacology what genomics is doing for correlative studies. We focus on studies in cell lines (which currently dominate mechanism-oriented analysis), but our arguments are equally valid for real tumors studied in short-term culture as xenografts and, perhaps some time in the future, in humans.


Assuntos
Antineoplásicos/uso terapêutico , Neoplasias/tratamento farmacológico , Farmacologia Clínica , Biologia de Sistemas , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/metabolismo , Sistemas de Liberação de Medicamentos , Meio Ambiente , Humanos
4.
Artigo em Inglês | MEDLINE | ID: mdl-21467141

RESUMO

Kinetochores are the elaborate protein assemblies that attach chromosomes to spindle microtubules in mitosis and meiosis. The kinetochores of point-centromere yeast appear to represent an elementary module, which repeats a number of times in kinetochores assembled on regional centromeres. Structural analyses of the discrete protein subcomplexes that make up the budding-yeast kinetochore have begun to reveal principles of kinetochore architecture and to uncover molecular mechanisms underlying functions such as transmission of tension and establishment and maintenance of bipolar attachment. The centromeric DNA is probably wrapped into a compact organization, not only by a conserved, centromeric nucleosome, but also by interactions among various other DNA-bound kinetochore components. The rod-like, heterotetrameric Ndc80 complex, roughly 600 Å long, appears to extend from the DNA-proximal assembly to the plus end of a microtubule, to which one end of the complex is known to bind. Ongoing structural studies will clarify the roles of a number of other well-defined complexes.


Assuntos
Cinetocoros/química , Cinetocoros/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Cinetocoros/ultraestrutura , Microtúbulos/metabolismo , Modelos Biológicos , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Complexos Multiproteicos/ultraestrutura , Ligação Proteica , Saccharomyces cerevisiae/ultraestrutura , Proteínas de Saccharomyces cerevisiae/ultraestrutura
5.
Mol Biol Cell ; 19(10): 4480-91, 2008 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-18701705

RESUMO

Mif2p is the budding-yeast orthologue of the mammalian centromere-binding protein CENP-C. We have mapped domains of Saccharomyces cerevisiae Mif2p and studied the phenotyptic consequences of their deletion. Using chromatin immunoprecipitation (ChIP) and electrophoretic mobility shift assays, we have further shown that Mif2p binds in the CDEIII region of the budding-yeast centromere, probably in close spatial association with Ndc10p. Moreover, ChIP experiments show that Mif2p recruits to yeast kinetochores a substantial subset of inner and outer kinetochore proteins, but not the Ndc80 or Spc105 complexes. We have determined the crystal structure of the C-terminal, dimerization domain of Mif2p. It has a "cupin" fold, extremely similar both in polypeptide chain conformation and in dimer geometry to the dimerization domain of a bacterial transcription factor. The Mif2p dimer seems to be part of an enhanceosome-like structure that nucleates kinetochore assembly in budding yeast.


Assuntos
Proteínas de Ligação a DNA/química , Regulação Fúngica da Expressão Gênica , Cinetocoros/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/fisiologia , Saccharomyces cerevisiae/metabolismo , Sequência de Aminoácidos , Cristalografia por Raios X/métodos , Proteínas de Ligação a DNA/fisiologia , Dimerização , Cinetocoros/química , Dados de Sequência Molecular , Fenótipo , Ligação Proteica , Estrutura Terciária de Proteína , Saccharomycetales , Homologia de Sequência de Aminoácidos
6.
Oncogene ; 27(8): 1142-54, 2008 Feb 14.
Artigo em Inglês | MEDLINE | ID: mdl-17700522

RESUMO

In the absence of mitogenic stimuli, cells normally arrest in G(1/0), because they fail to pass the G1-restriction point. However, abrogation of the G1-restriction point (by loss of the retinoblastoma gene family) reveals a second-restriction point that arrests cells in G2. Serum-starvation-induced G2 arrest is effectuated through inhibitory interactions of p27(KIP1) and p21(CIP1) with cyclins A and B1 and can be reversed through mitogen re-addition. In this study, we have investigated the pathways that allow cell cycle re-entry from this G2 arrest. We provide evidence that recovery from G2 arrest depends on the rat sarcoma viral oncogene (RAS) and phosphatidylinositol-3 kinase pathways and show that oncogenic hits, such as overexpression of c-MYC or mutational activation of RAS can abrogate the G2-restriction point. Together, our results provide new mechanistic insight into multistep carcinogenesis.


Assuntos
Fase G2/fisiologia , Oncogenes/fisiologia , Transdução de Sinais/fisiologia , Animais , Linhagem Celular , Transformação Celular Neoplásica/genética , Perfilação da Expressão Gênica , Genes ras/fisiologia , Camundongos , Família Multigênica/fisiologia , Fosfatidilinositol 3-Quinases/fisiologia , Proteínas ras/fisiologia
7.
Bioinformatics ; 23(13): 1666-73, 2007 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-17483508

RESUMO

MOTIVATION: Kinetochores are multiprotein complexes which mediate chromosome attachment to microtubules (MTs) of the mitotic spindle. They regulate MT dynamics during chromosome segregation. Our goal is to identify groups of kinetochore proteins with similar effects on MT dynamics, revealing pathways through which kinetochore proteins transform chemical and mechanical input signals into cues of MT regulation. RESULTS: We have developed a hierarchical, agglomerative clustering algorithm that groups Saccharomyces cerevisiae strains based on MT-mediated chromosome dynamics measured by high-resolution live cell microscopy. Clustering is based on parameters of autoregressive moving average (ARMA) models of the probed dynamics. We have found that the regulation of wildtype MT dynamics varies with cell cycle and temperature, but not with the chromosome an MT is attached to. By clustering the dynamics of mutants, we discovered that the three genes IPL1, DAM1 and KIP3 co-regulate MT dynamics. Our study establishes the clustering of chromosome and MT dynamics by ARMA descriptors as a sensitive framework for the systematic identification of kinetochore protein subcomplexes and pathways for the regulation of MT dynamics. AVAILABILITY: The clustering code, written in Matlab, can be downloaded from http://lccb.scripps.edu. ('download' hyperlink at bottom of website). SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.


Assuntos
Cinetocoros/fisiologia , Microtúbulos/fisiologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/classificação , Saccharomyces cerevisiae/fisiologia , Algoritmos , Análise por Conglomerados , Cinética , Modelos Biológicos , Mutação , Fenótipo , Saccharomyces cerevisiae/ultraestrutura , Proteínas de Saccharomyces cerevisiae/genética , Temperatura
8.
Syst Biol (Stevenage) ; 153(6): 425-32, 2006 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-17186704

RESUMO

Computational models aid in the quantitative understanding of cell signalling networks. One important goal is to ascertain how multiple network components work together to govern cellular responses, that is, to determine cell 'signal-response' relationships. Several methods exist to study steady-state signals in the context of differential equation-based models. However, many biological networks influence cell behaviour through time-varying signals operating during a transient activated state that ultimately returns to a basal steady-state. A computational approach adapted from dynamical systems analysis to discern how diverse transient signals relate to alternative cell fates is described. Direct finite-time Lyapunov exponents (DLEs) are employed to identify phase-space domains of high sensitivity to initial conditions. These domains delineate regions exhibiting qualitatively different transient activities that would be indistinguishable using steady-state analysis but which correspond to different outcomes. These methods are applied to a physicochemical model of molecular interactions among caspase-3, caspase-8 and X-linked inhibitor of apoptosis--proteins whose transient activation determines cell death against survival fates. DLE analysis enabled identification of a separatrix that quantitatively characterises network behaviour by defining initial conditions leading to apoptotic cell death. It is anticipated that DLE analysis will facilitate theoretical investigation of phenotypic outcomes in larger models of signalling networks.


Assuntos
Algoritmos , Proteínas Reguladoras de Apoptose/metabolismo , Apoptose/fisiologia , Caspase 3/metabolismo , Caspase 8/metabolismo , Fenômenos Fisiológicos Celulares , Modelos Biológicos , Simulação por Computador , Cinética , Fatores de Tempo
9.
EMBO J ; 25(12): 2814-27, 2006 Jun 21.
Artigo em Inglês | MEDLINE | ID: mdl-16763565

RESUMO

The correct formation of stable but dynamic links between chromosomes and spindle microtubules (MTs) is essential for accurate chromosome segregation. However, the molecular mechanisms by which kinetochores bind MTs and checkpoints monitor this binding remain poorly understood. In this paper, we analyze the functions of six kinetochore-bound MT-associated proteins (kMAPs) using RNAi, live-cell microscopy and quantitative image analysis. We find that RNAi-mediated depletion of two kMAPs, the adenomatous polyposis coli protein (APC) and its binding partner, EB1, are unusual in affecting the movement and orientation of paired sister chromatids at the metaphase plate without perturbing kinetochore-MT attachment per se. Quantitative analysis shows that misorientation phenotypes in metaphase are uniform across chromatid pairs even though chromosomal loss (CIN) during anaphase is sporadic. However, errors in kinetochore function generated by APC or EB1 depletion are detected poorly if at all by the spindle checkpoint, even though they cause chromosome missegregation. We propose that impaired EB1 or APC function generates lesions invisible to the spindle checkpoint and thereby promotes low levels of CIN expected to fuel aneuploidy and possibly tumorigenesis.


Assuntos
Proteína da Polipose Adenomatosa do Colo/deficiência , Segregação de Cromossomos , Cromossomos Humanos/metabolismo , Cinetocoros/metabolismo , Proteínas Associadas aos Microtúbulos/deficiência , Genes Dominantes/genética , Células HeLa , Humanos , Metáfase , Proteínas Associadas aos Microtúbulos/química , Modelos Biológicos , Mutação/genética , Não Disjunção Genética , Fenótipo
10.
Biophys J ; 89(4): 2835-54, 2005 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-16192284

RESUMO

We have probed single kinetochore microtubule (k-MT) dynamics in budding yeast in the G1 phase of the cell cycle by automated tracking of a green fluorescent protein tag placed proximal to the centromere on chromosome IV and of a green fluorescent protein tag fused to the spindle pole body protein Spc42p. Our method reliably distinguishes between different dynamics in wild-type and mutant strains and under different experimental conditions. Using our methods we established that in budding yeast, unlike in metazoans, chromosomes make dynamic attachments to microtubules in G1. This makes it possible to interpret measurements of centromere tag dynamics as reflecting k-MT dynamics. We have examined the sensitivity of our assay by studying the effect of temperature, exposure to benomyl, and a tubulin mutation on k-MT dynamics. We have found that lowering the temperature and exposing cells to benomyl attenuate k-MT dynamics in a similar manner. We further observe that, in contrast to previous reports, the mutant tub2-150 forms k-MTs that depolymerize faster than wild type. Based on these findings, we propose high-resolution light microscopy of centromere dynamics in G1 yeast cells as a sensitive assay for the regulation of single k-MT dynamics.


Assuntos
Aumento da Imagem/métodos , Interpretação de Imagem Assistida por Computador/métodos , Imageamento Tridimensional/métodos , Cinetocoros/metabolismo , Microscopia de Fluorescência/métodos , Microtúbulos/metabolismo , Saccharomycetales/metabolismo , Algoritmos , Cinética , Cinetocoros/ultraestrutura , Microtúbulos/ultraestrutura , Transporte Proteico/fisiologia , Saccharomycetales/citologia
11.
J Microsc ; 211(Pt 3): 230-48, 2003 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-12950472

RESUMO

We present an algorithm for the three-dimensional (3D) tracking of multiple fluorescent subresolution tags with super-resolution in images of living cells. Recently, we described an algorithm for the automatic detection of such tags in single frames and demonstrated its potential in a biological system. The algorithm presented here adds to the tag detector a module for relative tracking of the signals between frames. As with tag detection, the main problem in relative tracking arises when signals of multiple tags interfere. We propose a novel multitemplate matching framework that exploits knowledge of the microscope point spread function to separate the intensity contribution of each tag in image regions with signal interferences. We use this intensity splitting to reconstruct a template for each tag in the source frame and a patch in the target frame, which are both free of intensity contributions from other tag signals. Tag movements between frames are then tracked by seeking, for each template-patch pair, the displacement vector providing the best signal match in terms of the sum of squared intensity differences. Because template and patch generation of tags with overlapping signals are interdependent, the matching is carried out simultaneously for all tags, and in an iterative manner. We have examined the performance of our approach using synthetic 3D data and observed a significant increase in resolution and robustness as compared with our previously described detector. It is now possible to localize and track tags separated by a distance three times smaller than the Rayleigh limit with a relative positional accuracy of better than 50 nm. We have applied the new tracking system to extract metaphase trajectories of fluorescently tagged chromosomes relative to the spindle poles in budding yeast.


Assuntos
Algoritmos , Cromossomos Fúngicos/metabolismo , Proteínas Luminescentes/metabolismo , Fuso Acromático/metabolismo , Centrômero/genética , Cromossomos Fúngicos/genética , Proteínas de Fluorescência Verde , Processamento de Imagem Assistida por Computador , Imageamento Tridimensional , Proteínas Luminescentes/genética , Microscopia/métodos , Fuso Acromático/genética , Leveduras/genética , Leveduras/metabolismo
12.
J Microsc ; 208(Pt 1): 49-64, 2002 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-12366597

RESUMO

In this paper, we describe an algorithmic framework for the automatic detection of diffraction-limited fluorescent spots in 3D optical images at a separation below the Rayleigh limit, i.e. with super-resolution. We demonstrate the potential of super-resolution detection by tracking fluorescently tagged chromosomes during mitosis in budding yeast. Our biological objective is to identify and analyse the proteins responsible for the generation of tensile force during chromosome segregation. Dynamic measurements in living cells are made possible by green fluorescent protein (GFP)-tagging chromosomes and spindle pole bodies to generate cells carrying four fluorescent spots, and observe the motion of the spots over time using 3D-fluorescence microscopy. The central problem in spot detection arises with the partial or complete overlap of spots when tagged objects are separated by distances below the resolution of the optics. To detect multiple spots under these conditions, a set of candidate mixture models is built, and the best candidate is selected from the set based on chi2-statistics of the residuals in least-square fits of the models to the image data. Even with images having a signal-to-noise ratio (SNR) as low as 5-10, we are able to increase the resolution two-fold below the Rayleigh limit. In images with a SNR of 5-10, the accuracy with which isolated tags can be localized is less than 5 nm. For two tags separated by less than the Rayleigh limit, the localization accuracy is found to be between 10 and 20 nm, depending on the effective point-to-point distance. This indicates the intimate relationship between resolution and localization accuracy.


Assuntos
Algoritmos , Cromossomos Fúngicos/fisiologia , Imageamento Tridimensional/métodos , Microscopia de Fluorescência/métodos , Leveduras/citologia , Corantes Fluorescentes/análise , Proteínas de Fluorescência Verde , Proteínas Luminescentes , Fuso Acromático
13.
Dev Cell ; 1(2): 162-4, 2001 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-11702776

RESUMO

Most current models of spindle assembly checkpoint signaling involve inhibition of the Cdc20-APC by Mad2 protein. Interestingly, a paper from Hongtao Yu and colleagues in this issue of Developmental Cell suggests that the Cdc20/APC can also be inhibited in a Mad2-independent manner by a complex of proteins that includes BubR1.


Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Transporte , Proteínas de Ciclo Celular/metabolismo , Ciclo Celular , Proteínas Fúngicas/metabolismo , Cinetocoros/fisiologia , Proteínas Quinases/metabolismo , Proteínas de Saccharomyces cerevisiae , Animais , Proteínas Cdc20 , Humanos , Proteínas Mad2 , Modelos Biológicos , Proteínas Nucleares , Ligação Proteica , Proteínas Serina-Treonina Quinases , Saccharomyces cerevisiae/fisiologia , Transdução de Sinais
14.
Cell ; 106(2): 195-206, 2001 Jul 27.
Artigo em Inglês | MEDLINE | ID: mdl-11511347

RESUMO

The complex series of movements that mediates chromosome segregation during mitosis is dependent on the attachment of microtubules to kinetochores, DNA-protein complexes that assemble on centromeric DNA. We describe the use of live-cell imaging and chromatin immunoprecipitation in S. cerevisiae to identify ten kinetochore subunits, among which are yeast homologs of microtubule binding proteins in animal cells. By analyzing conditional mutations in several of these proteins, we show that they are required for the imposition of tension on paired sister kinetochores and for correct chromosome movement. The proteins include both molecular motors and microtubule associated proteins (MAPs), implying that motors and MAPs function together in binding chromosomes to spindle microtubules.


Assuntos
Cromossomos Fúngicos/metabolismo , Cinetocoros/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/metabolismo , Transporte Biológico , Núcleo Celular/metabolismo , Cromátides/química , Cromátides/genética , Cromátides/metabolismo , Cromatina/química , Cromatina/genética , Cromatina/metabolismo , Segregação de Cromossomos , Cromossomos Fúngicos/química , Cromossomos Fúngicos/genética , DNA Fúngico/genética , DNA Fúngico/metabolismo , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Cinetocoros/química , Proteínas Associadas aos Microtúbulos/química , Proteínas Associadas aos Microtúbulos/genética , Microtúbulos/química , Proteínas Motores Moleculares/química , Proteínas Motores Moleculares/genética , Proteínas Motores Moleculares/metabolismo , Mutação/genética , Testes de Precipitina , Ligação Proteica , Saccharomyces cerevisiae/genética , Fuso Acromático/química , Fuso Acromático/metabolismo
15.
Proc Natl Acad Sci U S A ; 98(16): 8944-9, 2001 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-11481466

RESUMO

DNA microarrays represent an important new method for determining the complete expression profile of a cell. In "spotted" microarrays, slides carrying spots of target DNA are hybridized to fluorescently labeled cDNA from experimental and control cells and the arrays are imaged at two or more wavelengths. In this paper, we perform statistical analysis on images of microarrays and show that quantitating the amount of fluorescent DNA bound to microarrays is subject to considerable uncertainty because of large and small-scale intensity fluctuations within spots, nonadditive background, and fabrication artifacts. Pixel-by-pixel analysis of individual spots can be used to estimate these sources of error and establish the precision and accuracy with which gene expression ratios are determined. Simple weighting schemes based on these estimates are effective in improving significantly the quality of microarray data as it accumulates in a multiexperiment database. We propose that error estimates from image-based metrics should be one component in an explicitly probabilistic scheme for the analysis of DNA microarray data.


Assuntos
Processamento de Imagem Assistida por Computador , Análise de Sequência com Séries de Oligonucleotídeos , DNA Complementar , Corantes Fluorescentes , Perfilação da Expressão Gênica , Hibridização de Ácido Nucleico
16.
Nat Cell Biol ; 3(4): 429-32, 2001 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-11283619

RESUMO

Mutations in the Adenomatous Polyposis Coli (APC) gene are responsible for familial colon cancer and also occur in the early stages of sporadic colon cancer. APC functions in the Wnt signalling pathway to regulate the degradation of beta-catenin (reviewed in refs 1-3). APC also binds to and stabilizes microtubules in vivo and in vitro, localizes to clusters at the ends of microtubules near the plasma membrane of interphase cells, and is an important regulator of cytoskeletal function. Here we show that cells carrying a truncated APC gene (Min) are defective in chromosome segregation. Moreover, during mitosis, APC localizes to the ends of microtubules embedded in kinetochores and forms a complex with the checkpoint proteins Bub1 and Bub3. In vitro, APC is a high-affinity substrate for Bub kinases. Our data are consistent with a role for APC in kinetochore-microtubule attachment and suggest that truncations in APC that eliminate microtubule binding may contribute to chromosomal instability in cancer cells.


Assuntos
Proteínas de Ciclo Celular , Segregação de Cromossomos , Proteínas do Citoesqueleto/fisiologia , Proteínas de Neoplasias/fisiologia , Proteína da Polipose Adenomatosa do Colo , Animais , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Linhagem Celular , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Quinase 3 da Glicogênio Sintase , Células HT29 , Células HeLa , Humanos , Cinetocoros/metabolismo , Microtúbulos/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases , Proteínas/genética , Proteínas/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Spodoptera
17.
Nature ; 409(6818): 355-9, 2001 Jan 18.
Artigo em Inglês | MEDLINE | ID: mdl-11201745

RESUMO

The mitotic checkpoint protein hsMad2 is required to arrest cells in mitosis when chromosomes are unattached to the mitotic spindle. The presence of a single, lagging chromosome is sufficient to activate the checkpoint, producing a delay at the metaphase-anaphase transition until the last spindle attachment is made. Complete loss of the mitotic checkpoint results in embryonic lethality owing to chromosome mis-segregation in various organisms. Whether partial loss of checkpoint control leads to more subtle rates of chromosome instability compatible with cell viability remains unknown. Here we report that deletion of one MAD2 allele results in a defective mitotic checkpoint in both human cancer cells and murine primary embryonic fibroblasts. Checkpoint-defective cells show premature sister-chromatid separation in the presence of spindle inhibitors and an elevated rate of chromosome mis-segregation events in the absence of these agents. Furthermore, Mad2+/- mice develop lung tumours at high rates after long latencies, implicating defects in the mitotic checkpoint in tumorigenesis.


Assuntos
Anáfase , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Transporte , Aberrações Cromossômicas , Proteínas Fúngicas/metabolismo , Genes cdc , Neoplasias Pulmonares/genética , Animais , Antineoplásicos/farmacologia , Proteínas de Ligação ao Cálcio/antagonistas & inibidores , Proteínas de Ciclo Celular , Células Cultivadas , Segregação de Cromossomos , Proteínas Fúngicas/antagonistas & inibidores , Deleção de Genes , Humanos , Cariotipagem , Proteínas Mad2 , Camundongos , Mitose/genética , Mitose/fisiologia , Nocodazol/farmacologia , Proteínas Nucleares , Células Tumorais Cultivadas
18.
Cell ; 101(6): 635-45, 2000 Jun 09.
Artigo em Inglês | MEDLINE | ID: mdl-10892650

RESUMO

The initiation of chromosome segregation at anaphase is linked by the spindle assembly checkpoint to the completion of chromosome-microtubule attachment during metaphase. To determine the function of the mitotic checkpoint protein Mad2 during normal cell division and when mitosis goes awry, we have knocked out Mad2 in mice. We find that E5.5 embryonic cells lacking Mad2, like mad2 yeast, grow normally but are unable to arrest in response to spindle disruption. At E6.5, the cells of the epiblast begin rapid cell division and the absence of a checkpoint results in widespread chromosome missegregation and apoptosis. In contrast, the postmitotic trophoblast giant cells survive without Mad2. Thus, the spindle assembly checkpoint is required for accurate chromosome segregation in mitotic mouse cells, and for embryonic viability, even in the absence of spindle damage.


Assuntos
Apoptose/genética , Proteínas de Ligação ao Cálcio/genética , Proteínas de Transporte , Segregação de Cromossomos , Proteínas Fúngicas/genética , Sequência de Aminoácidos , Animais , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Ciclo Celular , Proteínas Fúngicas/metabolismo , Regulação da Expressão Gênica , Proteínas Mad2 , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Proteínas Nucleares , Homologia de Sequência de Aminoácidos
19.
Cell ; 101(7): 763-75, 2000 Jun 23.
Artigo em Inglês | MEDLINE | ID: mdl-10892747

RESUMO

The accurate segregation of chromosomes at mitosis requires that all pairs of chromatids bind correctly to microtubules prior to the dissolution of sister cohesion and the initiation of anaphase. By analyzing the motion of GFP-tagged S. cerevisiae chromosomes, we show that kinetochore-microtubule attachments impose sufficient tension on sisters during prometaphase to transiently separate centromeric chromatin toward opposite sides of the spindle. Transient separations of 2-10 min duration occur in the absence of cohesin proteolysis, are characterized by independent motion of the sisters along the spindle, and are followed by the apparent reestablishment of sister linkages. The existence of transient sister separation in yeast explains the unusual bilobed localization of kinetochore proteins and supports an alternative model for spindle structure. By analogy with animal cells, we propose that yeast centromeric chromatin acts as a tensiometer.


Assuntos
Cromátides , Cromossomos , Mitose , Segregação de Cromossomos , Cromossomos/química , Cromossomos/ultraestrutura , Elasticidade , Saccharomyces cerevisiae
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