RESUMEN

We developed a genetically encoded, highly specific fluorescent probe for detecting hydrogen peroxide (H(2)O(2)) inside living cells. This probe, named HyPer, consists of circularly permuted yellow fluorescent protein (cpYFP) inserted into the regulatory domain of the prokaryotic H(2)O(2)-sensing protein, OxyR. Using HyPer we monitored H(2)O(2) production at the single-cell level in the cytoplasm and mitochondria of HeLa cells treated with Apo2L/TRAIL. We found that an increase in H(2)O(2) occurs in the cytoplasm in parallel with a drop in the mitochondrial transmembrane potential (DeltaPsi) and a change in cell shape. We also observed local bursts in mitochondrial H(2)O(2) production during DeltaPsi oscillations in apoptotic HeLa cells. Moreover, sensitivity of the probe was sufficient to observe H(2)O(2) increase upon physiological stimulation. Using HyPer we detected temporal increase in H(2)O(2) in the cytoplasm of PC-12 cells stimulated with nerve growth factor.

Asunto(s)

Proteínas Bacterianas/química , Técnicas Biosensibles/métodos , Peróxido de Hidrógeno/análisis , Membranas Intracelulares/metabolismo , Proteínas Luminiscentes/química , Animales , Proteínas Reguladoras de la Apoptosis/farmacología , Proteínas Bacterianas/genética , Citoplasma/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas de Escherichia coli/metabolismo , Código Genético , Células HeLa/metabolismo , Células HeLa/ultraestructura , Humanos , Peróxido de Hidrógeno/metabolismo , Proteínas Luminiscentes/genética , Mitocondrias/metabolismo , Factores de Crecimiento Nervioso/farmacología , Células PC12/metabolismo , Células PC12/ultraestructura , Células Procariotas/metabolismo , Ratas , Proteínas Represoras/metabolismo , Sensibilidad y Especificidad , Factores de Tiempo , Factores de Transcripción/metabolismo