GFP Variants Research Articles

High-level expression of a synthetic red-shifted GFP coding region incorporated into transgenic chloroplasts.

We describe here a synthetic red-shifted variant of GFP that can be introduced into tobacco plastid genomes and is highly expressed in regenerated plants that appear normal and fertile. The variant contains the S65G and S72A mutations which shift the absorption maximum from the 395 nm of wild-type GFP closer to 488 nm, a wavelength emitted by a laser commonly used in confocal microscopy. In addition to enhanced fluorescence, the removal of significant absorption below 450 nm will potentially facilitate double-labelling experiments. The variant GFP encoded by the synthetic gene can be expressed at a high level, forming approximately 5% of total leaf protein.

Development of gfp Vectors for Expression in Listeria monocytogenes and Other Low G+C Gram Positive Bacteria.

The gfp (green fluorescent protein) gene has previously been used to construct a variety of reporter plasmids for Gram-positive bacteria for bacterial localization and gene expression studies. When a native red-shifted gfp variant (gfp3) was cloned into an expression vector using the Pxyn promoter and used to transform the soil-borne pathogen Listeria monocytogenes, only a small proportion of the population was seen to fluoresce when examined by epifluorescence microscopy. When the Pxyn promoter was replaced with the PxylA promoter, with accompanying modification of the translation initiation region of the gfp3 gene, a homogeneously fluorescent population of cells was obtained. When expressed in other Gram-positive organisms, such as Staphylococcus aureus and Bacillus subtilis, the translationally enhanced gene also resulted in high-level and homogeneous GFP production within the bacterial population. High-level expression of these reporter constructs in L. monocytogenes was evaluated to determine if it had any detrimental biological effect during intracellular infection of eukaryotic cell lines. The gfp3+ Listeria were found to invade equally as well as the wild-type cells; showing that these expression systems can be used to monitor the bacterium in natural environments. Based on these results, similar translationally enhanced vectors were also developed using unstable GFP3 variants, which retain their short-half life characteristics in L. monocytogenes and therefore can be used as a sensitive monitor of gene expression.

GFP imaging: methodology and application to investigate cellular compartmentation in plants

The cloning of the jellyfish gfp (green fluorescent protein) gene and its alteration for expression in subcellular locations in transformed plant cells have resulted in new views of intracellular organization and dynamics. Fusions of GFP with entire proteins of known or unknown function have shown where the proteins are located and whether the proteins move from one compartment to another. GFP and variants with different spectral properties have been deliberately targeted to separate compartments to determine their size, shape, mobility, and dynamic changes during development or environmental response. Fluorescence Resonance Energy Transfer (FRET) between GFP variants can discern protein/ protein interactions. GFP has been used as a sensor to detect changes or differences in calcium, pH, voltage, metal, and enzyme activity. Photobleaching and photoactivation of GFP as well as fluorescence correlation spectroscopy can measure rates of diffusion and movement of GFP within or between compartments. This review covers past applications of these methods as well as promising developments in GFP imaging for understanding the functional organization of plant cells.

GFP imaging: methodology and application to investigate cellular compartmentation in plants

The cloning of the jellyfish gfp (green fluorescent protein) gene and its alteration for expression in subcellular locations in transformed plant cells have resulted in new views of intracellular organization and dynamics. Fusions of GFP with entire proteins of known or unknown function have shown where the proteins are located and whether the proteins move from one compartment to another. GFP and variants with different spectral properties have been deliberately targeted to separate compartments to determine their size, shape, mobility, and dynamic changes during development or environmental response. Fluorescence Resonance Energy Transfer (FRET) between GFP variants can discern protein/ protein interactions. GFP has been used as a sensor to detect changes or differences in calcium, pH, voltage, metal, and enzyme activity. Photobleaching and photoactivation of GFP as well as fluorescence correlation spectroscopy can measure rates of diffusion and movement of GFP within or between compartments. This review covers past applications of these methods as well as promising developments in GFP imaging for understanding the functional organization of plant cells.

Improved plasmid vectors for the production of multiple fluorescent protein fusions in Bacillus subtilis

The intrinsically fluorescent green fluorescent protein has been used in many laboratories as a cytological marker to monitor protein localisation in live cells. Multiple spectrally modified mutant versions and novel fluorescent proteins from other species have subsequently been reported and used for labelling cells with multiple fluorescent protein fusions. In this work we report the design and use of vectors containing some of these spectral variants of GFP for use in the Gram positive bacterium Bacillus subtilis. These vectors complement those previously described (Lewis and Marston, 1999. Gene 227, 101–109) to provide a large suite of plasmid vectors for use in this and other related Gram positive organisms. Using these vectors we have been able to directly demonstrate the sequential assembly/disassembly of proteins involved in the generation of cellular asymmetry during development.

Enhanced fluorescence resonance energy transfer between spectral variants of green fluorescent protein through zinc-site engineering.

Although spectral variants of GFP should in theory be suited for fluorescence resonance energy transfer (FRET) and therefore suited for studies of protein-protein interactions, the unfavorable location of the fluorophore 15 A deep inside the GFP molecule has especially impaired this application. Here, metal-ion site engineering around the dimerization interface known from the X-ray structure of GFP is applied to the cyan and the yellow spectral variant of GFP to stabilize the heterodimeric form of these molecules and thereby increase FRET signaling. The FRET signal, determined as the ratio between the maximal emission for the yellow variant, 530 nm, and the cyan variant, 475 nm, during excitation of the cyan variant at 433 nm was increased up to 8-10-fold in the presence of 10(-4) M ZnCl2 by engineering of two symmetric metal-ion sites being either bidentate or tridentate. A similar increase in FRET signaling was however obtained in a pair of molecules in which a single bidentate metal-ion site was generated by introducing a zinc-binding residue in each of the two spectral variants of GFP and therefore creating an obligate heterodimeric pair. It is concluded that FRET signaling between spectral variants of GFP can be increased by stabilizing dimer formation and especially by favoring heterodimer formation in this case performed by metal-ion site engineering.

Improved gfp and inaZ broad-host-range promoter-probe vectors.

A new set of broad-host-range promoter-probe vectors has been constructed. One subset contains the pVS1 and p15a replicons and confers resistance to either gentamicin or kanamycin. The other set contains the broad-host-range replicon from pBBR1 and confers resistance to kanamycin, tetracycline, ampicillin, or spectinomycin/streptomycin. Both plasmid sets are highly stable and are maintained without selection for more than 30 generations in several bacterial taxa. Each plasmid contains a promoter-probe cassette that consists of a multicloning site, containing several unique restriction sites, and gfp or inaZ as a reporter gene. The cassette is bound by transcriptional terminators to permit the insertion of strong promoters and to insulate the cassette from external transcription enabling the detection of weak or moderate promoters. The vector suite was augmented with derivatives of the kanamycin-resistant gfp promoter-probe plasmids that encode Gfp variants with different half-life times.

Imaging Neuronal Subsets in Transgenic Mice Expressing Multiple Spectral Variants of GFP

We generated transgenic mice in which red, green, yellow, or cyan fluorescent proteins (together termed XFPs) were selectively expressed in neurons. All four XFPs labeled neurons in their entirety, including axons, nerve terminals, dendrites, and dendritic spines. Remarkably, each of 25 independently generated transgenic lines expressed XFP in a unique pattern, even though all incorporated identical regulatory elements (from the thy1 gene). For example, all retinal ganglion cells or many cortical neurons were XFP positive in some lines, whereas only a few ganglion cells or only layer 5 cortical pyramids were labeled in others. In some lines, intense labeling of small neuronal subsets provided a Golgi-like vital stain. In double transgenic mice expressing two different XFPs, it was possible to differentially label 3 neuronal subsets in a single animal.

Green fluorescent protein functions as a reporter for protein localization in Escherichia coli.

The use of green fluorescent protein (GFP) as a reporter for protein localization in Escherichia coli was explored by creating gene fusions between malE (encoding maltose-binding protein [MBP]) and a variant of gfp optimized for fluorescence in bacteria (GFPuv). These constructs encode hybrid proteins composed of GFP fused to the carboxy-terminal end of MBP. Fluorescence was not detected when the hybrid protein was synthesized with the MBP signal sequence. In contrast, when the MBP signal sequence was deleted, fluorescence was observed. Cell fractionation studies showed that the fluorescent MBP-GFP hybrid protein was localized in the cytoplasm, whereas the nonfluorescent version was localized to the periplasmic space. Smaller MBP-GFP hybrid proteins, however, exhibited abnormal fractionation. Expression of the gene fusions in different sec mutants, as well as signal sequence processing assays, confirmed that the periplasmically localized hybrid proteins were exported by the sec-dependent pathway. The distinction between fluorescent and nonfluorescent colonies was exploited as a scorable phenotype to isolate malE signal sequence mutations. While expression of hybrid proteins comprised of full-length MBP did not result in overproduction lethality characteristic of some exported beta-galactosidase hybrid proteins, synthesis of shorter, exported hybrid proteins was toxic to the cells. Purification of MBP-GFP hybrid protein from the different cellular compartments indicated that GFP is improperly folded when localized outside of the cytoplasm. These results suggest that GFP could serve as a useful reporter for genetic analysis of bacterial protein export and of protein folding.

The immunological synapse.

What is it? The immunological synapse is an area of close membrane apposition observed during the interaction between a presenting cell bearing a specific antigen and a T lymphocyte.Why is it important? The synapse is thought to be the type of intercellular contact through which antigenic peptides — bound to major histocompatibility complex (MHC) molecules on the surface of antigen-presenting cells (APCs) — are recognized by the antigen receptors on T lymphocytes. This binding leads to intracellular signals that are critical for lymphocyte activation. It is thought to be essential for effective recognition of surface-bound antigen.How does it form? First, the recruitment into the contact zone of some adhesion molecules such as CD2 and LFA-1 helps to bring the membrane bilayers of the two interacting cells together. This facilitates the interactions of the T-cell receptor (TCR) with its ligand, neither of which extend very far from the membrane surface. Second, this high density of molecules may help overcome the intrinsic weak affinity of the TCR for the small number of specific MHC—peptide complexes usually borne by APCs.What is it made of? Many molecules expressed in T lymphocytes or in APCs accumulate in the area of the synapse. There are some integral membrane proteins (such as the TCR, MHC molecules, CD3-ϵ, CD4, CD2, LFA-1, ICAM-1, CD48, CDS8 and CD45), some cytoskeletal proteins (talin and CD2-associated protein) and some signaling molecules (PKC-θ, Lck, Fyn and MEKK2).Are the molecules homogeneously distributed in the synapse? Surprisingly, no. Recent studies have shown that the molecules are arranged in two distinct microdomains; the synapse Consists of a central core surrounded by a ring (see FigureFigure). There is intriguing, but still incomplete, information about the segregation of some of the T-cell molecules within the synapse; for this reason, some proteins are not shown in the FigureFigure.Figure 1Cross-section of the immunological synapse 30 mm after the initial contact, showing the different molecules that partition in the synapse in two discrete areas — also called supramolecular activation clusters' (SMACs). The T-cell molecules TCR/CD3-g (dark blue), CD2 (purple) and PKC-9 (pink) are in the central SMAC (cSMAC), whereas LFA-1 (orange) and talin (red) are found in a concentric peripheral zone (pSMAC). The transmembrane molecules are illustrated in proportion to their actual molecular dimensions. Antigenic peptides bound to MHC molecules are coloured grey, CD48/CD58 molecules are coloured brown and ICAM-1 is coloured yellow.View Large Image | View Hi-Res Image | Download PowerPoint SlideSo, is everything important clustered into the synapse? It's true that most molecules known to be involved in TCR-dependent signalling are found in the synapse, so that immunologists now tend to ask of any particular molecule in which they are interested: ‘Tell me if you are in the synapse and I'll tell you if you are important for T-cell activation’. Nevertheless, it turns out that many proteins are not present in the synapse and, in some cases, only particular isoforms accumulate in the synapse. The only protein that has been shown to be specifically excluded from the synapse is CD43.How do proteins get moved to the synapse? The T-cell cytoskeleton —in particular, actin rnicrofilaments and myosin motors — is thought to have a key role in driving the redistribution of synapse molecules.What remains to be discovered? A lot. Imaging of GFP-tagged molecules is now being used to follow the redistribution of molecules in living cells. This provides both precise timing and spatial information. Interestingly, the architectural changes can be correlated with the intensity of T-cell activation through the measurement of the rise in intracellular calcium concentration that occurs within seconds after antigen recognition. With the development of new GFP variants and the possibility of transducing normal T lymphocytes using retroviruses, there's bound to be plenty more information soon.Where can I find out more?Brown MJ, Shaw S: T-cell activation: interplay at the interface. Curr Biol 1999, 9:R26-R28.Grakoui A, Bromley SK, Sumen C, Davis MM, Shaw AS, Allen PM, Dustin ML: The immunological synapse: a molecular machine controlling T cell activation.Science 1999, 285:221-227.Monks CRF, Freiberg BA, Kupfer H, Sciaky N, Kupfer A: Three-dimensional segregation of supramolecular activation clusters in T cells.Nature 1998, 395:82-86.Wulfing C, Davis MM: A receptor/cytoskeletal movement triggered by costimulation during T cell activation. Science 1998, 282:2266-2269.

New ways to look at axons inCaenorhabditis elegans

In the nematode Caenorhabditis elegans, a well-established model organism for the analysis of nervous system development and function, nerve processes can be labelled in the intact animal with markers based on the "Green Fluorescent Protein" (GFP). The generation of GFP variants with improved brightness and modified emission spectra potentiated the use of this marker for in vivo labelling of subcellular structures. This made it possible to label different groups of neurons and their axons in the same animal with GFP variants of different spectral characteristics. Here I show with double labelling experiments that spatial relationships of axons in small axon bundles can now be resolved at the light microscopic level. In the future this will largely circumvent the need for time-consuming electron microscopic reconstructions to detect local defects in axon outgrowth. Furthermore, I demonstrate that neuronal processes can now be traced even in the head ganglia, an area of the nervous system that was previously almost inaccessible for analysis due to the compact arrangement of cell bodies and axons.

Ca2+-Induced Deprotonation of Peptide Hormones Inside Secretory Vesicles in Preparation for Release

The acidic environment inside secretory vesicles ensures that neuropeptides and peptide hormones are packaged in a concentrated condensed form. Although this is optimal for storage, decondensation limits release. Thus, it would be advantageous to alter the physical state of peptides in preparation for exocytosis. Here, we report that depolarization of the plasma membrane rapidly increases enhanced green fluorescent protein (EGFP)-tagged hormone fluorescence inside secretory vesicles. This effect requires Ca2+ influx and persists when exocytosis is inhibited by N-ethylmaleimide. Peptide deprotonation appears to produce this response, because it is not seen when the vesicle pH gradient is collapsed or when a pH-insensitive GFP variant is used. These data demonstrate that Ca2+ evokes alkalinization of the inside of secretory vesicles before exocytosis. Thus, Ca2+ influx into the cytoplasm alters the physical state of intravesicular contents in preparation for release.

GFP Fusions for Fluorescence Detection of Ca2+ and Ca2+-Calmodulin in Living Cells

Abstract We have previously described fluorescent indicators for Ca2+ (FIP-CAs) and (Ca2+)4-calmodulin (FIP-CBs) whose responses are based on a ligand-dependent decrease in fluorescence energy transfer (FRET) between GFP variants. The indicators for (Ca2+)4-calmodulin contain calmodulin-binding domains, those for Ca2+ also contain an integral calmodulin (CaM) domain. We have developed new versions of these indicators constructed with enhanced blue- and red-shifted GFPs suitable for stable and transient expression in mammalian cells, and have begun to use them to investigate the relationships between the free intracellular concentrations of Ca2+ ([Ca2+]i) and (Ca2+)4-CaM ([(Ca2+)4-CaM]i). When the blue-shifted fluorophore is excited at 380 nm these constructs exhibit an emission peak at 505 nm due to FRET to the red-shifted fluorophore. We have made FIP-CBs with dissociation constants for (Ca2+)4-CaM of 0.5 nM, 20 nM, 300 nM and > 20 μM by introducing R →Q substitutions in the CaM-binding sequence, and have stablyexpressed them in HEK-293 cells (Fig. 1).

New unstable variants of green fluorescent protein for studies of transient gene expression in bacteria.

Use of the green fluorescent protein (Gfp) from the jellyfish Aequorea victoria is a powerful method for nondestructive in situ monitoring, since expression of green fluorescence does not require any substrate addition. To expand the use of Gfp as a reporter protein, new variants have been constructed by the addition of short peptide sequences to the C-terminal end of intact Gfp. This rendered the Gfp susceptible to the action of indigenous housekeeping proteases, resulting in protein variants with half-lives ranging from 40 min to a few hours when synthesized in Escherichia coli and Pseudomonas putida. The new Gfp variants should be useful for in situ studies of temporal gene expression.

Chapter 6 Enlightening Mitosis: Construction and Expression of Green Fluorescent Fusion Proteins

Publisher Summary This chapter discusses the properties of green fluorescent fusion (GFP) as a fluorochrome and strategies for constructing functional biofluorescent probes and for the expression of GFP chimeras in cells. The fluorescence spectrum of GFP is obviously the key functional property of the protein and has turned out to be remarkably mutable, leading to the generation of several useful spectral variants of GFP. Wild-type GFP is maximally excited in the near UV (396 nm) and possesses a secondary excitation peak at 475 nm. Emission at 508 nm produces the characteristic bright green color. A key feature of GFP is the rate and extent of protein folding and chromophore formation, which does not take place optimally at 37° C for the wild-type protein. Many early workers reported no or low fluorescence yield with wild-type GFP in cells cultured at 37°C, which could be compensated by growth at lower temperatures. The humanized GFP coding sequence is ideal for expression in a wide variety of mammalian species. As its excitation and emission spectra are quite similar to fluorescein, special filters are not required for efficient visualization of S65T derivatives. Similarly, these GFP derivatives are ideal for confocal microscopy as they are very efficiently excited by the 488 nm argon laser line. For dual-receptor experiments, the second GFP of choice is an enhanced blue derivative as this provides the best spectral resolution from GFP.

Chapter 9: GFP Variants for Multispectral Imaging of Living Cells

Unlike enzyme markers, green fluorescent protein can be visualized at high resolution in living cells using confocal microscopy. The images are not prone to fixation or staining artifacts, and can be of exceptional clarity. Moreover, the activities of living cells, such as cytoplasmic streaming, are clearly evident during microscopy. Ordinarily, movement within a sample is a nuisance, placing constraints on the use of sometimes lengthy techniques for noise reduction during confocal microscopy, such as frame averaging. However, it is possible to monitor dynamic events by time-lapse confocal microscopy, and this combination of a vital fluorescent reporter with high-resolution optical techniques shows much promise for use in cell biological and physiological experiments. Genetic systems such as that of Arabidopsis provide a large resource of potentially informative mutants, and there has been much recent improvement in techniques for determining the molecular basis of a particular phenotype. The use of fluorescent proteins will provide further tools for examining the biology of mutant cells. The precision with which particular cellular structures can be decorated with GFP and the ease with which subcellular traffic can be monitored indicate that this approach will be very useful for cell biological and physiological observations, particularly for detailed examination of plant mutant phenotypes.