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
The parasite Trypanasoma brucei causes African trypanosomiasis, known as sleeping sickness in humans and nagana in domestic animals. These diseases are a major burden in the 36 sub-Saharan African countries where the tsetse fly vector is endemic. Untreated trypanosomiasis is fatal and the current treatments are stage-dependent and can be problematic during the meningoencephalitic stage, where no new therapies have been developed in recent years and the current drugs have a low therapeutic index. There is a need for more effective treatments and a better understanding of how these parasites evade the host immune response will help in this regard. The bloodstream form of T. brucei excretes significant amounts of aromatic ketoacids, including indolepyruvate, a transamination product of tryptophan. This study demonstrates that this process is essential in bloodstream forms, is mediated by a specialized isoform of cytoplasmic aminotransferase and, importantly, reveals an immunomodulatory role for indolepyruvate. Indolepyruvate prevents the LPS-induced glycolytic shift in macrophages. This effect is the result of an increase in the hydroxylation and degradation of the transcription factor hypoxia-inducible factor-1α (HIF-1α). The reduction in HIF-1α levels by indolepyruvate, following LPS or trypanosome activation, results in a decrease in production of the proinflammatory cytokine IL-1ß. These data demonstrate an important role for indolepyruvate in immune evasion by T. brucei.