Dual-color Fluorescence Fluctuation Spectroscopy Research Articles

Determining Protein Stoichiometry of Cellular Vesicles by Fluorescence Brightness Analysis

Fluorescence fluctuation spectroscopy (FFS) has been used to study concentration, transport parameters and interactions of fluorescently labeled molecules. One particularly important FFS parameter is brightness, which determines the stoichiometry of protein complexes inside living cells. Recent studies demonstrated that brightness analysis can be extended to characterize the copy number of viral-like particles that contain hundreds to thousands of proteins. Here we explore extending brightness analysis to study protein-carrying vesicles with significantly lower copy numbers than encountered with viral-like particles. Vesicles fulfill many important functions inside the cell. They store, transport, excrete, and digest cellular products. Characterizing vesicles by FFS is challenging, because the low concentration and brightness of the sample limits the achievable signal-to-noise. We present studies that identify the lowest brightness and concentration limit accessible by FFS using a model system. In addition, we present data on vesicles carrying the kinase PI4KIIbeta, both in vivo and in vitro. The experiments described so far utilize single-color detection, which is sufficient for determining the copy number for a single protein species. We perform dual-color FFS experiments on particles and vesicles to investigate its potential to characterize the copy number of two distinct protein species present on the same vesicle. This work is supported by NIH grants GM064589 and GM075401.

Heterospecies partition analysis reveals binding curve and stoichiometry of protein interactions in living cells

Measuring the binding curve and stoichiometry of protein complexes in living cells is a prerequisite for quantitative modeling of cellular processes. Dual-color fluorescence fluctuation spectroscopy provides a general framework for detecting protein interactions, but lacks suitable methods for quantifying protein heterointeractions in the cell. We address this challenge by introducing heterospecies partition (HSP) analysis for protein heterointeractions of the type D + nA<-->DA(n). HSP directly identifies the heterointeracting species from the sample mixture and determines the binding curve and stoichiometry of the protein complex. The HSP method is applied to provide the first direct characterization of the ligand-dependent binding of the retinoic X receptor to the coactivator transcription intermediate factor 2. A previous study based on protein fragments observed a higher binding stoichiometry than biologically expected. We address this difference in stoichiometry by measuring the binding curves of the full-length proteins in living cells. This study provides proof-of-principle experiments that illustrate the potential of HSP as a general and robust analysis tool for the quantitative characterization of protein heterointeractions by dual-color fluorescence fluctuation spectroscopy in living cells.

Protein-mRNA Interactions Observed in Living Cells By Dual-Color Fluorescence Fluctuation Spectroscopy

RNA binding protein controls the localization, translation and degradation of mRNA of specific proteins, therefore exerting a key influence on the cell metabolism, motility and differentiation. The zipcode binding protein 1 (ZBP1) binds to the 3′ untranslated region (UTR) of beta-actin mRNA and regulates its translation. Quantitative characterization of the interaction between mRNA and protein is crucial to dissect this regulation mechanism. We have developed a dual-color fluorescence fluctuation spectroscopy (FFS) technique to study the interaction between mRNA and protein directly in living cells. FFS determines the brightness, concentration and diffusion time of fluorescent particles from the intensity bursts generated by individual particles passing through a small observation volume. Dual-color FFS distinguishes fluorescent species by brightness and diffusion time as well as the fluorescence color. In this study, we apply dual-color FFS to study the interaction between beta-actin mRNA and ZBP1. The endogenous mouse beta-actin mRNA is visualized by incorporating 24 MS2 stem-loops in the 3′ UTR, which are specifically bound by fluorescently labeled MS2 coat protein (MCP). We have developed a tandem dimeric MCP system that is particularly suitable for FFS brightness analysis. Since multiple MCPs bind to a single mRNA, the brightness of mRNA is significantly higher than that of free MCP, which allows us to readily resolve mRNA from free MCPs. Furthermore, ZBP1 is labeled with a different color and dual-color FFS directly extracts the interacting mRNA-ZBP1 species. The interaction is visualized by brightness signatures and the binding affinity is extracted by measuring different cells with varying concentrations of ZBP1. This work is supported by National Institute of Health EB2060.

Observing Nuclear Receptor / Coactivator Interactions in Live Cells by Hetero-Species Partition Analysis

Measuring the binding curve and stoichiometry of protein complexes in living cells is a prerequisite for quantitative modeling of cellular processes. Dual-color fluorescence fluctuation spectroscopy provides a general framework for detecting protein interactions. However, quantitative characterization of protein hetero-interactions remains a difficult task. To address this challenge we introduce hetero-species partition (HSP) analysis for measuring protein hetero-interactions of the type D + nA -> DAn. HSP directly identifies the hetero-interacting species from the sample mixture and determines the binding curve and stoichiometry in the cellular environment. The method is applied to measure the ligand-dependent binding curve of the nuclear receptor retinoic X receptor to the coactivator transcription intermediate factor 2. The binding stoichiometry of this protein system has not been directly measured yet. A previous study using protein fragments observed a higher binding stoichiometry than biologically expected. We address this difference in stoichiometry by measuring the binding curves of the full-length proteins in living cells. This study provides proof-of-principle experiments that illustrate the potential of HSP as a general and robust analysis tool for the quantitative characterization of protein hetero-interactions in living cells.

Quantitative Comparison of Intracellular Unpacking Kinetics of Polyplexes by a Model Constructed From Quantum Dot-FRET

A major challenge for non-viral gene delivery is gaining a mechanistic understanding of the rate-limiting steps. A critical barrier in polyplex-mediated gene delivery is the timely unpacking of polyplexes within the target cell to liberate DNA for efficient gene transfer. In this study, the component plasmid DNA and polymeric gene carrier were individually labeled with quantum dots (QDs) and Cy5 dyes, respectively, as a donor and acceptor pair for fluorescence resonance energy transfer (FRET). The high signal-to-noise ratio in QD-mediated FRET enabled sensitive detection of discrete changes in polyplex stability. The intracellular uptake and dissociation of polyplexes through QD-FRET was captured over time by confocal microscopy. From quantitative image-based analysis, distributions of released plasmid within the endo/lysosomal, cytosolic, and nuclear compartments formed the basis for constructing a three-compartment first-order kinetics model. Polyplex unpacking kinetics for chitosan, polyethylenimine, and polyphosphoramidate were compared and found to correlate well with transfection efficiencies. Thus, QD-FRET-enabled detection of polyplex stability combined with image-based quantification is a valuable method for studying mechanisms involved in polyplex unpacking and trafficking within live cells. We anticipate that this method will also aid the design of more efficient gene carriers.

Studying the Intracellular Dissociation of Polymer−Oligonucleotide Complexes by Dual Color Fluorescence Fluctuation Spectroscopy and Confocal Imaging

To transfect cells, cationic polymers as well as cationic liposomes are widely investigated as carriers for both oligonucleotides and plasmid DNA. A major step in the successful intracellular delivery of the DNA is the release from its carrier. In this study, dual color fluorescence fluctuation spectroscopy (dual color FFS) was explored in order to characterize the intracellular dissociation of cationic polymer/oligonucleotide complexes. As a model, rhodamine green-labeled oligonucleotides (RhGr-ONs) were complexed with Cy5-labeled polymers of either high molar mass (Cy5-graft-pDMAEMA, 1700 kDa) or low molar mass [Cy5-poly(l-lysine), Cy5-pLL, 30 kDa]. The FFS results were compared with confocal laser scanning microscopy (CLSM) observations. CLSM proved that Cy5-graft-pDMAEMA/RhGr-ON complexes endocytosed by Vero cells dissociate in the cytoplasm: the polymer was only detected in the cytoplasm whereas the (released) RhGr-ONs accumulated in the nucleus. Transfecting Vero cells with Cy5-pLL/RhGr-ON complexes resulted, however, in colocalization of polymer and oligonucleotides in the nucleus. In the latter case, CLSM was not able to prove whether intact Cy5-pLL/RhGr-ON complexes were present in the nucleus or whether both components were located together in the nucleus without being associated. Dual color FFS, which monitors the movement of (dual labeled) fluorescent molecules, was able to answer this question. As a Cy5-pLL/RhGr-ON complex is multimolecular, i.e., it consists of many RhGr-ONs associated with many Cy5-pLL chains, it is both highly green and red fluorescent. Consequently, when Cy5-pLL/RhGr-ON complexes move through the excitation volume, the (green and red) detectors of the FFS instrument detect simultaneously a strong green and red fluorescence peak. Upon transfecting the Vero cells with Cy5-pLL/RhGr-ON complexes, FFS was indeed able to detect simultaneously green and red fluorescence peaks in the cytoplasm but never in the nucleus. From these results we conclude that the Cy5-pLL and RhGr-ONs present in the nucleus after transfection were not associated.

774. Studies on the Intracellular Release of Genetic Drugs from Pharmaceutical Carriers

To reveal how DNA/cationic carrier complexes physicochemically behave in cells, dual color fluorescence fluctuation spectroscopy (dual color FFS) is a promising tool. FFS monitors the fluorescence fluctuations caused by the diffusion of fluorescently labeled molecules through the excitation volume of a microscope. Dual color FFS answers the question whether the DNA and the cationic carrier move together (i.e. when they are associated) or separately (i.e. when they are dissociated) through the excitation volume.

Towards a better understanding of the dissociation behavior of liposome-oligonucleotide complexes in the cytosol of cells

To obtain real breakthroughs in antisense therapy, it is necessary to understand the cellular behavior of antisense delivery systems. Fluorescence fluctuation spectroscopy (FFS), which measures in time fluorescence fluctuations in the excitation volume of a microscope and which can thus be applied on a cellular scale, shows potential for this purpose. In this study dual color FFS was explored to characterize the complexation (association and dissociation) between Cy5-labeled oligonucleotides (Cy5-ONs) and FITC-labeled cationic liposomes (FITC-liposomes) in respectively buffer, cell lysate and the cytosol of Vero cells. In Hepes buffer the association of the Cy5-ONs to the FITC-liposomes could be clearly observed from the high peaks of Cy5- and FITC-fluorescence, which appeared simultaneously in the excitation volume. This was explained by the fact that in the complexed state many Cy5-ONs and FITC-liposomes are bound to each other and thus move together through the excitation volume thereby resulting in high fluorescence ‘FITC/Cy5-peaks’. FFS measurements on FITC-liposome/Cy5-ONs complexes in cell lysate revealed that a minor part of the Cy5-ONs was released from the complexes. The major part of the Cy5-ONs remained in the complexes, which also seemed to aggregate in cell lysate. In agreement with the measurements in cell lysate, after microinjection of FITC-liposome/Cy5-ONs complexes in the cytosol of Vero cells a part of the Cy5-ONs was released (as Cy-ONs were detected by FFS in the nuclei) while the other part remained bound (as Cy5-peaks were frequently observed in the cytosol). As will be explained, the Cy5-peaks could be due both to Cy5-ONs clustered with cytosol components and Cy5-ONs still complexed to FITC-liposomes with quenched FITC-fluorescence.

Studying Pegylated DNA Complexes by Dual Color Fluorescence Fluctuation Spectroscopy

In this study, dual color fluorescence fluctuation spectroscopy (dual color FFS) was explored to characterize the association of oligonucleotides (ONs) to pegylated cationic block copolymers, both diblock poly(ethylene glycol)-poly(ethylenimine) (pEG-pEI) and multiblock pEG-pEI. The resulting polyelectrolyte complexes consist of a core of polyion strands, surrounded by a shell of pEG chains. To study the association of the ONs to pEG-pEI by dual color FFS, the oligonucleotides and the cationic block copolymers were labeled with spectrally different fluorescent dyes. Two alternative approaches were executed to label the pEG-pEI's: the dye was attached either to the pEG segment or to the cationic pEI segment of pEG-pEI. When the (diblock and multiblock) pEG-pEI's were labeled on the pEG segment, dual color FFS on the doubly labeled FITC-pEG-pEI/Cy5-ONs complexes revealed the simultaneous appearance of peaks of high fluorescence intensity in both detector channels. This indicates that numerous Cy5-ONs and numerous FITC-pEG-pEI chains move together through the detection volume of the FFS instrument, proving the interaction between many Cy5-ONs and many FITC-pEG-pEI strands to form one multimolecular complex. However, when the fluorescent dye was bound to the cationic pEI segment of diblock or multiblock pEG-pEI, these simultaneously occurring peaks were never observed. Gel electrophoresis experiments proved that diblock pEG-pEI labeled at the pEI segment could not complex the ONs anymore. Multiblock pEG-pEI labeled at the pEI segment was still able to fully complex the oligonucleotides (as shown by gel electrophoresis). The absence of peaks of high fluorescence intensity in FFS measurements was attributed to the quenching of the fluorophores in the core of the complexes. From these results, it could be concluded that dual color FFS allows studying the association and dissociation of pEG-pEI/ONs provided that the fluorescent label of the polymer is attached to the pEG segment and not to the pEI segment.

Dual-Color Fluorescence Fluctuation Spectroscopy To Study the Complexation between Poly-l-lysine and Oligonucleotides

In this study, dual-color fluorescence fluctuation spectroscopy (FFS) was explored to characterize the association of oligonucleotides (ONs) to cationic polymers. The results from dual-color FFS, in which both the ONs and the cationic polymers were fluorescently labeled, were compared with data obtained from single-color FFS in which only the ONs or the cationic polymers were fluorescently marked. As a model, the association of negatively charged 20-mer ONs to positively charged poly-l-lysine (pLL) was studied. The binding of rhodamine green-labeled ONs (RhGr-ONs) to nonlabeled pLL could be clearly observed in the fluorescence fluctuation profiles. Especially, highly intense fluorescence peaks appeared in the fluorescence fluctuations. A highly intense fluorescence peak was considered to originate from one complex in which a number of RhGr-ONs were present. Complexing nonlabeled ONs to rhodamine green-labeled pLL (RhGr-pLL) also resulted in highly intense fluorescence peaks, indicating not only that the complexes consisted of a number of ONs but also that numerous RhGr-pLL chains were present. Upon complexation of red-labeled pLL (Cy5-pLL) to green-labeled ONs (RhGr-ONs), highly intense fluorescence peaks occurred simultaneously in the red and green detector. These data proved the multimolecular composition of the polyplexes and agreed with the observations from single-color FFS.