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A simple, highly sensitive, and facile method to quantify ceramide at the plasma membrane
The role of ceramide in biological functions is typically based on the elevation of cellular ceramide, measured by LC-MS in the total cell lysate. However, it has become increasingly appreciated that ceramide in different subcellular organelles regulates specific functions. In the plasma membrane, c...
Autores principales: | , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
American Society for Biochemistry and Molecular Biology
2022
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9853358/ https://www.ncbi.nlm.nih.gov/pubmed/36549592 http://dx.doi.org/10.1016/j.jlr.2022.100322 |
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author | Greene, Meaghan Hernandez-Corbacho, Maria Jose Ostermeyer-Fay, Anne G. Hannun, Yusuf A. Canals, Daniel |
author_facet | Greene, Meaghan Hernandez-Corbacho, Maria Jose Ostermeyer-Fay, Anne G. Hannun, Yusuf A. Canals, Daniel |
author_sort | Greene, Meaghan |
collection | PubMed |
description | The role of ceramide in biological functions is typically based on the elevation of cellular ceramide, measured by LC-MS in the total cell lysate. However, it has become increasingly appreciated that ceramide in different subcellular organelles regulates specific functions. In the plasma membrane, changes in ceramide levels might represent a small percentage of the total cellular ceramide, evading MS detection but playing a critical role in cell signaling. Importantly, there are currently no efficient techniques to quantify ceramide in the plasma membrane. Here, we developed a method to measure the mass of ceramide in the plasma membrane using a short protocol that is based on the hydrolysis of plasma membrane ceramide into sphingosine by the action of exogenously applied bacterial recombinant neutral ceramidase. Plasma membrane ceramide content can then be determined by measuring the newly generated sphingosine at a stoichiometry of 1:1. A key step of this protocol is the chemical fixation of cells to block cellular sphingolipid metabolism, especially of sphingosine to sphingosine 1-phosphate. We confirmed that chemical fixation does not disrupt the lipid composition at the plasma membrane, which remains intact during the time of the assay. We illustrate the power of the approach by applying this protocol to interrogate the effects of the chemotherapeutic compound doxorubicin. Here we distinguished two pools of ceramide, depending on the doxorubicin concentration, consolidating different reports. In summary, we have developed the first approach to quantify ceramide in the plasma membrane, allowing the study of new avenues in sphingolipid compartmentalization and function. |
format | Online Article Text |
id | pubmed-9853358 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | American Society for Biochemistry and Molecular Biology |
record_format | MEDLINE/PubMed |
spelling | pubmed-98533582023-01-24 A simple, highly sensitive, and facile method to quantify ceramide at the plasma membrane Greene, Meaghan Hernandez-Corbacho, Maria Jose Ostermeyer-Fay, Anne G. Hannun, Yusuf A. Canals, Daniel J Lipid Res Methods The role of ceramide in biological functions is typically based on the elevation of cellular ceramide, measured by LC-MS in the total cell lysate. However, it has become increasingly appreciated that ceramide in different subcellular organelles regulates specific functions. In the plasma membrane, changes in ceramide levels might represent a small percentage of the total cellular ceramide, evading MS detection but playing a critical role in cell signaling. Importantly, there are currently no efficient techniques to quantify ceramide in the plasma membrane. Here, we developed a method to measure the mass of ceramide in the plasma membrane using a short protocol that is based on the hydrolysis of plasma membrane ceramide into sphingosine by the action of exogenously applied bacterial recombinant neutral ceramidase. Plasma membrane ceramide content can then be determined by measuring the newly generated sphingosine at a stoichiometry of 1:1. A key step of this protocol is the chemical fixation of cells to block cellular sphingolipid metabolism, especially of sphingosine to sphingosine 1-phosphate. We confirmed that chemical fixation does not disrupt the lipid composition at the plasma membrane, which remains intact during the time of the assay. We illustrate the power of the approach by applying this protocol to interrogate the effects of the chemotherapeutic compound doxorubicin. Here we distinguished two pools of ceramide, depending on the doxorubicin concentration, consolidating different reports. In summary, we have developed the first approach to quantify ceramide in the plasma membrane, allowing the study of new avenues in sphingolipid compartmentalization and function. American Society for Biochemistry and Molecular Biology 2022-12-20 /pmc/articles/PMC9853358/ /pubmed/36549592 http://dx.doi.org/10.1016/j.jlr.2022.100322 Text en © 2022 The Authors https://creativecommons.org/licenses/by/4.0/This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Methods Greene, Meaghan Hernandez-Corbacho, Maria Jose Ostermeyer-Fay, Anne G. Hannun, Yusuf A. Canals, Daniel A simple, highly sensitive, and facile method to quantify ceramide at the plasma membrane |
title | A simple, highly sensitive, and facile method to quantify ceramide at the plasma membrane |
title_full | A simple, highly sensitive, and facile method to quantify ceramide at the plasma membrane |
title_fullStr | A simple, highly sensitive, and facile method to quantify ceramide at the plasma membrane |
title_full_unstemmed | A simple, highly sensitive, and facile method to quantify ceramide at the plasma membrane |
title_short | A simple, highly sensitive, and facile method to quantify ceramide at the plasma membrane |
title_sort | simple, highly sensitive, and facile method to quantify ceramide at the plasma membrane |
topic | Methods |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9853358/ https://www.ncbi.nlm.nih.gov/pubmed/36549592 http://dx.doi.org/10.1016/j.jlr.2022.100322 |
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