Nanoscale fluorescence correlation spectroscopy on intact living cell membranes with NSOM probes

Characterization of molecular dynamics on living cell membranes at the nanoscale is fundamental to unravel the mechanisms of membrane organization and compartmentalization. Here we demonstrate the feasibility of fluorescence correlation spectroscopy (FCS) based on the nanometric illumination of near-field scanning optical microscopy (NSOM) probes on intact living cells. NSOM-FCS applied to fluorescent lipid analogs allowed us to reveal details of the diffusion hidden by larger illumination areas. Moreover, the technique offers the unique advantages of evanescent axial illumination and straightforward implementation of multiple color excitation. As such, NSOM-FCS represents a powerful tool to study a variety of dynamic processes occurring at the nanometer scale on cell membranes.     

Detection of motional heterogeneities in lipid bilayer membranes by dual probe fluorescence correlation spectroscopy

We report the detection of heterogeneities in the diffusion of lipid molecules for the three-component mixture dipalmitoyl-PC/dilauroyl-PC/cholesterol, a chemically simple lipid model for the mammalian plasma membrane outer leaflet. Two-color fluorescence correlation spectroscopy (FCS) was performed on giant unilamellar vesicles (GUVs) using fluorescent probes that have differential lipid phase partition behavior—DiO-C18:2 favors disordered fluid lipid phases, whereas DiI-C20:0 prefers spatially ordered lipid phases. Simultaneously-obtained fluorescence autocorrelation functions from the same excitation volume for each dye showed that, depending on the lipid composition of this ternary mixture, the two dyes exhibited different lateral mobilities in regions of the phase diagram with previously proposed submicroscopic two-phase coexistence. In one-phase regions, both dyes reported identical diffusion coefficients. Two-color FCS thus may be detecting local membrane heterogeneities at size scales below the optical resolution limit, either due to short-range order in a single phase or due to submicroscopic phase separation.     

Fluorescence correlation spectroscopy with single-molecule sensitivity on cell and model membranes.

We report on the successful application of fluorescence correlation spectroscopy (FCS) to the analysis of single fluorescently labeled lipid analogue molecules diffusing laterally in lipid bilayers, as exemplified by time traces of fluorescence bursts of individual molecules entering and leaving the excitation area. FCS measurements performed on lipid probes in rat basophilic leukemia cell membranes showed deviations from two-dimensional Brownian motion with a single uniform diffusion constant. Giant unilamellar vesicles were employed as model systems to characterize diffusion of fluorescent lipid analogues in both homogeneous and mixed lipid phases with diffusion heterogeneity. Comparing the results of cell membrane diffusion with the findings on the model systems suggests possible explanations for the observations: (a) anomalous subdiffusion in which evanescent attractive interactions with disparate mobile molecules modifies the diffusion statistics; (b) alternatively, probe molecules are localized in microdomains of submicroscopic size, possibly in heterogeneous membrane phases.     

Recent progress on lipid lateral heterogeneity in plasma membranes: From rafts to submicrometric domains

The concept of transient nanometric domains known as lipid rafts has brought interest to reassess the validity of the Singer–Nicolson model of a fluid bilayer for cell membranes. However, this new view is still insufficient to explain the cellular control of surface lipid diversity or membrane deformability. During the past decades, the hypothesis that some lipids form large (submicrometric/mesoscale vs nanometric rafts) and stable (> min vs s) membrane domains has emerged, largely based on indirect methods. Morphological evidence for stable submicrometric lipid domains, well-accepted for artificial and highly specialized biological membranes, was further reported for a variety of living cells from prokaryot es to yeast and mammalian cells. However, results remained questioned based on limitations of available fluorescent tools, use of poor lipid fixatives, and imaging artifacts due to non-resolved membrane projections. In this review, we will discuss recent evidence generated using powerful and innovative approaches such as lipid-specific toxin fragments that support the existence of submicrometric domains. We will integrate documented mechanisms involved in the formation and maintenance of these domains, and provide a perspective on their relevance on membrane deformability and regulation of membrane protein distribution.     

STED nanoscopy reveals molecular details of cholesterol- and cytoskeleton-modulated lipid interactions in living cells

Details about molecular membrane dynamics in living cells, such as lipid-protein interactions, are often hidden from the observer because of the limited spatial resolution of conventional far-field optical microscopy. The superior spatial resolution of stimulated emission depletion (STED) nanoscopy can provide new insights into this process. The application of fluorescence correlation spectroscopy (FCS) in focal spots continuously tuned down to 30 nm in diameter distinguishes between free and anomalous molecular diffusion due to, for example, transient binding of lipids to other membrane constituents, such as lipids and proteins. We compared STED-FCS data recorded on various fluorescent lipid analogs in the plasma membrane of living mammalian cells. Our results demonstrate details about the observed transient formation of molecular complexes. The diffusion characteristics of phosphoglycerolipids without hydroxyl-containing headgroups revealed weak interactions. The strongest interactions were observed with sphingolipid analogs, which showed cholesterol-assisted and cytoskeleton-dependent binding. The hydroxyl-containing headgroup of gangliosides, galactosylceramide, and phosphoinositol assisted binding, but in a much less cholesterol- and cytoskeleton-dependent manner. The observed anomalous diffusion indicates lipid-specific transient hydrogen bonding to other membrane molecules, such as proteins, and points to a distinct connectivity of the various lipids to other membrane constituents. This strong interaction is different from that responsible for forming cholesterol-dependent, liquid-ordered domains in model membranes.     

Heterogeneous nanoscopic lipid diffusion in the live cell membrane and its dependency on cholesterol

Cholesterol plays a unique role in the regulation of membrane organization and dynamics by modulating the membrane phase transition at the nanoscale. Unfortunately, due to their small sizes and dynamic nature, the effects of cholesterol-mediated membrane nanodomains on membrane dynamics remain elusive. Here, using ultrahigh-speed single-molecule tracking with advanced optical microscope techniques, we investigate the diffusive motion of single phospholipids in the live cell plasma membrane at the nanoscale and its dependency on the cholesterol concentration. We find that both saturated and unsaturated phospholipids undergo anomalous subdiffusion on the length scale of 10–100 nm. The diffusion characteristics exhibit considerable variations in space and in time, indicating that the nanoscopic lipid diffusion is highly heterogeneous. Importantly, through the statistical analysis, apparent dual-mobility subdiffusion is observed from the mixed diffusion behaviors. The measured subdiffusion agrees well with the hop diffusion model that represents a diffuser moving in a compartmentalized membrane created by the cytoskeleton meshwork. Cholesterol depletion diminishes the lipid mobility with an apparently smaller compartment size and a stronger confinement strength. Similar results are measured with temperature reduction, suggesting that the more heterogeneous and restricted diffusion is connected to the nanoscopic membrane phase transition. Our conclusion supports the model that cholesterol depletion induces the formation of gel-phase, solid-like membrane nanodomains. These nanodomains undergo restricted diffusion and act as diffusion obstacles to the membrane molecules that are excluded from the nanodomains. This work provides the experimental evidence that the nanoscopic lipid diffusion in the cell plasma membrane is heterogeneous and sensitive to the cholesterol concentration and temperature, shedding new light on the regulation mechanisms of nanoscopic membrane dynamics.     

Sphingolipid-cholesterol rafts diffuse as small entities in the plasma membrane of mammalian cells

To probe the dynamics and size of lipid rafts in the membrane of living cells, the local diffusion of single membrane proteins was measured. A laser trap was used to confine the motion of a bead bound to a raft protein to a small area (diam < or = 100 nm) and to measure its local diffusion by high resolution single particle tracking. Using protein constructs with identical ectodomains and different membrane regions and vice versa, we demonstrate that this method provides the viscous damping of the membrane domain in the lipid bilayer. When glycosylphosphatidylinositol (GPI) -anchored and transmembrane proteins are raft-associated, their diffusion becomes independent of the type of membrane anchor and is significantly reduced compared with that of nonraft transmembrane proteins. Cholesterol depletion accelerates the diffusion of raft-associated proteins for transmembrane raft proteins to the level of transmembrane nonraft proteins and for GPI-anchored proteins even further. Raft-associated GPI-anchored proteins were never observed to dissociate from the raft within the measurement intervals of up to 10 min. The measurements agree with lipid rafts being cholesterol-stabilized complexes of 26 +/- 13 nm in size diffusing as one entity for minutes.     

FLUIDITY OF THE SURFACE OF CULTURED MUSCLE FIBERS : Rapid Lateral Diffusion of Marked Surface Antigens

Fluorescent antibody fragments of anti-muscle plasma membrane antibody bound as small fluorescent spots when applied by micropipetting to cultured myotubes. The spots were observed to enlarge with time. The rate of enlargement of fluorescent spots was greater when fragments were applied than when divalent antibody was used. It was also greater at 23°–25°C than at 0°–4°C. With glutaraldehyde-fixed cells no increase in the size of the spots was seen. The observations are consistent with the spread of fluorescent spots due to diffusion of surface protein antigens within the plane of a fluid membrane. From measurements of spot size against time, a diffusion constant of 1–3 x 10^-9 cm² s^-1 can be calculated for muscle plasma membrane proteins of mol wt approximately 200,000. This value is consistent with other observations on the diffusion of surface antigens and of labeled lipid molecules in synthetic and natural membranes.     

Comparative study of the lateral motion of extrinsic probes and anthracene-labelled constitutive phospholipids in the plasma membrane of Chinese hamster ovary cells

9-(2-Anthryl)-nonanoic acid is a new photoactivatable fluorescent probe which has been designed for the study of the lateral diffusion and distribution of lipids in biological membranes by means of the anthracene photodimerization reaction. This anthracene fatty acid can be incorporated metabolically into the glycerophospholipids (phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol) of Chinese hamster ovary (CHO) cells in culture. The diffusion coefficient of intrinsic lipids in the plasma membrane of these eukaryotic cells can thus be measured using the fluorescence recovery after a photobleaching technique, since illumination of the fluorescent anthracene groups yields non-fluorescent photodimers. For the sake of comparison, the extrinsic lipophilic probes 5-(N-hexadecanoyl)-aminofluorescein, 12-(9-anthroyloxy)-stearic acid, 9-(2-anthryl)-nonanoic acid and a synthetic anthracene-phosphatidylcholine were also used to label the plasma membrane of CHO cells. The diffusion coefficients for the extrinsic and intrinsic probes ranged over 1 - 2 x 10(-9) cm2/s. Small but significant differences were observed between the various probes reflecting differences they exhibit in size and polarity. All the extrinsic probes were free to diffuse, with a mobile fraction close to 100%. In contrast, a fractional recovery of only 75% was observed for the intrinsic anthracene-labelled phospholipids, suggesting that the anthracene fatty acid was metabolically incorporated into membrane lipid regions which were inaccessible to the extrinsic probes.     

High spatial resolution observation of single-molecule dynamics in living cell membranes

Self-organized lipid bilayers together with proteins are the essential building blocks of biological membranes. Membranes are associated with all living systems as they make up cell boundaries and provide basic barriers to cellular organelles. It is of interest to study the dynamics of individual molecules in cell membranes as the mechanism of how biological membranes function at the single molecule remains to be elucidated. In this letter we describe a study in which we incubate rat basophilic leukemia cells with a fluorescently labeled cell membrane component on a surface containing zero-mode waveguides (ZMWs). We used the ZMW to confine fluorescent excitation to an approximately 100-nm region of the membrane to monitor lipid diffusion along the cellular membrane. We showed that confinement with a ZMW largely reduced fluorescent contributions from the cytosolic pool that is present when using a more standard technique such as laser-induced confocal microscopy. We show that optical confinement with ZMWs is a facile way to probe dynamic processes on the membrane surface.     

Single-lipid tracking on nanoscale membrane buds: The effects of curvature on lipid diffusion and sorting

Nanoscale membrane curvature in cells is critical for endocytosis/exocytosis and membrane trafficking. However, the biophysical ramifications of nanoscale membrane curvature on the behavior of lipids remain poorly understood. Here, we created an experimental model system of membrane curvature at a physiologically-relevant scale and obtained nanoscopic information on single-lipid distributions and dynamics. Supported lipid bilayers were created over 50 and 70 nm radius nanoparticles to create membrane buds. Single-molecule localization microscopy was performed with diverse mixtures of fluorescent and non-fluorescent lipids. Variations in lipid acyl tales length, saturation, head-group, and fluorescent labeling strategy were tested while maintaining a single fluid lipid phase throughout the membrane. Monte Carlo simulations were used to fit our experimental results and quantify the effects of curvature on the lipid diffusion and sorting. Whereas varying the composition of the non-fluorescent lipids yielded minimal changes to the curvature effects, the labeling strategy of the fluorescent lipids yielded highly varying effects of curvature. Most conditions yield single-population Brownian diffusion throughout the membrane; however, curvature-induced lipid sorting, slowing, and aggregation were observed in some conditions. Head-group labeled lipids such as DPPE-Texas Red and POPE-Rhodamine diffused >2.4× slower on the curved vs. the planar membranes; tail-labeled lipids such as NBD-PPC, TopFluor-PPC, and TopFluor-PIP2, as well as DiIC₁₂ and DiIC₁₈ displayed no significant changes in diffusion due to the membrane curvature. This article is part of a Special Issue entitled: Emergence of Complex Behavior in Biomembranes edited by Marjorie Longo.     

Detection of motional heterogeneities in lipid bilayer membranes by dual probe fluorescence correlation spectroscopy

We report the detection of heterogeneities in the diffusion of lipid molecules for the three-component mixture dipalmitoyl-PC/dilauroyl-PC/cholesterol, a chemically simple lipid model for the mammalian plasma membrane outer leaflet. Two-color fluorescence correlation spectroscopy (FCS) was performed on giant unilamellar vesicles (GUVs) using fluorescent probes that have differential lipid phase partition behavior--DiO-C18:2 favors disordered fluid lipid phases, whereas DiI-C20:0 prefers spatially ordered lipid phases. Simultaneously-obtained fluorescence autocorrelation functions from the same excitation volume for each dye showed that, depending on the lipid composition of this ternary mixture, the two dyes exhibited different lateral mobilities in regions of the phase diagram with previously proposed submicroscopic two-phase coexistence. In one-phase regions, both dyes reported identical diffusion coefficients. Two-color FCS thus may be detecting local membrane heterogeneities at size scales below the optical resolution limit, either due to short-range order in a single phase or due to submicroscopic phase separation.     

A new FRAP/FRAPa method for three-dimensional diffusion measurements based on multiphoton excitation microscopy

We present a new convenient method for quantitative three-dimensionally resolved diffusion measurements based on the photobleaching (FRAP) or photoactivation (FRAPa) of a disk-shaped area by the scanning laser beam of a multiphoton microscope. Contrary to previously reported spot-photobleaching protocols, this method has the advantage of full scalability of the size of the photobleached area and thus the range of diffusion coefficients, which can be measured conveniently. The method is compatible with low as well as high numerical aperture objective lenses, allowing us to perform quantitative diffusion measurements in three-dimensional extended samples as well as in very small volumes, such as cell nuclei. Furthermore, by photobleaching/photoactivating a large area, diffusion along the optical axis can be measured separately, which is convenient when studying anisotropic diffusion. First, we show the rigorous mathematical derivation of the model, leading to a closed-form formula describing the fluorescence recovery/redistribution phase. Next, the ability of the multiphoton FRAP method to correctly measure absolute diffusion coefficients is tested thoroughly on many test solutions of FITC-dextrans covering a wide range of diffusion coefficients. The same is done for the FRAPa method on a series of photoactivatable green fluorescent protein solutions with different viscosities. Finally, we apply the method to photoactivatable green fluorescent protein diffusing freely in the nucleus of living NIH-3T3 mouse embryo fibroblasts.     

Lateral diffusion measurement at high spatial resolution by scanning microphotolysis in a confocal microscope.

Fluorescence photobleaching methods have been widely used to study diffusion processes in the plasma membrane of single living cells and other membrane systems. Here we describe the application of a new photobleaching technique, scanning microphotolysis. Employing a recently developed extension module to a commercial confocal microscope, an intensive laser beam was switched on and off during scanning according to a user definable image mask. Thereby the location, geometry, and number of photolysed spots could be chosen arbitrarily, their size ranging from tens of micrometers down to the diffraction limit. Therewith we bleached circular areas on the surface of single living 3T3 cells labeled with the fluorescent lipid analog NBD-HPC. Subsequently, the fluorescence recovery process was observed using the attenuated laser beam for excitation. This yielded image stacks representing snapshots of the spatial distribution of fluorescent molecules. From these we computed the radial distribution functions of the photobleached dye molecules. The variance of these distributions is linearly related to the diffusion constant, time, and the mobile fraction of the diffusing species. Furthermore, we compared directly the theoretically expected and measured distribution functions, and could thus determine the diffusion coefficient from each single image. The results of these two new evaluation methods (D = 0.3 +/- 0.1 micron 2/s) agreed well with the outcome of conventional fluorescence recovery measurements. We show that by scanning microphotolysis information on dynamical processes such as diffusion of lipids or proteins can be acquired at the superior spatial resolution of a confocal laser scanning microscope.     

Fluorescence photobleaching recovery using total internal reflection interference fringes

Lateral diffusion measurements on cell membrane molecules, most commonly accomplished through fluorescence photobleaching recovery (FPR or FRAP), provide information on such molecules' size, environment, and participation in intermolecular interactions. However, difficulties arise in FPR measurements of lateral dynamics of materials, such as visible fluorescent protein (VFP) fusion proteins, where fluorescent intracellular species contribute to the fluorescence recovery signal and thus distort measurements intended to reflect surface molecules only. A new method helps eliminate these difficulties. In total internal reflection interference fringe FPR, interfering laser beams enter a 1.65-numercial aperture (NA) Olympus objective at the periphery of the back focal plane where the NA exceeds 1.38. This creates an extended interference pattern totally internally reflected at the coverslip-medium interface which excites fluorescence only from fluorescent molecules located where the cell contacts the coverslip. The large illuminated area interrogates many more membrane receptors than spot methods and hence obtains more diffusion information per measurement while rejecting virtually all interfering intracellular fluorescence. We report successful measurements of membrane dynamics of both VFP-containing and conventionally labeled molecules by this technique and compare them with results of other FPR methods.     

Lipid diffusion in the thylakoid membranes of the cyanobacterium Synechococcus sp.: effect of fatty acid desaturation

Thylakoid membranes are crucial to photosynthesis in cyanobacteria and plants. In cyanobacteria, genetic modification of membrane lipid composition strongly influences cold tolerance and susceptibility to photoinhibition. We have used fluorescence recovery after photobleaching to measure the diffusion of a lipid-soluble fluorescent marker in cells of the cyanobacterium Synechococcus sp. PCC 7942. We have compared the wild-type strain with a transformant with an increased level of fatty acid unsaturation. The transformant showed a six-fold increase in the diffusion coefficient for the fluorescent marker at growth temperature. This is the first direct measurement of lipid diffusion in a photosynthetic membrane.     

Lateral diffusion in inhomogeneous membranes. Model membranes containing cholesterol.

The problem of lateral diffusion in inhomogeneous membranes is illustrated by a theoretical calculation of the lateral diffusion of a fluorescent lipid probe in binary mixtures of phosphatidylcholine and cholesterol under conditions of temperature and composition such that this lipid mixture consists of alternating parallel domains of fluid and solid lipid, having separations that are small compared with the distance scale employed in photobleaching experiments. The theoretical calculations clearly illustrate how inhomogeneities in membrane composition affecting the lateral motion of membrane components on a small (10-100 nm) distance scale can give complex diffusive responses in experiments such as fluorescence photobleaching that employ comparatively macroscopic distances (10-100 micrometers) for the measurement of diffusive recovery. The theoretical calculations exhibit the unusual dependence of the apparent lateral diffusion coefficient of a fluorescent lipid probe on lipid composition in binary mixtures of cholesterol and phosphatidylcholines as reported by Rubenstein et al. (1979, Proc. Natl. Acad. Sci. U.S.A., 76:15-18).     

Lateral diffusion in model membranes is independent of the size of the hydrophobic region of molecules.

We have systematically investigated the probe size and shape dependence of lateral diffusion in model dimyristoyl phosphatidylcholine membranes. Linear hydrophobic polymers, which differ in length by an order of magnitude, were used to explore the effect on the lateral diffusion coefficient of hydrodynamic restrictions in the bilayer interior. The polymers employed are isoprenoid alcohols--citronellol, solanesol, and dolichol. Tracer lateral diffusion coefficients were measured by fluorescence photobleaching recovery. Despite the large difference in lengths, the nitrobenzoxadiazole labelled alcohols all diffuse at the rate of lipid self-diffusion (5.0 x 10(-12) m2 s-1, 29 degrees C) in the liquid crystal phase. Companion measurements in isotropic polymer solution, in gel phase lipid membranes and with nonpolar fluorescent polyaromatic hydrocarbons, show a marked dependence of the lateral diffusion coefficient on the probe molecule size. Our results in the liquid crystal phase are in accord with free area theory which asserts that lateral diffusion in the membrane is restricted by the surface-free area. Probe molecules which are significantly longer than the host phospholipid, seven times longer in the case of dolichol, are still restricted in their lateral motion by the surface properties of the bilayer in the liquid crystal phase. Fluorescence quenching experiments indicate that the nitrobenzoxadiazole label does not reside at the aqueous interface, although it must reside in close proximity according to the diffusion measurements.     

Weak effect of membrane diffusion on the rate of receptor accumulation at adhesive contacts

To assess if membrane diffusion could affect the kinetics of receptor recruitment at adhesive contacts, we transfected neurons with green fluorescent protein-tagged immunoglobin cell adhesion molecules of varying length (25-180 kD), and measured the lateral mobility of single quantum dots bound to those receptors at the cell surface. The diffusion coefficient varied within a physiological range (0.1-0.5 microm(2)/s), and was inversely proportional to the size of the receptor. We then triggered adhesive contact formation by placing anti-green fluorescent protein-coated microspheres on growth cones using optical tweezers, and measured surface receptor recruitment around microspheres by time-lapse fluorescence imaging. The accumulation rate was rather insensitive to the type of receptor, suggesting that the long-range membrane diffusion of immunoglobin cell adhesion molecules is not a limiting step in the initiation of neuronal contacts.