Fluorescence Recovery After Photobleaching Recovery Research Articles

The Utility of Fluorescence Recovery after Photobleaching (FRAP) to Study the Plasma Membrane.

The plasma membrane of mammalian cells is involved in a wide variety of cellular processes, including, but not limited to, endocytosis and exocytosis, adhesion and migration, and signaling. The regulation of these processes requires the plasma membrane to be highly organized and dynamic. Much of the plasma membrane organization exists at temporal and spatial scales that cannot be directly observed with fluorescence microscopy. Therefore, approaches that report on the membrane's physical parameters must often be utilized to infer membrane organization. As discussed here, diffusion measurements are one such approach that has allowed researchers to understand the subresolution organization of the plasma membrane. Fluorescence recovery after photobleaching (or FRAP) is the most widely accessible method for measuring diffusion in a living cell and has proven to be a powerful tool in cell biology research. Here, we discuss the theoretical underpinnings that allow diffusion measurements to be used in elucidating the organization of the plasma membrane. We also discuss the basic FRAP methodology and the mathematical approaches for deriving quantitative measurements from FRAP recovery curves. FRAP is one of many methods used to measure diffusion in live cell membranes; thus, we compare FRAP with two other popular methods: fluorescence correlation microscopy and single-particle tracking. Lastly, we discuss various plasma membrane organization models developed and tested using diffusion measurements.

Localization, induction, and cellular effects of tau phosphorylated at threonine 217.

Tau phosphorylation at T217 is a promising Alzheimer's disease (AD) biomarker, but its functional consequences were unknown. Human brain and cultured mouse neurons were analyzed by immunoblotting and immunofluorescence for total tau, taupT217 , taupT181 , taupT231 , and taupS396/pS404 . Direct stochastic optical reconstruction microscopy (dSTORM) super resolution microscopy was used to localize taupT217 in cultured neurons. Enhanced green fluorescent protein (EGFP)-tau was expressed in fibroblasts as wild type and T217E pseudo-phosphorylated tau, and fluorescence recovery after photobleaching (FRAP) reported tau turnover rates on microtubules. In the brain, taupT217 appears in neurons at Braak stages I and II, becomes more prevalent later, and co-localizes partially with other phospho-tau epitopes. In cultured neurons, taupT217 is increased by extracellular tau oligomers (xcTauOs) and is associated with developing post-synaptic sites. FRAP recovery was fastest for EGFP-tauT217E . TaupT217 increases in the brain as AD progresses and is induced by xcTauOs. Post-synaptic taupT217 suggests a role for T217 phosphorylation in synapse impairment. T217 phosphorylation reduces tau's affinity for microtubules. Validation of anti-tau phosphorylated at threonine-217 (taupT217 ) specificity is essential due to epitope redundancy. taupT217 increases as Alzheimer's disease progresses and is found throughout diseased neurons. taupT217 is associated with developing post-synaptic sites in cultured neurons. Extracellular oligomers of tau, but not amyloid beta, increase intracellular taupT217 . T217E pseudo-phosphorylation reduces tau's affinity for microtubules.

Microstructure and biomolecules mobility of human milk fat globules by fluorescence recovery after photobleaching with confocal scanning laser microscope

This work demonstrates the use of fluorescence recovery after photobleaching (FRAP) for direct measurement of the microstructure and biomolecules mobility of human milk fat globules (HMFGs). In particular, five fluorescent probes (three types), Nile Red, Rh-DOPE, NBD-PC, NBD-SM, and Nile Blue, were evaluated in a Confocal scanning laser microscope combined with FRAP for the detection of the molecular diffusion of neutral lipids, polar lipids and membrane proteins in HMFGs. Mobile fraction (Mf), half-life recovery time (t1/2) and diffusion coefficient (K) were calculated from the FRAP recovery curve of five fluorescent molecules. The Mf of fluorescent probes labeled neutral lipids, polar lipids, membrane proteins, phosphatidylcholine, sphingomyelin, and on HMFGs were 63%, 18%, 45%, 28% and 5%, respectively. The recovery times of fluorescent molecules labeled with membrane biomolecules were longer as the size of the milk fat globules decreased and the photobleaching area increased. This method was applied to the HMFG membrane cytoplasmic remnants (CR) region showing a similar trend and much higher fluorescence intensity to the membrane region, which suggests that CR might contain multiple layers of membrane lipids.

Parameter estimation in fluorescence recovery after photobleaching: quantitative analysis of protein binding reactions and diffusion

Fluorescence recovery after photobleaching (FRAP) is a common experimental method for investigating rates of molecular redistribution in biological systems. Many mathematical models of FRAP have been developed, the purpose of which is usually the estimation of certain biological parameters such as the diffusivity and chemical reaction rates of a protein, this being accomplished by fitting the model to experimental data. In this article, we consider a two species reaction–diffusion FRAP model. Using asymptotic analysis, we derive new FRAP recovery curve approximation formulae, and formally re-derive existing ones. On the basis of these formulae, invoking the concept of Fisher information, we predict, in terms of biological and experimental parameters, sufficient conditions to ensure that the values all model parameters can be estimated from data. We verify our predictions with extensive computational simulations. We also use computational methods to investigate cases in which some or all biological parameters are theoretically inestimable. In these cases, we propose methods which can be used to extract the maximum possible amount of information from the FRAP data.

Altered Intracellular Trafficking of Mutant Thyroid Hormone Receptors in Resistance to Thyroid Hormone Syndrome

Resistance to Thyroid Hormone α (RTHα), a reduced sensitivity to thyroid hormone (T3) in peripheral tissues, is caused by mutations in thyroid hormone receptor α (TRα), a nuclear receptor that mediates T3-responsive gene expression. All mutations characterized in RTHα, to date, are in the ligand binding domain (LBD), resulting in reduced affinity for T3. In addition, some mutations result in truncated proteins lacking all or part of helix 12. Previously, we have used fluorescence recovery after photobleaching (FRAP) to examine the effects of select RTHα mutations on the intracellular trafficking of TRα. After transfecting HeLa cells with expression plasmids for green fluorescent protein (GFP)-tagged wild-type TRα and each of the mutants, we first assessed their intracellular distribution and initial intranuclear mobility. Although wild-type TRα is known to shuttle between the nucleus and cytoplasm, it is primarily localized to the nucleus at a steady state. We showed that TR-E403X, Ala382ProfsX7, and F397fs406X are also primarily localized to the nucleus, and FRAP revealed that wild-type TRα and the RTHα mutants are highly dynamic within the nucleus, indicating that the receptors rapidly dissociate and reassociate with DNA binding sites and/or other nuclear binding sites, independent of T3 (1). Some studies have shown that Nuclear Receptor Corepressor 1 (NCoR1), which interacts with the hinge region of TRα (the region between the DNA-binding domain and LBD) has a higher affinity for RTHα mutants compared to wild-type TRα, supporting the hypothesis that helix 12 of the LBD also functions to disassociate NCoR1 from TRα when it is bound to T3 (2). We proposed that this increased affinity for NCoR1 alters the mobility of TRα in the nucleus, impacting its function. Here, we show that NCoR1 has slower FRAP recovery kinetics compared with TRα, and we evaluate the intranuclear dynamics of RTH TRα1 mutants ΤR-E403X, Ala382ProfsX7, and F397fs406X in response to increased levels of NCoR1. Investigation of the effects of overexpression of NCoR1 will provide further insight into the impact of altered binding affinity for NCoR1 on the intranuclear dynamics of RTHα mutants.

Amyloid-like Assembly Activates a Phosphatase in the Developing Drosophila Embryo.

Prion-like proteins can assume distinct conformational and physical states in the same cell. Sequence analysis suggests that prion-like proteins are prevalent in various species; however, it remains unclear what functional space they occupy in multicellular organisms. Here, we report the identification of a prion-like protein, Herzog (CG5830), through a multimodal screen in Drosophila melanogaster. Herzog functions as a membrane-associated phosphatase and controls embryonic patterning, likely being involved in TGF-β/BMP and FGF/EGF signaling pathways. Remarkably, monomeric Herzog is enzymatically inactive and becomes active upon amyloid-like assembly. The prion-like domain of Herzog is necessary for both its assembly and membrane targeting. Removal of the prion-like domain impairs activity, while restoring assembly on the membrane using a heterologous prion-like domain and membrane-targeting motif can restore phosphatase activity. This study provides an example of a prion-like domain that allows an enzyme to gain essential functionality via amyloid-like assembly to control animal development.

Evaluation of Lateral Diffusion of Lipids in Continuous Membranes between Freestanding and Supported Areas by Fluorescence Recovery after Photobleaching.

The lateral diffusion of lipids is a key factor when functionalizing artificial planar bilayer lipid membranes (BLMs). Fluorescence recovery after photobleaching (FRAP) is an established method for evaluating the fluidity of BLMs by the quantitative determination of the diffusion coefficient. However, a BLM with a uniform diffusion coefficient is usually assumed for analysis. In addition, when the BLM to be evaluated is small, the spread of a bleached circle caused by lateral diffusion during the bleaching process and the divergence of the laser used for bleaching interfere with the quantitative analysis. In this study, a numerical calculation was adopted to make it possible to analyze the continuous BLM between freestanding and supported areas, where the diffusion coefficients change depending on the presence or absence of an interaction with the substrate. A quantitative evaluation independent of such bleaching conditions as the bleaching diameter was also ensured by incorporating the spreading effect of the bleached circle in the calculation, which was employed to analyze a freestanding BLM with a diameter of only a few micrometers. By comparing calculations and experiments on FRAP recovery curves, we found that there are multiple diffusion elements and high diffusion barriers at the boundary between the freestanding and supported areas in a BLM over a SiO2/Si microwell substrate.

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.

Microfluidic immobilization and subcellular imaging of developing Caenorhabditis elegans

Caenorhabditis elegans has been an essential model organism in the fields of developmental biology, neuroscience, and aging. However, these areas have been limited by our ability to visualize and track individual C. elegans worms, especially at the subcellular scale, over the course of their lifetime. Here we present a microfluidic device to culture individual C. elegans in parallel throughout post-embryonic development. The device allows for periodic mechanical immobilization of the worm, enabling 3D imaging at subcellular precision. The immobilization is sufficient to enable fluorescence recovery after photobleaching (FRAP) measurements on organelles and other substructures within the same specific cells throughout larval development, without the use of chemical anesthetics. Using this device, we measure FRAP recovery of two nucleolar proteins in specific intestinal cells within the same worms during larval development. We show that these proteins exhibit different fluorescence recovery as the worm grows, suggesting differential protein interactions during development. We anticipate that this device will help expand the possible uses of C. elegans as a model organism, enabling its use in addressing fundamental questions at the subcellular scale.

Transition from double to single diffusive behavior with increase in polymer concentration for oppositely charged guest – host systems of green fluorescent protein diffusing inside poly-l-lysine solutions

The effect of crowding, background and probe charges and chain flexibility on probe dynamics of Green Fluorescent Protein in polyelectrolyte solutions of poly-l-lysine was investigated using Fluorescence Recovery After Photobleaching (FRAP). An interesting double diffusive behavior in FRAP recovery curve was observed at low polymer concentration resulting in two relaxation modes, which disappears with rise in polymer concentration. The fast relaxation mode attributes to diffusion of free protein molecules alone, where as slow mode is credited to polymer adsorbed protein molecules. Absence of double diffusive behavior at higher polymer concentration is argued in terms of varying host chain conformation and the only relaxation mode present is due to movement of free probes alone rather than that of adsorbed proteins. We noticed only a marginal decrease in diffusion coefficient with rise in salt concentration and the trend is reversed when variation in sample viscosity with salt is taken into account. In addition a small but systematic decrease in diffusion coefficient is seen with increase in magnitude of probe charge. Comparison of results with ideal Stoke – Einstein relation brings out the importance of polyelectrolyte effect and indicates ∼200 – fold positive deviations from predicted value for both variation in ionic strength and solution pH.

Analysis of Active Transport by Fluorescence Recovery after Photobleaching

Fluorescence recovery after photobleaching (FRAP) is a well-established experimental technique to study binding and diffusion of molecules in cells. Although a large number of analytical and numerical models have been developed to extract binding and diffusion rates from FRAP recovery curves, active transport of molecules is typically not included in the existing models that are used to estimate these rates. Here we present a validated numerical method for estimating diffusion, binding/unbinding rates, and active transport velocities using FRAP data that captures intracellular dynamics through partial differential equation models. We apply these methods to transport and localization of mRNA molecules in Xenopus laevis oocytes, where active transport processes are essential to generate developmental polarity. By providing estimates of the effective velocities and diffusion, as well as expected run times and lengths, this approach can help quantify dynamical properties of localizing and nonlocalizing RNA. Our results confirm the distinct transport dynamics in different regions of the cytoplasm, and suggest that RNA movement in both the animal and vegetal directions may influence the timescale of RNA localization in Xenopus oocytes. We also show that model initial conditions extracted from FRAP postbleach intensities prevent underestimation of diffusion, which can arise from the instantaneous bleaching assumption. The numerical and modeling approach presented here to estimate parameters using FRAP recovery data is a broadly applicable tool for systems where intracellular transport is a key molecular mechanism.

Injectable pH-responsive supramolecular hydrogels for sustained drug delivery

Event Abstract Back to Event Injectable pH-responsive supramolecular hydrogels for sustained drug delivery Maarten Bakker1*, Patricia Y. Dankers1* and Dan Jing Wu1* 1 Eindhoven University of Technology, Department of Biomedical Engineering, Netherlands Introduction: Macroscale drug delivery vehicles are applied to locally deliver various drugs. The challenges for these vehicles to optimally function are their injectability, tunability of mechanical properties and spatiotemporal control of drug release[1]. In our group, supramolecular hydrogels have been developed based on poly(ethylene glycol) PEG polymers functionalized on both ends with quadruple hydrogen bonding ureido-pyrimidinone (UPy) units (UPy-PEG). These UPy-PEG hydrogels have been demonstrated to be biocompatible after implantation in the kidney and to erode over time, presumably due to dissolution of the supramolecular polymer aggregates[2]. Uniquely, this system shows a pH-responsiveness that allows for catheter-injection of the hydrogelator as viscous solution at pH > 8.5 with a fast sol-gel transition upon contact with tissues at physiological pH. We showed the feasibility of this approach by injection of the UPy-PEG hydrogel in combination with growth factors in the infarcted region of a pig heart[3],[4]. Theoretically, any drug or bioactive compound can be dissolved in the hydrogelator solution before injection. More recently we expanded our research to incorporation of RNAi therapeutics and chemotherapeutic compounds (Fig 1). A convenience of the UPy system is that new features can be added by supramolecular incorporation of functionalities. One example is the incorporation of amines to induce electrostatic interactions with RNAi molecules to prevent free diffusion from the hydrogel and to obtain a sustained release. Furthermore, we propose enhanced transfection efficiencies because of the presence of charges. Fig. 1| PEG polymers functionalized with UPy units form supramolecular injectable hydrogels that can serve as delivery depots after mixing with desired drugs or RNAi therapeutics. Materials and Methods: UPy-hydrogelator powder is dissolved in PBS pH 11.7 by heating to 70 °C for 1 hour. After cooling to RT the mixture reaches a pH of 9.0. A drug or biomolecule is subsequently added and gelation is triggered by adding HCl to decrease the pH to 7.4. Release studies are performed in Transwell 8 µm inserts at 37 ˚C with PBS as release medium. Release of molecules and degradation of hydrogel is quantified via fluorescence spectroscopy and UV-Vis spectroscopy. Material properties of the viscous solution (shear viscosity) and of the hydrogel (storage and loss moduli) are recorded on an Anton-Paar Rheometer. Fluorescence recovery after photobleaching (FRAP) experiments were performed on a Leica confocal microscopy system. Results: Drugs can be conveniently entrapped within the hydrogel by employing the pH switch method. Rheology measurements show that at pH 9.0 the solution has a shear viscosity below 1 Pa.s which allows for injection via a catheter. When brought to neutral pH, the material shows a solid-like response observed by a storage modulus G′ which is larger than the loss modulus G″. In vitro release studies show that the hydrogel slowly degrades and that RNAi therapeutics and chemotherapeutics are released from the hydrogel. With fluorescent model molecules a correlation was observed between FRAP recovery time and release from the hydrogel. Conclusion: UPy supramolecular hydrogels hold promise for in vivo application as sustained drug delivery depot. Unlike many other materials, it shows catheter compatibility, which allows minimal invasive treatments. New features can be added by supramolecular incorporation of functionalities, for example to slow down drug release.

Simultaneous FRAP, FLIM and FAIM for measurements of protein mobility and interaction in living cells.

We present a novel integrated multimodal fluorescence microscopy technique for simultaneous fluorescence recovery after photobleaching (FRAP), fluorescence lifetime imaging (FLIM) and fluorescence anisotropy imaging (FAIM). This approach captures a series of polarization-resolved fluorescence lifetime images during a FRAP recovery, maximizing the information available from a limited photon budget. We have applied this method to analyse the behaviour of GFP-labelled coxsackievirus and adenovirus receptor (CAR) in living human epithelial cells. Our data reveal that CAR exists in oligomeric states throughout the cell, and that these complexes occur in conjunction with high immobile fractions of the receptor at cell-cell junctions. These findings shed light on previously unknown molecular associations between CAR receptors in intact cells and demonstrate the power of combined FRAP, FLIM and FAIM microscopy as a robust method to analyse complex multi-component dynamics in living cells.

Dynamic actin filaments control the mechanical behavior of the human red blood cell membrane.

Short, uniform-length actin filaments function as structural nodes in the spectrin-actin membrane skeleton to optimize the biomechanical properties of red blood cells (RBCs). Despite the widespread assumption that RBC actin filaments are not dynamic (i.e., do not exchange subunits with G-actin in the cytosol), this assumption has never been rigorously tested. Here we show that a subpopulation of human RBC actin filaments is indeed dynamic, based on rhodamine-actin incorporation into filaments in resealed ghosts and fluorescence recovery after photobleaching (FRAP) analysis of actin filament mobility in intact RBCs (~25-30% of total filaments). Cytochalasin-D inhibition of barbed-end exchange reduces rhodamine-actin incorporation and partially attenuates FRAP recovery, indicating functional interaction between actin subunit turnover at the single-filament level and mobility at the membrane-skeleton level. Moreover, perturbation of RBC actin filament assembly/disassembly with latrunculin-A or jasplakinolide induces an approximately twofold increase or ~60% decrease, respectively, in soluble actin, resulting in altered membrane deformability, as determined by alterations in RBC transit time in a microfluidic channel assay, as well as by abnormalities in spontaneous membrane oscillations (flickering). These experiments identify a heretofore-unrecognized but functionally important subpopulation of RBC actin filaments, whose properties and architecture directly control the biomechanical properties of the RBC membrane.

On Data Space Selection and Data Processing for Parameter Identification in a Reaction-Diffusion Model Based on FRAP Experiments

Fluorescence recovery after photobleaching (FRAP) is a widely used measurement technique to determine the mobility of fluorescent molecules within living cells. While the experimental setup and protocol for FRAP experiments are usually fixed, data (pre)processing represents an important issue. The aim of this paper is twofold. First, we formulate and solve the problem ofrelevantFRAP data selection. The theoretical findings are illustrated by the comparison of the results of parameter identification when the full data set was used and the case when theirrelevant data set(data with negligible impact on the confidence interval of the estimated parameters) was removed from the data space. Second, we analyze and compare two approaches of FRAP data processing. Our proposition, surprisingly for the FRAP community, claims that the data set represented by the FRAP recovery curves in form of a time series (integrated data approachcommonly used by the FRAP community) leads to a larger confidence interval compared to thefull(spatiotemporal)data approach.

Inference of protein kinetics by stochastic modeling and simulation of fluorescence recovery after photobleaching experiments.

Fluorescence recovery after photobleaching (FRAP) is a functional live cell imaging technique that permits the exploration of protein dynamics in living cells. To extract kinetic parameters from FRAP data, a number of analytical models have been developed. Simplifications are inherent in these models, which may lead to inexhaustive or inaccurate exploitation of the experimental data. An appealing alternative is offered by the simulation of biological processes in realistic environments at a particle level. However, inference of kinetic parameters using simulation-based models is still limited. We introduce and demonstrate a new method for the inference of kinetic parameter values from FRAP data. A small number of in silico FRAP experiments is used to construct a mapping from FRAP recovery curves to the parameters of the underlying protein kinetics. Parameter estimates from experimental data can then be computed by applying the mapping to the observed recovery curves. A bootstrap process is used to investigate identifiability of the physical parameters and determine confidence regions for their estimates. Our method circumvents the computational burden of seeking the best-fitting parameters via iterative simulation. After validation on synthetic data, the method is applied to the analysis of the nuclear proteins Cdt1, PCNA and GFPnls. Parameter estimation results from several experimental samples are in accordance with previous findings, but also allow us to discuss identifiability issues as well as cell-to-cell variability of the protein kinetics. All methods were implemented in MATLAB R2011b. Monte Carlo simulations were run on the HPC cluster Brutus of ETH Zurich. lygeros@control.ee.ethz.ch or lygerou@med.upatras.gr Supplementary data are available at Bioinformatics online.

Assessing cellular efficacy of bromodomain inhibitors using fluorescence recovery after photobleaching.

BackgroundAcetylation of lysine residues in histone tails plays an important role in the regulation of gene transcription. Bromdomains are the readers of acetylated histone marks, and, consequently, bromodomain-containing proteins have a variety of chromatin-related functions. Moreover, they are increasingly being recognised as important mediators of a wide range of diseases. The first potent and selective bromodomain inhibitors are beginning to be described, but the diverse or unknown functions of bromodomain-containing proteins present challenges to systematically demonstrating cellular efficacy and selectivity for these inhibitors. Here we assess the viability of fluorescence recovery after photobleaching (FRAP) assays as a target agnostic method for the direct visualisation of an on-target effect of bromodomain inhibitors in living cells.ResultsMutation of a conserved asparagine crucial for binding to acetylated lysines in the bromodomains of BRD3, BRD4 and TRIM24 all resulted in reduction of FRAP recovery times, indicating loss of or significantly reduced binding to acetylated chromatin, as did the addition of known inhibitors. Significant differences between wild type and bromodomain mutants for ATAD2, BAZ2A, BRD1, BRD7, GCN5L2, SMARCA2 and ZMYND11 required the addition of the histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA) to amplify the binding contribution of the bromodomain. Under these conditions, known inhibitors decreased FRAP recovery times back to mutant control levels. Mutation of the bromodomain did not alter FRAP recovery times for full-length CREBBP, even in the presence of SAHA, indicating that other domains are primarily responsible for anchoring CREBBP to chromatin. However, FRAP assays with multimerised CREBBP bromodomains resulted in a good assay to assess the efficacy of bromodomain inhibitors to this target. The bromodomain and extraterminal protein inhibitor PFI-1 was inactive against other bromodomain targets, demonstrating the specificity of the method.ConclusionsViable FRAP assays were established for 11 representative bromodomain-containing proteins that broadly cover the bromodomain phylogenetic tree. Addition of SAHA can overcome weak binding to chromatin, and the use of tandem bromodomain constructs can eliminate masking effects of other chromatin binding domains. Together, these results demonstrate that FRAP assays offer a potentially pan-bromodomain method for generating cell-based assays, allowing the testing of compounds with respect to cell permeability, on-target efficacy and selectivity.

PRC1 components exhibit different binding kinetics in Polycomb bodies

Polycomb group (PcG) proteins keep the memory of cell identity by maintaining the repression of numerous target genes. They accumulate into nuclear foci called Polycomb bodies, which function in Drosophila cells as silencing compartments where PcG target genes convene. PcG proteins also exert their activities elsewhere in the nucleoplasm. In mammalian cells, the dynamic organisation and function of Polycomb bodies remain to be determined. Fluorescently tagged PcG proteins CBXs and their partners BMI1 and RING1 form foci of different sizes and intensities in human U2OS cells. Fluorescence recovery after photobleaching (FRAP) analysis reveals that PcG dynamics outside foci is governed by diffusion as complexes and transient binding. In sharp contrast, recovery curves inside foci are substantially slower and exhibit large variability. The slow binding component amplitudes correlate with the intensities and sizes of these foci, suggesting that foci contained varying numbers of binding sites. CBX4-green fluorescent protein (GFP) foci were more stable than CBX8-GFP foci; yet the presence of CBX4 or CBX8-GFP in the same focus had a minor impact on BMI1 and RING1 recovery kinetics. We propose that FRAP recovery curves provide information about PcG binding to their target genes outside foci and about PcG spreading onto chromatin inside foci.

Responses to Cell Loss Become Restricted as the Supporting Cells in Mammalian Vestibular Organs Grow Thick Junctional Actin Bands That Develop High Stability

Sensory hair cell (HC) loss is a major cause of permanent hearing and balance impairments for humans and other mammals. Yet, fish, amphibians, reptiles, and birds readily replace HCs and recover from such sensory deficits. It is unknown what prevents replacement in mammals, but cell replacement capacity declines contemporaneously with massive postnatal thickening of F-actin bands at the junctions between vestibular supporting cells (SCs). In non-mammals, SCs can give rise to regenerated HCs, and the bands remain thin even in adults. Here we investigated the stability of the F-actin bands between SCs in ears from chickens and mice and Madin-Darby canine kidney cells. Pharmacological experiments and fluorescence recovery after photobleaching (FRAP) of SC junctions in utricles from mice that express a γ-actin-GFP fusion protein showed that the thickening F-actin bands develop increased resistance to depolymerization and exceptional stability that parallels a sharp decline in the cell replacement capacity of the maturing mammalian ear. The FRAP recovery rate and the mobile fraction of γ-actin-GFP both decreased as the bands thickened with age and became highly stabilized. In utricles from neonatal mice, time-lapse recordings in the vicinity of dying HCs showed that numerous SCs change shape and organize multicellular actin purse strings that reseal the epithelium. In contrast, adult SCs appeared resistant to deformation, with resealing responses limited to just a few neighboring SCs that did not form purse strings. The exceptional stability of the uniquely thick F-actin bands at the junctions of mature SCs may play an important role in restricting dynamic repair responses in mammalian vestibular epithelia.

Remodeling of Clots without Proteolytic Digestion

Abstract 1184Fibrin polymerization is a necessary part of hemostasis but thrombi can obstruct blood vessels and cause heart attacks and strokes, so the possibility of reversing clot formation has significant clinical implications. It is generally assumed that fibrin polymerization is an irreversible process and that clots and thrombi are stable structures until proteolytic digestion, but this supposition has not been critically tested. Using the technique of fluorescence recovery after photobleaching (FRAP) of individual fluorescently labeled fibrin fibers in a clot, we demonstrate here for the first time that there is turnover of fibrin in an uncrosslinked clot. These conditions occur in the vasculature during early stages of clotting or thrombosis before extensive Factor XIIIa-induced crosslinking. Increase of fluorescence in the bleached area was observed as soon as 1 second after photobleaching and the intensity increased rapidly. The association/dissociation of fibrin was characterized quantitatively, with the mobile fraction representing an average of 14.1% ± 5.1% of the total fibrin. Analyses of recovery curves showed that there was a linear relationship between both the percentage of fluorescence recovery and the rate of recovery and the surface to volume ratio of the fibers. Thus, it appears that fibrin monomers or oligomers located on or near the surface of the fibers are more likely to dissociate and re-associate. The mechanism of fibrin turnover was further investigated by using the peptide GPRP, mimicking the knobs ‘A' involved in polymerization. Surprisingly, only 0.1 mM of GPRP was necessary to dissolve completely a preformed fibrin clot, and perfusion of 0.01 mM of GPRP through a clot decreased the mobile fraction of fibrin in FRAP experiments to 6.5% ± 1.7%. The kinetic parameters of fibrin turnover characterized from FRAP recovery curves revealed that the off rate was not affected by the GPRP concentration, but the equilibrium concentration of the bound complex decreased with increasing peptide concentration, likely because the free GPRP competes with fibrin knobs ‘A' for holes ‘a.' As a result of the enhanced dissociation induced by this peptide, striking rearrangements in clot structure were visualized by confocal light microscopy and scanning electron microscopy. Transmission electron microscopy of structures released from the clot revealed both monomers and larger aggregates. The implications of this research are that clots and thrombi appear to be much more dynamic structures than has been previously believed. The observed modulation of clot structure in vitro suggests that in vivo clots and thrombi may be dissolved or remodeled as a result of changing the environmental conditions surrounding them. These findings may also be relevant to embolization, which could in part be a consequence of the reversibility of polymerization. Furthermore, such modulation of clot structure could be a target for potential therapeutic intervention. Disclosures:No relevant conflicts of interest to declare.