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 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

Relationships, gene flow and species boundaries among New Zealand <i>Fuscospora</i> (Nothofagaceae: southern beech)

<div><p>We analysed DNA profiles at eight simple sequence repeat (SSR) loci for 449 individuals of <i>Fuscospora</i> species from New Zealand. The two species with serrated leaves (<i>F. fusca</i> and <i>F. truncata</i>) are clearly distinct genetically despite the occurrence of wild hybrids. Further, from our results, we infer that they are no more closely related to each other than they are to either of the entire-leaved species (<i>F. cliffortioides</i> and <i>F. solandri</i>). Although genetic groups corresponding with <i>F. cliffortioides</i> and <i>F. solandri</i> can be distinguished, our data suggest that considerable admixture may occur between them in some areas. Nonetheless, the broadly sympatric distribution of these two genetic groups, and their consistency with morphological and ecological groups, support their recognition at species rank rather than their treatment as varieties of one variable species.</p></div

Absence of hybridisation between <i>Fuscospora</i> species at a site in Arthur's Pass National Park, New Zealand

<div><p>We analysed simple sequence repeat markers for a selection of red beech (<i>Fuscospora fusca</i>) and mountain beech (<i>Fuscospora cliffortioides</i>) trees growing together and for seeds collected from them. Our analysis detected neither hybrid trees nor hybrid seeds within our sample. We suggest pre-zygotic reproductive isolating mechanisms such as differences in flowering time may be important in maintaining boundaries between <i>Fuscospora</i> species despite extensive sympatry.</p></div