Distribution of ether lipids and composition of the archaeal community in terrestrial geothermal springs: impact of environmental variables

Archaea can respond to changes in the environment by altering the composition of their membrane lipids, for example, by modification of the abundance and composition of glycerol dialkyl glycerol tetraethers (GDGTs). Here, we investigated the abundance and proportions of polar GDGTs (P‐GDGTs) and core GDGTs (C‐GDGTs) sampled in different seasons from Tengchong hot springs (Yunnan, China), which encompassed a pH range of 2.5–10.1 and a temperature range of 43.7–93.6°C. The phylogenetic composition of the archaeal community (reanalysed from published work) divided the Archaea in spring sediment samples into three major groups that corresponded with spring pH: acidic, circumneutral and alkaline. Cluster analysis showed correlation between spring pH and the composition of P‐ and C‐GDGTs and archaeal 16S rRNA genes, indicating an intimate link between resident Archaea and the distribution of P‐ and C‐GDGTs in Tengchong hot springs. The distribution of GDGTs in Tengchong springs was also significantly affected by temperature; however, the relationship was weaker than with pH. Analysis of published datasets including samples from Tibet, Yellowstone and the US Great Basin hot springs revealed a similar relationship between pH and GDGT content. Specifically, low pH springs had higher concentrations of GDGTs with high numbers of cyclopentyl rings than neutral and alkaline springs, which is consistent with the predominance of high cyclopentyl ring‐characterized Sulfolobales and Thermoplasmatales present in some of the low pH springs. Our study suggests that the resident Archaea in these hot springs are acclimated if not adapted to low pH by their genetic capacity to effect the packing density of their membranes by increasing cyclopentyl rings in GDGTs at the rank of community.     

Thermophilic archaeon orchestrates temporal expression of GDGT ring synthases in response to temperature and acidity stress

Glycerol dibiphytanyl glycerol tetraethers (GDGTs) are unique archaeal membrane-spanning lipids with 0–8 cyclopentane rings on the biphytanyl chains. The cyclization pattern of GDGTs is affected by many environmental factors, such as temperature and pH, but the underlying molecular mechanism remains elusive. Here, we find that the expression regulation of GDGT ring synthase genes grsA and grsB in thermophilic archaeon Sulfolobus acidocaldarius is temperature- and pH-dependent. Moreover, the presence of functional GrsA protein, or more likely its products cyclic GDGTs rather than the accumulation of GrsA protein itself, is required to induce grsB expression, resulting in temporal regulation of grsA and grsB expression. Our findings establish a molecular model of GDGT cyclization regulated by environment factors in a thermophilic ecosystem, which could be also relevant to that in mesophilic marine archaea. Our study will help better understand the biological basis for GDGT-based paleoclimate proxies. Archaea inhabit a wide range of terrestrial and marine environments. In response to environment fluctuations, archaea modulate their unique membrane GDGTs lipid composition with different strategies, in particular GDGTs cyclization significantly alters membrane permeability. However, the regulation details of archaeal GDGTs cyclization in response to different environmental factor changes remain unknown. We demonstrated, for the first time, thermophilic archaea orchestrate the temporal expression of GDGT ring synthases, leading to delicate control of GDGTs cyclization to respond environmental temperature and acidity stress. Our study provides insight into the regulation of archaea membrane plasticity, and the survival strategy of archaea in fluctuating environments.     

Temperature and pH controls on glycerol dibiphytanyl glycerol tetraether lipid composition in the hyperthermophilic crenarchaeon <Emphasis Type="Italic">Acidilobus sulfurireducens</Emphasis>

Cyclization in glycerol dibiphytanyl glycerol tetraethers (GDGTs) results in internal cyclopentane moieties which are believed to confer thermal stability to crenarchaeal membranes. While the average number of rings per GDGT lipid (ring index) is positively correlated with temperature in many temperate environments, poor correlations are often observed in geothermal environments, suggesting that additional parameters may influence GDGT core lipid composition in these systems. However, the physical and chemical parameters likely to influence GDGT cyclization which are often difficult to decouple in geothermal systems, making it challenging to assess their influence on lipid composition. In the present study, the influence of temperature (range 65–81°C), pH (range 3.0–5.0), and ionic strength (range 10.1–55.7 mM) on GDGT core lipid composition was examined in the hyperthermoacidophile Acidilobus sulfurireducens, a crenarchaeon originally isolated from a geothermal spring in Yellowstone National Park, Wyoming. When cultivated under defined laboratory conditions, the composition of individual and total GDGTs varied significantly with temperature and to a lesser extent with the pH of the growth medium. Ionic strength over the range of values tested did not influence GDGT composition. The GDGT core lipid ring index was positively correlated with temperature and negatively correlated with pH, suggesting that A. sulfurireducens responds to increasing temperature and acidity by increasing the number of cyclopentyl rings in GDGT core membrane lipids.     

Multiple environmental parameters impact lipid cyclization in Sulfolobus acidocaldarius

Adaptation of lipid membrane composition is an important component of archaeal homeostatic response. Historically, the number of cyclopentyl and cyclohexyl rings in the glycerol dibiphytanyl glycerol tetraether (GDGT) Archaeal lipids has been linked to variation in environmental temperature. However, recent work with GDGT-making archaea highlight the roles of other factors, such as pH or energy availability, in influencing the degree of GDGT cyclization. To better understand the role of multiple variables in a consistent experimental framework and organism, we cultivated the model Crenarchaeon Sulfolobus acidocaldarius DSM639 at different combinations of temperature, pH, oxygen flux, or agitation speed. We quantified responses in growth rate, biomass yield, and core lipid compositions, specifically the degree of core GDGT cyclization. The degree of GDGT cyclization correlated with growth rate under most conditions. The results suggest the degree of cyclization in archaeal lipids records a universal response to energy availability at the cellular level, both in thermoacidophiles, and in other recent findings in the mesoneutrophilic Thaumarchaea. Although we isolated the effects of key individual parameters, there remains a need for multi-factor experiments (e.g., pH + temperature + redox) in order to more robustly establish a framework to better understand homeostatic membrane responses.     

Crenarchaeol: the characteristic core glycerol dibiphytanyl glycerol tetraether membrane lipid of cosmopolitan pelagic crenarchaeota

The basic structure and stereochemistry of the characteristic glycerol dibiphytanyl glycerol tetraether (GDGT) membrane lipid of cosmopolitan pelagic crenarchaeota has been identified by high field two-dimensional (2D)-NMR techniques. It contains one cyclohexane and four cyclopentane rings formed by internal cyclisation of the biphytanyl chains. Its structure is similar to that of GDGTs biosynthesized by (hyper)thermophilic crenarchaeota apart from the cyclohexane ring. These findings are consistent with the close phylogenetic relationship of (hyper)thermophilic and pelagic crenarchaeota based 16S rRNA. The latter group inherited the biosynthetic capabilities for a membrane composed of cyclopentane ring-containing GDGTs from the (hyper)thermophilic crenarchaeota. However, to cope with the much lower temperature of the ocean, a small but key step in their evolution was the adjustment of the membrane fluidity by making a kink in one of the bicyclic biphytanyl chains by the formation of a cyclohexane ring. This prevents the dense packing characteristic for the cyclopentane ring-containing GDGTs membrane lipids used by hyperthermophilic crenarchaeota to adjust their membrane fluidity to high temperatures.     

Factors controlling the distribution of archaeal tetraethers in terrestrial hot springs

Glycerol dialkyl glycerol tetraethers (GDGTs) found in hot springs reflect the abundance and community structure of Archaea in these extreme environments. The relationships between GDGTs, archaeal communities, and physical or geochemical variables are underexamined to date and when reported often result in conflicting interpretations. Here, we examined profiles of GDGTs from pure cultures of Crenarchaeota and from terrestrial geothermal springs representing a wide distribution of locations, including Yellowstone National Park (United States), the Great Basin of Nevada and California (United States), Kamchatka (Russia), Tengchong thermal field (China), and Thailand. These samples had temperatures of 36.5 to 87 degrees C and pH values of 3.0 to 9.2. GDGT abundances also were determined for three soil samples adjacent to some of the hot springs. Principal component analysis identified four factors that accounted for most of the variance among nine individual GDGTs, temperature, and pH. Significant correlations were observed between pH and the GDGTs crenarchaeol and GDGT-4 (four cyclopentane rings, m/z 1,294); pH correlated positively with crenarchaeol and inversely with GDGT-4. Weaker correlations were observed between temperature and the four factors. Three of the four GDGTs used in the marine TEX(86) paleotemperature index (GDGT-1 to -3, but not crenarchaeol isomer) were associated with a single factor. No correlation was observed for GDGT-0 (acyclic caldarchaeol): it is effectively its own variable. The biosynthetic mechanisms and exact archaeal community structures leading to these relationships remain unknown. However, the data in general show promise for the continued development of GDGT lipid-based physiochemical proxies for archaeal evolution and for paleo-ecology or paleoclimate studies.     

A combined lipidomic and 16S rRNA gene amplicon sequencing approach reveals archaeal sources of intact polar lipids in the stratified Black Sea water column

Archaea are important players in marine biogeochemical cycles, and their membrane lipids are useful biomarkers in environmental and geobiological studies. However, many archaeal groups remain uncultured and their lipid composition unknown. Here, we aim to expand the knowledge on archaeal lipid biomarkers and determine the potential sources of those lipids in the water column of the euxinic Black Sea. The archaeal community was evaluated by 16S rRNA gene amplicon sequencing and by quantitative PCR. The archaeal intact polar lipids (IPLs) were investigated by ultra‐high‐pressure liquid chromatography coupled to high‐resolution mass spectrometry. Our study revealed both a complex archaeal community and large changes with water depth in the IPL assemblages. In the oxic/upper suboxic waters (<105 m), the archaeal community was dominated by marine group (MG) I Thaumarchaeota, coinciding with a higher relative abundance of hexose phosphohexose crenarchaeol, a known marker for Thaumarchaeota. In the suboxic waters (80–110 m), MGI Nitrosopumilus sp. dominated and produced predominantly monohexose glycerol dibiphytanyl glycerol tetraethers (GDGTs) and hydroxy‐GDGTs. Two clades of MGII Euryarchaeota were present in the oxic and upper suboxic zones in much lower abundances, preventing the detection of their specific IPLs. In the deep sulfidic waters (>110 m), archaea belonging to the DPANN Woesearchaeota, Bathyarchaeota, and ANME‐1b clades dominated. Correlation analyses suggest that the IPLs GDGT‐0, GDGT‐1, and GDGT‐2 with two phosphatidylglycerol (PG) head groups and archaeol with a PG, phosphatidylethanolamine, and phosphatidylserine head groups were produced by ANME‐1b archaea. Bathyarchaeota represented 55% of the archaea in the deeper part of the euxinic zone and likely produces archaeol with phospho‐dihexose and hexose‐glucuronic acid head groups.     

Membrane lipids of mesophilic anaerobic bacteria thriving in peats have typical archaeal traits

The 16S ribosomal DNA based distinction between the bacterial and archaeal domains of life is strongly supported by the membrane lipid composition of the two domains; Bacteria generally contain dialkyl glycerol diester lipids, whereas Archaea produce isoprenoid dialkyl glycerol diether and membrane-spanning glycerol dialkyl glycerol tetraether (GDGT) lipids. Here we show that a new group of ecologically abundant membrane-spanning GDGT lipids, containing branched instead of isoprenoid carbon skeletons, are of a bacterial origin. This was revealed by examining the stereochemistry of the glycerol moieties of those branched tetraether membrane lipids, which was found to be the bacterial 1,2-di-O-alkyl-sn-glycerol stereoconfiguration and not the 2,3-di-O-alkyl-sn-glycerol stereoconfiguration as in archaeal membrane lipids. In addition, unequivocal evidence for the presence of cyclopentyl moieties in these bacterial membrane lipids was obtained by NMR. The biochemical traits of biosynthesis of tetraether membrane lipids and the formation of cyclopentyl moieties through internal cyclization, which were thought to be specific for the archaeal lineage of descent, thus also occur in the bacterial domain of life.     

Constraints on the Biological Source(s) of the Orphan Branched Tetraether Membrane Lipids

A soil profile from the Saxnäs Mosse peat bog, Sweden, has been analysed for glycerol dialkyl glycerol tetraether (GDGT) membrane lipids and 16S rRNA genes in order to constrain the source of the yet ‘orphan,’ but supposedly bacterial, branched GDGTs. Branched GDGT lipids dominate over archaeal membrane lipids. The Acidobacteria comprise the dominant bacterial group, accounting for the majority of total Bacteria, and are generally more abundant than methanogenic archaea. Analysed acidobacterial strains did not contain branched GDGT lipids. Thus, the source organism must likely be searched for in other acidobacterial phyla or in another abundant group within the remaining bacteria.     

Distribution of intact and core membrane lipids of archaeal glycerol dialkyl glycerol tetraethers among size-fractionated particulate organic matter in hood canal, puget sound

There is great interest in the membrane lipids of archaea (glycerol dialkyl glycerol tetraethers [GDGTs]) as tracers of archaeal biomass because of their utility as paleoproxies and because of the biogeochemical importance of archaea. While core GDGTs (formed by hydrolysis of polar head groups of intact GDGTs after cell death) are appropriate for paleostudies, they have also been used to trace archaeal populations. Also, despite the small size (0.2 by 0.7 μm) of cultivated marine archaea, 0.7-μm glass-fiber filters (GFFs) are typically used to collect GDGTs from natural waters. We quantified both core and intact GDGTs in free-living (0.2- to 0.7-μm), suspended (0.7- to 60-μm), and aggregate (>60-μm) particle size fractions in Puget Sound (Washington State). On average, the free-living fraction contained 36% of total GDGTs, 90% of which were intact. The intermediate-size fraction contained 62% of GDGTs, and 29% of these were intact. The aggregate fraction contained 2% of the total GDGT pool, and 29% of these were intact. Our results demonstrate that intact GDGTs are largely in the free-living fraction. Because only intact GDGTs are present in living cells, protocols that target this size fraction and analyze the intact GDGT pool are necessary to track living populations in marine waters. Core GDGT enrichment in larger-size fractions indicates that archaeal biomass may quickly become attached or entrained in particles once the archaea are dead or dying. While the concentrations of the two pools were generally not correlated, the similar sizes of the core and intact GDGT pools suggest that core GDGTs are removed from the water column on timescales similar to those of cell replication, on timescales of days to weeks.     

Membrane adaptation in the hyperthermophilic archaeon Pyrococcus furiosus relies upon a novel strategy involving glycerol monoalkyl glycerol tetraether lipids

Microbes preserve membrane functionality under fluctuating environmental conditions by modulating their membrane lipid composition. Although several studies have documented membrane adaptations in Archaea, the influence of most biotic and abiotic factors on archaeal lipid compositions remains underexplored. Here, we studied the influence of temperature, pH, salinity, the presence/absence of elemental sulfur, the carbon source and the genetic background on the lipid core composition of the hyperthermophilic neutrophilic marine archaeon Pyrococcus furiosus. Every growth parameter tested affected the lipid core composition to some extent, the carbon source and the genetic background having the greatest influence. Surprisingly, P. furiosus appeared to only marginally rely on the two major responses implemented by Archaea, i.e. the regulation of the ratio of diether to tetraether lipids and that of the number of cyclopentane rings in tetraethers. Instead, this species increased the ratio of glycerol monoalkyl glycerol tetraethers (GMGT, aka. H-shaped tetraethers) to glycerol dialkyl glycerol tetraethers in response to decreasing temperature and pH and increasing salinity, thus providing for the first time evidence of adaptive functions for GMGT. Besides P. furiosus, numerous other species synthesize significant proportions of GMGT, which suggests that this unprecedented adaptive strategy might be common in Archaea.     

Intact membrane lipids of "Candidatus Nitrosopumilus maritimus," a cultivated representative of the cosmopolitan mesophilic group I Crenarchaeota

In this study we analyzed the membrane lipid composition of "Candidatus Nitrosopumilus maritimus," the only cultivated representative of the cosmopolitan group I crenarchaeota and the only mesophilic isolate of the phylum Crenarchaeota. The core lipids of "Ca. Nitrosopumilus maritimus" consisted of glycerol dialkyl glycerol tetraethers (GDGTs) with zero to four cyclopentyl moieties. Crenarchaeol, a unique GDGT containing a cyclohexyl moiety in addition to four cyclopentyl moieties, was the most abundant GDGT. This confirms unambiguously that crenarchaeol is synthesized by species belonging to the group I.1a crenarchaeota. Intact polar lipid analysis revealed that the GDGTs have hexose, dihexose, and/or phosphohexose head groups. Similar polar lipids were previously found in deeply buried sediments from the Peru margin, suggesting that they were in part synthesized by group I crenarchaeota.     

Intact polar and core glycerol dibiphytanyl glycerol tetraether lipids of group I.1a and I.1b thaumarchaeota in soil

Ecological studies of thaumarchaeota often apply glycerol dibiphytanyl glycerol tetraether (GDGT)-based intact membrane lipids. However, these components have only been characterized for thaumarchaeota from aquatic environments. Thaumarchaeota have been shown to play an important role in the nitrogen cycle in soil as ammonium oxidizers, and GDGTs are common lipids encountered in soil. We report the core and intact polar lipid (IPL) GDGTs produced by three newly available thaumarchaeota isolated from grassland soil in Austria ("Nitrososphaera viennensis," group I.1b) and enriched from agricultural soils in South Korea ("Candidatus Nitrosoarchaeum koreensis" MY1, group I.1a; and "Candidatus Nitrososphaera" strain JG1, group I.1b). The soil thaumarchaeota all synthesize crenarchaeol as their major core GDGT, in agreement with the fact that crenarchaeol has also been detected in thaumarchaeota from aquatic environments. The crenarchaeol regioisomer apparently is produced in significant quantities only by soil thaumarchaeota of the I.1b subgroup. In addition, GDGTs with 0 to 4 cyclopentane moieties and GDGTs containing an additional hydroxyl group were detected. The IPL head groups of their membrane lipids comprised mainly monohexose, dihexose, trihexose, phosphohexose, and hexose-phosphohexose moieties. The hexose-phosphohexose head group bound to crenarchaeol occurred in all soil thaumarchaeota, and this IPL is at present the only lipid that is detected in all thaumarchaeota analyzed so far. This specificity and its lability indicate that it is the most suitable biomarker lipid to trace living thaumarchaeota. This study, in combination with previous studies, also suggests that hydroxylated GDGTs occur in the I.1a, but not in the I.1b, subgroup of the thaumarchaeota.     

Fossilization and degradation of archaeal intact polar tetraether lipids in deeply buried marine sediments (Peru Margin)

Glycerol dibiphytanyl glycerol tetraether (GDGT) lipids are part of the cellular membranes of Thaumarchaeota, an archaeal phylum composed of aerobic ammonia oxidizers, and are used in the paleotemperature proxy TEX₈₆. GDGTs in live cells possess polar head groups and are called intact polar lipids (IPL‐GDGTs). Their transformation to core lipids (CL) by cleavage of the head group was assumed to proceed rapidly after cell death, but it has been suggested that some of these IPL‐GDGTs can, just like the CL‐GDGTs, be preserved over geological timescales. Here, we examined IPL‐GDGTs in deeply buried (0.2–186 mbsf, ~2.5 Myr) sediments from the Peru Margin. Direct measurements of the most abundant IPL‐GDGT, IPL‐crenarchaeol, specific for Thaumarchaeota, revealed depth profiles, which differed per head group. Shallow sediments (<1 mbsf) contained IPL‐crenarchaeol with both glycosidic and phosphate head groups, as also observed in thaumarchaeal enrichment cultures, marine suspended particulate matter and marine surface sediments. However, hexose, phosphohexose‐crenarchaeol is not detected anymore below 6 mbsf (~7 kyr), suggesting a high lability. In contrast, IPL‐crenarchaeol with glycosidic head groups is preserved over timescales of Myr. This agrees with previous analyses of deeply buried (>1 m) marine sediments, which only reported glycosidic and no phosphate‐containing IPL‐GDGTs. TEX₈₆ values of CL‐GDGTs did not markedly change with depth, and the TEX₈₆ of IPL‐derived GDGTs decreased only when the proportions of monohexose‐ to dihexose‐GDGTs changed, likely due to the enhanced preservation of the monohexose GDGTs. Our results support the hypothesis that in situ GDGT production and differential IPL degradation in sediments is not substantially affecting TEX₈₆ paleotemperature estimations based on CL–GDGTs and indicates that likely only a small amount of IPL‐GDGTs present in deeply buried sediments is part of cell membranes of active archaea. The amount of archaeal biomass in the deep biosphere based on these IPLs may have been substantially overestimated.     

Effects of high temperature and low pH on photosystem 2 photochemistry in spinach thylakoid membranes

The effects of temperature (25–45 °C) and pH (7.5–5.5) on photosystem (PS) 2 was studied in spinach (Spinacia oleracea L.) thylakoid membranes using chlorophyll a fluorescence induction kinetics. In high temperature and low pH treated thylakoid membranes a decline in the variable to maximum fluorescence ratio (F_v/F_m) and PS 2 electron transport rate were observed. More stacking in thylakoid membranes, studied by digitonin fractionation method, was observed at low pH, while the degree of unstacking increased under high temperature conditions. We conclude that the change in pH does not significantly affect the donor/acceptor side of PS 2 while high temperature does. Fluorescence emission spectra at 77 K indicated that low pH is associated with energy redistribution between the two photosystems while high temperature induced changes do not involve energy re-distribution. We suggest that both, high temperature and low pH, show an inhibitory effect on PS 2 but their mechanisms of action are different.     

Tetraether membrane lipids of <Emphasis Type="Italic">Candidatus “Aciduliprofundum boonei”</Emphasis>, a cultivated obligate thermoacidophilic euryarchaeote from deep-sea hydrothermal vents

The lipid composition of Candidatus “Aciduliprofundum boonei”, the only cultivated representative of archaea falling in the DHVE2 phylogenetic cluster, a group of microorganisms ubiquitously occurring at hydrothermal vents, was studied. The predominant core membrane lipids in this thermophilic euryarchaeote were found to be composed of glycerol dibiphytanyl glycerol tetraethers (GDGTs) containing 0–4 cyclopentyl moieties. In addition, GDGTs with an additional covalent bond between the isoprenoid hydrocarbon chains, so-called H-shaped GDGTs, were present. The latter core lipids have been rarely reported previously. Intact polar lipid analysis revealed that they predominantly consist of GDGTs with a phospho-glycerol headgroup.     

13,16-Dimethyl octacosanedioic acid (iso-diabolic acid), a common membrane-spanning lipid of Acidobacteria subdivisions 1 and 3

The distribution of membrane lipids of 17 different strains representing 13 species of subdivisions 1 and 3 of the phylum Acidobacteria, a highly diverse phylum of the Bacteria, were examined by hydrolysis and gas chromatography-mass spectrometry (MS) and by high-performance liquid chromatography-MS of intact polar lipids. Upon both acid and base hydrolyses of total cell material, the uncommon membrane-spanning lipid 13,16-dimethyl octacosanedioic acid (iso-diabolic acid) was released in substantial amounts (22 to 43% of the total fatty acids) from all of the acidobacteria studied. This lipid has previously been encountered only in thermophilic Thermoanaerobacter species but bears a structural resemblance to the alkyl chains of bacterial glycerol dialkyl glycerol tetraethers (GDGTs) that occur ubiquitously in peat and soil and are suspected to be produced by acidobacteria. As reported previously, most species also contained iso-C(15) and C(16:1ω7C) as major fatty acids but the presence of iso-diabolic acid was unnoticed in previous studies, most probably because the complex lipid that contained this moiety was not extractable from the cells; it could only be released by hydrolysis. Direct analysis of intact polar lipids in the Bligh-Dyer extract of three acidobacterial strains, indeed, did not reveal any membrane-spanning lipids containing iso-diabolic acid. In 3 of the 17 strains, ether-bound iso-diabolic acid was detected after hydrolysis of the cells, including one branched GDGT containing iso-diabolic acid-derived alkyl chains. Since the GDGT distribution in soils is much more complex, branched GDGTs in soil likely also originate from other (acido)bacteria capable of biosynthesizing these components.     

Membrane stabilization during freezing: the role of two natural cryoprotectants, trehalose and proline

The relative effectiveness of two natural cryoprotectants, proline and trehalose, in preserving membrane structure and function during freezing was studied. Isolated vesicles of sarcoplasmic reticulum (SR) from lobster muscle (Homarus americanus) were employed to study changes in structure and function during rapid freeze-thaw conditions. Both proline and trehalose were shown to effectively preserve the structure (assessed with freeze fracture) and function (assessed by the ability of the membranes to transport calcium) in the frozen vesicles. As a first step toward determining the mechanism of cryoprotection by these compounds, we have investigated their effectiveness in inhibiting freezing induced fusion between phospholipid vesicles. Pamiltoyloleoyl-phosphatidylcholine: phosphatidylserine (85:15 mole ratio) small unilamellar vesicles (SUVs) were made incorporating one of the following fluorescent probes, and energy donor, cholesteryl anthracene-9-carboxylate, or an energy acceptor, nitrobenzo-2-oxa-1,3-diazole phosphatidylethanolamine to investigate the amount of membrane mixing during rapid freeze-thaw cycles, and storage at -20 degrees C. Membrane mixing was measured as an energy transfer from donor to acceptor when donor vesicles and acceptor vesicles were mixed before a particular freezing treatment. Membrane mixing was correlated with structural changes in these membranes by freeze-fracture analysis. Both trehalose and proline were found to be more effective in preventing membrane mixing between SUVs than the standard protectants, glycerol and dimethylsulfoxide.     

Protein mediated glycolipid transfer is inhibited FROM sphingomyelin membranes but enhanced TO sphingomyelin containing raft like membranes

The mammalian glycolipid transfer protein, GLTP, catalyzes the transfer in vitro of glycolipids between membranes. In this study we have examined on one hand the effect of the variations in the donor vesicle composition and on the other hand the effects of variations in the acceptor vesicle composition on the GLTP-catalyzed transfer kinetics of galactosylceramide between bilayer vesicles. For this purpose a resonance energy transfer assay was used, the energy donor being anthrylvinyl-galactosylceramide and the energy acceptor DiO-C₁₆. First, we show that the transfer of anthrylvinyl-galactosylceramide from palmitoyl-oleoyl-phosphatidylcholine donor vesicles was faster than from dipalmitoyl-phosphatidylcholine vesicles, and that there is no transfer from palmitoyl-sphingomyelin vesicles regardless of the cholesterol amount. In this setup the acceptor vesicles were always 100% palmitoyl-oleoyl-phosphatidylcholine. We also showed that the transfer in general is faster from small highly curved vesicles compared to that from larger vesicles. Secondly, by varying the acceptor vesicle composition we showed that the transfer is faster to mixtures of sphingomyelin and cholesterol compared to mixtures of phosphatidylcholines and cholesterol. Based on these experiments we conclude that the GLTP mediated transfer of anthrylvinyl-galactosylceramide is sensitive to the matrix lipid composition and membrane bending. We postulate that a tightly packed membrane environment is most effective in preventing GLTP from accessing its substrates, and cholesterol is not required to protect the glycosphingolipid in the membrane from being transferred by GLTP. On the other hand GLTP can more easily transfer glycolipids to ‘lipid raft’ like membranes, suggesting that the protein could be involved in raft assembly.