A high-throughput platform for single-molecule tracking identifies drug interaction and cellular mechanisms

• DOI: 10.7554/elife.93183.2
• Publication Date: 2024-05-13T15:22:14Z
• Authors: David T. McSwiggen; Helen Liu; Ruensern Tan; Sebastia Agramunt...; Lakshmi B. Akella; Russell Berman; Mason Bretan; Hanzhe Chen; Xavier Darzacq; Kelsey Ford; Ruth Godbey; Eric Gonzalez; Adi Hanuka; Alec Heckert; Jaclyn J. Ho; Stephanie L. Johnson; Reed Kelso; Aaron Klammer; Ruchira Krishnamu...; Jifu Li; Kevin Lin; Brian Margolin; Patrick McNamara; Laurence Meyer; Sarah E. Pierce; Akshay Sule; Connor Stashko; Yangzhong Tang; Daniel J. Anderson; Hilary P. Beck
• Keywords: 0301 basic medicine; single-molecule imaging; QH301-705.5; Science; live-cell imaging; Cell Line; drug discovery; 03 medical and health sciences; cell biology; physics of living systems; Drug Discovery; Receptors; high-throughput imaging; Humans; Drug Interactions; human; Biology (General); Tumor; Q; R; Cell Biology; Estrogen; Single Molecule Imaging; High-Throughput Screening Assays; Medicine; protein motion; estrogen receptor

The regulation of cell physiology depends largely upon interactions of functionally distinct proteins and cellular components. These interactions may be transient or long-lived, but often affect protein motion. Measurement of protein dynamics within a cellular environment, particularly while perturbing protein function with small molecules, may enable dissection of key interactions and facilitate drug discovery; however, current approaches are limited by throughput with respect to data acquisition and analysis. As a result, studies using super-resolution imaging are typically drawing conclusions from tens of cells and a few experimental conditions tested. We addressed these limitations by developing a high-throughput single-molecule tracking (htSMT) platform for pharmacologic dissection of protein dynamics in living cells at an unprecedented scale (capable of imaging > 10 6 cells/day and screening > 10 4 compounds). We applied htSMT to measure the cellular dynamics of fluorescently tagged estrogen receptor (ER) and screened a diverse library to identify small molecules that perturbed ER function in real time. With this one experimental modality, we determined the potency, pathway selectivity, target engagement, and mechanism of action for identified hits. Kinetic htSMT experiments were capable of distinguishing between on-target and on-pathway modulators of ER signaling. Integrated pathway analysis recapitulated the network of known ER interaction partners and suggested potentially novel, kinase-mediated regulatory mechanisms. The sensitivity of htSMT revealed a new correlation between ER dynamics and the ability of ER antagonists to suppress cancer cell growth. Therefore, measuring protein motion at scale is a powerful method to investigate dynamic interactions among proteins and may facilitate the identification and characterization of novel therapeutics.