title: Flipping the Switch on Clathrin-Mediated Endocytosis using Thermally Responsive Protein Microdomains.
abstract: A ubiquitous approach to study protein function is to knock down activity (gene deletions, siRNA, small molecule inhibitors, etc) and study the cellular effects. Using a new methodology, this manuscript describes how to rapidly and specifically switch off cellular pathways using thermally responsive protein polymers. A small increase in temperature stimulates cytosolic elastin-like polypeptides (ELPs) to assemble microdomains. We hypothesize that ELPs fused to a key effector in a target macromolecular complex will sequester the complex within these microdomains, which will bring the pathway to a halt. To test this hypothesis, we fused ELPs to clathrin-light chain (CLC), a protein associated with clathrin-mediated endocytosis. Prior to thermal stimulation, the ELP fusion is soluble and clathrin-mediated endocytosis remains 'on.' Increasing the temperature induces the assembly of ELP fusion proteins into organelle-sized microdomains that switches clathrin-mediated endocytosis 'off.' These microdomains can be thermally activated and inactivated within minutes, are reversible, do not require exogenous chemical stimulation, and are specific for components trafficked within the clathrin-mediated endocytosis pathway. This temperature-triggered cell switch system represents a new platform for the temporal manipulation of trafficking mechanisms in normal and disease cell models and has applications for manipulating other intracellular pathways.
authors.affiliation: Department of Pharmacology and Pharmaceutical Sciences, University of Southern California. 1985 Zonal Avenue, Los Angeles 90033-9121.
authors.forename: Martha K
authors.initials: MK
authors.lastname: Pastuszka
authors.affiliation: Department of Pharmacology and Pharmaceutical Sciences, University of Southern California. 1985 Zonal Avenue, Los Angeles 90033-9121.
authors.forename: Curtis T
authors.initials: CT
authors.lastname: Okamoto
authors.affiliation: Department of Pharmacology and Pharmaceutical Sciences, University of Southern California. 1985 Zonal Avenue, Los Angeles 90033-9121.
authors.forename: Sarah F
authors.initials: SF
authors.lastname: Hamm-Alvarez
authors.affiliation: Department of Pharmacology and Pharmaceutical Sciences, University of Southern California. 1985 Zonal Avenue, Los Angeles 90033-9121.
authors.forename: J Andrew
authors.initials: JA
authors.lastname: MacKay
keywords: Synthetic biology; clathrin-light chain; clathrin-mediated endocytosis; elastin-like polypeptide; inverse phase transition temperature