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Imaging Plasma Membrane Deformations With pTIRFM
To gain novel insights into the dynamics of exocytosis, our group focuses on the changes in lipid bilayer shape that must be precisely regulated during the fusion of vesicle and plasma membranes. These rapid and localized changes are achieved by dynamic interactions between lipids and specialized pr...
Autores principales: | , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MyJove Corporation
2014
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4161200/ https://www.ncbi.nlm.nih.gov/pubmed/24747638 http://dx.doi.org/10.3791/51334 |
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author | Passmore, Daniel R. Rao, Tejeshwar C. Peleman, Andrew R. Anantharam, Arun |
author_facet | Passmore, Daniel R. Rao, Tejeshwar C. Peleman, Andrew R. Anantharam, Arun |
author_sort | Passmore, Daniel R. |
collection | PubMed |
description | To gain novel insights into the dynamics of exocytosis, our group focuses on the changes in lipid bilayer shape that must be precisely regulated during the fusion of vesicle and plasma membranes. These rapid and localized changes are achieved by dynamic interactions between lipids and specialized proteins that control membrane curvature. The absence of such interactions would not only have devastating consequences for vesicle fusion, but a host of other cellular functions that involve control of membrane shape. In recent years, the identity of a number of proteins with membrane-shaping properties has been determined. What remains missing is a roadmap of when, where, and how they act as fusion and content release progress. Our understanding of the molecular events that enable membrane remodeling has historically been limited by a lack of analytical methods that are sensitive to membrane curvature or have the temporal resolution to track rapid changes. PTIRFM satisfies both of these criteria. We discuss how pTIRFM is implemented to visualize and interpret rapid, submicron changes in the orientation of chromaffin cell membranes during dense core vesicle (DCV) fusion. The chromaffin cells we use are isolated from bovine adrenal glands. The membrane is stained with a lipophilic carbocyanine dye,1,1'-dioctadecyl-3,3,3',3'-tetramethylindodicarbocyanine, 4-chlorobenzenesulfonate, or diD. DiD intercalates in the membrane plane with a "fixed" orientation and is therefore sensitive to the polarization of the evanescent field. The diD-stained cell membrane is sequentially excited with orthogonal polarizations of a 561 nm laser (p-pol, s-pol). A 488 nm laser is used to visualize vesicle constituents and time the moment of fusion. Exocytosis is triggered by locally perfusing cells with a depolarizing KCl solution. Analysis is performed offline using custom-written software to understand how diD emission intensity changes relate to fusion pore dilation. |
format | Online Article Text |
id | pubmed-4161200 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2014 |
publisher | MyJove Corporation |
record_format | MEDLINE/PubMed |
spelling | pubmed-41612002014-09-17 Imaging Plasma Membrane Deformations With pTIRFM Passmore, Daniel R. Rao, Tejeshwar C. Peleman, Andrew R. Anantharam, Arun J Vis Exp Biochemistry To gain novel insights into the dynamics of exocytosis, our group focuses on the changes in lipid bilayer shape that must be precisely regulated during the fusion of vesicle and plasma membranes. These rapid and localized changes are achieved by dynamic interactions between lipids and specialized proteins that control membrane curvature. The absence of such interactions would not only have devastating consequences for vesicle fusion, but a host of other cellular functions that involve control of membrane shape. In recent years, the identity of a number of proteins with membrane-shaping properties has been determined. What remains missing is a roadmap of when, where, and how they act as fusion and content release progress. Our understanding of the molecular events that enable membrane remodeling has historically been limited by a lack of analytical methods that are sensitive to membrane curvature or have the temporal resolution to track rapid changes. PTIRFM satisfies both of these criteria. We discuss how pTIRFM is implemented to visualize and interpret rapid, submicron changes in the orientation of chromaffin cell membranes during dense core vesicle (DCV) fusion. The chromaffin cells we use are isolated from bovine adrenal glands. The membrane is stained with a lipophilic carbocyanine dye,1,1'-dioctadecyl-3,3,3',3'-tetramethylindodicarbocyanine, 4-chlorobenzenesulfonate, or diD. DiD intercalates in the membrane plane with a "fixed" orientation and is therefore sensitive to the polarization of the evanescent field. The diD-stained cell membrane is sequentially excited with orthogonal polarizations of a 561 nm laser (p-pol, s-pol). A 488 nm laser is used to visualize vesicle constituents and time the moment of fusion. Exocytosis is triggered by locally perfusing cells with a depolarizing KCl solution. Analysis is performed offline using custom-written software to understand how diD emission intensity changes relate to fusion pore dilation. MyJove Corporation 2014-04-02 /pmc/articles/PMC4161200/ /pubmed/24747638 http://dx.doi.org/10.3791/51334 Text en Copyright © 2014, Journal of Visualized Experiments http://creativecommons.org/licenses/by-nc-nd/3.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported License. To view a copy of this license, visithttp://creativecommons.org/licenses/by-nc-nd/3.0/ |
spellingShingle | Biochemistry Passmore, Daniel R. Rao, Tejeshwar C. Peleman, Andrew R. Anantharam, Arun Imaging Plasma Membrane Deformations With pTIRFM |
title | Imaging Plasma Membrane Deformations With pTIRFM |
title_full | Imaging Plasma Membrane Deformations With pTIRFM |
title_fullStr | Imaging Plasma Membrane Deformations With pTIRFM |
title_full_unstemmed | Imaging Plasma Membrane Deformations With pTIRFM |
title_short | Imaging Plasma Membrane Deformations With pTIRFM |
title_sort | imaging plasma membrane deformations with ptirfm |
topic | Biochemistry |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4161200/ https://www.ncbi.nlm.nih.gov/pubmed/24747638 http://dx.doi.org/10.3791/51334 |
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