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

Abstract

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.

Understanding the Phase Behavior of a Multistimuli-Responsive Elastin-like Polymer: Insights from Dynamic Light Scattering Analysis

“…Key areas for exploration include the effect of cosolute molecules on rates of ELP assembly, the kinetics and hysteresis of the phase transition, how the sensitivity of an ELP to a given cosolute is encoded in its sequence, the influence of molecular crowding, and the thermodynamics of the various competing interactions present in the system. Along similar lines, we celebrate recent methodological advances in single live-cell imaging for the intracellular measurement of ELP T t . A deeper understanding of how cosolutes modulate ELP LCST behavior will facilitate their use as stimuli-responsive polymeric materials in various and dynamic environments and will be especially important to fully realize their promise within biological and industrial sensing applications.…”

“…Along similar lines, we celebrate recent methodological advances in single live-cell imaging for the intracellular measurement of ELP T t . 61 A deeper understanding of how cosolutes modulate ELP LCST behavior will facilitate their use as stimuli-responsive polymeric materials in various and dynamic environments and will be especially important to fully realize their promise within biological and industrial sensing applications.…”

Hydra-Elastin-like Polypeptides Increase Rapamycin Potency When Targeting Cell Surface GRP78

“…Poly-D lysine (Sigma, #P0899)-treated cover slips (VWR, #483830-046) were plated with BT474 cells in complete Hybri-Care media and cultured to 70% confluence. Cells were starved without FBS for 24 h before treatment with 5 μM of either 5FA, L-5FA, or neither at 4 °C for 1 h. Cells were either fixed or incubated at 37 °C for 3 h and then fixed as previously described; , cells were fixed in 4% paraformaldehyde (Alfa Aesar, #43368) and neutralized in 50 mM ammonium chloride (Alfa Aesar, #L05181). Cells were permeabilized with triton X-100 (Sigma, #T8787) and then blocked in 1% BSA (Sigma, #A9647) before an overnight incubation in the 1:100 mouse α-ELP primary antibody at 4 °C .…”