Dataset Information

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.

SUBMITTER: Pastuszka MK

PROVIDER: S-EPMC4235962 | biostudies-literature | 2014 Sep

REPOSITORIES: biostudies-literature

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

Pastuszka Martha K MK Okamoto Curtis T CT Hamm-Alvarez Sarah F SF MacKay J Andrew JA

Advanced functional materials 20140901 34

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 macromol ...[more]

PMID: 25419208

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Project description:In eukaryotic cells, the internalization of extracellular cargo via the endocytic machinery is an important regulatory process required for many essential cellular functions. The role of cooperative protein-protein and protein-membrane interactions in the ubiquitous endocytic pathway in mammalian cells, namely the clathrin-dependent endocytosis, remains unresolved. We employ the Helfrich membrane Hamiltonian together with surface evolution methodology to address how the shapes and energetics of vesicular-bud formation in a planar membrane are stabilized by presence of the clathrin-coat assembly. Our results identify a unique dual role for the tubulating protein epsin: multiple epsins localized spatially and orientationally collectively play the role of a curvature inducing capsid; in addition, epsin serves the role of an adapter in binding the clathrin coat to the membrane. Our results also suggest an important role for the clathrin lattice, namely in the spatial- and orientational-templating of epsins. We suggest that there exists a critical size of the coat above which a vesicular bud with a constricted neck resembling a mature vesicle is stabilized. Based on the observed strong dependence of the vesicle diameter on the bending rigidity, we suggest that the variability in bending stiffness due to variations in membrane composition with cell type can explain the experimentally observed variability on the size of clathrin-coated vesicles, which typically range 50-100 nm. Our model also provides estimates for the number of epsins involved in stabilizing a coated vesicle, and without any direct fitting reproduces the experimentally observed shapes of vesicular intermediates as well as their probability distributions quantitatively, in wildtype as well as CLAP IgG injected neuronal cell experiments. We have presented a minimal mesoscale model which quantitatively explains several experimental observations on the process of vesicle nucleation induced by the clathrin-coated assembly prior to vesicle scission in clathrin dependent endocytosis.

Dataset Information

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

Publications

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

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