Journal of Experimental Botany, Vol. 52, No. 356, pp. 529-539,
April 2001
© 2001 Oxford University Press
GFP imaging: methodology and application to investigate cellular compartmentation in plants
Department of Molecular Biology and Genetics, Cornell University, Biotechnology Building, Ithaca, NY 14853, USA
Received 31 March 2000; Accepted 19 September 2000
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Abstract |
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 Top Abstract IntroductionDetermination of the...Compartment labelling with GFPFluorescent labelling of...GFP for measurement of...ConclusionsSupplementary MaterialReferences |
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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.
Key words: Green fluorescent protein, microscopy, organelle, localization, photobleaching, plastid tubule, confocal microscopy.
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Introduction |
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TopAbstractIntroductionDetermination of the...Compartment labelling with GFPFluorescent labelling of...GFP for measurement of...ConclusionsSupplementary MaterialReferences |
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Expression of genes encoding green fluorescent protein (GFP) within cells has engendered experiments that could not be imagined ten years ago. Among the many applications are ones which permit new inquiries into the organization of cellular activities. This review will specifically focus on the GFP expression and imaging methods that have and will continue to produce advances in understanding compartmentation within plant cells. Details of the use of particular fluorescence, confocal laser-scanning, or multiphoton microscopes to visualize GFP-derived fluorescent proteins will not be covered, but can be found in many cited references. Research that has exploited GFP imaging to gain new insights will be briefly described in order to illustrate the experiments that are feasible.
At the present time, GFP imaging has revealed more about animal and fungal cells than about plant cells, partly because of early problems with the use of the jellyfish GFP gene in plants. The original animal gene contains sequences that are recognized erroneously as an intron in plants, leading to poor expression levels (Haseloff et al., 1997
; Rouwendal et al., 1997). The production and distribution of modified, thermostable, brightly fluorescent GFPs suitable for expression in plant cells (Reichel et al., 1996; Chiu et al., 1996; Pang et al., 1996; Davis and Vierstra, 1998; Haseloff et al., 1997; Haseloff, 1999) has stimulated many experiments with plant systems. Additional genes suitable for use in vascular plants have become available with the recoding of GFP for expression in mammals and other organisms (Chiu et al., 1996; Muldoon et al., 1997; Fuhrmann et al., 1999), as the codon alterations often have fortuitously destroyed the cryptic splice site sequence. Thus some, though not all, of the new GFP variants with altered spectral properties may be functional in plants without further alterations. The many different variants of GFP are discussed in a number of reviews and papers (Cubitt et al., 1995; Cormack et al., 1996; Leffel et al., 1997; Patterson et al., 1997; Misteli and Spector, 1997; Haseloff and Siemering, 1998; Haseloff, 1999) and will not be covered here. Nevertheless, the selection of a plant-expressable GFP variant with appropriate features is obviously a critical decision during experimental design.
One of the chief advantages of GFP as a fluorescent probe is its lack of a requirement for an exogenous cofactor (Chalfie et al., 1994). GFP can be expressed within intact tissues and processes monitored without the disturbance caused by introduction of reagents. Because of the impermeability of the plant cell to many stains and dyes readily taken up by animal cells, GFP technology may ultimately prove to be a more important stimulus to plant than to animal cell biology.
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Determination of the intracellular location of proteins |
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TopAbstractIntroductionDetermination of the...Compartment labelling with GFPFluorescent labelling of...GFP for measurement of...ConclusionsSupplementary MaterialReferences |
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To understand how the plant cell is functionally organized, it is necessary to know where enzymes and regulatory proteins are located in specific plant cells at particular times in development and under particular environmental conditions. Where a particular enzyme is located is essential information for understanding intracellular compartmentation of metabolism. Compartments prevent entry of particular proteins, ions and compounds to prevent undesirable reactions, and sequester participants in enzymatic reactions to facilitate cellular processes. A number of known examples exist in which the enzyme and substrate for a particular reaction are located in one compartment, but the product is then utilized in another, raising questions concerning how products and substrates move from one compartment to another to complete a metabolic pathway. Plant cells also harbour many enzymes whose intracellular location is unknown. Furthermore, the enzymatic reactions carried out by many previously unknown proteins, discovered through brute-force genomic sequencing, may not be readily elucidated unless the subcellular compartmentation of the unknown enzyme is determined.
Predictions of intracellular location from DNA sequence alone by current computer methods are helpful but not conclusive. Furthermore, some plant proteins have been observed to be targeted to more than one location (Small et al., 1998). Cell fractionation and purification of a protein for verification of intracellular location is often technically challenging, and antibody production for immunodetection of a protein in sectioned tissues can be time-consuming and laborious. Fusion of GFP coding sequences to coding regions of genes of unknown location has therefore become an extremely valuable tool for determining where a protein, and thus a biochemical or regulatory process, resides within the plant cell.
GFP coding sequences have been fused at either the 5' or 3' end of the coding region of a DNA sequence of interest, leading to production of N-terminal or C-terminal fusions of GFP. Such chimeric genes have been incorporated into stable transgenic plants, or introduced into plant cells for transient assays. Production of stable transgenic plants has the advantage that many different cell types can be examined in which the GFP/protein fusion is expressed, while not all cell types are technically suitable for transient expression. Furthermore, because of the damage that often occurs during DNA incorporation and the variability in amount of gene delivered and GFP transcript produced in transient assays, analysis of healthy stable transformants may sometimes be more reliable than analysis of cells that have been disrupted for DNA uptake. Nevertheless, the rapidity and simplicity of transient assays makes them a valuable tool, especially before investing the time in production of stable transformants. Onion epidermis, which has an exceptionally clear cytoplasm and consists of a single layer of living cells, appears to be a particularly useful material for transient assay. Following particle bombardment with various constructs, cell wall, chloroplast, cytoplasm, nucleus, and endoplasmic reticulum (ER) could be labelled by different transiently expressed GFP fusions (von Arnim et al., 1998; Scott et al., 1999).
Because of its small size, GFP can enter nuclear pores, so that GFP not fused to other polypeptides is found in both cytoplasm and nucleus in plant, animal and yeast cells (Grebenok et al., 1997a, b; Köhler et al., 1997b; Haseloff and Siemering, 1998; Fig. 1A). Fusion of several plant gene sequences results in GFP fluorescence in the nucleus only, leading to the conclusion that the fused sequence codes for a nuclear-localized protein. Among the GFP/plant protein fusions localized to the nucleus are the cryptochrome CRY2, a blue light photoreceptor (Kleiner et al., 1999) and the ROOT HAIRLESS 1 gene, which is needed for primary root hair formation and seedling viability (Schneider et al., 1998). Fusion of GFP with a geminivirus movement protein revealed that it serves as a nuclear shuttle protein (Lazarowitz and Beachy, 1999).
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Fusion of other proteins of uncertain destination has revealed their compartmentation in plastids, mitochondria, or cytosol. Proteins with geranylgeranyl diphosphate synthase activity (Zhu et al., 1997
) and a NADP-dependent isocitrate dehydrogenase (Galvez et al., 1998) were shown to be located in mitochondria. Fusion of genes homologous to phage-type RNA polymerases revealed that one was mitochondrial-localized and the other was transported to the chloroplast (Hedtke et al., 1999). Three nuclear-encoded RNA polymerase sigma factors were all shown to be plastid-localized by fusion of putative targeting signals to GFP (Kanamaru et al., 1999). The Arabidopsis serine acetyltransferases encoded by three different cDNA clones were assigned to three different compartments—cytosol, chloroplast, and mitochondria—based on localization of GFP fusion proteins (Noji et al., 1998). GFP fusions also verified that the product of the Arabidopsis PALE CRESS nuclear gene, required for normal chloroplast development (Meurer et al., 1998) and AST82, a sulphate transporter (Takahashi et al., 1999), are normally found in the chloroplast. The success of GFP fusions in determining subcellular localization of proteins encoded by unknown cDNAs has led to the development of a method termed ‘Motif Trap’ in which random DNA fragments are fused to gfp, cells expressing GFP in various subcellular compartments are isolated, and the expressed DNA fragments are then cloned by RT-PCR (Bejarno and Gonzalez, 1999).
GFP fusions may also be used to determine whether a protein moves from one location to another within the plant cell as the result of an environmental stimulus or developmental cue. GFP sequences were fused to sequences encoding phyA and phyB, members of the phytochrome family of photoreceptors (Kircher et al., 1999; Yamaguchi et al., 1999). Upon irradiation with red light, phyA and phyB were translocated to the nucleus. Treatment with far-red light, which can inhibit photomorphogenic responses to red light, was found to reduce red light-induced movement of phyB but not phyA to the nucleus (Kircher et al., 1999). Similarly, by fusion with GFP, the movement of parsley bZIP transcription factor CPRF2 was also shown to be under phytochrome control. GFP fusions have also been used to study both the nuclear export pathway in plants (Haasen et al., 1999) and trafficking of protein through plasmodesmata (Imlau et al., 1999).
Fusion with GFP is not necessarily a foolproof method of determining the intracellular location of a protein. Because fusion of GFP to an enzyme has often not inhibited enzyme activity, GFP is commonly thought to be an innocuous tag. However, some evidence exists that high levels of expression in certain locations can be detrimental to the cell. For example, Haseloff et al. found it difficult to obtain highly fluorescent transgenic plant lines unless the GFP was sequestered in the ER (Haseloff et al., 1997). While by far most reports of GFP expression in animal cells describe no toxic effect of GFP expression, detrimental effects have occasionally been noted (Hanazono et al., 1997; Liu et al., 1999). Furthermore, it remains possible that, in certain cases, the GFP/protein fusion and the wild-type protein will differ in their subcellular locations. The presence of GFP could possibly hinder proper localization encoded by a transit sequence on the attached protein. Most passenger proteins were efficiently incorporated into the organelle in the many studies of mitochondrial targeting by transit sequences in fungi and plants, but incorrect or incomplete localization has sometimes been observed (Kimura et al., 1993). Conversely, if fusion of GFP causes a conformational change in the attached protein, then a localization signal could become active though it is normally sequestered in the absence of GFP or when it is lacking some endogenous ligand.
Another potential problem is that the fusion of the gene sequence of interest with gfp might result in improper folding or instability of the encoded chimeric GFP, so that little or no fluorescence is detectable. Negative results of this type are rarely described in the literature, but anecdotal evidence suggests that some investigators have encountered difficulties in obtaining brightly fluorescent protein with certain chimeric GFP constructions. Proper folding of amino-terminal attached protein has been shown to affect folding of C-terminal GFP, thereby affecting the level of its fluorescence (Waldo et al., 1999). Fusion of the signal sequence of the maltose-binding protein to GFP was shown to cause improper folding and lack of fluorescence of the hybrid protein in E. coli (Feilmeier et al., 2000).
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Compartment labelling with GFP |
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TopAbstractIntroductionDetermination of the...Compartment labelling with GFPFluorescent labelling of...GFP for measurement of...ConclusionsSupplementary MaterialReferences |
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As well as determining where particular proteins are located, GFP fusions with transit sequences or entire proteins can be used for deliberate labelling of particular compartments (Köhler, 1998
). The purposes of such experiments may be to study one or more compartments with regard to number, size, shape, mobility, interaction with other organelles, and observation of dynamic changes during development or environmental response. Localization of GFP has allowed visualization of the organization of the plant cell as has not been previously possible (Figs 1
, 2).
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Several investigators have deliberately labelled nuclei to study the compartment. Chytilova et al. have designed a chimeric gene resulting in expression of a GFP/ ß-glucuronidase (GUS) fusion that contains a NLS in order to produce transgenic plants carrying labelled nuclei for studies of nuclear shape and movement during the cell cycle (Chytilova et al., 1999
). Increasing the size of the protein by fusion with GUS allows retention of the GFP in the nucleus (Grebenok et al., 1997a, b; von Arnim et al., 1998). The nuclear localization signal (NLS) from SV40 attached to GFP allowed production of transgenic plants with green fluorescent nuclei (Fig. 1B) but a detectable amount of this smaller GFP fusion protein remains in the cytoplasm (RH Köhler and MR Hanson, unpublished results). Additional compartments that have been labelled in plant cells are the vacuole (Di Sansebasiano et al., 1998), peroxisomes (Mano et al., 1999), Golgi (Boevink et al., 1998; Essl et al., 1999), mitochondria (Köhler et al., 1997b; Yasuo et al., 1999; Logan and Leaver, 2000; Figs 1C, 2D), plastids (Köhler et al., 1997a, c; Hedtke et al., 1999; Jang et al., 1999; Tirlapur et al., 1999; Köhler and Hanson, 2000; Figs 2A, 3), cytoskeleton (Kost et al., 1999; Ueda et al., 1999; Hasezawa et al., 2000), plasma membrane (Bischoff et al., 2000), cell wall (Scott et al., 1999) and ER (Haseloff and Siemering, 1998; Figs 1D, 2C).
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The identity of some of the cellular structures labelled by a GFP fusion—such as nucleus or cell wall can be determined by simple comparison to the light microscopic image of the cell. The identity of other fluorescent organelles or structures visualized is not always obvious, however, and requires experimental determination. A common method is to label a convenient cell type with a specific chemical stain and compare the image to the pattern of green fluorescence. For example, specific labelling of mitochondria can be verified with mitochondrial-specific fluorescent dyes (Köhler et al., 1997
b), and co-localization of GFP with the red signal of autofluorescent chlorophyll can verify chloroplast localization.
Another type of intracellular ‘compartment’ that has been labelled with GFP is plant virus. The movement of fluorescent viruses through plasmodesmata and from one cell to another has provided many new insights into virus propagation and transmission (Oparka et al., 1997). Furthermore, GFP can be used to label viral proteins, such as the movement protein (Epel et al., 1996) to observe the association of the viral-encoded protein with structures within the cell. Further discussion of this topic can be found in several recent papers (Itaya et al., 1998; Lazarowitz and Beachy, 1999).
While most subcellular compartments must be labelled by introducing the chimeric gfp into the nucleus and targeting the protein to the desired location, an alternative strategy is possible with plastids of a few species, those in which plastid genomes can be transformed (Svab and Maliga, 1993). A GFP coding region was introduced into tobacco or potato chloroplasts by particle bombardment (ML Reed, S Wilson, CA Sutton, MR Hanson, unpublished results; Sidorov et al., 1999) or by protoplast DNA uptake (Gray et al., 1999), resulting in transgenic plants containing brightly fluorescent plastids (Fig. 3). Khan and Maliga have fused a GFP coding region to an antibiotic resistance selectable marker in a chloroplast DNA transformation vector and have reported transgenic plants with fluorescent plastids (Khan and Maliga, 1999). GFP can also be expressed transiently from within chloroplasts following particle bombardment (Hibberd et al., 1998) or chloroplast microinjection (Knoblauch et al., 1999).
In addition to discerning features of compartments in normal plant cells, labelling of compartments will be useful for analysing cell function disrupted by mutation or pathogen attack. For example, transgenic plant cells with GFP-labelled ER could be used to observe changes in the ER caused by infection with tobacco mosaic virus (Reichel and Beachy, 1998) or cowpea mosaic virus (Carette et al., 2000). GFP labelling of plastids should be particularly valuable for examination of mutant chlorophyll-deficient plants, in which abnormal plastids are difficult to observe by light microscopy.
One more application of compartment labelling that remains to be further exploited in plant systems is tagging of compartments to facilitate their isolation. Galbraith et al. demonstrated that GFP-labelled nuclei could be separated from cell debris by flow sorting (Galbraith et al., 1999). It may become possible to use GFP fluorescence as a marker to isolate GFP-labelled organelles and compartments that are not easily separated by more traditional means.
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Fluorescent labelling of multiple compartments |
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TopAbstractIntroductionDetermination of the...Compartment labelling with GFPFluorescent labelling of...GFP for measurement of...ConclusionsSupplementary MaterialReferences |
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A number of biosynthetic and metabolic processes require co-operation of different organelles or compartments (reviews in Tobin, 1992
). Positioning of subcellular structures can affect diffusion efficiency, so changes in distance and contact between compartments during development or environmental response may be significant for efficient function. Observations of changes in positions and movement of compartments can be made by labelling them with fluorescent probes whose signals can be distinguished from one another.
A few plant compartments and structures are naturally autofluorescent at wavelengths distinct from GFP excitation and emission, allowing visualization of a GFP-labelled structure along with the autofluorescent body. To date, autofluorescence in combination with GFP has been chiefly exploited to visualize red-fluorescing chloroplasts in combination with GFP-labelled mitochondria, nuclei, or ER (Köhler et al., 1997a, b; Haseloff and Siemering, 1998; Fig. 1).
Only a few reports have appeared concerning double labelling of cells with GFP variants, but successful applications of this method are likely to increase rapidly in the next few years. Cyan and yellow fluorescent proteins that differ in excitation wavelength could be expressed in different compartments for visualization of multiple structures within one cell (Haseloff and Siemering, 1998, Haseloff, 1999). Homologues of GFP with different spectral properties, proteins that fluoresce red and yellow, have been isolated and are promising for double labelling experiments (Matz et al., 1999). In animal cells, both mitochondria and nuclei (Rizzuto et al., 1996), or both mitochondria and ER (Pinton et al., 1996) could be visualized, and chromatin and nuclear membrane were both labelled to analyse events during cell division (Ellenberg et al., 1999).
Presently, several fluorescent chemical probes are available to image compartments (such as mitochondria or cell wall) separately from GFP-labelled compartments (Haseloff, 1999). Another future development may be the use of other fluorescent proteins in combination with GFP variants. Phytochromes can be adapted for use as fluorescent labels (Murphy and Lagiarias, 1997), and their red fluorescence is easily separated from the green of GFP. These so-called ‘phytofluors’ do have the drawback that an exogenous cofactor must be added, making them not as versatile as GFP. Another peptide tag that requires a co-factor, a peptide which binds to a fluorescein derivative (Griffin et al., 1998), may also eventually find application in plant cells in combination with GFP.
Further improvement in microscopes and spectroscopy methods will also facilitate separation of signals from GFP variants. Fluorescence lifetime imaging microscopy (FLIM) has successfully been utilized to separate some spectrally similar GFP variants because their fluorescence lifetimes could be distinguished (Pepperkok et al., 1999). FLIM can also be used in combination with FRET specifically to detect the donor versus acceptor fluorescence signal (Wouters and Bastiaens, 1999).
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GFP for measurement of parameters within compartments |
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TopAbstractIntroductionDetermination of the...Compartment labelling with GFPFluorescent labelling of...GFP for measurement of...ConclusionsSupplementary MaterialReferences |
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Cell fractionation has revealed that compartments differ from each other in concentration of molecules. A few molecular probes have also been available to discern gradients and dynamic changes of certain parameters within different compartments in vivo. GFP variants will provide new means to evaluate and compare the contents of different compartments. Modified GFPs have been synthesized that can serve as indicators of pH, membrane voltage, proteases, metals or calcium. GFP variants that display pH-dependent absorbance and fluorescent emission were produced. pH in the cytosol, nucleus, and mitochondrial matrix of HeLa cells could be measured following incorporation of the GFPs into these compartments (Llopis et al., 1998
). Measurement of voltage changes was possible in animal cells by fusion of GFP with a voltage-sensitive K+ channel (Siegel and Isacoff, 1997). To observe protease activity, a blue fluorescent GFP variant (BFP) and a GFP variant were linked by a protease domain in a hybrid protein, and Fluorescence Resonance Energy Transfer (FRET) could occur, with BFP as donor and GFP as receptor, unless protease cleavage occurred, separating the donor and acceptor GFPs (Heim and Tsien, 1996). Metal ions could be assayed by metal-binding GFP variants, a technique based on the quenching of fluorescence caused by proximity of metals to chromophores (Richmond et al., 2000) or by using FRET occurring in a rearranged GFP containing a metal-binding domain (Baird et al., 1999). Similarly, hybrid GFP/calmodulin proteins (‘cameleons’) have been made to detect intracellular calcium. In the cameleon, increase in Ca2+ concentration results in energy transfer between a cyan fluorescent protein and a yellow fluorescent protein (Miyawaki et al., 1999). Of these GFP sensors, only the use of the cameleons has been reported in plants as yet. They have been exploited to examine calcium changes in Arabidopsis guard cells (Allen et al., 1999). Additional sensors based on GFP will likely become available, with the discovery that several GFP variants with rearranged coding regions remain fluorescent when expressed, allowing addition of sensing domains (Baird et al., 1999).
FRET can also potentially be utilized to observe the interactions of two proteins within one compartment or to detect movement of a protein from one compartment to another (review by Pollok and Heim, 1999). Though such a use has not yet been reported in plant systems, the interaction of two proteins in mammalian mitochondria during apoptosis has been observed by GFP-FRET (Mahajan et al., 1998).
Information about protein concentration and mobility within the living cell can be gained by deliberate photobleaching of GFP variants. Fluorescent molecules such as GFP undergo photodamage when irradiated, an undesirable phenomenon if it occurs rapidly before images can be acquired. Most GFP variants in use have been selected for slow photobleaching so that material may be viewed microscopically for adequate time periods. Localized intense irradiation by a laser, however, can be used to photobleach the GFP molecules within a cell. The movement of GFP into the irradiated region from other parts of the cell can then be observed, and the rate of Fluorescence Recovery after Photobleaching (FRAP) will give an estimate of the diffusion coefficient and an indication of the fraction of the GFP that is mobile (White and Stelzer, 1999; Swaminathan et al., 1997). A related technique is GFP photoactivation, in which GFP in a small region is given a pulse of blue light, causing the irradiated GFP molecules to emit red, and the rate of movement of the red-emitting molecules is observed (Elowitz et al., 1997). These techniques have been used to measure the diffusion of GFP and GFP fusion proteins within animal mitochondria (Partikian et al., 1998) and within a single E.coli cell (Elowitz et al., 1999), and therefore should be applicable to plant plastids, mitochondria, and nuclei. Recently, the method has been improved by the use of multiphoton excitation, which gives a more defined photobleached volume to facilitate calculation of the diffusion coefficient (Brown et al., 1999).
Both FRAP and a related technique, Fluorescence Loss in Photobleaching (FLIP) can reveal whether flow occurs between two compartments or not. FRAP and FLIP have been quite valuable for studies of Golgi/ER trafficking in animal cells (Cole et al., 1996; Zaal et al., 1999). In plants, the first application of FRAP and FLIP was to examine whether plastids could exchange protein molecules through narrow connecting structures (‘stromules’) observed when GFP was targeted to the plastid stroma (Köhler et al., 1997b). A laser irradiated the GFP in one of two connected plastids (Fig. 4A), causing loss of fluorescence initially only in the irradiated plastid. Six seconds later, the fluorescence of the irradiated plastid had increased and that of the unirradiated plastid had decreased. In a FLIP experiment, the bridge between two connected plastids was irradiated repeatedly, so that molecules passing through the bridge would be photobleached (Fig. 4B). The irradiation resulted in loss of fluorescence in both plastids, indicating that molecules are flowing between them (Köhler et al., 1997b). More recently, FLIP was used in tissue-cultured cells to determine the degree of autonomy of plastids that are clustered around the nucleus and have extraordinarily long thin tubules extending outward to the cell periphery (Fig. 2A). Photobleaching of plastid bodies near the nucleus resulted in loss of fluorescence only in the irradiated plastids and a few tubules extending from the irradiated plastids. The retention of GFP fluorescence in most tubules and unirradiated plastids indicates that, despite the striking appearance of a network (Fig. 2A), most of the plastids within the tissue cultured cell do not exchange GFP molecules (Köhler and Hanson, 2000). This result contrasts with images obtained with a true interconnected network, animal ER. Photobleaching of a small region of ER resulted in loss of fluorescence over the entire ER within an animal cell that contained GFP fused to a Golgi protein that flows through the ER (Zaal et al., 1999).
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An alternative to FRAP or GFP photoactivation for measuring diffusion coefficients and flow rates is Fluorescence Correlation Spectroscopy (FCS). In this method, fluorescence of a small number of molecules moving in and out of a very small volume is recorded, increasing the range of diffusion rate that can be observed (Thompson, 1991
; Xu et al., 1996; Schwille et al., 1999a, b). This technique could be applied to measure the rate of diffusion of stroma-targeted GFP in vivo (Köhler et al., 2000).
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Conclusions |
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TopAbstractIntroductionDetermination of the...Compartment labelling with GFPFluorescent labelling of...GFP for measurement of...ConclusionsSupplementary MaterialReferences |
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Knowledge of the mechanisms which allow metabolites and macromolecules to move through the cell to perform biochemical or regulatory processes is presently incomplete. GFP-based methods will be instrumental for learning more about the function of compartmentation and how organization is achieved. From large-scale genome projects, soon all of the sequences of proteins present in plant cells of several species will be predictable from DNA sequence. The location(s) of these proteins can be identified by GFP fusion methods. Interactions of two proteins can be probed by GFP/FRET methods. Movement of proteins within the cell and plant can be traced. Compartment labelling with GFP will facilitate analysis of the size, shape and number of different compartments and has already revealed previously unknown features of compartment morphology and interaction with other compartments. GFP sensors can be used to determine gradients in ions and metal concentration, pH and voltage, or to detect activity of particular enzymes. FRAP, FLIP and FCS can reveal the existence of intercompartmental communication and rates of movement of molecules within or between compartments. All of this data will generate a much more complete picture of the functional organization of the plant cell.
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Supplementary Material |
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TopAbstractIntroductionDetermination of the...Compartment labelling with GFPFluorescent labelling of...GFP for measurement of...ConclusionsSupplementary MaterialReferences |
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Supplementary Material is available at JXB Online.
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Acknowledgments |
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Research by the authors described here was supported by the Energy Biosciences Program of the Department of Energy (DE FG02-89ER14030) and the National Science Foundation (MCB-9808101). We thank Susan Wilson and Martha Reed for providing their unpublished image of chloroplasts containing plastid-encoded GFP.
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Notes |
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1 To whom correspondence should be addressed. Fax: +1 607 255 6249. E-mail: mrh5{at}cornell.edu
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TopAbstractIntroductionDetermination of the...Compartment labelling with GFPFluorescent labelling of...GFP for measurement of...ConclusionsSupplementary MaterialReferences |
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