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Segregation of Fluorescent Membrane Lipids into Distinct Micrometric Domains: Evidence for Phase Compartmentation of Natural Lipids?
BACKGROUND: We recently reported that sphingomyelin (SM) analogs substituted on the alkyl chain by various fluorophores (e.g. BODIPY) readily inserted at trace levels into the plasma membrane of living erythrocytes or CHO cells and spontaneously concentrated into micrometric domains. Despite sharing...
Autores principales: | , , , , |
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Formato: | Texto |
Lenguaje: | English |
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Public Library of Science
2011
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3046177/ https://www.ncbi.nlm.nih.gov/pubmed/21386970 http://dx.doi.org/10.1371/journal.pone.0017021 |
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author | D′auria, Ludovic Van Der Smissen, Patrick Bruyneel, Frédéric Courtoy, Pierre J. Tyteca, Donatienne |
author_facet | D′auria, Ludovic Van Der Smissen, Patrick Bruyneel, Frédéric Courtoy, Pierre J. Tyteca, Donatienne |
author_sort | D′auria, Ludovic |
collection | PubMed |
description | BACKGROUND: We recently reported that sphingomyelin (SM) analogs substituted on the alkyl chain by various fluorophores (e.g. BODIPY) readily inserted at trace levels into the plasma membrane of living erythrocytes or CHO cells and spontaneously concentrated into micrometric domains. Despite sharing the same fluorescent ceramide backbone, BODIPY-SM domains segregated from similar domains labelled by BODIPY-D-e-lactosylceramide (D-e-LacCer) and depended on endogenous SM. METHODOLOGY/PRINCIPAL FINDINGS: We show here that BODIPY-SM further differed from BODIPY-D-e-LacCer or -glucosylceramide (GlcCer) domains in temperature dependence, propensity to excimer formation, association with a glycosylphosphatidylinositol (GPI)-anchored fluorescent protein reporter, and lateral diffusion by FRAP, thus demonstrating different lipid phases and boundaries. Whereas BODIPY-D-e-LacCer behaved like BODIPY-GlcCer, its artificial stereoisomer, BODIPY-L-t-LacCer, behaved like BODIPY- and NBD-phosphatidylcholine (PC). Surprisingly, these two PC analogs also formed micrometric patches yet preferably at low temperature, did not show excimer, never associated with the GPI reporter and showed major restriction to lateral diffusion when photobleached in large fields. This functional comparison supported a three-phase micrometric compartmentation, of decreasing order: BODIPY-GSLs > -SM > -PC (or artificial L-t-LacCer). Co-existence of three segregated compartments was further supported by double labelling experiments and was confirmed by additive occupancy, up to ∼70% cell surface coverage. Specific alterations of BODIPY-analogs domains by manipulation of corresponding endogenous sphingolipids suggested that distinct fluorescent lipid partition might reflect differential intrinsic propensity of endogenous membrane lipids to form large assemblies. CONCLUSIONS/SIGNIFICANCE: We conclude that fluorescent membrane lipids spontaneously concentrate into distinct micrometric assemblies. We hypothesize that these might reflect preexisting compartmentation of endogenous PM lipids into non-overlapping domains of differential order: GSLs > SM > PC, resulting into differential self-adhesion of the two former, with exclusion of the latter. |
format | Text |
id | pubmed-3046177 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2011 |
publisher | Public Library of Science |
record_format | MEDLINE/PubMed |
spelling | pubmed-30461772011-03-08 Segregation of Fluorescent Membrane Lipids into Distinct Micrometric Domains: Evidence for Phase Compartmentation of Natural Lipids? D′auria, Ludovic Van Der Smissen, Patrick Bruyneel, Frédéric Courtoy, Pierre J. Tyteca, Donatienne PLoS One Research Article BACKGROUND: We recently reported that sphingomyelin (SM) analogs substituted on the alkyl chain by various fluorophores (e.g. BODIPY) readily inserted at trace levels into the plasma membrane of living erythrocytes or CHO cells and spontaneously concentrated into micrometric domains. Despite sharing the same fluorescent ceramide backbone, BODIPY-SM domains segregated from similar domains labelled by BODIPY-D-e-lactosylceramide (D-e-LacCer) and depended on endogenous SM. METHODOLOGY/PRINCIPAL FINDINGS: We show here that BODIPY-SM further differed from BODIPY-D-e-LacCer or -glucosylceramide (GlcCer) domains in temperature dependence, propensity to excimer formation, association with a glycosylphosphatidylinositol (GPI)-anchored fluorescent protein reporter, and lateral diffusion by FRAP, thus demonstrating different lipid phases and boundaries. Whereas BODIPY-D-e-LacCer behaved like BODIPY-GlcCer, its artificial stereoisomer, BODIPY-L-t-LacCer, behaved like BODIPY- and NBD-phosphatidylcholine (PC). Surprisingly, these two PC analogs also formed micrometric patches yet preferably at low temperature, did not show excimer, never associated with the GPI reporter and showed major restriction to lateral diffusion when photobleached in large fields. This functional comparison supported a three-phase micrometric compartmentation, of decreasing order: BODIPY-GSLs > -SM > -PC (or artificial L-t-LacCer). Co-existence of three segregated compartments was further supported by double labelling experiments and was confirmed by additive occupancy, up to ∼70% cell surface coverage. Specific alterations of BODIPY-analogs domains by manipulation of corresponding endogenous sphingolipids suggested that distinct fluorescent lipid partition might reflect differential intrinsic propensity of endogenous membrane lipids to form large assemblies. CONCLUSIONS/SIGNIFICANCE: We conclude that fluorescent membrane lipids spontaneously concentrate into distinct micrometric assemblies. We hypothesize that these might reflect preexisting compartmentation of endogenous PM lipids into non-overlapping domains of differential order: GSLs > SM > PC, resulting into differential self-adhesion of the two former, with exclusion of the latter. Public Library of Science 2011-02-28 /pmc/articles/PMC3046177/ /pubmed/21386970 http://dx.doi.org/10.1371/journal.pone.0017021 Text en D′auria et al. http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited. |
spellingShingle | Research Article D′auria, Ludovic Van Der Smissen, Patrick Bruyneel, Frédéric Courtoy, Pierre J. Tyteca, Donatienne Segregation of Fluorescent Membrane Lipids into Distinct Micrometric Domains: Evidence for Phase Compartmentation of Natural Lipids? |
title | Segregation of Fluorescent Membrane Lipids into Distinct Micrometric Domains: Evidence for Phase Compartmentation of Natural Lipids? |
title_full | Segregation of Fluorescent Membrane Lipids into Distinct Micrometric Domains: Evidence for Phase Compartmentation of Natural Lipids? |
title_fullStr | Segregation of Fluorescent Membrane Lipids into Distinct Micrometric Domains: Evidence for Phase Compartmentation of Natural Lipids? |
title_full_unstemmed | Segregation of Fluorescent Membrane Lipids into Distinct Micrometric Domains: Evidence for Phase Compartmentation of Natural Lipids? |
title_short | Segregation of Fluorescent Membrane Lipids into Distinct Micrometric Domains: Evidence for Phase Compartmentation of Natural Lipids? |
title_sort | segregation of fluorescent membrane lipids into distinct micrometric domains: evidence for phase compartmentation of natural lipids? |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3046177/ https://www.ncbi.nlm.nih.gov/pubmed/21386970 http://dx.doi.org/10.1371/journal.pone.0017021 |
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