Suman Ranjit

Cyanine dyes in biophysical research: the photophysics of polymethine fluorescent dyes in biomolecular environments

The breakthroughs in single molecule spectroscopy of the last decade and the recent advances in super resolution microscopy have boosted the popularity of cyanine dyes in biophysical research. These applications have motivated the investigation of the reactions and relaxation processes that cyanines undergo in their electronically excited states. Studies show that the triplet state is a key intermediate in the photochemical reactions that limit the photostability of cyanine dyes. The removal of oxygen greatly reduces photobleaching, but induces rapid intensity fluctuations (blinking). The existence of non-fluorescent states lasting from milliseconds to seconds was early identified as a limitation in single-molecule spectroscopy and a potential source of artifacts. Recent studies demonstrate that a combination of oxidizing and reducing agents is the most efficient way of guaranteeing that the ground state is recovered rapidly and efficiently. Thiol-containing reducing agents have been identified as the source of long-lived dark states in some cyanines that can be photochemically switched back to the emissive state. The mechanism of this process is the reversible addition of the thiol-containing compound to a double bond in the polymethine chain resulting in a non-fluorescent molecule. This process can be reverted by irradiation at shorter wavelengths. Another mechanism that leads to non-fluorescent states in cyanine dyes is cis-trans isomerization from the singlet-excited state. This process, which competes with fluorescence, involves the rotation of one-half of the molecule with respect to the other with an efficiency that depends strongly on steric effects. The efficiency of fluorescence of most cyanine dyes has been shown to depend dramatically on their molecular environment within the biomolecule. For example, the fluorescence quantum yield of Cy3 linked covalently to DNA depends on the type of linkage used for attachment, DNA sequence and secondary structure. Cyanines linked to the DNA termini have been shown to be mostly stacked at the end of the helix, while cyanines linked to the DNA internally are believed to partially bind to the minor or major grooves. These interactions not only affect the photophysical properties of the probes but also create a large uncertainty in their orientation.

Photophysics of Backbone Fluorescent DNA Modifications: Reducing Uncertainties in FRET

We have investigated the photophysical properties of backbone fluorescent DNA modifications with the goal of reducing many of the sources of uncertainty commonly encountered in Forster resonance energy transfer (FRET) measurements. We show that backbone modifications constrain rotational motions, providing a way by which the orientation of the dye can be controlled in a predictable manner, and reduce the uncertainties in donor-acceptor distance associated with the flexible linkers commonly used in conjugate chemistry. Rotational rigidity also prevents undesirable dye-DNA interactions, which have been shown to affect the photophysical properties of the dye. Unusually large FRET efficiencies for donor-acceptor pairs separated by 102 A (three helical turns) were measured and attributed to the favorable relative orientation of the dipoles. The same FRET efficiency was measured for a sample in which the donor-acceptor separation was 12 A shorter, demonstrating the important role of relative orientation in FRET experiments.

Imaging Fibrosis and Separating Collagens using Second Harmonic Generation and Phasor Approach to Fluorescence Lifetime Imaging

In this paper we have used second harmonic generation (SHG) and phasor approach to auto fluorescence lifetime imaging (FLIM) to obtain fingerprints of different collagens and then used these fingerprints to observe bone marrow fibrosis in the mouse femur. This is a label free approach towards fast automatable detection of fibrosis in tissue samples. FLIM has previously been used as a method of contrast in different tissues and in this paper phasor approach to FLIM is used to separate collagen I from collagen III, the markers of fibrosis, the largest groups of disorders that are often without any effective therapy. Often characterized by an increase in collagen content of the corresponding tissue, the samples are usually visualized by histochemical staining, which is pathologist dependent and cannot be automated.

Probing the Interaction Between Fluorophores and DNA Nucleotides by Fluorescence Correlation Spectroscopy and Fluorescence Quenching

We have investigated the association interactions between the fluorescent dyes TAMRA, Cy3B and Alexa‐546 and the DNA deoxynucleoside monophosphates by means of fluorescence quenching and fluorescence correlation spectroscopy (FCS). The interactions of Cy3B and TAMRA with the nucleotides produce a decrease in the apparent diffusion coefficient of the dyes, which result in a shift toward longer times in the FCS autocorrelation decays. Our results with Cy3B demonstrate the existence of Cy3B‐nucleotide interactions that do not affect the fluorescence intensity or lifetime of the dye significantly. The same is true for TAMRA in the presence of dAMP, dCMP and dTMP. In contrast, the diffusion coefficient of Alexa 546 remains practically unchanged even at high concentrations of nucleotide. These results demonstrate that interactions between this dye and the four dNMPs are not significant. The presence of the negatively charged sulfonates and the bulky chlorine atoms in the phenyl group of Alexa 546 possibly prevent strong interactions that are otherwise possible for TAMRA. The characterization of dye–DNA interactions is important in biophysical research because they play an important role in the interpretation of energy transfer experiments, and because they can potentially affect the structure and dynamics of the DNA.

Mapping diffusion in a living cell via the phasor approach.

Diffusion of a fluorescent protein within a cell has been measured using either fluctuation-based techniques (fluorescence correlation spectroscopy (FCS) or raster-scan image correlation spectroscopy) or particle tracking. However, none of these methods enables us to measure the diffusion of the fluorescent particle at each pixel of the image. Measurement using conventional single-point FCS at every individual pixel results in continuous long exposure of the cell to the laser and eventual bleaching of the sample. To overcome this limitation, we have developed what we believe to be a new method of scanning with simultaneous construction of a fluorescent image of the cell. In this believed new method of modified raster scanning, as it acquires the image, the laser scans each individual line multiple times before moving to the next line. This continues until the entire area is scanned. This is different from the original raster-scan image correlation spectroscopy approach, where data are acquired by scanning each frame once and then scanning the image multiple times. The total time of data acquisition needed for this method is much shorter than the time required for traditional FCS analysis at each pixel. However, at a single pixel, the acquired intensity time sequence is short; requiring nonconventional analysis of the correlation function to extract information about the diffusion. These correlation data have been analyzed using the phasor approach, a fit-free method that was originally developed for analysis of FLIM images. Analysis using this method results in an estimation of the average diffusion coefficient of the fluorescent species at each pixel of an image, and thus, a detailed diffusion map of the cell can be created.

Intrinsic stability and oligomerization dynamics of DNA processivity clamps

Sliding clamps are ring-shaped oligomeric proteins that are essential for processive deoxyribonucleic acid replication. Although crystallographic structures of several clamps have been determined, much less is known about clamp structure and dynamics in solution. Here, we characterized the intrinsic solution stability and oligomerization dynamics of the homodimeric Escherichia coli β and the homotrimeric Saccharomyces cerevisiae proliferating cell nuclear antigen (PCNA) clamps using single-molecule approaches. We show that E. coli β is stable in solution as a closed ring at concentrations three orders of magnitude lower than PCNA. The trimeric structure of PCNA results in slow subunit association rates and is largely responsible for the lower solution stability. Despite this large difference, the intrinsic lifetimes of the rings differ by only one order of magnitude. Our results show that the longer lifetime of the E. coli β dimer is due to more prominent electrostatic interactions that stabilize the subunit interfaces.

Spatial dynamics of SIRT1 and the subnuclear distribution of NADH species

Significance Environmental and nutritional cues are crucial to determine genomic responses. They generally proceed through modulation of epigenetic mechanisms. Nuclear sirtuin 1 (SIRT1) is a well-known epigenetic modifier, because it deacetylates histones, and nutrient sensor, because its enzymatic activity is coupled to hydrolysis of NAD+. Compartmentalization of NAD+ metabolism makes it difficult to predict the pace of NAD+-dependent reactions in cells. Here, we use nonlinear optics in live cells to define subnuclear distribution of free and bound NADH, which determines local enzymatic activity. We define subnuclear dynamics of SIRT1 and establish a biophysical signature for SIRT1 activity in live cells. These findings have far-reaching implications, because they describe unique aspects of SIRT1 activity and delineate subnuclear territories of metabolic cues. Sirtuin 1 (SIRT1) is an NAD+-dependent deacetylase that functions as metabolic sensor of cellular energy and modulates biochemical pathways in the adaptation to changes in the environment. SIRT1 substrates include histones and proteins related to enhancement of mitochondrial function as well as antioxidant protection. Fluctuations in intracellular NAD+ levels regulate SIRT1 activity, but how SIRT1 enzymatic activity impacts on NAD+ levels and its intracellular distribution remains unclear. Here, we show that SIRT1 determines the nuclear organization of protein-bound NADH. Using multiphoton microscopy in live cells, we show that free and bound NADH are compartmentalized inside of the nucleus, and its subnuclear distribution depends on SIRT1. Importantly, SIRT6, a chromatin-bound deacetylase of the same class, does not influence NADH nuclear localization. In addition, using fluorescence fluctuation spectroscopy in single living cells, we reveal that NAD+ metabolism in the nucleus is linked to subnuclear dynamics of active SIRT1. These results reveal a connection between NAD+ metabolism, NADH distribution, and SIRT1 activity in the nucleus of live cells and pave the way to decipher links between nuclear organization and metabolism.

3D fluorescence anisotropy imaging using selective plane illumination microscopy

Fluorescence anisotropy imaging is a popular method to visualize changes in organization and conformation of biomolecules within cells and tissues. In such an experiment, depolarization effects resulting from differences in orientation, proximity and rotational mobility of fluorescently labeled molecules are probed with high spatial resolution. Fluorescence anisotropy is typically imaged using laser scanning and epifluorescence-based approaches. Unfortunately, those techniques are limited in either axial resolution, image acquisition speed, or by photobleaching. In the last decade, however, selective plane illumination microscopy has emerged as the preferred choice for three-dimensional time lapse imaging combining axial sectioning capability with fast, camera-based image acquisition, and minimal light exposure. We demonstrate how selective plane illumination microscopy can be utilized for three-dimensional fluorescence anisotropy imaging of live cells. We further examined the formation of focal adhesions by three-dimensional time lapse anisotropy imaging of CHO-K1 cells expressing an EGFP-paxillin fusion protein.

Characterizing fibrosis in UUO mice model using multiparametric analysis of phasor distribution from FLIM images

Phasor approach to fluorescence lifetime microscopy is used to study development of fibrosis in the unilateral ureteral obstruction model (UUO) of kidney in mice. Traditional phasor analysis has been modified to create a multiparametric analysis scheme that splits the phasor points in four equidistance segments based on the height of peak of the phasor distribution and calculates six parameters including average phasor positions, the shape of each segment, the angle of the distribution and the number of points in each segment. These parameters are used to create a spectrum of twenty four points specific to the phasor distribution of each sample. Comparisons of spectra from diseased and healthy tissues result in quantitative separation and calculation of statistical parameters including AUC values, positive prediction values and sensitivity. This is a new method in the evolving field of analyzing phasor distribution of FLIM data and provides further insights. Additionally, the progression of fibrosis with time is detected using this multiparametric approach to phasor analysis.

FXR/TGR5 Dual Agonist Prevents Progression of Nephropathy in Diabetes and Obesity

Bile acids are ligands for the nuclear hormone receptor farnesoid X receptor (FXR) and the G protein-coupled receptor TGR5. We have shown that FXR and TGR5 have renoprotective roles in diabetes- and obesity-related kidney disease. Here, we determined whether these effects are mediated through differential or synergistic signaling pathways. We administered the FXR/TGR5 dual agonist INT-767 to DBA/2J mice with streptozotocin-induced diabetes, db/db mice with type 2 diabetes, and C57BL/6J mice with high-fat diet-induced obesity. We also examined the individual effects of the selective FXR agonist obeticholic acid (OCA) and the TGR5 agonist INT-777 in diabetic mice. The FXR agonist OCA and the TGR5 agonist INT-777 modulated distinct renal signaling pathways involved in the pathogenesis and treatment of diabetic nephropathy. Treatment of diabetic DBA/2J and db/db mice with the dual FXR/TGR5 agonist INT-767 improved proteinuria and prevented podocyte injury, mesangial expansion, and tubulointerstitial fibrosis. INT-767 exerted coordinated effects on multiple pathways, including stimulation of a signaling cascade involving AMP-activated protein kinase, sirtuin 1, PGC-1α, sirtuin 3, estrogen-related receptor-α, and Nrf-1; inhibition of endoplasmic reticulum stress; and inhibition of enhanced renal fatty acid and cholesterol metabolism. Additionally, in mice with diet-induced obesity, INT-767 prevented mitochondrial dysfunction and oxidative stress determined by fluorescence lifetime imaging of NADH and kidney fibrosis determined by second harmonic imaging microscopy. These results identify the renal signaling pathways regulated by FXR and TGR5, which may be promising targets for the treatment of nephropathy in diabetes and obesity.