A fluorescence recovery after photobleaching protocol to measure surface diffusion of DAGLα in primary cultured cortical mouse neurons.
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ABSTRACT:
SUBMITTER: Yoon S
PROVIDER: S-EPMC8783203 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The multiphoton fluorescence recovery after photobleaching (MP-FRAP) technique has been developed to measure the three-dimensional (3D) solute diffusion within biological systems. However, current 3D MP-FRAP models are based on isotropic diffusion and spatial domain analysis. The 3D anisotropic diffusion and frequency domain analysis for MP-FRAP measurements are rarely studied. In this study, a new technique is demonstrated for the quantitative and non-destructive determination of 3D anisotropic solute diffusion tensors within biological fibrosis tissues by multiphoton photobleaching and spatial Fourier analysis (SFA). Compared to the spatial domain analysis based MP-FRAP techniques, this SFA-based method has the capability for determining the 3D anisotropic diffusion tensors as well as the flexibility for satisfying initial and boundary conditions. First, a close-form solution of the 3D anisotropic diffusion equation is derived by solely using SFA. Next, this new method is validated by computer-simulated MP-FRAP experiments with pre-defined 3D anisotropic diffusion tensors as well as experimental diffusion measurements of FITC-Dextran (FD) molecules in aqueous glycerol solutions. Finally, this MP-FRAP technique is applied to the measurement of 3D anisotropic diffusion tensors of FD molecules within porcine tendon tissues. This study provides a new tool for complete determination of 3D anisotropic solute diffusion tensor in biological tissues.
| S-EPMC3943478 | biostudies-literature
Project description:Fluorescence recovery after photobleaching (FRAP) enables characterization of quantitative dynamic properties such as diffusion coefficients of fluorescent molecules in living cells by analyzing the recovery of fluorescence intensity after photobleaching in a specific cellular compartment or area. To quantitatively determine high intracellular diffusion coefficients, a suitable optical system as well as an appropriate model for fast diffusion analysis is necessary. Here, we propose a procedure to quantify the diffusion coefficient of rapidly-diffusing fluorescent molecules that makes use of an epi-fluorescence microscope with a photobleaching laser in combination with established models for diffusion analysis. Analysis for the diffusion coefficients of tandemly oligomerized green flurescent proteins (GFPs) in living cells when changing the photobleaching times showed that photobleaching with shorter times than the diffusion speed indicated not the only way to obtain appropriate diffusion coefficients of fast-moving molecules. Our results also showed that the apparent spreading of the effective radius of the photobleached area works as a correction factor for determining appropriate diffusion coefficients of fast-moving molecules like monomeric GFPs. Our procedure provides a useful approach for quantitative measurement of diffusion coefficients in living cells. This procedure is relevant for characterizing dynamic molecular interactions, especially of fast-moving molecules, and is relevant for studies in many biological fields.
| S-EPMC5812315 | biostudies-literature
Project description:Fluorescence recovery after photobleaching (FRAP) and fluorescence correlation spectroscopy (FCS) are the two most direct methods to measure the diffusion of molecules in intact living cells. Ideally, these methods should produce similar results for an identical system. We have used these methods to monitor the diffusion of two G-protein-coupled receptors and their associated proteins in the plasma membranes of cells that do not or do contain invaginated protein domains called caveolae. FRAP studies show that caveolae domains increase the immobile fraction of receptors without significantly changing their mobility. On the other hand, FCS studies show an unexpected increase the mobility of caveolae-associated proteins. Our data suggest that the geometry of caveolae domains gives rise to a confined diffusion of its attached proteins, resulting in an apparent increase in mobility.
| S-EPMC3770895 | biostudies-literature
Project description:Fluorescence recovery after photobleaching (FRAP) is a microscopy technique for measuring the kinetics of fluorescently labeled molecules and can be applied both in vitro and in vivo for two- and three-dimensional systems. This introduction discusses the three basic FRAP methods: traditional FRAP, multiphoton FRAP (MPFRAP), and FRAP with spatial Fourier analysis (SFA-FRAP). Each discussion is accompanied by a description of the mathematical analysis appropriate for situations in which the recovery kinetics is dictated by free diffusion. In some experiments, the recovery kinetics is dictated by the boundary conditions of the system, and FRAP is then used to quantify the connectivity of various compartments. Because the appropriate mathematical analysis is independent of the bleaching method, the analysis of compartmental connectivity is discussed last, in a separate section.
| S-EPMC4399237 | biostudies-other
Project description: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.
| S-EPMC5406284 | biostudies-literature
Project description:Fluorescence recovery after photobleaching (FRAP) is now widely used to investigate binding interactions in live cells. Although various idealized solutions have been identified for the reaction-diffusion equations that govern FRAP, there has been no comprehensive analysis or systematic approach to serve as a guide for extracting binding information from an arbitrary FRAP curve. Here we present a complete solution to the FRAP reaction-diffusion equations for either single or multiple independent binding interactions, and then relate our solution to the various idealized cases. This yields a coherent approach to extract binding information from FRAP data which we have applied to the question of transcription factor mobility in the nucleus. We show that within the nucleus, the glucocorticoid receptor is transiently bound to a single state, with each molecule binding on average 65 sites per second. This rapid sampling is likely to be important in finding a specific promoter target sequence. Further we show that this predominant binding state is not the nuclear matrix, as some studies have suggested. We illustrate how our analysis provides several self-consistency checks on a FRAP fit. We also define constraints on what can be estimated from FRAP data, show that diffusion should play a key role in many FRAP recoveries, and provide tools to test its contribution. Overall our approach establishes a more general framework to assess the role of diffusion, the number of binding states, and the binding constants underlying a FRAP recovery.
| S-EPMC1304253 | biostudies-literature
Project description:The determination of diffusion coefficients from fluorescence recovery data is often complicated by geometric constraints imposed by the complex shapes of intracellular compartments. To address this issue, diffusion of proteins in the lumen of the endoplasmic reticulum (ER) is studied using cell biological and computational methods. Fluorescence recovery after photobleaching (FRAP) experiments are performed in tissue culture cells expressing GFP-KDEL, a soluble, fluorescent protein, in the ER lumen. The three-dimensional (3D) shape of the ER is determined by confocal microscopy and computationally reconstructed. Within these ER geometries diffusion of solutes is simulated using the method of particle strength exchange. The simulations are compared to experimental FRAP curves of GFP-KDEL in the same ER region. Comparisons of simulations in the 3D ER shapes to simulations in open 3D space show that the constraints imposed by the spatial confinement result in two- to fourfold underestimation of the molecular diffusion constant in the ER if the geometry is not taken into account. Using the same molecular diffusion constant in different simulations, the observed speed of fluorescence recovery varies by a factor of 2.5, depending on the particular ER geometry and the location of the bleached area. Organelle shape considerably influences diffusive transport and must be taken into account when relating experimental photobleaching data to molecular diffusion coefficients. This novel methodology combines experimental FRAP curves with high accuracy computer simulations of diffusion in the same ER geometry to determine the molecular diffusion constant of the solute in the particular ER lumen.
| S-EPMC1366654 | biostudies-literature
Project description: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.
| S-EPMC4115480 | biostudies-literature
Project description: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.
| S-EPMC6818185 | biostudies-literature
Project description:Diffusion in the extracellular space (ECS) is crucial for normal central nervous system physiology. The determinants of ECS diffusion include viscous interactions with extracellular matrix/plasma membranes ("viscosity") and ECS geometry ("tortuosity"). To resolve viscosity versus tortuosity effects, we measured direction-dependent (anisotropic) diffusion in ECS in mouse spinal cord by photobleaching using an elliptical spot produced by a cylindrical lens in the excitation path. Anisotropic diffusion slowed fluorescence recovery when the long axis of the ellipse was parallel versus perpendicular to the direction of faster diffusion. A mathematical model was constructed to deduce diffusion coefficients (D(x), D(y)) from fluorescence recovery measured for parallel and perpendicular orientations of the long axis of the ellipse. Elliptical spot photobleaching was validated by photobleaching aqueous-phase fluorophores on a diffraction grating, where diffusion is one-dimensional. Measurement of the diffusion of 70 kDa FITC-dextran in spinal cord in living mice indicated that viscosity slows diffusion by approximately 1.8-fold compared with its diffusion in solution. ECS geometry hinders diffusion across (but not along) axonal fibers in spinal cord further by approximately fivefold. In cerebral cortex, however, approximately 50% of the hindrance to ECS diffusion comes from viscosity and approximately 50% from tortuosity. We suggest that the extracellular matrix might have evolved to facilitate rather than hinder diffusion even for large molecules.
| S-EPMC1366858 | biostudies-literature