RESUMO
To expand the range of experiments that are accessible with optogenetics, we developed a photocleavable protein (PhoCl) that spontaneously dissociates into two fragments after violet-light-induced cleavage of a specific bond in the protein backbone. We demonstrated that PhoCl can be used to engineer light-activatable Cre recombinase, Gal4 transcription factor, and a viral protease that in turn was used to activate opening of the large-pore ion channel Pannexin-1.
Assuntos
Optogenética/métodos , Engenharia de Proteínas/métodos , Proteínas Recombinantes/metabolismo , Conexinas/genética , Conexinas/metabolismo , Evolução Molecular Direcionada , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Proteínas de Choque Térmico HSP90/genética , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Sinais de Localização Nuclear/genética , Técnicas de Patch-Clamp , Fotoquímica/métodos , Proteínas Recombinantes/genética , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismo , Proteína Vermelha FluorescenteRESUMO
In the online version of the article [ 1 ], Figure S1 was mistakenly replaced with Figure 1.
RESUMO
BACKGROUND: Genetically encoded calcium ion (Ca2+) indicators (GECIs) are indispensable tools for measuring Ca2+ dynamics and neuronal activities in vitro and in vivo. Red fluorescent protein (RFP)-based GECIs have inherent advantages relative to green fluorescent protein-based GECIs due to the longer wavelength light used for excitation. Longer wavelength light is associated with decreased phototoxicity and deeper penetration through tissue. Red GECI can also enable multicolor visualization with blue- or cyan-excitable fluorophores. RESULTS: Here we report the development, structure, and validation of a new RFP-based GECI, K-GECO1, based on a circularly permutated RFP derived from the sea anemone Entacmaea quadricolor. We have characterized the performance of K-GECO1 in cultured HeLa cells, dissociated neurons, stem-cell-derived cardiomyocytes, organotypic brain slices, zebrafish spinal cord in vivo, and mouse brain in vivo. CONCLUSION: K-GECO1 is the archetype of a new lineage of GECIs based on the RFP eqFP578 scaffold. It offers high sensitivity and fast kinetics, similar or better than those of current state-of-the-art indicators, with diminished lysosomal accumulation and minimal blue-light photoactivation. Further refinements of the K-GECO1 lineage could lead to further improved variants with overall performance that exceeds that of the most highly optimized red GECIs.
Assuntos
Cálcio/análise , Substâncias Luminescentes/análise , Proteínas Luminescentes/análise , Proteínas Luminescentes/genética , Animais , Células Cultivadas , Cristalografia/métodos , Células HeLa , Humanos , Substâncias Luminescentes/química , Proteínas Luminescentes/química , Camundongos , Técnicas de Cultura de Órgãos , Estrutura Secundária de Proteína , Ratos , Anêmonas-do-Mar , Peixe-Zebra , Proteína Vermelha FluorescenteRESUMO
The tetrameric red fluorescent protein from Discosoma sp. coral (DsRed) has previously been engineered to produce dimeric and monomeric fluorescent variants with excitation and emission profiles that span the visible spectrum. The brightest of the effectively monomeric DsRed variants is tdTomato-a tandem fusion of a dimeric DsRed variant. Here we describe the engineering of brighter red (RRvT), green (GGvT), and green-red heterodimeric (GRvT) tdTomato variants. GRvT exhibited 99 % intramolecular FRET efficiency, resulting in long Stokes shift red fluorescence. These new variants could prove useful for multicolor live-cell imaging applications.
Assuntos
Transferência Ressonante de Energia de Fluorescência , Proteínas Luminescentes/química , Animais , Antozoários/metabolismo , Dimerização , Células HeLa , Humanos , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Microscopia de Fluorescência , Modelos Moleculares , Mutagênese Sítio-Dirigida , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteína Vermelha FluorescenteRESUMO
Mapping the protein-protein interaction (PPi) landscape is of critical importance to furthering our understanding how cells and organisms function. Optogenetic methods, that is, approaches that utilize genetically encoded fluorophores or fluorogenic enzyme reactions, uniquely enable the visualization of biochemical phenomena in live cells with high spatial and temporal accuracy. Applying optogenetic methods to the detection of PPis requires the engineering of protein-based systems in which an optical signal undergoes a substantial change when the two proteins of interest interact. In recent years, researchers have developed a number of creative and effective optogenetic methods that achieve this goal, and used them to further elaborate our map of the PPi landscape. In this review, we provide an introduction to the general principles of optogenetic PPi detection, and then provide a number of representative examples of how these principles have been applied. We have organized this review by categorizing methods based on whether the signal generated is reversible or irreversible in nature, and whether the signal is localized or nonlocalized at the subcellular site of the PPi. We discuss these techniques giving both their benefits and drawbacks to enable rational choices about their potential use. This article is categorized under: Laboratory Methods and Technologies > Imaging Laboratory Methods and Technologies > Macromolecular Interactions, Methods Analytical and Computational Methods > Analytical Methods.
Assuntos
Optogenética/métodos , Proteínas/química , Proteínas/metabolismoRESUMO
Creative engineering of fluorescent proteins has yielded a variety of tools for visualization of biochemical events in vivo. In this issue of Cell Chemical Biology, To et al. (2016) describe a fluorogenic green fluorescent protein that is activated by caspase-3 activity and enables imaging of apoptosis in developing zebrafish embryos (To et al., 2016).