Segregation of Fluorescent Membrane Lipids into Distinct Micrometric Domains: Evidence for Phase Compartmentation of Natural Lipids?

Abstract

BackgroundWe 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 FindingsWe 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/SignificanceWe 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.

Micrometric segregation of fluorescent membrane lipids: relevance for endogenous lipids and biogenesis in erythrocytes

“…The latter observation also indicated that patches revealed by CTxB were not induced by exogenous GM1BODIPY insertion. These results supported the interpretation that micrometric BODIPY lipid domains could refl ect endogenous lipid compartmentation, as previously suggested for BODIPY-SM domains in CHO cells based on endogenous SM depletion ( 19,20 ).…”

“…IIIB). Thus, cholesterol depletion specifi cally impaired the less-packed BODIPY-PC and BODIPY-SM domains and spared the mostpacked BODIPY-GlcCer domains, packing of domains being previously suggested by the propensity of BODIPYlipids to form excimers ( 20 ). To further test whether cholesterol depletion could instead favor other domains, we looked at the artifi cial dialkylindocarbocyanine DiIC18, a well-established membrane probe that homogenously labels untreated RBCs ( 19,42 ), but which is recognized to preferentially partition into the liquid-disordered phase in giant unilamellar vesicles, segregated from the liquid-ordered phase decorated by fl uor escent cholera toxin that binds ganglioside GM1 ( 8 ).…”

“…Although the concept remains debated ( 7 ), micrometric membrane lipid domains have been visualized with a variety of fl uorescent lipids on erythrocytes ( 19,20 ), blood platelets ( 16 ), and nucleated cells ( 15,(17)(18)(19). Micrometric fl uorescent lipid domains were found to depend on cholesterol ( 19,27 ) and to show distinct phase behavior in response to changes of temperature ( 19,20 ).…”

“…We recently compared by vital imaging the lateral organization of fl uorescent boron dipyrromethene (BODIPY) lipid analogs of GlcCer, SM, and PC at the outer PM leafl et of Chinese hamster ovary (CHO) cells and human red blood cells (RBCs) ( 19,20 ). These studies visualized welldefi ned brilliant spots corresponding to submicrometric domains ( ‫ف‬ 0.5-1 m; i.e., well-above the resolution limit of confocal microscopy), with 5-8-fold enrichment over the rest of the labeled cell surface.…”

“…RBCs were then spread on PLKcoverslips and washed twice with DMEM/BSA. For confocal imaging, coverslips were placed bottom-up into Lab-Tek chambers and examined with a Zeiss LSM510 confocal microscope using a plan-Apochromat 63× NA 1.4 oil immersion objective in a thermostatically controlled cabinet set at 37 ± 1°C (XL/LSM incubator, Zeiss; Tempcontrol 37-2, PeCon) ( 19,20 ). FRAP and scanning electron microscopy were performed as described ( 19,20 ).…”