Melittin Induces Local Order Changes in Artificial and Biological Membranes as Revealed by Spectral Analysis of Laurdan Fluorescence

Antimicrobial peptides (AMPs) are a class of molecules widely used in applications on eukaryotic and prokaryotic cells. Independent of the peptide target, all of them need to first pass or interact with the plasma membrane of the cells. In order to have a better image of the peptide action mechanism with respect to the particular features of the membrane it is necessary to better understand the changes induced by AMPs in the membranes. Laurdan, a lipid membrane probe sensitive to polarity changes in the environment, is used in this study for assessing changes induced by melittin, a well-known peptide, both in model and natural lipid membranes. More importantly, we showed that generalized polarization (GP) values are not always efficient or sufficient to properly characterize the changes in the membrane. We proved that a better method to investigate these changes is to use the previously described log-normal deconvolution allowing us to infer other parameters: the difference between the relative areas of elementary peak (ΔSr), and the ratio of elementary peaks areas (Rs). Melittin induced a slight decrease in local membrane fluidity in homogeneous lipid membranes. The addition of cholesterol stabilizes the membrane more in the presence of melittin. An opposite response was observed in the case of heterogeneous lipid membranes in cells, the local order of lipids being diminished. RS proved to be the most sensitive parameter characterizing the local membrane order, allowing us to distinguish among the responses to melittin of both classes of membrane we investigated (liposomes and cellular membranes). Molecular simulation of the melittin pore in homogeneous lipid bilayer suggests that lipids are more closely packed in the proximity of the melittin pore (a smaller area per lipid), supporting the experimental observation.

CHO/LY-B cell growth under limiting sphingolipid supply: correlation between lipid composition and biophysical properties of sphingolipid-restricted cell membranes+

ABSTRACTSphingolipids (SL) are ubiquitous in mammalian cell membranes, yet there is little data on the behavior of cells under SL-restriction conditions. LY-B cells derive from a CHO line in which serine palmitoyl transferase (SPT), thus de novo SL synthesis, is suppressed, while maintaining the capacity of taking up and metabolizing exogenous sphingoid bases from the culture medium. In the present study LY-B cells were adapted to grow in a fetal bovine serum (FBS)-deficient medium to avoid external uptake of lipids. The lowest FBS concentration that allowed LY-B cell growth, though at a slow rate, under our conditions was 0.04%, i.e. 250-fold less than the standard (10%) concentration. Cells grown under limiting SL concentrations remained viable for at least 72 h. Enriching with sphingomyelin the SL-deficient medium allowed the recovery of control LY-B cell growth rates. Studies including whole cells, plasma membrane preparations, and derived lipid vesicles were carried out. Laurdan fluorescence was recorded to measure membrane molecular order, showing a significant decrease in the rigidity of LY-B cells, not only in plasma membrane but also in whole cell lipid extract, as a result of SL limitation in the growth medium. Plasma membrane preparations and whole cell lipid extracts were also studied using atomic force microscopy in the force spectroscopy mode. Force measurements demonstrated that lower breakthrough forces were required to penetrate samples obtained from SL-poor LY-B cells than those obtained from control cells. Mass-spectroscopic analysis was also a helpful tool to understand the rearrangement undergone by the LY-B cell lipid metabolism. The most abundant SL in LY-B cells, sphingomyelin, decreased by about 85% as a result of SL limitation in the medium, the bioactive lipid ceramide and the ganglioside precursor hexosylceramide decreased similarly, together with cholesterol. Quantitative SL analysis showed that a 250-fold reduction in sphingolipid supply to LY-B cells led to a 6-fold decrease in membrane sphingolipids, underlining the resistance to changes in composition of these cells. Plasma membrane compositions exhibited similar changes, at least qualitatively, as the whole cells with SL restriction. A linear correlation was observed between the sphingomyelin concentration in the membranes, the degree of lipid order as measured by laurdan fluorescence, and membrane breakthrough forces assessed by atomic force microscopy. Concomitant changes were detected in glycerophospholipids under SL-restriction conditions.

Multiple interfacial hydration of dihydro-sphingomyelin bilayer reported by the Laurdan fluorescence

The hydration properties of the lipid bilayer interface are important for determining membrane characteristics. The hydration properties of different lipid bilayer species were evaluated using the solvent sensitive fluorescence probe, 6-lauroyl-2-dimethylamino naphthalene (Laurdan). Sphingolipids, D-erythro-N-palmitoyl-sphingosylphosphorylcholine (PSM) and D-erythro-N-palmitoyl-dihydrosphingomyelin (DHPSM) showed specific, interfacial hydration properties stemming from their intra- and intermolecular hydrogen bonds. As control, the bilayers of glycerophospholipids, such as 1-palmitoyl-2-palmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1-oleoyl-2-oleoyl-sn-glycero-3-phosphocholine (DOPC), were also evaluated. The fluorescence properties of Laurdan in sphingolipids indicated multiple excited states according to the results obtained from the emission spectra, fluorescence anisotropy, and the center of mass spectra during the decay time. Deconvolution of the Laurdan emission spectra into four components enabled us to identify the variety of hydration and the configurational states derived from intermolecular hydrogen bonding in sphingolipids. Particularly, the Laurdan in DHPSM revealed more hydrated properties compared to the case in PSM, even though DHPSM has a higher Tm than PSM. Since DHPSM forms hydrogen bonds with water molecules (in 2NH configurational functional groups) and the different flexibility among the head groups compared with PSM, which could modulate space to retain a high amount of water molecules. The careful analysis of Laurdan such as the deconvolution of emission spectra into four components performed in this study gives the important view for understanding the membrane hydration property.