RESUMO
Imaging the dynamics of proteins in living cells is a powerful means for understanding cellular functions at a deeper level. Here, we report a versatile method for spatiotemporal imaging of specific endogenous proteins in living mammalian cells. The method employs a bifunctional aptamer capable of selective protein recognition and fluorescent probe-binding, which is induced only when the aptamer specifically binds to its target protein. An aptamer for ß-actin protein preferentially recognizes its monomer forms over filamentous forms, resulting in selective G-actin staining in both fixed and living cells. Through actin-drug treatment, the method permitted direct monitoring of the intracellular concentration change of endogenous G-actin. This protein-labeling method, which is highly selective and non-covalent, provides rich insights into the study of spatiotemporal protein dynamics in living cells.
Assuntos
Aptâmeros de Nucleotídeos , Imagem Óptica/métodos , Proteínas/análise , Actinas/análise , Aptâmeros de Nucleotídeos/química , Corantes Fluorescentes , Células HeLa , Humanos , Imagem Molecular/métodos , RNA/química , Imagem com Lapso de TempoRESUMO
Here, we developed two pairs of high-contrast chemical probes and their RNA aptamers with distinct readout channels that permitted simultaneous live-cell imaging of endogenous ß-actin and cortactin mRNAs. Application of this technology allowed the direct observation of the formation process of stress granules, protein-RNA assemblies essential for cellular response to the environment.