Probing the Link between Pancratistatin and Mitochondrial Apoptosis through Changes in the Membrane Dynamics on the Nanoscale.

Pancratistatin (PST) is a natural antiviral alkaloid that has demonstrated specificity toward cancerous cells and explicitly targets the mitochondria. PST initiates apoptosis while leaving healthy, noncancerous cells unscathed. However, the manner by which PST induces apoptosis remains elusive and impedes the advancement of PST as a natural anticancer therapeutic agent. Herein, we use neutron spin-echo (NSE) spectroscopy, molecular dynamics (MD) simulations, and supporting small angle scattering techniques to study PST's effect on membrane dynamics using biologically representative model membranes. Our data suggests that PST stiffens the inner mitochondrial membrane (IMM) by being preferentially associated with cardiolipin, which would lead to the relocation and release of cytochrome c. Second, PST has an ordering effect on the lipids and disrupts their distribution within the IMM, which would interfere with the maintenance and functionality of the active forms of proteins in the electron transport chain. These previously unreported findings implicate PST's effect on mitochondrial apoptosis.

A Mechanical Mechanism for Vitamin E Acetate in E-cigarette/Vaping-Associated Lung Injury.

The outbreak of electronic-cigarette/vaping-associated lung injury (EVALI) has made thousands ill. This lung injury has been attributed to a physical interaction between toxicants from the vaping solution and the pulmonary surfactant. In particular, studies have implicated vitamin E acetate as a potential instigator of EVALI. Pulmonary surfactant is vital to proper respiration through the mechanical processes of adsorption and interface stability to achieve and maintain low surface tension at the air-liquid interface. Using neutron spin echo spectroscopy, we investigate the impact of vitamin E acetate on the mechanical properties of two lipid-only pulmonary surfactant mimics: pure 1,2-dipalmitoyl-sn-glycero-3-phosphocholine and a more comprehensive lipid mixture. It was found that increasing vitamin E acetate concentration nonlinearly increased membrane fluidity and area compressibility to a plateau. Softer membranes would promote adsorption to the air-liquid interface during inspiration as well as collapse from the interface during expiration. These findings indicate the potential for the failure of the pulmonary surfactant upon expiration, attributed to monolayer collapse. This collapse could contribute to the observed EVALI signs and symptoms, including shortness of breath and pneumonitis.

Methanol Accelerates DMPC Flip-Flop and Transfer: A SANS Study on Lipid Dynamics.

Methanol is a common solubilizing agent used to study transmembrane proteins/peptides in biological and synthetic membranes. Using small angle neutron scattering and a strategic contrast-matching scheme, we show that methanol has a major impact on lipid dynamics. Under increasing methanol concentrations, isotopically distinct 1,2-dimyristoyl-sn-glycero-3-phosphocholine large unilamellar vesicle populations exhibit increased mixing. Specifically, 1,2-dimyristoyl-sn-glycero-3-phosphocholine transfer and flip-flop kinetics display linear and exponential rate enhancements, respectively. Ultimately, methanol is capable of influencing the structure-function relationship associated with bilayer composition (e.g., lipid asymmetry). The use of methanol as a carrier solvent, despite better simulating some biological conditions (e.g., antimicrobial attack), can help misconstrue lipid scrambling as the action of proteins or peptides, when in actuality it is a combination of solvent and biological agent. As bilayer compositional stability is crucial to cell survival and protein reconstitution, these results highlight the importance of methanol, and solvents in general, in biomembrane and proteolipid studies.

Peptide-Induced Lipid Flip-Flop in Asymmetric Liposomes Measured by Small Angle Neutron Scattering.

Despite the prevalence of lipid transbilayer asymmetry in natural plasma membranes, most biomimetic model membranes studied are symmetric. Recent advances have helped to overcome the difficulties in preparing asymmetric liposomes in vitro, allowing for the examination of a larger set of relevant biophysical questions. Here, we investigate the stability of asymmetric bilayers by measuring lipid flip-flop with time-resolved small-angle neutron scattering (SANS). Asymmetric large unilamellar vesicles with inner bilayer leaflets containing predominantly 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and outer leaflets composed mainly of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) displayed slow spontaneous flip-flop at 37 ◦C (half-time, t1/2 = 140 h). However, inclusion of peptides, namely, gramicidin, alamethicin, melittin, or pHLIP (i.e., pH-low insertion peptide), accelerated lipid flip-flop. For three of these peptides (i.e., pHLIP, alamethicin, and melittin), each of which was added externally to preformed asymmetric vesicles, we observed a completely scrambled bilayer in less than 2 h. Gramicidin, on the other hand, was preincorporated during the formation of the asymmetric liposomes and showed a time resolvable 8-fold increase in the rate of lipid asymmetry loss. These results point to a membrane surface-related (e.g., adsorption/insertion) event as the primary driver of lipid scrambling in the asymmetric model membranes of this study. We discuss the implications of membrane peptide binding, conformation, and insertion on lipid asymmetry.

Transverse lipid organization dictates bending fluctuations in model plasma membranes.

Membrane undulations play a vital role in many biological processes, including the regulation of membrane protein activity. The asymmetric lipid composition of most biological membranes complicates theoretical description of these bending fluctuations, yet experimental data that would inform any such a theory is scarce. Here, we used neutron spin-echo (NSE) spectroscopy to measure the bending fluctuations of large unilamellar vesicles (LUV) having an asymmetric transbilayer distribution of high- and low-melting lipids. The asymmetric vesicles were prepared using cyclodextrin-mediated lipid exchange, and were composed of an outer leaflet enriched in egg sphingomyelin (ESM) and an inner leaflet enriched in 1-palmitoyl-2-oleoyl-phosphoethanolamine (POPE), which have main transition temperatures of 37 °C and 25 °C, respectively. The overall membrane bending rigidity was measured at three temperatures: 15 °C, where both lipids are in a gel state; 45 °C, where both lipids are in a fluid state; and 30 °C, where there is gel-fluid co-existence. Remarkably, the dynamics for the fluid asymmetric LUVs (aLUVs) at 30 °C and 45 °C do not follow trends predicted by their symmetric counterparts. At 30 °C, compositional asymmetry suppressed the bending fluctuations, with the asymmetric bilayer exhibiting a larger bending modulus than that of symmetric bilayers corresponding to either the outer or inner leaflet. We conclude that the compositional asymmetry and leaflet coupling influence the internal dissipation within the bilayer and result in membrane properties that cannot be directly predicted from corresponding symmetric bilayers.

The antioxidant vitamin E as a membrane raft modulator: Tocopherols do not abolish lipid domains.

The antioxidant vitamin E is a commonly used vitamin supplement. Although the multi-billion dollar vitamin and nutritional supplement industry encourages the use of vitamin E, there is very little evidence supporting its actual health benefits. Moreover, vitamin E is now marketed as a lipid raft destabilizing anti-cancer agent, in addition to its antioxidant behaviour. Here, we studied the influence of vitamin E and some of its vitamers on membrane raft stability using phase separating unilamellar lipid vesicles in conjunction with small-angle scattering techniques and fluorescence microscopy. We find that lipid phase behaviour remains unperturbed well beyond physiological concentrations of vitamin E (up to a mole fraction of 0.10). Our results are consistent with a proposed line active role of vitamin E at the domain boundary. We discuss the implications of these findings as they pertain to lipid raft modification in native membranes, and propose a new hypothesis for the antioxidant mechanism of vitamin E.