Interaction of high density lipoprotein particles with membranes containing cholesterol

In this study, free cholesterol (FC) efflux mediated by human HDL was investigated using fluorescence methodologies. The accessibility of FC to HDL may depend on whether it is located in regions rich in unsaturated phospholipids or in domains containing high levels of FC and sphingomyelin, known as "lipid rafts." Laurdan generalized polarization and two-photon microscopy were used to quantify FC removal from different pools in the bilayer of giant unilamellar vesicles (GUVs). GUVs made of POPC and FC were observed after incubation with reconstituted particles containing apolipoprotein A-I and POPC [78Å diameter reconstituted high density lipoprotein (rHDL)]. Fluorescence correlation spectroscopy data show an increase in rHDL size during the incubation period. GUVs made of two "raft-like" mixtures [DOPC/DPPC/FC (1:1:1) and POPC/SPM/FC (6:1:1)] were used to model liquid-ordered/liquid-disordered phase coexistence. Through these experiments, we conclude that rHDL preferentially removes cholesterol from the more fluid phases. These data, and their extrapolation to in vivo systems, show the significant role that phase separation plays in the regulation of cholesterol homeostasis.Instituto de Investigaciones Bioquímicas de La Plat

Laurdan generalized polarization fluctuations measures membrane packing micro-heterogeneity in vivo

Cellular membranes are heterogeneous in composition, and the prevailing theory holds that the structures responsible for this heterogeneity in vivo are small structures (10-200 nm), sterol- and sphingolipid-enriched, of different sizes, highly dynamic denominated rafts. Rafts are postulated to be platforms, which by sequestering different membrane components can compartmentalize cellular processes and regulate signaling pathways. Despite an enormous effort in this area, the existence of these domains is still under debate due to the characteristics of the structures itself: small in size and highly mobile, which from the technical point of view implies using techniques with high spatial and temporal resolution. In this report we measured rapid fluctuations of the normalized ratio of the emission intensity at two wavelengths of Laurdan, a membrane fluorescent dye sensitive to local membrane packing. We observed generalized polarization fluctuations in the plasma membrane of intact rabbit erythrocytes and Chinese hamster ovary cells that can be explained by the existence of tightly packed micro-domains moving in a more fluid background phase. These structures, which display different lipid packing, have different sizes; they are found in the same cell and in the entire cell population. The small size and characteristic high lipid packing indicate that these micro-domains have properties that have been proposed for lipid rafts.Instituto de Investigaciones Bioquímicas de La Plat

Lipid packing determines protein-membrane interactions: Challenges for apolipoprotein A-I and high density lipoproteins

Protein and protein-lipid interactions, with and within specific areas in the cell membrane, are critical in order to modulate the cell signaling events required to maintain cell functions and viability. Biological bilayers are complex, dynamic platforms, and thus in vivo observations usually need to be preceded by studies on model systems that simplify and discriminate the different factors involved in lipid-protein interactions. Fluorescence microscopy studies using giant unilamellar vesicles (GUVs) as membrane model systems provide a unique methodology to quantify protein binding, interaction, and lipid solubilization in artificial bilayers. The large size of lipid domains obtainable on GUVs, together with fluorescence microscopy techniques, provides the possibility to localize and quantify molecular interactions. Fluorescence Correlation Spectroscopy (FCS) can be performed using the GUV model to extract information on mobility and concentration. Two-photon Laurdan Generalized Polarization (GP) reports on local changes in membrane water content (related to membrane fluidity) due to protein binding or lipid removal from a given lipid domain. In this review, we summarize the experimental microscopy methods used to study the interaction of human apolipoprotein A-I (apoA-I) in lipid-free and lipid-bound conformations with bilayers and natural membranes. Results described here help us to understand cholesterol homeostasis and offer a methodological design suited to different biological systems.Instituto de Investigaciones Bioquímicas de La PlataFacultad de Ciencias Médica

Effect of Sublethal Copper Overload on Cholesterol De Novo Synthesis in Undifferentiated Neuronal Cells

Although copper (Cu) is an essential trace metal for cells, it can induce harmful effects as it participates in the Fenton reaction. Involuntary exposure to Cu overload is much more common than expected and has been linked with neurodegeneration, particularly with Alzheimer's disease (AD) evidenced by a positive correlation between free Cu in plasma and the severity of the disease. It has been suggested that Cu imbalance alters cholesterol (Chol) homeostasis and that high membrane Chol promotes the amyloidogenic processing of the amyloid precursor protein (APP) secreting the β-amyloid (Aβ) peptide. Despite the wide knowledge on the effects of Cu in mature brain metabolism, the consequence of its overload on immature neurons remains unknown. Therefore, we used an undifferentiated human neuroblastoma cell line (SH-SY5Y) to analyze the effect of sublethal concentrations of Cu on 1? de novo Chol synthesis and membrane distribution; 2?APP levels in cells and its distribution in membrane rafts; 3?the levels of Aβ in the culture medium. Our results demonstrated that Cu increases reactive oxygen species (ROS) and favors Chol de novo synthesis in both ROS-dependent and independent manners. Also, at least part of these effects was due to the activation of 3-hydroxy-3-methyl glutaryl CoA reductase (HMGCR). In addition, Cu increases the Chol/PL ratio in the cellular membranes, specifically Chol content in membrane rafts. We found no changes in total APP cell levels; however, its presence in membrane rafts increases with the consequent increase of Aβ in the culture medium. We conclude that Cu overload favors Chol de novo synthesis in both ROS-dependent and independent manners, being at least in part, responsible for the high Chol levels found in the cell membrane and membrane rafts. These may promote the redistribution of APP into the rafts, favoring the amyloidogenic processing of this protein and increasing the levels of Aβ.Fil: Zubillaga, Marlene. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner". Universidad Nacional de la Plata. Facultad de Ciencias Médicas. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner"; ArgentinaFil: Rosa, Diana. Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias. Departamento de Ciencias Básicas. Laboratorio de Nutrición Mineral y Fisiología Reproductiva; ArgentinaFil: Astiz, Mariana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner". Universidad Nacional de la Plata. Facultad de Ciencias Médicas. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner"; ArgentinaFil: Tricerri, Maria Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner". Universidad Nacional de la Plata. Facultad de Ciencias Médicas. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner"; ArgentinaFil: Arnal, Nathalie. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner". Universidad Nacional de la Plata. Facultad de Ciencias Médicas. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner"; Argentin

Interaction of high density lipoprotein particles with membranes containing cholesterol

In this study, free cholesterol (FC) efflux mediated by human HDL was investigated using fluorescence methodologies. The accessibility of FC to HDL may depend on whether it is located in regions rich in unsaturated phospholipids or in domains containing high levels of FC and sphingomyelin, known as "lipid rafts." Laurdan generalized polarization and two-photon microscopy were used to quantify FC removal from different pools in the bilayer of giant unilamellar vesicles (GUVs). GUVs made of POPC and FC were observed after incubation with reconstituted particles containing apolipoprotein A-I and POPC [78Å diameter reconstituted high density lipoprotein (rHDL)]. Fluorescence correlation spectroscopy data show an increase in rHDL size during the incubation period. GUVs made of two "raft-like" mixtures [DOPC/DPPC/FC (1:1:1) and POPC/SPM/FC (6:1:1)] were used to model liquid-ordered/liquid-disordered phase coexistence. Through these experiments, we conclude that rHDL preferentially removes cholesterol from the more fluid phases. These data, and their extrapolation to in vivo systems, show the significant role that phase separation plays in the regulation of cholesterol homeostasis.Instituto de Investigaciones Bioquímicas de La Plat

Propiedades de la membrana y viabilidad celular: Importancia de la fluidez

Numerosos estudios sugieren que las vías de señalización y por ende la funcionalidad celular dependen de la organización de dominios en la membrana, que a su vez está determinada por la composición lipídica de la misma. El colesterol (Col) interviene en la regulación de la fluidez al particionar de manera selectiva en dominios específicos de la membrana, y se ha demostrado que su homeostasis es crucial para la viabilidad celular. Además, se sabe que el exceso de Col puede resultar citotóxico. Este lípido no puede ser degradado o utilizado como combustible, por lo que su exceso debe ser removido por aceptores o almacenado en compartimientos intracelulares. Las lipoproteínas de alta densidad (HDL) y en particular su apolipoproteína mayoritaria, la apoA-I, cumplen un rol fundamental en el transporte reverso del Col, que consiste en transportar el excedente desde los tejidos periféricos hacia el hígado para su eliminación en forma de sales biliares, o para ser redirigido desde los hepatocitos hacia los tejidos esteroidogénicos

Laurdan generalized polarization fluctuations measures membrane packing micro-heterogeneity in vivo

Cellular membranes are heterogeneous in composition, and the prevailing theory holds that the structures responsible for this heterogeneity in vivo are small structures (10-200 nm), sterol- and sphingolipid-enriched, of different sizes, highly dynamic denominated rafts. Rafts are postulated to be platforms, which by sequestering different membrane components can compartmentalize cellular processes and regulate signaling pathways. Despite an enormous effort in this area, the existence of these domains is still under debate due to the characteristics of the structures itself: small in size and highly mobile, which from the technical point of view implies using techniques with high spatial and temporal resolution. In this report we measured rapid fluctuations of the normalized ratio of the emission intensity at two wavelengths of Laurdan, a membrane fluorescent dye sensitive to local membrane packing. We observed generalized polarization fluctuations in the plasma membrane of intact rabbit erythrocytes and Chinese hamster ovary cells that can be explained by the existence of tightly packed micro-domains moving in a more fluid background phase. These structures, which display different lipid packing, have different sizes; they are found in the same cell and in the entire cell population. The small size and characteristic high lipid packing indicate that these micro-domains have properties that have been proposed for lipid rafts.Instituto de Investigaciones Bioquímicas de La Plat

Lipid packing determines protein-membrane interactions: Challenges for apolipoprotein A-I and high density lipoproteins

Protein and protein-lipid interactions, with and within specific areas in the cell membrane, are critical in order to modulate the cell signaling events required to maintain cell functions and viability. Biological bilayers are complex, dynamic platforms, and thus in vivo observations usually need to be preceded by studies on model systems that simplify and discriminate the different factors involved in lipid-protein interactions. Fluorescence microscopy studies using giant unilamellar vesicles (GUVs) as membrane model systems provide a unique methodology to quantify protein binding, interaction, and lipid solubilization in artificial bilayers. The large size of lipid domains obtainable on GUVs, together with fluorescence microscopy techniques, provides the possibility to localize and quantify molecular interactions. Fluorescence Correlation Spectroscopy (FCS) can be performed using the GUV model to extract information on mobility and concentration. Two-photon Laurdan Generalized Polarization (GP) reports on local changes in membrane water content (related to membrane fluidity) due to protein binding or lipid removal from a given lipid domain. In this review, we summarize the experimental microscopy methods used to study the interaction of human apolipoprotein A-I (apoA-I) in lipid-free and lipid-bound conformations with bilayers and natural membranes. Results described here help us to understand cholesterol homeostasis and offer a methodological design suited to different biological systems.Instituto de Investigaciones Bioquímicas de La PlataFacultad de Ciencias Médica

Methyl-β-cyclodextrins preferentially remove cholesterol from the liquid disordered phase in giant unilamellar vesicles

Methyl-β-cyclodextrins (MβCDs) are molecules that are extensively used to remove and to load cholesterol (Chol) from artificial and natural membranes; however, the mechanism of Chol extraction by MβCD from pure lipids or from complex mixtures is not fully understood. One of the outstanding questions in this field is the capability of MβCD to remove Chol from lipid domains having different packing. Here, we investigated the specificity of MβCD to remove Chol from coexisting macrodomains with different lipid packing. We used giant unilamellar vesicles (GUVs) made of 1,2- dioleoylphosphatidylcholine:1,2-dipalmitoylphatidylcholine:free cholesterol, 1:1:1 molar ratio at 27°C. Under these conditions, individual GUVs present Chol distributed into l o and l d phases. The two phases can be distinguished and visualized using Laurdan generalized polarization and two-photon excitation fluorescence microscopy. Our data indicate that MβCD removes Chol preferentially from the more disordered phase. The process of selective Chol removal is dependent on the MβCD concentration. At high concentrations, MβCD also removes phospholipids.Instituto de Investigaciones Bioquímicas de La Plat

Dataset of the construction and characterization of stable biological nanoparticles

This article shows the dataset of clearance assays and the reconstitution of stable biological nano-complexes using both detergent-assisted and spontaneous solubilization of phospholipids by the recombinant purified apolipoprotein A-I (apoA-I). Protein was intra-chain crosslinked in order to introduce steric constrains. Then, native and crosslinked protein function was evaluated by a data collection of dimiristoyl phosphatidyl choline (DMPC) micellization curves. Additionally, resulting particles from spontaneous or detergent-assisted lipid solubilization were characterized by transmission electron microscopy (TEM), size exclusion chromatography (SEC), and native polyacrylamide gel electrophoresis (PAGE). Here we set up an experimental design that may help study protein structure based on its function, since interaction with biological membranes and lipids is an intrinsic activity attributed to many proteins in circulation. In addition, by t-test analysis of collected-data, we examined the formation of lipoprotein particles by native and intra-chain crosslinked proteins under different conditions like temperature and time incubation. Thus, data shown here strengthen the usefulness of an easy, rapid, accessible and inexpensive approach to test protein flexibility related to its function.Fil: Gisonno, Romina Antonela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner". Universidad Nacional de la Plata. Facultad de Ciencias Médicas. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner"; ArgentinaFil: Tricerri, Maria Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner". Universidad Nacional de la Plata. Facultad de Ciencias Médicas. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner"; ArgentinaFil: Gonzalez, Marina Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner". Universidad Nacional de la Plata. Facultad de Ciencias Médicas. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner"; ArgentinaFil: Garda, Horacio Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner". Universidad Nacional de la Plata. Facultad de Ciencias Médicas. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner"; ArgentinaFil: Ramella, Nahuel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner". Universidad Nacional de la Plata. Facultad de Ciencias Médicas. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner"; ArgentinaFil: Díaz Ludovico, Ivo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner". Universidad Nacional de la Plata. Facultad de Ciencias Médicas. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner"; Argentin