GFP Reconstitution Across Synaptic Partners (GRASP) defines cell contacts and synapses in living nervous systems

The identification of synaptic partners is challenging in dense nerve bundles, where many processes occupy regions beneath the resolution of conventional light microscopy. To address this difficulty, we have developed GRASP, a system to label membrane contacts and synapses between two cells in living animals. Two complementary fragments of GFP are expressed on different cells, tethered to extracellular domains of transmembrane carrier proteins. When the complementary GFP fragments are fused to ubiquitous transmembrane proteins, GFP fluorescence appears uniformly along membrane contacts between the two cells. When one or both GFP fragments are fused to synaptic transmembrane proteins, GFP fluorescence is tightly localized to synapses. GRASP marks known synaptic contacts in C. elegans, correctly identifies changes in mutants with altered synaptic specificity, and can uncover new information about synaptic locations as confirmed by electron microscopy. GRASP may prove particularly useful for defining connectivity in complex nervous systems.     

Exceptional total and functional yields of the human adenosine (A2a) receptor expressed in the yeast Saccharomyces cerevisiae

The yeast Saccharomyces cerevisiae was used to express a medically relevant G-protein coupled receptor (GPCR), the human adenosine (A2a) receptor, with a C-terminal green fluorescent protein (GFP) fusion tag. In prior studies, we established an expression system for A2a-GFP. Here, we quantified the total A2a-GFP expression levels by correlating GFP levels as detected by fluorescence and densitometry to A2a-GFP molecules overexpressed in the system. We also quantified A2a-GFP functional levels by classical radioligand binding assays. Approximately, 120,000 functional A2a-GFP molecules per cell were present on the plasma membrane as determined by radioligand binding. Using whole cell GFP fluorescence, 340,000 A2a-GFP molecules per cell were detected; approximately 70% of those molecules were plasma membrane localized, as determined by using confocal microscopy analysis. These results show that a significant portion of the total expressed protein is functional. In addition, the quick and inexpensive whole cell fluorescence appears to provide a good approximation of functional receptor numbers for this case. Importantly, the amount of functionally expressed A2a-GFP per culture ( approximately 4 mg/L) is among the highest reported for any GPCR in any expression system.     

Display of green fluorescent protein on Escherichia coli cell surface

In this study, expression of green fluorescence protein (GFP) on the external surface of Escherichia coli was achieved by construction of a fusion protein between Lpp-OmpA hybrid and a GFP variant, GFPmut2. The GFP was fused in frame to the carboxyl-terminus of Lpp-OmpA fusion previously shown to direct various other heterologous proteins to E. coli cell surface. Western blot analysis of membrane fractions identified the Lpp-OmpA-GFP fusion protein with the expected size (43 kDa). Immunofluorescence microscopy, immunoelectron microscopy, protease and extracellular pH sensitivity assays further confirmed that GFP is anchored on the outer membrane. The GFP displayed on the E. coli outer surface retained its fluorescence and was not susceptible to the indigenous outer membrane protease OmpT even though there are two putative OmpT proteolytic sites present in GFP. Optimization of the expression conditions was conducted using fluorometry, eliminating cumbersome immuno-labeling procedures. Surface-displayed GFP could be used in a variety of applications including screening of polypeptide libraries, development of live vaccines, construction of whole cell allosteric biosensors, and signal transduction studies.     

Rapid and sensitive detection method of a bacterium by using a GFP reporter phage

A rapid, sensitive, and convenient method for detecting a specific bacterium was developed by using a GFP phage. Here we describe a model system that utilizes the temperate Escherichia coli-restricted bacteriophage lambda, which was genetically modified to express a reporter gene for GFP to identify the colon bacillus E. coli in the specimen. E. coli infected with GFP phage was detected by GFP fluorescence after 4-6 hr of incubation. The results show that a few bacteria in a specimen can be detected under fluorescence microscopy equipped with a sensitive cooled CCD camera. When E. coli and Mycobacterium smegmatis were mixed in a solution containing GFP phage, only E. coli was infected, indicating the specificity of this method. The method has the following advantages: 1) Bacteria from biological samples need not be purified unless they contain fluorescent impurities; 2) The infection of GFP phage to bacteria is specific; 3) The fluorescence of GFP within infected bacteria enables highly sensitive detection; 4) Exogenous substrates and cofactors are not required for fluorescence. Therefore this method is suitable for any phage-bacterium system when bacteria-specific phages are available.     

Confocal quantification of cis-regulatory reporter gene expression in living sea urchin

Quantification of GFP reporter gene expression at single cell level in living sea urchin embryos can now be accomplished by a new method of confocal laser scanning microscopy (CLSM). Eggs injected with a tissue-specific GFP reporter DNA construct were grown to gastrula stage and their fluorescence recorded as a series of contiguous Z-section slices that spanned the entire embryo. To measure the depth-dependent signal decay seen in the successive slices of an image stack, the eggs were coinjected with a freely diffusible internal fluorescent standard, rhodamine dextran. The measured rhodamine fluorescence was used to generate a computational correction for the depth-dependent loss of GFP fluorescence per slice. The intensity of GFP fluorescence was converted to the number of GFP molecules using a conversion constant derived from CLSM imaging of eggs injected with a measured quantity of GFP protein. The outcome is a validated method for accurately counting GFP molecules in given cells in reporter gene transfer experiments, as we demonstrate by use of an expression construct expressed exclusively in skeletogenic cells.     

New perspectives on the assembly process of mitochondrial respiratory chain complex cytochrome c oxidase

The assembly of cytochrome c oxidase (COX) is a complicated process and requires a number of assembly factors to put all the necessary subunits in the correct position. Defects in COX assembly lead in particular to serious neuromuscular disorders. We demonstrated that COX-deficient patients can be associated with different assembly patterns. To obtain more insight in the biogenesis of COX in a living cell, we used yeast as a model organism to design a way to pulse label holo-COX with green fluorescent protein (GFP). Using blue native electrophoresis, we showed that the GFP-tagged subunit is incorporated into fully assembled COX and this GFP tagged complex still has enzymatic activity. This allows us to correlate the GFP fluorescence signal detected in vivo by microscopy with the synthesis, turnover and assembly of COX.     

Design and synthesis of cell-permeable fluorescent nitrilotriacetic acid derivatives

Fluorescence labeling of the target molecules using a small molecule-based probe is superior than a method using genetically expressed green fluorescence protein (GFP) in terms of convenience in its preparation and functionalization. Fluorophore-nitrilotriacetic acid (NTA) conjugates with several ester protecting groups were synthesized and evaluated for their cell membrane permeability by fluorescence microscopy analysis. One of the derivatives, acetoxymethyl (AM)-protected NTA conjugate is hydrolyzed, resulting in intracellular accumulation, thus providing localized fluorescence intensity in cells. This modification is expected as an effective method for converting a non-cell membrane permeable NTA-BODIPY conjugates to a cell membrane permeable derivatives.     

Correlation functions quantify super-resolution images and estimate apparent clustering due to over-counting

We present an analytical method using correlation functions to quantify clustering in super-resolution fluorescence localization images and electron microscopy images of static surfaces in two dimensions. We use this method to quantify how over-counting of labeled molecules contributes to apparent self-clustering and to calculate the effective lateral resolution of an image. This treatment applies to distributions of proteins and lipids in cell membranes, where there is significant interest in using electron microscopy and super-resolution fluorescence localization techniques to probe membrane heterogeneity. When images are quantified using pair auto-correlation functions, the magnitude of apparent clustering arising from over-counting varies inversely with the surface density of labeled molecules and does not depend on the number of times an average molecule is counted. In contrast, we demonstrate that over-counting does not give rise to apparent co-clustering in double label experiments when pair cross-correlation functions are measured. We apply our analytical method to quantify the distribution of the IgE receptor (FcεRI) on the plasma membranes of chemically fixed RBL-2H3 mast cells from images acquired using stochastic optical reconstruction microscopy (STORM/dSTORM) and scanning electron microscopy (SEM). We find that apparent clustering of FcεRI-bound IgE is dominated by over-counting labels on individual complexes when IgE is directly conjugated to organic fluorophores. We verify this observation by measuring pair cross-correlation functions between two distinguishably labeled pools of IgE-FcεRI on the cell surface using both imaging methods. After correcting for over-counting, we observe weak but significant self-clustering of IgE-FcεRI in fluorescence localization measurements, and no residual self-clustering as detected with SEM. We also apply this method to quantify IgE-FcεRI redistribution after deliberate clustering by crosslinking with two distinct trivalent ligands of defined architectures, and we evaluate contributions from both over-counting of labels and redistribution of proteins.     

Visualizing single molecules inside living cells using total internal reflection fluorescence microscopy

Over the past 10 years, advances in laser and detector technologies have enabled single fluorophores to be visualized in aqueous solution. Here, we describe methods based on total internal reflection fluorescence microscopy (TIRFM) that we have developed to study the behavior of individual protein molecules within living mammalian cells. We have used cultured myoblasts that were transiently transfected with DNA plasmids encoding a target protein fused to green fluorescent protein (GFP). Expression levels were quantified from confocal images of control dilutions of GFP and cells with 1-100 nM GFP were then examined using TIRFM. An evanescent field was produced by a totally internally reflected, argon ion laser beam that illuminated a shallow region (50-100 nm deep) at the glass-water interface. Individual GFP-tagged proteins that entered the evanescent field appeared as individual, diffraction-limited spots of light, which were clearly resolved from background fluorescence. Molecules that bound to the basal cell membrane remained fixed in position for many seconds, whereas those diffusing freely in the cytoplasm disappeared within a few milliseconds. We developed automated detection and tracking methods to recognize and characterize the behavior of single molecules in recorded video sequences. This enabled us to measure the kinetics of photobleaching and lateral diffusion of membrane-bound molecules.     

Reverse-engineering of biochemical reaction networks from spatio-temporal correlations of fluorescence fluctuations

Recent developments of fluorescence labeling and highly advanced microscopy techniques have enabled observations of activities of biosignaling molecules in living cells. The high spatial and temporal resolutions of these video microscopy experiments allow detection of fluorescence fluctuations at the timescales approaching those of enzymatic reactions. Such fluorescence fluctuation patterns may contain information about the complex reaction-diffusion system driving the dynamics of the labeled molecule. Here, we have developed a method of identifying the reaction-diffusion system of fluorescently labeled signaling molecules in the cell, by combining spatio-temporal correlation function analysis of fluctuating fluorescent patterns, stochastic reaction-diffusion simulations, and an iterative system identification technique using a simulated annealing algorithm. In this report, we discuss the validity and usability of spatio-temporal correlation functions in characterizing the reaction-diffusion dynamics of biomolecules, and demonstrate application of our reaction-diffusion system identification method to a simple conceptual model for small GTPase activation.     

Septin filaments exhibit a dynamic, paired organization that is conserved from yeast to mammals

The septins are conserved, GTP-binding proteins important for cytokinesis, membrane compartmentalization, and exocytosis. However, it is unknown how septins are arranged within higher-order structures in cells. To determine the organization of septins in live cells, we developed a polarized fluorescence microscopy system to monitor the orientation of GFP dipole moments with high spatial and temporal resolution. When GFP was fused to septins, the arrangement of GFP dipoles reflected the underlying septin organization. We demonstrated in a filamentous fungus, a budding yeast, and a mammalian epithelial cell line that septin proteins were organized in an identical highly ordered fashion. Fluorescence anisotropy measurements indicated that septin filaments organized into pairs within live cells, just as has been observed in vitro. Additional support for the formation of pairs came from the observation of paired filaments at the cortex of cells using electron microscopy. Furthermore, we found that highly ordered septin structures exchanged subunits and rapidly rearranged. We conclude that septins assemble into dynamic, paired filaments in vivo and that this organization is conserved from yeast to mammals.     

A new,long-wavelength borondipyrromethene sphingosine for studying sphingolipid dynamics in live cells

Sphingolipids function as cell membrane components and as signaling molecules that regulate critical cellular processes. To study unacylated and acylated sphingolipids in cells with fluorescence microscopy, the fluorophore in the analog must be located within the sphingoid backbone and not the N-acyl fatty acid side chain. Although such fluorescent sphingosine analogs have been reported, they either require UV excitation or their emission overlaps with that of the most common protein label, green fluorescent protein (GFP). We report the synthesis and use of a new fluorescent sphingolipid analog, borondipyrromethene (BODIPY) 540 sphingosine, which has an excitation maximum at 540 nm and emission that permits its visualization in parallel with GFP. Mammalian cells readily metabolized BODIPY 540 sphingosine to more complex fluorescent sphingolipids, and subsequently degraded these fluorescent sphingolipids via the native sphingolipid catabolism pathway. Visualization of BODIPY 540 fluorescence in parallel with GFP-labeled organelle-specific proteins showed the BODIPY 540 sphingosine metabolites were transported through the secretory pathway and were transiently located within lysosomes, mitochondria, and the nucleus. The reported method for using BODIPY 540 sphingosine to visualize sphingolipids in parallel with GFP-labeled proteins within living cells may permit new insight into sphingolipid transport, metabolism, and signaling.     

Regeneration of plasmalemma and surface properties in macrophages

The regeneration of surface anionic groups in mouse peritoneal macrophages was investigated by electron microscopy, using cationized ferritin (CF) as a tool for the localization and evaluation of negative charge density on the cell surface. In vitro interaction of living macrophages with CF resulted in removal of most anionic groups, either by concentration of their receptor sites to a part of the membrane which is subsequently internalized, or by detachment of the aggregated label from the surface. After incubation of macrophages lacking surface anionic groups in tissue culture medium without the ligand, regeneration of the binding capacity for CF took place within 3 h. The first regenerated parts of the membrane can be visualized within 1 h on the upper part of the adherent cells; there is a discontinuous coating of ferritin, with the lateral regions of the plasmalemma free of label. The attached CF particles on the regenerated membrane are closer to the membrane and their density is considerably higher than on the normal control macrophages. The results indicate that the turnover of the plasmalemma is regional and not dispersed; the mechanism involved is insertion of membrane patches into the pre-existing plasma membrane.     

Expression and use of superfolder green fluorescent protein at high temperatures in vivo: a tool to study extreme thermophile biology

Superfolder GFP (sGFP) is a variant of the Green Fluorescent Protein that folds efficiently when fused to poorly folded proteins. In this study, we show that sGFP, but not enhanced GFP, is functional in vivo at 70°C in the extreme thermophile Thermus thermophilus ( Tth ); thus, permitting the use of sGFP as a localization tag in vivo . We created a suite of plasmids that allow the expression of carboxy-terminal sGFP fusion proteins in both Escherichia coli and Tth . In order to demonstrate the facility of sGFP as an in vivo localization tag in Tth , we tagged GroES (the small subunit of the bacterial GroES/GroEL chaperone), NarC (a membrane component of the nitrate respiration apparatus) and PhoA (a TAT-secreted periplasmic protein), and visualized the distribution of the sGFP fusion proteins using confocal microscopy. Fusions to NarC and PhoA produced enzymatically active proteins that complemented both the narC and the phoA strains respectively. Observation of the distribution of the GroES-sGFP protein by confocal microscopy revealed a homogeneous fluorescence in the cells, which is in full agreement with the cytoplasmic nature of GroES, whereas the NarC-sGFP protein was localized to the membrane. Finally, a combination of confocal microscopy and biochemistry revealed that PhoA is localized in the periplasm. We suggest that sGFP will be broadly applicable in characterizing various extreme thermophile systems.     

Fluorometric quantification of green fluorescent protein in tobacco leaf extracts

The main use of green fluorescent protein (GFP) is as a reporter system, where the existence of the protein is usually determined visually using fluorescent microscopy. Although fluorescence-based quantification of GFP is possible, background fluorescence in plants and in plant extracts was observed by our group. Another phenomenon we observed that makes quantification difficult is the increased level of GFP fluorescence in Nicotiana benthamiana leaf extracts, probably the result of dimerization of GFP molecules promoted by interaction with some component(s) of tobacco extracts. In the current work, the background fluorescence was minimized and the enhancement of GFP fluorescence in tobacco extracts was eliminated with the addition of urea to the measured solution so that a simple quantification assay for the GFP in the tobacco extracts could be established.     

GFP-tagged proteins visualized by freeze-fracture immuno-electron microscopy: a new tool in cellular and molecular medicine

GFP-tagging is widely used as a molecular tool to localize and visualize the trafficking of proteins in cells but interpretation is frequently limited by the low resolution afforded by fluorescence light microscopy. Although complementary thin-section immunogold electron microscopic techniques go some way in aiding interpretation, major limitations, such as relatively poor structural preservation of membrane systems, low labelling efficiency and the two-dimensional nature of the images, remain. Here we demonstrate that the electron microscopic technique freeze-fracture replica immunogold labelling overcomes these disadvantages and can be used to define, at high resolution, the precise location of GFP-tagged proteins in specific membrane systems and organelles of the cell. Moreover, this technique provides information on the location of the protein within the phospholipid bilayer, potentially providing insight into mis-orientation of tagged proteins compared to their untagged counterparts. Complementary application of the freeze-fracture replica immunogold labelling technique alongside conventional fluorescence microscopy is seen as a novel and valuable approach to verification, clarification and extension of the data obtained using fluorescent-tagged proteins. The application of this approach is illustrated by new findings on PAT-family proteins tagged with GFP transfected into fibroblasts from patients with Niemann-Pick type C disease.     

Clustering and dynamics of cytochrome bd‐I complexes in the Escherichia coli plasma membrane in vivo

The cytochrome bd‐I complex of Escherichia coli is a respiratory terminal oxidase and an integral component of the cytoplasmic membrane. As with other respiratory components, the organization and dynamics of this complex in living membranes is unknown. We set out to visualize the distribution and dynamics of this complex in vivo. By exchanging cydB for cydB–gfpgcn4 on the E. coli chromosome, we produced a strain (YTL01) that expresses functional GFP‐tagged cytochrome bd‐I terminal oxidase complexes under wild‐type genetic control. We imaged live YTL01 cells using video‐rate epifluorescence and total internal reflection fluorescence (TIRF) microscopy in combination with fluorescence recovery after photobleaching (FRAP) and saw mobile spots of GFP fluorescence in plasma membranes. Numbers of GFP molecules per spot were quantified by step‐wise photobleaching giving a broad distribution with a mean of ～76, indicating that cytochrome bd‐I is concentrated in mobile patches in the E. coli plasma membrane. We hypothesize that respiration occurs in mobile membrane patches which we call ‘respirazones’.     

Export of active green fluorescent protein to the periplasm by the twin-arginine translocase (Tat) pathway in Escherichia coli

The twin-arginine translocation (Tat) system targets cofactor-containing proteins across the Escherichia coli cytoplasmic membrane via distinct signal peptides bearing a twin-arginine motif. In this study, we have analysed the mechanism and capabilities of the E. coli Tat system using green fluorescent protein (GFP) fused to the twin-arginine signal peptide of TMAO reductase (TorA). Fractionation studies and fluorescence measurements demonstrate that GFP is exported to the periplasm where it is fully active. Export is almost totally blocked in tat deletion mutants, indicating that the observed export in wild-type cells occurs predominantly, if not exclusively, by the Tat pathway. Imaging studies reveal a halo of fluorescence in wild-type cells corresponding to the exported periplasmic form; the GFP is distributed uniformly throughout the cytoplasm in a tat mutant. Because previous work has shown GFP to be incapable of folding in the periplasm, we propose that GFP is exported in a fully folded, active state. These data also show for the first time that heterologous proteins can be exported in an active form by the Tat pathway.     

Location of Ferritin-Labeled Concanavalin A Binding to Influenza Virus and Tumor Cell Surfaces

Concanavalin A (Con-A) was linked to ferritin with glutaraldehyde and chromatographed on Sepharose 6B to separate unconjugated Con-A and ferritin from covalently cross-linked molecules. Ehrlich ascites tumor cells were infected with WSA influenza virus, stained at intervals with the ferritin-labeled Con-A and examined by electron microscopy. The surfaces of most mature viruses were specifically stained, providing direct evidence that influenza viruses maturing in this cell type have exposed Con-A receptor sites. The ferritin cores of the staining reagent were found at an average distance of 21.3 nm from the virus membrane and 10.8 nm from the uninfected cell membrane. This finding was interpreted to mean that the population of Con-A receptor sites on influenza virus particles is located at an average distance from the virus membrane twice that of the population of Con-A receptor sites found on uninfected cells. The structural elements of viral membranes can provide a reliable means for evaluating electron microscopy staining reagents, thereby enhancing their usefulness as probes for the study of membrane relationships.