Visual detection of submicroscopic protein clusters with a phase-separation-based fluorescent reporter

Abstract

ABSTRACTProtein clustering plays numerous roles in cell physiology and disease. However, protein oligomers can be difficult to detect because they are often small and fall below the detection limits of conventional fluorescence microscopy. Existing techniques to visualize such aggregates require specialized microscopy and may require overexpression of the protein of interest, which can introduce clustering artifacts that are not representative of the endogenous protein. Here we describe a fluorescent reporter strategy that detects endogenous protein clustering with high sensitivity, called CluMPS (Clusters Magnified by Phase Separation). A CluMPS reporter detects and visually amplifies even small clusters of a binding partner, generating large, easily quantifiable phase-separated condensates as a readout. We use optogenetic clustering to show that the CluMPS approach can reliably report on target clusters as small as tetramers. Experiments and simulations showed that CluMPS activation depends on the affinity for the target, the target cluster size, and the cluster size distribution. CluMPS detected small aggregates of pathological proteins where the corresponding GFP fusions appeared diffuse. Uniquely, CluMPS permitted visualization of clusters of endogenous proteins, allowing the measurement of drug response kinetics of oncogenic condensates in patient-derived cancer cells. Finally, CluMPS could be multiplexed to report on distinct clustered species in the same cell. CluMPS thus provides a powerful yet straightforward approach to observe higher-order protein assembly in its native cellular context.