Nanodomains can persist at physiologic temperature in plasma membrane vesicles and be modulated by altering cell lipids

Abstract

The formation and properties of liquid-ordered (Lo) lipid domains (rafts) in the plasma membrane are still poorly understood. This limits our ability to manipulate ordered lipid domain-dependent biological functions. Giant plasma membrane vesicles (GPMVs) undergo large-scale phase separations into coexisting Lo and liquid-disordered lipid domains. However, large-scale phase separation in GPMVs detected by light microscopy is observed only at low temperatures. Comparing Förster resonance energy transfer-detected versus light microscopy-detected domain formation, we found that nanodomains, domains of nanometer size, persist at temperatures up to 20°C higher than large-scale phases, up to physiologic temperature. The persistence of nanodomains at higher temperatures is consistent with previously reported theoretical calculations. To investigate the sensitivity of nanodomains to lipid composition, GPMVs were prepared from mammalian cells in which sterol, phospholipid, or sphingolipid composition in the plasma membrane outer leaflet had been altered by cyclodextrin-catalyzed lipid exchange. Lipid substitutions that stabilize or destabilize ordered domain formation in artificial lipid vesicles had a similar effect on the thermal stability of nanodomains and large-scale phase separation in GPMVs, with nanodomains persisting at higher temperatures than large-scale phases for a wide range of lipid compositions. This indicates that it is likely that plasma membrane nanodomains can form under physiologic conditions more readily than large-scale phase separation. We also conclude that membrane lipid substitutions carried out in intact cells are able to modulate the propensity of plasma membranes to form ordered domains. This implies lipid substitutions can be used to alter biological processes dependent upon ordered domains. The formation and properties of liquid-ordered (Lo) lipid domains (rafts) in the plasma membrane are still poorly understood. This limits our ability to manipulate ordered lipid domain-dependent biological functions. Giant plasma membrane vesicles (GPMVs) undergo large-scale phase separations into coexisting Lo and liquid-disordered lipid domains. However, large-scale phase separation in GPMVs detected by light microscopy is observed only at low temperatures. Comparing Förster resonance energy transfer-detected versus light microscopy-detected domain formation, we found that nanodomains, domains of nanometer size, persist at temperatures up to 20°C higher than large-scale phases, up to physiologic temperature. The persistence of nanodomains at higher temperatures is consistent with previously reported theoretical calculations. To investigate the sensitivity of nanodomains to lipid composition, GPMVs were prepared from mammalian cells in which sterol, phospholipid, or sphingolipid composition in the plasma membrane outer leaflet had been altered by cyclodextrin-catalyzed lipid exchange. Lipid substitutions that stabilize or destabilize ordered domain formation in artificial lipid vesicles had a similar effect on the thermal stability of nanodomains and large-scale phase separation in GPMVs, with nanodomains persisting at higher temperatures than large-scale phases for a wide range of lipid compositions. This indicates that it is likely that plasma membrane nanodomains can form under physiologic conditions more readily than large-scale phase separation. We also conclude that membrane lipid substitutions carried out in intact cells are able to modulate the propensity of plasma membranes to form ordered domains. This implies lipid substitutions can be used to alter biological processes dependent upon ordered domains. brain SM Dulbecco's PBS 1,6-diphenyl-1,3,5-hexatriene 3,3′-dilinoleyloxacarbocyanine perchlorate Förster resonance energy transfer giant plasma membrane vesicle liquid-disordered liquid-ordered methyl-α-cyclodextrin methyl-β-cyclodextrin octadecyl rhodamine B phosphatidylcholine paraformaldehyde 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine transition endpoint temperature transition midpoint temperature The conditions under which sphingolipid- and cholesterol-rich ordered domains form in the plasma membrane of mammalian cells remain unclear. With a few notable exceptions (1LaRocca T.J. Pathak P. Chiantia S. Toledo A. Silvius J.R. Benach J.L. London E. Proving lipid rafts exist: membrane domains in the prokaryote Borrelia burgdorferi have the same properties as eukaryotic lipid rafts.PLoS Pathog. 2013; 9: e1003353Crossref PubMed Scopus (77) Google Scholar, 2Toulmay A. Prinz W.A. Direct imaging reveals stable, micrometer-scale lipid domains that segregate proteins in live cells.J. Cell Biol. 2013; 202: 35-44Crossref PubMed Scopus (142) Google Scholar), ordered domains remain difficult to directly observe in intact cells. Under many conditions, ordered domains may not even be a constitutive feature of the plasma membrane, but rather one induced in the presence of a stimulus (3Sohn H.W. Tolar P. Pierce S.K. Membrane heterogeneities in the formation of B cell receptor-Lyn kinase microclusters and the immune synapse.J. Cell Biol. 2008; 182: 367-379Crossref PubMed Scopus (106) Google Scholar, 4Holowka D. Baird B. Roles for lipid heterogeneity in immunoreceptor signaling.Biochim. Biophys. Acta. 2016; 1861: 830-836Crossref PubMed Scopus (17) Google Scholar). Giant plasma membrane vesicles (GPMVs) represent a natural membrane system in which the domain-forming properties of plasma membrane lipids and proteins can be investigated much more readily than in intact cells (5Sengupta P. Hammond A. Holowka D. Baird B. Structural determinants for partitioning of lipids and proteins between coexisting fluid phases in giant plasma membrane vesicles.Biochim. Biophys. Acta. 2008; 1778: 20-32Crossref PubMed Scopus (163) Google Scholar, 6Baumgart T. Hammond A.T. Sengupta P. Hess S.T. Holowka D.A. Baird B.A. Webb W.W. Large-scale fluid/fluid phase separation of proteins and lipids in giant plasma membrane vesicles.Proc. Natl. Acad. Sci. USA. 2007; 104: 3165-3170Crossref PubMed Scopus (585) Google Scholar). They have the advantage over artificial lipid vesicles that they contain a complex natural mixture of lipids and proteins. At low temperatures, the lipids in GPMVs undergo a phase separation in which classical large-scale coexisting liquid-disordered (Ld) and liquid-ordered (Lo) phases form and are easily visualized by light microscopy. These phases have properties very similar to those formed in phase-separating artificial vesicles with simple lipid mixtures in terms of their properties and association with specific proteins. Although large-scale phase separation is only seen in GPMVs at lower temperatures, it has been predicted that nanodomains that decrease in size as temperature increases would persist to much higher temperatures in GPMVs (7Veatch S.L. P. Sengupta P. A. Holowka D. Baird B. in plasma membrane Biol. 2008; PubMed Scopus Google Scholar). with in artificial lipid nanodomains under conditions that large-scale phase separation is not and domain size can decrease as temperature increases P. London E. of lipid formation and size in vesicles and domain size by nanodomains but not domain PubMed Scopus Google Scholar, with a transition from to phase domains in Biophys. 1861: PubMed Scopus Google Scholar, S.L. of a phases in giant PubMed Scopus Google Scholar). GPMVs, have that the conditions under which cells are or that can as the stability of ordered domains in GPMVs P. T. phase separation in giant PubMed Scopus Google Scholar, S.L. lipid heterogeneity in membranes at 2013; 104: PubMed Scopus Google Scholar, S.L. transition temperature with temperature in a cell PubMed Scopus Google Scholar, E. S.L. lower temperatures in plasma membrane 2013; PubMed Scopus Google Scholar). that GPMVs are a system for the lipid domain formation in natural we investigated the between and large-scale phase separation in GPMVs and the formation of large-scale phases and nanodomains is by of plasma membrane lipid We found that ordered nanodomains persist to much higher temperatures than large-scale phase to plasma membrane composition, we found that large-scale phase separation and thermal stability to lipid composition in a to has been previously reported for domain formation in artificial However, in nanodomains to a than large-scale phase separations into the ability of plasma membrane lipids to form ordered nanodomains and the properties of domains are altered by lipid substitutions in intact cells. SM 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine and were from and were from 1,6-diphenyl-1,3,5-hexatriene and methyl-β-cyclodextrin were from from The Dulbecco's PBS and and and with were from from rhodamine B paraformaldehyde and 3,3′-dilinoleyloxacarbocyanine perchlorate were from from Cell cell were from were from were at and were as at to by as previously A. D.A. London E. The effect of upon and Cell Sci. PubMed Scopus Google Scholar). cells were a from Baird cells were from the cells were in with and and cells were in with cells were in a with at were to formation and were the were on for Förster resonance energy transfer and on for microscopy and were prepared and carried out to A. D.A. London E. The effect of upon and Cell Sci. PubMed Scopus Google Scholar, J.R. D.A. London E. of plasma membrane outer leaflet and in cells with Natl. Acad. Sci. USA. 2016; PubMed Scopus Google Scholar). and were prepared to or were under by for at They were with or for or for in in a for were or or or were for or were at for and at temperature or at in also prepared the for cells were with or and or of the prepared or were The were at temperature for or at for cells were with and or of were were in a for and cells were used for GPMVs were prepared E. T. P. membrane and giant plasma membrane PubMed Scopus Google Scholar). that had been as or were and were and at a of and We as GPMVs were from and cells. were with and with and for or for of were and at for with found to be for cells. The by to of intact and at for to which The from used for the the a at for to the microscopy the which to to a of the were in GPMVs were used for or microscopy on the same as they were to from GPMVs from a at for The and were The GPMVs that were to by microscopy. The Lipid in the and vesicle with the of lipid in the versus temperature for the and as Lipid in GPMVs for by of under conditions in which in E. and for vesicles PubMed Scopus (77) Google Scholar, S. P. D. S. London E. plasma membrane outer leaflet lipids with lipid membrane Google Scholar). lipid of lipid vesicles prepared to a To of and of were and under by for the lipids were in of and of PBS and and at a for of were to from the to or a range of vesicle from the vesicle to to with for at temperature in the the of as a of lipid at and GPMVs from cells of lipid and had a of from one lipid prepared from cells and had a of from one vesicles were prepared of lipid or The lipids were and under by for the lipids were at a for in of PBS and in a at for to vesicles from the the vesicles were to of a mixture of and by to temperature and were were to with PBS and to cell of and as for GPMVs were with as the and as the The with prepared by from the in to of GPMVs or lipid vesicles that had been to cell and at for in the The also at for not to or not not from a in to the and and at temperature for in the of To were in a and to a and to temperature at up to or lipid temperature at a of The of in the presence of to that in the of the temperature which of lipids into ordered and domains the of ordered domains is by the of a to the and at in the of domains is a decrease in increases as temperature is London E. of on ordered membrane domain stability in and vesicles.Biochim. Biophys. 1861: PubMed Scopus Google Scholar, London E. Lipid and composition of in PubMed Scopus Google that the of to GPMVs by of GPMVs and at and that in at temperatures not in to GPMVs not for light microscopy were GPMVs were prepared from cells or GPMVs were from cells. GPMVs were only from cells cells were to in cells or were with and the by a in in of were to the cells and at for of by the cells were with and with and GPMVs were prepared as or from their in to of or GPMVs and at temperature for The GPMVs were by at for of the in up to for microscopy of GPMVs were in of a Cell cell and with a to in the with temperature with a system used to of GPMVs were over a range of at of and for the the temperature to the same that GPMVs were seen GPMVs were for up to at not at the temperatures at which but not GPMVs had the domain of GPMVs for The are for To the transition from temperatures at which domains form to those at which they not the of GPMVs with domains as a of to a and the at which of the GPMVs and which as the at which of the GPMVs were or GPMVs were by at at for and the lipids in the of were by The under and lipids were in for were in and detected as previously J.R. D.A. London E. of plasma membrane outer leaflet and in cells with Natl. Acad. Sci. USA. 2016; PubMed Scopus Google Scholar). Lo and in GPMVs can be visualized by light microscopy. domains can be detected as in a that into Lo domains in that of GPMVs are and the used to domain is the of GPMVs coexisting ordered and domains domains of properties and in plasma membrane vesicles.Proc. Natl. Acad. Sci. USA. PubMed Scopus Google Scholar). temperature is the of GPMVs with domains to from to transition midpoint temperature or as the temperature at which of GPMVs coexisting ordered and domains domains of properties and in plasma membrane vesicles.Proc. Natl. Acad. Sci. USA. PubMed Scopus Google Scholar). The transition endpoint as the temperature which of GPMVs ordered domains. To nanodomains in GPMVs, to ordered domains in (1LaRocca T.J. Pathak P. Chiantia S. Toledo A. Silvius J.R. Benach J.L. London E. Proving lipid rafts exist: membrane domains in the prokaryote Borrelia burgdorferi have the same properties as eukaryotic lipid rafts.PLoS Pathog. 2013; 9: e1003353Crossref PubMed Scopus (77) Google Scholar), as and as to in GPMVs with membranes with coexisting ordered and is of to between ordered and domains of phase and in phosphatidylcholine PubMed Scopus Google from in domains A. of a from the cholesterol-rich phase in Biophys. Acta. PubMed Scopus Google a the of by the which the of in the presence of to that in the of is to in membranes in the of the of with coexisting domains at lower temperatures a decrease in at temperatures. The is that the between and Lo domains as they with lipids at temperatures. Lo domains are we to the to transition with at temperature as a To the ability of to ordered domain formation, in artificial lipid vesicles mixtures of phosphatidylcholine and to form coexisting Lo and domains P. London E. The effect of membrane lipid composition on the formation of lipid PubMed Scopus Google Scholar, P. and domain size in lipid Biophys. Acta. 2013; PubMed Scopus Google in at low temperatures and at temperatures. the vesicles in which lipids were temperature GPMVs were prepared from cells the and domain formation by microscopy and with domains of properties and in plasma membrane vesicles.Proc. Natl. Acad. Sci. USA. PubMed Scopus Google Scholar), and the of GPMVs in that GPMVs formed coexisting Lo and phases at low temperatures, and phases as temperature and as the of used to the GPMVs in with domains of properties and in plasma membrane vesicles.Proc. Natl. Acad. Sci. USA. PubMed Scopus Google Scholar). The as as and the as as GPMVs were prepared with At the used of the and the These are to those reported previously domains of properties and in plasma membrane vesicles.Proc. Natl. Acad. Sci. USA. PubMed Scopus Google and for large-scale phase separation and formation for GPMVs prepared with at were to a of GPMVs with domains versus the at low temperature would of vesicles domains. phase as temperature at which of the vesicles would have and the temperature which than of vesicles contain domains. nanodomains, as the temperature at which a and from the from are in a The were to a of GPMVs with domains versus the at low temperature would of vesicles domains. phase as temperature at which of the vesicles would have and the temperature which than of vesicles contain domains. nanodomains, as the temperature at which a and from the from are similar to that by microscopy in the that higher in formation and thermal stability of ordered domains as by were to by much higher than that detected by as at with This that nanodomains persist to much higher temperatures than large-scale phase separation. that the between domain stability by microscopy and not of in the used in microscopy to large-scale phase separation to than detected by be under conditions, can domain formation S.L. Webb W.W. and Biophys. Acta. PubMed Scopus Google Scholar), but the large-scale phase separations observed were as as the were that not a in their We found that the for and vesicles in the were very similar to those for the easily GPMVs This that the between by microscopy and not a in properties of and The formation of large-scale phase separation and nanodomains in phase by the that in domain formation were upon and upon However, the transition temperature a few and a The transition temperature also not were visualized up to at GPMVs were prepared not versus temperature were also a with the temperature a decrease in thermal stability have one of and J.R. D.A. London E. of plasma membrane outer leaflet and in cells with Natl. Acad. Sci. USA. 2016; PubMed Scopus Google Scholar, S. P. D. S. London E. plasma membrane outer leaflet lipids with lipid membrane Google Scholar), we used to manipulate plasma membrane and sphingolipid composition in a that stabilize or destabilize domain formation predicted by the effect of lipid composition upon domain formation in artificial To investigate lipids in cells plasma membrane domain formation, GPMVs were prepared from cells in which composition had been altered by with mixtures under conditions that in of outer leaflet and SM with lipid J.R. D.A. London E. of plasma membrane outer leaflet and in cells with Natl. Acad. Sci. USA. 2016; PubMed Scopus Google Scholar, S. P. D. S. London E. plasma membrane outer leaflet lipids with lipid membrane Google Scholar). not and not cell London E. of artificial plasma membrane vesicles with lipid 9: PubMed Scopus Google and that of plasma membrane outer leaflet lipids with SM to large-scale ordered domain formation in GPMVs, with the as predicted from the effect of lipids upon domain formation in artificial lipid These are consistent with that the stability of large-scale domain formation in GPMVs from cells at temperatures is in a that likely in lipid composition S.L. transition temperature with temperature in a cell PubMed Scopus Google Scholar). of lipid on formation were observed by of the lipids of GPMVs prepared from cells and that the altered as the in the lipid mixtures not large-scale domain formation This indicates that the in domain-forming properties are for the This is not a as of in lipid composition only lipid J.R. D.A. London E. of plasma membrane outer leaflet and in cells with Natl. Acad. Sci. USA. 2016; PubMed Scopus Google Scholar, S. P. D. S. London E. plasma membrane outer leaflet lipids with lipid membrane Google and for large-scale phase separation and formation for GPMVs prepared and substitutions or and from the from are for lipid for which in a and from the from are for lipid for which were also carried out to the a altered ordered domain formation in a similar to which it ordered domain formation in artificial lipid by our London E. of on ordered membrane domain stability in and vesicles.Biochim. Biophys. 1861: PubMed Scopus Google Scholar, domains of properties and in plasma membrane vesicles.Proc. Natl. Acad. Sci. USA. PubMed Scopus Google Scholar, London E. The effect of on membrane lipid domains reveals can lipid domain PubMed Scopus Google Scholar, D. London E. of the of natural and on the formation of ordered domains (rafts) of to and and of and Biol. PubMed Scopus Google Scholar, London E. between and in ordered lipid domains for lipid and PubMed Scopus Google and for or lipid PubMed Scopus Google Scholar, S.L. S.L. versus in lipid PubMed Scopus Google have that can stabilize or destabilize ordered domain formation in artificial vesicles on in cells with a range of or properties has been used in many to membrane domain formation a biological A. D.A. London E. The effect of upon and Cell Sci. PubMed Scopus Google Scholar, London E. to the of membrane domains in membrane PubMed Scopus Google Scholar). carried out as previously A. D.A. London E. The effect of upon and Cell Sci. PubMed Scopus Google Scholar), cells with at low to A. P. of with PubMed Scopus Google Scholar). We previously found that to of with A. D.A. London E. The effect of upon and Cell Sci. PubMed Scopus Google Scholar). GPMVs were prepared from the cells. and and that altered the thermal stability of large-scale phase to the in large-scale domain stability were observed with and previously to ordered domain formation London E. of on ordered membrane domain stability in and vesicles.Biochim. Biophys. 1861: PubMed Scopus Google Scholar, London E. The effect of on membrane lipid domains reveals can lipid domain PubMed Scopus Google Scholar). with and or simple of the thermal stability of large-scale domain formation to a which can membrane A. D.A. London E. The effect of upon and Cell Sci. PubMed Scopus Google Scholar), or with large-scale domain be that the of or in GPMVs upon large-scale phase separation are in with P. T. phase separation in giant PubMed Scopus Google Scholar, S.L. lipid heterogeneity in membranes at 2013; 104: PubMed Scopus Google Scholar). The effect of substitutions upon formation also The observed for the similar to that for large-scale domain formation, but also observed for phase formation much more than and and to similar to the observed with and to similar to those but not stabilize formation to GPMVs from effect upon for large-scale phase separation. be that between the of formation and large-scale phase separation can also be seen in artificial lipid vesicles in which have been London E. The effect of on membrane lipid domains reveals can lipid domain PubMed Scopus Google Scholar, D. London E. of the of natural and on the formation of ordered domains (rafts) of to and and of and Biol. PubMed Scopus Google Scholar, S.L. S.L. versus in lipid PubMed Scopus Google Scholar). the of on stability not phase is very to that observed for ordered domain stability is in artificial lipid vesicles London E. of on ordered membrane domain stability in and vesicles.Biochim. 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They were more than those formed by cells The effect of lipid upon the stability of formation also in cells and stability than in cells. large-scale phase separation and ordered formation as with stability upon with and stability upon with that the for cell GPMVs to be a higher than that for cells This is consistent with the higher of SM to in cells to cells the between for large-scale phase formation and formation in cells under the conditions is a between for phase and stability with between and higher for nanodomains than for large-scale domain The lipid of the between that the are they to in lipid The of are ordered lipid nanodomains are in GPMVs at temperatures those in which is large-scale phase of plasma membrane lipid composition and properties in intact cells in in the composition and properties of the and lipid composition thermal stability than that of phase separation. plasma membrane lipid composition the thermal stability of ordered domains in GPMVs prepared from cells to lipid exchange. The in stability as a of lipid composition were similar to the effect of the same in lipid composition upon the stability of ordered domains in artificial lipid vesicles London E. Lipid and composition of in PubMed Scopus Google Scholar). found to stabilize or destabilize ordered domain formation in lipid vesicles had the effect upon ordered stability in GPMVs London E. of on ordered membrane domain stability in and vesicles.Biochim. Biophys. 1861: PubMed Scopus Google Scholar). it is that the effect of upon nanodomains the for the effect of upon A. D.A. London E. The effect of upon and Cell Sci. PubMed Scopus Google Scholar). is that can be in the of for A. D.A. London E. The effect of upon and Cell Sci. PubMed Scopus Google Scholar), which of the of altered stability in GPMVs to that of artificial vesicles with compositions. the that the ordered domain formation by lipids in simple they have that in are likely to to the more complex lipid in natural The that nanodomains, of lipid composition properties that are in the nanometer size can under conditions in which large-scale domains are not is consistent with the of lipids in artificial lipid under which domains form but is large-scale phase separation have been reported for a of lipid mixtures P. London E. of lipid formation and size in vesicles and domain size by nanodomains but not domain PubMed Scopus Google Scholar, with a transition from to phase domains in Biophys. 1861: PubMed Scopus Google Scholar, S.L. of a phases in giant PubMed Scopus Google Scholar). The that nanodomains not into domains has been much in the has been with a transition from to phase domains in Biophys. 1861: PubMed Scopus Google Scholar, P. S.L. S.L. and in lipid membranes 2008; PubMed Scopus Google Scholar, S. liquid-disordered liquid-ordered domain size transition in lipid PubMed Scopus Google Scholar, rafts in of B. PubMed Scopus Google Scholar). has been that the of nanodomains between Lo and domains with a transition from to phase domains in Biophys. 1861: PubMed Scopus Google Scholar, S. liquid-disordered liquid-ordered domain size transition in lipid PubMed Scopus Google Scholar). has also been found by that not lipid mixtures a decrease in ordered domain size as temperature is P. London E. of lipid formation and size in vesicles and domain size by nanodomains but not domain PubMed Scopus Google Scholar). decrease in domain size as temperature is has also been observed as a lipid mixture a and it has been that domains the the of GPMVs (7Veatch S.L. P. Sengupta P. A. Holowka D. Baird B. in plasma membrane Biol. 2008; PubMed Scopus Google Scholar, P. S.L. S.L. and in lipid membranes 2008; PubMed Scopus Google Scholar). The for the temperature of domain size the temperature is in with our has been that domains of size would be at in GPMVs, higher than the which would be to The higher observed for the persistence of nanodomains the temperature may the ability of to domains than in can domains than the for for the for the used in P. London E. of lipid formation and size in vesicles and domain size by nanodomains but not domain PubMed Scopus Google Scholar). be that large-scale phases and are by nanodomains at domain a nanodomains for in membranes with lipid compositions. the of membranes that are the ordered and domain and properties and at domain may the large-scale phases to nanodomains form the temperature is lipid mixtures may have low at domain over a wide range of may only have low at domain The that as temperature is large-scale domain formation in GPMVs nanodomains that in biological membranes nanodomains are more likely to form than large-scale domains. The that lipids in intact cells by not only lipid composition, but also that plasma membrane lipid composition is by the of the plasma membrane to form domains is also likely to be in the of of lipid composition the to which nanodomains are altered in GPMVs by lipid be a of the to which the to form domains in intact cells is altered by lipid from the effect of lipid composition upon large-scale phase separation to lipid in cells may be of the more lipid can form nanodomains but the to form large-scale phase even in artificial lipid vesicles S.L. of a phases in giant PubMed Scopus Google Scholar). that the ability of nanodomains to form from the that nanodomains to into domains. the of also that the in plasma membrane composition and for the lipid This that on plasma membranes carried out a lipid still the of the lipid This the of lipid as a to the of membrane domains in the in of the of GPMVs, it be in that the that nanodomains to temperature in not that they are in intact cells. This in the lipid found in plasma At it is that at of the of is in GPMVs P. the formation of membrane PubMed Scopus Google Scholar). it is not lipids represent a of plasma membrane to GPMVs and in intact cells be to investigate with

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