Single-fluorophore biosensors for sensitive and multiplexed detection of signalling activities

Abstract

Unravelling the dynamic molecular interplay behind complex physiological processes such as neuronal plasticity requires the ability to both detect minute changes in biochemical states in response to physiological signals and track multiple signalling activities simultaneously. Fluorescent protein-based biosensors have enabled the real-time monitoring of dynamic signalling processes within the native context of living cells, yet most commonly used biosensors exhibit poor sensitivity (for example, due to low dynamic range) and are limited to imaging signalling activities in isolation. Here, we address this challenge by developing a suite of excitation ratiometric kinase activity biosensors that offer the highest reported dynamic range and enable the detection of subtle changes in signalling activity that could not be reliably detected previously, as well as a suite of single-fluorophore biosensors that enable the simultaneous tracking of as many as six distinct signalling activities in single living cells.

Genetically encoded fluorescent biosensors illuminate kinase signaling in cancer

“…Continuing with this same substrate-cpFP-PAABD configuration, Mehta et al (35) were also able to develop single-color KARs based on cp-T-sapphire, which exhibits single excitation and emission maxima at ϳ400 and ϳ513 nm, respectively, and cpBFP, which displays single excitation and emission peaks at ϳ385 and ϳ450 nm, respectively. By utilizing an alternative design based on FP dimerization (36), they were further able to develop red fluorescent single-color KARs (Fig.…”

“…However, although numerous single-fluorophore sensors have been developed to monitor different cellular analytes, such as calcium (31,32), cAMP (33), and metabolic products (34), the application of this sensor design to visualize kinase activity has been largely unexplored. To this end, Mehta et al (35) recently engineered a series of single-fluorophore biosensors in which cpGFP is sandwiched between a kinase substrate peptide and PAABD, giving rise to a novel suite of excitation ratiometric kinase activity reporters (ExRai-KARs), wherein the maximum excitation peak of cpGFP undergoes a phosphorylation-dependent shift from ϳ400 to ϳ488 nm, with ϳ515-nm (green) emission, in response to elevated kinase activity (Fig. 1B).…”

“…Given that complex cell behaviors are regulated by highly integrated kinase signaling networks, these single-color kinase sensors are highly desirable tools for simultaneously examining multiple kinase activities, as well as the cross-regulation between kinases, in single cells. Indeed, through the use of their singlecolor KARs, Mehta et al (35) were able to successfully perform 4 -6-parameter multiplexed imaging to simultaneously monitor changes in kinases activities, as well as cAMP and Ca 2ϩ elevations, in single cells.…”

Control of Akt activity and substrate phosphorylation in cells

“…Discriminating between these two scenarios is important, as they involve alternative mechanisms of Akt activation. The first scenario, where phosphorylated Akt is imported into the nucleus from the cytosol, is traditionally supported by the delayed phosphorylation of Akt FRET reporters in the nucleus and several imaging studies of Akt localization in fixed cells . Yet, the evidence regarding the dependence of Akt nuclear accumulation on its phosphorylation state is at best contradictory .…”

Live-imaging of endothelial Erk activity reveals dynamic and sequential signalling events during regenerative angiogenesis

“…The first ERK FRETbased biosensor (ERK activity reporter (EKAR)) was developed in 2008 (Harvey et al, 2008). Since then, modifications had been made to improve its sensitivity and dynamic range to generate other ERK FRET-based biosensors such as EKAR-EV, RAB-EKARev, and sREACh (Komatsu et al, 2011;Ding et al, 2015;Tang and Yasuda, 2017;Mehta et al, 2018). Importantly, these ERK FRET-based biosensors had been applied in vivo to visualise ERK-signalling dynamics in various cell types during development, cell migration, and wound healing (Kamioka et al, 2012;Mizuno et al, 2014;Goto et al, 2015;Hiratsuka et al, 2015;Kamioka et al, 2017;Takeda and Kiyokawa, 2017;Sano et al, 2018;Wong et al, 2018).…”