Sandra N. Pinto

Effect of ceramide structure on membrane biophysical properties: the role of acyl chain length and unsaturation.

Ceramide is an important bioactive sphingolipid involved in a variety of biological processes. The mechanisms by which ceramide regulates biological events are not fully understood, but may involve alterations in the biophysical properties of membranes. We now examine the properties of ceramide with different acyl chains including long chain (C16- and C18-), very long chain (C24-) and unsaturated (C18:1- and C24:1-) ceramides, in phosphatidylcholine model membranes. Our results show that i) saturated ceramides have a stronger impact on the fluid membrane, increasing its order and promoting gel/fluid phase separation, while their unsaturated counterparts have a lower (C24:1-) or no (C18:1-) ability to form gel domains at 37°C; ii) differences between saturated species are smaller and are mainly related to the morphology and size of the gel domains, and iii) very long chain ceramides form tubular structures likely due to their ability to form interdigitated phases. These results suggest that generation of different ceramide species in cell membranes has a distinct biophysical impact with acyl chain saturation dictating membrane lateral organization, and chain asymmetry governing interdigitation and membrane morphology.

Membrane Domain Formation, Interdigitation, and Morphological Alterations Induced by the Very Long Chain Asymmetric C24:1 Ceramide ☆

Ceramide (Cer) is involved in the regulation of several biological processes, such as apoptosis and cell signaling. The alterations induced by Cer in the biophysical properties of membranes are thought to be one of the major routes of Cer action. To gain further knowledge about the alterations induced by Cer, membrane reorganization by the very long chain asymmetric nervonoylceramide (NCer) was studied. The application of an established fluorescence multiprobe approach, together with x-ray diffraction, differential scanning calorimetry, and confocal fluorescence microscopy, allowed the characterization of NCer and the determination of the phase diagram of palmitoyloleoylphosphatidylcholine (POPC)/NCer binary mixtures. Nervonoylceramide undergoes a transition from a mixed interdigitated gel phase to a partially interdigitated gel phase at approximately 20 degrees C, and a broad main transition to the fluid phase at approximately 52 degrees C. The solubility of NCer in the fluid POPC is low, driving gel-fluid phase separation, and the binary-phase diagram is characterized by multiple and large coexistence regions between the interdigitated gel phases and the fluid phase. At 37 degrees C, the relevant phases are the fluid and the partially interdigitated gel. Moreover, the formation of NCer interdigitated gel phases leads to strong morphological alterations in the lipid vesicles, driving the formation of cochleate-type tubular structures.

A V-shaped cationic dye for nonlinear optical bioimaging

A symmetric cationic molecule with D-π-A(+)-π-D architecture was synthesized with high two-photon absorption cross-section (σ(2) ≈ 1140 GM). Application as a marker in fluorescence microscopy of living cells revealed its presence inside the cell staining vesicular shape organelles in the cytoplasm. Fluorescence lifetime imaging microscopy shows that it is also able to penetrate within the nucleus.

A combined fluorescence spectroscopy, confocal and 2-photon microscopy approach to re-evaluate the properties of sphingolipid domains

The aim of this study is to provide further insight about the interplay between important signaling lipids and to characterize the properties of the lipid domains formed by those lipids in membranes containing distinct composition. To this end, we have used a combination of fluorescence spectroscopy, confocal and two-photon microscopy and a stepwise approach to re-evaluate the biophysical properties of sphingolipid domains, particularly lipid rafts and ceramide (Cer)-platforms. By using this strategy we were able to show that, in binary mixtures, sphingolipids (Cer and sphingomyelin, SM) form more tightly packed gel domains than those formed by phospholipids with similar acyl chain length. In more complex lipid mixtures, the interaction between the different lipids is intricate and is strongly dictated by the Cer-to-Chol ratio. The results show that in quaternary phospholipid/SM/Chol/Cer mixtures, Cer forms gel domains that become less packed as Chol is increased. Moreover, the extent of gel phase formation is strongly reduced in these mixtures, even though Cer molar fraction is increased. These results suggest that in biological membranes, lipid domains such as rafts and ceramide platforms, might display distinctive biophysical properties depending on the local lipid composition at the site of the membrane where they are formed, further highlighting the potential role of membrane biophysical properties as an underlying mechanism for mediating specific biological processes.

Changes in membrane biophysical properties induced by sphingomyelinase depend on the sphingolipid N-acyl chain

Ceramide (Cer) is involved in the regulation of several cellular processes by mechanisms that depend on Cer-induced changes on membrane biophysical properties. Accumulating evidence shows that Cers with different N-acyl chain composition differentially impact cell physiology, which may in part be due to specific alterations in membrane biophysical properties. We now address how the sphingolipid (SL) N-acyl chain affects membrane properties in cultured human embryonic kidney cells by overexpressing different Cer synthases (CerSs). Our results show an increase in the order of cellular membranes in CerS2-transfected cells caused by the enrichment in very long acyl chain SLs. Formation of Cer upon treatment of cells with bacterial sphingomyelinase promoted sequential changes in the properties of the membranes: after an initial increase in the order of the fluid plasma membrane, reorganization into domains with gel-like properties whose characteristics are dependent on the acyl chain structure of the Cer was observed. Moreover, the extent of alterations of membrane properties correlates with the amount of Cer formed. These data reinforce the significance of Cer-induced changes on membrane biophysical properties as a likely molecular mechanism by which different acyl chain Cers exert their specific biological actions.

Development of functionalized nanoparticles for vaccine delivery to dendritic cells: a mechanistic approach

AIM Produce biodegradable nanoparticles to target antigen-presenting cells (APCs) and evaluate their potential to be used as a vaccine delivery system. MATERIALS & METHODS Untargeted PEGylated poly(d,l-lactic-co-glycolide)-based nanoparticles and mannose-grafted nanoparticles were formulated and physicochemically characterized. Immortalized and primary APCs were used to study nanoparticle internalization patterns. The endocytic pathways and intracellular trafficking followed by nanoparticles were also investigated. RESULTS & DISCUSSION Nanoparticles displayed mannose residues available for binding at the nanoparticle surface. Different nanoparticle internalization patterns by immortalized and primary APCs were verified. Macropinocytosis, clathrin-mediated endocytosis, caveolin- and lipid raft-dependent endocytosis are involved in nanoparticles internalization. Nanoparticles demonstrate both endolysosomal and cytosolic localizations and a tendency to accumulate nearby the endoplasmic reticulum. CONCLUSION The developed nanoparticles might drive antigens to be presented through MHC class I and II molecules to both CD8(+) and CD4(+) T cells, favoring a complete and coordinated immune response.

Immobilization and characterization of giant unilamellar vesicles (GUVs) within porous silica glasses

Immobilization of cells or artificial liposomes has interesting applications in protein biology, membrane biophysics, biomedicine, biosensor technology and new materials development. In this work for the first time we have entrapped giant unilamellar vesicles (GUVs) in silica glasses prepared by the sol–gel process. Results show that GUVs are successfully confined in the porous matrix retaining their structural integrity for at least fifteen days, allowing single-vesicle studies to be performed. Using different fluorescence microscopy approaches, we have studied the effect of the encapsulation on membrane properties, such as their size and shape, hydration degree, domain coexistence and lipid lateral mobility. Results reveal that these properties are altered to a more or less degree after immobilization, but most of vesicles are affected in a similar fashion and no different populations are distinguished. Such effects are attributed to the increase in lateral packing induced by changes in the hydrostatic and/or osmotic pressure occurring during the sol–gel process, as well as to the establishment of interactions between the polar head of the phospholipids and the negatively charged silica surface of the porous matrix.

Nonlinear emission of quinolizinium-based dyes with application in fluorescence lifetime imaging.

Charged molecules based on the quinolizinum cation have potential applications as labels in fluorescence imaging in biological media under nonlinear excitation. A systematic study of the linear and nonlinear photophysics of derivatives of the quinolizinum cation substituted by either dimethylaniline or methoxyphenyl electron donors is performed. The effects of donor strength, conjugation length, and symmetry in the two-photon emission efficiency are analyzed in detail. The best performing nonlinear fluorophore, with two-photon absorption cross sections of 1140 GM and an emission quantum yield of 0.22, is characterized by a symmetric D-π-A(+)-π-D architecture based on the methoxyphenyl substituent. Application of this molecule as a fluorescent marker in optical microscopy of living cells revealed that, under favorable conditions, the fluorophore can be localized in the cytoplasmatic compartment of the cell, staining vesicular shape organelles. At higher dye concentrations and longer staining times, the fluorophore can also penetrate into the nucleus. The nonlinearly excited fluorescence lifetime imaging shows that the fluorophore lifetime is sensitive to its location in the different cell compartments. Using fluorescence lifetime microscopy, a multicolor map of the cell is drafted with a single dye.

Deoxycholic acid modulates cell death signaling through changes in mitochondrial membrane properties

Cytotoxic bile acids, such as deoxycholic acid (DCA), are responsible for hepatocyte cell death during intrahepatic cholestasis. The mechanisms responsible for this effect are unclear, and recent studies conflict, pointing to either a modulation of plasma membrane structure or mitochondrial-mediated toxicity through perturbation of mitochondrial outer membrane (MOM) properties. We conducted a comprehensive comparative study of the impact of cytotoxic and cytoprotective bile acids on the membrane structure of different cellular compartments. We show that DCA increases the plasma membrane fluidity of hepatocytes to a minor extent, and that this effect is not correlated with the incidence of apoptosis. Additionally, plasma membrane fluidity recovers to normal values over time suggesting the presence of cellular compensatory mechanisms for this perturbation. Colocalization experiments in living cells confirmed the presence of bile acids within mitochondrial membranes. Experiments with active isolated mitochondria revealed that physiologically active concentrations of DCA change MOM order in a concentration- and time-dependent manner, and that these changes preceded the mitochondrial permeability transition. Importantly, these effects are not observed on liposomes mimicking MOM lipid composition, suggesting that DCA apoptotic activity depends on features of mitochondrial membranes that are absent in protein-free mimetic liposomes, such as the double-membrane structure, lipid asymmetry, or mitochondrial protein environment. In contrast, the mechanism of action of cytoprotective bile acids is likely not associated with changes in cellular membrane structure.

Characterization of BCAM0224, a multifunctional trimeric autotransporter from the human pathogen Burkholderia cenocepacia

Members of the trimeric autotransporter adhesin (TAA) family play a crucial role in adhesion of Gram-negative pathogens to host cells. Moreover, these proteins are multifunctional virulence factors involved in several other biological traits, including invasion into host cells and evasion of the host immune system. In cystic fibrosis epidemic Burkholderia cenocepacia strain J2315, we identified a unique TAA (BCAM0224)-encoding gene, previously described as being implicated in virulence. Here, we characterized this multifunctional protein, trying to establish its role in B. cenocepacia pathogenicity. We show that BCAM0224 occurs on the bacterial surface and adopts a trimeric conformation. Furthermore, we demonstrated that BCAM0224 is needed for earlier stages of biofilm formation and is required for swarming motility. In addition, BCAM0224 plays an important role in evasion of the human innate immune system, providing resistance against the bactericidal activity of serum via the complement classical pathway. Finally, BCAM0224 mediates bacterial adhesion to and invasion of cultured human bronchial epithelial cells. Together, these data reveal the high versatility of the BCAM0224 protein as a virulence factor in the pathogenic bacterium B. cenocepacia.