RESUMO
Cadherins play important roles in cell-cell adhesion during tissue differentiation. Cadherins are linked to the actin cytoskeleton by catenins (beta-catenin/armadillo, plakoglobin, and alpha-catenin). Recent results show that beta-catenin also binds to another cytoskeletal complex containing the adenomatous polyposis coli protein and microtubules, and interacts with several signaling pathways that include tyrosine kinases and phosphatases and Wnt/Wingless. Interplay between these cytoskeletal complexes and signaling pathways may regulate morphogenesis.
Assuntos
Caderinas/fisiologia , Proteínas do Citoesqueleto/metabolismo , Citoesqueleto/metabolismo , Proteínas de Drosophila , Transdução de Sinais/fisiologia , Animais , Proteínas Proto-Oncogênicas/fisiologia , Proteína Wnt1RESUMO
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
Truncation mutations in the adenomatous polyposis coli protein (APC) are responsible for familial and sporadic colonic tumours. APC is best known for its role in regulating beta-catenin, an important mediator of cell adhesion and a transcriptional activator. However, recent studies indicate that APC has additional roles in cytoskeletal regulation. It binds to microtubules directly and indirectly. Furthermore, indirect connections between APC and the actin cytoskeleton have also been described. Here, we integrate recent information describing the association between APC and the cytoskeleton to illustrate how this multifaceted protein might link different cytoskeletal elements to each other and to cellular signaling pathways.
Assuntos
Proteínas do Citoesqueleto/metabolismo , Citoesqueleto/metabolismo , Microtúbulos/metabolismo , Proteínas de Peixe-Zebra , Proteína da Polipose Adenomatosa do Colo , Animais , Proteínas do Citoesqueleto/genética , Genes APC/fisiologia , Genes Reporter , Humanos , Modelos Biológicos , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais/fisiologia , Proteínas WntRESUMO
Calcium-dependent cell-cell adhesion is mediated by the cadherin family of cell adhesion proteins. Transduction of cadherin adhesion into cellular reorganization is regulated by cytosolic proteins, termed alpha-, beta-, and gamma-catenin (plakoglobin), that bind to the cytoplasmic domain of cadherins and link them to the cytoskeleton. Previous studies of cadherin/catenin complex assembly and organization relied on the coimmunoprecipitation of the complex with cadherin antibodies, and were limited to the analysis of the Triton X-100 (TX-100)-soluble fraction of these proteins. These studies concluded that only one complex exists which contains cadherin and all of the catenins. We raised antibodies specific for each catenin to analyze each protein independent of its association with E-cadherin. Extracts of Madin-Darby canine kidney epithelial cells were sequentially immunoprecipitated and immunoblotted with each antibody, and the results showed that there were complexes of E-cadherin/alpha-catenin, and either beta-catenin or plakoglobin in the TX-100-soluble fraction. We analyzed the assembly of cadherin/catenin complexes in the TX-100-soluble fraction by [35S]methionine pulse-chase labeling, followed by sucrose density gradient fractionation of proteins. Immediately after synthesis, E-cadherin, beta-catenin, and plakoglobin cosedimented as complexes. alpha-Catenin was not associated with these complexes after synthesis, but a subpopulation of alpha-catenin joined the complex at a time coincident with the arrival of E-cadherin at the plasma membrane. The arrival of E-cadherin at the plasma membrane coincided with an increase in its insolubility in TX-100, but extraction of this insoluble pool with 1% SDS disrupted the cadherin/catenin complex. Therefore, to examine protein complex assembly in both the TX-100-soluble and -insoluble fractions, we used [35S]methionine labeling followed by chemical cross-linking before cell extraction. Analysis of cross-linked complexes from cells labeled to steady state indicates that, in addition to cadherin/catenin complexes, there were cadherin-independent pools of catenins present in both the TX-100-soluble and -insoluble fractions. Metabolic labeling followed by chase showed that immediately after synthesis, cadherin/beta-catenin, and cadherin/plakoglobin complexes were present in the TX-100-soluble fraction. Approximately 50% of complexes were titrated into the TX-100-insoluble fraction coincident with the arrival of the complexes at the plasma membrane and the assembly of alpha-catenin. Subsequently, > 90% of labeled cadherin, but no additional labeled catenin complexes, entered the TX-100-insoluble fraction.(ABSTRACT TRUNCATED AT 400 WORDS)
Assuntos
Caderinas/metabolismo , Proteínas do Citoesqueleto/metabolismo , Transativadores , Animais , Células Cultivadas , Centrifugação com Gradiente de Concentração , Reagentes de Ligações Cruzadas , Proteínas do Citoesqueleto/imunologia , Desmoplaquinas , Cães , Substâncias Macromoleculares , Modelos Biológicos , Octoxinol , Testes de Precipitina , Ligação Proteica , Succinimidas , alfa Catenina , beta Catenina , gama CateninaRESUMO
The cadherin/catenin complex plays important roles in cell adhesion, signal transduction, as well as the initiation and maintenance of structural and functional organization of cells and tissues. In the preceding study, we showed that the assembly of the cadherin/catenin complex is temporally regulated, and that novel combinations of catenin and cadherin complexes are formed in both Triton X-100-soluble and -insoluble fractions; we proposed a model in which pools of catenins are important in regulating assembly of E-cadherin/catenin and catenin complexes. Here, we sought to determine the spatial distributions of E-cadherin, alpha-catenin, beta-catenin, and plakoglobin, and whether different complexes of these proteins accumulate at steady state in polarized Madin-Darby canine kidney cells. Protein distributions were visualized by wide field, optical sectioning, and double immunofluorescence microscopy, followed by reconstruction of three-dimensional images. In cells that were extracted with Triton X-100 and then fixed (Triton X-100-insoluble fraction), more E-cadherin was concentrated at the apical junction relative to other areas of the lateral membrane. alpha-Catenin and beta-catenin colocalize with E-cadherin at the apical junctional complex. There is some overlap in the distribution of these proteins in the lateral membrane, but there are also areas where the distributions are distinct. Plakoglobin is excluded from the apical junctional complex, and its distribution in the lateral membrane is different from that of E-cadherin. Cells were also fixed and then permeabilized to reveal the total cellular pool of each protein (Triton X-100-soluble and -insoluble fractions). This analysis showed lateral membrane localization of alpha-catenin, beta-catenin, and plakoglobin, and it also revealed that they are distributed throughout the cell. Chemical cross-linking of proteins and analysis with specific antibodies confirmed the presence at steady state of E-cadherin/catenin complexes containing either beta-catenin or plakoglobin, and catenin complexes devoid of E-cadherin. Complexes containing E-cadherin/beta-catenin and E-cadherin/alpha-catenin are present in both the Triton X-100-soluble and -insoluble fractions, but E-cadherin/plakoglobin complexes are not detected in the Triton X-100-insoluble fraction. Taken together, these results show that different complexes of cadherin and catenins accumulate in fully polarized epithelial cells, and that they distribute to different sites. We suggest that cadherin/catenin and catenin complexes at different sites have specialized roles in establishing and maintaining the structural and functional organization of polarized epithelial cells.
Assuntos
Caderinas/metabolismo , Compartimento Celular , Polaridade Celular , Proteínas do Citoesqueleto/metabolismo , Transativadores , Animais , Caderinas/isolamento & purificação , Adesão Celular/fisiologia , Células Cultivadas , Proteínas do Citoesqueleto/isolamento & purificação , Desmoplaquinas , Cães , Células Epiteliais , Imunofluorescência , Modelos Biológicos , Modelos Estruturais , Transdução de Sinais/fisiologia , alfa Catenina , beta Catenina , gama CateninaRESUMO
Mutations in the adenomatous polyposis coli (APC) gene are linked to polyp formation in familial and sporadic colon cancer, but the functions of the protein are not known. We show that APC protein localizes mainly to clusters of puncta near the ends of microtubules that extend into actively migrating regions of epithelial cell membranes. This subcellular distribution of APC protein requires microtubules, but not actin filaments. APC protein-containing membranes are actively involved in cell migration in response to wounding epithelial monolayers, addition of the motorgen hepatocyte growth factor, and during the formation of cell-cell contacts. In the intestine, APC protein levels increase at the crypt/villus boundary, where cell migration is crucial for enterocyte exit from the crypt and where cells accumulate during polyp formation that is linked to mutations in the microtubule-binding domain of APC protein. Together, these data indicate that APC protein has a role in directed cell migration.
Assuntos
Membrana Celular/ultraestrutura , Movimento Celular , Proteínas do Citoesqueleto/fisiologia , Epitélio/ultraestrutura , Microtúbulos/ultraestrutura , Transativadores , Proteína da Polipose Adenomatosa do Colo , Animais , Compartimento Celular , Linhagem Celular , Membrana Celular/fisiologia , Proteínas do Citoesqueleto/metabolismo , Cães , Células Epiteliais , Técnica Indireta de Fluorescência para Anticorpo , Genes Supressores de Tumor , Fator de Crescimento de Hepatócito/farmacologia , Mucosa Intestinal/ultraestrutura , Gravação em Vídeo , Cicatrização , beta CateninaRESUMO
Beta-catenin is a cytosolic protein originally identified through its association with the cadherin class of cell-adhesion proteins. However, recent studies have demonstrated that there are cadherin-independent pools of beta-catenin and that beta-catenin binds at least one other protein, the product of the tumor-suppressor gene APC. Furthermore, beta-catenin is the target of two signal transduction pathways mediated by the proto-oncogenes src and wnt-1. This raises the possibility that beta-catenin plays a pivotal role in balancing cellular responses to both adhesive and proliferative signals.
Assuntos
Adesão Celular/fisiologia , Proteínas do Citoesqueleto/fisiologia , Proteína Oncogênica pp60(v-src)/fisiologia , Proteínas Proto-Oncogênicas/fisiologia , Transdução de Sinais/fisiologia , Transativadores , Proteínas de Peixe-Zebra , Animais , Proteínas Wnt , Proteína Wnt1 , beta CateninaRESUMO
The adenomatous polyposis coli gene (Apc) is mutated in most colorectal cancers. The multifunctional character of the Apc protein in the regulation of beta-catenin-mediated gene transcription and cytoskeletal proteins has been well described. An important question is how this protein affects the behaviour of cells within a tumour and how its mutational status influences the prognosis for these tumours. Here we provide an overview of the functions of Apc and examine how this information can be used in the prognosis and development of directed therapy in colorectal cancer.
Assuntos
Neoplasias Colorretais/genética , Genes APC/fisiologia , Anti-Inflamatórios não Esteroides/farmacologia , Aspirina/farmacologia , Citoesqueleto/genética , Humanos , Prognóstico , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Proteínas Wnt/genética , beta Catenina/metabolismoRESUMO
Truncation mutations in the adenomatous polyposis coli protein (APC) are responsible for familial polyposis, a form of inherited colon cancer. In addition to its role in mediating beta-catenin degradation in the Wnt signaling pathway, APC plays a role in regulating microtubules. This was suggested by its localization to the end of dynamic microtubules in actively migrating areas of cells and by the apparent correlation between the dissociation of APC from polymerizing microtubules and their subsequent depolymerization [1, 2]. The microtubule binding domain is deleted in the transforming mutations of APC [3, 4]; however, the direct effect of APC protein on microtubules has never been examined. Here we show that binding of APC to microtubules increases microtubule stability in vivo and in vitro. Deleting the previously identified microtubule binding site from the C-terminal domain of APC does not eliminate its binding to microtubules but decreases the ability of APC to stabilize them significantly. The interaction of APC with microtubules is decreased by phosphorylation of APC by GSK3 beta. These data confirm the hypothesis that APC is involved in stabilizing microtubule ends. They also suggest that binding of APC to microtubules is mediated by at least two distinct sites and is regulated by phosphorylation.
Assuntos
Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Proteínas do Citoesqueleto/metabolismo , Microtúbulos/metabolismo , Proteína da Polipose Adenomatosa do Colo , Quinase 3 da Glicogênio Sintase , Humanos , Fosforilação , Ligação ProteicaRESUMO
Altered tissue structure is a feature of many disease states and is usually measured by microscopic methods, limiting analysis to small areas. Means to rapidly and quantitatively measure the structure and organisation of large tissue areas would represent a major advance not just for research but also in the clinic. Here, changes in tissue organisation that result from heterozygosity in Apc, a precancerous situation, are comprehensively measured using microultrasound and three-dimensional high-resolution microscopy. Despite its normal appearance in conventionally examined cross-sections, both approaches revealed a significant increase in the variability of tissue organisation in Apc heterozygous tissue. These changes preceded the formation of aberrant crypt foci or adenoma. Measuring these premalignant changes using microultrasound provides a potential means to detect microscopically abnormal regions in large tissue samples, independent of visual examination or biopsies. Not only does this provide a powerful tool for studying tissue structure in experimental settings, the ability to detect and monitor tissue changes by microultrasound could be developed into a powerful adjunct to screening endoscopy in the clinic.
Assuntos
Focos de Criptas Aberrantes/diagnóstico por imagem , Proteína da Polipose Adenomatosa do Colo/genética , Imageamento Tridimensional/métodos , Intestinos/diagnóstico por imagem , Intestinos/patologia , Focos de Criptas Aberrantes/patologia , Animais , Sobrevivência Celular , Feminino , Humanos , Masculino , Camundongos , Microscopia , Microtecnologia , Mutação , UltrassonografiaRESUMO
Mutations in the adenomatous polyposis coli (APC) tumour suppressor are the key initiating event of colorectal cancer. Although the control of WNT signalling is well established as a central tumour-suppressive function, the significance of APC in regulating chromosome instability is less well established. In this study, we test whether APC-deficient cells have a functional spindle assembly checkpoint (SAC) in vivo by examining the response of these cells to Taxol and Vinorelbine. We also show for the first time that APC deficiency compromises the arrest response to Taxol in vivo. This effect is independent of the role that APC has in WNT signalling. At higher levels of Taxol, APC-deficient cells arrest as efficiently as wild-type cells. Importantly, this dose of Taxol strongly suppresses intestinal tumourigenesis in models of benign (APC(Min/+) mouse) and invasive (AhCreER(+)APC(fl/+)PTEN(fl/fl)) cancer. In contrast to intestinal enterocytes with a general SAC defect because of Bub1 (budding uninhibited by benzimidazole 1) deletion, APC-deficient enterocytes arrest equivalently to wild type when treated with Vinorelbine. This suggests that the failed arrest in response to Taxol is because of a specific defect in microtubule stabilization following Taxol treatment rather than a general role of the APC protein in the mitotic spindle checkpoint. In summary, this study clarifies the role of APC as a mitotic spindle checkpoint protein in vivo and shows that APC-deficient cells have a compromised response to Taxol.
Assuntos
Proteína da Polipose Adenomatosa do Colo/fisiologia , Polipose Adenomatosa do Colo/tratamento farmacológico , Antineoplásicos Fitogênicos/uso terapêutico , Resistencia a Medicamentos Antineoplásicos/genética , Paclitaxel/uso terapêutico , Fuso Acromático/metabolismo , Polipose Adenomatosa do Colo/patologia , Proteína da Polipose Adenomatosa do Colo/genética , Animais , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/genética , Transformação Celular Neoplásica/efeitos dos fármacos , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/patologia , Instabilidade Cromossômica/genética , Enterócitos/efeitos dos fármacos , Enterócitos/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Serina-Treonina Quinases/genética , Deleção de Sequência , Fuso Acromático/genética , Vimblastina/análogos & derivados , Vimblastina/uso terapêutico , Vinorelbina , Proteínas Wnt/metabolismoRESUMO
Mutations in the adenomatous polyposis coli (APC) gene are associated with most colorectal cancers. The APC protein has been implicated in many aspects of tumour development. This article will discuss recent data suggesting that APC may have multiple functions in the cell. First, APC is a component of the Wnt signalling pathway; second, APC may have a role in cell migration; finally, APC may regulate proliferation and apoptosis.
Assuntos
Proteínas do Citoesqueleto/fisiologia , Proteína da Polipose Adenomatosa do Colo , Fenômenos Fisiológicos Celulares , Neoplasias Colorretais/fisiopatologia , Humanos , Proteínas de Neoplasias/fisiologia , Transdução de Sinais/fisiologiaRESUMO
Epithelial cell adhesion is principally regulated by calcium-dependent cell adhesion proteins, termed cadherins. Recent studies indicate that cadherin function is modulated by a class of proteins, termed catenins, that bind to the cytoplasmic domain of cadherin. Here we review the evidence that catenins regulate cadherin function in cell-cell adhesion, and discuss their role in initiating cell surface polarity in epithelial cells.
Assuntos
Caderinas/fisiologia , Adesão Celular/fisiologia , Polaridade Celular/fisiologia , Sequência de Aminoácidos , Animais , Caderinas/genética , Comunicação Celular/fisiologia , Proteínas do Citoesqueleto/fisiologia , Células Epiteliais , Epitélio/metabolismo , Dados de Sequência Molecular , Sistemas do Segundo Mensageiro/fisiologia , Transdução de Sinais/fisiologiaRESUMO
The triskelion shape of the clathrin molecule enables it to form the polyhedral protein network that covers clathrin-coated pits and vesicles. Domains within the clathrin heavy chain that are responsible for maintaining triskelion shape and function were identified and localized. Sequences that mediate trimerization are distal to the carboxyl terminus and are adjacent to a domain that mediates both light chain binding and clathrin assembly. Structural modeling predicts that within this domain, the region of heavy chain-light chain interaction is a bundle of three or four alpha helices. These studies establish a low resolution model of clathrin subunit folding in the central portion (hub) of the triskelion, thus providing a basis for future mutagenesis experiments.