Fluorescence Correlation Spectroscopy Data Research Articles

Fission yeast Myo2: Molecular organization and diffusion in the cytoplasm.

Myosin-II is required for the assembly and constriction of cytokinetic contractile rings in fungi and animals. We used electron microscopy, fluorescence recovery after photobleaching (FRAP), and fluorescence correlation spectroscopy (FCS) to characterize the physical properties of Myo2 from fission yeast Schizosaccharomyces pombe. By electron microscopy, Myo2 has two heads and a coiled-coiled tail like myosin-II from other species. The first 65 nm of the tail is a stiff rod, followed by a flexible, less-ordered region up to 30 nm long. Myo2 sediments as a 7 S molecule in high salt, but aggregates rather than forming minifilaments at lower salt concentrations; this is unaffected by heavy chain phosphorylation. We used FRAP and FCS to observe the dynamics of Myo2 in live S. pombe cells and in cell extracts at different salt concentrations; both show that Myo2 with an N-terminal mEGFP tag has a diffusion coefficient of ∼ 3 µm2 s-1 in the cytoplasm of live cells during interphase and mitosis. Photon counting histogram analysis of the FCS data confirmed that Myo2 diffuses as doubled-headed molecules in the cytoplasm. FCS measurements on diluted cell extracts showed that mEGFP-Myo2 has a diffusion coefficient of ∼ 30 µm2 s-1 in 50 to 400 mM KCl concentrations.

Determination of Equilibrium Constant and Relative Brightness in FRET-FCS by Including the Third-Order Correlations.

Fluorescence correlation spectroscopy (FCS) encodes the information on the equilibrium constant (K), the relative fluorescence brightness of fluorophore (Q), and the forward and backward reaction rate constants (k+ and k-) on a physical or chemical relaxation. However, it has been a long-standing problem to completely resolve the FCS data to get the thermodynamic and kinetic information. Recently, we have solved the problem for fluorescence autocorrelation spectroscopy (FACS). Here, we extend the method to fluorescence cross-correlation spectroscopy (FCCS), which appears when FCS is coupled with fluorescence resonance energy transfer (FRET). Among 12 total second-order and third-order pre-exponential factors in a relaxation process probed by the FRET-FCS technique, 3 are independent. We presented and discussed 3 sets of explicit solutions to use these pre-exponential factors to calculate K and Q. Together with the relaxation time, the acquired K will allow people to obtain k+ and k-, so that the goal of deciphering the FRET-FCS data will be fully reached. The theory is verified by extensive computer simulations and tested experimentally on a system of oligonucleotide hybridization.

Reconceptualizing Fluorescence Correlation Spectroscopy for Monitoring and Analyzing Periodically Passing Objects.

Fluorescence correlation spectroscopy (FCS) is a sensitive technique commonly applied for studying the dynamics of nanoscale-labeled objects in solution. Current analysis of FCS data is largely based on the assumption that the labeled objects are stochastically displaced due to Brownian motion. However, this assumption is often invalid for microscale objects, since the motion of these objects is dominated by Stokes drag and settling or rising effects, rather than stochastic Brownian motion. To utilize the power of FCS for systems with nonstochastic displacements of objects, the collection and analysis of FCS data have to be reconceptualized. Here, we extended the applicability of FCS for the detection and analysis of periodically passing objects. Toward this end, we implemented droplet-based microfluidics, in which monodispersed droplets containing fluorescent marker are flowing equally spaced within microchannels. We show by simulations and experiments that FCS can sensitively quantify the flow-rates, variability, and content of rapidly passing droplets. This information can be derived at high temporal resolution, based on the intensity fluctuations generated by only 5–10 passing droplets. Moreover, by utilizing the periodicity of the flowing droplets for noise reduction by averaging, FCS can monitor accurately the droplets flow even if their fluorescence intensity is negligible. Hence, extending FCS for periodically passing objects converts it into a powerful analytical tool for high-throughput droplet-based microfluidics. Moreover, based on the principles described here, FCS can be straightforwardly applied for a variety of systems in which the passing of objects is periodic rather than stochastic.

Apparent Anomalous Diffusion in the Cytoplasm of Human Cells: The Effect of Probes' Polydispersity.

This work, based on in vivo and in vitro measurements, as well as in silico simulations, provides a consistent analysis of diffusion of polydisperse nanoparticles in the cytoplasm of living cells. Using the example of fluorescence correlation spectroscopy (FCS), we show the effect of polydispersity of probes on the experimental results. Although individual probes undergo normal diffusion, in the ensemble of probes, an effective broadening of the distribution of diffusion times occurs-similar to anomalous diffusion. We introduced fluorescently labeled dextrans into the cytoplasm of HeLa cells and found that cytoplasmic hydrodynamic drag, exponentially dependent on probe size, extraordinarily broadens the distribution of diffusion times across the focal volume. As a result, the in vivo FCS data were effectively fitted with the anomalous subdiffusion model while for a monodisperse probe the normal diffusion model was most suitable. Diffusion time obtained from the anomalous diffusion model corresponds to a probe whose size is determined by the weight-average molecular weight of the polymer. The apparent anomaly exponent decreases with increasing polydispersity of the probes. Our results and methodology can be applied in intracellular studies of the mobility of nanoparticles, polymers, or oligomerizing proteins.

Size and Structure of Cytochrome-c bound to Gold nano-clusters: Effect of Ethanol

Size and structure of cytochrome c (Cyt C) bound to gold nano-clusters (AuNC) were studied using fluorescence correlation spectroscopy (FCS) and circular dichroism (CD) spectroscopy. The CD spectra of Cyt C indicate that the ellipticity is almost completely lost on binding to AuNC which indicates unfolding. Addition of ethanol causes partial restoration of ellipticity and hence, structure of Cyt C. FCS data indicate that size (hydrodynamic radius, rH) of free Cyt C is 17 Å which increases to 24 Å on binding to AuNC. This too suggests unfolding of Cyt C upon binding to AuNCs. Both the size and conformational relaxation time of Cyt C bound to AuNC vary non-monotonically with increase in ethanol content. Size, conformation dynamics and structure of cytochrome c (Cyt C) bound to gold nanoclusters (AuNC) have been studied using fluorescence correlation spectroscopy (FCS) and circular dichroism (CD). The CD spectra of Cyt C indicate that the helical structure is almost completely lost on binding to AuNC signifying unfolding. Addition of ethanol causes partial restoration of structure of Cyt C. FCS data indicate that both the size and conformational relaxation time of Cyt C bound to AuNC vary non-monotonically with increase in ethanol content.

Exploring the Stability and Cardiolipin Affinity of Cytochrome C's Domain Swapped Dimer Conformation

Cytochrome c's (cytc) oxidation of the lipid cardiolipin (CL) is a proposed switch involved in initiation of the intrinsic pathway of apoptosis. Recently, there has been an increased number of reports in the literature of cytc domain-swapped dimers (DSD). The Hirota lab has reported that the horse DSD has increased peroxidase activity compared to the monomeric form. The Bowler lab has solved DSD crystal structures with various detergents binding in the hydrophobic tube of the proposed “C” cytc-CL binding site. We postulate that the DSD conformation of cytc is an evolutionary switch allowing for tighter regulation of the intrinsic pathway of apoptosis. In order to examine the stability of the DSD, samples were incubated temporally at fixed temperatures and then analyzed using size-exclusion chromatography. The data, presented here, indicate that the human DSD is more kinetically stable than horse or yeast DSD, suggesting evolution of a more kinetically stable conformation in primates. Additionally, fluorescence correlation spectroscopy (FCS) data, also presented, show the binding of Zn-cytc to CL nanodiscs (ND). ND are discoidal lipid bilayers maintained by two alpha helical belt proteins and provide a nice model system to study protein-lipid interactions. Initial FCS results indicate that the Kd of cytc for CL NDs is in the micromolar range. This poster will compare the relative CL binding affinities of monomer and DSD cytc.

Examination of the Oligomerization Mechanism of SOD1 In Vitro and in Live Cells

The dimeric β-barrel protein Cu, Zn superoxide dismutase (SOD1) is an optimal model system for investigating the molecular basis for toxicity in ALS. Mutations throughout the β-barrel sequence are causative for ALS and implicated in the gain-of-function toxicity. The occurrence of mutations at dozens of sites suggests a common mechanism of toxicity for SOD1 disease causing variants. Previous biophysical and structural data on SOD1 and its variants have established a fairly detailed understanding of its folding energy landscape. A key finding is that ALS-variants of SOD1 lead to enhanced populations of both folded and unfolded monomeric forms of SOD1. An unresolved question, however, is the mechanism of oligomerization and toxicity of SOD1 variants. To address this question, we present an aggregation assay that matches physiological conditions as much as possible, avoiding denaturants, fluorinated alcohols and agitation. Our results, based on the analysis of the WT protein and several variants, suggest that the oligomerization rate is correlated with the population of the unfolded monomer. Furthermore, although a secondary nucleation mechanism has been proposed as a key step in SOD1 aggregation our results, obtained without agitation, are best fitted with a crystallization-like nucleation model without fragmentation. We test the consistency of in vitro and live cell data by performing fluorescence correlation spectroscopy (FCS) measurements on live HEK293 cells expressing EGFP fusion constructs of WT and disease causing SOD1 variants. Preliminary quantitative modeling of the FCS data suggest consistency in the propagation rates between in vitro and live cell data. The implication of the unfolded monomer of SOD1 suggests that small-molecules stabilizing the dimeric native state can be used as a potential therapeutic strategy.This research was supported by NIH grant GM54836.

Background suppression in fluorescence nanoscopy with stimulated emission double depletion

Stimulated emission depletion (STED) fluorescence nanoscopy is a powerful super-resolution imaging technique based on the confinement of fluorescence emission to the central subregion of an observation volume through de-excitation of fluorophores in the periphery via stimulated emission. Here, we introduce stimulated emission double depletion (STEDD) as a method to selectively remove artificial background intensity. In this approach, a first, conventional STED pulse is followed by a second, delayed Gaussian STED pulse that specifically depletes the central region, thus leaving only background. Thanks to time-resolved detection we can remove this background intensity voxel by voxel by taking the weighted difference of photons collected before and after the second STED pulse. STEDD thus yields background-suppressed super-resolved images as well as STED-based fluorescence correlation spectroscopy data. Furthermore, the proposed method is also beneficial when considering lower-power, less redshifted depletion pulses. Stimulated emission double depletion addresses the issue of background in super-resolution imaging and quantitative microscopy through implementation of a two-pulse sequence in a modified stimulated emission depletion set-up. The measured background intensity is removed from each voxel in the acquired images thanks to time-resolved detection.

Dynamic studies of the interaction of a pH responsive, amphiphilic polymer with a DOPC lipid membrane.

Deeper understanding of the molecular interactions between polymeric materials and the lipid membrane is important across a range of applications from permeation for drug delivery to encapsulation for immuno-evasion. Using highly fluidic microcavity supported lipid bilayers, we studied the interactions between amphiphilic polymer PP50 and a DOPC lipid bilayer. As the PP50 polymer is pH responsive the studies were carried out at pH 6.5, 7.05 and 7.5, corresponding to fully, partly protonated (pH = pKa = 7.05) and fully ionized states of the polymer, respectively. Fluorescence correlation spectroscopy (FCS) using both labelled lipid and polymer revealed the PP50 associates with the bilayer interface across all pHs where its diffusion along the interface is impeded. Both FCS and electrochemical impedance spectroscopy (EIS) data indicate that the PP50 does not penetrate fully into the bilayer core but rather forms a layer at the bilayer aqueous interface reflected in increased resistance and decreased capacitance of the bilayer on PP50 binding. The extent of these effects and the dynamics of binding are influenced by pH, increasing with decreasing pH. These experimental trends concurred with coarse grained Monte Carlo simulations of polymer-bilayer interactions wherein a model hydrophilic polymer backbone grafted with side chains of varying hydrophobicity, to mimic the effect of varying pH, was simulated based on the bond fluctuation model with explicit solvent. Simulation results showed that with increasing hydrophobicity, the polymer penetrated deeper into the contacting bilayer leaflet of the membrane suppressing, consistent with EIS data, solvent permeation and that a full insertion of the polymer into the bilayer core is not necessary for suppression of permeability.

Nuclear magnetic resonance, fluorescence correlation spectroscopy and time-resolved fluorescence anisotropy studies of intermolecular interactions in bis(1-methyl-1H-imidazol-3-ium-3-yl)dihydroborate bis(trifluoromethylsulfonyl)amide and its mixtures with various cosolvents

Keeping in mind the potential usefulness of mixed ionic liquid (IL)-cosolvents systems in several industrial applications, intermolecular interactions between a borate-based IL, bis(1-methyl-1H-imidazol-3-ium-3-yl)dihydroborate bis(trifluoromethylsulfonyl)amide ([BIMIMDBA][TF2N]), and its binary mixtures with several molecular solvents has been investigated through NMR and fluorescence spectroscopy. Analysis of the 1H chemical shifts (δ/ppm) and translational diffusion coefficients (D) of the IL in different solvent mixtures demonstrate interplay of nonspecific (ion-dipole) and specific (hydrogen bonding) interactions in governing the properties of these mixtures. Fluorescence correlation spectroscopy (FCS) and time-resolved fluorescence anisotropy data provide evidence in favour of different IL-solvent interaction for different IL-cosolvent systems.

What information is contained in the fluorescence correlation spectroscopy curves, and where.

We discuss the application of fluorescence correlation spectroscopy (FCS) for characterization of anomalous diffusion of tracer particles in crowded environments. While the fact of anomaly may be detected by the standard fitting procedure, the value of the exponent α of anomalous diffusion may be not reproduced correctly for non-Gaussian anomalous diffusion processes. The important information is however contained in the asymptotic behavior of the fluorescence autocorrelation function at long and at short times. Thus, analysis of the short-time behavior gives reliable values of α and of lower moments of the distribution of particles' displacement, which allows us to confirm or reject its Gaussian nature. The method proposed was tested on the FCS data obtained in artificial crowded fluids and in living cells.

Fluctuations in reactive networks subject to extrinsic noise studied in the framework of the chemical Langevin equation.

Theoretical and experimental studies have shown that the fluctuations of in vivo systems break the fluctuation-dissipation theorem. One can thus ask what information is contained in the correlation functions of protein concentrations and how they relate to the response of the reactive network to a perturbation. Answers to these questions are of prime importance to extract meaningful parameters from the in vivo fluorescence correlation spectroscopy data. In this paper we study the fluctuations of the concentration of a reactive species involved in a cyclic network that is in a nonequilibrium steady state perturbed by a noisy force, taking into account both the breaking of detailed balance and extrinsic noises. Using a generic model for the network and the extrinsic noise, we derive a chemical Langevin equation that describes the dynamics of the system, we determine the expressions of the correlation functions of the concentrations, and we estimate the deviation of the fluctuation-dissipation theorem and the range of parameters in which an effective temperature can be defined.

Protonation Dynamics on Lipid Nanodiscs: Influence of the Membrane Surface Area and External Buffers

Lipid membrane surfaces can act as proton-collecting antennae, accelerating proton uptake by membrane-bound proton transporters. We investigated this phenomenon in lipid nanodiscs (NDs) at equilibrium on a local scale, analyzing fluorescence fluctuations of individual pH-sensitive fluorophores at the membrane surface by fluorescence correlation spectroscopy (FCS). The protonation rate of the fluorophores was ∼100-fold higher when located at 9- and 12-nm diameter NDs, compared to when in solution, indicating that the proton-collecting antenna effect is maximal already for a membrane area of ∼60 nm2. Fluorophore-labeled cytochrome c oxidase displayed a similar increase when reconstituted in 12 nm NDs, but not in 9 nm NDs, i.e., an acceleration of the protonation rate at the surface of cytochrome c oxidase is found when the lipid area surrounding the protein is larger than 80 nm2, but not when below 30 nm2. We also investigated the effect of external buffers on the fluorophore proton exchange rates at the ND membrane-water interfaces. With increasing buffer concentrations, the proton exchange rates were found to first decrease and then, at millimolar buffer concentrations, to increase. Monte Carlo simulations, based on a simple kinetic model of the proton exchange at the membrane-water interface, and using rate parameter values determined in our FCS experiments, could reconstruct both the observed membrane-size and the external buffer dependence. The FCS data in combination with the simulations indicate that the local proton diffusion coefficient along a membrane is ∼100 times slower than that observed over submillimeter distances by proton-pulse experiments (Ds ∼ 10−5cm2/s), and support recent theoretical studies showing that proton diffusion along membrane surfaces is time- and length-scale dependent.

Effect of anchor positioning on binding and diffusion of elongated 3D DNA nanostructures on lipid membranes

DNA origami is a state-of-the-art technology that enables the fabrication of nano-objects with defined shapes, to which functional moieties, such as lipophilic anchors, can be attached with a nanometre scale precision. Although binding of DNA origami to lipid membranes has been extensively demonstrated, the specific requirements necessary for membrane attachment are greatly overlooked. Here, we designed a set of amphipathic rectangular-shaped DNA origami structures with varying placement and number of chol-TEG anchors used for membrane attachment. Single- and multiple-cholesteryl-modified origami nanostructures were produced and studied in terms of their membrane localization, density and dynamics. We show that the positioning of at least two chol-TEG moieties near the corners is essential to ensure efficient membrane binding of large DNA nanostructures. Quantitative fluorescence correlation spectroscopy data further confirm that increasing the number of corner-positioned chol-TEG anchors lowers the dynamics of flat DNA origami structures on freestanding membranes. Taken together, our approach provides the first evidence of the importance of the location in addition to the number of hydrophobic moieties when rationally designing minimal DNA nanostructures with controlled membrane binding.

Structural Ensembles of Intrinsically Disordered Proteins Depend Strongly on Force Field: A Comparison to Experiment

Intrinsically disordered proteins (IDPs) are notoriously challenging to study both experimentally and computationally. The structure of IDPs cannot be described by a single conformation, but must instead be described as an ensemble of interconverting conformations. Atomistic simulations are increasingly used to obtain such IDP conformational ensembles. Here, we have compared the IDP ensembles generated by nine all-atom empirical force fields against primary small angle x-ray scattering (SAXS), NMR, and fluorescence correlation spectroscopy (FCS) data. Ensembles obtained with different force fields exhibit marked differences in chain dimensions, hydrogen-bonding, and secondary structure content. These differences are unexpectedly large: changing the force field is found to have a stronger effect on secondary structure content than changing the entire peptide sequence. The CHARMM 22∗ ensemble performs best in this force field comparison: it has the lowest error in chemical shifts and J- couplings, and agrees well with the SAXS and FCS data. A high population of left-handed alpha-helix is present in the CHARMM 36 ensemble, which is inconsistent with measured scalar couplings. To eliminate inadequate sampling as a reason for differences between force fields, extensive simulations were carried out (1.351 ms in total); the remaining small sampling uncertainty is shown to be much smaller than the observed differences. Our findings highlight how IDPs, with their rugged energy landscapes, are highly-sensitive test systems that are capable of revealing force field deficiencies and, therefore, contribute to force field development.

Application of Fluorescence Correlation Spectroscopy to Study Dynamics of Proteins Involved in Neuronal Synapse-to-Nucleus Signaling

Activity-dependent gene transcription is required for the long-term stabilization of synaptic plasticity, a process that is key to learning and memory. We are studying the transcription factor, NFAT, which has been shown to be actuated by postsynaptic signaling. Ca2+ influx through voltage-gated L-type calcium channels plays an exclusive role in initiating NFAT signaling via the action of the Ca2+/calmodulin- phosphatase calcineurin (CaN). CaN dephosphorylates multiple residues on NFAT, resulting in the exposure of its nuclear localization sequence and subsequent translocation to the nucleus. A required component of this cascade is the A-kinase anchoring protein (AKAP) 79/150, which serves to recruit CaN to the channel. New evidence from our group indicates that NFAT is also tethered to this complex via a transient association with AKAP.This scenario suggests a model wherein NFAT signaling is instigated by local synaptic activity and the actuated transcription factor is then conveyed to the nucleus over long intracellular distances via diffusion or active transport. However, the molecular mechanisms of this process have yet to be elucidated. A potentially powerful approach to investigate the dynamics of the signaling molecules in this pathway is fluorescence correlation spectroscopy (FCS). This technique yields quantitative information on protein mobility in living cells by measuring intensity fluctuations produced by fluorescent (or fluorescently tagged) molecules diffusing through a confined detection volume.Here we present FCS measurements characterizing NFAT dynamics in response to induced synaptic activity in cultured hippocampal neurons, utilizing models that account for dendritic morphology. Additionally, we have found that there is a prominent component in the autocorrelation curve due to GFP photophysics (triplet state transitions and dynamic quenching) in FCS data from these cells, which requires further modifications to the standard fitting models.

The Effect of Small Molecules on Early and Late Events of Alpha Synuclein Aggregation in Solution and Inside Living Cells

Parkinson disease (PD) is the most common motor-neurodegenerative disease, affecting 1-2% of the population older than 65 years and 4-5% of those aged over 85 years. PD is a movement disorder, associated with different motor and nonmotor syndromes, arising from degeneration of dopaminergic neurons of substantia nigra. The hallmark of PD is the observation of intracellular protein aggregates, Lewy Bodies (LB). Alpha-synuclein is one of the major components of LB. The heterogeneity of aggregation process and the presence of large number of triggering mechanisms results in the difficulty to devise therapeutic strategies against these toxic inclusions formation. There have been continuing efforts to search for small molecules against alpha synuclein aggregation.With the help of fluorescence correlation spectroscopy (FCS), confocal imaging, FRAP and other biophysical experiments we have studied the early and late events of alpha-synuclein aggregation. Subsequently, we have explored the possibilities of identifying possible small molecules, which would prevent early oligomer formation. For the FCS experiments, we have labeled a single cysteine mutant of alpha synuclein (G132C) using Alexa488Maleimide. Our FCS data have shown significant changes in the conformation of the protein in the presence of an inhibiting small molecule. To obtain additional insights into these molecules, we have studied their effects inside two mammalian model cell lines, namely HeLa and SH-SY5Y. For this purpose, we have transiently transfected the cells using a tetra-cysteine tagged alpha-synuclein, following which the cells have been stained using Flash-EDT2 dye for fluorescence imaging and FRAP studies. The results show that the electrostatic differences between the protein and the small molecules contribute significantly towards the ability of the small molecules to modulate the early and late stages of aggregation.

Measuring Size, Size Distribution, and Polydispersity of Water-in-Oil Microemulsion Droplets using Fluorescence Correlation Spectroscopy: Comparison to Dynamic Light Scattering.

Water-in-oil microemulsion droplets (MEDs) are thermodynamically stable supramolecular structures formed in a mixture of water and oil, stabilized by surfactant layer. Here we use fluorescence correlation spectroscopy (FCS) to measure the diffusion, and the size, size distribution, and polydispersity of MEDs prepared in ternary mixtures of water/oil/sodium bis(2-ethylhexyl) sulfosuccinate (AOT) in heptane, isooctane, and nonane at (near) single droplet level. We compare FCS data directly to dynamic light scattering (DLS) data, which shows that the optical matching point (OMP) conditions of MEDs in different oils (where excess optical polarizability of droplets vanish) severely influence DLS data, while FCS extracts the accurate size, size distribution, and polydispersity of AOT-MEDs in all three oils. This suggests that extreme precaution must be taken in acquiring and explaining DLS data of MEDs in solution. FCS data show nearly identical W0-dependent (peak) size variations of AOT-MEDs in all three oils, though a subtle increase in (average) polydispersity of droplets is observed with increase in carbon chain length of oils. Establishing the accuracy of FCS data for AOT-MEDs, we further apply FCS to measure the size parameters of MEDs prepared in a quaternary mixture of water/oil/cetyltrimethylammonium bromide (CTAB)/1-butanol in hexane, heptane, and isooctane. Unlike AOT-MEDs, FCS data show substantial effect of added cosurfactant (1-butanol) and external oil on size, size distribution and polydispersity of quaternary CTAB-MEDs. Analysis of size distributions reveals large variation of polydispersity which possibly indicates the existence of larger shape heterogeneity, together with size heterogeneity, of CTAB-MEDs compared to AOT-MEDs in solution.

Determination of equilibrium and rate constants for complex formation by fluorescence correlation spectroscopy supplemented by dynamic light scattering and Taylor dispersion analysis.

The equilibrium and rate constants of molecular complex formation are of great interest both in the field of chemistry and biology. Here, we use fluorescence correlation spectroscopy (FCS), supplemented by dynamic light scattering (DLS) and Taylor dispersion analysis (TDA), to study the complex formation in model systems of dye-micelle interactions. In our case, dyes rhodamine 110 and ATTO-488 interact with three differently charged surfactant micelles: octaethylene glycol monododecyl ether C12E8 (neutral), cetyltrimethylammonium chloride CTAC (positive) and sodium dodecyl sulfate SDS (negative). To determine the rate constants for the dye-micelle complex formation we fit the experimental data obtained by FCS with a new form of the autocorrelation function, derived in the accompanying paper. Our results show that the association rate constants for the model systems are roughly two orders of magnitude smaller than those in the case of the diffusion-controlled limit. Because the complex stability is determined by the dissociation rate constant, a two-step reaction mechanism, including the diffusion-controlled and reaction-controlled rates, is used to explain the dye-micelle interaction. In the limit of fast reaction, we apply FCS to determine the equilibrium constant from the effective diffusion coefficient of the fluorescent components. Depending on the value of the equilibrium constant, we distinguish three types of interaction in the studied systems: weak, intermediate and strong. The values of the equilibrium constant obtained from the FCS and TDA experiments are very close to each other, which supports the theoretical model used to interpret the FCS data.

Evidence for Homodimerization of the c-Fos Transcription Factor in Live Cells Revealed by Fluorescence Microscopy and Computer Modeling.

The c-Fos and c-Jun transcription factors, members of the activator protein 1 (AP-1) complex, form heterodimers and bind to DNA via a basic leucine zipper and regulate the cell cycle, apoptosis, differentiation, etc. Purified c-Jun leucine zipper fragments could also form stable homodimers, whereas c-Fos leucine zipper homodimers were found to be much less stable in earlier in vitro studies. The importance of c-Fos overexpression in tumors and the controversy in the literature concerning c-Fos homodimerization prompted us to investigate Fos homodimerization. Förster resonance energy transfer (FRET) and molecular brightness analysis of fluorescence correlation spectroscopy data from live HeLa cells transfected with fluorescent-protein-tagged c-Fos indicated that c-Fos formed homodimers. We developed a method to determine the absolute concentrations of transfected and endogenous c-Fos and c-Jun, which allowed us to determine dissociation constants of c-Fos homodimers (Kd = 6.7 ± 1.7 μM) and c-Fos-c-Jun heterodimers (on the order of 10 to 100 nM) from FRET titrations. Imaging fluorescence cross-correlation spectroscopy (SPIM-FCCS) and molecular dynamics modeling confirmed that c-Fos homodimers were stably associated and could bind to the chromatin. Our results establish c-Fos homodimers as a novel form of the AP-1 complex that may be an autonomous transcription factor in c-Fos-overexpressing tissues and could contribute to tumor development.