Mapping the Direction of Nucleocytoplasmic Transport of Glucocorticoid Receptor (GR) in Live Cells Using Two-Foci Cross-Correlation in Massively Parallel Fluorescence Correlation Spectroscopy (mpFCS).

Nucleocytoplasmic transport of transcription factors is vital for normal cellular function, and its breakdown is a major contributing factor in many diseases. The glucocorticoid receptor (GR) is an evolutionarily conserved, ligand-dependent transcription factor that regulates homeostasis and response to stress and is an important target for therapeutics in inflammation and cancer. In unstimulated cells, the GR resides in the cytoplasm bound to other molecules in a large multiprotein complex.

Dynamic Cellular Cartography: Mapping the Local Determinants of Oligodendrocyte Transcription Factor 2 (OLIG2) Function in Live Cells Using Massively Parallel Fluorescence Correlation Spectroscopy Integrated with Fluorescence Lifetime Imaging Microscopy (mpFCS/FLIM).

Compartmentalization and integration of molecular processes through diffusion are basic mechanisms through which cells perform biological functions. To characterize these mechanisms in live cells, quantitative and ultrasensitive analytical methods with high spatial and temporal resolution are needed. Here, we present quantitative scanning-free confocal microscopy with single-molecule sensitivity, high temporal resolution (∼10 μs/frame), and fluorescence lifetime imaging capacity, developed by integrating massively parallel fluorescence correlation spectroscopy with fluorescence lifetime imaging microscopy (mpFCS/FLIM); we validate the method, use it to map in live cell location-specific variations in the concentration, diffusion, homodimerization, DNA binding, and local environment of the oligodendrocyte transcription factor 2 fused with the enhanced Green Fluorescent Protein (OLIG2-eGFP), and characterize the effects of an allosteric inhibitor of OLIG2 dimerization on these determinants of OLIG2 function.

A strategy for designing allosteric modulators of transcription factor dimerization.

Transcription factors (TFs) are fundamental in the regulation of gene expression in the development and differentiation of cells. They may act as oncogenes and when overexpressed in tumors become plausible targets for the design of antitumor agents. Homodimerization or heterodimerization of TFs are required for DNA binding and the association interface between subunits, for the design of allosteric modulators, appears as a privileged structure for the pharmacophore-based computational strategy.

Functional Fluorescence Microscopy Imaging: Quantitative Scanning-Free Confocal Fluorescence Microscopy for the Characterization of Fast Dynamic Processes in Live Cells.

Functional fluorescence microscopy imaging (fFMI), a time-resolved (21 μs/frame) confocal fluorescence microscopy imaging technique without scanning, is developed for quantitative characterization of fast reaction-transport processes in solution and in live cells. The method is based on massively parallel fluorescence correlation spectroscopy (FCS). Simultaneous excitation of fluorescent molecules in multiple spots in the focal plane is achieved using a diffractive optical element (DOE).

Amyloidogenic Nanoplaques in Blood Serum of Patients with Alzheimer's Disease Revealed by Time-Resolved Thioflavin T Fluorescence Intensity Fluctuation Analysis.

Background: Biomarkers are central to current research on molecular mechanisms underlying Alzheimer's disease (AD). Their further development is of paramount importance for understanding pathophysiological processes that eventually lead to disease onset. Biomarkers are also crucial for early disease detection, before clinical manifestation, and for development of new disease modifying therapies.

Control of Hox transcription factor concentration and cell-to-cell variability by an auto-regulatory switch.

The variability in transcription factor concentration among cells is an important developmental determinant, yet how variability is controlled remains poorly understood. Studies of variability have focused predominantly on monitoring mRNA production noise. Little information exists about transcription factor protein variability, as this requires the use of quantitative methods with single-molecule sensitivity.

Fluorescence-based monitoring of electronic state and ion exchange kinetics with FCS and related techniques: from T-jump measurements to fluorescence fluctuations.

In this review, we give a historical view of how our research in the development and use of fluorescence correlation spectroscopy (FCS) and related techniques has its roots and how it originally evolved from the pioneering work of Manfred Eigen, his colleagues, and coworkers. Work on temperature-jump (T-jump) experiments, conducted almost 50 years ago, led on to the development of the FCS technique. The pioneering work in the 1970s, introducing and demonstrating the concept for FCS, in turn formed the basis for the breakthrough use of FCS more than 15 years later.

Time-resolved homo-FRET studies of biotin-streptavidin complexes.

Förster resonance energy transfer is a mechanism of fluorescence quenching that is notably useful for characterizing properties of biomolecules and/or their interactions. Here we study water-solutions of Biotin-Streptavidin complexes, in which Biotin is labeled with a rigidly-bound fluorophore that can interact by Förster resonance energy transfer with the fluorophores labeling the other, up to three, Biotins of the same complex. The fluorophore, Atto550, is a Rhodamine analogue.

Probing the kinetic landscape of Hox transcription factor-DNA binding in live cells by massively parallel Fluorescence Correlation Spectroscopy.

Hox genes encode transcription factors that control the formation of body structures, segment-specifically along the anterior-posterior axis of metazoans. Hox transcription factors bind nuclear DNA pervasively and regulate a plethora of target genes, deploying various molecular mechanisms that depend on the developmental and cellular context. To analyze quantitatively the dynamics of their DNA-binding behavior we have used confocal laser scanning microscopy (CLSM), single-point fluorescence correlation spectroscopy (FCS), fluorescence cross-correlation spectroscopy (FCCS) and bimolecular fluorescence complementation (BiFC).

Boar spermatozoa successfully predict mitochondrial modes of toxicity: implications for drug toxicity testing and the 3R principles.

Replacement of animal testing by in vitro methods (3-R principles) requires validation of suitable cell models, preferably obtained non-invasively, defying traditional use of explants. Ejaculated spermatozoa are highly dependent on mitochondrial production and consumption of ATP for their metabolism, including motility display, thus becoming a suitable model for capturing multiple modes of action of drugs and other chemicals acting via mitochondrial disturbance. In this study, a hypothesis was tested that the boar spermatozoon is a suitable cell type for toxicity assessment, providing a protocol for 3R-replacement of animals for research and drug-testing.

Fluorescence and single molecule analysis in cell biology.

An overview is presented which describes the development of fluorescence spectroscopy at the cellular level from its beginning as a quantitative tool to determine the content of cellular components to its present use. Analysis of individual biomolecules, their transport and kinetics within a single cell is now possible.

Function and specificity of synthetic Hox transcription factors in vivo.

Homeotic (Hox) genes encode transcription factors that confer segmental identity along the anteroposterior axis of the embryo. However the molecular mechanisms underlying Hox-mediated transcription and the differential requirements for specificity in the regulation of the vast number of Hox-target genes remain ill-defined. Here we show that synthetic Sex combs reduced (Scr) genes that encode the Scr C terminus containing the homedomain (HD) and YPWM motif (Scr-HD) are functional in vivo.

Quantitative study of synthetic Hox transcription factor-DNA interactions in live cells.

Transcription factor-DNA interactions are life sustaining and therefore the subject of intensive research. In spite of vast effort, quantitative in vivo studies of the molecular mechanisms underlying these fundamental interactions remain challenging. In the preceding paper, we designed synthetic Sex combs reduced (Scr) peptides and validated genetically their function as transcriptional regulators.

Ethanol/naltrexone interactions at the mu-opioid receptor. CLSM/FCS study in live cells.

Background: Alcoholism is a widespread chronic disorder of complex aetiology with a significant negative impact on the individual and the society. Mechanisms of ethanol action are not sufficiently well understood at the molecular level and the pharmacotherapy of alcoholism is still in its infancy. Our study focuses at the cellular and molecular level on ethanol-induced effects that are mediated through the micro-opioid receptor (MOP) and on the effects of naltrexone, a well-known antagonist at MOP that is used clinically to prevent relapse in alcoholism.

Quantitative single-molecule imaging by confocal laser scanning microscopy.

A new approach to quantitative single-molecule imaging by confocal laser scanning microscopy (CLSM) is presented. It relies on fluorescence intensity distribution to analyze the molecular occurrence statistics captured by digital imaging and enables direct determination of the number of fluorescent molecules and their diffusion rates without resorting to temporal or spatial autocorrelation analyses. Digital images of fluorescent molecules were recorded by using fast scanning and avalanche photodiode detectors.

Mu-opioid receptor activation in live cells.

Interaction of the mu-opioid receptor (MOP) with selected ligands was investigated in live cells using advanced imaging by confocal laser scanning microscopy integrated with fluorescence correlation spectroscopy and fluorescence cross-correlation spectroscopy. In PC12 cells stably transformed to express the fluorescently labeled MOP-enhanced green fluorescent protein construct, two pools of MOP were identified that could be discriminated by differences in their lateral mobility in the cell membrane. The majority of MOP receptors (80+/-10%) were characterized by a diffusion coefficient D(MOP,1) = (4+/-2) x 10(-11) m(2) s(-1), compared with the slowly moving fraction, D(MOP,2) = (4+/-2) x 10(-12) m(2) s(-1).

Dynamic disorder in horseradish peroxidase observed with total internal reflection fluorescence correlation spectroscopy.

This paper discusses the application of objective-type total internal reflection fluorescence correlation spectroscopy (TIR-FCS) to the study of the kinetics of immobilized horseradish peroxidase on a single molecule level. Objective-type TIR-FCS combines the advantages of FCS with TIRF microscopy in a way that allows for simultaneous ultra-sensitive spectroscopic measurements using a single-point detector and convenient localization of single molecules on a surface by means of parallel imaging.

Parallel dual-color fluorescence cross-correlation spectroscopy using diffractive optical elements.

Dual-color cross-correlation spectroscopy allows the detection and quantification of labeled biomolecules at ultra-low concentrations, whereby the sensitivity of the assay correlates with the measurement time. We now describe a parallel multifocal dual-color spectroscopic configuration employing multiple avalanche photodiodes and hardware correlators. Cross-correlation curves are obtained from several dual-color excitation foci simultaneously.

Total internal reflection fluorescence correlation spectroscopy (TIR-FCS) with low background and high count-rate per molecule.

We designed a fluorescence correlation spectroscopy (FCS) system for measurements on surfaces. The system consists of an objective-type total internal reflection fluorescence (TIRF) microscopy setup, adapted to measure FCS. Here, the fluorescence exciting evanescent wave is generated by epi-illumination through the periphery of a high NA oil-immersion objective.

Functional conformational motions in the turnover cycle of cholesterol oxidase.

Reexamining experimental data of single-molecule fluorescence correlation spectroscopy for cholesterol oxidase, we find that the existing Michaelis-Menten models with dynamical disorder cannot explain strong correlations between subsequent turnover cycles revealed in the diagonal feature in the joint statistical distribution of adjacent "on" times of this enzyme. We suggest that functional conformational motions representing ordered sequences of transitions between a set of conformational substates are involved, along with equilibrium conformational fluctuations in the turnover cycle of cholesterol oxidase. A two-channel model of single-enzyme dynamics, including a slow functional conformational motion in one of the channels, is proposed that allows us to reproduce such strong correlations.

CD8 kinetically promotes ligand binding to the T-cell antigen receptor.

The mechanism of CD8 cooperation with the TCR in antigen recognition was studied on live T cells. Fluorescence correlation measurements yielded evidence of the presence of two TCR and CD8 subpopulations with different lateral diffusion rate constants. Independently, evidence for two subpopulations was derived from the experimentally observed two distinct association phases of cognate peptide bound to class I MHC (pMHC) tetramers and the T cells.

Ultrasensitive reporter protein detection in genetically engineered bacteria.

We demonstrate the use of laser-induced fluorescence confocal spectroscopy to measure analyte-stimulated enhanced green fluorescent protein (egfp) synthesis by genetically modified Escherichia coli bioreporter cells. Induction is measured in cell lysates and, since the spectroscopic focal volume is approximately the size of one bioreporter cell, also in individual live bacteria. This is, to our knowledge, the first ever proof-of-concept work utilizing instrumentation with single-molecule detection capability to monitor bioreporter response.

Study of molecular events in cells by fluorescence correlation spectroscopy.

To understand processes in a living cell, sophisticated and creative approaches are required that can be used for gathering quantitative information about large number of components interacting across temporal and spatial scales without major disruption of the integral network of processes. A physical method of analysis that can meet these requirements is fluorescence correlation spectroscopy (FCS), which is an ultrasensitive and non-invasive detection method capable of single-molecule and real-time resolution. Since its introduction about 3 decades ago, this until recently emerging technology has reached maturity.

High count rates with total internal reflection fluorescence correlation spectroscopy.

We achieved photon count rates per molecule as high as with commonly used confocal fluorescence correlation spectroscopy instruments using a new total internal reflection fluorescence correlation spectroscopy system based on an epi-illumination configuration.

Fluorescence correlation spectroscopy of molecular motions and kinetics.

The foundations for fluorescence correlation spectroscopy (FCS) were already laid in the early 1970s, but this technique did not become widely used until single-molecule detection was established almost 20 years later with the use of diffraction-limited confocal volume element. The analysis of molecular noise from the GHz- to the Hz-region facilitates measurements over a large dynamic range covering photophysics, conformational transitions and interactions as well as transport properties of fluorescent biomolecules. From the Poissonian nature of the noise spectrum the absolute number of molecules is obtainable.