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/metabolismoRESUMO
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 , ProteômicaRESUMO
The mitotic spindle is a self-organizing structure that is constructed primarily from microtubules. Among the most important spindle microtubules are those that bind to kinetochores and form the fibers along which chromosomes move. Chemotherapeutics such as taxol and the vinca alkaloids perturb kinetochore-microtubule attachment and disrupt chromosome segregation. This activates a checkpoint pathway that delays cell cycle progression and induces programmed cell death. Recent work has identified at least four mammalian spindle assembly checkpoint proteins.
Assuntos
Apoptose/fisiologia , Divisão Celular/fisiologia , Microtúbulos/fisiologia , Aneuploidia , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Divisão Celular/efeitos dos fármacos , Divisão Celular/genética , Microtúbulos/efeitos dos fármacos , Modelos Biológicos , Fuso Acromático/fisiologiaRESUMO
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 , SpodopteraRESUMO
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 SinaisRESUMO
Kinetochores are structures that assemble on centromeric DNA and mediate the attachment of chromosomes to the microtubules of the mitotic spindle. The protein components of kinetochores are poorly understood, but the simplicity of the S. cerevisiae kinetochore makes it an attractive candidate for molecular dissection. Mutations in genes encoding CBF1 and CBF3, proteins that bind to yeast centromeres, interfere with chromosome segregation in vivo. To determine the roles played by these factors and by various regions of centromeric DNA in kinetochore function, we have developed a method to partially reassemble kinetochores on exogenous centromeric templates in vitro and to visualize the attachment of these reassembled kinetochore complexes to microtubules. In this assay, single reassembled complexes appear to mediate microtubule binding. We find that CBF3 is absolutely essential for this attachment but, contrary to previous reports (Hyman, A. A., K. Middleton, M. Centola, T.J. Mitchison, and J. Carbon. 1992. Microtubule-motor activity of a yeast centromere-binding protein complex. Nature (Lond.). 359:533-536) is not sufficient. Additional cellular factors interact with CBF3 to form active microtubule-binding complexes. This is mediated primarily by the CDEIII region of centromeric DNA but CDEII plays an essential modulatory role. Thus, the attachment of kinetochores to microtubules appears to involve a hierarchy of interactions by factors that assemble on a core complex consisting of DNA-bound CBF3.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas Fúngicas/metabolismo , Cinetocoros/metabolismo , Microtúbulos/metabolismo , Proteínas Nucleares , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Sequência de Bases , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos , Centrômero/metabolismo , Primers do DNA , DNA Fúngico/metabolismo , Proteínas de Ligação a DNA/isolamento & purificação , Proteínas Fúngicas/isolamento & purificação , Cinética , Dados de Sequência Molecular , Mutagênese , Reação em Cadeia da Polimerase , Ligação Proteica , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismoRESUMO
In budding yeast, accurate chromosome segregation requires that one and only one kinetochore assemble per chromosome. In this paper, we report the use of DNA-protein crosslinking and nondenaturing gel analysis to study the structure of CBF3, a four-protein complex that binds to the essential CDEIII region of Saccharomyces cerevisiae centromeres. We find that three subunits of CBF3 are in direct contact with CDEIII over a region of DNA that spans 80 bp. A highly asymmetric core complex containing p58(CTF13) p64(CEP3) and p110(NDC10) in direct contact with DNA forms at the genetically defined center of CDEIII. This core complex spans approximately 56 bp of CEN3. An extended complex comprising the core complex and additional DNA-bound p110(NDC10) also forms. It spans approximately 80 bp of DNA. CBF3 makes sequence-specific and -nonspecific contacts with DNA. Both contribute significantly to the energy of CBF3-DNA interaction. Moreover, important sequence-specific contacts are made with bases that are not conserved among yeast centromeres. These findings provide a foundation for understanding the organization of the CBF3-centromere complex, a structure that appears to initiate the formation of microtubule attachment sites at yeast kinetochores. These results also have implications for understanding centromere-binding proteins in higher cells.
Assuntos
Cromossomos Fúngicos/química , DNA Fúngico/química , Proteínas de Ligação a DNA/química , Proteínas Fúngicas/química , Cinetocoros/química , Proteínas Nucleares , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/fisiologia , Sequência de Bases , Sítios de Ligação , Cromossomos Fúngicos/ultraestrutura , Reagentes de Ligações Cruzadas , Cinetocoros/ultraestrutura , Substâncias Macromoleculares , Modelos Moleculares , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Ligação ProteicaRESUMO
Kinetochores are smaller and more accessible experimentally in budding yeast than in any other eukaryote. Believing that simple and complex kinetochores have important structural and functional properties in common, we characterized the structure of CBF3, the essential centromere-binding complex that initiates kinetochore formation in Saccharomyces cerevisiae. We find that the four subunits of CBF3 are multimeric in solution: p23(Skp1) and p58(Ctf13) form a heterodimer, and p64(Cep3) and p110(Ndc10) form homodimers. Subcomplexes involving p58 and each of the other CBF3 subunits can assemble in the absence of centromeric DNA. In these subcomplexes, p58 appears to function as a structural core mediating stable interactions among other CBF3 proteins. p58 has a short half-life in yeast, being subject to ubiquitin-dependent proteolysis, but we find that it is much more stable following association with p64. We propose that p23(Skp1)-p58-p64 complexes constitute the primary pool of active p58 in yeast cells. These complexes can either dissociate, reexposing p58 to the degradation pathway, or can bind to p110 and centromeric DNA, forming a functional CBF3 complex in which p58 is fully protected from degradation. This pathway may constitute an editing mechanism preventing the formation of ectopic kinetochores and ensuring the fidelity of chromosome segregation.
Assuntos
Proteínas de Ligação a DNA/química , Proteínas Fúngicas/química , Cinetocoros/química , Proteínas Nucleares/química , Proteínas de Saccharomyces cerevisiae , Sequência de Aminoácidos , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Dimerização , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Cinetocoros/metabolismo , Dados de Sequência Molecular , Mapeamento de Peptídeos , Saccharomyces cerevisiaeRESUMO
Clathrin triskelions form polyhedral cages with hexagonal and pentagonal faces when dialyzed against suitable assembly buffers. However, when the buffer is made 12% saturated in ammonium sulfate and the dialysis is performed at 4 degrees C, clathrin polymerizes into cubes. The cube is constructed from eight triskelions with one at each corner. The edge length of the cube is approximately 45 nm, equivalent to the length of the leg of a triskelion. Thus, each edge of the cube is composed of two antiparallel legs overlapping over their whole length. The interactions between the legs in the cube are a subset of those postulated to occur in cages. Indeed, the cube can be derived from a pentagonal dodecahedron by removing 12 of the 20 triskelions with only slight adjustment of the legs of the remaining triskelions. The cube forms regular arrays and appears to be a favorable species for crystallization of clathrin.
Assuntos
Clatrina/isolamento & purificação , Cristalização , Microscopia Eletrônica , Conformação ProteicaRESUMO
Genetic selections were used to find peptides that inhibit biological pathways in budding yeast. The peptides were presented inside cells as peptamers, surface loops on a highly expressed and biologically inert carrier protein, a catalytically inactive derivative of staphylococcal nuclease. Peptamers that inhibited the pheromone signaling pathway, transcriptional silencing, and the spindle checkpoint were isolated. Putative targets for the inhibitors were identified by a combination of two-hybrid analysis and genetic dissection of the target pathways. This analysis identified Ydr517w as a component of the spindle checkpoint and reinforced earlier indications that Ste50 has both positive and negative roles in pheromone signaling. Analysis of transcript arrays showed that the peptamers were highly specific in their effects, which suggests that they may be useful reagents in organisms that lack sophisticated genetics as well as for identifying components of existing biological pathways that are potential targets for drug discovery.
Assuntos
Peptídeos/farmacologia , Feromônios/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Seleção Genética , Transdução de Sinais , Fuso Acromático/metabolismo , Sequência de Aminoácidos , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Proteínas Fúngicas/metabolismo , Fase G1 , Galactose/metabolismo , Lipoproteínas/metabolismo , Fator de Acasalamento , Nuclease do Micrococo , Mitose , Dados de Sequência Molecular , Biblioteca de Peptídeos , Peptídeos/genética , Peptídeos/metabolismo , Ligação Proteica , Proteínas Serina-Treonina Quinases , Proteínas Tirosina Quinases , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Fuso Acromático/efeitos dos fármacos , Transcrição GênicaRESUMO
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 , TemperaturaRESUMO
The equal division of chromosomes among daughter cells at mitosis involves a complex series of kinetochore-dependent chromosome movements. The kinetochore-associated CENP-E motor protein is critical for the sustained movement of chromosomes towards the metaphase plate during chromosome congression.
Assuntos
Cromossomos/fisiologia , Cinetocoros/fisiologia , Animais , Proteínas Cromossômicas não Histona/fisiologia , Microtúbulos/fisiologia , Mitose/fisiologia , Modelos Biológicos , Movimento/fisiologia , Fuso Acromático/fisiologiaRESUMO
We report the sequence of a cDNA clone that encodes the C-terminal half of the hamster 94 X 10(3) Mr glucose-regulated protein, grp94. The amino acid sequence of this protein is about 50% homologous to Drosophila hsp83 and yeast hsp90, suggesting that grp94 and hsp90 have similar functional properties. Unlike hsp90, grp94 is associated with the endoplasmic reticulum. It has the same C-terminal tetrapeptide as two other luminal endoplasmic reticulum proteins, grp78 and protein disulphide isomerase. We suggest that this sequence forms part of a signal for retention of proteins in the lumen of the endoplasmic reticulum.
Assuntos
Proteínas de Choque Térmico HSP70 , Proteínas de Choque Térmico , Proteínas de Membrana , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Cricetinae , DNA , Proteínas de Choque Térmico/genética , Proteínas de Membrana/genética , Homologia de Sequência do Ácido NucleicoRESUMO
Dissociation of turnip crinkle virus (TCV) at elevated pH and ionic strength produces free dimers of the coat protein and a ribonucleoprotein complex that contains the viral RNA, six coat-protein subunits, and the minor protein species, p80 (a covalently linked coat-protein dimer). This "rp-complex" is stable for several days in high salt at pH 8.5. Reassembly of TCV can be accomplished under physiological conditions, using isolated coat protein and either rp-complex or protein-free RNA. If rp-complex is used in reassembly, the same subunits remain bound to RNA on subsequent dissociation; if free RNA is used, rp-complex is regenerated. In both cases, the assembly is selective for viral RNA in competition experiments with heterologous RNA. Electron microscopy shows that assembly proceeds by continuous growth of a shell from an initiating structure, rather than by formation of distinct intermediates. We suggest that rp-complex is the initiating structure, suggest a model based on the organization of the TCV particle, and propose a mechanism for TCV assembly.
Assuntos
Vírus de Plantas/crescimento & desenvolvimento , Vírus de RNA/crescimento & desenvolvimento , Capsídeo , Centrifugação com Gradiente de Concentração , Concentração de Íons de Hidrogênio , Técnicas In Vitro , Microscopia Eletrônica , Modelos Biológicos , Vírus de Plantas/ultraestrutura , Vírus de RNA/ultraestrutura , RNA Viral/metabolismoRESUMO
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 , HumanosRESUMO
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/ultraestruturaRESUMO
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/fisiologiaRESUMO
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 AminoácidosRESUMO
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 , FenótipoRESUMO
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.