Segregation of fluorescent membrane lipids into distinct micrometric domains: evidence for phase compartmentation of natural lipids?

<h4>Background</h4>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 domai...

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Autores principales:	Ludovic D'auria, Patrick Van der Smissen, Frédéric Bruyneel, Pierre J Courtoy, Donatienne Tyteca
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2011
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/cbc427ea56be4a18811b219e13c53f27
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spelling	oai:doaj.org-article:cbc427ea56be4a18811b219e13c53f272021-11-18T06:58:05ZSegregation of fluorescent membrane lipids into distinct micrometric domains: evidence for phase compartmentation of natural lipids?1932-620310.1371/journal.pone.0017021https://doaj.org/article/cbc427ea56be4a18811b219e13c53f272011-02-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/21386970/?tool=EBIhttps://doaj.org/toc/1932-6203<h4>Background</h4>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.<h4>Methodology/principal findings</h4>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.<h4>Conclusions/significance</h4>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.Ludovic D'auriaPatrick Van der SmissenFrédéric BruyneelPierre J CourtoyDonatienne TytecaPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 6, Iss 2, p e17021 (2011)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Ludovic D'auria Patrick Van der Smissen Frédéric Bruyneel Pierre J Courtoy Donatienne Tyteca Segregation of fluorescent membrane lipids into distinct micrometric domains: evidence for phase compartmentation of natural lipids?
description	<h4>Background</h4>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.<h4>Methodology/principal findings</h4>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.<h4>Conclusions/significance</h4>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	article
author	Ludovic D'auria Patrick Van der Smissen Frédéric Bruyneel Pierre J Courtoy Donatienne Tyteca
author_facet	Ludovic D'auria Patrick Van der Smissen Frédéric Bruyneel Pierre J Courtoy Donatienne Tyteca
author_sort	Ludovic D'auria
title	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_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_sort	segregation of fluorescent membrane lipids into distinct micrometric domains: evidence for phase compartmentation of natural lipids?
publisher	Public Library of Science (PLoS)
publishDate	2011
url	https://doaj.org/article/cbc427ea56be4a18811b219e13c53f27
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Segregation of fluorescent membrane lipids into distinct micrometric domains: evidence for phase compartmentation of natural lipids?

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