Fluorescence Recovery After Photobleaching Analysis Research Articles

Long-Term Fluorescence Recovery After Photobleaching (FRAP).

Numerous models have been developed for the analysis of fluorescence recovery after photobleaching (FRAP), by which intracellular diffusion and turnover rate are quantitatively evaluated. FRAP analyses typically focus on such events that occur within several minutes, but to precisely evaluate a slow turnover rate of particularly actin stress fibers, achieving long-term FRAP observations of more than 10min is necessary. In such long-term observations, the effect of intracellular advection is no longer ignored, which motivated us to develop a novel method to decouple the multiple factors associated with the long FRAP response. This method allows us to distinguish the origin of mechanobiological responses of stress fibers that come from either the level of individual actin filaments or that of actin monomers.

Analysis of chemomechanical behavior of stress fibers by continuum mechanics-based FRAP

Fluorescence recovery after photobleaching (FRAP) is a common technique to analyze the turnover of molecules in living cells. Numerous physicochemical models have been developed to quantitatively evaluate the rate of turnover driven by chemical reaction and diffusion that occurs in a few seconds to minutes. On the other hand, they have limitations in interpreting long-term FRAP responses where intracellular active movement inevitably provides target molecular architectures with additional effects other than chemical reaction and diffusion, namely directed transport and structural deformation. To overcome the limitations, we develop a continuum mechanics-based model that allows for decoupling FRAP response into the intrinsic turnover rate and subcellular mechanical characteristics such as displacement vector and strain tensor. Our approach was validated using fluorescently labeled β-actin in an actomyosin-mediated contractile apparatus called stress fibers, revealing spatially distinct patterns of the multi-physicochemical events, in which the turnover rate, which represents effective off-rate of β-actin, was significantly higher at the center of the cell. We also found that the turnover rate is negatively correlated with the rate of displacement or velocity along stress fibers but, interestingly, not with the absolute magnitude of strain. Moreover, stress fibers are subjected to centripetal flow that is facilitated by the circulation of actin molecules. Taken together, this novel framework for long-term FRAP analysis allows for unveiling the contribution of overlooked microscopic mechanics to molecular turnover in living cells.

Regression Analysis of Confocal FRAP and its Application to Diffusion in Membranes.

In most biological processes, diffusion plays a critical role in transferring various bio-molecules to transfer desirable locations in an effective and energy-efficient manner. How fast molecules are transferred is measured by diffusion coefficients. Since each bio-molecules, in particular, signaling molecules have their unique diffusion coefficients and quantifying the diffusion coefficients help us to understand various time scales of both physiological and pathological processes in biological systems. Moreover, since diffusion profiles of a diffusant vary in different micro-environments of cell membranes, accurate diffusion coefficient also can provide a good picture of membrane landscapes as well as interactions of different membrane constituents. Currently, only a few experimental methods are available to assess the diffusion coefficient of a biomolecule of interest in live cells including Fluorescence Recovery After Photobleaching (FRAP). FRAP was developed to study diffusion processes of biomolecules in the cell membranes in the 1970s. Albeit its long history, the main principle of FRAP analysis has remained unchanged since its inception: fitting FRAP data to a theoretical diffusion model for the best fitting diffusion coefficient or using the relation between the half time of recovery and ROI size. In this study, we developed a flexible yet versatile confocal FRAP data analysis framework based on linear regression analysis which allows FRAP users to determine the diffusion from either single or multiple FRAP data points without data fitting. We also validated this approach for a series of fluorescently labeled soluble and membrane-bound proteins and lipids.

Mobility of kinetochore proteins measured by FRAP analysis in living cells

The kinetochore is essential for faithful chromosome segregation during mitosis and is assembled through dynamic processes involving numerous kinetochore proteins. Various experimental strategies have been used to understand kinetochore assembly processes. Fluorescence recovery after photobleaching (FRAP) analysis is also a useful strategy for revealing the dynamics of kinetochore assembly. In this study, we introduced fluorescence protein-tagged kinetochore protein cDNAs into each endogenous locus and performed FRAP analyses in chicken DT40 cells. Centromeric protein (CENP)-C was highly mobile in interphase, but immobile during mitosis. CENP-C mutants lacking the CENP-A-binding domain became mobile during mitosis. In contrast to CENP-C, CENP-T and CENP-H were immobile during both interphase and mitosis. The mobility of Dsn1, which is a component of the Mis12 complex and directly binds to CENP-C, depended on CENP-C mobility during mitosis. Thus, our FRAP assays provide dynamic aspects of how the kinetochore is assembled.

Endocrine disrupting chemicals differentially alter intranuclear dynamics and transcriptional activation of estrogen receptor-α

SummaryTranscription is a highly regulated sequence of stochastic processes utilizing many regulators, including nuclear receptors (NR) that respond to stimuli. Endocrine disrupting chemicals (EDCs) in the environment can compete with natural ligands for nuclear receptors to alter transcription. As nuclear dynamics can be tightly linked to transcription, it is important to determine how EDCs affect NR mobility. We use an EPA-assembled set of 45 estrogen receptor-α (ERα) ligands and EDCs in our engineered PRL-Array model to characterize their effect upon transcription using fluorescence in situ hybridization and fluorescence recovery after photobleaching (FRAP). We identified 36 compounds that target ERα-GFP to a transcriptionally active, visible locus. Using a novel method for multi-region FRAP analysis we find a strong negative correlation between ERα mobility and inverse agonists. Our findings indicate that ERα mobility is not solely tied to transcription but affected highly by the chemical class binding the receptor.

Interclaudin Antagonism: A New Mechanism Of Tight Junction Dependent Barrier Regulation

Tight junctions form selectively‐permeable barriers that regulate paracellular flux. Individual members of the claudin protein family have been categorized as having either pore‐forming or sealing functions. For example, claudin‐2 forms paracellular cation‐selective pores. Conversely, overexpression of claudin‐4 can enhance the paracellular barrier, i.e., reduce permeability, in vitro; claudin‐4 has therefore been classified as a sealing claudin. Although specific residues that form paracellular pores, e.g., within claudin‐2, have been defined, the molecular mechanisms by which sealing claudins, e.g., claudin‐4, regulate the barrier have not. The goal of these studies was to determine the means by which the representative sealing protein claudin‐4 reduces paracellular permeability.METHODSClaudin‐4 was knocked out in Madin‐Darby Canine Kidney I (MDCK I) epithelial cells, which do not express claudin‐2 or other pore‐forming claudins, e.g., claudin‐15. These Cldn4‐KO MDCK I cells were stably transfected to inducibly express mCherry‐claudin‐4 with or without constitutive expression of EGFP‐claudin‐2. Transepithelial resistance (TER) and bi‐ionic potential measurements were used to assess barrier function. Protein anchoring and exchange were determined by Fluorescence Recovery After Photobleaching (FRAP). Widefield, confocal, and super‐resolution microscopy were used for live‐cell imaging of fluorescent claudin proteins in MDCK I and undifferentiated, fibroblast‐like cells (U2OS).RESULTSSurprisingly, claudin‐4 knockout had no effect on the paracellular barrier. Inducible mCherry‐claudin‐4 expression was also insufficient to modify TER. In contrast, EGFP‐claudin‐2 expression reduced TER 10‐fold (p<0.0001). Induction of mCherry‐claudin‐4 expression in EGFP‐claudin‐2‐expressing, but not claudin‐2‐deficient, MDCK I increased TER by 92±18% (p<0.001). FRAP analysis showed that mCherry‐claudin‐4 doubled the mobile fraction of tight junction‐associated EGFP‐claudin‐2 from 16±3% to 32±4% (p<0.001). In U2OS cells, mCherry‐claudin‐4 alone was incapable of forming tight junction‐like strands whereas EGFP‐claudin‐2 assembled into polymeric strands. Induction of mCherry‐claudin‐4 expression disrupted strands formed by EGFP‐claudin‐2, which was subsequently internalized and trafficked to LAMP2‐positive late endosomes. Although MitMAB (dynamin inhibitor) prevented EGFP‐claudin‐2 removal from the junction after mCherry‐claudin‐4 induction, monolayer TER was still increased by mCherry‐claudin‐4 expression.CONCLUSIONSThese data refute the widely‐held view that claudin‐4 forms polymers that seal the paracellular pathway by demonstrating that i) neither knockout nor overexpression of claudin‐4 affect barrier function; and ii) claudin‐4 does not assemble into tight junction‐like strands (polymers). Instead, the results support an alternative model where claudin‐4 enhances barrier function by destabilizing and inhibiting flux via claudin‐2 pores. This challenges existing dogma regarding claudin function and reveals interclaudin antagonism as an unsuspected mechanism of barrier regulation.Support or Funding InformationNIH 5R01DK06 (JRT), CCF RFA 622459 (NS)

Real-time visualization of titin dynamics reveals extensive reversible photobleaching in human induced pluripotent stem cell-derived cardiomyocytes.

Fluorescence recovery after photobleaching (FRAP) has been useful in delineating cardiac myofilament biology, and innovations in fluorophore chemistry have expanded the array of microscopic assays used. However, one assumption in FRAP is the irreversible photobleaching of fluorescent proteins after laser excitation. Here we demonstrate reversible photobleaching regarding the photoconvertible fluorescent protein mEos3.2. We used CRISPR/Cas9 genome editing in human induced pluripotent stem cells (hiPSCs) to knock-in mEos3.2 into the COOH terminus of titin to visualize sarcomeric titin incorporation and turnover. Upon cardiac induction, the titin-mEos3.2 fusion protein is expressed and integrated in the sarcomeres of hiPSC-derived cardiomyocytes (CMs). STORM imaging shows M-band clustered regions of bound titin-mEos3.2 with few soluble titin-mEos3.2 molecules. FRAP revealed a baseline titin-mEos3.2 fluorescence recovery of 68% and half-life of ~1.2 h, suggesting a rapid exchange of sarcomeric titin with soluble titin. However, paraformaldehyde-fixed and permeabilized titin-mEos3.2 hiPSC-CMs surprisingly revealed a 55% fluorescence recovery. Whole cell FRAP analysis in paraformaldehyde-fixed, cycloheximide-treated, and untreated titin-mEos3.2 hiPSC-CMs displayed no significant differences in fluorescence recovery. FRAP in fixed HEK 293T expressing cytosolic mEos3.2 demonstrates a 58% fluorescence recovery. These data suggest that titin-mEos3.2 is subject to reversible photobleaching following FRAP. Using a mouse titin-eGFP model, we demonstrate that no reversible photobleaching occurs. Our results reveal that reversible photobleaching accounts for the majority of titin recovery in the titin-mEos3.2 hiPSC-CM model and should warrant as a caution in the extrapolation of reliable FRAP data from specific fluorescent proteins in long-term cell imaging.

Quantifying Dynamics in Phase-Separated Condensates Using Fluorescence Recovery after Photobleaching

Cells contain numerous membraneless organelles that assemble by intracellular liquid-liquid phase separation. The viscous properties and associated biomolecular mobility within these condensed phase droplets, or condensates, are increasingly recognized as important for cellular function and also dysfunction, for example, in protein aggregation pathologies. Fluorescence recovery after photobleaching (FRAP) is widely used to assess condensate fluidity and to estimate protein diffusion coefficients. However, the models and assumptions utilized in FRAP analysis of protein condensates are often not carefully considered. Here, we combine FRAP experiments on both in vitro reconstituted droplets and intracellular condensates with systematic examination of different models that can be used to fit the data and evaluate the impact of model choice on measured values. A key finding is that model boundary conditions can give rise to widely divergent measured values. This has important implications, for example, in experiments that bleach subregions versus the entire condensate, two commonly employed experimental approaches. We suggest guidelines for determining the appropriate modeling framework and highlight emerging questions about the molecular dynamics at the droplet interface. The ability to accurately determine biomolecular mobility both in the condensate interior and at the interface is important for obtaining quantitative insights into condensate function, a key area for future research.

Determination of δ-opioid receptor molecules mobility in living cells plasma membrane by novel method of FRAP analysis

Fluorescence recovery after photobleaching (FRAP) is the preferred method for analyzing the lateral mobility of fluorescently-tagged proteins in the plasma membranes (PMs) of live cells. FRAP experiments are described as being easy to perform; however, the analysis of the acquired data can be difficult. The evaluation procedure must be properly combined with the imaging setup of the confocal microscope to provide unbiased results.With the aim of increasing the accuracy of determining the diffusion coefficient (D) and mobile fraction (Mf) of PM proteins, we developed a novel method for FRAP analysis in the equatorial plane of the cell. This method is based on the calculation of photobleaching characteristics, derived from the light intensity profile and optical parameters of the confocal microscope, and on the model of fluorescent molecule diffusion in PM regions outside of the focal plane. Furthermore, cell movement artifacts in the FRAP data are ameliorated by using a region of interest, which is not fixed but instead moves adaptively in coordination with the movement of cells.When this method was used to determine the mobility of the δ-opioid receptor-eYFP in HEK293 cells, a highly significant decrease in receptor mobility was detected in cholesterol-depleted cells. This decrease was fully reversible by the replenishment of cholesterol levels. Our results demonstrate the crucial role played by cholesterol in the dynamic organization of δ-opioid receptors in the PM under in vivo conditions. Our method may be applied for the determination of the D and Mf values of other PM proteins.

Quantitative diffusion measurements using the open-source software PyFRAP

Fluorescence Recovery After Photobleaching (FRAP) and inverse FRAP (iFRAP) assays can be used to assess the mobility of fluorescent molecules. These assays measure diffusion by monitoring the return of fluorescence in bleached regions (FRAP), or the dissipation of fluorescence from photoconverted regions (iFRAP). However, current FRAP/iFRAP analysis methods suffer from simplified assumptions about sample geometry, bleaching/photoconversion inhomogeneities, and the underlying reaction-diffusion kinetics. To address these shortcomings, we developed the software PyFRAP, which fits numerical simulations of three-dimensional models to FRAP/iFRAP data and accounts for bleaching/photoconversion inhomogeneities. Using PyFRAP we determined the diffusivities of fluorescent molecules spanning two orders of magnitude in molecular weight. We measured the tortuous effects that cell-like obstacles exert on effective diffusivity and show that reaction kinetics can be accounted for by model selection. These applications demonstrate the utility of PyFRAP, which can be widely adapted as a new extensible standard for FRAP analysis.

Characterization of Cell Boundary and Confocal Effects Improves Quantitative FRAP Analysis

Fluorescence recovery after photobleaching (FRAP) is an important tool used by cell biologists to study the diffusion and binding kinetics of vesicles, proteins, and other molecules in the cytoplasm, nucleus, or cell membrane. Although many FRAP models have been developed over the past decades, the influence of the complex boundaries of 3D cellular geometries on the recovery curves, in conjunction with regions of interest and optical effects (imaging, photobleaching, photoswitching, and scanning), has not been well studied. Here, we developed a 3D computational model of the FRAP process that incorporates particle diffusion, cell boundary effects, and the optical properties of the scanning confocal microscope, and validated this model using the tip-growing cells of Physcomitrella patens. We then show how these cell boundary and optical effects confound the interpretation of FRAP recovery curves, including the number of dynamic states of a given fluorophore, in a wide range of cellular geometries—both in two and three dimensions—namely nuclei, filopodia, and lamellipodia of mammalian cells, and in cell types such as the budding yeast, Saccharomyces pombe, and tip-growing plant cells. We explored the performance of existing analytical and algorithmic FRAP models in these various cellular geometries, and determined that the VCell VirtualFRAP tool provides the best accuracy to measure diffusion coefficients. Our computational model is not limited only to these cells types, but can easily be extended to other cellular geometries via the graphical Java-based application we also provide. This particle-based simulation—called the Digital Confocal Microscopy Suite or DCMS—can also perform fluorescence dynamics assays, such as number and brightness, fluorescence correlation spectroscopy, and raster image correlation spectroscopy, and could help shape the way these techniques are interpreted.

TRH receptor mobility in the plasma membrane is strongly affected by agonist binding and by interaction with some cognate signaling proteins

Objectives: Extensive research has been dedicated to elucidating the mechanisms of signal transduction through different G protein-coupled receptors (GPCRs). However, relatively little is known about the regulation of receptor movement within the cell membrane upon ligand binding. In this study we focused our attention on the thyrotropin-releasing hormone (TRH) receptor that typically couples to Gq/11 proteins.Methods: We monitored receptor diffusion in the plasma membrane of HEK293 cells stably expressing yellow fluorescent protein (YFP)-tagged TRH receptor (TRHR-YFP) by fluorescence recovery after photobleaching (FRAP).Results: FRAP analysis indicated that the lateral movement of the TRH receptor was markedly reduced upon TRH binding as the value of its diffusion coefficient fell down by 55%. This effect was prevented by the addition of the TRH receptor antagonist midazolam. We also found that siRNA-mediated knockdown of Gq/11α, Gβ, β-arrestin2 and phospholipase Cβ1, but not of Giα1, β-arrestin1 or G protein-coupled receptor kinase 2, resulted in a significant decrease in the rate of TRHR-YFP diffusion, indicating the involvement of the former proteins in the regulation of TRH receptor behavior. The observed partial reduction of the TRHR-YFP mobile fraction caused by down-regulation of Giα1 and β-arrestin1 suggests that these proteins may also play distinct roles in THR receptor-mediated signaling.Conclusion: These results demonstrate for the first time that not only agonist binding but also abundance of some signaling proteins may strongly affect TRH receptor dynamics in the plasma membrane.

FRAP analysis of chromatin looseness in mouse zygotes that allows full-term development.

Chromatin looseness, which can be analyzed by fluorescence recovery after photobleaching (FRAP) using eGFP-tagged core histone proteins, is an important index of the differentiation potential of blastomere cells and embryonic stem cells. Whether chromatin looseness is a reliable index of the developmental potential of embryos during ontogenesis is not known. As a necessary first step toward answering this question, we investigated whether FRAP-analyzed embryos are capable of normal preimplantation and full-term development. All tested concentrations (50, 100, and 250 ng/μL) of microinjected eGFP-H2B mRNA were sufficient for detecting differences in chromatin looseness between male and female pronuclei. After FRAP analysis, most of the zygotes developed into blastocysts. Importantly, a considerable number of offspring developed from the FRAP analyzed zygotes (32/78; 41.0%) and grew into healthy adults. The offspring of zygotes injected with 250 ng/μL of eGFP-H2B mRNA and bleached using 110 μW laser power for 5 s were not genetically modified. Interestingly, bleaching using a 3-fold stronger laser intensity for a 6-fold longer time did not cause toxicity during preimplantation development, indicating that bleaching did not critically affect preimplantation development. Finally, we confirmed that similar results were obtained using two different types of confocal laser-scanning microscopes. This FRAP protocol would be useful for investigating the association between chromatin looseness and development.

Validation of Normalizations, Scaling, and Photofading Corrections for FRAP Data Analysis.

Fluorescence Recovery After Photobleaching (FRAP) has been a versatile tool to study transport and reaction kinetics in live cells. Since the fluorescence data generated by fluorescence microscopy are in a relative scale, a wide variety of scalings and normalizations are used in quantitative FRAP analysis. Scaling and normalization are often required to account for inherent properties of diffusing biomolecules of interest or photochemical properties of the fluorescent tag such as mobile fraction or photofading during image acquisition. In some cases, scaling and normalization are also used for computational simplicity. However, to our best knowledge, the validity of those various forms of scaling and normalization has not been studied in a rigorous manner. In this study, we investigate the validity of various scalings and normalizations that have appeared in the literature to calculate mobile fractions and correct for photofading and assess their consistency with FRAP equations. As a test case, we consider linear or affine scaling of normal or anomalous diffusion FRAP equations in combination with scaling for immobile fractions. We also consider exponential scaling of either FRAP equations or FRAP data to correct for photofading. Using a combination of theoretical and experimental approaches, we show that compatible scaling schemes should be applied in the correct sequential order; otherwise, erroneous results may be obtained. We propose a hierarchical workflow to carry out FRAP data analysis and discuss the broader implications of our findings for FRAP data analysis using a variety of kinetic models.

Dissecting protein reaction dynamics in living cells by fluorescence recovery after photobleaching.

Proteins within most macromolecular complexes or organelles continuously turn over. This turnover results from association and dissociation reactions that are mediated by each of the protein's functional domains. Thus, studying organelle or macromolecular formation from the bottom up using theoretical and computational modeling approaches will necessitate the determination of all of these reaction rates in vivo. Yet current methods for examining protein dynamics either necessitate highly specialized equipment or limit themselves to basic measurements. In this protocol, we describe a broadly applicable method based on fluorescence recovery after photobleaching (FRAP) for determining how many reaction processes participate in the turnover of any given protein of interest, for characterizing their apparent association and dissociation rates, and for determining their relative importance in the turnover of the overall protein population. Experiments were performed in melanoma M2 cells expressing mutant forms of ezrin that provide a link between the plasma membrane and the cortical actin cytoskeleton. We also describe a general strategy for the identification of the protein domains that mediate each of the identified turnover processes. Our protocol uses widely available laser-scanning confocal microscopes, open-source software, graphing software and common molecular biology techniques. The entire FRAP experiment preparation, data acquisition and analysis require 3-4 d.

Dynamic regulation of a cell adhesion protein complex including CADM1 by combinatorial analysis of FRAP with exponential curve-fitting.

Protein components of cell adhesion machinery show continuous renewal even in the static state of epithelial cells and participate in the formation and maintenance of normal epithelial architecture and tumor suppression. CADM1 is a tumor suppressor belonging to the immunoglobulin superfamily of cell adhesion molecule and forms a cell adhesion complex with an actin-binding protein, 4.1B, and a scaffold protein, MPP3, in the cytoplasm. Here, we investigate dynamic regulation of the CADM1-4.1B-MPP3 complex in mature cell adhesion by fluorescence recovery after photobleaching (FRAP) analysis. Traditional FRAP analysis were performed for relatively short period of around 10min. Here, thanks to recent advances in the sensitive laser detector systems, we examine FRAP of CADM1 complex for longer period of 60 min and analyze the recovery with exponential curve-fitting to distinguish the fractions with different diffusion constants. This approach reveals that the fluorescence recovery of CADM1 is fitted to a single exponential function with a time constant (τ) of approximately 16 min, whereas 4.1B and MPP3 are fitted to a double exponential function with two τs of approximately 40-60 sec and 16 min. The longer τ is similar to that of CADM1, suggesting that 4.1B and MPP3 have two distinct fractions, one forming a complex with CADM1 and the other present as a free pool. Fluorescence loss in photobleaching analysis supports the presence of a free pool of these proteins near the plasma membrane. Furthermore, double exponential fitting makes it possible to estimate the ratio of 4.1B and MPP3 present as a free pool and as a complex with CADM1 as approximately 3:2 and 3:1, respectively. Our analyses reveal a central role of CADM1 in stabilizing the complex with 4.1B and MPP3 and provide insight in the dynamics of adhesion complex formation.

RNA imaging: seeing is believing.

Twenty years ago, in the middle 1990s, a pending debate in the RNA cell biology field concerned the nuclear organization of pre-mRNA splicing. Localization studies using antibodies to detect proteins of the spliceosome and oligonucleotide probes that hybridized with the spliceosomal small nuclear RNAs had revealed that practically all building blocks of the splicing machinery were not uniformly distributed in the nucleus but rather appeared concentrated in defined regions. These regions were termed “nuclear speckles” or “splicing domains” due to the local enrichment in splicing factors. Whether or not pre-mRNA splicing occurred within these domains remained controversial. To address this question, it was critical to visualize the transition from nascent unspliced transcripts to the spliced forms. This was made possible through successive optimizations of in situ hybridization, a microscopic technique pioneered independently by Gall and Jones back in the late 1960s. An important advance was made by Lawrence, who introduced a procedure for fluorescent detection of nuclear RNA using biotin-labeled DNA probes and fluorophore-conjugated avidin. During the 1990s, the method was further optimized for the simultaneous detection of DNA, unspliced precursors and spliced mRNA. Using these tools, several laboratories showed that spliced mRNA localized in close proximity to the DNA from which it was transcribed, consistent with earlier electron micrographs obtained by Beyer and Osheim suggesting that the spliceosome formed shortly after synthesis of the 3′ splice site and that splicing of pre-mRNA could occur while the pre-mRNA was still tethered to the gene locus via RNA polymerase. A subsequent imaging breakthrough resulted from the development of genetically encoded fluorescent tags that, combined with fluorescence-based microscopic approaches of increasingly higher spatial and temporal resolution, made it possible to analyze protein movement in living cells. Tom Misteli in the Spector lab reported the first time-series recordings of cells expressing the green fluorescent protein (GFP) fused to an essential splicing protein. These studies provided direct evidence that nuclear speckles supply spliceosomal components to nearby activated sites of transcription and splicing. Further developments of FRAP (fluorescence recovery after photobleaching) methods unravelled the kinetic properties of splicing proteins in the nucleus of live cells. FRAP studies showed that spliceosomal proteins are continuously moving in the nucleus, shuttling in and out of nuclear speckles within seconds. Since actively transcribed genes are intimately associated with the periphery of nuclear speckles, one possibility is that spatial confinement of spliceosomal components in dedicated compartments increases their local concentration in the neighborhood of nascent transcripts, thus enhancing spliceosome assembly on newly synthesized introns. This could be a reason why splicing is so much faster in vivo than in vitro. FRAP analysis further indicated that the trafficking kinetics of splicing factors in and out of nuclear speckles is independent of ongoing transcription and splicing, arguing that spliceosomal components are not stored in nuclear speckles until a signal triggers their recruitment to nascent introns; rather, it is more likely that components of the spliceosome are constantly roaming the nuclear space until they collide and transiently interact with either a nascent pre-mRNA, to form a spliceosome, or with other splicing proteins at nuclear speckles. In 2004, two seminal reports from the Singer and Spector labs revolutionized the RNA imaging field by pioneering an approach to visualize mRNA synthesis and transport in living mammalian cells. The method consists in genetically inserting binding sites for the MS2 bacteriophage coat protein in a gene of interest. The resulting reporter transgene is then integrated in the genome of mammalian cells that express the MS2 coat protein fused to a fluorescent protein such as GFP. Upon transcription, binding of fluorescent coat protein to its target sequence makes that particular mRNA visible in living cells. With this approach it was possible to track, in real-time, the life cycle of an mRNA tagged in the 3′ UTR, from transcription to transport and localization in the cytoplasm. The results showed unequivocally that mRNAs diffuse throughout the nucleus and hit nuclear pores in a stochastic manner. This came as a shock to many cell biologists that advocated the existence of an internal nuclear scaffold (or “matrix”) composed of a fibrillar system that would guide mRNAs from the site of transcription directly to the pores. Another surprising revelation of RNA imaging studies was the occurrence of random fluctuations in transcription of individual genes. Using either the MS2-GFP system or variants of fluorescence in situ hybridization (FISH) methodologies capable of detecting individual mRNA molecules in single cells, different laboratories demonstrated that many genes in both prokaryotes and eukaryotes are transcribed in a pulsatile fashion. Potential biological implications of such transcriptional bursts are just starting to be experimentally explored. While accuracy in all steps of gene expression is vital for cellular and organismal integrity, stochastic fluctuations are likely important for biological plasticity. To date, many studies link stochastic gene expression with cell-fate decisions during development of higher eukaryotes. There is also evidence that aging is correlated with increased noise in gene expression. A fascinating line of future research will be to determine the molecular mechanisms involved in cell-to-cell variability in gene expression patterns and to understand how cells control and tolerate noise in each step of gene expression, from transcription to pre-mRNA processing and translation. This will likely rely on the development of new imaging strategies capable of monitoring in real time the dynamic behavior of single endogenous molecules in living cells and tissues. If randomness plays a fundamental role in the establishment of gene expression patterns, as anticipated, future discoveries on this topic may prove important in ways that are unpredictable today.

Disruption of NAD(+) binding site in glyceraldehyde 3-phosphate dehydrogenase affects its intranuclear interactions.

To characterize phosphorylation of human glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and mobility of GAPDH in cancer cells treated with chemotherapeutic agents. We used proteomics analysis to detect and characterize phosphorylation sites within human GAPDH. Site-specific mutagenesis and alanine scanning was then performed to evaluate functional significance of phosphorylation sites in the GAPDH polypeptide chain. Enzymatic properties of mutated GAPDH variants were assessed using kinetic studies. Intranuclear dynamics parameters (diffusion coefficient and the immobile fraction) were estimated using fluorescence recovery after photobleaching (FRAP) experiments and confocal microscopy. Molecular modeling experiments were performed to estimate the effects of mutations on NAD(+) cofactor binding. Using MALDI-TOF analysis, we identified novel phosphorylation sites within the NAD(+) binding center of GAPDH at Y94, S98, and T99. Using polyclonal antibody specific to phospho-T99-containing peptide within GAPDH, we demonstrated accumulation of phospho-T99-GAPDH in the nuclear fractions of A549, HCT116, and SW48 cancer cells after cytotoxic stress. We performed site-mutagenesis, and estimated enzymatic properties, intranuclear distribution, and intranuclear mobility of GAPDH mutated variants. Site-mutagenesis at positions S98 and T99 in the NAD(+) binding center reduced enzymatic activity of GAPDH due to decreased affinity to NAD(+) (Km = 741 ± 257 μmol/L in T99I vs 57 ± 11.1 µmol/L in wild type GAPDH. Molecular modeling experiments revealed the effect of mutations on NAD(+) binding with GAPDH. FRAP (fluorescence recovery after photo bleaching) analysis showed that mutations in NAD(+) binding center of GAPDH abrogated its intranuclear interactions. Our results suggest an important functional role of phosphorylated amino acids in the NAD(+) binding center in GAPDH interactions with its intranuclear partners.

Universal Approach to FRAP Analysis of Arbitrary Bleaching Patterns

Fluorescence recovery after photobleaching (FRAP) is a classical biophysical method, that has been used extensively in the study of numerous systems, particularly cell membranes. It consists of irreversibly photobleaching a fluorescently labeled diffusing species, and then following the fluorescence recovery over time. By analyzing the fluorescence recovery dynamics it is possible to extract the diffusion coefficient of the fluorescent species. While the experimental setup is relatively simple, the interpretation of the results is complex, severely limiting the quantitative use of the technique.With the introduction of digital imaging and the Laser Scanning Confocal Microscope (LSCM), a multitude of variations of the technique became possible. While these modern options offer high flexibility and extend the availability of the method, the initial and boundary conditions are too complex to allow a precise closed, analytical solution of the diffusion problem, and thus quantitative extraction of the diffusion coefficient.We have developed a fast algorithm that allows the extraction of diffusion coefficients given a stack of images representing a FRAP experiment, and acquired with any method, for a completely arbitrary geometry of the initially bleached area. The algorithm treats the first post-bleach frame as the initial and boundary conditions, and simulates the diffusion of the fluorescent molecules. The lack of closed analytical solutions for complex geometries is not a limiting factor, since the diffusion equation is effectively solved numerically by iterative simulation.We validated our approach using a well characterized diffusing molecule (DiIC18) and against the well-established analytical procedures for Gaussian (Axelrod, Koppel et al. 1976) and box bleaching geometries (Ellenberg, Siggia et al. 1997). Furthermore, we show that the algorithm can deduce the diffusion coefficient for an arbitrary bleaching geometrys.The authors gratefully acknowledge a grant from the US-Israel Binational Science Foundation, #2009345.

Nuclear Actin Dynamics Regulate Nuclear Organization and Transcription

2716-Pos Board B146 Nuclear Actin Dynamics Regulate Nuclear Organization and Transcription Leo Serebryannyy 1 , Megan Parilla 1 , Paolo Annibale 2 , Christina Cruz 1 , Dmitri Kudryashov 3 , Enrico Gratton 2 , Cara J. Gottardi 4 , Primal de Lanerolle 1 . Physiology and Biophysics, University of Illinois at Chicago, Chicago, IL, USA, 2 Laboratory of Fluorescence Dynamics, University of California at Irvine, Irvine, CA, USA, 3 Chemistry and Biochemistry, Ohio State University, Columbus, OH, USA, 4 Medicine, Northwestern University, Chicago, IL, USA. Actin is an important and elegant mechanotransducer that transfers messages from the extracellular environment into the nucleus, thus conferring changes in both the physical properties and the genetic program of the cell. Although actin in the nucleus has been established in a growing number of functions, the form of nuclear actin remains poorly understood as there are no apparent actin filaments in the nucleus. Therefore, we tested how disrupting the form of nuclear actin impacts function and what effects this may have on the nucleus. To do so, we formed stable nuclear actin filaments using a variety of methods, including the nuclear enrichment of actin binding proteins supervillin and a-E-catenin and the expression of a mutant form of skeletal a-actin. Using fluorescence recovery after photobleaching (FRAP), we found that stabiliza- tion of nuclear actin filaments significantly impairs actin dynamics within the nucleus. The formation of nuclear actin filaments coincides with striking changes in nuclear structure and overall nuclear topography as determined by confocal microscopy and raster image correlation spectroscopy. Using a com- bination of FRAP analysis, structured illumination microscopy and immuno- logical assays, we found that nuclear actin filaments reduce the association of actin with RNA polymerase II and this correlates with impaired RNA poly- merase II dynamics, localization and gene recruitment. Moreover, we were able to recapitulate our findings in purified nuclear extract by using in vitro transcription assays with the covalent actin crosslinking domain (ACD) of V. cholerae MARTX toxin. Based on our data, which help explain the absence of nuclear actin filaments in the interphase mammalian nucleus, we propose a model where nuclear actin dynamics are critical for maintaining proper nu- clear function.