Plasma membrane effects of sphingolipid-synthesis inhibition by myriocin in CHO cells: a biophysical and lipidomic study.

Suppression of a specific gene effect can be achieved by genetic as well as chemical methods. Each approach may hide unexpected drawbacks, usually in the form of side effects. In the present study, the specific inhibitor myriocin was used to block serine palmitoyltransferase (SPT), the first enzyme in the sphingolipid synthetic pathway, in CHO cells. The subsequent biophysical changes in plasma membranes were measured and compared with results obtained with a genetically modified CHO cell line containing a defective SPT (the LY-B cell line). Similar effects were observed with both approaches: sphingomyelin values were markedly decreased in myriocin-treated CHO cells and, in consequence, their membrane molecular order (measured as laurdan general polarization) and mechanical resistance (AFM-measured breakthrough force values) became lower than in the native, non-treated cells. Cells treated with myriocin reacted homeostatically to maintain membrane order, synthesizing more fully saturated and less polyunsaturated GPL than the non-treated ones, although they achieved it only partially, their plasma membranes remaining slightly more fluid and more penetrable than those from the control cells. The good agreement between results obtained with very different tools, such as genetically modified and chemically treated cells, reinforces the use of both methods and demonstrates that both are adequate for their intended use, i.e. the complete and specific inhibition of sphingolipid synthesis in CHO cells, without apparent unexpected effects.

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

Sphingolipids (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 linein whichserine 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 this 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%, that is, 250-fold less than the standard (10%) concentration. Cells grown under limiting SL concentrations remained viable for at least 72 hours. Enriching with sphingomyelin the SL-deficient medium allowed the recovery of growth rates analogous to those of control LY-B cells. 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 only to a sixfold 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. Smaller, though significant, changes were also detected in glycerophospholipids under SL-restriction conditions.

Phase-selective staining of model and cell membranes, lipid droplets and lipoproteins with fluorescent solvatochromic pyrene probes.

The push-pull solvatochromic pyrene derivatives PA and PK have been applied to the study of model membrane vesicles, cells and purified human serum lipoproteins, using both confocal fluorescence microscopy and fluorescence spectroscopy. These polarity-sensitive probes provide information similar to that obtained by Laurdan or Prodan, i.e. mainly lipid order in biomembranes, but they have the essential advantage of being excitable by a standard 405 nm laser light, bypassing the use of multiphoton excitation. In addition, they are brighter and much more photostable than those dimethylamino naphthalene derivatives. Our results with model membrane spectroscopy (multilamellar vesicles) and with microscopy (giant unilamellar vesicles) showed the capacity of PA and PK to report differently on liquid-disordered, liquid-ordered and gel phase bilayers. Moreover, a ratiometric parameter, the Red/Blue Intensity Ratio (RBIR) could be used for inter-domain, inter-vesicle and even inter-technique comparison, and the appropriate microscopy-spectroscopy conversion coefficients could be estimated. In studies at the cellular level, PA probe stained almost exclusively the plasma membrane of red blood cells, revealing its high degree of lipid order. Using Chinese Hamster Ovary cells PA was shown to be an excellent probe for the detection of cytoplasmic lipid droplets, superior to Nile Red in that PA provides simultaneously a detailed information of membrane order in the whole cell, in which the lipid droplets appear with a very good contrast. Moreover, spectrofluorometric data of PA-stained serum lipoproteins indicated an essentially identical value of RBIR for lipid droplets and for high-density lipoproteins.

Patches and Blebs: A Comparative Study of the Composition and Biophysical Properties of Two Plasma Membrane Preparations from CHO Cells.

This study was aimed at preparing and characterizing plasma membranes (PM) from Chinese Hamster Ovary (CHO) cells. Two methods of PM preparation were applied, one based on adhering cells to a poly-lysine-coated surface, followed by hypotonic lysis and removal of intracellular components, so that PM patches remain adhered to each other, and a second one consisting of bleb induction in cells, followed by separation of giant plasma membrane vesicles (GPMV). Both methods gave rise to PM in sufficient amounts to allow biophysical and biochemical characterization. Laurdan generalized polarization was used to measure molecular order in membranes, PM preparations were clearly more ordered than the average cell membranes (GP ≈0.450 vs. ≈0.20 respectively). Atomic force microscopy was used in the force spectroscopy mode to measure breakthrough forces of PM, both PM preparations provided values in the 4-6 nN range, while the corresponding value for whole cell lipid extracts was ≈2 nN. Lipidomic analysis of the PM preparations revealed that, as compared to the average cell membranes, PM were enriched in phospholipids containing 30-32 C atoms in their acyl chains but were relatively poor in those containing 34-40 C atoms. PM contained more saturated and less polyunsaturated fatty acids than the average cell membranes. Blebs (GPMV) and patches were very similar in their lipid composition, except that blebs contained four-fold the amount of cholesterol of patches (≈23 vs. ≈6 mol% total membrane lipids) while the average cell lipids contained 3 mol%. The differences in lipid composition are in agreement with the observed variations in physical properties between PM and whole cell membranes.

Photoacoustic effect applied on model membranes and living cells: direct observation with multiphoton excitation microscopy and long-term viability analysis.

The photoacoustic effect is generated when a variable light interacts with a strongly light-absorbing material. In water, it may produce hot bubbles and shock waves that could affect the integrity of nearby cellular membranes, opening transient pores (photoporation). In this study, we have evaluated the effect of pulsed laser-irradiated carbon nanoparticles (cNP) on model membranes and on Chinese hamster ovary (CHO) cells. Fluorescence lifetime measurements of calcein-loaded liposomes support the notion that the photoacoustic effect causes transient openings in membranes, allowing diffusion fluxes driven by gradient concentrations. With CHO cells, we have shown that this effect can induce either intracellular delivery of calcein, or release of cellular compounds. The latter process has been recorded live with multiphoton excitation microscopy during pulsed infrared laser irradiation. Calcein loading and cell viability were assayed by flow cytometry, measuring necrotic cells as well as those in early apoptosis. To further assess long-term cell recovery after the rather harsh treatment, cells were reseeded and their behaviour recorded for 48 h. These extended studies on cell viability show that pulsed laser cNP photoporation may be considered an adequate intracellular delivery technique only if employed with soft irradiation conditions (below 50 mJ/cm2).

Fast and slow biomembrane solubilizing detergents: Insights into their mechanism of action.

Detergents are water-soluble amphiphiles. Above a critical concentration they self-organize in micelles and in the presence of phospholipids mixed micelles are formed. Much information is available on the structure of these self-assemblies and on the thermodynamics of their formation. The aim of this study was to deepen our understanding of the mechanisms of solubilization. Solubilization of lipid vesicles made of egg phosphatidylcholine (PC) by twenty one commercially available, structurally heterogeneous detergents, has been assessed by a decrease in turbidity of the vesicle suspension. Both steady-state and time-resolved measurements have been performed. The results show that the detergents under study fall into one of two categories, namely fast-solubilizing and slow-solubilizing detergents. This categorization is independent of detergent concentration, i.e. a "slow" cannot be converted into a "fast" surfactant by increasing its bulk concentration. 31P-NMR spectra indicate that slow-acting detergents cause either a gradual, monotonic micellization of bilayers (sodium dodecyl sulphate), or formation of more complex, perhaps non-lamellar, non-micellar intermediates (dodecylmaltoside). In contrast, fast detergents (e.g. Triton X-100) cause lysis and reassembly of vesicles before bulk solubilization takes place. These results support the idea that membrane solubilization by detergents is rapid only when surfactant transbilayer (flipping) motion is easy.

Pb(II) Induces Scramblase Activation and Ceramide-Domain Generation in Red Blood Cells.

The mechanisms of Pb(II) toxicity have been studied in human red blood cells using confocal microscopy, immunolabeling, fluorescence-activated cell sorting and atomic force microscopy. The process follows a sequence of events, starting with calcium entry, followed by potassium release, morphological change, generation of ceramide, lipid flip-flop and finally cell lysis. Clotrimazole blocks potassium channels and the whole process is inhibited. Immunolabeling reveals the generation of ceramide-enriched domains linked to a cell morphological change, while the use of a neutral sphingomyelinase inhibitor greatly delays the process after the morphological change, and lipid flip-flop is significantly reduced. These facts point to three major checkpoints in the process: first the upstream exchange of calcium and potassium, then ceramide domain formation, and finally the downstream scramblase activation necessary for cell lysis. In addition, partial non-cytotoxic cholesterol depletion of red blood cells accelerates the process as the morphological change occurs faster. Cholesterol could have a role in modulating the properties of the ceramide-enriched domains. This work is relevant in the context of cell death, heavy metal toxicity and sphingolipid signaling.

Coating Graphene Oxide with Lipid Bilayers Greatly Decreases Its Hemolytic Properties.

Toxicity evaluation for the proper use of graphene oxide (GO) in biomedical applications involving intravenous injections is crucial, but the GO circulation time and blood interactions are largely unknown. It is thought that GO may cause physical disruption (hemolysis) of red blood cells. The aim of this work is to characterize the interaction of GO with model and cell membranes and use this knowledge to improve GO hemocompatibility. We have found that GO interacts with both neutral and negatively charged lipid membranes; binding is decreased beyond a certain concentration of negatively charged lipids and favored in high-salt buffers. After this binding occurs, some of the vesicles remain intact, while others are disrupted and spread over the GO surface. Neutral membrane vesicles tend to break down and extend over the GO, while vesicles with negatively charged membranes are mainly bound to the GO without disruption. GO also interacts with red blood cells and causes hemolysis; hemolysis is decreased when GO is previously coated with lipid membranes, particularly with pure phosphatidylcholine vesicles.

Polyamine-RNA-membrane interactions: From the past to the future in biology.

Biogenic polyamines (PAs), spermine, spermidine and putrescine are widely spread amino acid derivatives, present in living cells throughout the whole evolutionary scale. Their amino groups confer them a marked basic character at the cellular pH. We have tested the interaction of PAs with negatively-charged phospholipids in the absence and presence of nucleic acids (tRNA was mainly used for practical reasons). PAs induced aggregation of lipid vesicles containing acidic phospholipids. Aggregation was detected using both spectroscopic and fluorescence microscopy methods (the latter with giant unilamellar vesicles). PA-liposome complexes were partially disaggregated when nucleic acids were added to the mixture, indicating a competition between lipids and nucleic acids for PAs in a multiple equilibrium phenomenon. Equivalent observations could be made when vesicles composed of oleic acid and 1-decanol (1:1mol ratio) were used instead of phospholipid liposomes. The data could evoke putative primitive processes of proto-biotic evolution. At the other end of the time scale, this system may be at the basis of an interesting tool in the development of nanoscale drug delivery.