RESUMO
Smad proteins are the major signal transducers for the Transforming Growth Factor superfamily of cytokines and their serine/threonine kinase receptors. Smads mediate the signal from the membrane into the nucleus. Bone Morphogenetic Protein-4 stimulates phosphorylation of Smad1, which interacts with Smad4. This complex translocates into the nucleus and regulates transcription of target genes. Here, we report our development of cellular fluorescence biosensors for direct visualization of Smad signaling in live mammalian cells. Fluorescence resonance energy transfer between cyan and yellow fluorescent proteins fused to the Smad1 and Smad4 proteins was used to unravel the temporal aspects of BMP/Smad signaling. A rate-limiting delay of 2-5 min occurred between BMP activation and Smad1 activity. A similar delay was observed in the Smad1/Smad4 complexation. Further experimentation indicated that the delay is dependent on the MH1 domain and linker of Smad1. These results give new insights into the dynamics of the BMP receptor -Smad1/4 signaling process and provide a new tool for studying Smads.
Assuntos
Técnicas Biossensoriais/métodos , Proteínas Morfogenéticas Ósseas/farmacologia , Transferência Ressonante de Energia de Fluorescência/métodos , Transdução de Sinais , Proteína Smad1/metabolismo , Proteína Smad4/metabolismo , Sequência de Aminoácidos , Animais , Proteína Morfogenética Óssea 4 , Células COS , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Chlorocebus aethiops , Células HeLa , Humanos , Cinética , Proteínas Luminescentes/metabolismo , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Transporte Proteico/efeitos dos fármacos , Proteínas Recombinantes de Fusão/metabolismo , Proteína Smad1/química , Transcrição Gênica/efeitos dos fármacosRESUMO
Three promising variants of autofluorescent proteins have been analyzed photophysically for their proposed use in single-molecule microscopy studies in living cells to compare their superiority to other fluorescent proteins previously reported regarding the number of photons emitted. The first variant under investigation the F46L mutant of eYFP has a 10% greater photon emission rate and >50% slower photobleaching rate on average than the standard eYFP fluorophore. The monomeric red fluorescent protein (mRFP) has a fivefold lower photon emission rate, likely due to the monomeric content, and also a tenfold faster photobleaching rate than the DsRed fluorescent protein. In contrast, the previously reported eqfp611 has a 50% lower emission rate yet photobleaches more than a factor 2 slowly. We conclude that the F46L YFP and the eqfp611 are superior new options for single molecule imaging and tracking studies in living cells. Studies were also performed on the effects of forced quenching of multiple fluorescent proteins in sub-micrometer regions that would show the effects of dimerization at low concentration levels of fluorescent proteins and also indicate corrections to stoichiometry patterns with fluorescent proteins previously in print. We also introduce properties at the single molecule level of new FRET pairs with combinations of fluorescent proteins and artificial fluorophores.