Single-molecule anisotropy imaging

A novel method, single-molecule anisotropy imaging, has been employed to simultaneously study lateral and rotational diffusion of fluorescence-labeled lipids on supported phospholipid membranes. In a fluid membrane composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, in which the rotational diffusion time is on the order of the excited-state lifetime of the fluorophore rhodamine, a rotational diffusion constant, D(rot) = 7 x 10(7) rad(2)/s, was determined. The lateral diffusion constant, measured by direct analysis of single-molecule trajectories, was D(lat) = 3.5 x 10(-8) cm(2)/s. As predicted from the free-volume model for diffusion, the results exhibit a significantly enhanced mobility on the nanosecond time scale. For membranes of DPPC lipids in the L(beta) gel phase, the slow rotational mobility permitted the direct observation of the rotation of individual molecules characterized by D(rot) = 1.2 rad(2)/s. The latter data were evaluated by a mean square angular displacement analysis. The technique developed here should prove itself profitable for imaging of conformational motions of individual proteins on the time scale of milliseconds to seconds.     

Statistical Analysis of Lateral Diffusion and Multistate Kinetics in Single-Molecule Imaging

Single-molecule trajectories of molecules on the membrane of living cells have indicated the possibility that the lateral mobility of individual molecules is variable with time. Such temporal variation in mobility may indicate intrinsic kinetics of multiple molecular states. To clarify the mechanisms of signal processing on the membrane, quantitative characterizations of such temporal variations are necessary. Here we propose a method to analyze and characterize the multiple states in lateral mobility and their transition kinetics from single-molecule trajectories based on a displacement probability density function and an autocorrelation function of squared displacements. We performed our method for three cases: a molecule with a single diffusion coefficient (D), a mixture of molecules in two states with different D-values, and a molecule switching between two states with different D-values. Our analysis of numerically generated trajectories successfully distinguished the three cases and estimated the characteristic parameters for mobility and the kinetics of state transitions. This method is applicable to single-molecule tracking analysis of molecules in multiple functional states with different lateral mobility on the membrane of living cells.     

Lowering the barriers to random walks on the cell surface

We used fluorescence recovery after photobleaching (FRAP) and single particle tracking (SPT) techniques to compare diffusion of class I major histocompatibility complex molecules (MHC) on normal and alpha-spectrin-deficient murine erythroleukemia (MEL) cells. Because the cytoskeleton mesh acts as a barrier to lateral mobility of membrane proteins, we expected that diffusion of membrane proteins in alpha-spectrin-deficient MEL cells would differ greatly from that in normal MEL cells. In the event, diffusion coefficients derived from either FRAP or SPT analysis were similar for alpha-spectrin-deficient and normal MEL cells, differing by a factor of approximately 2, on three different timescales: tens of seconds, 1-10 s, and 100 ms. SPT analysis showed that the diffusion of most class I MHC molecules was confined on both cell types. On the normal MEL cells, the mean diagonal length of the confined area was 330 nm with a mean residency time of 40s. On the alpha-spectrin-deficient MEL cells, the mean diagonal length was 650 nm with a mean residency time of 45s. Thus there are fewer barriers to lateral diffusion on cytoskeleton mutant MEL cells than on normal MEL cells, but this difference does not strongly affect lateral diffusion on the scales measured here.     

Methods to measure the lateral diffusion of membrane lipids and proteins

In this chapter, we discuss methods to measure lateral mobility of membrane lipids and proteins using techniques based on the light microscope. These methods typically sample lateral mobility in very small, micron-sized regions of the membrane so that they can be used to measure diffusion in regions of single cells. The methods are based on fluorescence from the molecules of interest or from light scattered from particles attached to single or small groups of membrane lipids or proteins. Fluorescence recovery after photobleaching (FRAP), fluorescence correlation spectroscopy (FCS) and Single particle tracking (SPT) are presented in that order. FRAP and FCS methodologies are described for a dedicated wide field microscope although many confocal microscopes now have software permitting these measurement to be made; nevertheless, the principles of the measurement are the same for a wide field or confocal microscope. SPT can be applied to trace the movements of single fluorescent molecules in membranes but this aspect will not be treated in detail.     

Free brownian motion of individual lipid molecules in biomembranes

The mobility of phospolipids in free-standing and supported membranes was investigated on the level of individual molecules. For the analysis of trajectories a new statistical treatment was developed that permitted us to clearly distinguish different types of diffusional motion. A freely diffusing subfraction of lipids within supported membranes was identified. Its mobility was characterized by a mean lateral diffusion constant of D(supp) = 4.6 &mgr;m(2)/s. In comparison, the mobility of lipids embedded in "free-standing" planar membranes yielded an increase in the mean diffusion constant by a factor of 4.5, D(free) = 20.6 &mgr;m(2)/s. This increase is attributed to the ultrathin (</=1 nm) lubricating water layer between membranes and glass support.     

Lateral diffusion anisotropy and membrane lipid/skeleton interaction in outer hair cells

The organization of the plasma membrane of cells in lipid domains affects the way the membrane interacts with the underlying protein skeleton, which in turn affects the lateral mobility of lipid and protein molecules in the membrane. Membrane fluidity properties can be monitored by various approaches, the most versatile of which is fluorescence recovery after photobleaching (FRAP). We extended previous FRAP experiments on isolated cochlear outer hair cells (OHCs) by analyzing the two-dimensional pattern of lipid diffusion in the lateral membrane of these cells. We found that membrane lipid mobility in freshly isolated OHCs is orthotropic, diffusion being faster in the axial direction of the cell and slower in the circumferential direction. Increasing the cell's turgor pressure by osmotic challenge reduced the axial diffusion constant, but had only a slight effect on circumferential diffusion. Our results suggest that lipid mobility in the OHC plasma membrane is affected by the presence of the cell's orthotropic membrane skeleton. This effect could reflect interaction with spectrin filaments or with other membrane skeletal proteins. We also performed a number of FRAP measurements in temporal bone preparations preserving the structural integrity of the hearing organ. The diffusion rates measured for OHCs in this preparation were in good agreement with those obtained in isolated OHCs, and comparable to the mobility rates measured on the sensory inner hair cells. These observations support the idea that the plasma membranes of both types of hair cells share similar highly fluid phases in the intact organ. Lipid mobility was significantly slower in the membranes of supporting cells of the organ of Corti, which could reflect differences in lipid phase or stronger hindrance by the cytoskeleton in these membranes.     

巨噬细胞吞噬过程中膜磷脂流动性和受体流动性的关系

本文用ＦＲＡＰ（ｆｌｕｏｒｅｓｃｅｎｃｅｒｅｃｏｖｅｒｙａｆｔｅｒｐｈｏｔｏｂｌｅａｃｈｉｎｇ）技术,测量了静息状态和刀豆素Ａ刺激不同时间后巨噬细胞膜磷脂、ＣｏｎＡ受体扩散系数和荧光恢复率的变化。结果显示ＣｏｎＡ刺激后膜磷脂和ＣｏｎＡ受体的扩散系数和荧光恢复率均较静息状态的巨噬细胞明显降低,磷脂流动性的变化与ＣｏｎＡ受体流动性的变化呈正相关。提示受体介导内吞导致的膜磷脂流动性的降低,可能是由于配体与细胞膜上受体结合形成配体－受体复合体,增加了受体的负荷,使受体的流动性降低,进而使膜磷脂的流动性降低。巨噬细胞内吞过程中膜磷脂和ＣｏｎＡ受体流动性的降低,可能还与ＣｏｎＡ刺激后巨噬细胞胞浆ｐＨ值有关。     

The polymer-supported phospholipid bilayer: tethering as a new approach to substrate-membrane stabilization

We present a new molecular engineering approach in which a polymer-supported phospholipid bilayer is vertically stabilized by controlled covalent tethering at both the polymer-substrate and polymer-bilayer interfaces. This approach is based on lipopolymer molecules, which not only form a polymer cushion between the phospholipid bilayer and a solid glass substrate but also act as covalent connections (tethers) between the bilayer and cushion. Our approach involves Langmuir-Blodgett transfer of a phospholipid-lipopolymer monolayer followed by Schaefer transfer of a pure phospholipid monolayer and is capable of varying the tethering density between the polymer layer and the phospholipid bilayer in a very controlled manner. Further stabilization is achieved if the glass substrate is surface-functionalized with a benzophenone silane. In this case, a photocross-linking reaction between the polymer and benzophenone group allows for the covalent attachment of the polymer cushion to the glass substrate. This approach is similar to that recently reported by Wagner and Tamm in which double tethering is achieved via lipopolymer silanes (Wagner, M. L.; Tamm, L. K. Biophys. J. 2000, 79, 1400). To obtain a deeper understanding of how the covalent tethering affects the lateral mobility of the bilayer, we performed fluorescence recovery after photobleaching (FRAP) experiments on polymer-tethered bilayers at different tethering densities (lipopolymer/phospholipid molar ratios). The FRAP data clearly indicate that the hydrophobic lipopolymer moieties act as rather immobile obstacles within the phospholipid bilayer, thereby leading to hindered diffusion of phospholipids. Whereas the high lateral diffusion coefficient of D = 17.7 mum(2)/s measured at low tethering density (5 mol % lipopolymer) indicates rather unrestricted motion within the bilayer, corresponding values at moderate (10 mol % lipopolymer) and high (30 mol % lipopolymer) tethering densities of D = 9.7 mum(2)/s and D = 1.1 mum(2)/s, respectively, show significant hindered diffusion. These results are contrary to the recent findings on similar membrane systems reported by Wagner and Tamm in which no significant change in phospholipid diffusion was found between 0 and 10 mol % lipopolymer. Our experimental report leads to a deeper understanding of the complex problem of interlayer coupling and offers a path toward a compromise between stability of the whole system and lateral mobility within the bilayer. Furthermore, the FRAP measurements show that polymer-tethered membranes are very interesting model systems for studying problems of restricted diffusion within two-dimensional fluids.     

FRAP analysis of membrane-associated proteins: lateral diffusion and membrane-cytoplasmic exchange

Obtaining quantitative kinetic parameters from fluorescence recovery after photobleaching (FRAP) experiments generally requires a theoretical analysis of protein mobility and appropriate solutions for FRAP recovery derived for a given geometry. Here we provide a treatment of FRAP recovery for a molecule undergoing a combined process of reversible membrane association and lateral diffusion on the plasma membrane for two commonly used bleach geometries: stripes, and boxes. Such analysis is complicated by the fact that diffusion of a molecule during photobleaching can lead to broadening of the bleach area, resulting in significant deviations of the actual bleach shape from the desired bleach geometry, which creates difficulty in accurately measuring kinetic parameters. Here we overcome the problem of deviations between actual and idealized bleach geometries by parameterizing, more accurately, the initial postbleach state. This allows for reconstruction of an accurate and analytically tractable approximation of the actual fluorescence distribution. Through simulated FRAP experiments, we demonstrate that this method can be used to accurately measure a broad range of combinations of diffusion constants and exchange rates. Use of this method to analyze the plextrin homology domain of PLC-δ1 in Caenorhabditis elegans results in quantitative agreement with prior analysis of this domain in other cells using other methods. Because of the flexibility, relative ease of implementation, and its use of standard, easily obtainable bleach geometries, this method should be broadly applicable to investigation of protein dynamics at the plasma membrane.     

Characterization of Horizontal Lipid Bilayers as a Model System to Study Lipid Phase Separation

Artificial lipid membranes are widely used as a model system to study single ion channel activity using electrophysiological techniques. In this study, we characterize the properties of the artificial bilayer system with respect to its dynamics of lipid phase separation using single-molecule fluorescence fluctuation and electrophysiological techniques. We determined the rotational motions of fluorescently labeled lipids on the nanosecond timescale using confocal time-resolved anisotropy to probe the microscopic viscosity of the membrane. Simultaneously, long-range mobility was investigated by the lateral diffusion of the lipids using fluorescence correlation spectroscopy. Depending on the solvent used for membrane preparation, lateral diffusion coefficients in the range D_lat = 10-25 μm²/s and rotational diffusion coefficients ranging from D_rot = 2.8 − 1.4 × 10⁷ s⁻¹ were measured in pure liquid-disordered (L_d) membranes. In ternary mixtures containing saturated and unsaturated phospholipids and cholesterol, liquid-ordered (L_o) domains segregated from the L_d phase at 23°C. The lateral mobility of lipids in L_o domains was around eightfold lower compared to those in the L_d phase, whereas the rotational mobility decreased by a factor of 1.5. Burst-integrated steady-state anisotropy histograms, as well as anisotropy imaging, were used to visualize the rotational mobility of lipid probes in phase-separated bilayers. These experiments and fluorescence correlation spectroscopy measurements at different focal diameters indicated a heterogeneous microenvironment in the L_o phase. Finally, we demonstrate the potential of the optoelectro setup to study the influence of lipid domains on the electrophysiological properties of ion channels. We found that the electrophysiological activity of gramicidin A (gA), a well-characterized ion-channel-forming peptide, was related to lipid-domain partitioning. During liquid-liquid phase separation, gA was largely excluded from L_o domains. Simultaneously, the number of electrically active gA dimers increased due to the increased surface density of gA in the L_d phase.     

Diffusion measurement of fluorescence-labeled amphiphilic molecules with a standard fluorescence microscope.

The lateral diffusion of fluorescence-labeled amphiphilic tracer molecules dissolved within a two-dimensional matrix of lipids was measured by continuous illumination of an elongated rectangular region. The resulting spatial concentration profile of unbleached tracer molecules was observed with a standard epifluorescence microscope and analyzed with digital image-processing techniques. These concentration profiles are governed by the mobility of the tracers, their rate of photolysis, and the geometry of the illuminated area. For the case of a long and narrow illuminated stripe, a mathematical analysis of the process is given. After prolonged exposure, the concentration profile can be approximated by a simple analytical function. This fact was used to measure the quotient of the rate of photolysis, and the diffusion constant of the fluorescent probe. With an additional measurement of the rate of photolysis, the mobility of the tracer was determined. As prototype experiments we studied the temperature dependence of the lateral diffusion of N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-dipalmitoylphosphatidyl++ + ethanolamine in glass-supported bilayers of L-alpha-dimyristoylphosphatidylcholine. Because of its simple experimental setup, this technique represents a very useful method of determining the lateral diffusion of fluorescence-labeled membrane molecules.     

Coupling optical and electrical measurements in artificial membranes: Lateral diffusion of lipids and channel forming peptides in planar bilayers

Planar lipid bilayers (PLB) were prepared by the Montal-Mueller technique in a FRAP system designed to simultaneously measure conductivity across, and lateral diffusion of, the bilayer. In the first stage of the project the FRAP system was used to characterise the lateral dynamics of bilayer lipids with regards to phospholipid composition (headgroup, chain unsaturation etc.), presence of cholesterol and the effect of divalent cations on negatively-charged bilayers. In the second stage of the project, lateral diffusion of two fluorescently-labelled voltage-dependent pore-forming peptides (alamethicin and S4s from Shaker K⁺ channel) was determined at rest and in the conducting state. This study demonstrates the feasibility of such experiments with PLBs, amenable to physical constraints, and thus offers new opportunities for systematic studies of structure-function relationships in membrane-associating molecules.     

Simplified Equation to Extract Diffusion Coefficients from Confocal FRAP Data

Quantitative measurements of diffusion can provide important information about how proteins and lipids interact with their environment within the cell and the effective size of the diffusing species. Confocal fluorescence recovery after photobleaching (FRAP) is one of the most widely accessible approaches to measure protein and lipid diffusion in living cells. However, straightforward approaches to quantify confocal FRAP measurements in terms of absolute diffusion coefficients are currently lacking. Here, we report a simplified equation that can be used to extract diffusion coefficients from confocal FRAP data using the half time of recovery and effective bleach radius for a circular bleach region, and validate this equation for a series of fluorescently labeled soluble and membrane‐bound proteins and lipids. We show that using this approach, diffusion coefficients ranging over three orders of magnitude can be obtained from confocal FRAP measurements performed under standard imaging conditions, highlighting its broad applicability.     

Anomalously slow mobility of fluorescent lipid probes in the plasma membrane of the yeastSaccharomyces cerevisiae

Summary We measured the lateral mobility of two fluorescent lipid probes dioctadecylindocarbocyanine (dil) and tetramethyl rhodamine phosphatidylethanolamine (R-PE) in the plasma mem branesof Saccharomyces cerevisiae inol andopi 3 spheroplasts. These are well-characterized strains with mutations in the inositol and phosphatidylcholine biosynthetic pathways. Membrane phospholipid composition was altered by growing these mutants in the presence or absence of inositol and choline. Lateral mobil ity was measured by fluorescence recovery after photobleaching (FRAP). Microscopic fluorescence polarization employing CCD digital imaging produced an ordered orientation distribution of the lipid probe dil, confirming that at least one of the probes was largely incorporated into the bilayer membrane. Our results demonstrated anomalously slow mobility of both lipid probes for both mutants, regardless of whether the lipid composition was near normal or dramatically altered in relative composition of phosphatidylinositol and phosphatidylcholine. Trypsinization of the spheroplasts to remove surface proteins resulted in markedly increased lateral mobility. However, even in trypsinized sphero plasts, mobility was still somewhat lower than the mobility ob served in the membrane of mammalian cells, such as rat smooth muscle culture cells tested here for comparison.     

Interaction of lipopolysaccharide and phospholipid in mixed membranes: solid-state 31P-NMR spectroscopic and microscopic investigations

Lipopolysaccharide (LPS), which constitutes the outermost layer of Gram-negative bacterial cells as a typical component essential for their life, induces the first line defense system of innate immunity of higher animals. To understand the basic mode of interaction between bacterial LPS and phospholipid cell membranes, distribution patterns were studied by various physical methods of deep rough mutant LPS (ReLPS) of Escherichia coli incorporated in phospholipid bilayers as simple models of cell membranes. Solid-state ³¹P-NMR spectroscopic analysis suggested that a substantial part of ReLPS is incorporated into 1,2-dimyristoyl-sn-glycero-3-phosphocholine lipid bilayers when multilamellar vesicles were prepared from mixtures of these. In egg L-α-phosphatidylcholine (egg-PC)-rich membranes, ReLPS undergoes micellization. In phosphatidylethanolamine-rich membranes, however, micellization was not observed. We studied by microscopic techniques the location of ReLPS in membranes of ReLPS/egg-PC (1:10 M/M) and ReLPS/egg-PC/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) (1:9:1 M/M/M). The influence of ReLPS on the physicochemical properties of the membranes was studied as well. Microscopic images of both giant unilamellar vesicles and supported planar lipid bilayers showed that LPS was uniformly incorporated in the egg-PC lipid bilayers. In the egg-PC/POPG (9:1 M/M) lipid bilayers, however, ReLPS is only partially incorporated and becomes a part of the membrane in a form of aggregates (or as mixed aggregates with the lipids) on the bilayer surface. The lipid lateral diffusion coefficient measurements at various molar ratios of ReLPS/egg-PC/POPG indicated that the incorporated ReLPS reduces the diffusion coefficients of the phospholipids in the membrane. The retardation of diffusion became more significant with increasing POPG concentrations in the membrane at high ReLPS/phospholipid ratios. This work demonstrated that the phospholipid composition has critical influence on the distribution of added ReLPS in the respective lipid membranes and also on the morphology and physicochemical property of the resulting membranes. A putative major factor causing these phenomena is reasoned to be the miscibility between ReLPS and individual phospholipid compositions.     

Lateral diffusivity of lipid analogue excimeric probes in dimyristoylphosphatidylcholine bilayers.

The lateral mobility of pyrenyl phospholipid probes in dimyristoylphosphatidylcholine (DMPC) vesicles was determined from the dependence of the pyrene monomeric and excimeric fluorescence yields on the molar probe ratio. The analysis of the experimental data makes use of the milling crowd model for two-dimensional diffusivity and the computer simulated random walks of probes in an array of lipids. The fluorescence yields for 1-palmitoyl-2-(1'-pyrenedecanoyl)phosphatidylcholine (py10PC) in DMPC bilayers are well fitted by the model both below and above the fluid-gel phase transition temperature (Tc) and permit the evaluation of the probe diffusion rate (f), which is the frequency with which probes take random steps of length L, the host membrane lipid-lipid spacing. The lateral diffusion coefficient is then obtained from the relationship D = fL2/4. In passing through the fluid-gel phase transition of DMPC (Tc = 24 degrees C), the lateral mobility of py10PC determined in this way decrease only moderately, while D measured by fluorescence photobleaching recovery (FPR) experiments is lowered by two or more orders of magnitude in gel phase. This difference in gel phase diffusivities is discussed and considered to be related either to (a) the diffusion length in FPR experiments being about a micrometer or over 100 times greater than that of excimeric probes (approximately 1 nm), or (b) to nonrandomicity in the distribution of the pyrenyl probes in gel phase DMPC. At 35 degrees C, in fluid DMPC vesicles, the diffusion rate is f = 1.8 x 10(8) s-1, corresponding to D = 29 microns2 s-1, which is about three times larger than the value obtained in FPR experiments. The activation energy for lateral diffusion in fluid DMPC was determined to be 8.0 kcal/mol.     

Differential dynamics of membrane proteins in yeast

Lateral diffusion of lipids and proteins in yeast plasma membranes has been reported to be anomalously slow, and implicated as a possible reason for polarization in yeast. In order to gain insight into the observed slow diffusion in yeast membranes, we explored lateral diffusion of two proteins of different origin. We compared lateral dynamics of the Candida drug resistance protein-1 (Cdr1p), and the human serotonin_1A receptor (5-HT_1AR) by fluorescence recovery after photobleaching (FRAP). Our results show that while Cdr1p-GFP displays slow diffusion, the diffusion of 5-HT_1AR-EYFP is significantly faster. Interestingly, upon ergosterol depletion, the mobility of Cdr1p-GFP did not exhibit appreciable change, while 5-HT_1AR-EYFP mobility showed an increase. On the other hand, upon actin cytoskeleton destabilization, the mobile fraction of 5-HT_1AR-EYFP showed considerable increase, while the mobility of Cdr1p-GFP was not altered. Our results represent the first report on the dynamics of the important drug resistance protein Cdr1p and provide novel insight on diffusion of membrane proteins in yeast membranes.     

Phospholipids diffusion on the surface of model lipid droplets

Lipid droplets (LD) are organelles localized in the membrane of the Endoplasmic Reticulum (ER) that play an important role in metabolic functions. They consist of a core of neutral lipids surrounded by a monolayer of phosphoplipids and proteins resembling an oil-in-water emulsion droplet. Many studies have focused on the biophysical properties of these LDs. However, despite numerous efforts, we are lacking information on the mobility of phospholipids on the LDs surface, although they may play a key role in the protein distribution. In this article, we developed a microfluidic setup that allows the formation of a triolein–buffer interface decorated with a phospholipid monolayer. Using this setup, we measured the motility of phospholipid molecules by performing Fluorescent Recovery After Photobleaching (FRAP) experiments for different lipidic compositions. The results of the FRAP measurements reveal that the motility of phospholipids is controlled by the monolayer packing decorating the interface.     

Characterization of membrane domains by frap experiments at variable observation areas

In this paper we show that FRAP experiments at variable beam radii provide an experimental approach for investigating membrane organization and dynamics, with great potential for identifying micrometer-sized domains and determining their size and the diffusion coefficient of the lipid and protein molecules they contain. Monte Carlo simulations of FRAP experiments at variable beam radii R on models of compartmentalized membranes have allowed us to establish the relationships (i) between the mobile fraction M of a diffusing particle and the size r of the domains, and (ii) between the apparent diffusion coefficient D_app and the real diffusion coefficient D₀ of this particle inside the domains. Furthermore, in its present stage of development, this approach allows us to specify whether these domains are strictly closed or not. This approach was first validated on an experimental model of a strictly compartmentalized membrane consisting of a monolayer of apposed spherical phospholipid bilayers supported by silica beads of known radius (0.83 μm). To prevent fusion between the spherical bilayers 5 mol% of a polymer-grafted phospholipid was added to the lipids. Analysis of the M versus R data yielded a radius r of 0.92±0.09 μm for the spherical bilayers, close to that of the supporting silica beads. When applied to the experimental data available for lipids and proteins in the plasma membrane of living cells, this approach suggests the existence of domains within these membranes with a radius of about 0.4 – 0.7 μm for the lipids and 0.25 μm for the proteins. These domains are not strictly closed and they are believed to be delineated by fluctuating barriers which are more or less permeable to lipid and protein molecules. Received: 4 September 1997 / Revised version: 19 January 1998 / Accepted: 19 January 1998