RESUMEN
Identifying the interacting partners and the dynamics of the molecular networks constitutes the key point in understanding cellular processes. Different methods often based on energy transfer strategies have been developed to examine the molecular dynamics of protein complexes. However, these methods suffer a couple of drawbacks: a single complex can be studied at a time, and its localization and tracking cannot generally be investigated. Here, we report a multicolor time-resolved Förster resonance energy transfer microscopy method that allows the identification of up to 3 different complexes simultaneously, their localization in cells, and their tracking after activation. Using this technique, we studied GPCR oligomerization and internalization in human embryonic kidney 293 cells. We definitively show that receptors can internalize as oligomers and that receptor coexpression deeply impacts oligomer internalization processes.
Asunto(s)
Endocitosis , Transferencia Resonante de Energía de Fluorescencia/métodos , Microscopía Fluorescente/métodos , Multimerización de Proteína , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/metabolismo , Animales , Células CHO , Cricetinae , Cricetulus , Transferencia Resonante de Energía de Fluorescencia/instrumentación , Células HEK293 , Humanos , Microscopía Fluorescente/instrumentación , Receptores de Vasopresinas/agonistas , Receptores de Vasopresinas/química , Receptores de Vasopresinas/metabolismo , Reproducibilidad de los Resultados , Imagen de Lapso de Tiempo/instrumentación , Imagen de Lapso de Tiempo/métodosRESUMEN
We report the design and evaluation of pH responsive luminescent europium(iii) probes that allow conjugation to targeting vectors to monitor receptor internalisation in cells. The approach adopted here can be used to tag proteins selectively and to monitor uptake into more acidic organelles, thereby enhancing the performance of time-resolved internalisation assays that require pH monitoring in real time.