FLIPing heterokaryons to analyze nucleo-cytoplasmic shuttling of yeast proteins

Nucleo-cytoplasmic shuttling is an important feature of proteins involved in nuclear export/import of RNAs, proteins, and also large ribonucleoprotein complexes such as ribosomes. The vast amount of proteomic data available shows that many of these processes are highly dynamic. Therefore, methods are needed to reliably assess whether a protein shuttles between nucleus and cytoplasm, and the kinetics with which it exchanges. Here we describe a combination of the classical heterokaryon assay with fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP) techniques, which allows an assessment of the kinetics of protein shuttling in the yeast Saccharomyces cerevisiae.     

Dynamic interaction between BAF and emerin revealed by FRAP, FLIP, and FRET analyses in living HeLa cells

Barrier-to-autointegration factor (BAF) is a conserved 10 kDa DNA-binding protein. BAF interacts with LEM-domain proteins including emerin, LAP2 beta, and MAN1 in the inner nuclear membrane. Using fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP), we compared the mobility of BAF to its partners emerin, LAP2 beta, and MAN1 in living HeLa cells. Like endogenous BAF, GFP-BAF was enriched at the nuclear envelope, and found inside the nucleus and in the cytoplasm during interphase. At every location, FRAP and FLIP analysis showed that GFP-BAF diffused rapidly; the halftimes for recovery in a 0.8 microm square area were 260 ms at the nuclear envelope, and even faster inside the nucleus and in the cytoplasm. GFP-fused emerin, LAP2 beta, and MAN1 were all relatively immobile, with recovery halftimes of about 1 min, for a 2 microm square area. Thus, BAF is dynamic and mobile during interphase, in stark contrast to its nuclear envelope partners. FLIP results further showed that rapidly diffusing cytoplasmic and nuclear pools of GFP-BAF were distinctly regulated, with nuclear GFP-BAF unable to replenish cytoplasmic BAF. Fluorescence resonance energy transfer (FRET) results showed that CFP-BAF binds directly to YFP-emerin at the inner nuclear membrane of living cells. We propose a "touch-and-go" model in which BAF binds emerin frequently but transiently during interphase. These findings contrast with the slow mobility of both GFP-BAF and GFP-emerin during telophase, when they colocalized at the 'core' region of telophase chromosomes at early stages of nuclear assembly.     

Fluorescence-based methods to image palmitoylated proteins

A well known function of palmitoylation is to promote protein binding to cell membranes. Until recently, it was unclear what additional roles, if any, palmitoylation has in controlling protein localization in cells. Recent studies of palmitoylated forms of the small GTPase Ras have now revealed that palmitoylation plays multiple roles in the regulation of protein trafficking, including targeting proteins into the secretory pathway and recycling proteins between the plasma membrane and Golgi complex. We here describe how quantitative fluorescence microscopy and photobleaching approaches can be used to study the intracellular targeting and trafficking of GFP-tagged palmitoylated proteins in living cells. We discuss (1) general considerations for fluorescence recovery after photobleaching (FRAP) measurements of GFP-tagged proteins; (2) FRAP-based assays to test the strength of binding of palmitoylated proteins to cell membranes; (3) methods to establish the kinetics and mechanisms of recycling of palmitoylated proteins between the Golgi complex and the plasma membrane; (4) the use of the palmitoylation inhibitor 2-bromo-palmitate as a tool to study the dynamic regulation of protein targeting and trafficking by palmitate turnover.     

Demonstration of phycobilisome mobility by the time- and space-correlated fluorescence imaging of a cyanobacterial cell

The cell-wide mobility of PBSs was confirmed by synchronously monitoring the fluorescence recovery after photobleaching (FRAP) and the fluorescence loss in photobleaching (FLIP). On the other hand, a fluorescence recovery was still observed even if PBSs were immobile (PBSs fixed on the membranes by betaine and isolated PBSs fixed on the agar plate) or PBS mobility was unobservable (cell wholly bleached). Furthermore, it was proved that some artificial factors were involved not only in FRAP but also in FLIP, including renaturation of the reversibly denatured proteins, laser scanning-induced fluorescence loss and photo-damage to the cell. With consideration of the fast renaturation component in fluorescence recovery, the diffusion coefficient was estimated to be tenfold smaller than that without the component. Moreover, it was observed that the fluorescence intensity on the bleached area was always lower than that on the non-bleached area, even after 20 min, while it should be equal if PBSs were mobile freely. Based on the increasing proportion of the PBSs anti-washed to Triton X-100 (1%) with prolonged laser irradiation to the cells locked in light state 1 by PBQ, it was concluded that some PBSs became immobile due to photo-linking to PSII.     

Demonstration of phycobilisome mobility by the time- and space-correlated fluorescence imaging of a cyanobacterial cell

The cell-wide mobility of PBSs was confirmed by synchronously monitoring the fluorescence recovery after photobleaching (FRAP) and the fluorescence loss in photobleaching (FLIP). On the other hand, a fluorescence recovery was still observed even if PBSs were immobile (PBSs fixed on the membranes by betaine and isolated PBSs fixed on the agar plate) or PBS mobility was unobservable (cell wholly bleached). Furthermore, it was proved that some artificial factors were involved not only in FRAP but also in FLIP, including renaturation of the reversibly denatured proteins, laser scanning-induced fluorescence loss and photo-damage to the cell. With consideration of the fast renaturation component in fluorescence recovery, the diffusion coefficient was estimated to be tenfold smaller than that without the component. Moreover, it was observed that the fluorescence intensity on the bleached area was always lower than that on the non-bleached area, even after 20 min, while it should be equal if PBSs were mobile freely. Based on the increasing proportion of the PBSs anti-washed to Triton X-100 (1%) with prolonged laser irradiation to the cells locked in light state 1 by PBQ, it was concluded that some PBSs became immobile due to photo-linking to PSII.     

Fluorescence Recovery After Photobleaching (FRAP) of Fluorescence Tagged Proteins in Dendritic Spines of Cultured Hippocampal Neurons

FRAP has been used to quantify the mobility of GFP-tagged proteins. Using a strong excitation laser, the fluorescence of a GFP-tagged protein is bleached in the region of interest. The fluorescence of the region recovers when the unbleached GFP-tagged protein from outside of the region diffuses into the region of interest. The mobility of the protein is then analyzed by measuring the fluorescence recovery rate. This technique could be used to characterize protein mobility and turnover rate.In this study, we express the (enhanced green fluorescent protein) EGFP vector in cultured hippocampal neurons. Using the Zeiss 710 confocal microscope, we photobleach the fluorescence signal of the GFP protein in a single spine, and then take time lapse images to record the fluorescence recovery after photobleaching. Finally, we estimate the percentage of mobile and immobile fractions of the GFP in spines, by analyzing the imaging data using ImageJ and Graphpad softwares.This FRAP protocol shows how to perform a basic FRAP experiment as well as how to analyze the data.     

ER retention may play a role in sorting of the nuclear pore membrane protein POM121

Integral membrane proteins of the nuclear envelope (NE) are synthesized on the rough endoplasmic reticulum (ER) and following free diffusion in the continuous ER/NE membrane system are targeted to their proper destinations due to interactions of specific domains with other components of the NE. By studying the intracellular distribution and dynamics of a deletion mutant of an integral membrane protein of the nuclear pores, POM121, which lacks the pore-targeting domain, we investigated if ER retention plays a role in sorting of integral membrane proteins to the nuclear envelope. A nascent membrane protein lacking sorting determinants is believed to diffuse laterally in the continuous ER/NE lipid bilayer and expected to follow vesicular traffic to the plasma membrane. The GFP-tagged deletion mutant, POM121(1-129)-GFP, specifically distributed within the ER membrane, but was completely absent from the Golgi compartment and the plasma membrane. Experiments using fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP) demonstrated that despite having very high mobility within the whole ER network (D = 0.41 +/- 0.11 micro m(2)/s) POM121(1-129)-GFP was unable to exit the ER. It was also not detected in post-ER compartments of cells incubated at 15 degrees C. Taken together, these experiments show that amino acids 1-129 of POM121 are able to retain GFP in the ER membrane and suggest that this retention occurs by a direct mechanism rather than by a retrieval mechanism. Our data suggest that ER retention might be important for sorting of POM121 to the nuclear pores.     

Reversible photobleaching of enhanced green fluorescent proteins

Color variants of green fluorescent protein (GFP) are increasingly used for multicolor imaging, fluorescence resonance energy transfer (FRET), and fluorescence recovery after photobleaching (FRAP). Here we show that experimental settings commonly used in these imaging experiments may induce an as yet uncharacterized reversible photobleaching of fluorescent proteins, which is more pronounced at acidic pH. Whereas the reversible photobleaching spectrum of eCFP corresponds to its absorption spectrum, reversible photobleaching spectra of yellow variants resemble absorption spectra of their protonated states. Fluorescence intensities recover spontaneously with time constants of 25-58 s. The recovery of eCFP can be further accelerated by illumination. The resulting steady-state fluorescence reflects a variable equilibrium between reversible photobleaching, spontaneous recovery, and light-induced recovery. These processes can cause significant artifacts in commonly applied imaging techniques, photobleach-based FRET determinations, and FRAP assays.     

Live-cell nucleocytoplasmic protein shuttle assay utilizing laser confocal microscopy and FRAP.

In eukaryotes, protein trafficking to and from the nucleus, or shuttling, has been demonstrated to be an important function for proteins that have vital roles in one or both subcellular compartments. Current techniques of detecting protein nuclear shuttling are extremely labor intensive and only statically visualize evidence of shuttling. Fluorescence recovery after photobleaching (FRAP), or fluorescence microphotolysis, has proven to be an effective method of analyzing protein dynamics in live cells, especially when coupled to GFP technology. Here, we describe a relatively simple in vivo protein nuclear shuttling assay that utilizes red fluorescent and green fluorescent protein fusions as substrates for FRAP using a laser confocal microscope. This technique is less time consuming than established shuttle assays, is internally controlled, and visualizes nucleocytoplasmic shuttling in living cells of the same species and cell type. This technique can be potentially used to detect the ability of any nuclear protein to shuttle from the nucleus to any other subcellular compartment for any eukaryotic species in which GFP or dsRed1 fusion protein can be expressed.     

Simplified Equation to Extract Diffusion Coefficients from Confocal FRAP Data

Quantitative measurements of diffusion can provide important information about how proteins and lipids interact with their environment within the cell and the effective size of the diffusing species. Confocal fluorescence recovery after photobleaching (FRAP) is one of the most widely accessible approaches to measure protein and lipid diffusion in living cells. However, straightforward approaches to quantify confocal FRAP measurements in terms of absolute diffusion coefficients are currently lacking. Here, we report a simplified equation that can be used to extract diffusion coefficients from confocal FRAP data using the half time of recovery and effective bleach radius for a circular bleach region, and validate this equation for a series of fluorescently labeled soluble and membrane‐bound proteins and lipids. We show that using this approach, diffusion coefficients ranging over three orders of magnitude can be obtained from confocal FRAP measurements performed under standard imaging conditions, highlighting its broad applicability.     

Genetic tags for labelling live cells: gap junctions and beyond

The availability of green fluorescent protein (GFP) as a tracer for observing proteins in living cells has revolutionized cell biology and spurred an intensive search for GFP variants with novel characteristics, additional autofluorescent proteins and alternative techniques of protein labelling. Two recent studies - one on tagging with tetracysteine motifs and labelling with biarsenic fluorophores of different colours, and the other on GFP tagging and fluorescence recovery after photobleaching (FRAP) - show how membrane channels are added and removed from gap junctions by using different fluorescent tags to distinguish between newly synthesized and older protein populations.     

Visualization of Endoplasmic Reticulum Subdomains in Cultured Cells

The lipids and proteins in eukaryotic cells are continuously exchanged between cell compartments, although these retain their distinctive composition and functions despite the intense interorganelle molecular traffic. The techniques described in this paper are powerful means of studying protein and lipid mobility and trafficking in vivo and in their physiological environment. Fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP) are widely used live-cell imaging techniques for studying intracellular trafficking through the exo-endocytic pathway, the continuity between organelles or subcompartments, the formation of protein complexes, and protein localization in lipid microdomains, all of which can be observed under physiological and pathological conditions. The limitations of these approaches are mainly due to the use of fluorescent fusion proteins, and their potential drawbacks include artifactual over-expression in cells and the possibility of differences in the folding and localization of tagged and native proteins. Finally, as the limit of resolution of optical microscopy (about 200 nm) does not allow investigation of the fine structure of the ER or the specific subcompartments that can originate in cells under stress (i.e. hypoxia, drug administration, the over-expression of transmembrane ER resident proteins) or under pathological conditions, we combine live-cell imaging of cultured transfected cells with ultrastructural analyses based on transmission electron microscopy.     

基于激光共聚焦同步双扫描技术的LC3脱乙酰化修饰调控自噬的机理研究

激光共聚焦同步双扫描(simultaneous,SIM)技术在常规扫描单元的基础上,引入一个同步扫描单元(SIM scanner),该技术独立控制了两个激光束,一个用于激光光刺激,另一个用于同步成像。本实验中,采用激光共聚焦同步双扫描系统的405 nm和488 nm激光分别对细胞的特定部位进行刺激和同步成像,实时检测了LC3复合物的形成,记录并分析了乙酰化前后LC3的光动力学变化过程,证实了LC3的脱乙酰化修饰是自噬性降解所必须的,本实验体系为激光共聚焦双扫描技术的推广提供了一个很好的平台。SIM技术的应用,解决了刺激过程无法成像的问题,为漂白后荧光恢复(fluorescence recovery after photobleaching,FRAP)、漂白后荧光损失(fluorescence loss in photobleaching,FLIP)和光诱导激活等研究提供了最佳的解决方案,可作为光刺激的一种实验模式在很多实验设计中进行延伸应用。     

Existence of periplasmic barriers preventing green fluorescent protein diffusion from cell to cell in the cyanobacterium Anabaena sp. strain PCC 7120

When deprived of combined nitrogen, the filamentous cyanobacterium Anabaena PCC 7120 relies on intercellular cooperation involving two cell types: nitrogen-fixing heterocysts and photosynthetic vegetative cells. Heterocysts send fixed nitrogen to vegetative cells over long distances along the filament, receiving a reduced carbon source from them. These intercellular exchanges might involve a continuous periplasm along the filament or cytoplasm-to-cytoplasm conduits or both. In the present study, the green fluorescent protein (GFP) was fused to a twin-arginine translocation signal sequence, which exported GFP to the periplasm of either a heterocyst using the heterocyst-specific promoters PhepA and PpatB or to the periplasm of vegetative cells using the vegetative cell-specific promoter PrbcL. Using the techniques of FRAP (fluorescence recovery after photobleaching) and FLIP (fluorescence loss in photobleaching), we found no evidence for intercellular diffusion of GFP through the periplasm, either from a heterocyst to vegetative cells or vice versa, or among vegetative cells. GFP could diffuse within the periplasm of the producing cell, but the diffusion stopped at the cell border. GFP diffusion could occur between two dividing cells before septum closure. This study indicates that barriers exist at the periplasmic space to prevent free GFP diffusion across cell border along the filament.     

Spatial Fourier analysis of video photobleaching measurements. Principles and optimization.

The major use of the fluorescence recovery after photobleaching (FRAP) technique is to measure the translational motion of the molecular components in various condensed media. In a conventional laser spot photobleaching experiment, a photomultiplier is used to measure the total brightness levels of the bleached region in the sample, so no spatial information can be directly obtained. In video-FRAP, a series of images after photobleaching is acquired, allowing the spatial character of the recovery to be determined; this permits direct detection of both anisotropic diffusion and flow. To utilize all of the available image data to determine the transport coefficients, a two-dimensional spatial Fourier transform analysis of the images after photobleaching was employed. The change in the transform between two time points reflects the action of diffusion during the interim. An important advantage of this method, which involves taking the ratio of image transforms at different time points, is that it does not require a specific initial condition to be created by laser photobleaching. The ability of the analysis to extract transport coefficients from computer-simulated diffusional recovery is assessed in the presence of increasing amounts of noise. Experimental data analysis from the diffusion of proteins in viscous solutions and from the diffusion of protein receptors on cell surfaces demonstrate the feasibility of the Fourier analysis to obtain transport coefficients from the video FRAP measurement.     

Lateral Diffusion and Exocytosis of Membrane Proteins in Cultured Neurons Assessed using Fluorescence Recovery and Fluorescence-loss Photobleaching

Membrane proteins such as receptors and ion channels undergo active trafficking in neurons, which are highly polarised and morphologically complex. This directed trafficking is of fundamental importance to deliver, maintain or remove synaptic proteins.Super-ecliptic pHluorin (SEP) is a pH-sensitive derivative of eGFP that has been extensively used for live cell imaging of plasma membrane proteins^1-2. At low pH, protonation of SEP decreases photon absorption and eliminates fluorescence emission. As most intracellular trafficking events occur in compartments with low pH, where SEP fluorescence is eclipsed, the fluorescence signal from SEP-tagged proteins is predominantly from the plasma membrane where the SEP is exposed to a neutral pH extracellular environment. When illuminated at high intensity SEP, like every fluorescent dye, is irreversibly photodamaged (photobleached)^3-5. Importantly, because low pH quenches photon absorption, only surface expressed SEP can be photobleached whereas intracellular SEP is unaffected by the high intensity illumination^6-10. FRAP (fluorescence recovery after photobleaching) of SEP-tagged proteins is a convenient and powerful technique for assessing protein dynamics at the plasma membrane. When fluorescently tagged proteins are photobleached in a region of interest (ROI) the recovery in fluorescence occurs due to the movement of unbleached SEP-tagged proteins into the bleached region. This can occur via lateral diffusion and/or from exocytosis of non-photobleached receptors supplied either by de novo synthesis or recycling (see Fig. 1). The fraction of immobile and mobile protein can be determined and the mobility and kinetics of the diffusible fraction can be interrogated under basal and stimulated conditions such as agonist application or neuronal activation stimuli such as NMDA or KCl application^8,10. We describe photobleaching techniques designed to selectively visualize the recovery of fluorescence attributable to exocytosis. Briefly, an ROI is photobleached once as with standard FRAP protocols, followed, after a brief recovery, by repetitive bleaching of the flanking regions. This ''FRAP-FLIP'' protocol, developed in our lab, has been used to characterize AMPA receptor trafficking at dendritic spines¹⁰, and is applicable to a wide range of trafficking studies to evaluate the intracellular trafficking and exocytosis.     

A Customized Light Sheet Microscope to Measure Spatio-Temporal Protein Dynamics in Small Model Organisms

We describe a customizable and cost-effective light sheet microscopy (LSM) platform for rapid three-dimensional imaging of protein dynamics in small model organisms. The system is designed for high acquisition speeds and enables extended time-lapse in vivo experiments when using fluorescently labeled specimens. We demonstrate the capability of the setup to monitor gene expression and protein localization during ageing and upon starvation stress in longitudinal studies in individual or small groups of adult Caenorhabditis elegans nematodes. The system is equipped to readily perform fluorescence recovery after photobleaching (FRAP), which allows monitoring protein recovery and distribution under low photobleaching conditions. Our imaging platform is designed to easily switch between light sheet microscopy and optical projection tomography (OPT) modalities. The setup permits monitoring of spatio-temporal expression and localization of ageing biomarkers of subcellular size and can be conveniently adapted to image a wide range of small model organisms and tissue samples.