Flipping the Switch on Clathrin-Mediated Endocytosis using Thermally Responsive Protein Microdomains.

Pastuszka, Martha K.; Okamoto, Curtis T.; Hamm‐Alvarez, Sarah F.; MacKay, J. Andrew

A ubiquitous approach to studying protein function is to knock down activity (gene deletions, siRNA, small molecule inhibitors, etc.) and to study the cellular effects. Using a new methodology, this article 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. It is hypothesized 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, ELPs are fused 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.

ENDOCYTOSIS; PROTEINS; POLYPEPTIDES; TEMPERATURE control; SYNTHETIC biology; CYTOLOGY

Advanced Functional Materials, 2014, Vol 24, Issue 34, p5340

1616-301X

Academic Journal

10.1002/adfm.201400715