Abstract
Ras is a signaling protein involved in a variety of cellular processes. Hence, studying Ras signaling with high spatiotemporal resolution is crucial to understanding the roles of Ras in many important cellular functions. Previously, fluorescence lifetime imaging (FLIM) of fluorescent resonance energy transfer (FRET)-based Ras activity sensors, FRas and FRas-F, have been demonstrated to be useful for measuring the spatiotemporal dynamics of Ras signaling in subcellular micro-compartments. However the predominantly nuclear localization of the sensors' acceptor has limited its sensitivity. Here, we have overcome this limitation and developed two variants of the existing FRas sensor with different affinities: FRas2-F (Kd∼1.7 µM) and FRas2-M (Kd∼0.5 µM). We demonstrate that, under 2-photon fluorescence lifetime imaging microscopy, FRas2 sensors provide higher sensitivity compared to previous sensors in 293T cells and neurons.
Highlights
Ras is a member of a large family of small GTPase proteins that bind to and hydrolyze guanosine triphosphate (GTP) into guanosine diphosphate (GDP) [1]
It is crucial to measure the spatiotemporal dynamics of Ras signaling to understand how it regulates its diverse downstream targets
When 293T cells were transfected with FRas-F (Figure 1C), we observed that mEGFP-HRas was localized at the plasma membrane and internal membranes (Figure 1C), to endogenous HRas [26]
Summary
Ras is a member of a large family of small GTPase proteins that bind to and hydrolyze guanosine triphosphate (GTP) into guanosine diphosphate (GDP) [1]. Major subtypes include H-, N- and K-Ras, and all of these subtypes express ubiquitously [1]. When bound to GTP, Ras is active and able to bind and activate downstream effectors; whereas when bound to GDP, it is inactive [5]. Tight spatiotemporal regulation of Ras activity is central to the activation of specific signaling pathways in order to achieve appropriate biological outcomes [2,9]. It is crucial to measure the spatiotemporal dynamics of Ras signaling to understand how it regulates its diverse downstream targets
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