RESUMO
Genetically encoded fluorescent biosensors have revolutionized the study of signal transduction by enabling the real-time tracking of signaling activities in live cells. Investigating the interaction between signaling networks has become increasingly important to understanding complex cellular phenomena, necessitating an update of the biosensor toolkit to allow monitoring and perturbing multiple activities simultaneously in the same cell. We therefore developed a new class of fluorescent biosensors based on homo-FRET, deemed FLuorescence Anisotropy REporters (FLAREs), which combine the multiplexing ability of single-color sensors with a quantitative, ratiometric readout. Using an array of color variants, we were able to demonstrate multiplexed imaging of three activity reporters simultaneously in the same cell. We further demonstrate the compatibility of FLAREs for use with optogenetic tools as well as intravital two-photon imaging.
Assuntos
Técnicas Biossensoriais , Polarização de Fluorescência/métodos , Transferência Ressonante de Energia de Fluorescência/métodos , Corantes Fluorescentes/metabolismo , Transdução de Sinais , Análise de Célula Única/métodos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Núcleo Celular/metabolismo , Núcleo Celular/ultraestrutura , Cor , Proteínas Quinases Dependentes de AMP Cíclico/genética , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Citosol/metabolismo , Citosol/ultraestrutura , Transferência Ressonante de Energia de Fluorescência/instrumentação , Corantes Fluorescentes/síntese química , Regulação da Expressão Gênica , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Células HeLa , Humanos , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Plasmídeos/química , Plasmídeos/metabolismo , Transfecção , Quinases da Família src/genética , Quinases da Família src/metabolismo , Proteína Vermelha FluorescenteRESUMO
BACKGROUND: Clinical studies implementing late gadolinium-enhanced (LGE) cardiovascular magnetic resonance (CMR) studies suggest that the peri-infarct zone (PIZ) contains a mixture of viable and non-viable myocytes, and is associated with greater susceptibility to ventricular tachycardia induction and adverse cardiac outcomes. However, CMR data assessing the temporal formation and functional remodeling characteristics of this complex region are limited. We intended to characterize early temporal changes in scar morphology and regional function in the PIZ. METHODS AND RESULTS: CMR studies were performed at six time points up to 90 days after induction of myocardial infarction (MI) in eight minipigs with reperfused, anterior-septal infarcts. Custom signal density threshold algorithms, based on the remote myocardium, were applied to define the infarct core and PIZ region for each time point. After the initial post-MI edema subsided, the PIZ decreased by 54% from day 10 to day 90 (p = 0.04). The size of infarct scar expanded by 14% and thinned by 56% from day 3 to 12 weeks (p = 0.004 and p < 0.001, respectively). LVEDV increased from 34.7. ± 2.2 ml to 47.8 ± 3.0 ml (day 3 and week 12, respectively; p < 0.001). At 30 days post-MI, regional circumferential strain was increased between the infarct scar and the PIZ (-2.1 ± 0.6 and -6.8 ± 0.9, respectively;* p < 0.05). CONCLUSIONS: The PIZ is dynamic and decreases in mass following reperfused MI. Tensile forces in the PIZ undergo changes following MI. Remodeling characteristics of the PIZ may provide mechanistic insights into the development of life-threatening arrhythmias and sudden cardiac death post-MI.
