Single-Cell Quantification of the Transition Temperature of Intracellular Elastin-like Polypeptides

Elastin-like polypeptides (ELPs) are modular, stimuli-responsive materials that self-assemble into protein-rich microdomains in response to heating. By cloning ELPs to effector proteins, expressed intracellular fusions can even modulate cellular pathways. A critical step in engineering these fusions is to determine and control their intracellular phase transition temperature (Tt). To do so, this Method paper describes a simple live-cell imaging technique to estimate the Tt of non-fluorescent ELP fusion proteins by co-transfection with a fluorescent ELP marker. Intracellular microdomain formation can then be visualized in live cells through the co-assembly of the non-fluorescent and fluorescent ELP fusion proteins. If the two ELP fusions have different Tt, the intracellular ELP mixture phase separates at the temperature corresponding to the fusion with the lower Tt. In addition, co-assembled ELP microdomains often exhibit pronounced differences in size or number, compared to single transfected treatments. These features enable live-cell imaging experiments and image analysis to determine the intracellular Tt of a library of related ELP fusions. As a case study, we employ the recently reported Caveolin1-ELP library (CAV1-ELPs). In addition to providing a detailed protocol, we also report the development of a useful FIJI plugin named SIAL (Simple Image Analysis Library), which contains programs for image randomization and blinding, phenotype scoring, and ROI selection. These tasks are important parts of the protocol detailed here and are also commonly employed in other image analysis workflows.