Juha M. Holopainen

Vectorial budding of vesicles by asymmetrical enzymatic formation of ceramide in giant liposomes.

Sphingomyelin is an abundant component of eukaryotic membranes. A specific enzyme, sphingomyelinase can convert this lipid to ceramide, a central second messenger in cellular signaling for apoptosis (programmed cell death), differentiation, and senescence. We used microinjection and either Hoffman modulation contrast or fluorescence microscopy of giant liposomes composed of 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC), N-palmitoyl-sphingomyelin (C16:0-SM), and Bodipy-sphingomyelin as a fluorescent tracer (molar ratio 0.75:0.20:0.05, respectively) to observe changes in lipid lateral distribution and membrane morphology upon formation of ceramide. Notably, in addition to rapid domain formation (capping), vectorial budding of vesicles, i.e., endocytosis and shedding, can be induced by the asymmetrical sphingomyelinase-catalyzed generation of ceramide in either the outer or the inner leaflet, respectively, of giant phosphatidylcholine/sphingomyelin liposomes. These results are readily explained by 1) the lateral phase separation of ceramide enriched domains, 2) the area difference between the adjacent monolayers, 3) the negative spontaneous curvature, and 4) the augmented bending rigidity of the ceramide-containing domains, leading to membrane invagination and vesiculation of the bilayer.

Lipid microdomains in dimyristoylphosphatidylcholine-ceramide liposomes.

Binary membranes composed of dimyristoylphosphatidylcholine (DMPC) and natural ceramide (up to a mole fraction Xcer = 0.25) were investigated by measuring the excimer:monomer fluorescence emission intensity ratio IE:IM for the pyrene labeled phospholipid probe 1-palmitoyl-2[(pyren-1-yl)]decanoyl-sn-glycero-3-phosphocholine (PPDPC), by monitoring fluorescence polarization of diphenylhexatriene (DPH), as well as using differential scanning calorimetry (DSC). Increasing Xcer > 0.10 both below and above the main transition temperature Tm increased IE:IM for PPDPC maximally approximately 1.6-fold at Xcer = 0.25. Above Tm, and when Xcer approaches 0.10, fluorescence polarization P for DPH increases steeply, reflecting an overall decrease in acyl chain motions. At Xcer = 0.10 there is a discontinuity in P and upon further increase in the content of ceramide a smaller, yet significant increase in P is evident. DSC revealed ceramide to increase in the pretransition temperature until at Xcer exceeding 0.07 this transition was no longer evident. Simultaneously, increasing Xcer up to 0.05 increased Tm from 23.9 to 24.6 degrees C. Total enthalpy delta Hm of the main transition diminished progressively upon increase in Xcer up to approximately 0.10. Above this concentration of ceramide a new endotherm became evident at 22.5 degrees C, and exceeding Xcer = 0.14 this endotherm became dominant. Our results indicate an enrichment of the pyrene labeled phospholipid analog into microdomains concomitant with the formation of a distinct ceramide-enriched phase at Xcer > 0.10.

Interfacial Interactions of Ceramide with Dimyristoylphosphatidylcholine: Impact of the N-Acyl Chain

The mixing behavior of dimyristoylphosphatidylcholine (DMPC) with either N-palmitoyl-sphingosine (C16:0-ceramide) or N-nervonoyl-sphingosine (C24:1-ceramide) was examined using monomolecular films. While DMPC forms highly elastic liquid-expanded monolayers, both neat C16:0-ceramide and C24:1-ceramide yield stable solid condensed monomolecular films with small areas and low interfacial elasticity. Compression isotherms of mixed C16:0-ceramide/DMPC films exhibit an apparent condensation upon increasing X(cer16:0) at all surface pressures. The average area isobars, coupled with the lack of a liquid-expanded to condensed phase transition as X(cer16:0) is increased, are indicative of immiscibility of the lipids at all surface pressures. In contrast, isobars for C24:1-ceramide/DMPC mixtures show surface pressure-dependent apparent condensation or expansion and surface pressure-area isotherms show a composition and surface pressure-dependent phase transition. This suggests miscibility, albeit non-ideal, of C24:1-ceramide and DMPC in both liquid and condensed surface phases. The above could be verified by fluorescence microscopy of the monolayers and measurements of surface potential, which revealed distinctly different domain morphologies and surface potential values for the DMPC/C16:0- and DMPC/C24:1-ceramide monolayers. Taken together, whereas C16:0-ceramide and DMPC form immiscible pseudo-compounds, C24:1-ceramide and DMPC are partially miscible in both the liquid-expanded and condensed phases, and a composition and lateral pressure-dependent two-phase region is evident between the liquid-expanded and condensed regimes. Our results provide novel understanding of the regulation of membrane properties by ceramides and raise the possibility that ceramides with different acyl groups could serve very different functions in cells, relating to their different physicochemical properties.

Evidence for the formation of microdomains in liquid crystalline large unilamellar vesicles caused by hydrophobic mismatch of the constituent phospholipids.

The excimer-to-monomer fluorescence emission intensity ratio (IE/IM) of the fluorescent probe 1-palmitoyl-2-[(pyren-1-yl)]decanoyl-sn-glycero-3-phosphocholine (PPDPC, 1 mol%) was measured at 30 degrees C as a function of the thickness of fluid liposomal membranes composed of phosphatidylcholines (PCs) with homologous monounsaturated acyl chains of varying lengths N (= number of carbon atoms). Upon decreasing N from di-24:1 PC to di-14:1 PC, the rate of excimer formation was sigmoidally augmented from 0.02 to 0.06. This increase in IE/IM can arise either from enhanced lateral mobility or from the lateral enrichment of PPDPC into domains, or both. Direct evidence for partial lateral segregation of PPDPC being involved is provided by experiments where 1.6 mol% of 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamino-N- (5-fluoresceinthiocarbamoyl) (DPPF) was included together with PPDPC into the bilayers. Notably, because of spectral overlap DPPF can function as a resonance energy transfer acceptor for pyrene excimer. Fluorescence intensity ratio (F/Fo) measured at 480 nm for PPDPC/DPPF (yielding F) and PPDPC (yielding Fo) containing membranes as a function of N reveals a sharp maximum for di-20:1 PC, i.e., the quenching of pyrene excimer fluorescence by DPPF is least efficient in this lipid and is enhanced upon either decrease or increase in N. This is compatible with colocalization of DPPF in PPDPC enriched domains when N not equal to 20, whereas in di-20:1 PC these probes appear to be effectively dispersed. The driving force for the enrichment of PPDPC in thin (N < 20) and thick (N > 20) PC matrices is likely to be hydrophobic mismatch of the effective lengths of the matrix phospholipids and the fluorescent probes. We also measured fluorescence polarization (P) for 1,6-diphenyl-1,3,5-hexatriene (DPH) as well as the IE/IM for the intramolecular excimer forming probe 1,2-bis[(pyren-1-yl)]decanoyl-sn-glycero-3-phosphocholine (bisPDPC) as a function of N. In brief, neither the fluorescence polarization data and nor the measurements of intramolecular chain dynamics using bisPDPC concur with enhanced lateral diffusion as the sole cause for the increase in the IE/IM for PPDPC in thin membranes. Our findings suggest hydrophobic mismatch as the cause of microdomain formation of lipids in fluid, liquid crystalline bilayers, while simultaneously allowing for a high rates of lateral diffusion. Such hydrophobic mismatch-induced compositional fluctuations would also offer one plausible explanation for the chain length diversity observed for biological membranes.

Comparison of the membrane association of two antimicrobial peptides, magainin 2 and indolicidin.

Interactions of two antimicrobial peptides, magainin 2 and indolicidin, with three different model biomembranes, namely, monolayers, large unilamellar vesicles (LUVs), and giant liposomes, were studied. Insertion of both peptides into lipid monolayers was progressively enhanced when the content of an acidic phospholipid, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) in a film of 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC) was increased. Indolicidin and magainin 2 penetrated also into lipid monolayers containing cholesterol (mole fraction, X = 0.1). Membrane association of magainin 2 attenuated lipid lateral diffusion in POPG-containing LUVs as revealed by the decrease in the excimer/monomer fluorescence ratio I(e)/I(m) for the pyrene fatty-acid-containing phospholipid derivative 1-palmitoyl-2-[10-(pyren-1-yl) decanoyl]-sn-glycero-3-phospho-rac-glycerol (PPDPG). Likewise, an increase in steady-state fluorescence anisotropy of the membrane-incorporated diphenylhexatriene (DPH) was observed, revealing magainin 2 to increase acyl chain order and induce segregation of acidic phospholipids. Similar effects were observed for indolicidin. The topological effects of magainin 2 and indolicidin on phospholipid membranes were investigated using optical microscopy of giant vesicles. Magainin 2 had essentially no influence on either SOPC or SOPC:cholesterol (X = 0.1) giant liposomes. However, effective vesiculation was observed when acidic phospholipid (X(PG) = 0.1) was included in the giant vesicles. Indolicidin caused only a minor shrinkage of giant SOPC vesicles whereas the formation of endocytotic vesicles was observed when the giant liposome contained POPG (X(PG) = 0.1). Interestingly, for indolicidin, vesiculation was also observed for giant vesicles composed of SOPC/cholesterol (X(chol) = 0.1). Possible mechanisms of membrane transformation induced by these two peptides are discussed.

Evidence for the Lack of a Specific Interaction between Cholesterol and Sphingomyelin

The putative specific interaction and complex formation by sphingomyelin and cholesterol was investigated. Accordingly, low contents (1 mol % each) of fluorescently labeled derivatives of these lipids, namely 1-palmitoyl-2[10-(pyren-1-yl)]decanoyl-sn-glycero-3-phosphocholine (PyrPC), n-[10-(1-pyrenyl)decanoyl]sphingomyelin (PyrSM), and increasing concentrations of cholesterol (up to 5 mol %), were included in large unilamellar vesicles composed of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) or 1,2-dinervonoyl-sn-glycero-3-phosphocholine (DNPC), and the excimer/monomer fluorescence emission ratio (I(e)/I(m)) was measured. In DNPC below the main phase transition, the addition of up to 5 mol % cholesterol reduced I(e)/I(m) significantly. Except for this, cholesterol had only a negligible effect in both matrices and for both probes. We then compared the efficiency of resonance energy transfer from PyrPC and PyrSM to 22-(n-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-23,24-bisnor-5-cholen-3beta-ol (NBDchol). An augmenting colocalization of the latter resonance energy transfer pair with temperature was observed in a DMPC matrix below the main phase transition. In contrast, compared to PyrSM the colocalization of PyrPC with NBDchol was more efficient in the longer DNPC matrix. These results could be confirmed using 5,6-dibromo-cholestan-3beta-ol as a collisional quencher for the pyrene-labeled lipids. The results indicate lack of a specific interaction between sphingomyelin and cholesterol, and further imply that hydrophobic mismatch between the lipid constituents could provide the driving force for the cosegregation of sphingomyelin and cholesterol in fluid phospholipid bilayers of thicknesses comparable to those found for biomembranes.

Human Tear Fluid Lipidome: From Composition to Function

We have explored human aqueous tear fluid lipidome with an emphasis to identify the major lipids. We also address the physiological significance of the lipidome. The tears were analysed using thin layer chromatographic, enzymatic and mass spectrometric techniques. To emphasize the physiological aspect of the lipidome, we modelled the spreading of the non-polar tear fluid lipids at air-water interface in macroscopic scale with olive oil and egg yolk phosphatidylcholine. Based on enzymatic analysis the respective concentrations of choline-containing lipids, triglycerides, and cholesteryl esters were 48±14, 10±0, and 21±18 µM. Ultra performance liquid chromatography quadrupole time of flight mass spectrometry analysis showed that phosphatidylcholine and phosphatidylethanolamine were the two most common polar lipids comprising 88±6% of all identified lipids. Triglycerides were the only non-polar lipids detected in mass spectrometric analysis i.e. no cholesteryl or wax esters were identified. The spreading experiments show that the presence of polar lipids is an absolute necessity for a proper spreading of non-polar tear fluid lipids. We provide evidence that polar lipids are the most common lipid species. Furthermore, we provide a physiological rationale for the observed lipid composition. The results open insights into the functional role of lipids in the tear fluid and also aids in providing new means to understand and treat diseases of the ocular surface.

Study on liposomes by capillary electrophoresis.

Liposomes made of mixtures of zwitterionic and anionic lipids were investigated by means of capillary electrophoresis and dynamic light scattering. The influence of the molar lipid ratio and of the buffers, used in the running electrolyte solution, on the physical characteristics of the liposomes were investigated. Data on effective electrophoretic mobilities, total charges as well as sizes of the liposomes are given. In addition, examples on the use of liposomes as carriers in electrokinetic capillary electrophoresis for the separation of benzene derivatives, steroids, and phenols are shown.

Multifactor effects and evidence of potential interaction between complement factor H Y402H and LOC387715 A69S in age-related macular degeneration.

Background Variants in the complement cascade genes and the LOC387715/HTRA1, have been widely reported to associate with age-related macular degeneration (AMD), the most common cause of visual impairment in industrialized countries. Methods/Principal Findings We investigated the association between the LOC387715 A69S and complement component C3 R102G risk alleles in the Finnish case-control material and found a significant association with both variants (OR 2.98, p = 3.75×10−9; non-AMD controls and OR 2.79, p = 2.78×10−19, blood donor controls and OR 1.83, p = 0.008; non-AMD controls and OR 1.39, p = 0.039; blood donor controls), respectively. Previously, we have shown a strong association between complement factor H (CFH) Y402H and AMD in the Finnish population. A carrier of at least one risk allele in each of the three susceptibility loci (LOC387715, C3, CFH) had an 18-fold risk of AMD when compared to a non-carrier homozygote in all three loci. A tentative gene-gene interaction between the two major AMD-associated loci, LOC387715 and CFH, was found in this study using a multiplicative (logistic regression) model, a synergy index (departure-from-additivity model) and the mutual information method (MI), suggesting that a common causative pathway may exist for these genes. Smoking (ever vs. never) exerted an extra risk for AMD, but somewhat surprisingly, only in connection with other factors such as sex and the C3 genotype. Population attributable risks (PAR) for the CFH, LOC387715 and C3 variants were 58.2%, 51.4% and 5.8%, respectively, the summary PAR for the three variants being 65.4%. Conclusions/Significance Evidence for gene-gene interaction between two major AMD associated loci CFH and LOC387715 was obtained using three methods, logistic regression, a synergy index and the mutual information (MI) index.

Mechanisms of Initiation of Membrane Fusion: Role of Lipids

Main emphasis in studies on the mechanisms of fusion of cellular membranes has been in the roles of various proteins, with far less interest in the properties of lipids. Yet, on a molecular level fusion involves the merging of lipid bilayers. Studies so far have revealed lipids forming inverted non-lamellar phases to be important in controlling membrane fusion. However, the underlying molecular level mechanisms have remained controversial. While this review is focused on presenting one possible mechanism, involving so-called extended lipid conformation, we are also advocating the view, that in order to obtain a more complete understanding of this process it is necessary to merge the relevant physicochemical properties of lipids with the models describing the specific functions of proteins. To this end, taking into account the central importance of fusion in a wide range of cellular processes, we may anticipate its control to open novel possibilities also for therapeutic intervention.