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

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

Uso de simuladores para la adquisición de habilidades: ¿y si usamos más la imaginación?

La enseñanza en Ciencias de la Salud enfrenta al alumno al permanente desafío de transformar los contenidos teóricos adquiridos en acciones concretas que determinan su labor diaria. Para acercar al alumno a las prácticas desde los primeros años de la carrera de Medicina, desde la Cátedra de Fisiología y Física Biológica de la Facultad de Ciencias Médicas UNLP, se propuso realizar una actividad optativa que consistió en la extracción de sangre venosa. Para ello se armó manualmente un simulador con material sencillo, a fin de que los estudiantes puedan ejercitar la técnica las veces que sean necesarias antes de enfrentarse con el paciente Objetivos: se planteó como objetivo fundamental que los alumnos puedan adquirir esta destreza en un simulador, para que al momento de la práctica frente al paciente se genere el menor daño posible, sin perder de vista la brecha que existe entre una simulación y una situación real.Facultad de Ciencias Médica

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

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

La extensión universitaria como camino para repensar la docencia y la investigación hacia la inclusión social

El Instituto de Investigaciones Bioquímicas de La Plata (INIBIOLP) desarrolla exitosamente desde hace varios años un programa de pasantías para estudiantes próximos a egresar del colegio secundario. Está especialmente destinado a alumnos con vocación en el área de salud y cuyos medios sociales y económicos les implica un alejamiento al sentimiento de “posibilidad” de transitar un ambiente universitario. El objetivo del programa es colaborar para que estos alumnos se conciban capaces y esto les ayude a tomar la decisión de inscribirse en el nivel superior. La ejecución de una actividad de investigación es la excusa para convocar a un tránsito periódico en un ambiente de elaboración de pensamientos críticos, de aprehensión de destrezas prácticas y estímulo a la inserción en la academia. Dado el espíritu de este proyecto, los destinatarios por excelencia son estudiantes próximos a egresar del Centro de Especialización Bachillerato de Adultos en Salud (CEBAS No 1 Floreal Ferrara). La experiencia en estos años es altamente estimulante, ya que varios de ellos logran la percepción de inscribirse, algunos en Medicina, Obstetricia y gran parte en Enfermería. Consideramos por tanto que gran parte del logro de este proyecto es el fomentar la posibilidad de inserción en la educación superior en áreas de salud a un grupo poblacional con alta vocación y esfuerzo de superación, en especial en estos momentos históricos de reivindicación social de estas profesiones. El proyecto involucra articulación de distintos actores (Docentes, No docentes, investigadores, alumnos y graduados jóvenes) que participan como instructores. Todos ellos pertenecen al INIBIOLP ó a la Facultad de Ciencias Médicas (incluso alumnos avanzados de esta carrera). Se implementan experiencias que, si bien son parte del quehacer en su desempeño profesional ya sea en las prácticas docentes o de investigación, son resignificadas para que sean reconocidas y entendible por los alumnos mediante un lenguaje accesible, en un entorno contenedor, y que avalen el recrear como posible la futura práctica a la que estos pasantes puedan enfrentarse en su campo laboral. Por tanto consideramos que un segundo logro de este proyecto es el estimular en el plantel de instructores el espíritu solidario e integral de la educación y el desarrollo interrelacionado de los 3 pilares de la universidad: investigación, docencia y extensión.Secretaría de Asuntos Académico