RESUMEN
BACKGROUND: Mad1 and Mad2 are constituents of the spindle-assembly checkpoint, a device coupling the loss of sister-chromatid cohesion at anaphase to the completion of microtubule attachment of the sister chromatids at metaphase. Fluorescence recovery after photobleaching (FRAP) revealed that the interaction of cytosolic Mad2 with kinetochores is highly dynamic, suggesting a mechanism of catalytic activation of Mad2 at kinetochores followed by its release in a complex with Cdc20. The recruitment of cytosolic Mad2 to kinetochores has been attributed to a stable receptor composed of a distinct pool of Mad2 tightly bound to Mad1. Whether specifically this interaction accounts for the kinetochore dynamics of Mad2 is currently unknown. RESULTS: To gain a precise molecular understanding of the interaction of Mad2 with kinetochores, we reconstituted the putative Mad2 kinetochore receptor and developed a kinetochore recruitment assay with purified components. When analyzed by FRAP in vitro, this system faithfully reproduced the previously described in vivo dynamics of Mad2, providing an unequivocal molecular account of the interaction of Mad2 with kinetochores. Using the same approach, we dissected the mechanism of action of p31(comet), a spindle-assembly checkpoint inhibitor. CONCLUSIONS: In vitro FRAP is a widely applicable approach to dissecting the molecular bases of the interaction of a macromolecule with an insoluble cellular scaffold. The combination of in vitro fluorescence recovery after photobleaching with additional fluorescence-based assays in vitro can be used to unveil mechanism, stoichiometry, and kinetic parameters of a macromolecular interaction, all of which are important for modeling protein interaction networks.
Asunto(s)
Proteínas de Unión al Calcio/metabolismo , Proteínas de Ciclo Celular/metabolismo , Recuperación de Fluorescencia tras Fotoblanqueo , Cinetocoros/metabolismo , Proteínas Represoras/metabolismo , Proteínas de Unión al Calcio/química , Proteínas Cdc20 , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/fisiología , Proteínas Mad2 , Proteínas Nucleares/metabolismo , Proteínas Represoras/química , Huso Acromático/metabolismoRESUMEN
A major obstacle for the development of effective immunotherapy is the ability of tumors to escape the immune system. The possibility to kill tumor cells because they are recognized as infected rather than as malignant could help overcome immune escape mechanisms. Here we report a conceptually new approach of cancer immunotherapy based on in vivo infection of tumors and killing of infected tumor cells. Attenuated but still invasive, Salmonella typhimurium can be successfully exploited to invade melanoma cells that can present antigenic determinants of bacterial origin and become targets for anti-Salmonella-specific T cells. However, to fully appreciate the anticancer therapeutic properties of S. typhimurium, tumor-bearing mice need to be vaccinated against S. typhimurium before intratumoral Salmonella injection. Tumor infection when coupled to anti-Salmonella vaccination leads to 50% to 100% tumor-free mice with a better outcome on larger tumors. Invasive Salmonella also exert an indirect toxic effect on tumor cells through the recruitment of inflammatory cells and the cross-presentation of tumor antigens, which allow induction of tumor-specific immune response. This is effective in retarding the growth of untreated established distant tumors and in protecting the mice from subsequent tumor challenges.
Asunto(s)
Inmunoterapia/métodos , Melanoma/microbiología , Melanoma/terapia , Infecciones por Salmonella/inmunología , Salmonella typhimurium/inmunología , Animales , Antígenos Bacterianos/inmunología , Epítopos de Linfocito T/inmunología , Femenino , Humanos , Melanoma/inmunología , Melanoma Experimental/inmunología , Melanoma Experimental/microbiología , Melanoma Experimental/terapia , Ratones , Ratones Endogámicos C57BL , Ovalbúmina/inmunología , Vacunas contra la Salmonella/inmunología , Vacunas contra la Salmonella/farmacología , Linfocitos T Citotóxicos/inmunologíaRESUMEN
Plasma membrane receptors can be endocytosed through clathrin-dependent and clathrin-independent pathways. Here, we show that the epidermal growth factor (EGF) receptor (EGFR), when stimulated with low doses of EGF, is internalized almost exclusively through the clathrin pathway, and it is not ubiquitinated. At higher concentrations of ligand, however, a substantial fraction of the receptor is endocytosed through a clathrin-independent, lipid raft-dependent route, as the receptor becomes ubiquitinated. An ubiquitination-impaired EGFR mutant was internalized through the clathrin pathway, whereas an EGFR/ubiquitin chimera, that can signal solely through its ubiquitin (Ub) moiety, was internalized exclusively by the non-clathrin pathway. Non-clathrin internalization of ubiquitinated EGFR depends on its interaction with proteins harboring the Ub-interacting motif, as shown through the ablation of three Ub-interacting motif-containing proteins, eps15, eps15R, and epsin. Thus, eps15s and epsin perform an important function in coupling ubiquitinated cargo to clathrin-independent internalization.
Asunto(s)
Clatrina/fisiología , Endocitosis , Receptores ErbB/metabolismo , Ubiquitina/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Animales , Células CHO , Proteínas de Unión al Calcio/fisiología , Cricetinae , Células HeLa , Humanos , Péptidos y Proteínas de Señalización Intracelular , Fosfoproteínas/fisiologíaRESUMEN
Our knowledge of the genetic mechanisms controlling cell proliferation and differentiation usually originates from in vitro cultured cell line models. However, the definition of the molecular switches involved in control of homeostasis and the understanding of the changes occurring in neoplastic transformation require looking at single cells as the components of a complex tissue network. Histological examination of tissue samples can gain a substantial amount of information from high-resolution fluorescence analysis. In particular, confocal microscopy can help in the definition of functional pathways using multiparameter analysis. In this report, we present acquisition and analysis procedures to obtain high-resolution data from tissue sections. Confocal microscopy coupled to computational restoration, statistical evaluation of spatial correlations, and morphological analysis over large tissue areas were applied to colorectal samples providing a molecular fingerprint of the biological differences inferred from classical histological examination.
Asunto(s)
Colon/citología , Microscopía Confocal/métodos , Microscopía Fluorescente/métodos , Recto/citología , Animales , Colon/metabolismo , Colon/ultraestructura , Técnicas de Preparación Histocitológica , Histona Desacetilasa 1 , Histona Desacetilasa 2 , Histona Desacetilasas/metabolismo , Humanos , Procesamiento de Imagen Asistido por Computador , Microtomía , Proteínas de Neoplasias/metabolismo , Proteínas Nucleares/metabolismo , Proteína de la Leucemia Promielocítica , Recto/metabolismo , Recto/ultraestructura , Proteínas Represoras/metabolismo , Factores de Transcripción/metabolismo , Proteínas Supresoras de TumorRESUMEN
Budding yeast Sgt1 is required for kinetochore assembly, and its homologues have a role in cAMP signalling in fungi and pathogen resistance in plants. The function of mammalian Sgt1 is unknown. We report that RNA interference-mediated depletion of Sgt1 from HeLa cells causes dramatic alterations of the mitotic spindle and problems in chromosome alignment. Cells lacking Sgt1 undergo a mitotic delay due to activation of the spindle checkpoint. The checkpoint response, however, is significantly weakened in Sgt1-depleted cells, and this correlates with a dramatic reduction in kinetochore levels of Mad1, Mad2 and BubR1. These effects are explained by a problem in kinetochore assembly that prevents the localization of Hec1, CENP-E, CENP-F, CENP-I, but not CENP-C, to mitotic kinetochores. Our studies implicate Sgt1 as an essential protein and a critical assembly factor for the mammalian kinetochore, and lend credit to the hypothesis of a kinetochore assembly pathway that is conserved from yeast to man.
Asunto(s)
Proteínas de Ciclo Celular/fisiología , Cinetocoros/ultraestructura , Interferencia de ARN , Huso Acromático/genética , Proteínas de Unión al Calcio/análisis , Proteínas de Unión al Calcio/inmunología , Proteínas de Ciclo Celular/genética , Centrómero/inmunología , Centrómero/metabolismo , Proteínas Cromosómicas no Histona/análisis , Proteínas Cromosómicas no Histona/inmunología , Proteínas de Unión al ADN/análisis , Proteínas de Unión al ADN/inmunología , Células HeLa , Humanos , Cinetocoros/metabolismo , Proteínas Mad2 , Mitosis/genética , Mitosis/fisiología , Proteínas Nucleares , Fosfoproteínas/análisis , Fosfoproteínas/inmunología , Proteínas Quinasas/análisis , Proteínas Quinasas/inmunología , Proteínas Serina-Treonina Quinasas , ARN Interferente Pequeño/metabolismo , Proteínas Represoras/análisis , Proteínas Represoras/inmunología , Huso Acromático/metabolismoRESUMEN
1. The present study was aimed to investigate the effect of benzydamine, an anti-inflammatory drug devoid of activity on arachidonic acid metabolism, on monocyte chemotaxis and to define the possible biochemical correlates of activity. 2. Benzydamine inhibited monocyte chemotaxis in response to three classes of chemoattractants: the prototypic CC-chemokine CCL2 (MCP-1), the microbial product fMLP and the complement cascade component C5a. The effect was dose-dependent with IC50's of 100, 50 and 45 microm for MCP-1/CCL2, fMLP and C5a, respectively. At the dose of 100 microm, the effect resulted in a 50+/-10% inhibition of MCP-1/CCL2-induced chemotaxis and 53+/-6 and 54+/-5% inhibitions of chemotaxis in response of fMLP and C5a, respectively (n=3). 3. Receptor expression as well as calcium fluxes in response to chemoattractants were not affected by benzydamine. 4. Benzydamine strongly inhibited chemoattractant-induced activation of the mitogen-activated protein kinase (MAPK) ERK1/2, and of its upstream activator kinase MEK1/2. ERK1/12 activation in response to chemoattractants was 89-98% inhibited by a 100 microm concentration of benzydamine with an IC50 of 30 microm. 5. Under the same experimental conditions, pretreatment with 100 microm benzydamine caused a 75-89% inhibition of p38 activation (IC50 25 microm). 6. These results indicate that the anti-inflammatory activity of benzydamine is exerted at multiple levels, including monocyte migration to chemotactic factors associated to a blockage of ERK and p38 MAPK pathways.