Open AccessSingle-Cell Quantification of the Transition Temperature of Intracellular Elastin-like Polypeptides
Author(s) -
David R. Tyrpak,
Yaocun Li,
Siqi Lei,
Hugo Avila,
J. Andrew MacKay
Publication year - 2021
Publication title -
acs biomaterials science and engineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.082
H-Index - 50
ISSN - 2373-9878
DOI - 10.1021/acsbiomaterials.0c01117
Subject(s) - intracellular , transfection , fusion protein , microbiology and biotechnology , green fluorescent protein , lipid microdomain , elastin , biology , computational biology , biophysics , chemistry , cell culture , biochemistry , recombinant dna , genetics , membrane , gene
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 ( T t ). To do so, this Method paper describes a simple live-cell imaging technique to estimate the T t 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 T t , the intracellular ELP mixture phase separates at the temperature corresponding to the fusion with the lower T t . 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 T t 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.