Fluorescence spectroscopic studies on phase heterogeneity in lipid bilayer membranes (original) (raw)
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PLoS ONE, 2011
Background: We recently reported that sphingomyelin (SM) analogs substituted on the alkyl chain by various fluorophores (e.g. BODIPY) readily inserted at trace levels into the plasma membrane of living erythrocytes or CHO cells and spontaneously concentrated into micrometric domains. Despite sharing the same fluorescent ceramide backbone, BODIPY-SM domains segregated from similar domains labelled by BODIPY-D-e-lactosylceramide (D-e-LacCer) and depended on endogenous SM. Methodology/Principal Findings: We show here that BODIPY-SM further differed from BODIPY-D-e-LacCer orglucosylceramide (GlcCer) domains in temperature dependence, propensity to excimer formation, association with a glycosylphosphatidylinositol (GPI)-anchored fluorescent protein reporter, and lateral diffusion by FRAP, thus demonstrating different lipid phases and boundaries. Whereas BODIPY-D-e-LacCer behaved like BODIPY-GlcCer, its artificial stereoisomer, BODIPY-L-t-LacCer, behaved like BODIPY-and NBD-phosphatidylcholine (PC). Surprisingly, these two PC analogs also formed micrometric patches yet preferably at low temperature, did not show excimer, never associated with the GPI reporter and showed major restriction to lateral diffusion when photobleached in large fields. This functional comparison supported a three-phase micrometric compartmentation, of decreasing order: BODIPY-GSLs .-SM .-PC (or artificial L-t-LacCer). Coexistence of three segregated compartments was further supported by double labelling experiments and was confirmed by additive occupancy, up to ,70% cell surface coverage. Specific alterations of BODIPY-analogs domains by manipulation of corresponding endogenous sphingolipids suggested that distinct fluorescent lipid partition might reflect differential intrinsic propensity of endogenous membrane lipids to form large assemblies. Conclusions/Significance: We conclude that fluorescent membrane lipids spontaneously concentrate into distinct micrometric assemblies. We hypothesize that these might reflect preexisting compartmentation of endogenous PM lipids into non-overlapping domains of differential order: GSLs. SM. PC, resulting into differential self-adhesion of the two former, with exclusion of the latter.
Methods in molecular biology, 2021
Fluorescence-based techniques have been an integral factor in the study of cellular and model membranes. Fluorescence studies carried out on model membranes have provided valuable structural information and have helped reveal mechanistic detail regarding the formation and properties of ordered lipid domains, commonly known as lipid rafts. This chapter focuses on four techniques, based on fluorescence spectroscopy or microscopy, which are commonly used to analyze lipid rafts. The techniques described in this chapter may be used in a variety of ways and in combination with other techniques to provide valuable information regarding lipid order and domain formation, especially in model membranes.
Controlling and measuring local composition and properties in lipid bilayer membranes
Journal of biological physics, 2002
Local composition, structure, morphology, and phase are interrelated in lipid bilayer membranes. This gives us the opportunity to control one or more of these properties by manipulating others. We investigate theserelationships with combinations of simultaneous two-color widefield fluorescence imaging, three-dimensional rendering of vesicle domains, andmanipulation of the vesicle morphology via optical trapping and micropipetteaspiration. We describe methods to modulate, to measure, and to probe thelocal structure of model membranes through control of membrane curvature inliposomes.
Monitoring Biophysical Properties of Lipid Membranes by Environment-Sensitive Fluorescent Probes
Biophysical Journal, 2009
We review the main trends in the development of fluorescence probes to obtain information about the structure, dynamics, and interactions in biomembranes. These probes are efficient for studying the microscopic analogs of viscosity, polarity, and hydration, as well as the molecular order, environment relaxation, and electrostatic potentials at the sites of their location. Progress is being made in increasing the information content and spatial resolution of the probe responses. Multichannel environment-sensitive probes that can distinguish between different membrane physicochemical properties through multiple spectroscopic parameters show considerable promise.
Fluorescence studies of lipid regular distribution in membranes
Chemistry and Physics of Lipids, 2002
This article reviews the use of fluorescent lipids and free probes in the studies of lipid regular distribution in model membranes. The first part of this article summarizes the evidence and physical properties for lipid regular distribution in pyrene-labeled phosphatidylcholine (PC)/unlabeled PC binary mixtures as revealed by the fluorescence of pyrene-labeled PC. The original and the extended hexagonal superlattice model are discussed. The second part focuses on the fluorescence studies of sterol regular distributions in membranes. The experimental evidence for sterol superlattice formation obtained from the fluorescent sterol (i.e. dehydroergosterol) and non-sterol fluorescent probes (e.g. DPH and Laurdan) are evaluated. Prospects and concerns are given with regard to the sterol regular distribution. The third part deals briefly with the evidence for polar headgroup superlattices. The emphasis of this article is placed on the new concept that membrane properties and activities, including the activities of surface acting enzymes, drug partitioning, and membrane free volume, are fine-tuned by minute changes in the concentration of bulky lipids (e.g. sterols and pyrene-containing acyl chains) in the vicinities of the critical mole fractions for superlattice formation.
Yves Mély and Guy Duportail (Eds.): Fluorescent methods to study biological membranes
Analytical and Bioanalytical Chemistry, 2013
Book's topic Cellular membranes are a fundamental element of living cells, providing a boundary that separates them from their environment, controlling molecular transport into and out of the cytosol, and allowing fundamental cellular functions such as motility, adhesion and signaling. Beyond these structural functions, the membrane was long regarded as an undifferentiated fluid of lipids with little functional significance, where membrane proteins are dissolved. This view was a consequence of the fact that the highly dynamic nature of the lipid bilayer hampered its detailed structural characterization. However, over the years, compelling experimental evidence has demonstrated the presence of significant lateral and transbilayer heterogeneities, and of selective interactions of membrane proteins with some lipid components. Much of this progress has been gained through the application of spectroscopic and microscopic fluorescence techniques, which are uniquely suited to study membrane systems thanks to their high sensitivity and intrinsic nanoseconds timescale. The impressive development of these methods in the last years has witnessed a dramatic progress in the space and time resolution of microscopic methods, the introduction of new fluorescent labels with unique sensitivity to different membrane properties and a progressive improvement of techniques for a detailed analysis of membrane dynamics. These methods now reached a stage of maturity where they can be applied to living cells to understand the significance of membrane structure and dynamics in controlling vital cellular functions. The book "Fluorescent Methods to Study Biological Membranes", edited by Yves Mély and Guy Duportail as the 13 th volume of the Springer Series on Fluorescence, is therefore a very timely comprehensive reference on this topic, providing a complete overview of the different experimental approaches available and of their state of the art, through a collection of in depth review articles written by renowned experts in the field.
Journal of Fluorescence, 2003
Time-resolved fluorescence of eight fluorescence probes were studied in EggPC bilayer membrane vesicles. Emission wavelength dependent fluorescence decays were analyzed in a model-independent way to obtain time resolved area normalized emission spectra (TRANES). The TRANES spectra of the probes studied were classified into four types: (1) spectra that are identical at all time (one emissive species), (2) spectra that show an isoemissive point (two emissive species), (3) spectra that shift continuously with time (slow solvation dynamics or multiple species), and (4) spectra that shift for a short time and thereafter one or two emissive species are indicated. The TRANES spectra of these eight probes, except RH421, belong to the type 1, 2, or 4. The continuous shift of the TRANES spectra that was observed for the probe RH421 is attributed to multiple ground state species and not due to slow solvation dynamics.
Direct observation of lipid domains in free standing bilayers: from simple to complex lipid mixtures
Chemistry and Physics of Lipids, 2003
The direct observation of temperature-dependent lipid phase equilibria, using two-photon excitation fluorescence microscopy on giant unilamellar vesicles (GUVs) composed of different lipid mixtures, provides novel information about the physical characteristics of lipid domain coexistence. Physical characteristics such as the shape, size, and time evolution of different lipid domains are not directly accessible from the traditional experimental approaches that employ either small and large unilamellar vesicles or multilamellar vesicles. In this review article, we address the most relevant findings reported from our laboratory regarding the direct observation of lipid domain coexistence at the level of single vesicles in artificial and natural lipid mixtures. In addition, key points concerning our experimental approach will be discussed. The unique advantages of the fluorescent probe 6dodecanoyl-2-dimethylaminonaphthalene (LAURDAN) under two-photon excitation fluorescence microscopy is particularly addressed, especially, the possibility of obtaining information on the phase state of different lipid domains directly from the fluorescent images.