Tracer Diffusivity in Amphiphilic Polymer Model Co-Networks

Amphiphilic polymer Co-networks based on cross-linked tetra-PEG-b-PCL star block copolymers

Fluorescence recovery after photobleaching (FRAP) is a fluorescence microscope technique to measure molecular diffusion and transport. FRAP is a valuable technique in cell biological research and evolved conjointly with microscope and fluorescent probes advancements. Although developed in the 1970s, the discovery and further development of fluorescent proteins revolutionised FRAP. After the discovery of green fluorescence protein and its application as a noninvasive and genetically coded protein‐tag, in vivo studies of protein dynamics and interactions became possible. FRAP is based on irreversibly bleaching a pool of fluorescent probes and monitoring the recovery in fluorescence due to movement of surrounding intact probes into the bleached spot. Although measurements are straightforward, quantitative FRAP requires careful experimental design, solid controls, data collection, and analysis. Over the past years, several FRAP‐related techniques have been tailored to suit particular cell biological questions, including inverse FRAP, fluorescence loss in photobleaching, and fluorescence localisation after photobleaching. Key Concepts: Fluorescence recovery after photobleaching (FRAP) is a method to qualitatively and quantitatively study biomolecule dynamics in living cells. FRAP is based on irreversibly bleaching a pool of fluorescent probes with high intensity light and monitoring the recovery in fluorescence due to movement of surrounding intact probes into the bleached spot. FRAP experiments are often conducted on confocal microscopes. To derive quantitative results from such experiments, several parameters and controls need to be considered and utilised in the analysis. There are several FRAP‐related methods that have been developed for specific applications and biological questions. FRAP is a versatile and popular method in modern biomedical research. Its application is broad and is increasingly applied in pharmacological, therapeutic and diagnostic areas.

Fluorescence recovery after photobleaching (FRAP) has been used to study the dynamics of the cyanobacterial photosynthesis apparatus since 1997. Fluorescence recovery of cyanobacteria during FRAP was conventionally interpreted as a result of phycobilisome (PBS) diffusion on the surface of the thylakoid membrane. The mechanism of state transition in cyanobacteria has been widely attributed to PBS diffusion. However, in red algae, another PBS-containing group, the intrinsic photoprocess was found to contribute greatly to the fluorescence recovery of PBS, which raises questions concerning the role of FRAP in red algal PBS. Therefore, it is important to re-evaluate the nature of PBS fluorescence recovery in cyanobacteria. In the present study, four cyanobacterial strains with different phenotypes and PBS compositions were used to investigate their FRAP characteristics. Fluorescence recovery of PBS was observed in wholly photobleached cells in all four cyanobacterial strains, in which the contribution of PBS diffusion to the fluorescence recovery was not possible. Moreover, the fluorescence recovered in isolated PBSs and PBS-thylakoid membranes after photobleaching further demonstrated the intrinsic photoprocess nature of fluorescence recovery. These findings suggest that the intrinsic photoprocess contributed to the fluorescence recovery following photobleaching when measured by the FRAP method.

Homeostatic mechanisms maintaining high levels of adhesion molecules in synapses over prolonged periods of time remain incompletely understood. We used fluorescence recovery after photobleaching experiments to analyze the steady state turnover of the immobile pool of green fluorescent protein-labeled NCAM180, the largest postsynaptically accumulating isoform of the neural cell adhesion molecule (NCAM). We show that there is a continuous flux of NCAM180 to the postsynaptic membrane from nonsynaptic regions of dendrites by diffusion. In the postsynaptic membrane, the newly delivered NCAM180 slowly intermixes with the immobilized pool of NCAM180. Preferential immobilization and accumulation of NCAM180 in the postsynaptic membrane is reduced after disruption of the association of NCAM180 with the spectrin cytoskeleton and in the absence of the homophilic interactions of NCAM180 in synapses. Our observations indicate that the homophilic interactions and binding to the cytoskeleton promote immobilization of NCAM180 and its accumulation in the postsynaptic membrane. Flux of NCAM180 from extrasynaptic regions and its slow intermixture with the immobile pool of NCAM180 in the postsynaptic membrane may be important for the continuous homeostatic replenishment of NCAM180 protein at synaptic contacts without compromising the long term synaptic contact stability.

Fluorescence recovery after photobleaching (FRAP) is a widely used imaging technique for measuring protein dynamics in live cells that has provided many important biological insights. Although FRAP presumes that the conversion of a fluorophore from a bright to a dark state is irreversible, GFP as well as other genetically encoded fluorescent proteins now in common use can also exhibit a reversible conversion known as photoswitching. Various studies have shown how photoswitching can cause at least four different artifacts in FRAP, leading to false conclusions about various biological phenomena, including the erroneous identification of anomalous diffusion or the overestimation of the freely diffusible fraction of a cellular protein. Unfortunately, identifying and then correcting these artifacts is difficult. Here we report a new characteristic of an organic fluorophore tetramethylrhodamine bound to the HaloTag protein (TMR-HaloTag), which like GFP can be genetically encoded, but which directly and simply overcomes the artifacts caused by photoswitching in FRAP. We show that TMR exhibits virtually no photoswitching in live cells under typical imaging conditions for FRAP. We also demonstrate that TMR eliminates all of the four reported photoswitching artifacts in FRAP. Finally, we apply this photoswitching-free FRAP with TMR to show that the chromatin decondensation following UV irradiation does not involve loss of nucleosomes from the damaged DNA. In sum, we demonstrate that the TMR Halo label provides a genetically encoded fluorescent tag very well suited for accurate FRAP experiments.

Cell polarity reflected by asymmetric distribution of proteins at the plasma membrane is a fundamental feature of unicellular and multicellular organisms. It remains conceptually unclear how cell polarity is kept in cell wall-encapsulated plant cells. We have used super-resolution and semi-quantitative live-cell imaging in combination with pharmacological, genetic, and computational approaches to reveal insights into the mechanism of cell polarity maintenance in Arabidopsis thaliana. We show that polar-competent PIN transporters for the phytohormone auxin are delivered to the center of polar domains by super-polar recycling. Within the plasma membrane, PINs are recruited into non-mobile membrane clusters and their lateral diffusion is dramatically reduced, which ensures longer polar retention. At the circumventing edges of the polar domain, spatially defined internalization of escaped cargos occurs by clathrin-dependent endocytosis. Computer simulations confirm that the combination of these processes provides a robust mechanism for polarity maintenance in plant cells. Moreover, our study suggests that the regulation of lateral diffusion and spatially defined endocytosis, but not super-polar exocytosis have primary importance for PIN polarity maintenance.

Measurement of the permeability of microcapsules with Fluorescence Recovery After Photobleaching (FRAP) enables analysis of individual microcapsules, the study of their batch heterogeneity, and the survey of changes in an individual capsule’s transport properties under different environmental conditions. We present a modified protocol and analysis which expands the parameter space of microcapsule sizes and permeabilities that can be accurately measured with FRAP. Simulations and experiments show that the hybrid analysis, which accounts for both permeation through the shell and free diffusion, precisely captures the kinetics of typical fluorescence recovery curves. The modified approach eliminates the need for painstaking controls to determine the FRAP measurement volume and the spatial distribution of bleached tracers, while accurately extracting the permeability of the microcapsule from FRAP data acquired using a straightforward procedure.

Fluorescence recovery after photobleaching (FRAP)is a microscopy technique that can be used to quantify protein mobility in live cells. In a typical FRAP experiment, steady-state fluorescence is observed by repeated imaging with low-intensity laser light. Subsequently, the fluorescent molecules are rapidly and irreversibly impaired via brief exposure to high-intensity laser light. Information about protein mobility is obtained by monitoring the recovery of fluorescence. We used FRAP to determine the mobility of p62 in aggresome-like induced structures (ALIS) in murine macrophages after stimulation with lipopolysaccharide (LPS). Because many existing FRAP protocols are either incomplete or complex, our goal was to provide a comprehensive, practical, and straightforward step-by-step protocol for FRAP experiments with live cells. Here, we describe RAW264.7 macrophage transfection with yellow fluorescent protein-p62 (YFP-p62), induction of ALIS by exposing the cells to LPS, and a step-by-step method for collecting prebleach and postbleach FRAP images and data analysis. Finally, we discuss important factors to consider when conducting a FRAP experiment.

GABAB (gamma-aminobutyric acid, type B) is a heterodimeric G-protein-coupled receptor. The GABAB1 subunit, which contains an endoplasmic reticulum retention sequence, is only transported to the cell surface when it is associated with the GABAB2 subunit. Fluorescence recovery after photobleaching studies in transfected COS-7 cells and hippocampal neurons revealed that GABAB2 diffuses slowly within the plasma membrane whether expressed alone or with the GABAB1 subunit. Treatment of cells with brefeldin A revealed that GABAB2 moves freely within the endoplasmic reticulum, suggesting that slow movement of GABAB2 is a result of its plasma membrane insertion. Disruption of the cytoskeleton did not affect the mobility of GABAB2, indicating that its restricted diffusion is not due to direct interactions with actin or tubulin. To determine whether the C terminus of GABAB2 regulates its diffusion, this region of the subunit was attached to the lymphocyte membrane protein, CD2, which then exhibited a slower rate of lateral diffusion. Furthermore, co-expression of a cytoplasmically expressed soluble form of the GABAB2 C terminus increased movement of the GABAB2 subunit. We constructed forms of GABAB2 with various C-terminal truncations. Truncation of GABAB2 after residue 862, but not residue 886, caused a dramatic increase in its mobility, suggesting that the region between these two residues is critical for restricting GABAB2 diffusion. Finally, we investigated whether activation of GABAB might modulate its movement. Treatment of COS-7 cells with the GABAB receptor agonist baclofen significantly increased its mobile fraction. These data show that the restricted movement of GABAB at the cell surface is regulated by a region within its C terminus.

Fluorescence recovery after photobleaching (FRAP) has remained a powerful tool to probe intracellular dynamics. FRAP relies on two aspects: (1) localized excitation resulting in photobleaching, and (2) fluorescence recovery of the bleached volume which provides insight into kinetics. Existing FRAP systems are limited by a trade-off between time and spatial multiplexing due to galvanometric scanning methods, precluding the study of multiple independent positions simultaneously as well as advanced widefield imaging modes. Hence, they may not capture the dynamic and non-isotropic environments in biological studies. In this paper, we utilize phase profiles corresponding to an array of Fresnel lenses and diffractive masks to switch between simultaneous FRAP of independent spatial positions whilst maintaining epifluorescence, highly inclined laminated optical sheet (HILO), and total internal reflection fluorescence (TIRF) microscopy modalities. Our approach bridges high contrast fluorescence imaging (HILO and TIRF) and a multi-position FRAP technique using a single spatial light modulator. As such, this technique enables high contrast bleaching and screening across volumes, which we envision will be of value to areas such as single particle tracking and single molecule imaging where dynamic photobleaching is necessary to measure fast events across the field of view in a single versatile instrument.

Fluorescence recovery after photobleaching (FRAP) and single particle tracking (SPT) techniques determine the diffusion coefficient from average diffusive motion of high-concentration molecules and from trajectories of low-concentration single molecules, respectively. Lateral diffusion coefficients measured by FRAP and SPT techniques for the same biomolecule on cell membrane have exhibited inconsistent values across laboratories and platforms with larger diffusion coefficient determined by FRAP, but the sources of the inconsistency have not been investigated thoroughly. Here, we designed an image-based FRAP-SPT system and made a direct comparison between FRAP and SPT for diffusion coefficient of submicron particles with known theoretical values derived from Stokes–Einstein equation in aqueous solution. The combined [Formula: see text]FRAP-SPT technique allowed us to measure the diffusion coefficient of the same fluorescent particle by utilizing both techniques in a single platform and to scrutinize inherent errors and artifacts of FRAP. Our results reveal that diffusion coefficient overestimated by FRAP is caused by inaccurate estimation of the bleaching spot size and can be corrected by simple image analysis. Our [Formula: see text]FRAP-SPT technique can be potentially used for not only cellular membrane dynamics but also for quantitative analysis of the spatiotemporal distribution of the solutes in small scale analytical devices.

Analysis of Active Transport by Fluorescence Recovery after Photobleaching

We report the simultaneous combination of three powerful techniques in uorescence microscopy: Fluorescence Lifetime Imaging (FLIM), Fluorescence Anisotropy Imaging (FAIM) and Fluorescence Recovery After Photobleaching (FRAP), also called F3 microscopy. An exhaustive calibration of the setup was carried out with several rhodamine 6G (R6G) solutions in water-glycerol and from the combination of the FAIM and FRAP data, the hydrodynamic radius of the dye was directly calculated. The F3 data was analyzed with a home-built MATLAB script, and the setup is currently explored further with Green Fluorescent Protein (GFP). Some molecular dynamic (MD) simulations are currently being run in order to help with the interpretation of the experimental anisotropy data.

Molecular transport mechanisms in membranes of smooth muscle cells (SMC) are of interest for a better understanding of atherogenesis. Atherosclerotic plaque material from the coronary arteries of 5 patients was extracted by thromboendarterectomy. After enzymatic disaggregation of plaque cells, isolated SMC were identified with monoclonal antibodies against muscle cell α-actin. Confluent cultures of SMC were stained with the low molecular lipid analogon Nile Red, a lipophil fluorescent dye which is largely specific for the hydrophobic inner domains of biomembranes. After irradiation of the labeled living cells in an inverted microscope with a laser beam, bleaching due to irreversible loss of fluorescent molecules is observed in the exposed region. Immediately thereafter the fluorescence recovers due to lateral movement of dye molecules from the neigbouring membrane regions. This effect is called fluorescence recovery after photobleaching (FRAP). Due to a modification of the usual FRAP technique, in which we made use of total internal reflection of the laser beam, we were able to measure largely specifically the FRAP kinetics of the outer cell membrane. The results showed complete fluorescence recovery already shortly after bleaching. This observation implies that 100 % of the lipid molecules belong to the mobile fraction, thus they are not firmly anchored in the membrane structure to a measurable extent. Moreover, the kinetics of FRAP could be closely approximated by the equation of a pure diffusion process in two dimensions. This fact indicates that the lateral mobility of the lipid molecule is due to passive diffusion only, without participation of active transport processes. Finally, the data allow a quantification of the speed of diffusion by determination of the quantity T1/2, that is the time within which half of the fluorescence intensity has recovered. The method offers perspectives for the investigation of lateral mobility of all kinds of molecules and could provide new insights into the action of drugs on the cell membrane.

The fluorescence recovery after photobleaching (FRAP) technique has been used for decades to measure movements of molecules in two-dimension (2D). Data obtained by FRAP experiments in cell plasma membranes are assumed to be described through a means of two parameters, a diffusion coefficient, D (as defined in a pure Brownian model) and a mobile fraction, M. Nevertheless, it has also been shown that recoveries can be nicely fit using anomalous subdiffusion. Fluorescence recovery after photobleaching (FRAP) at variable radii has been developed using the Brownian diffusion model to access geometrical characteristics of the surrounding landscape of the molecule. Here, we performed numerical simulations of continuous time random walk (CTRW) anomalous subdiffusion and interpreted them in the context of variable radii FRAP. These simulations were compared to experimental data obtained at variable radii on living cells using the pleckstrin homology (PH) domain of the membrane binding protein EFA6 (exchange factor for ARF6, a small G protein). This protein domain is an excellent candidate to explore the structure of the interface between cytosol and plasma membrane in cells. By direct comparison of our numerical simulations to the experiments, we show that this protein does not exhibit anomalous diffusion in baby hamster kidney (BHK) cells. The non Brownian PH-EFA6 dynamics observed here are more related to spatial heterogeneities such as cytoskeleton fence effects.