A Tethered Vesicle Assay for High-Throughput Quantification of Membrane Fission.
| Autor: | Snead WT; Department of Biomedical Engineering, The University of Texas at Austin, Austin, TX, United States., Stachowiak JC; Department of Biomedical Engineering, The University of Texas at Austin, Austin, TX, United States; Institute for Cellular and Molecular Biology, The University of Texas at Austin, Austin, TX, United States. Electronic address: jcstach@austin.utexas.edu. |
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| Jazyk: | angličtina |
| Zdroj: | Methods in enzymology [Methods Enzymol] 2018; Vol. 611, pp. 559-582. Date of Electronic Publication: 2018 Sep 25. |
| DOI: | 10.1016/bs.mie.2018.08.014 |
| Abstrakt: | 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. (© 2018 Elsevier Inc. All rights reserved.) |
| Databáze: | MEDLINE |
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