Visualization of Ceramide-Associated Proteins in Ceramide-Rich Platforms Using a Cross-Linkable Ceramide Analog and Proximity Ligation Assays With Anti-ceramide Antibody
Ceramide-rich platforms (CRPs) mediate association of proteins with the sphingolipid ceramide and may regulate protein interaction in membrane contact sites to the cytoskeleton, organelles, and infectious pathogens. However, visualization of ceramide association to proteins is one of the greatest ch...
Autores principales: | , , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Frontiers Media S.A.
2019
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6706757/ https://www.ncbi.nlm.nih.gov/pubmed/31475148 http://dx.doi.org/10.3389/fcell.2019.00166 |
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author | Jiang, Xue Zhu, Zhihui Qin, Haiyan Tripathi, Priyanka Zhong, Liansheng Elsherbini, Ahmed Karki, Sanjib Crivelli, Simone M. Zhi, Wenbo Wang, Guanghu Spassieva, Stefanka D. Bieberich, Erhard |
author_facet | Jiang, Xue Zhu, Zhihui Qin, Haiyan Tripathi, Priyanka Zhong, Liansheng Elsherbini, Ahmed Karki, Sanjib Crivelli, Simone M. Zhi, Wenbo Wang, Guanghu Spassieva, Stefanka D. Bieberich, Erhard |
author_sort | Jiang, Xue |
collection | PubMed |
description | Ceramide-rich platforms (CRPs) mediate association of proteins with the sphingolipid ceramide and may regulate protein interaction in membrane contact sites to the cytoskeleton, organelles, and infectious pathogens. However, visualization of ceramide association to proteins is one of the greatest challenges in understanding the cell biology of ceramide. Here we introduce a novel labeling technique for ceramide-associated proteins (CAPs) by combining photoactivated cross-linking of a bioorthogonal and bifunctional ceramide analog, pacFACer with proximity ligation assays (PLAs). pacFACer cross-linked to CAPs is covalently attached to a fluorophore using click chemistry. PLAs use antibodies to: (1) the candidate CAP and the fluorophore (PLA1); and (2) the CAP and ceramide (PLA2). PLA1 shows the subcellular localization of a particular CAP that is cross-linked to pacFACer, while PLA2 tests if the cross-linked CAP forms a complex with endogenous ceramide. Two proteins, tubulin and voltage-dependent anion channel 1 (VDAC1), were cross-linked to pacFACer and showed PLA signals for a complex with ceramide and pacFACer, which were predominantly colocalized with microtubules and mitochondria, respectively. Binding of tubulin and VDAC1 to ceramide was confirmed by coimmunoprecipitation assays using anti ceramide antibody. Cross-linking to pacFACer was confirmed using click chemistry-mediated attachment of biotin and streptavidin pull-down assays. Inhibition of ceramide synthases with fumonisin B1 (FB1) reduced the degree of pacFACer cross-linking and complex formation with ceramide, while it was enhanced by amyloid beta peptide (Aβ). Our results show that endogenous ceramide is critical for mediating cross-linking of CAPs to pacFACer and that a combination of cross-linking with PLAs (cross-link/PLA) is a novel tool to visualize CAPs and to understand the regulation of protein interaction with ceramide in CRPs. |
format | Online Article Text |
id | pubmed-6706757 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2019 |
publisher | Frontiers Media S.A. |
record_format | MEDLINE/PubMed |
spelling | pubmed-67067572019-08-30 Visualization of Ceramide-Associated Proteins in Ceramide-Rich Platforms Using a Cross-Linkable Ceramide Analog and Proximity Ligation Assays With Anti-ceramide Antibody Jiang, Xue Zhu, Zhihui Qin, Haiyan Tripathi, Priyanka Zhong, Liansheng Elsherbini, Ahmed Karki, Sanjib Crivelli, Simone M. Zhi, Wenbo Wang, Guanghu Spassieva, Stefanka D. Bieberich, Erhard Front Cell Dev Biol Cell and Developmental Biology Ceramide-rich platforms (CRPs) mediate association of proteins with the sphingolipid ceramide and may regulate protein interaction in membrane contact sites to the cytoskeleton, organelles, and infectious pathogens. However, visualization of ceramide association to proteins is one of the greatest challenges in understanding the cell biology of ceramide. Here we introduce a novel labeling technique for ceramide-associated proteins (CAPs) by combining photoactivated cross-linking of a bioorthogonal and bifunctional ceramide analog, pacFACer with proximity ligation assays (PLAs). pacFACer cross-linked to CAPs is covalently attached to a fluorophore using click chemistry. PLAs use antibodies to: (1) the candidate CAP and the fluorophore (PLA1); and (2) the CAP and ceramide (PLA2). PLA1 shows the subcellular localization of a particular CAP that is cross-linked to pacFACer, while PLA2 tests if the cross-linked CAP forms a complex with endogenous ceramide. Two proteins, tubulin and voltage-dependent anion channel 1 (VDAC1), were cross-linked to pacFACer and showed PLA signals for a complex with ceramide and pacFACer, which were predominantly colocalized with microtubules and mitochondria, respectively. Binding of tubulin and VDAC1 to ceramide was confirmed by coimmunoprecipitation assays using anti ceramide antibody. Cross-linking to pacFACer was confirmed using click chemistry-mediated attachment of biotin and streptavidin pull-down assays. Inhibition of ceramide synthases with fumonisin B1 (FB1) reduced the degree of pacFACer cross-linking and complex formation with ceramide, while it was enhanced by amyloid beta peptide (Aβ). Our results show that endogenous ceramide is critical for mediating cross-linking of CAPs to pacFACer and that a combination of cross-linking with PLAs (cross-link/PLA) is a novel tool to visualize CAPs and to understand the regulation of protein interaction with ceramide in CRPs. Frontiers Media S.A. 2019-08-16 /pmc/articles/PMC6706757/ /pubmed/31475148 http://dx.doi.org/10.3389/fcell.2019.00166 Text en Copyright © 2019 Jiang, Zhu, Qin, Tripathi, Zhong, Elsherbini, Karki, Crivelli, Zhi, Wang, Spassieva and Bieberich. http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. |
spellingShingle | Cell and Developmental Biology Jiang, Xue Zhu, Zhihui Qin, Haiyan Tripathi, Priyanka Zhong, Liansheng Elsherbini, Ahmed Karki, Sanjib Crivelli, Simone M. Zhi, Wenbo Wang, Guanghu Spassieva, Stefanka D. Bieberich, Erhard Visualization of Ceramide-Associated Proteins in Ceramide-Rich Platforms Using a Cross-Linkable Ceramide Analog and Proximity Ligation Assays With Anti-ceramide Antibody |
title | Visualization of Ceramide-Associated Proteins in Ceramide-Rich Platforms Using a Cross-Linkable Ceramide Analog and Proximity Ligation Assays With Anti-ceramide Antibody |
title_full | Visualization of Ceramide-Associated Proteins in Ceramide-Rich Platforms Using a Cross-Linkable Ceramide Analog and Proximity Ligation Assays With Anti-ceramide Antibody |
title_fullStr | Visualization of Ceramide-Associated Proteins in Ceramide-Rich Platforms Using a Cross-Linkable Ceramide Analog and Proximity Ligation Assays With Anti-ceramide Antibody |
title_full_unstemmed | Visualization of Ceramide-Associated Proteins in Ceramide-Rich Platforms Using a Cross-Linkable Ceramide Analog and Proximity Ligation Assays With Anti-ceramide Antibody |
title_short | Visualization of Ceramide-Associated Proteins in Ceramide-Rich Platforms Using a Cross-Linkable Ceramide Analog and Proximity Ligation Assays With Anti-ceramide Antibody |
title_sort | visualization of ceramide-associated proteins in ceramide-rich platforms using a cross-linkable ceramide analog and proximity ligation assays with anti-ceramide antibody |
topic | Cell and Developmental Biology |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6706757/ https://www.ncbi.nlm.nih.gov/pubmed/31475148 http://dx.doi.org/10.3389/fcell.2019.00166 |
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