RESUMO
Reversibly switchable proteins are powerful tools with which to explore protein function in vitro and in vivo. For example, the activity of many proteins fused to the hormone-binding domain of the modified oestrogen receptor (ER(TAM)) can be regulated by provision or removal of 4-hydroxytamoxifen (4-OHT). Despite the widespread use of ER(TAM) fusions in vivo, inadequate data are available as to the most efficacious routes for systemic tamoxifen delivery. In this study, we have used two well-characterized ER(TAM) fusion proteins, both reversibly activated by 4-OHT, to compare the effectiveness and kinetics of 4-OHT delivery in mice in vivo by either tamoxifen in food or by intraperitoneal injection. Our data indicate that dietary tamoxifen offers an effective, facile and ethically preferable means for long-term activation of ER(TAM) fusion proteins in vivo.
Assuntos
Antineoplásicos/administração & dosagem , Receptores de Estrogênio/genética , Tamoxifeno/análogos & derivados , Administração Oral , Animais , Antineoplásicos/farmacologia , Genes Reporter , Injeções Intraperitoneais , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/metabolismo , Cinética , Camundongos , Ratos , Receptores de Estrogênio/biossíntese , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Tamoxifeno/administração & dosagem , Tamoxifeno/farmacologia , Ativação Transcricional/efeitos dos fármacos , Proteína Supressora de Tumor p53/biossíntese , Proteína Supressora de Tumor p53/genéticaRESUMO
Cell adhesion, a process mediated by the formation of discrete structures known as focal adhesions (FAs), is pivotal to many biological events including cell motility. Much is known about the molecular composition of FAs, although our knowledge of the spatio-temporal recruitment and the relative occupancy of the individual components present in the FAs is still incomplete. To fill this gap, an essential prerequisite is a highly reliable procedure for the recognition, segmentation and tracking of FAs. Although manual segmentation and tracking may provide some advantages when done by an expert, its performance is usually hampered by subjective judgement and the long time required in analysing large data sets. Here, we developed a model-based segmentation and tracking algorithm that overcomes these problems. In addition, we developed a dedicated computational approach to correct segmentation errors that may arise from the analysis of poorly defined FAs. Thus, by achieving accurate and consistent FA segmentation and tracking, our work establishes the basis for a comprehensive analysis of FA dynamics under various experimental regimes and the future development of mathematical models that simulate FA behaviour.
