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.