Unraveling signal transduction: extended single-molecule visualization in living cells

The National Institutes of Health Office of the Director awarded The Broad Institute, Inc. $831,250 on September 1, 2026, under the Trans-NIH Research Support program (CFDA 93.310) to develop novel nanoprobes and advanced imaging techniques that extend single-molecule fluorescence microscopy capabilities in living cells.

The project addresses fundamental limitations in visualizing G-protein-coupled receptor (GPCR) signal transduction. Current single-molecule imaging methods photobleach within tens of seconds and support fewer than four simultaneous colors, preventing observation of protein dynamics over extended timescales and in native cellular environments. The Broad Institute will develop nanoprobes and cutting-edge imaging techniques to enable single-molecule visualization with more than 20 colors and essentially infinite imaging duration, allowing continuous real-time monitoring of GPCR activation and signal transduction in living cells. This capability directly supports drug discovery, as GPCRs are the target of approximately 35% of FDA-approved drugs.

The award period runs from September 1, 2026, through August 31, 2031. Work is performed in Cambridge, Massachusetts. This is a project grant, a form of assistance funding supporting research in a defined scientific area for a fixed period.

Abstract

Cellular signaling involves cascades of protein-protein interactions and relies on precise spatial organization and temporal dynamics in cells. Specifically, g-protein-coupled receptors (gpcrs) mediate the signaling of many hormones and neurotransmitters, and dysregulation of gpcr signaling can lead to diseases ranging from neuropsychological disorders, immunoinflammatory disease, and cardiovascular disease. Consequently, gpcrs are the most-drugged family of proteins with about 35% of FDA-approved drugs targeting them. Despite the importance, how gpcrs are differentially activated and how gpcrs transduce signals in living cells is still incompletely understood. Most studies have relied on bulk biochemical assays or structural techniques. However, these methods only measure population-averaged snapshots, and cannot monitor the sequence of events with sufficient time resolution. More importantly, they are unable to measure protein dynamics in the native environment - living cells. In contrast, single-molecule fluorescence microscopy is a powerful tool to visualize molecular interactions and transient events in real-time and in living cells. However, our ability to continuously record single-molecule movies using traditional probes is limited by their photo-instability. Dyes or fluorescent proteins photobleach within tens of seconds, preventing US from obtaining essential dynamic information that occur on a much longer timescale. Moreover, the substantial overlap between the broad emission spectra of these probes limits the number of colors one can simultaneously image to fewer than four. This proposal will overcome this long-standing challenge through (1) the development of novel nanoprobes and two cutting-edge imaging techniques to advance single-molecule microscopy in living cells with more than 20 colors and essentially infinite imaging duration; and (2) apply these techniques to interrogate live-cell dynamics of gpcr signaling. I will develop multiplexed ultralong-term single-particle tracking (SPT) and long-range single-molecule förster resonant energy transfer (smfret) leveraging our recently developed upconverting nanoparticles (ucnps) whose luminescence emission is stable over years. These techniques will enable various investigations such as: (a) the dynamics of receptor dimerization and higher-order assembly in live cells; (b) the stability and turnover of these dimers and how they are regulated by different ligands; (c) the dynamics of intracellular effector protein recruitment; and (d) the non-canonical gpcr signaling pathway. Overall, we will develop novel probes and microscopy that open new avenues for monitoring the complete life cycle of individual biomolecules with essentially unlimited imaging duration and exceptional spatial and temporal resolution, providing an unprecedented view into molecular processes in living systems. More importantly, these new techniques can be broadly applied to unravel the molecular mechanisms underlying various gpcr signaling pathways and other biological processes associated with human diseases.