Probes In Model Membranes Research Articles

Aggregation and mobility of membrane proteins interplay with local lipid order in the plasma membrane of T cells.

To disentangle the elusive lipid-protein interactions in T-cell activation, we investigate how externally imposed variations in mobility of key membrane proteins (T-cell receptor [TCR], kinase Lck, and phosphatase CD45) affect the local lipid order and protein colocalisation. Using spectral imaging with polarity-sensitive membrane probes in model membranes and live Jurkat T cells, we find that partial immobilisation of proteins (including TCR) by aggregation or ligand binding changes their preference towards a more ordered lipid environment, which can recruit Lck. Our data suggest that the cellular membrane is poised to modulate the frequency of protein encounters upon alterations of their mobility, for example in ligand binding, which offers new mechanistic insight into the involvement of lipid-mediated interactions in membrane-hosted signalling events.

Targeted Solvatochromic Fluorescent Probes for Imaging Lipid Order in Organelles under Oxidative and Mechanical Stress.

Biomembranes constitute a basis for all compartments of live cells, and therefore, the monitoring of their lipid organization is essential for understanding cell status and activity. However, the sensing and imaging of lipid organization specifically in different organelles of live cells remain challenging. Here, we designed an array of solvatochromic probes based on Nile Red bearing ligands for specific targeting of the endoplasmic reticulum, mitochondria, lysosomes, Golgi apparatus, plasma membranes, and lipid droplets. These polarity-sensitive probes detected variations in the lipid order by changing their emission maximum, as evidenced by fluorescence spectroscopy in model membranes. In colocalization microscopy experiments with reference organelle markers, they exhibited good organelle selectivity. Using two-color fluorescence microscopy, the new probes enabled imaging of the local polarity of organelles in live cells. To exclude the biased effect of the probe design on the sensitivity to the membrane properties, we calibrated all probes in model membranes under the microscope, which enabled the first quantitative description of the lipid order in each organelle of interest. Cholesterol extraction/enrichment confirmed the capacity of the probes to sense the lipid order, revealing that organelles poor in cholesterol are particularly affected by its enrichment. The probes also revealed that oxidative and mechanical stresses produced changes in the local polarity and lipid order that were characteristic for each organelle, with mitochondria and lysosomes being particularly stress sensitive. The new probes constitute a powerful toolbox for monitoring the response of the cells to physical and chemical stimuli at the level of membranes of individual organelles, which remains an underexplored direction in cellular research.

Organization and dynamics of NBD-labeled lipids in lipid bilayer analyzed by FRET using the small membrane fluorescent probe AHBA as donor

Fluorescent probes are employed to investigate natural and model membranes. It is important to know probe location and extent of perturbations they cause into the lipid bilayer. Förster Resonance Energy Transfer (FRET) is a useful tool to investigate phenomena involving plasma membranes, and reports in literature used relatively large fluorophores like 1,6-diphenylhexatriene, located at the center of the hydrophobic region, 4-aminophthalimide-based molecules located at lipid/water interfaces and BODIPY-labeled phosphatidylcholine. In this work we explored FRET process in 1,2-dimyristoyl-L-α-GPC large unilamellar vesicles, in gel and fluid phase, using as donor the very small group o-Abz bound to hexadecyl chain (2-amino-N-hexadecyl-benzamide - AHBA) and 7-nitro-2-1,3-benzoxadiazol-4-yl (NBD) labeled lipids as acceptor. From the intensity decay of donor in presence of acceptors, the FRET efficiency was calculated, and used to fit the model proposed by Fung and Stryer to that efficiency. Using lipid bilayer structural data, the procedure allowed the determination of Förster distance for each donor-acceptor pair in vesicles, without imposing any value for the orientational factor κ2. From distance distributions between o-Abz in AHBA and NBD in lipid bilayer obtained using the program CONTIN, we obtained donor-acceptor populations having different separation distances. The populations reflect the occurrence of FRET involving probes in the same or in opposite leaflet. A dynamic picture emerged showing how relative position of the probes is dependent on the structural thermal phase of the DMPC bilayer. The results emphasize the need of careful analysis in order to understand processes involving fluorescent probes in model membranes.

New BODIPY lipid probes for fluorescence studies of membranes

Many fluorescent lipid probes tend to loop back to the membrane interface when attached to a lipid acyl chain rather than embedding deeply into the bilayer. To achieve maximum embedding of BODIPY (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene) fluorophore into the bilayer apolar region, a series of sn-2 acyl-labeled phosphatidylcholines was synthesized bearing 4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene-8-yl (Me(4)-BODIPY-8) at the end of C(3)-, C(5)-, C(7)-, or C(9)-acyl. A strategy was used of symmetrically dispersing the methyl groups at BODIPY ring positions 1, 3, 5, and 7 to decrease fluorophore polarity. Iodide quenching of the phosphatidylcholine probes in bilayer vesicles confirmed that the Me(4)-BODIPY-8 fluorophore was embedded in the bilayer. Parallax analysis of Me(4)-BODIPY-8 fluorescence quenching by phosphatidylcholines containing iodide at different positions along the sn-2 acyl chain indicated that the penetration depth of Me(4)-BODIPY-8 into the bilayer was determined by the length of the linking acyl chain. Evaluation using monolayers showed minimal perturbation of <10 mol% probe in fluid-phase and cholesterol-enriched phosphatidylcholine. Spectral characterization in monolayers and bilayers confirmed the retention of many features of other BODIPY derivatives (i.e., absorption and emission wavelength maxima near 498 nm and approximately 506-515 nm) but also showed the absence of the 620-630 nm peak associated with BODIPY dimer fluorescence and the presence of a 570 nm emission shoulder at high Me(4)-BODIPY-8 surface concentrations. We conclude that the new probes should have versatile utility in membrane studies, especially when precise location of the reporter group is needed.

Fluorescence Resonance Energy Transfer between Lipid Probes Detects Nanoscopic Heterogeneity in the Plasma Membrane of Live Cells

Fluorescence resonance energy transfer (FRET) between matched carbocyanine lipid analogs in the plasma membrane outer leaflet of RBL mast cells was used to investigate lateral distributions of lipids and to develop a general method for quantitative measurements of lipid heterogeneity in live cell membranes. FRET measured as fluorescence quenching of long-chain donor probes such as DiO-C 18 is greater with long-chain, saturated acceptor probes such as DiI-C 16 than with unsaturated or shorter-chain acceptors with the same chromophoric headgroup compared at identical concentrations. FRET measurements between these lipid probes in model membranes support the conclusion that differential donor quenching is not caused by nonideal mixing or spectroscopic differences. Sucrose gradient analysis of plasma membrane-labeled, Triton X-100-lysed cells shows that proximity measured by FRET correlates with the extent of lipid probe partitioning into detergent-resistant membranes. FRET between DiO-C 16 and DiI-C 16 is sensitive to cholesterol depletion and disruption of liquid order (Lo) by short-chain ceramides, and it is enhanced by cross linking of Lo-associated proteins. Consistent results are obtained when homo-FRET is measured by decreased fluorescence anisotropy of DiI-C 16. These results support the existence of nanometer-scale Lo/liquid disorder heterogeneity of lipids in the outer leaflet of the plasma membrane in live cells.

Determination of the depth of BODIPY probes in model membranes by parallax analysis of fluorescence quenching

The location of a series of lipophilic and lipid-attached BODIPY (4,4-difluoro-4-bora-3a,4a-diaza- s-indacene) membrane probes was analyzed by the quenching of BODIPY fluorescence by a series of nitroxide-labeled lipids in which the depth of the nitroxide group is varied. When attached to the polar headgroup of PE the BODIPY remained near the polar headgroup in depth. However, when attached at the end of free or phospholipid-attached fatty acyl chains, or when attached to two hydrocarbon chains, we observed two probe populations. One, usually dominant, population of BODIPY groups ‘looped back’ towards the surface, but a second population remained deeply embedded within the bilayer. When attached to a fatty acid or fatty acyl chain, the deep population appeared to locate at a depth related to its point of attachment to the acyl chain. In BODIPY linked to free fatty acids, the location of the deep population responded to the ionization of the carboxyl group. Because, unlike NBD (7-nitro-2,1,3-benzoxadiazol-4-yl) and most dansyl groups, acyl chain linked BODIPY groups can exist in a deeply buried form we conclude that BODIPY linked acyl chains are superior to NBD or dansyl linked acyl chains as membrane probes.

Control of the depth of molecules within membranes by polar groups: determination of the location of anthracene-labeled probes in model membranes by parallax analysis of nitroxide-labeled phospholipid induced fluorescence quenching.

The location of anthracene-labeled molecules incorporated into model membranes was measured by fluorescence quenching. The depth of the anthracene group was calculated from the degree of quenching by lipids carrying a nitroxide at different depths, using the parallax analysis (Chattopadhyay & London (1987) Biochemistry 26, 39-45). A series of anthracene derivatives was examined in order to determine what polar functional groups would anchor at the membrane surface, and at what depth anchoring would occur. An anthracene with only a methyl group was not anchored at the membrane surface, but derivatives with polar or charged groups did anchor near the membrane surface as demonstrated by a shallower anthracene depth. Based on anthracene depths, protonated primary amine, secondary amine, and hydroxyl groups appear to be located 15-16 A from the center of the membrane. A quaternary amino locates more shallowly, at 18 A from the bilayer center. A protonated carboxyl group is slightly deeper, at 14 A from the center of the bilayer. Ester groups are found to be weakly anchoring, having a location dependent on the structure of the molecule to which they are attached. In methyl 9-anthracenepropionate, the ester group is located about 13 A from the bilayer center. Anthracene esters attached to cholesterol or cholesterol esters showed various depths. An anthracene ester attached to the tail of cholesterol was located 1-6 A from the center of the bilayer for a cholesterol derivative, but at 12 A from the bilayer center for a cholesterol oleate derivative.(ABSTRACT TRUNCATED AT 250 WORDS)

Spectral inhomogeneity and wavelength-dependent rotation of probe molecules in membranes

Using subnanosecond laser spectrofluorometry, the spectral and polarization time-resolved characteristics of 1-phenylnaphthylamine (1-AN) fluorescent probe in phospholipid bilayer and red blood cell (RBC) membranes have been studied. It is shown that the electronic spectra of the probe in model-membranes are inhomogeneously broadened. In contrast to low-molecular weight solvents, there are two reasons for inhomogeneous broadening. The first is connected with different levels of location of probe molecules in the membrane. Inhomogeneous broadening due to the first factor has a static character. The second reason is similar to that of solutions and linked with fluctuations of solvate structure. This type of broadening has a dynamic character. The process of intermolecular relaxation in membranes is accompanied by the release of the free energy excess, which results in wavelength-dependent rotation of probe.

19] In situ resonance energy transfer microscopy: Monitoring membrane fusion in living cells

Publisher Summary The resolution limit of fluorescence light microscopy (about 400 nm) can be exceeded indirectly by using resonance energy transfer (RET) microscopy. This technique is able to visualize the spatial location of two different, fluorescently labeled membrane probes and determine if they are in the same membrane, or in physically adjacent but separate bilayers (10 to 400 nm apart. Resonance energy transfer microscopy was developed to study the spatial and temporal distribution of fluorescent probes in model membranes and in living cells. Particular attention was paid to the mechanics of visualizing definitively the colocalization of both membrane probes in the same bilayer. This microscope configuration is used to study the ATP-dependent liposome fusion with the Golgi apparatus of permeabilized, cultured skin fibroblasts. However, combining RET microscopy with low light-level detector technology and digital image analysis will facilitate both image acquisition and kinetic studies. The use of two-stage or three-stage charge coupled cameras will allow low illumination levels to be used and reduce photo bleaching to undetectable levels. Microprocessor-controlled digital imaging will quantitatively determine fluorescence intensity at discrete locations in the microscopic field of interest and will calculate wavelength ratios

A test of discreteness-of-charge effects in phospholipid vesicles: measurements using paramagnetic amphiphiles.

A new series of negatively charged, paramagnetic alkylsulfonate probes was synthesized and can be used to measure both the internal and the external surface potentials of model membrane systems. We tested for discreteness-of-charge effects in lipid membranes by comparing the surface potentials, estimated by use of these negatively charged amphiphiles, with that of a series of positively charged alkylammonium nitroxides in charged membranes. From the partitioning of these probes, the membrane surface potential was estimated in phosphatidylcholine membranes containing either phosphatidylserine or didodecyldimethylammonium bromide. The surface potentials, estimated with either positive or negative probes, were identical, within experimental error, in either positive or negative membranes, and they were well accounted for by a simple Gouy-Chapman-Stern theory. This symmetry, with respect to the sign of the charge, indicates that discreteness-of-charge effects are not significant in determining the potential-sensitive phase partitioning of these probes in model membranes. Thus, despite the fact that charge on membranes is discrete, models that assume a uniform density of charge in the plane of the membrane adequately account for the potentials measured by these amphiphilic probes.

Resonance energy transfer microscopy: observations of membrane-bound fluorescent probes in model membranes and in living cells.

A conventional fluorescence microscope was modified to observe the sites of resonance energy transfer (RET) between fluorescent probes in model membranes and in living cells. These modifications, and the parameters necessary to observe RET between membrane-bound fluorochromes, are detailed for a system that uses N-4-nitrobenzo-2-oxa-1,3-diazole (NBD) or fluorescein as the energy donor and sulforhodamine as the energy acceptor. The necessary parameters for RET in this system were first optimized using liposomes. Both quenching of the energy donor and sensitized fluorescence of the energy acceptor could be directly observed in the microscope. RET microscopy was then used in cultured fibroblasts to identify those intracellular organelles labeled by the lipid probe, N-SRh-decylamine (N-SRh-C10). This was done by observing the sites of RET in cells doubly labeled with N-SRh-C10 and an NBD-labeled lipid previously shown to label the endoplasmic reticulum, mitochondria, and nuclear envelope. RET microscopy was also used in cells treated with fluorescein-labeled Lens culinaris agglutinin and a sulforhodamine derivative of phosphatidylcholine to examine the internalization of plasma membrane lipid and protein probes. After internalization, the fluorescent lectin resided in most, but not all of the intracellular compartments labeled by the fluorescent lipid, suggesting sorting of the membrane-bound lectin into a subset of internal compartments. We conclude that RET microscopy can co-localize different membrane-bound components at high resolution, and may be particularly useful in examining temporal and spatial changes in the distribution of fluorescent molecules in membranes of the living cell.

The use of wide-line [ 14N]nitrogen NMR as a probe in model membranes

1. Introduction A major aim of bioiogi~al membrane research is to understand the relationship between membrane struc- ture and function at the molecular level. The presently accepted fluid mosaic model [ 1] indicates that the membrane is basically an anisotropic fluid composed of a lipid bilayer in which proteins are embedded. At the molecular level it is necessary to describe both the average conformation of the molecules and their molecular dynamics. There is both translation and rotation of the lipid and protein molecules within the bilayer structure. For the lipids in particular, there are many internal degrees of freedom. Of the many techniques [Z] being used to probe the molecular environment within membranes, nuclear magnetic resonance (NMR) [3,4] has proven to be one of the most useful methods for investigating the dynamical state of the membrane. Work has con- centrated on describing the dynamical behaviour of the lipid molecules and the interaction of these molecules with other membrane components such as various proteins [5,6] and cholesteroi [7,8]. To do this, it is essential to probe various positions in the lipid molecules. Because of the overabundance of hydrogen atoms, proton NMR spectra have been difficult to interpret [9,10] and give little information about the motion of a single segment of the lipid molecule. 3’P NMR [l I] has been successfully used to probe the head group region of phospholipids. Selective incorporation of ‘II [12], 13C [ 131 and tgF [14] into lipid molecules has been the most fruitful method of probing the molecular dynamics. However, considerable time and expertise are necessary for the synthesis of the labelled molecules required for a complete investigation of the lipid dynamics. The use of naturally abundant NMR probes is there- fore advantageous whenever possible. One naturally

Fluorescent probes in model membranes I: anthroyl fatty acid derivatives in monolayers and liposomes of dipalmitoylphosphatidylcholine.

A study of three fluorescent anthroyl probes has been carried out using pure and mixed monomolecular films with dipalmitoylphosphatidylcholine. In addition, fluorescence depolarization and differential scanning calorimetry data were obtained from dipalmitoylphosphatidylcholine vesicles with incorporated anthroyl probes. The three probes used were 2-(9-anthroyl)palmitic acid. 12-(9-anthroyl)stearic acid, and 16-(9-anthroyl)palmitic acid. The latter probe was synthesized for these studies. In monolayers the probes shifted the onset of the liquid-condensed/liquid-expanded monolayer phase transition with the extent of the shift decreasing in the order: 2-(9-anthroyl)palmitic acid greater than 12-(9-anthroyl)stearic acid greater than 16-(9-anthroyl)stearic acid. A corresponding decrease in the gel-liquid crystalline bilayer transition temperature (Tc) showed the same order of perturbation in both the fluorescence depolarization and differential scanning calorimetry data. Locating the anthroyl entity in the center of the bilayer would appear to provide a minimum perturbation.

Orientation and motion of a fluorescent probe in model membranes

A method has been developed, based on polarized fluorescence intensity measurements, for studying the orientation and motion of fluorescent probes incorporated into lipid bilayers. Its use is illustrated in a study of the effect of cholesterol in egg lecithin multibilayers. When no cholesterol is present the probes are constrained to angles between 0° and 80° to the normal to the plane and show, superimposed, a rapid but restricted motion. The angular distribution decreases to a limiting range of 0° to 67° as cholesterol is increased to 25–30 mole % but the rapid motion is unaffected.