Investigating Liquid-Liquid Phase Separation in Virus-Generated Inclusion Bodies Using Fluorescence Recovery After Photobleaching of Fluorescently Labeled Host Proteins.

Many negative-sense single-stranded RNA viruses within the order Mononegavirales harm humans. A common feature shared among cells infected by these viruses is the formation of subcellular membraneless structures called biomolecular condensates, also known as inclusion bodies (IBs), that form through a process called liquid-liquid phase separation (LLPS). Like many other membraneless organelles, viral IBs enrich a specific subset of viral and host proteins involved in the formation of viral particles. Elucidation of the properties and regulation of these IBs as they mature throughout the viral replication process are important for our understanding of viral replication, which may also lead to the development of alternative antiviral treatments. The protocol outlined in this chapter aims to characterize the intrinsic properties of LLPS within the measles virus (MeV, a member of Mononegavirales) IBs by using an imaging approach that fluorescently tags an IB-associated host protein. This method uses common laboratory techniques and is generalizable to any host factors as well as other viral systems.

Expanding the applicability of multiphoton fluorescence recovery after photobleaching by incorporating shear stress in laminar flow.

Significance: Multi-photon fluorescence recovery after photobleaching (MPFRAP) is a nonlinear microscopy technique used to measure the diffusion coefficient of fluorescently tagged molecules in solution. Previous MPFRAP fitting models calculate the diffusion coefficient in systems with diffusion or diffusion in laminar flow. Aim: We propose an MPFRAP fitting model that accounts for shear stress in laminar flow, making it a more applicable technique for in vitro and in vivo studies involving diffusion. Approach: Fluorescence recovery curves are generated using high-throughput molecular dynamics simulations and then fit to all three models (diffusion, diffusion and flow, and diffusion and shear flow) to define the limits within which accurate diffusion coefficients are produced. Diffusion is simulated as a random walk with a variable horizontal bias to account for shear flow. Results: Contour maps of the accuracy of the fitted diffusion coefficient as a function of scaled velocity and scaled shear rate show the parameter space within which each model produces accurate diffusion coefficients; the shear-flow model covers a larger area than the previous models. Conclusion: The shear-flow model allows MPFRAP to be a viable optical tool for studying more biophysical systems than previous models.

Beyond analytic solution: Analysis of FRAP experiments by spatial simulation of the forward problem.

Fluorescence redistribution after photobleaching is a commonly used method to understand the dynamic behavior of molecules within cells. Analytic solutions have been developed for specific, well-defined models of dynamic behavior in idealized geometries, but these solutions are inaccurate in complex geometries or when complex binding and diffusion behaviors exist. We demonstrate the use of numerical reaction-diffusion simulations using the Virtual Cell software platform to model fluorescence redistribution after photobleaching experiments. Multiple simulations employing parameter scans and varying bleaching locations and sizes can help to bracket diffusion coefficients and kinetic rate constants in complex image-based geometries. This approach is applied to problems in membrane surface diffusion as well as diffusion and binding in cytosolic volumes in complex cell geometries. In addition, we model diffusion and binding within phase-separated biomolecular condensates (liquid droplets). These are modeled as spherical low-affinity binding domains that also define a high viscosity medium for exchange of the free fluorescently labeled ligand with the external cytosol.

What's past is prologue: FRAP keeps delivering 50 years later.

Fluorescence recovery after photobleaching (FRAP) has emerged as one of the most widely utilized techniques to quantify binding and diffusion kinetics of biomolecules in biophysics. Since its inception in the mid-1970s, FRAP has been used to address an enormous array of questions including the characteristic features of lipid rafts, how cells regulate the viscosity of their cytoplasm, and the dynamics of biomolecules inside condensates formed by liquid-liquid phase separation. In this perspective, I briefly summarize the history of the field and discuss why FRAP has proven to be so incredibly versatile and popular. Next, I provide an overview of the extensive body of knowledge that has emerged on best practices for quantitative FRAP data analysis, followed by some recent examples of biological lessons learned using this powerful approach. Finally, I touch on new directions and opportunities for biophysicists to contribute to the continued development of this still-relevant research tool.

CM-FRAP-Continuum Mechanics-Based Fluorescence Recovery After Photobleaching.

Fluorescence recovery after photobleaching (FRAP) is widely used to evaluate intracellular molecular turnover or repeated translocation of molecules using confocal laser scanning microscopy. While numerous models have been developed for the analysis of FRAP responses, in which chemical interactions and/or fast diffusion processes are involved, it is inherently difficult to evaluate the long-term behavior of molecular turnover because of the presence of intracellular flow and microscopic deformation of bleached regions. To overcome these difficulties, we have developed a novel continuum mechanics-based FRAP (CM-FRAP) approach that enables simultaneous evaluation of long-term molecular turnover and intracellular flow/deformation. Here we demonstrate the utility of CM-FRAP by using actin molecules associated with stress fibers in rat aortic smooth muscle cells with clarification of the experimental setup and data analysis. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Plasmid construction and sample preparation Basic Protocol 2: How to perform FRAP experiments Basic Protocol 3: Data analysis based on CM-FRAP.

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 10 min 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.

Long-term molecular turnover of actin stress fibers revealed by advection-reaction analysis in fluorescence recovery after photobleaching.

Fluorescence recovery after photobleaching (FRAP) is a versatile technique to evaluate the intracellular molecular exchange called turnover. Mechanochemical models of FRAP typically consider the molecular diffusion and chemical reaction that simultaneously occur on a time scale of seconds to minutes. Particularly for long-term measurements, however, a mechanical advection effect can no longer be ignored, which transports the proteins in specific directions within the cells and accordingly shifts the spatial distribution of the local chemical equilibrium. Nevertheless, existing FRAP models have not considered the spatial shift, and as such, the turnover rate is often analyzed without considering the spatiotemporally updated chemical equilibrium. Here we develop a new FRAP model aimed at long-term measurements to quantitatively determine the two distinct effects of the advection and chemical reaction, i.e., the different major sources of the change in fluorescence intensity. To validate this approach, we carried out FRAP experiments on actin in stress fibers over a time period of more than 900 s, and the advection rate was shown to be comparable in magnitude to the chemical dissociation rate. We further found that the actin-myosin interaction and actin polymerization differently affect the advection and chemical dissociation. Our results suggest that the distinction between the two effects is indispensable to extract the intrinsic chemical properties of the actin cytoskeleton from the observations of complicated turnover in cells.

Monitoring cell membrane recycling dynamics of proteins using whole-cell fluorescence recovery after photobleaching of pH-sensitive genetic tags.

Population behavior of signaling molecules on the cell surface is key to their adaptive function. Live imaging of proteins tagged with fluorescent molecules has been an essential tool in understanding this behavior. Typically, genetic or chemical tags are used to target molecules present throughout the cell, whereas antibody-based tags label the externally exposed molecular domains only. Both approaches could potentially overlook the intricate process of in-out membrane recycling in which target molecules appear or disappear on the cell surface. This limitation is overcome by using a pH-sensitive fluorescent tag, such as Super-Ecliptic pHluorin (SEP), because its emission depends on whether it resides inside or outside the cell. Here we focus on the main glial glutamate transporter GLT1 and describe a genetic design that equips GLT1 molecules with SEP without interfering with the transporter's main function. Expressing GLT1-SEP in astroglia in cultures or in hippocampal slices enables monitoring of the real-time dynamics of the cell-surface and cytosolic fractions of the transporter in living cells. Whole-cell fluorescence recovery after photobleaching and quantitative image-kinetic analysis of the resulting time-lapse images enables assessment of the rate of GLT1-SEP recycling on the cell surface, a fundamental trafficking parameter unattainable previously. The present protocol takes 15-20 d to set up cell preparations, and 2-3 d to carry out live cell experiments and data analyses. The protocol can be adapted to study different membrane molecules of interest, particularly those proteins whose lifetime on the cell surface is critical to their adaptive function.

Monitoring condensate dynamics in S. cerevisiae using fluorescence recovery after photobleaching.

This protocol describes the use of fluorescence recovery after photobleaching (FRAP) to investigate the dynamics of Matrin-3 (MATR3) condensates in live budding yeast. We detail how to generate yeast strains containing MATR3 with an enhanced green fluorescent protein (eGFP) tag and induce MATR3-eGFP expression. We provide steps to prepare slides of immobilized yeast cells and perform FRAP imaging and data analysis. This protocol can be broadly applied to study condensate dynamics of a range of proteins in different model systems. For complete details on the use and execution of this protocol, please refer to Sprunger et al. (2022).

Dynamic Mode Decomposition of Fluorescence Loss in Photobleaching Microscopy Data for Model-Free Analysis of Protein Transport and Aggregation in Living Cells.

The phase separation and aggregation of proteins are hallmarks of many neurodegenerative diseases. These processes can be studied in living cells using fluorescent protein constructs and quantitative live-cell imaging techniques, such as fluorescence recovery after photobleaching (FRAP) or the related fluorescence loss in photobleaching (FLIP). While the acquisition of FLIP images is straightforward on most commercial confocal microscope systems, the analysis and computational modeling of such data is challenging. Here, a novel model-free method is presented, which resolves complex spatiotemporal fluorescence-loss kinetics based on dynamic-mode decomposition (DMD) of FLIP live-cell image sequences. It is shown that the DMD of synthetic and experimental FLIP image series (DMD-FLIP) allows for the unequivocal discrimination of subcellular compartments, such as nuclei, cytoplasm, and protein condensates based on their differing transport and therefore fluorescence loss kinetics. By decomposing fluorescence-loss kinetics into distinct dynamic modes, DMD-FLIP will enable researchers to study protein dynamics at each time scale individually. Furthermore, it is shown that DMD-FLIP is very efficient in denoising confocal time series data. Thus, DMD-FLIP is an easy-to-use method for the model-free detection of barriers to protein diffusion, of phase-separated protein assemblies, and of insoluble protein aggregates. It should, therefore, find wide application in the analysis of protein transport and aggregation, in particular in relation to neurodegenerative diseases and the formation of protein condensates in living cells.

Quantifying Molecular Dynamics within Complex Cellular Morphologies using LLSM-FRAP.

Quantifying molecular dynamics within the context of complex cellular morphologies is essential toward understanding the inner workings and function of cells. Fluorescence recovery after photobleaching (FRAP) is one of the most broadly applied techniques to measure the reaction diffusion dynamics of molecules in living cells. FRAP measurements typically restrict themselves to single-plane image acquisition within a subcellular-sized region of interest due to the limited temporal resolution and undesirable photobleaching induced by 3D fluorescence confocal or widefield microscopy. Here, an experimental and computational pipeline combining lattice light sheet microscopy, FRAP, and numerical simulations, offering rapid and minimally invasive quantification of molecular dynamics with respect to 3D cell morphology is presented. Having the opportunity to accurately measure and interpret the dynamics of molecules in 3D with respect to cell morphology has the potential to reveal unprecedented insights into the function of 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.

Fluorescence Recovery after Photobleaching in Colloidal Science: Introduction and Application.

FRAP (fluorescence recovery after photo bleaching) is a method for determining diffusion in material science. In industrial applications such as medications, foods, Medtech, hygiene, and textiles, the diffusion process has a substantial influence on the overall qualities of goods. All these complex and heterogeneous systems have diffusion-based processes at the local level. FRAP is a fluorescence-based approach for detecting diffusion; in this method, a high-intensity laser is made for a brief period and then applied to the samples, bleaching the fluorescent chemical inside the region, which is subsequently filled up by natural diffusion. This brief Review will focus on the existing research on employing FRAP to measure colloidal system heterogeneity and explore diffusion into complicated structures. This description of FRAP will be followed by a discussion of how FRAP is intended to be used in colloidal science. When constructing the current Review, the most recent publications were reviewed for this assessment. Because of the large number of FRAP articles in colloidal research, there is currently a dearth of knowledge regarding the growth of FRAP's significance to colloidal science. Colloids make up only 2% of FRAP papers, according to ISI Web of Knowledge.

Determining vitreous viscosity using fluorescence recovery after photobleaching.

PURPOSE: Vitreous humor is a complex biofluid whose composition determines its structure and function. Vitreous viscosity will affect the delivery, distribution, and half-life of intraocular drugs, and key physiological molecules. The central pig vitreous is thought to closely match human vitreous viscosity. Diffusion is inversely related to viscosity, and diffusion is of fundamental importance for all biochemical reactions. Fluorescence Recovery After Photobleaching (FRAP) may provide a novel means of measuring intravitreal diffusion that could be applied to drugs and physiological macromolecules. It would also provide information about vitreous viscosity, which is relevant to drug elimination, and delivery. METHODS: Vitreous viscosity and intravitreal macromolecular diffusion of fluorescently labelled macromolecules were investigated in porcine eyes using fluorescence recovery after photobleaching (FRAP). Fluorescein isothiocyanate conjugated (FITC) dextrans and ficolls of varying molecular weights (MWs), and FITC-bovine serum albumin (BSA) were employed using FRAP bleach areas of different diameters. RESULTS: The mean (±standard deviation) viscosity of porcine vitreous using dextran, ficoll and BSA were 3.54 ± 1.40, 2.86 ± 1.13 and 4.54 ± 0.13 cP respectively, with an average of 3.65 ± 0.60 cP. CONCLUSIONS: FRAP is a feasible and practical optical method to quantify the diffusion of macromolecules through vitreous.

Use of Fluorescence Recovery After Photobleaching (FRAP) to Measure In Vivo Dynamics of Cell Junction-Associated Polarity Proteins.

Here, we present a detailed protocol for fluorescence recovery after photobleaching (FRAP) to measure the dynamics of junctional populations of proteins in living tissue. Specifically, we describe how to perform FRAP in Drosophila pupal wings on fluorescently tagged core planar polarity proteins, which exhibit relatively slow junctional turnover. We provide a step-by-step practical guide to performing FRAP, and list a series of controls and optimizations to do before conducting a FRAP experiment. Finally, we describe how to present the FRAP data for publication.

A fluorescence recovery after photobleaching protocol to measure surface diffusion of DAGLα in primary cultured cortical mouse neurons.

This protocol describes using fluorescence recovery after photobleaching (FRAP) of a superecliptic pHluorin (SEP)-diacylglycerol lipase α (DAGLα) to measure membrane-bound DAGLα mobility in dendritic shafts of primary cultured cortical mouse neurons. This could serve as an excellent tool to analyze endocannabinoid-mediated synaptic plasticity. We have used this protocol to show that DAGLα surface dynamics play an integral role in regulating the dendritic spine. We also detail how we test the qualities of generated SEP-DAGLα in HEK293T cells by FRAP assay. For complete details on the use and execution of this profile, please refer to Yoon et al. (2021a).

Deafness mutation in the MYO3A motor domain impairs actin protrusion elongation mechanism.

Class III myosins are actin-based motors proposed to transport cargo to the distal tips of stereocilia in the inner ear hair cells and/or to participate in stereocilia length regulation, which is especially important during development. Mutations in the MYO3A gene are associated with delayed onset deafness. A previous study demonstrated that L697W, a dominant deafness mutation, disrupts MYO3A ATPase and motor properties but does not impair its ability to localize to the tips of actin protrusions. In the current study, we characterized the transient kinetic mechanism of the L697W motor ATPase cycle. Our kinetic analysis demonstrates that the mutation slows the ADP release and ATP hydrolysis steps, which results in a slight reduction in the duty ratio and slows detachment kinetics. Fluorescence recovery after photobleaching (FRAP) of filopodia tip localized L697W and WT MYO3A in COS-7 cells revealed that the mutant does not alter turnover or average intensity at the actin protrusion tips. We demonstrate that the mutation slows filopodia extension velocity in COS-7 cells which correlates with its twofold slower in vitro actin gliding velocity. Overall, this work allowed us to propose a model for how the motor properties of MYO3A are crucial for facilitating actin protrusion length regulation.

Antioxidant activity of Haloxylon scoparium alkaloid extracts from Figuig region (southeastern of Morocco)

Abstract The aim of this paper is to study the chemical composition of alkaloids present in Haloxylon scoparium Pomel extracts and to evaluate their antioxidant capacity. The alkaloids were isolated from two parts of Haloxylon scoparium plant by two extraction protocols. and The quantitative study made it possible to propose the best protocol for the extraction of the alkaloids. Moreover, GC-MS analysis of alkaloid extracts allowed us to determine their chemical composition. Haloxylon scoparium contains four types of alkaloids: tetraisoquinolines, phenylethylamines, tryptolines and tryptamines. The main compounds are the tetraisoquinolines type, the predominant product of which was N-methylsalsoline. These compounds present a great interest for the researchers due to their various pharmacological and biological activities. The antioxidant effect of the different plant extracts was studied by two methods: the ferric reducing antioxidant power (FRAP) and 1,1-diphenyl-2-picryl hydrazyl free radical (DPPH·) scavenging tests. The results show that extracts of root part are more active than those from aerial part; the acetone/water extract is the most powerful. The interesting results obtained in this study will be supplemented by other analyses and biological tests in order to better valorize this plant.

Quantitative theory for the diffusive dynamics of liquid condensates.

Key processes of biological condensates are diffusion and material exchange with their environment. Experimentally, diffusive dynamics are typically probed via fluorescent labels. However, to date, a physics-based, quantitative framework for the dynamics of labeled condensate components is lacking. Here, we derive the corresponding dynamic equations, building on the physics of phase separation, and quantitatively validate the related framework via experiments. We show that by using our framework, we can precisely determine diffusion coefficients inside liquid condensates via a spatio-temporal analysis of fluorescence recovery after photobleaching (FRAP) experiments. We showcase the accuracy and precision of our approach by considering space- and time-resolved data of protein condensates and two different polyelectrolyte-coacervate systems. Interestingly, our theory can also be used to determine a relationship between the diffusion coefficient in the dilute phase and the partition coefficient, without relying on fluorescence measurements in the dilute phase. This enables us to investigate the effect of salt addition on partitioning and bypasses recently described quenching artifacts in the dense phase. Our approach opens new avenues for theoretically describing molecule dynamics in condensates, measuring concentrations based on the dynamics of fluorescence intensities, and quantifying rates of biochemical reactions in liquid condensates.

Isolation of adult mouse hippocampal neural stem cells for fluorescence loss in photobleaching assays.

This protocol describes the isolation and culturing of primary neural stem cells (NSCs) from the adult mouse hippocampus, followed by the experimental approach for fluorescence loss in photobleaching assays, previously used to characterize the presence of an endoplasmic reticulum (ER) membrane diffusion barrier. The assay described here can be used to study live asymmetry in the ER membrane or other organelles that is established in dividing NSCs. For complete details on the use and execution of this protocol, please refer to Clay et al. (2014); bin Imtiaz et al. (2021); Lee et al. (2016); Luedeke et al. (2005); Moore et al. (2015); Shcheprova et al. (2008).