Functional characterization of green fluorescent protein-profilin fusion proteins.

To clarify the role of profilins in cells, fusion proteins constructed with green fluorescent protein (GFP) should be extremely helpful. As profilins are considerably smaller than the GFP fusion partner (14-17 kDa compared with 27 kDa, respectively), we characterized the fusion proteins in vitro, to ascertain their biological function. We fused mouse profilin I and II to either the C-terminus or N-terminus of GFP. These fusion proteins were expressed in Escherichia coli and affinity-purified on polyproline-Sepharose. Interaction with vasodilator-stimulated phosphoprotein, a proline-rich ligand of profilin, was investigated by ELISA, as was binding to PtdIns(4,5)P2. The affinity for actin was quantitatively determined in polymerization assays. Our results show that fusion of GFP to the C-terminus of profilin I abolishes polyproline binding. In contrast, the other fusion proteins bound to polyproline-Sepharose and VASP. Binding to PtdIns(4,5)P2 was not significantly altered. Furthermore, fusion of either isoform with GFP did not decrease the affinity for actin. In localization studies with mammalian cells, all fusion proteins showed the localization expected for profilin in areas of high actin dynamics, such as leading lamellae and ruffles induced by epidermal growth factor. However, with regard to our in vitro data, we suspect that only a minor fraction of profilin I carrying the GFP at the C-terminus can target these sites. Therefore, other constructs should be preferred for further in vivo studies.     

Dual labeling with green fluorescent proteins for confocal microscopy

We report a dual labeling technique involving two green fluorescent protein (GFP) variants that is compatible with confocal microscopy. Two lasers were used to obtain images of (i) mixed cultures of cells, where one species contained GFPuv and another species contained GFPmut2 or GFPmut3, and (ii) a single species containing both GFPuv and GFPmut2 in the same cell. This method shows promise for monitoring gene expression and as a nondestructive and in situ technique for confocal microscopy of multispecies biofilms.     

Sweeping model of dynamin activity. Visualization of coupling between exocytosis and endocytosis under an evanescent wave microscope with green fluorescent proteins

Vesicle recycling through exocytosis and endocytosis is mediated by a coordinated cascade of protein-protein interactions. Previously, exocytosis and endocytosis were studied separately so that the coupling between them was understood only indirectly. We focused on the coupling of these processes by observing the secretory vesicle marker synaptobrevin and the endocytotic vesicle marker dynamin I tagged with green and red fluorescent proteins under an evanescent wave microscope in pheochromocytoma cells. In control cells, many synaptobrevin-expressing vesicles were found as fluorescent spots near the plasma membrane. Upon electrical stimulation, many of these vesicles showed an exocytotic response as a transient increase in fluorescence intensity followed by their disappearance. In contrast, fluorescent dynamin appeared as clusters increasing slowly in number upon stimulation. The clusters of fluorescent dynamin moved around beneath the plasma membrane for a significant distance. Simultaneous observations of green fluorescent dynamin and red fluorescent synaptobrevin indicated that more than 70% of the exocytotic responses of synaptobrevin had no immediate dynamin counterpart at the same site. From these findings it was concluded that dynamin-mediated recycling is not directly coupled to exocytosis but rather completed by a scanning movement of dynamin for the sites of invaginating membrane destined to endocytosis.     

A method for determining the individual optical characteristics of posttranslational fluorescent forms of fluorescent proteins with the use of nonlinear laser fluorimetry

A method for determining the individual optical characteristics (fluorescence quantum yield, the rate constant and quantum yield of singlet-triplet conversion, excitation of fluorescence cross-section, extinction coefficient) and concentration correlations between the fluorescent forms of fluorescent proteins arising in the reaction of posttranslational chromophore formation has been developed, which is based on combined application of absorption spectroscopy and classical and nonlinear laser fluorimretry. The method allows one to determine the share of fluorescent forms in the mixture of chromoproteins. The individual optical characteristics of the red form of the fluorescent protein mRFP1 has been determined: the fluorescence quantum yield eta = 0.24 +/- 0.03; the extinction coefficient in the maximum of absorbance band (584 nm) epsilon = 213 +/- 40 mM(-1) cm(-1) (the cross-section of absorbance sigma = (8.2 +/- 1.5).10(-16) cm2); the constant of singlet-triplet conversion rate K32 = (0 +/- 0.6)-10970 s(-1). The part of the red form in the mixture of chromoproteins is 26 +/- 6%.     

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.     

Automated live cell imaging of green fluorescent protein degradation in individual fibroblasts.

To accurately interpret the data from fluorescent proteins as reporters of gene activation within living cells, it is important to understand the kinetics of the degradation of the reporter proteins. We examined the degradation kinetics over a large number (>1,000) of single, living cells from a clonal population of NIH3T3 fibroblasts that were stably transfected with a destabilized, enhanced green fluorescent protein (eGFP) reporter driven by the tenascin-C promoter. Data collection and quantification of the fluorescence protein within a statistically significant number of individual cells over long times (14 h) by automated microscopy was facilitated by culturing cells on micropatterned arrays that confined their migration and allowed them to be segmented using phase contrast images. To measure GFP degradation rates unambiguously, protein synthesis was inhibited with cycloheximide. Results from automated live cell microscopy and image analysis indicated a wide range of cell-to-cell variability in the GFP fluorescence within individual cells. Degradation for this reporter was analyzed as a first order rate process with a degradation half-life of 2.8 h. We found that GFP degradation rates were independent of the initial intensity of GFP fluorescence within cells. This result indicates that higher GFP abundance in some cells is likely due to higher rates of gene expression, because it is not due to systematically lower rates of protein degradation. The approach described in this study will assist the quantification and understanding of gene activity within live cells using fluorescent protein reporters.     

Development of a method for screening short-lived proteins using green fluorescent protein

We have developed a screening technology for the identification of short-lived proteins. A green fluorescent protein (GFP)-fusion cDNA library was generated for monitoring degradation kinetics. Cells expressing a subset of the GFP-cDNA expression library were screened to recover those in which the fluorescence signal diminished rapidly when protein synthesis was inhibited. Thirty clones that met the screening criteria were characterized individually. Twenty-three (73%) proved to have a half-life of 4 hours or less.     

Design of generic biosensors based on green fluorescent proteins with allosteric sites by directed evolution.

Protein-engineering techniques have been adapted for the molecular design of biosensors that combine a molecular-recognition site with a signal-transduction function. The optical signal-transduction mechanism of green fluorescent protein (GFP) is most attractive, but hard to combine with a ligand-binding site. Here we describe a general method of creating entirely new molecular-recognition sites on GFPs. At the first step, a protein domain containing a desired molecular-binding site is inserted into a surface loop of GFP. Next, the insertional fusion protein is randomly mutated, and new allosteric proteins that undergo changes in fluorescence upon binding of target molecules are selected from the random library. We have tested this methodology by using TEM1 beta-lactamase and its inhibitory protein as our model protein-ligand system. 'Allosteric GFP biosensors' constructed by this method may be used in a wide range of applications including biochemistry and cell biology.     

Visualization of molecular and cellular events with green fluorescent proteins in developing embryos: a review

An Erratum has been published for this article in Luminescence (2003) 18(4) 243 During the past 5 years, green fluorescent protein (GFP) has become one of the most widely used in vivo protein markers for studying a number of different molecular processes during development, such as promoter activation, gene expression, protein trafficking and cell lineage determination. GFP fluorescence allows observation of dynamic developmental processes in real time, in both transiently and stably transformed cells, as well as in live embryos. In this review, we include the most up‐to‐date use of GFP during embryonic development and point out the unique contribution of GFP visualization, which resulted in novel discoveries. Copyright © 2002 John Wiley & Sons, Ltd.     

Evolutionary and functional diversity of green fluorescent proteins in cephalochordates

Green fluorescent protein (GFP) has been widely used as a molecular marker in modern biological research. Before the recent report of one GFP gene in Branchiostoma floridae, GFP family members were cloned only from other two groups of species: Cnidaria and Copepoda. Here we describe the complete GFP gene repertoire of B. floridae which includes 13 functional genes and 2 pseudogenes, representing the largest GFP family found so far. Coupling with nine other GFP sequences from another two species of genus Branchiostoma and the sequences from Cnidaria and Copepoda, we made a deep-level phylogenetic analysis for GFP genes in cephalochordates and found: 1) GFP genes have experienced a divergent evolution in cephalochordates; 2) all amphioxus GFP genes form four main clades on the tree which had diverged before the radiation of the last common ancestor of all extant cephalochordates; 3) GFP genes in amphioxus shared a common ancestor with that in Copepoda rather than being derived from horizontal gene transfer, which indicates that our ancestor was derived from a fluorescent organism and lost this ability after its separation from Cephalochordata, and also makes GFP a rare gene which has a rather unusual evolutionary path. In addition, we also provided evidence indicating that GFP genes have evolved divergent functions by specializing their expression profile, and different fluorescent spectra by changing their emission peaks. These findings spark two interesting issues: what are GFP in vivo functions in cephalochordates and why they are lost in other examined deuterostomes?     

Facilitating chromophore formation of engineered Ca binding green fluorescent proteins

Green fluorescent protein (GFP) containing a self-coded chromophore has been applied in protein trafficking and folding, gene expression, and as sensors in living cells. While the “cycle3” mutation denoted as C3 mutation (F99S/M153T/V163A) offers the ability to increase GFP fluorescence at 37 °C, it is not clear whether such mutations will also be able to assist the folding and formation of the chromophore upon the addition of metal ion binding sites. Here, we investigate in both bacterial and mammalian systems, the effect of C2 (M153T/V163A) and C3 (F99S/M153T/V163A) mutations on the folding of enhanced GFP (EGFP, includes F64L/S65T) and its variants engineered with two types of Ca²⁺ binding sites: (1) a designed discontinuous Ca²⁺ binding site and (2) a grafted continuous Ca²⁺ binding motif. We show that, for the constructed EGFP variants, the C2 mutation is sufficient to facilitate the production of fluorescence in both bacterial and mammalian cells. Further addition of the mutation F99S decreases the folding efficiency of these variants although a similar effect is not detectable for EGFP, likely due to the already greatly enhanced mutation F64L/S65T from the original GFP, which hastens the chromophore formation. The extinction coefficient and quantum yield of purified proteins of each construct were also examined to compare the effects of both C2 and C3 mutations on protein spectroscopic properties. Our quantitative analyses of the effect of C2 and C3 mutations on the folding and formation of GFP chromophore that undergoes different folding trajectories in bacterial versus mammalian cells provide insights into the development of fluorescent protein-based analytical sensors.     

Structural stability of green fluorescent proteins entrapped in polyelectrolyte nanocapsules

Molecules of a green fluorescent protein mutant, GFPmut2, have been immobilized in nanocapsules, assemblies of charged polyelectrolyte multilayers, with the aim to study the effect of protein‐polyelectrolyte interactions on the protein stability against chemical denaturation. GFPmut2 proteins turn out to be stabilized and protected against the denaturating action of small chemical compounds. The nanocapsule protective effect on GFPmut2 is likely due to protein interactions with the negatively charged polymers, that induce an increase in the local rigidity of the protein nano‐environment. This suggestion is supported by Fluorescence Polarization measurements on GFPmut2 proteins bound to the NC layers. (© 2008 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Trafficking of green fluorescent protein-tagged SNARE proteins in HSY cells

SNARE proteins are widely accepted to be involved in the docking and fusion process of intracellular vesicle trafficking. VAMP-2, syntaxin-4, and SNAP-23 are plausible candidate SNARE proteins for non-neuronal exocytosis. Thus, we examined the localization, protein-protein interaction, and intracellular trafficking of these proteins by expressing them as green fluorescent protein (GFP)- and FLAG-tagged fusion proteins in various cells, including HSY cells, a human parotid epithelial cell line. GFP-VAMP-2 was ex-pressed strongly in the Golgi area and weakly on the plasma membrane. Although GFP-SNAP-23 seemed to be expressed universally in the cytosol, the GFP signal was clearly seen on the plasma membrane, when soluble GFP-SNAP-23 was removed by treatment with saponin. GFP-syntaxin-4 was undetectable on the plasma membrane but was strongly expressed on unidentified unusually large vesicles. GFP-syntaxin-4 without its transmembrane domain was still incompletely soluble and observed as aggregates. When syntaxin-4 and munc18c were coexpressed, syntaxin-4 was translocated at least in part to the plasma membrane. The protein-protein interaction between syntaxin-4 and VAMP-2 with their transmembrane domains was markedly inhibited on coexpression of munc18c. These results suggest that munc18c plays an important role in the trafficking of syntaxin-4 to its proper destination by preventing premature interactions with other proteins, including SNARE proteins.     

Incomplete proteasomal degradation of green fluorescent proteins in the context of tandem fluorescent protein timers

Tandem fluorescent protein timers (tFTs) report on protein age through time-dependent change in color, which can be exploited to study protein turnover and trafficking. Each tFT, composed of two fluorescent proteins (FPs) that differ in maturation kinetics, is suited to follow protein dynamics within a specific time range determined by the maturation rates of both FPs. So far, tFTs have been constructed by combining slower-maturing red fluorescent proteins (redFPs) with the faster-maturing superfolder green fluorescent protein (sfGFP). Toward a comprehensive characterization of tFTs, we compare here tFTs composed of different faster-maturing green fluorescent proteins (greenFPs) while keeping the slower-maturing redFP constant (mCherry). Our results indicate that the greenFP maturation kinetics influences the time range of a tFT. Moreover, we observe that commonly used greenFPs can partially withstand proteasomal degradation due to the stability of the FP fold, which results in accumulation of tFT fragments in the cell. Depending on the order of FPs in the timer, incomplete proteasomal degradation either shifts the time range of the tFT toward slower time scales or precludes its use for measurements of protein turnover. We identify greenFPs that are efficiently degraded by the proteasome and provide simple guidelines for the design of new tFTs.     

M153R mutation in a pH-sensitive green fluorescent protein stabilizes its fusion proteins

Background

Green fluorescent protein (GFP) and its fusion proteins have been used extensively to monitor and analyze a wide range of biological processes. However, proteolytic cleavage often removes GFP from its fusion proteins, not only causing a poor signal-to-noise ratio of the fluorescent images but also leading to wrong interpretations.

Methodology/Principal Findings

Here, we report that the M153R mutation in a ratiometric pH-sensitive GFP, pHluorin, significantly stabilizes its fusion products while the mutant protein still retaining a marked pH dependence of 410/470 nm excitation ratio of fluorescence intensity. The M153R mutation increases the brightness in vivo but does not affect the 410/470-nm excitation ratios at various pH values.

Conclusions/Significance

Since the pHluorin(M153R) probe can be directly fused to the target proteins, we suggest that it will be a potentially powerful tool for the measurement of local pH in living cells as well as for the analysis of subcellular localization of target proteins.     

Reliable use of green fluorescent protein in fluorescent pseudomonads.

When fluorescent pseudomonads are cultured on standard solid media under iron limiting conditions, they produce fluorescent, pigmented iron collating agents (siderophores). Siderophores can be readily identified by strong fluorescence seen under UV/blue light. The application of the eukaryotic green fluorescent protein (GFP) as a bacterial marker in microbial ecology is increasingly being used, particularly as it is a powerful method for non-destructive monitoring in situ. As gfp expressing bacteria have to be detected under UV/blue light, the fluorescence of siderophore-producing Pseudomonas spp. masks normal levels of GFP fluorescence when colonies are viewed on standard bacterial agar. Here, we describe a simple but effective way of identifying gfp-expressing Pseudomonas fluorescens using media supplemented with 0.45 mM FeSO(4).7H(2)O. This is of relevance for the screening of insertion libraries and in the application of GFP transposons as promoter probes.     

Stable genetic transformation of Eschscholzia californica expressing synthetic green fluorescent proteins

An efficient protocol is described for the stable genetic transformation of Eschscholzia californica (California poppy) using Agrobacterium tumefaciens as a vector. We have employed the disarmed A. tumefaciens LBA4404 encoding a synthetic green fluorescent protein reporter gene that is further controlled by an enhanced cauliflower mosaic virus 35S promoter. Stably transformed E. californica cells appear 3 weeks after initial cocultivation of A. tumefaciens with poppy leaves, stems, or roots. Transformed poppy calli were visualized by exposure to long-wave UV or blue light and analyzed in detail by fluorescent microscopy and laser-scanning microscopy. Moreover, green fluorescent calli have been maintained through continual subculture and grow well either on Gamborg's B5 agarose or liquid medium.     

The green fluorescent protein targets secretory proteins to the yeast vacuole

The green fluorescent protein (GFP) was used as a marker to study the intracellular transport of vacuolar and secretory proteins in yeast. Therefore, the following gene constructs were expressed in Saccharomyces cerevisiae under control of the GAL1 promoter: GFP N-terminally fused to the yeast secretory invertase (INV-GFP), the plant vacuolar chitinase (CHN-GFP) and its secretory derivative (CHNDeltaVTP-GFP), which did not contain the vacuolar targeting peptide (VTP), both chitinase forms (CHN and CHNDeltaVTP), GFP without any targeting information and two secretory GFP variants with and without the VTP of chitinase (N-GFP-V and N-GFP). Whereas chitinase without VTP is accumulated in the culture medium the other gene products are retained inside the cell up to 48 h of induction. Independently of a known VTP they are transported to the vacuole, so far as they contain a signal peptide for entering the endoplasmic reticulum. This was demonstrated by confocal laser scanning microscopy, immunocytochemical analysis and subcellular fractionation experiments as well. The transport of the GFP fusion proteins is temporary delayed by a transient accumulation in electron-dense structures very likely derived from the ER, because they also contain the ER chaperone Kar2p/Bip. Our results demonstrate that GFP directs secretory proteins without VTP to the yeast vacuole, possibly by the recognition of an unknown vacuolar signal and demonstrates, therefore, a first limitation for the application of GFP as a marker for the secretory pathway in yeast.