Practical guidelines for dual-color fluorescence cross-correlation spectroscopy

Dual-color fluorescence cross-correlation spectroscopy (FCCS) allows for the determination of molecular mobility and concentrations and for the quantitative analysis of molecular interactions such as binding or cleavage at very low concentrations. This protocol discusses considerations for preparing a biological system for FCCS experiments and offers practical advice for performing FCCS on a commercially available setup. Although FCCS is closely related to two-color confocal microscopy, critical adjustments and test measurements are necessary to establish successful FCCS measurements, which are described in a step-by-step manner. Moreover, we discuss control experiments for a negative cross-correlation artifact, arising from a lack of detection volume overlap, and a positive artifact, arising from cross-talk. FCCS has been applied to follow molecular interactions in solutions, on membranes and in cells and to analyze dynamic colocalization during intracellular transport. It is a technique that is expected to see new applications in various fields of biochemical and cell biological research.     

A fluorescent variant of a protein from the stony coral Montipora facilitates dual-color single-laser fluorescence cross-correlation spectroscopy

Dual-color fluorescence cross-correlation spectroscopy (FCCS) is a promising technique for quantifying protein-protein interactions. In this technique, two different fluorescent labels are excited and detected simultaneously within a common measurement volume. Difficulties in aligning two laser lines and emission crossover between the two fluorophores, however, make this technique complex. To overcome these limitations, we developed a fluorescent protein with a large Stokes shift. This protein, named Keima, absorbs and emits light maximally at 440 nm and 620 nm, respectively. Combining a monomeric version of Keima with cyan fluorescent protein allowed dual-color FCCS with a single 458-nm laser line and complete separation of the fluorescent protein emissions. This FCCS approach enabled sensitive detection of proteolysis by caspase-3 and the association of calmodulin with calmodulin-dependent enzymes. In addition, Keima and a spectral variant that emits maximally at 570 nm might facilitate simultaneous multicolor imaging with single-wavelength excitation.     

Detection of prion protein immune complex for bovine spongiform encephalopathy diagnosis using fluorescence correlation spectroscopy and fluorescence cross-correlation spectroscopy

Fluorescence correlation spectroscopy (FCS) and fluorescence cross-correlation spectroscopy (FCCS) are powerful techniques to measure molecular interactions with high sensitivity in homogeneous solution and living cells. In this study, we developed methods for the detection of prion protein (PrP) using FCS and FCCS. A combination of a fluorescent-labeled Fab' fragment and another anti-PrP monoclonal antibody (mAb) enabled us to detect recombinant bovine PrP (rBoPrP) using FCS because there was a significant difference in the diffusion coefficients between the labeled Fab' fragment and the trimeric immune complex consisting of rBoPrP, labeled Fab' fragment, and another anti-PrP mAb. On the other hand, FCCS detected rBoPrP using two mAbs labeled with different fluorescence dyes. The detection limit for PrP in FCCS was approximately threefold higher than that in FCS. The sensitivity of FCCS in detection of abnormal isoform of PrP (PrP(Sc)) was comparable to that of enzyme-linked immunosorbent assay (ELISA). Because FCS and FCCS detect the PrP immune complex in homogeneous solution of only microliter samples with a single mixing step and without any washing steps, these features of measurement may facilitate automating bovine spongiform encephalopathy diagnosis.     

Direct detection of caspase-3 activation in single live cells by cross-correlation analysis

Dual color fluorescence cross-correlation spectroscopy (FCCS) provides information about the coincidence of spectrally well-defined two fluorescent molecules in a small observation area at the single-molecule level. To evaluate the activity of caspase-3 in vivo directly, FCCS was applied to single live cells. We constructed chimeric proteins that consisted of tandemly fused enhanced green FP (EGFP) and monomeric red FP (mRFP). In control experiments, the protease reaction was monitored in solution, where a decrease in cross-correlation amplitude was observed due to specific cleavage of the amino acid sequence between EGFP and mRFP. Moreover, a decrease in cross-correlation amplitude could be detected in a live cell, where caspase-3 activation was induced by apoptosis. This is the first report of FP-based in vivo cross-correlation analysis. FP-based FCCS may become the most versatile method for analysis of protein-protein interactions in live cells.     

Fluorescence cross-correlation spectroscopy in living cells

Cell biologists strive to characterize molecular interactions directly in the intracellular environment. The intrinsic resolution of optical microscopy, however, allows visualization of only coarse subcellular localization. By extracting information from molecular dynamics, fluorescence cross-correlation spectroscopy (FCCS) grants access to processes on a molecular scale, such as diffusion, binding, enzymatic reactions and codiffusion, and has become a valuable tool for studies in living cells. Here we review basic principles of FCCS and focus on seminal applications, including examples of intracellular signaling and trafficking. We consider FCCS in the context of fluorescence resonance energy transfer and multicolor imaging techniques and discuss application strategies and recent technical advances.     

Characterization of the control catabolite protein of gluconeogenic genes repressor by fluorescence cross-correlation spectroscopy and other biophysical approaches

Determination of the physical parameters underlying protein-DNA interactions is crucial for understanding the regulation of gene expression. In particular, knowledge of the stoichiometry of the complexes is a prerequisite to determining their energetics and functional molecular mechanisms. However, the experimental determination of protein-DNA complex stoichiometries remains challenging. We used fluorescence cross-correlation spectroscopy (FCCS) to investigate the interactions of the control catabolite protein of gluconeogenic genes, a key metabolic regulator in Gram-positive bacteria, with two oligonucleotides derived from its target operator sequences, gapB and pckA. According to our FCCS experiments, the stoichiometry of binding is twofold larger for the pckA target than for gapB. Correcting the FCCS data for protein self-association indicated that control catabolite protein of gluconeogenic genes forms dimeric complexes on the gapB target and tetrameric complexes on the pckA target. Analytical ultracentrifugation coupled with fluorescence anisotropy and hydrodynamic modeling allowed unambiguous confirmation of this result. The use of multiple complementary techniques to characterize these complexes should be employed wherever possible. However, there are cases in which analytical ultracentrifugation is precluded, due to protein stability, solubility, or availability, or, more obviously, when the studies are carried out in live cells. If information concerning the self-association of the protein is available, FCCS can be used for the direct and simultaneous determination of the affinity, cooperativity, and stoichiometry of protein-DNA complexes in a concentration range and conditions relevant to the regulation of these interactions.     

Unveiling mRNP composition by fluorescence correlation and cross-correlation spectroscopy using cell lysates

Understanding the mRNA life cycle requires information about the dynamics and macromolecular composition and stoichiometry of mRNPs. Fluorescence correlation and cross-correlation spectroscopy (FCS and FCCS) are appealing technologies to study these macromolecular structures because they have single molecule sensitivity and readily provide information about their molecular composition and dynamics. Here, we demonstrate how FCS can be exploited to study cytoplasmic mRNPs with high accuracy and reproducibility in cell lysates. Cellular lysates not only recapitulate data from live cells but provide improved readings and allow investigation of single mRNP analysis under particular conditions or following enzymatic treatments. Moreover, FCCS employing minute amounts of cells closely corroborated previously reported RNA dependent interactions and provided estimates of the relative overlap between factors in the mRNPs, thus depicting their heterogeneity. The described lysate-based FCS and FCCS analysis may not only complement current biochemical approaches but also provide novel opportunities for the quantitative analysis of the molecular composition and dynamics of single mRNPs.     

Monitoring the caspase cascade in single apoptotic cells using a three-color fluorescent protein substrate

Fluorescence cross-correlation spectroscopy (FCCS) reveals information about the spatiotemporal coincidence of two spectrally well-defined fluorescent molecules in a small observation area at the level of single-molecule sensitivity. To simultaneously evaluate the activities of caspase-3 and caspase-9, we constructed a chimeral protein that consisted of tandemly fused enhanced cyan fluorescent protein (ECFP), monomeric red fluorescent protein (mCherry) and monomeric yellow fluorescent protein (Venus). In HeLa cell lysates, a combination of tumor necrosis factor-α (TNF-α)- and cycloheximide (CHX-)-induced apoptosis was monitored. In this, decreases of cross-correlation amplitudes were observed between ECFP and mCherry and between mCherry and Venus. Moreover, time-dependent monitoring of single cells revealed decreases in the cross-correlation amplitudes between ECFP and mCherry and between mCherry and Venus before morphologic changes were observed by laser scanning fluorescence microscopy (LSM). Thus, our method could predict the fate of the cell in the early apoptotic stage.     

荧光相关谱技术及其应用

基于对处于平衡态少量荧光分子集合的强度涨落进行时间平均的技术,荧光相关谱fluoreswceance correlation spectroscopy,FCS)技术最近已经应用于细胞环境过程的研究。FCS优秀的灵敏特性为我们实时测量许多参数提供了途径,而且具有快速的时间特性和高空间分辨率。测量的参数包括扩散速率、局部浓度、聚合状态和分子间的相互作用。荧光互相关谱(fluorescence cross-correlation spectroscopy,FCCS)进一步扩展了FCS技术的应用,包括在活细胞中的广泛应用。本文介绍了FCS技术的原理、实验装置及其应用。     

Two-photon fluorescence cross-correlation spectroscopy as a potential tool for high-throughput screening of DNA repair activity

Several lines of evidence indicate that differences in DNA repair capacity are an important source of variability in cancer risk. However, traditional assays for measurement of DNA repair activity in human samples are laborious and time-consuming. DNA glycosylases are the first step in base excision repair of a variety of modified DNA bases. Here, we describe the development of a new sensitive DNA glycosylase assay based on fluorescence cross-correlation spectroscopy (FCCS) with two-photon excitation. FCCS was applied to the measurement of uracil DNA glycosylase activity of human cell extracts and validated by comparison with standard gel electrophoresis assay. Our results indicate that FCCS can be adapted to efficient assays for DNA glycosylase activity in protein extracts from human cells. This method has a potential for the development of automated screening of large number of samples.     

Simultaneous multicolor fluorescence cross-correlation spectroscopy to detect higher order molecular interactions using single wavelength laser excitation

Fluorescence cross-correlation spectroscopy is a powerful method for the study of molecular interactions and dynamics in solution and even in living cells. Usually, in the optical setup, either two laser beams have to be superimposed in their respective confocal volumes or two-photon excitation is used for a dual-color detection system. It has been shown recently that fluorescence cross correlation can be achieved with spectrally similar fluorophores using single wavelength excitation fluorescence cross-correlation spectroscopy (SW-FCCS). In this study, we show that SW-FCCS allows the simultaneous excitation of up to three fluorophores in which the cross correlation of their fluctuation signals is detected separately in three detection channels. The experimental and theoretical model to describe triple pairwise cross correlations incorporating cross talk and possible changes in emission characteristics such as quenching upon binding are outlined. The effectiveness of SW-FCCS to detect binding of three interacting partners is experimentally verified with a standard ligand-receptor model, biotin-streptavidin, where differently labeled biotin ligands and their binding to a third-color labeled streptavidin are studied. The cross-correlation amplitudes and their changes with stoichiometric binding are analyzed and the upper limits of dissociation constants are determined. Performed with appropriate negative controls, SW-FCCS can determine interaction patterns between ligands and receptors.     

Protein-protein interaction analysis by C-terminally specific fluorescence labeling and fluorescence cross-correlation spectroscopy

Here, we describe novel puromycin derivatives conjugated with iminobiotin and a fluorescent dye that can be linked covalently to the C-terminus of full-length proteins during cell-free translation. The iminobiotin-labeled proteins can be highly purified by affinity purification with streptavidin beads. We confirmed that the purified fluorescence-labeled proteins are useful for quantitative protein-protein interaction analysis based on fluorescence cross-correlation spectroscopy (FCCS). The apparent dissociation constants of model protein pairs such as proto-oncogenes c-Fos/c-Jun and archetypes of the family of Ca2+-modulated calmodulin/related binding proteins were in accordance with the reported values. Further, detailed analysis of the interactions of the components of polycomb group complex, Bmi1, M33, Ring1A and RYBP, was successfully conducted by means of interaction assay for all combinatorial pairs. The results indicate that FCCS analysis with puromycin-based labeling and purification of proteins is effective and convenient for in vitro protein-protein interaction assay, and the method should contribute to a better understanding of protein functions by using the resource of available nucleotide sequences.     

In vivo quantitative analysis of PKA subunit interaction and cAMP level by dual color fluorescence cross correlation spectroscopy

We employed dual color Fluorescence Cross Correlation Spectroscopy (FCCS) to measure the interaction between PKA regulatory (RII) and catalytic subunits (CAT) in living cells. Elevation of intracellular cAMP with forskolin decreased the cross-correlation amplitude between RFP-fused RII (RII-mRFP) and GFP-fused CAT (CAT-EGFP) by 50%, indicating that cAMP elevation leads to dissociation of RII-CAT complexes. Moreover, diffusion coefficient analysis showed that the diffusion rate of CAT-EGFP was significantly increased, suggesting that the decreased RII-CAT association caused by cAMP generated free CAT subunits. Our study demonstrates that in vivo FCCS measurements and their quantitative analysis permit one not only to directly quantify protein-protein interactions but also to estimate changes in the intracellular cAMP concentration.     

Factors affecting the quantification of biomolecular interactions by fluorescence cross-correlation spectroscopy

Fluorescence cross-correlation spectroscopy (FCCS) is used to determine interactions and dissociation constants (K_ds) of biomolecules. The determination of a K_d depends on the accurate measurement of the auto- and cross-correlation function (ACF and CCF) amplitudes. In the case of complete binding, the ratio of the CCF/ACF amplitudes is expected to be 1. However, measurements performed on tandem fluorescent proteins (FPs), in which two different FPs are linked, yield CCF/ACF amplitude ratios of ～0.5 or less for different FCCS schemes. We use single wavelength FCCS and pulsed interleaved excitation FCCS to measure various tandem FPs constituted of different red and green FPs and determine the causes for this suboptimal ratio. The main causes for the reduced CCF/ACF amplitude ratio are differences in observation volumes for the different labels, the existence of dark FPs due to maturation problems, photobleaching, and to a lesser extent Förster (or fluorescence) resonance energy transfer between the labels. We deduce the fraction of nonfluorescent proteins for EGFP, mRFP, and mCherry as well as the differences in observation volumes. We use this information to correct FCCS measurements of the interaction of Cdc42, a small Rho-GTPase, with its effector IQGAP1 in live cell measurements to obtain a label-independent value for the K_d.     

Escherichia coli ribosomal protein L20 binds as a single monomer to its own mRNA bearing two potential binding sites

Ribosomal protein L20 is crucial for the assembly of the large ribosomal subunit and represses the translation of its own mRNA. L20 mRNA carries two L20-binding sites, the first folding into a pseudoknot and the second into an imperfect stem and loop. These two sites and the L20-binding site on 23S ribosomal RNA are recognized similarly using a single RNA-binding site located on one face of L20. In this work, using gel filtration and fluorescence cross-correlation spectroscopy (FCCS) experiments, we first exclude the possibility that L20 forms a dimer, which would allow each monomer to bind one site of the mRNA. Secondly we show, using affinity purification and FCCS experiments, that only one molecule of L20 binds to the L20 mRNA despite the presence of two potential binding sites. Thirdly, using RNA chemical probing, we show that the two L20-binding sites are in interaction. This interaction provides an explanation for the single occupancy of the mRNA. The two interacting sites could form a single hybrid site or the binding of L20 to a first site may inhibit binding to the second. Models of regulation compatible with our data are discussed.     

Protein–protein interaction analysis by C-terminally specific fluorescence labeling and fluorescence cross-correlation spectroscopy

Here, we describe novel puromycin derivatives conjugated with iminobiotin and a fluorescent dye that can be linked covalently to the C-terminus of full-length proteins during cell-free translation. The iminobiotin-labeled proteins can be highly purified by affinity purification with streptavidin beads. We confirmed that the purified fluorescence-labeled proteins are useful for quantitative protein–protein interaction analysis based on fluorescence cross-correlation spectroscopy (FCCS). The apparent dissociation constants of model protein pairs such as proto-oncogenes c-Fos/c-Jun and archetypes of the family of Ca²⁺-modulated calmodulin/related binding proteins were in accordance with the reported values. Further, detailed analysis of the interactions of the components of polycomb group complex, Bmi1, M33, Ring1A and RYBP, was successfully conducted by means of interaction assay for all combinatorial pairs. The results indicate that FCCS analysis with puromycin-based labeling and purification of proteins is effective and convenient for in vitro protein–protein interaction assay, and the method should contribute to a better understanding of protein functions by using the resource of available nucleotide sequences.     

Applications of dual-color fluorescence cross-correlation spectroscopy in antibody binding studies

Two-photon dual-color fluorescence cross-correlation spectroscopy (DC-FCCS) was applied to study the binding interactions of monoclonal antibodies (mAbs) and protein antigens. We measured the binding constant of the interaction of a 32-amino acid brain natriuretic peptide (BNP) with a mAbs and demonstrated the utility of DC-FCCS in studies of antibody sandwiches, trimolecular formations, where two different antibodies bind the same antigen simultaneously. We also show the use of DC-FCCS for monitoring competitive displacement of the labeled antibody in antibody-antigen complexes and subsequent determination of the pertinent dissociation rate (off-rate). The off-rate measurements were performed for two mAbs toward tissue inhibitor 1 of metalloproteinases (TIMP-1). From a methodological perspective, selection of the best labeling protocols and careful optimization of the FCCS instrumentation are essential to achieve the highest cross-correlation signal. When working in vitro, it is practical to generate a complete binding curve using the normalized cross-correlation signal and then fit the experimental points to a binding model. DC-FCCS offers the sensitivity and all other advantages of a solution phase fluorescence-based technique. For systems containing proteins of a similar size that interact without substantial changes in the fluorescence intensity, DC-FCCS serves as a preferred means of measuring solution phase binding constants.     

Toward the detection of single virus particle in serum

There is a grand challenge for the detection of target molecules at single molecule sensitivity in a bulk body fluid for the early diagnosis of diseases. We report our progress on tackling this challenge via the combination of fluorescence cross-correlation spectroscopy (FCCS) and micro fabricated devices toward highly sensitive detection of the dengue virus. We demonstrate that by using a dengue-specific antibody, we can probe the individual dengue virus in a nanomolar bulk solution by following the specific association of dengue antibody using FCCS. Consequently, we designed and fabricated a microfluidic chamber array structure and were able to compartmentalize the bulk aqueous dengue sample into femtoliter volumes using such a device. More importantly we demonstrate that we can differentiate between the compartments containing the dengue virus and the virus-free compartments. Our experiment suggests that by expanding the throughput using microfluidic devices integrated with FCCS, both of which can be achieved practically, we should be able to detect single virus particle in human body fluids in the near future.