High-content imaging of unbiased chemical perturbations reveals that the phenotypic plasticity of the actin cytoskeleton is constrained

Although F-actin has a large number of binding partners and regulators, the number of phenotypic states available to the actin cytoskeleton is unknown. Here, we quantified 74 features defining F-actin and cellular morphology in >25 million cells after treatment with a library of 114,400 structurally diverse compounds. After reducing the dimensionality of these data, we found that only ~25 recurrent F-actin phenotypes emerged, each defined by distinct quantitative features that could be machine learned. We identified 2003 unknown compounds as inducers of actin-related phenotypes, including two that directly bind talin, a core regulator of integrin activity. Moreover, we observed that compounds with distinct molecular mechanisms could induce equivalent phenotypes and that initially divergent cellular responses could converge over time. These findings suggest a conceptual parallel between the actin cytoskeleton and gene regulatory networks; where the theoretical plasticity of interactions is nearly infinite, yet phenotypes observed in vivo are constrained into a limited subset of practicable configurations.