Probing GFP-actin diffusion in living cells using fluorescence correlation spectroscopy

The cytoskeleton of eukaryotic cells is continuously remodeled by polymerization and depolymerization of actin. Consequently, the relative content of polymerized filamentous actin (F-actin) and monomeric globular actin (G-actin) is subject to temporal and spatial fluctuations. Since fluorescence correlation spectroscopy (FCS) can measure the diffusion of fluorescently labeled actin it seems likely that FCS allows us to determine the dynamics and hence indirectly the structural properties of the cytoskeleton components with high spatial resolution. To this end we investigate the FCS signal of GFP-actin in living Dictyostelium discoideum cells and explore the inherent spatial and temporal signatures of the actin cytoskeleton. Using the free green fluorescent protein (GFP) as a reference, we find that actin diffusion inside cells is dominated by G-actin and slower than diffusion in diluted cell extract. The FCS signal in the dense cortical F-actin network near the cell membrane is probed using the cytoskeleton protein LIM and is found to be slower than cytosolic G-actin diffusion. Furthermore, we show that polymerization of the cytoskeleton induced by Jasplakinolide leads to a substantial decrease of G-actin diffusion. Pronounced fluctuations in the distribution of the FCS correlation curves can be induced by latrunculin, which is known to induce actin waves. Our work suggests that the FCS signal of GFP-actin in combination with scanning or spatial correlation techniques yield valuable information about the local dynamics and concomitant cytoskeletal properties.     

Anomalous protein diffusion in living cells as seen by fluorescence correlation spectroscopy

We investigate the challenges and limitations that are encountered when studying membrane protein dynamics in vivo by means of fluorescence correlation spectroscopy (FCS). Based on theoretical arguments and computer simulations, we show that, in general, the fluctuating fluorescence has a fractal dimension D(0) >or= 1.5, which is determined by the anomality alpha of the diffusional motion of the labeled particles, i.e., by the growth of their mean square displacement as (Deltax)(2) approximately t(alpha). The fractality enforces an initial power-law behavior of the autocorrelation function and related quantities for small times. Using this information, we show by FCS that Golgi resident membrane proteins move subdiffusively in the endoplasmic reticulum and the Golgi apparatus in vivo. Based on Monte Carlo simulations for FCS on curved surfaces, we can rule out that the observed anomalous diffusion is a result of the complex topology of the membrane. The apparent mobility of particles as determined by FCS, however, is shown to depend crucially on the shape of the membrane and its motion in time. Due to this fact, the hydrodynamic radius of the tracked particles can be easily overestimated by an order of magnitude.     

Counting nucleosomes in living cells with a combination of fluorescence correlation spectroscopy and confocal imaging

Although methods for light microscopy of chromatin are well established, there are no quantitative data for nucleosome concentrations in vivo. To establish such a method we used a HeLa clone expressing the core histone H2B fused to the enhanced yellow fluorescent protein (H2B-EYFP). Quantitative gel electrophoresis and fluorescence correlation spectroscopy (FCS) of isolated oligonucleosomes show that 5% of the total H2Bs carry the fluorescent tag and an increased nucleosome repeat length of 204 bp for the fluorescent cells. In vivo, the mobility and distribution of H2B-EYFP were studied with a combination of FCS and confocal imaging. With FCS, concentration and brightness of nascent molecules were measured in the cytoplasm, while in the nucleoplasm a background of mobile fluorescent histones was determined by continuous photobleaching. Combining these results allows converting confocal fluorescence images of nuclei into calibrated nucleosome density maps. Absolute nucleosome concentrations in interphase amount up to 250 microM locally, with mean values of 140(+/-28)microM, suggesting that a condensation-controlled regulation of site accessibility takes place at length scales well below 200 nm.     

Vesicle diffusion close to a membrane: intermembrane interactions measured with fluorescence correlation spectroscopy

The protein machinery controlling membrane fusion (or fission) has been well studied; however, the role of vesicle diffusion near membranes in these critical processes remains unclear. We experimentally and theoretically investigated the dynamics of small vesicles (∼50 nm in diameter) that are diffusing near supported planar bilayers acting as “target” membranes. Using total internal reflection-fluorescence correlation spectroscopy, we examined the validity of theoretical analyses of vesicle-membrane interactions. Vesicles were hindered by hydrodynamic drag as a function of their proximity to the planar bilayer. The population distributions and diffusion kinetics of the vesicles were further affected by changing the ionic strength and pH of the buffer, as well as the lipid composition of the planar membrane. Effective surface charges on neutral bilayers were also analyzed by comparing experimental and theoretical data, and we show the possibility that vesicle dynamics can be modified by surface charge redistribution of the planar bilayer. Based on these results, we hypothesize that the dynamics of small vesicles, diffusing close to biomembranes, may be spatially restricted by altering local physiological conditions (e.g., salt concentration, lipid composition, and pH), which may represent an additional mechanism for controlling fusion (or fission) dynamics.     

Characterizing diffusion dynamics of a membrane protein associated with nanolipoproteins using fluorescence correlation spectroscopy

Nanolipoprotein particles (NLPs) represent a unique nanometer-sized scaffold for supporting membrane proteins (MP). Characterization of their dynamic shape and association with MP in solution remains a challenge. Here, we present a rapid method of analysis by fluorescence correlation spectroscopy (FCS) to characterize bacteriorhodopsin (bR), a membrane protein capable of forming a NLP complex. By selectively labeling individual components of NLPs during cell-free synthesis, FCS enabled us to measure specific NLP diffusion times and infer size information for different NLP species. The resulting bR-loaded NLPs were shown to be dynamically discoidal in solution with a mean diameter of 7.8 nm. The insertion rate of bR in the complex was ～55% based on a fit model incorporating two separate diffusion properties to best approximate the FCS data. More importantly, based on these data, we infer that membrane protein associated NLPs are thermodynamically constrained as discs in solution, while empty NLPs appear to be less constrained and dynamically spherical.     

Benzodiazepine binding studies on living cells: application of small ligands for fluorescence correlation spectroscopy

We demonstrate the applicability of fluorescence correlation spectroscopy (FCS) for receptor binding studies using low molecular weight ligands on the membranes of living nerve cells. The binding of the benzodiazepine Ro 7-1986/602 (N-des-diethyl-fluorazepam), labeled with the fluorophore Alexa 532, to the benzodiazepine receptor was analyzed quantitatively at the membrane of single rat hippocampal neurons. The values obtained for the dissociation constant Kd = (9.9 +/- 1.9) nm and the rate constant for ligand-receptor dissociation kdisS = (1.28 +/- 0.08) x 10(-3) s(-1) show that there is a specific and high affinity interaction between the dye-labeled ligand (Ro-Alexa) and the receptor site. The binding was saturated at approx. 100 nM and displacement of 10 nM Ro-Alexa, with a 1,000-fold excess of midazolam, showed a non-specific binding of 7-10%. Additionally, two populations of the benzodiazepine receptor that differed in their lateral mobility were detected in the membrane of rat neurons. The diffusion coefficients for these two populations [D(bound1) = (1.32 +/- 0.26) microm2/s; D(bound2) = (2.63 +/- 0.63) x 10(-2) microm2/s] are related to binding sites, which shows a mono-exponential decay in a time-dependent dissociation of the ligand-receptor complex.     

Hierarchical organization of the plasma membrane: Investigations by single-molecule tracking vs. fluorescence correlation spectroscopy

Single-molecule tracking and fluorescence correlation spectroscopy (FCS) applied to the plasma membrane in living cells have allowed a number of unprecedented observations, thus fostering a new basic understanding of molecular diffusion, interaction, and signal transduction in the plasma membrane. It is becoming clear that the plasma membrane is a heterogeneous entity, containing diverse structures on nano-meso-scales (2-200 nm) with a variety of lifetimes, where certain membrane molecules stay together for limited durations. Molecular interactions occur in the time-dependent inhomogeneous two-dimensional liquid of the plasma membrane, which might be a key for plasma membrane functions.     

Detection of ligand-induced CNTF receptor dimers in living cells by fluorescence cross correlation spectroscopy

Ciliary neurotrophic factor (CNTF) signals via a receptor complex consisting of the specific CNTF receptor (CNTFR) and two promiscuous signal transducers, gp130 and leukemia inhibitory factor receptor (LIFR). Whereas earlier studies suggested that the signaling complex is a hexamer, more recent analyses strongly support a tetrameric structure. However, all studies so far analyzed the stoichiometry of the CNTF receptor complex in vitro and not in the context of living cells. We generated and expressed in mammalian cells acyl carrier protein-tagged versions of both CNTF and CNTFR. After labeling CNTF and CNTFR with different dyes we analyzed their diffusion behavior at the cell surface. Fluorescence (cross) correlation spectroscopy (FCS/FCCS) measurements reveal that CNTFR diffuses with a diffusion constant of about 2 × 10^− 9 cm² s^− 1 independent of whether CNTF is bound or not. FCS and FCCS measurements detect the formation of receptor complexes containing at least two CNTFs and CNTFRs. In addition, we measured Förster-type fluorescence resonance energy transfer between two differently labeled CNTFs within a receptor complex indicating a distance of 5-7 nm between the two. These findings are not consistent with a tetrameric structure of the CNTFR complex suggesting that either hexamers and or even higher-order structures (e.g. an octamer containing two tetramers) are formed.     

Interactions of two monoclonal antibodies with BNP: high resolution epitope mapping using fluorescence correlation spectroscopy

Structure-function studies of antibody-antigen systems include the identification of amino acid residues in the antigen that interact with an antibody and elucidation of their individual contributions to binding affinity. We used fluorescence correlation spectroscopy (FCS) and alanine-scanning mutagenesis to characterize the interactions of brain natriuretic peptide (BNP) with two monoclonal antibodies. Human BNP is a 32 amino acid residue long cyclic polypeptide with the ring structure confined between cysteines in positions 10 and 26. It is an important cardiovascular hormone and a valuable diagnostic cardiac marker. We compare the binding strength of the N-terminus Alexa488-labeled BNP, native cyclic BNP, BNP alanine-substituted mutants, linear BNP, and its short fragments to determine the individual contributions of amino acid residues included in the continuous antigenic epitopes that are recognized by two different monoclonal antibodies raised toward BNP. Implementation of FCS for these studies offers all of the advantages of solution phase measurements, including high sensitivity, simplicity of manipulation with reagents, and elimination of solid phase interferences or separation steps. Significant differences in the molecular masses of the free and antibody bound BNP results in a substantial ( approximately 2.5-times) increase in the diffusion rates. Determination of the binding constants and inhibition effects by measuring the diffusion rates of the ligand at the single molecule level introduces the ultimate opportunity for researching systems where the fluorescence intensity and/or fluorescence anisotropy do not change upon interaction of the ligand with the protein. Monoclonal antibodies 106.3 and BC203 demonstrate high affinities to BNP and bind two distant epitopes forming robust antibody sandwiches. Both antibodies are used in Abbott diagnostic assays on AxSYM, IMx, and Architect platforms.     

TMA-DPH: a suitable fluorescence polarization probe for specific plasma membrane fluidity studies in intact living cells

Fluorescence intensity measurements and fluorescence microscopy data showed that TMA-DPH (trimethylammonium diphenylhexatriene), a cationic derivative of the fluorescence polarization probe DPH, has a considerably different behavior in L929 cultured cells than does its parent molecule. In contrast to DPH, it incorporates very rapidly in the plasma membranes of the treated cells, and remains specifically localized on the cell surface for at least 25 min. It can therefore be recommended for specific plasma membrane fluidity measurements in whole living cells. No relevant information about the localization of the probes could be obtained by other techniques used in parallel, namely: subcellular fractionation and fluorescence inhibition by trinitrobenzene sulfonate (TNBS).     

Ligand-receptor interactions in the membrane of cultured cells monitored by fluorescence correlation spectroscopy

We investigated the specific binding of epidermal growth factor (EGF) to its membrane-bound receptors in cultured cells. The specificity of the binding was attested by the consistent displacement of bound rhodamine-labeled EGF (Rh-EGF) following addition of 1000-fold molar excess of unlabeled EGF. The binding specificity of EGF was further confirmed when vascular EGF was unable to displace Rh-EGF binding, demonstrating no cross-reaction. Evidence for the specific interactions was verified by an equilibrium saturation binding experiment. EGF binding to the cell membranes is saturated at nanomolar concentration. The Scatchard plots show a binding process with K(ass) of 1.5 x 10(9) M(-1). The dissociation kinetics follow a single exponential function characteristic for a relatively slow dissociation process with k(diss) = 2.9 x 10(-4) s(-1). The appearance of two binding complexes through the distribution of diffusion times may suggest that these are representatives of two different forms or subtypes of EGF receptors. This study is of pharmaceutical significance as it provides evidence that fluorescence correlation spectroscopy can be used as a rapid technique for studying ligand-receptor interactions in cell cultures. This is a step forward toward large-scale drug screening in cell cultures.     

Toward a new picture of the living plasma membrane

Our understanding of the plasma membrane structure has undergone a major change since the proposal of the fluid mosaic model of Singer and Nicholson in the 1970s. In this model, the membrane, composed of over thousand lipid and protein species, is organized as a well‐equilibrated two‐dimensional fluid. Here, the distribution of lipids is largely expected to reflect a multicomponent system, and proteins are expected to be surrounded by an annulus of specialized lipid species. With the recognition that a multicomponent lipid membrane is capable of phase segregation, the membrane is expected to appear as patchwork quilt pattern of membrane domains. However, the constituents of a living membrane are far from being well equilibrated. The living cell membrane actively maintains a trans‐bilayer asymmetry of composition, and its constituents are subject to a number of dynamic processes due to synthesis, lipid transfer as well as membrane traffic and turnover. Moreover, membrane constituents engage with the dynamic cytoskeleton of a living cell, and are both passively as well as actively manipulated by this engagement. The extracellular matrix and associated elements also interact with membrane proteins contributing to another layer of interaction. At the nano‐ and mesoscale, the organization of lipids and proteins emerge from these encounters, as well as from protein–protein, protein–lipid, and lipid–lipid interactions in the membrane. New methods to study the organization of membrane components at these scales have also been developed, and provide an opportunity to synthesize a new picture of the living cell surface as an active membrane composite.     

Targeting membrane proteins to liquid-ordered phases: molecular self-organization explored by fluorescence correlation spectroscopy

The complex and dynamic architecture of biological membranes comprises of various heterogeneities, some of which may include lipid-based and/or protein-based microdomains called "rafts". Due to interactions among membrane components, several types of domains can form with different characteristics and mechanisms of formation. Model membranes, such as giant unilamellar vesicles (GUVs), provide a key system to study lipid-lipid and lipid-protein interactions, which are potentially relevant to raft formation, by (single-molecule) optical microscopy. Here, we review studies of combined confocal imaging and fluorescence correlation spectroscopy (FCS) on lipid dynamics and organization in domains assembled in GUVs, prepared from various lipid mixtures, which are relevant to the problem of raft formation. Finally, we summarize the results on lipid-protein interactions, which govern the targeting of several putative raft- and non-raft-associated membrane proteins to domain-exhibiting GUVs.     

Quantification of alpha-synuclein binding to lipid vesicles using fluorescence correlation spectroscopy

Alpha-synuclein (alphaS) is a soluble synaptic protein that is the major proteinaceous component of insoluble fibrillar Lewy body deposits that are the hallmark of Parkinson's disease. The interaction of alphaS with synaptic vesicles is thought to be critical both to its normal function as well as to its pathological role in Parkinson's disease. We demonstrate the use of fluorescence correlation spectroscopy as a tool for rapid and quantitative analysis of the binding of alphaS to large unilamellar vesicles of various lipid compositions. We find that alphaS binds preferentially to vesicles containing acidic lipids, and that this interaction can be blocked by increasing the concentration of NaCl in solution. Negative charge is not the only factor determining binding, as we clearly observe binding to vesicles composed entirely of zwitterionic lipids. Additionally, we find enhanced binding to lipids with less bulky headgroups. Quantification of the protein-to-lipid ratio required for binding to different lipid compositions, combined with other data in the literature, yields an upper bound estimate for the number of lipid molecules required to bind each individual molecule of alphaS. Our results demonstrate that fluorescence correlation spectroscopy provides a powerful tool for the quantitative characterization of alphaS-lipid interactions.     

Lateral mobility of membrane-binding proteins in living cells measured by total internal reflection fluorescence correlation spectroscopy

Total internal reflection fluorescence correlation spectroscopy (TIR-FCS) allows us to measure diffusion constants and the number of fluorescent molecules in a small area of an evanescent field generated on the objective of a microscope. The application of TIR-FCS makes possible the characterization of reversible association and dissociation rates between fluorescent ligands and their receptors in supported phospholipid bilayers. Here, for the first time, we extend TIR-FCS to a cellular application for measuring the lateral diffusion of a membrane-binding fluorescent protein, farnesylated EGFP, on the plasma membranes of cultured HeLa and COS7 cells. We detected two kinds of diffusional motion-fast three-dimensional diffusion (D(1)) and much slower two-dimensional diffusion (D(2)), simultaneously. Conventional FCS and single-molecule tracking confirmed that D(1) was free diffusion of farnesylated EGFP close to the plasma membrane in cytosol and D(2) was lateral diffusion in the plasma membrane. These results suggest that TIR-FCS is a powerful technique to monitor movement of membrane-localized molecules and membrane dynamics in living cells.     

Molecular dynamics in living cells observed by fluorescence correlation spectroscopy with one- and two-photon excitation.

Multiphoton excitation (MPE) of fluorescent probes has become an attractive alternative in biological applications of laser scanning microscopy because many problems encountered in spectroscopic measurements of living tissue such as light scattering, autofluorescence, and photodamage can be reduced. The present study investigates the characteristics of two-photon excitation (2PE) in comparison with confocal one-photon excitation (1PE) for intracellular applications of fluorescence correlation spectroscopy (FCS). FCS is an attractive method of measuring molecular concentrations, mobility parameters, chemical kinetics, and fluorescence photophysics. Several FCS applications in mammalian and plant cells are outlined, to illustrate the capabilities of both 1PE and 2PE. Photophysical properties of fluorophores required for quantitative FCS in tissues are analyzed. Measurements in live cells and on cell membranes are feasible with reasonable signal-to-noise ratios, even with fluorophore concentrations as low as the single-molecule level in the sampling volume. Molecular mobilities can be measured over a wide range of characteristic time constants from approximately 10(-3) to 10(3) ms. While both excitation alternatives work well for intracellular FCS in thin preparations, 2PE can substantially improve signal quality in turbid preparations like plant cells and deep cell layers in tissue. At comparable signal levels, 2PE minimizes photobleaching in spatially restrictive cellular compartments, thereby preserving long-term signal acquisition.     

Fluorescence correlation spectroscopy close to a fluctuating membrane

Compartmentalization of the cytoplasm by membranes should have a strong influence on the diffusion of macromolecules inside a cell, and we have studied how this could be reflected in fluorescence correlation spectroscopy (FCS) experiments. We derived the autocorrelation function measured by FCS for fluorescent particles diffusing close to a soft membrane, and show it to be the sum of two contributions: short timescale correlations come from the diffusion of the particles (differing from free diffusion because of the presence of an obstacle), whereas long timescale correlations arise from fluctuations of the membrane itself (which create intensity fluctuations by modulating the number of detected particles). In the case of thermal fluctuations this second type of correlation depends on the elasticity of the membrane. To illustrate this calculation, we report the results of FCS experiments carried out close to a vesicle membrane. The measured autocorrelation functions display very distinctly the two expected contributions, and allow both to recover the diffusion coefficient of the fluorophore and to characterize the membrane fluctuations in term of a bending rigidity. Our results show that FCS measurements inside cells can lead to erroneous values of the diffusion coefficient if the influence of membranes is not recognized.     

Spatially resolved fluorescence correlation spectroscopy using a spinning disk confocal microscope

We develop an extension of fluorescence correlation spectroscopy (FCS) using a spinning disk confocal microscope. This approach can spatially map diffusion coefficients or flow velocities at up to approximately 10(5) independent locations simultaneously. Commercially available cameras with frame rates of 1000 Hz allow FCS measurements of systems with diffusion coefficients D~10(-7) cm(2)/s or smaller. This speed is adequate to measure small microspheres (200-nm diameter) diffusing in water, or hindered diffusion of macromolecules in complex media (e.g., tumors, cell nuclei, or the extracellular matrix). There have been a number of recent extensions to FCS based on laser scanning microscopy. Spinning disk confocal microscopy, however, has the potential for significantly higher speed at high spatial resolution. We show how to account for a pixel size effect encountered with spinning disk confocal FCS that is not present in standard or scanning FCS, and we introduce a new method to correct for photobleaching. Finally, we apply spinning disk confocal FCS to microspheres diffusing in Type I collagen, which show complex spatially varying diffusion caused by hydrodynamic and steric interactions with the collagen matrix.     

Spatial-temporal studies of membrane dynamics: scanning fluorescence correlation spectroscopy (SFCS)

Giant unilamellar vesicles (GUVs) have been widely used as a model membrane system to study membrane organization, dynamics, and protein-membrane interactions. Most recent studies have relied on imaging methods, which require good contrast for image resolution. Multiple sequential image processing only detects slow components of membrane dynamics. We have developed a new fluorescence correlation spectroscopy (FCS) technique, termed scanning FCS (i.e., SFCS), which performs multiple FCS measurements simultaneously by rapidly directing the excitation laser beam in a uniform (circular) scan across the bilayer of the GUVs in a repetitive fashion. The scan rate is fast compared to the diffusion of the membrane proteins and even small molecules in the GUVs. Scanning FCS outputs a "carpet" of timed fluorescence intensity fluctuations at specific points along the scan. In this study, GUVs were assembled from rat kidney brush border membranes, which included the integral membrane proteins. Scanning FCS measurements on GUVs allowed for a straightforward detection of spatial-temporal interactions between the protein and the membrane based on the diffusion rate of the protein. To test for protein incorporation into the bilayers of the GUVs, antibodies against one specific membrane protein (NaPi II cotransporter) were labeled with ALEXA-488. Fluorescence images of the GUVs in the presence of the labeled antibody showed marginal fluorescence enhancement on the GUV membrane bilayers (poor image contrast and resolution). With the application of scanning FCS, the binding of the antibody to the GUVs was detected directly from the analysis of diffusion rates of the fluorescent antibody. The diffusion coefficient of the antibody bound to NaPi II in the GUVs was approximately 200-fold smaller than that in solution. Scanning FCS provided a simple, quantitative, yet highly sensitive method to study protein-membrane interactions.