The actin cytoskeleton is pivotal to many cellular processes, from migration to proliferation and protein trafficking to polarisation. It critically underpins numerous physiological and pathological phenomena, including tumourigenesis and metastasis. Although drug-based modulation of this system is a clinically significant goal, actin itself is a poor drug target, being highly conserved and widely expressed. Instead, identifying drugs selectively targeting signalling pathways or binding partners that modulate actin may provide more precise clinical / research tools.
To this end, we performed a massive drug screen to identify molecules perturbing the actin cytoskeleton. Using fluorescence imaging of F-actin to detect phenotypic responses, we assessed > 114k structurally diverse drugs and > 10k positive / negative controls. Generating ~ 2M images of ~ 60M cells, quantitative image analyses extracted 95 phenotypic features per cell, totalling ~ 6B measurements.
To interrogate this actin-focused big data, we constructed a Systems Microscopy-based analytical framework linking images, image-derived quantitative data and drug structures via an integrated suite of statistical, machine learning and data visualization tools. Using this framework, we have, for example: identified 31 distinct actin phenotypes, including 7 naturally occurring and 24 drug-induced; delineated key features distinguishing these phenotypes; discovered > 250 new drugs that induce phenotypes matching known regulators (e.g. Latrunculin, Jasplakinolide, Y27632), thereafter using biochemistry to parse known vs novel mechanisms; discovered ~ 2500 drugs inducing 21 novel phenotypes; and visually matched published phenotypes to generate molecular-targeting hypotheses, discovering, for example, novel Tropomyosin- and Talin-targeting drugs.
Collectively, this drug library, screen-derived data and Systems Microscopy analysis framework constitute exciting tools for actin-focused research questions, from the systemic (what actin phenotypes are possible?) to the specific (what is the mechanism of drug ‘x’?). This exemplifies how Systems Microscopy approaches can catalyse rapid advances in basic and translational cell biology research.