Dataset Information

A Tethered Vesicle Assay for High-Throughput Quantification of Membrane Fission.

ABSTRACT: Membrane fission, which divides membrane surfaces into separate compartments, is essential to diverse cellular processes including membrane trafficking and cell division. Quantitative assays are needed to elucidate the physical mechanisms by which proteins drive membrane fission. Toward this goal, several experimental tools have been developed, including visualizing fission products using electron microscopy, measuring membrane shedding from a lipid reservoir, and observing fission of individual membrane tubes pulled from giant vesicles. However, no existing assay of membrane fission provides a quantitative, high-throughput measure of the distribution of vesicle curvatures generated by fission-driving proteins. Toward addressing this challenge, here we describe a novel approach that uses confocal fluorescence imaging to quantify the diameter distribution of membrane vesicles that have been tethered to a coverslip surface following exposure to fission-driving proteins. We employ this assay to measure the progressive appearance of high curvature fission products upon exposure of vesicles to increasing protein concentration. Results from this approach are in quantitative agreement with measurements from electron microscopy, but can be collected with considerably greater throughput, enabling examination of a broad range of experimental conditions. Using the tethered vesicle approach, we have recently found that membrane-bound intrinsically disordered proteins are surprisingly potent drivers of membrane fission. The capacity to drive fission arises from steric pressure generated when disordered domains with large hydrodynamic radii bind to membranes at high local densities. More broadly, the experimental tools described here have the potential to improve our mechanistic understanding of membrane fission in diverse biophysical contexts.

SUBMITTER: Snead WT

PROVIDER: S-EPMC6279246 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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A Tethered Vesicle Assay for High-Throughput Quantification of Membrane Fission.

Snead Wilton T WT Stachowiak Jeanne C JC

Methods in enzymology 20180925

Membrane fission, which divides membrane surfaces into separate compartments, is essential to diverse cellular processes including membrane trafficking and cell division. Quantitative assays are needed to elucidate the physical mechanisms by which proteins drive membrane fission. Toward this goal, several experimental tools have been developed, including visualizing fission products using electron microscopy, measuring membrane shedding from a lipid reservoir, and observing fission of individual ...[more]

PMID: 30471700

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Project description:Mitochondrial respiratory chain disease is caused by a wide range of individually rare genetic disorders that impair cellular energy metabolism. While fluorescence microscopy analysis of nematodes fed MitoTracker Green (MTG) and tetramethylrhodamine ethyl ester (TMRE) can reliably quantify relative mitochondrial density and membrane potential, respectively, in C. elegans models of mitochondrial dysfunction, it is a tedious process with limitations in the number and age of animals that can be studied. A novel, large particle, flow cytometry-based method reported here accelerates and automates the relative quantitation of mitochondrial physiology in nematode populations. Relative fluorescence profiles of nematode populations co-labeled with MTG and TMRE were obtained and analyzed by BioSorter (Union Biometrica). Variables tested included genetic mutation (wild-type N2 Bristol versus nuclear-encoded respiratory chain complex I mutant gas-1(fc21) worms), animal age (day 1 versus day 4 adults), classical respiratory chain inhibitor and uncoupler effects (oligomycin, FCCP), and pharmacologic therapy duration (24h versus 96h treatments with glucose or nicotinic acid). A custom MATLAB script, which can be run on any computer with MATLAB runtime, was written to automatically quantify and analyze results in large animal populations. BioSorter analysis independently validated relative MTG and TMRE changes that we had previously performed by fluorescence microscopy in a variety of experimental conditions, with notably greater animal population sizes and substantially reduced experimental time. Older, fragile animal populations that are difficult to study by microscopy approaches were readily amenable to analysis with the BioSorter method. Overall, this high-throughput method enables efficient relative quantitation of in vivo mitochondrial physiology over time in a living animal in response to gene mutations and candidate therapies, which can be used to accelerate the translation of basic research into optimization of clinical therapies for mitochondrial disease.

Dataset Information

A Tethered Vesicle Assay for High-Throughput Quantification of Membrane Fission.

Publications

A Tethered Vesicle Assay for High-Throughput Quantification of Membrane Fission.

Similar Datasets

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