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New tools for in vivo fluorescence tagging
New tools for in vivo fluorescence tagging Sean Chapman1, Karl J Oparka2 and Alison G Roberts1 Engineering of fluorescent proteins continues to produce new tools for in vivo studies. The current selection contains brighter, monomeric, spectral variants that will facilitate multiplex imaging and FRET, and a collection of optical highlighter proteins that might replace photoactivatable-GFP. These new highlighter proteins, which include proteins that have photoswitchable fluorescence characteristics and a protein whose fluorescence can be repeatedly turned on and off, should simplify refined analyses of protein dynamics and kinetics. Fluorescent protein-based systems have also been developed to allow facile detection of protein–protein interactions in planta. In addition, new tags in the form of peptides that bind fluorescent ligands and quantum dots offer the prospect of overcoming some of the limitations of fluorescent proteins such as excessive size and insufficient brightness. Addresses 1 Programme of Cell–Cell Communication, Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, UK 2 Institute of Molecular and Plant Sciences, King’s Buildings, University of Edinburgh, Edinburgh EH9 3JH, UK Corresponding author: Chapman, Sean ([email protected])
Current Opinion in Plant Biology 2005, 8:565–573 This review comes from a themed issue on Cell biology Edited by Patricia C Zambryski and Karl Oparka Available online 26th September 2005 1369-5266/$ – see front matter # 2005 Elsevier Ltd. All rights reserved. DOI 10.1016/j.pbi.2005.09.011
Introduction Since the cloning of the gene for the green fluorescent protein of Aequoria victoria (avGFP), efforts have been made to modify the spectral characteristics and biochemical properties of a range of marine invertebrate-derived fluorescent proteins to enhance their utility as biological reporters. The genes for about 100 proteins, fluorescent and non-fluorescent, that are capable of autocatalytic chromophore formation have now been cloned [1]. Desirable traits include brightness (a product of the extinction coefficient and quantum yield), photostability, rapid maturation, small size, pH insensitivity, tolerance of fusions and resistance to oligomerisation. The in vivo expression of fusions to avGFP-like fluorescent proteins is generally the first method of choice for studying protein localisations, movement and interactions with other cellular proteins. Fluorescent proteins that have different www.sciencedirect.com
spectral characteristics are useful for simultaneous imaging of multiple proteins and for fluorescence resonance energy transfer (FRET) studies. The use of FRET-based biosensors in cell biology is reviewed by Lalonde et al. in this issue. Here, we review recent advances in fluorescent tagging that should enhance the spatial and temporal resolution of future studies, and should facilitate the identification of interacting protein partners.
Novel fluorescent reporter proteins The range of available spectral variants has expanded with the cloning of many new Anthozoan-derived chromoproteins [2]. However, the fact that most are obligate tetramers, and many form higher molecular weight aggregates, necessitates their genetic engineering to create useful fluorescent reporter proteins. Much effort has concentrated on developing fluorescent proteins that have red-shifted emission spectra, as autofluorescence in most eukaryotic cells is reduced at longer wavelengths, and on developing new acceptors for FRET studies. However, the utility of these new reporters might be compromised in plant tissues where the main peak of chlorophyll autofluorescence overlays the emission of such proteins (see Figure 1). The monomeric red fluorescent protein (mRFP) [3], an evolved form of the Discosoma coral RFP, has a lower extinction coefficient, quantum yield and photostability than its progenitor, but matures much more rapidly and has red-shifted excitation and emission spectra. mRFP has been taken as a starting point for further mutagenesis [4] and in vivo evolution [5], resulting in the generation of a set of new, monomeric fluorescent proteins, whose emission maxima range from 537 to 649 nm. These proteins provide emission maxima beyond that of the avGFP-derived yellow fluorescent protein (YFP), and they offer improved brightness, fusion tolerance, maturation rates and photostability when compared with the popular mRFP.
Fluorescent peptide ligands The utility of all avGFP-like fluorescent proteins is limited by their size of circa 25 kDa [6]. Phage library screening has identified a peptide of only 38 amino acids that binds the fluorescent dye Texas red [7], and this has been used to localise a membrane-targeted GFP fusion protein in mouse embryo cells. The specificity of this peptide results from its binding to the xanthene core of Texas red, which is shared by fluorescent X-rhod calcium sensors; hence, this peptide might be useful for localised calcium sensing. The six amino acid, tetracysteine motif that covalently binds biarsenate derivatives of fluorescein and resorufin (Fluorescein Arsenical Helical binder [FlAsH] and Resorufin Arsenical Helical binder Current Opinion in Plant Biology 2005, 8:565–573
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Figure 1
Emission spectra of the main sources of autofluorescence in roots and leaves when excited with commonly used laser sources for confocal microscopes. Lignin (dotted lines) and chlorophyll (solid lines) were excited, in roots and leaves respectively, using nine different excitation wavelengths, and emission spectra were collected. All lasers were run at 50% maximal power output. Although the amplitude of autofluorescence varies between laser sources, it is clear that, in roots (or in the vascular tissue and cell walls of aerial plant parts), lignin fluorescence will cause most interference with imaging at wavelengths of between 490 and 620 nm. By contrast, chlorophyll fluorescence in leaves and stems is most prevalent between 650 and 770 nm. In practice, this means that the imaging of green fluorescent proteins is most problematic in roots, whereas red fluorescent proteins can be difficult to discriminate in tissues that have many chloroplasts, and are unsuitable as fusion partners for proteins that localise to, or near, chloroplasts.
[ReAsH]) is an even shorter peptide tag [8]. It has been used in mammalian [8], Drosophila [9] and yeast [10] cells, but not in plants. The bound, fluorescent labels can generate singlet oxygen upon illumination, and this oxygen species can be of utility in downstream electron microscopy studies by facilitating aminobenzidine polymerisation and the production of an electron-dense precipitate [11]. The tetracysteine tag needs a reducing environment, however, and there are concerns about endogenous thiol groups giving non-specific labelling [6]; for example, the Arabidopsis HMA3 Zn/Cd heavymetal transporter actually contains a tetracysteine motif. Signal background might be ameliorated by a recent fluorescence-assisted cell sorting (FACS)-based genetic screen that can be used to optimise the tag’s context [12]. This screen identified a twelve amino acid motif (FLNCCPGCCMEP) that produces more than 25 times the contrast produced by the earlier motif in living cells. The benefit of using the small tetracysteine motif is emphasised by a yeast study in which fusions between GFP, or between one and three repeats of this motif, and b-tubulin were all incorporated into microtubules, but only fusions with one or two tetracysteine repeats could functionally substitute for the wildtype protein [10].
Quantum dots Semiconductor nanocrystals or quantum dots are a group of fluorescent labels that have been the centre of much recent interest [13]. Their colloidal semiconductor cores, which are between 2 and 10 nm in diameter, confer several desirable traits, including photostability, broad Current Opinion in Plant Biology 2005, 8:565–573
absorption spectra, narrow emission spectra, a broad range of size- and composition-dependent emission maxima, long fluorescence lifetimes that allow time-delayed detection and extreme brightness that permits single molecule detection. These labels have already proven themselves in vivo in animal studies for cell tracking [14] and in studies of cell-surface proteins [15], and are likely to be of use in biomedical applications [16]. How useful these labels will be for in vivo plant studies is unclear as they are membrane impermeant. Furthermore, the semiconductor cores require the addition of a pacifying coat and some form of biological functionalisation (e.g. through the coupling of peptide ligands or antibodies), thereby increasing their size above 10 nm. Whether such large molecules can effectively traverse the plant extracellular matrix and then be taken up is unclear, limiting their in vivo utility, but they might be useful novel probes for endocytosis in plants. Protein transduction domains [17] might facilitate this process, and a nona-argenine peptide fusion to GFP has been shown to allow rapid internalisation of GFP (circa 5 nm) into plant tissues [18]. However, linkage of the same peptide sequence to quantum dots only resulted in their accumulation in endosomes and lysosomes in mammalian cells [19]. Nonetheless, antibody-coupled quantum dots have been successfully used to localise a pollen tube adhesion protein to the tube tip [20].
Novel highlighters Although fluorescent proteins such as avGFP can be used to study protein dynamics and kinetics through photowww.sciencedirect.com
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Table 1 Summary of spectroscopic data for highlighter proteins. Protein
l (ex)
l (em)
e
QY
Relative brightness
Oligomeric state
Conversion wavelength
pKa
PA–GFP (N) PA–GFP (P) Kaede (N) Kaede (P) Kikume (N) Kikume (P) Kindling (N) Kindling (P) 1 PA–mRFP1-1 (N) PA–mRFP1-1 (P) PS–CFP (N) PS–CFP (P) PS–CFP2 (N) PS–CFP2 (P) mEosFP (N) mEosFP (P) DRONPA (N)
400 504 508 572 507 583 580 580 588 578 402 490 400 490 505 569 503
515 517 518 580 517 593 600 600 602 605 468 511 468 511 516 581 518
20 700 17 400 98 800 60 400 28 200 32 600 123 000 59 000
0.13 0.79 0.88 0.33 0.70 0.65 <0.001 0.07
8 41 259 59 59 63 0 12
413
4.5
350–410
5.6 5.6 7.8 5.5
0.08 0.16 0.19 0.20 0.23 0.64 0.62 0.85
2 16 15 26 32 128 68 240
Monomer Monomer Tetramer Tetramer Tetramer Tetramer Tetramer Tetramer Monomer Monomer Monomer Monomer Monomer Monomer Monomer Monomer Monomer
10 34 27 43 47 67 37 95
000 000 000 000 000 200 000 000
350–420 530–560 430–490 375–385 405 405 400 490 (400)
4.4 4.0 6.0 4.3 6.1 5.8 5.8 5.0
Data are provided for the native (N) and photoconverted (P) states of the proteins. l (ex) and l (em) are the excitation and emission maxima, respectively, in units of nm. e is the extinction coefficient in units of M 1 cm 1. QY is the fluorescence quantum yield. Relative brightness is given as a percentage of the commonly used EGFP. 1 Figures are given for irreversibly activated, kindled, protein.
bleaching techniques [21], the advent of fluorescent proteins that undergo light-induced spectral changes, termed optical highlighters, should facilitate such studies. The first effective highlighter was an avGFP mutant that, following UV or violet illumination, displayed a 100-fold increase in emission upon 488 nm excitation (see Table 1 and Figure 2; [22]). However, the low quantum yield of photoactivatable GFP (PA-GFP) before activation can make targeting difficult. Furthermore, activation with short-wavelength light can cause damage: activation with a single-photon laser of PA-GFP fused to a histone variant prevented nuclear fission in Drosophila embryos [23]. Two-photon excitation reduced chromatin damage, but even so, longer wavelengths and lower energies were required to allow continued nuclear division. To date, PA-GFP has been used in cellular marking [24], and to study inter-organelle [22,25] and membrane-associated protein dynamics [26]. We have used PA-GFP to study plasmodesmatal connections, and plants that have a PAGFP transgene offer an elegant and facile system for studies of symplastic communication within and between tissues (see Figure 3a–c). Since the discovery of PA-GFP, several Anthozoan proteins, including Kaede and Kikume, that fluoresce green before UV or violet illumination and red subsequently have been cloned (see Figure 2; [2,27–29]). Irreversible photoconversion of Kaede results from extension of the chromophore in a reaction that involves the cleavage of the peptide backbone [30]. The utility of these Anthozoan-derived proteins as highlightable fusions is compromised by their oligomerisation, although fusions of Kaede to organelle-targeting sequences have been successfully used to monitor organelle dynamics in plants www.sciencedirect.com
[31]. Mutagenesis of the Anthozoan-derived mRFP has also produced photoactivatable mRFPs [32]. However, like an earlier Anemonia sulcata-derived protein that contains similar amino-acid substitutions and can be reversibly photoactivated with green light (Kindling fluorescent protein [33]), these variants, which are activated by more damaging UV light, are poorly fluorescent after photoactivation. Mutagenesis of the highly fluorescent aceGFP protein, which was derived from a non-fluorescent chromoprotein of the Hydrozoan Aequorea coerulescens, has produced a photoswitchable cyan fluorescent protein (PS-CFP) [34]. Upon violet illumination, the cyan fluorescent form converts to a green fluorescent form (Figure 2). Both forms give approximately 2.5-fold less fluorescence than activated PA-GFP but there is a 1500-fold ratiometric difference between the native and switched forms. PS-CFP has been used to monitor the dynamics of the membraneassociated human dopamine transporter, and there is now a faster-maturing commercial derivative, PS-CFP2 (EvrVgen), which is brighter and gives a ratiometric difference of over 2000-fold. One problem with PSCFP2 is that violet light is used both to image and to photoconvert the cyan fluorescent form, necessitating cautious imaging to prevent premature photoconversion. However, in our hands, PS-CFP2 is a useful protein for studies of the dynamics of different protein pools in plant cells (see Figure 3d,e), and has an advantage over PAGFP in that it is easily detectable pre-activation. The gene for a green-to-red switchable protein, EosFP, which has homology to Kaede, has been cloned from the coral Lobophyllia hemprichii, and dimeric and monomeric forms have been engineered from the tetrameric wildtype proCurrent Opinion in Plant Biology 2005, 8:565–573
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Figure 2
The photoconversion mechanisms for the most useful optical highlighters. The photoconversion sequence for each highlighter is shown running left to right across the page, beginning in each case with the native or inactivated form. Each element shows a leaf epidermal cell containing a nucleus, imaged under different fluorescence excitation wavelengths (in this example under 561, 488 and 405 nm laser light sources). Arrows represent the light used for photoconversion: high-and low-intensity illumination is indicated by large and small arrows respectively, and the colours of the arrows indicate the wavelength used. Emission colours appear in the nucleus and/or cytoplasm of each cell. (a) A cell containing photoactivatable green fluorescent protein (PA-GFP) appears non-fluorescent (grey) prior to photoconversion in the first image. After illuminating the nucleus with 405-nm light (purple arrow), the PA-GFP protein exhibits green fluorescence, which slowly diffuses out into the cytoplasm, as illustrated in subsequent cells. (b) The irradiation of Kaede and Kikume highlighters causes the native, green protein to convert to a red form. The green protein is imaged under 488 nm light and switched to the red form using 405 nm illumination. Once converted, the red form must be imaged using 561 nm illumination. A pool of non-switched protein will still be visible if 488 nm illumination is used. (c) Native PS-CFP protein is cyan fluorescent under violet illumination. Following illumination of the nucleus by high-intensity 405 nm light, cyan fluorescence is quenched and, when excited with 488 nm light, PS-CFP emits green light. The GFP diffuses out of the nucleus and into the surrounding cytoplasm. Non-switched cyan-fluorescent protein is still visible if the cell is imaged using violet excitation. (d) Photoswitching of the green to red form of the monomeric EosFP protein can be achieved, and imaged, using the same methods as described for Kaede and Kikume above. (e) DRONPA exists in two inter-convertible protonated and deprotonated forms. When irradiated at 488 nm, the deprotonated protein pool strongly emits green fluorescence. Following intense illumination with 488 nm light, the protein population is driven towards the almost non-fluorescent protonated form. Subsequent illumination with 405 nm light switches the protein back to the deprotonated, green-fluorescent form.
tein [35]. Like PS-CFP, EosFP is photoconverted by violet light, but the green and red forms are both excited by longer wavelengths (see Figures 2 and 3 Figures 2 and 3f-i). The various forms of EosFP have been used to precisely photoconvert and monitor various fusion proteins in mammalian cells with the sole caveat that the monomeric form is limited to temperatures below 30 8C. A near-ideal monomeric highlighter protein has recently been obtained through the cloning of the gene for a GFP from Pectiniidae coral, and its power demonstrated by Current Opinion in Plant Biology 2005, 8:565–573
monitoring the fast nucleocytoplasmic shuttling of a mitogen-activated protein kinase fusion [36]. Strong illumination of the protein, called DRONPA, with 490 nm light leads to loss of green fluorescence, but this can be repeatedly recovered by illumination with low levels of violet light. Consequently, this is the first protein to allow fluorescence to be switched off and on at will, providing a potentially excellent reporter for dynamic cell processes (see Figures 2 and 3 Figures 2 and 3m,n). DRONPA has a relatively fast bleaching rate under www.sciencedirect.com
New tools for in vivo fluorescence tagging Chapman, Oparka and Roberts 569
Figure 3
Imaging of fluorescent highlighters and bimolecular fluorescence complementation in plants. All images were collected using a Leica SP2 AOBS-spectral confocal laser scanning microscope (CLSM). (a–c) Photoactivation of PA-GFP in a transgenic, PA-GFP-expressing, Arabidopsis apical meristem. (a) Before activation, no green signal is visible. (b) Following the localised photoactivation of a single cell at the tip of the meristem, GFP becomes visible and (c) subsequently moves symplastically into adjacent cells. The scale bars in (a–c) represent 20 mm. (d,e) Photoswitching of a Tobacco mosaic virus (TMV)-expressed fusion of PS-CFP and the viral movement protein (MP) in Nicotiana tabacum epidermal cells. (d) Before switching, the MP fusion is seen as aggregates on the vertices of the endoplasmic reticulum under low-power 405 nm illumination. (e) Following the irradiation of a square region of interest (ROI) with high-power 405 nm light, fluorescence emission changes from blue to green. The scale bars in (d,e) represent 50 mm. (f–i) Imaging and photoswitching of tetrameric EosFP in a Nicotiana benthamiana leaf. If plants are grown in the light, tEosFP undergoes slow photoswitching from the red- to the green-emitting form. This can be seen at the macroscopic scale in (f), in which a TMV vector that expresses tEosFP has unloaded from the veins of a systemically infected leaf. In cells near the veins, where the protein has been present for some time (3 days), the red, switched version of the protein can be seen. Further from the veins, in tissue more recently infected, the protein is detected in the green, un-switched form. The scale bar in (f) represents 2 mm. (g) Under the CLSM, tEosFP is visible in the cytosol and nuclei of infected cells. (h) Following switching of a circular ROI that surrounds the nucleus of one cell, the green emission is reduced throughout the cytosol and bleached in the nucleus, whereas (i) red emission is detected throughout the cytosol. The scale bar in (g) represents 50 mm. (j–l) BiFC detection and localisation of protein–protein interactions in epidermal
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490 nm illumination, but because its brightness is high, multiple images can be obtained using an attenuated excitation light source.
Novel reporters for protein interactions The discovery that avGFP could be circularly permuted led to the development of bimolecular fluorescence complementation (BiFC) assays in which the sites of protein interactions are localised in living cells [37,38]. In these assays, interacting pairs of proteins bring together two fragments of YFP (or other spectral variants [38]) to reconstitute a fluorescent complex. Following its development in mammalian cells, two groups simultaneously adapted the system for use in plants [39,40]. The reporter constructs that were developed by the two groups differ only in minor respects: one amino acid difference in the YFP sequences used, a single residue difference in the permutation site, and differences in the linkers and antibody tags added. These systems have been used to detect homodimeric [39] and heterodimeric interactions [40] in the nucleus and cytoplasm. The two systems should greatly facilitate the detection of protein–protein interactions in planta because they do not require the complex instrumentation and data analysis that are necessary for FRET. However, as the binding of the YFP fragments is essentially irreversible, BiFC cannot be used to monitor dynamic protein interactions. BiFC is a simple method to identify protein interactions in planta, however, producing a fluorescent complex that is easily detected without specialist equipment (see Figure 3j–l).
will reduce the efficiency of energy transfer. Other FRET pairs have been tested in the past, but have generally been flawed in some respect. Mutagenesis of the most commonly used ECFP has produced a variant, Cerulean, that has improved brightness that is reported to have mono-exponential decay kinetics [42], which would facilitate fluorescence lifetime data analysis. More recently a novel intramolecular ECFP–YFP FRET pair has been evolved through DNA shuffling and FACS [43]. The resulting pair of fluorescent proteins, CyPet and YPet, exhibited a 20-fold ratiometric FRET signal change between the on- and off-states; a significant improvement when compared to the three-fold change observed for the parental pair. The advantage in using small fluorophores rather than fluorescent proteins has been highlighted in a study that monitored the activation of the human adenosine A2A receptor using intramolecular FRET [44]. An A2A–ECFP–YFP construct allowed FRET monitoring of the receptor’s conformational change in activation but prevented coupling to adenylyl cyclase. By contrast, an A2A–CCXXCC–YFP construct bound to FlAsH gave a five-fold greater FRET signal upon receptor activation and produced adenylyl cyclase activation equivalent to that of wildtype A2A. A further recent improvement of FRET has been provided by the development of a threeway system, which is advantageous as many biological interactions involve more than two proteins. This novel technique, using ECFP, EYFP and mRFP, has been used successfully to detect and localise a three-way protein interaction in mammalian cells [45].
Protein inactivation For dynamic interactions, FRET, whether measured through intensity-based protocols or by fluorescence decay kinetics, remains a more rigorous method for quantification of in vivo interactions [41]. ECFP and EYFP, the pair of fluorescent proteins most commonly used in FRET studies, are less than ideal. They have suboptimal extinction coefficients and quantum yields, and also have broad excitation and emission spectra with a small Stoke’s shift (the difference between the excitation and emission maxima) for EYFP. Some of the new redshifted spectral variants, such as mRFP and its engineered derivatives [4], might provide better acceptor molecules. These new variants have a disadvantage, however, in that they have reduced overlap between donor emission and acceptor excitation spectra, which
Chromophore- and fluorophore-assisted light inactivation (CALI and FALI) are methods that allow specific protein inactivation with high spatial resolution. Small organic fluorophores that have genetically encoded binding sequences, rather than fluorescent proteins, have been more widely used in such studies [9,46,47]. The reason for this is that these small fluorophores, such as FlAsH and particularly ReAsH, are more efficient than GFP in producing active oxygen species and hence in inactivating the coupled protein [47]. Recently, multiphoton microscopy has been used for CALI of EGFP fusions that are involved in gap-junction function and nuclear division [48]. Significantly, it has been found that a high-power threshold is required for efficient protein inactivation. In wide-field illumination, this would be detrimental to cell
(Figure 3 Legend continued) cells of N. benthamiana plants. (j) Two days after the agro-infiltration of leaves with BiFC vectors that were designed to express the amino- and carboxy-terminal fragments of split YFP as fusions to the tobacco 14-3-3 protein T14-3c, yellow fluorescence was visible in the cytosol of abaxial epidermal cells. (j,k) At the same time point, self-interaction of the Arabidopsis ALY4 exon junction complex protein was detected in the nucleus and nucleolus following co-infiltration of BiFC binary vectors expressing fusions to the ALY4 protein. The scale bars in represent (j) 50 mm, (k) 20 mm, (l) 10 mm and (m) 10 mm. (m,n) Rapid nuclear import of a mitogen-activated protein kinase–DRONPA fusion protein in COS7 cells. In the absence of epidermal growth factor (EGF), ERK (extracellular signal-regulated kinase) is retained in the cytosol (m(i–v)), whereas after stimulation by EGF, ERK moves from the cytosol to the nucleus (n(i–v)). In each panel of images, image (i) was obtained after the erasure of the DRONPA signal to a background level with strong 488 nm light. DRONPA was then selectively activated in a portion of the cytosol using low intensity 405 nm illumination. Images (ii-v) are collected at 1, 11, 21 and 41 seconds after activation, respectively. The scale bar in (m)(i) represents 10 mm for (m–n). C, cytosol; Nu, nucleus. Current Opinion in Plant Biology 2005, 8:565–573
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viability, but this problem is avoided in multiphotonCALI because the excitation is restricted to a very small volume [49]. Multi-photon systems that permit the efficient use of genetically encoded fluorescent fusion proteins should allow more refined analyses in the future because such proteins confer greater specificity than small fluorophores, which may bind to other cellular components.
Conclusions AvGFP-like reporter proteins currently remain the method of choice for localising proteins in vivo. The latest collection of fluorescent proteins [4,5] broadens the spectral range of available reporters, allowing more proteins to be imaged simultaneously. They are likely to make the commonly used mRFP redundant as they offer increased brightness, better tolerance of fusions, faster maturation and greater photostability.
but as yet, there is no method for introducing them into plants other than through the technically challenging procedure of micro-injection. The development of BiFC systems for detecting protein– protein interactions in plants is likely to be the most important recent breakthrough. Although BiFC cannot monitor dynamic interactions, the regeneration of a fluorescent complex requires that the two parts of YFP are brought into a proximity similar to that required for FRET between two fluorescent proteins. Thus, BiFC systems offer a far more readily accessible method than FRET for the detection of proteins interactions in planta. Perhaps of greatest advantage of BiFC over FRET is that whereas FRET is not amenable to high-throughput studies, BiFC offers the prospect of screening for proteins interactions in planta rather than in a heterologous system such as yeast.
Although it is over three years since PA-GFP was discovered, there have been no published reports of its use in plants to date. Such highlighter proteins should be an invaluable tool for high-resolution, subcellular studies; in particular for monitoring protein trafficking and possibly, through the use of the fusions to ribonucleic acid binding proteins such as the MS2 bacteriophage coat protein, for monitoring RNA trafficking. The latest three highlighters (PS-CFP, EosFP and DRONPA) [34,35,36] should be easier to use than PA-GFP as they can be imaged easily before photoconversion, obviating the possible need to mark target sites with a second fluorescent label. Like PAGFP, these three highlighters are photoswitched by violet light, which can have deleterious side effects [23], although dual photon microscopy might alleviate such problems while additionally permitting photoswitching with higher spatial resolution. Given the rapid recent development of fluorescent proteins, it is probably just a matter of time before there are highlighters that can be switched by less damaging, longer wavelength light.
Acknowledgements
Although invaluable as reporters and in FRET studies, the size of avGFP-like proteins can lead to artefacts and can disrupt protein function [10,44]. Small peptides that bind fluorescent ligands are a possible alternative, but cannot yet confer the same absolute specificity of the entirely genetically encoded fluorescent proteins. There is no means to engineer smaller reporters from current fluorescent proteins because their b-barrel structure is intrinsic to their function. Thus, if smaller fluorescent proteins are to be developed, a novel source will have to be identified. It might be that technological advances in other areas of science will have an impact on the future of fluorescent-tagging methods; recent advances in light microscopy have provided resolutions that were previously thought unattainable [50] and the imaging of single molecules is now feasible. The extreme brightness of quantum dots makes them attractive for such studies, www.sciencedirect.com
We would like to acknowledge funding from the Scottish Executive for the Environment and Rural Affairs Development (grants SCR/0611 and SCR/0612). The authors would like to thank Atsushi Miyawaki, Petra Boevink, Kath Wright, Sabrina Da¨hne and Christian Lauterbach for providing some of the images in Figure 3, and Stuart MacFarlane and Joachim Uhrig for providing ALY4 clones.
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13. Michalet X, Pinaud FF, Bentolila LA, Tsay JM, Doose S, Li JJ, Sundaresan G, Wu AM, Gambhir SS, Weiss S: Quantum dots for live cells, in vivo imaging, and diagnostics. Science 2005, 307:538-544. 14. Dubertret B, Skourides P, Norris DJ, Noireaux V, Brivanlou AH, Libchader A: In vivo imaging of quantum dots encapsulated in phospholipid micelles. Science 2002, 298:1759-1762. 15. Lidke DS, Nagy P, Heintzmann R, Arndt-Jovin DJ, Post JN, Grecco HE, Jares-Erijman EA, Jovin TM: Quantum dot ligands provide new insights into erbB/HER receptor-mediated signal transduction. Nat Biotechnol 2004, 22:198-203. 16. Kim S, Lim YT, Soltesz EG, De Grand AM, Lee J, Nakayama A, Parker JA, Mihaljevic T, Laurence RG, Dor DM, Cohn LH, Bawendi MG, Frangioni JV: Near-infrared fluorescent type II quantum dots for sentinel lymph node mapping. Nat Biotechnol 2004, 22:93-97. 17. Wadia JS, Dowdy SF: Protein transduction technology. Curr Opin Biotechnol 2002, 13:52-56. 18. Chang M, Chou J-C, Lee H-J: Cellular internalization of fluorescent proteins via arginine-rich intracellular delivery peptide in plant cells. Plant Cell Physiol 2005, 46:482-488. 19. Lagerholm BC, Wang M, Ernst LA, Ly DH, Liu H, Bruchez MP, Waggoner AS: Multicolor coding of cells with cationic peptide coated quantum dots. Nano Lett 2004, 4:2019-2022. 20. Ravindran S, Kim S, Martin R, Lord EM, Ozkan CS: Quantum dots as bio-labels for the localization of a small plant adhesion protein. Nanotechnology 2005, 16:1-4. 21. Ko¨ster M, Frahm T, Hauser H: Nucleocytoplasmic shuttling revealed by FRAP and FLIP technologies. Curr Opin Biotechnol 2005, 16:28-34. 22. Patterson GH, Lippincott-Schwartz J: A photoactivatable GFP for selective photolabeling of proteins and cells. Science 2002, 297:1873-1877. 23. Post JN, Lidke KA, Rieger B, Arndt-Jovin DJ: One- and two-photon photoactivation of a paGFP–fusion protein in live Drosophila embryos. FEBS Lett 2005, 579:325-330. 24. Stark DA, Kulesa PM: Photoactivatable green fluorescent protein as a single-cell marker in living embryos. Dev Dyn 2005, 233:983-992. 25. Karbowski M, Arnoult D, Chen H, Chan DC, Smith CL, Youle RJ: Quantitation of mitochondrial dynamics by photolabeling of individual organelles show that mitochondrial fusion is blocked during the Bax activation phase of apoptosis. J Cell Biol 2004, 164:493-499. 26. O’Connell KMS, Tamkun MM: Targeting of voltage-gated potassium channel isoforms to distinct cell surface microdomains. J Cell Sci 2005, 118:2155-2166. 27. Ando R, Hama H, Yamamoto-Hino M, Mizuno H, Miyawaki A: An optical marker based on the UV-induced green-to-red photoconversion of a fluorescent protein. Proc Natl Acad Sci USA 2002, 99:12651-12656. 28. Tsutsui H, Karasawa S, Shimizu H, Nukina N, Miyawaki A: Semi-rational engineering of a coral fluorescent protein into an efficient highlighter. EMBO Rep 2005, 6:233-238. 29. Pakhomov AA, Martynova NY, Gurskaya NG, Balahova TA, Martynov VI: Photoconversion of the chromophore of a fluorescent protein from Dendronephthya sp. Biochemistry 2004, 69:901-908. Current Opinion in Plant Biology 2005, 8:565–573
33. Chudakov DM, Belousov VV, Zaraisky AG, Novoselov VV, Staroverov DB, Zorov DB, Lukyanov S, Lukyanov KA: Kindling fluorescent proteins for precise in vivo photolabeling. Nat Biotechnol 2003, 21:191-194. 34. Chudakov DM, Verkhusha VV, Staroverov DB, Souslova EA, Lukyanov S, Lukyanov KA: Photoswitchable cyan fluorescent protein for protein tracking. Nat Biotechnol 2004, 22:1435-1439. The authors mutagenised a bright monomeric GFP to produce a photoswitchable highlighter and used it to monitor human dopamine transport along the cell surface and between endosomes. 35. Wiedenmann J, Ivanchenko S, Oswald F, Schmitt F, Ro¨cker C, Salih A, Spindler K-D, Nienhaus GU: EosFP, a fluorescent marker protein with UV-inducible green-to-red fluorescence conversion. Proc Natl Acad Sci USA 2004, 101:15905-15910. This paper describes the cloning of an Anthozoan green-to-red switchable protein and its rational engineering to produce dimeric and monomeric derivatives. Bright green forms can be converted to bright red forms by illumination with light of a wavelength different to that used to image either form. The authors show precise photoconversion in different subcellular locations. 36. Ando R, Mizuno H, Miyawaki A: Regulated fast nucleocytoplasmic shuttling observed by reversible protein highlighting. Science 2004, 306:1370-1373. The authors clone a photochromic Anthozoan protein and engineer a monomeric form. The fluorescence of the protein can be repeatedly turned on and off by illuminations with appropriate wavelengths. The highlighter is used to monitor the fast nuclear import and export of protein fusions. 37. Hu C-D, Chinenov Y, Kerppola TK: Visualization of interactions among bZIP and Rel family proteins in living cells using bimolecular fluorescence complementation. Mol Cell 2002, 9:789-798. 38. Hu C-D, Kerppola TK: Simultaneous visualization of multiple protein interactions in living cells using multicolour fluorescence complementation analysis. Nat Biotechnol 2003, 21:539-545. 39. Walter M, Chaban C, Schu¨tze K, Batistic O, Weckermann K, Nake C, Blazevic D, Grefen C, Schumacher K, Oecking C et al.: Visualization of protein interactions in living plant cells using bimolecular fluorescence complementation. Plant J 2004, 40:428-438. The authors describe the construction of vectors to detect protein interactions through bimolecular fluorescence complementation in plants. They present vectors for both transient expression and stable transformation. The efficacy of the system to localise protein interactions is demonstrated with homodimerising proteins that accumulate in the nucleus or cytoplasm, with mutants that are impaired in homo-dimerisation being used as negative controls. 40. Bracha-Drori K, Shichrur K, Katz A, Oliva M, Angelovici R, Yalovsky S, Ohad N: Detection of protein–protein interactions in plants using bimolecular fluorescence complementation. Plant J 2004, 40:419-427. As in [39], the authors describe vectors for carrying out BiFC assays in plants. The vectors were created to test fusions to both the amino- and carboxy-temini of the YFP fragments. Using transient Agrobacteriummediated expression from binaries, the authors show the cytoplasmic interaction of different subunits from an Arabidopsis farnesyl transferase and the nuclear and cytoplasmic interaction between two polycomb proteins. 41. Voss TC, Demarco IA, Day RN: Quantitative imaging of protein interactions. Biotechniques 2005, 38:413-424. 42. Rizzo MA, Springer GH, Granada B, Piston DW: An improved cyan fluorescent protein variant useful for FRET. Nat Biotechnol 2004, 22:445-449. www.sciencedirect.com
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43. Nguyen AW, Daugherty PS: Evolutionary optimization of fluorescent proteins for intracellular FRET. Nat Biotechnol 2005, 23:355-360.
47. Tour O, Meijer RM, Zacharias DA, Adams SR, Tsien RY: Genetically targeted chromophore-assisted light inactivation. Nat Biotechnol 2003, 21:1505-1508.
44. Hoffmann C, Gaietta G, Bu¨nemann M, Adams SR, Oberdorff-Maass S, Behr B, Vilardaga J-P, Tsien RY, Ellisman MH, Lohse MJ: A FlAsH-based FRET approach to determine G protein-coupled receptor activation in living cells. Nat Methods 2005, 2:171-176.
48. Tanabe T, Oyamada M, Fujita K, Dai P, Tanaka H, Takamatsu T: Multiphoton excitation-evoked chromophore-assisted laser inactivation using green fluorescent protein. Nat Methods 2005, 2:503-505.
45. Galperin E, Verkhusha VV, Sorkin A: Three-chromophore FRET microscopy to analyze multiprotein interactions in living cells. Nat Methods 2004, 1:209-217. 46. Marek KW, Davis GW: Transgenically encoded protein photoinactivation (FlAsH–FALI): acute inactivation of synaptotagmin I. Neuron 2002, 36:805-813.
49. Zipfel WR, Williams RM, Webb WW: Nonlinear magic: multiphoton microscopy in the biosciences. Nat Biotechnol 2003, 21:1369-1377. 50. Garini Y, Vermolen BJ, Young IT: From micro to nano: recent advances in high-resolution microscopy. Curr Opin Biotechnol 2005, 16:3-12.
Congratulations to the winners of the Current Opinion in Plant Biology poster prizes at the 2nd Tri-national Arabidopsis meeting A jury of the conference committee awarded prizes for the best poster in each of four sessions: Photosynthesis and metabolism Nu´ria Sa´nchez Coll, Antoine Danon and Klaus Apel Characterization of the signal transduction pathway activated by singlet oxygen. Epigenetics and gene silencing Claudia Kerzendorfer, Svetlana Akimcheva, Dieter Schweizer and Peter Schlo¨gelhofer Characterisation of ATFANCD2 in Arabidopsis thaliana. Development and evolution Patricia Torres-Galea and Cordelia Bolle The role of GRAS proteins in light signaling Biotic and abiotic stress Khaoula Belhaj, Baiqing Lin and Felix Mauch RPH1, a novel Arabidopsis gene involved in resistance to Phytophthora brassicae.
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Current Opinion in Plant Biology 2005, 8:565–573
Current Opinion in Plant Biology 2005, 8:565–573 This review comes from a themed issue on Cell biology Edited by Patricia C Zambryski and Karl Oparka Available online 26th September 2005 1369-5266/$ – see front matter # 2005 Elsevier Ltd. All rights reserved. DOI 10.1016/j.pbi.2005.09.011
Introduction Since the cloning of the gene for the green fluorescent protein of Aequoria victoria (avGFP), efforts have been made to modify the spectral characteristics and biochemical properties of a range of marine invertebrate-derived fluorescent proteins to enhance their utility as biological reporters. The genes for about 100 proteins, fluorescent and non-fluorescent, that are capable of autocatalytic chromophore formation have now been cloned [1]. Desirable traits include brightness (a product of the extinction coefficient and quantum yield), photostability, rapid maturation, small size, pH insensitivity, tolerance of fusions and resistance to oligomerisation. The in vivo expression of fusions to avGFP-like fluorescent proteins is generally the first method of choice for studying protein localisations, movement and interactions with other cellular proteins. Fluorescent proteins that have different www.sciencedirect.com
spectral characteristics are useful for simultaneous imaging of multiple proteins and for fluorescence resonance energy transfer (FRET) studies. The use of FRET-based biosensors in cell biology is reviewed by Lalonde et al. in this issue. Here, we review recent advances in fluorescent tagging that should enhance the spatial and temporal resolution of future studies, and should facilitate the identification of interacting protein partners.
Novel fluorescent reporter proteins The range of available spectral variants has expanded with the cloning of many new Anthozoan-derived chromoproteins [2]. However, the fact that most are obligate tetramers, and many form higher molecular weight aggregates, necessitates their genetic engineering to create useful fluorescent reporter proteins. Much effort has concentrated on developing fluorescent proteins that have red-shifted emission spectra, as autofluorescence in most eukaryotic cells is reduced at longer wavelengths, and on developing new acceptors for FRET studies. However, the utility of these new reporters might be compromised in plant tissues where the main peak of chlorophyll autofluorescence overlays the emission of such proteins (see Figure 1). The monomeric red fluorescent protein (mRFP) [3], an evolved form of the Discosoma coral RFP, has a lower extinction coefficient, quantum yield and photostability than its progenitor, but matures much more rapidly and has red-shifted excitation and emission spectra. mRFP has been taken as a starting point for further mutagenesis [4] and in vivo evolution [5], resulting in the generation of a set of new, monomeric fluorescent proteins, whose emission maxima range from 537 to 649 nm. These proteins provide emission maxima beyond that of the avGFP-derived yellow fluorescent protein (YFP), and they offer improved brightness, fusion tolerance, maturation rates and photostability when compared with the popular mRFP.
Fluorescent peptide ligands The utility of all avGFP-like fluorescent proteins is limited by their size of circa 25 kDa [6]. Phage library screening has identified a peptide of only 38 amino acids that binds the fluorescent dye Texas red [7], and this has been used to localise a membrane-targeted GFP fusion protein in mouse embryo cells. The specificity of this peptide results from its binding to the xanthene core of Texas red, which is shared by fluorescent X-rhod calcium sensors; hence, this peptide might be useful for localised calcium sensing. The six amino acid, tetracysteine motif that covalently binds biarsenate derivatives of fluorescein and resorufin (Fluorescein Arsenical Helical binder [FlAsH] and Resorufin Arsenical Helical binder Current Opinion in Plant Biology 2005, 8:565–573
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Figure 1
Emission spectra of the main sources of autofluorescence in roots and leaves when excited with commonly used laser sources for confocal microscopes. Lignin (dotted lines) and chlorophyll (solid lines) were excited, in roots and leaves respectively, using nine different excitation wavelengths, and emission spectra were collected. All lasers were run at 50% maximal power output. Although the amplitude of autofluorescence varies between laser sources, it is clear that, in roots (or in the vascular tissue and cell walls of aerial plant parts), lignin fluorescence will cause most interference with imaging at wavelengths of between 490 and 620 nm. By contrast, chlorophyll fluorescence in leaves and stems is most prevalent between 650 and 770 nm. In practice, this means that the imaging of green fluorescent proteins is most problematic in roots, whereas red fluorescent proteins can be difficult to discriminate in tissues that have many chloroplasts, and are unsuitable as fusion partners for proteins that localise to, or near, chloroplasts.
[ReAsH]) is an even shorter peptide tag [8]. It has been used in mammalian [8], Drosophila [9] and yeast [10] cells, but not in plants. The bound, fluorescent labels can generate singlet oxygen upon illumination, and this oxygen species can be of utility in downstream electron microscopy studies by facilitating aminobenzidine polymerisation and the production of an electron-dense precipitate [11]. The tetracysteine tag needs a reducing environment, however, and there are concerns about endogenous thiol groups giving non-specific labelling [6]; for example, the Arabidopsis HMA3 Zn/Cd heavymetal transporter actually contains a tetracysteine motif. Signal background might be ameliorated by a recent fluorescence-assisted cell sorting (FACS)-based genetic screen that can be used to optimise the tag’s context [12]. This screen identified a twelve amino acid motif (FLNCCPGCCMEP) that produces more than 25 times the contrast produced by the earlier motif in living cells. The benefit of using the small tetracysteine motif is emphasised by a yeast study in which fusions between GFP, or between one and three repeats of this motif, and b-tubulin were all incorporated into microtubules, but only fusions with one or two tetracysteine repeats could functionally substitute for the wildtype protein [10].
Quantum dots Semiconductor nanocrystals or quantum dots are a group of fluorescent labels that have been the centre of much recent interest [13]. Their colloidal semiconductor cores, which are between 2 and 10 nm in diameter, confer several desirable traits, including photostability, broad Current Opinion in Plant Biology 2005, 8:565–573
absorption spectra, narrow emission spectra, a broad range of size- and composition-dependent emission maxima, long fluorescence lifetimes that allow time-delayed detection and extreme brightness that permits single molecule detection. These labels have already proven themselves in vivo in animal studies for cell tracking [14] and in studies of cell-surface proteins [15], and are likely to be of use in biomedical applications [16]. How useful these labels will be for in vivo plant studies is unclear as they are membrane impermeant. Furthermore, the semiconductor cores require the addition of a pacifying coat and some form of biological functionalisation (e.g. through the coupling of peptide ligands or antibodies), thereby increasing their size above 10 nm. Whether such large molecules can effectively traverse the plant extracellular matrix and then be taken up is unclear, limiting their in vivo utility, but they might be useful novel probes for endocytosis in plants. Protein transduction domains [17] might facilitate this process, and a nona-argenine peptide fusion to GFP has been shown to allow rapid internalisation of GFP (circa 5 nm) into plant tissues [18]. However, linkage of the same peptide sequence to quantum dots only resulted in their accumulation in endosomes and lysosomes in mammalian cells [19]. Nonetheless, antibody-coupled quantum dots have been successfully used to localise a pollen tube adhesion protein to the tube tip [20].
Novel highlighters Although fluorescent proteins such as avGFP can be used to study protein dynamics and kinetics through photowww.sciencedirect.com
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Table 1 Summary of spectroscopic data for highlighter proteins. Protein
l (ex)
l (em)
e
QY
Relative brightness
Oligomeric state
Conversion wavelength
pKa
PA–GFP (N) PA–GFP (P) Kaede (N) Kaede (P) Kikume (N) Kikume (P) Kindling (N) Kindling (P) 1 PA–mRFP1-1 (N) PA–mRFP1-1 (P) PS–CFP (N) PS–CFP (P) PS–CFP2 (N) PS–CFP2 (P) mEosFP (N) mEosFP (P) DRONPA (N)
400 504 508 572 507 583 580 580 588 578 402 490 400 490 505 569 503
515 517 518 580 517 593 600 600 602 605 468 511 468 511 516 581 518
20 700 17 400 98 800 60 400 28 200 32 600 123 000 59 000
0.13 0.79 0.88 0.33 0.70 0.65 <0.001 0.07
8 41 259 59 59 63 0 12
413
4.5
350–410
5.6 5.6 7.8 5.5
0.08 0.16 0.19 0.20 0.23 0.64 0.62 0.85
2 16 15 26 32 128 68 240
Monomer Monomer Tetramer Tetramer Tetramer Tetramer Tetramer Tetramer Monomer Monomer Monomer Monomer Monomer Monomer Monomer Monomer Monomer
10 34 27 43 47 67 37 95
000 000 000 000 000 200 000 000
350–420 530–560 430–490 375–385 405 405 400 490 (400)
4.4 4.0 6.0 4.3 6.1 5.8 5.8 5.0
Data are provided for the native (N) and photoconverted (P) states of the proteins. l (ex) and l (em) are the excitation and emission maxima, respectively, in units of nm. e is the extinction coefficient in units of M 1 cm 1. QY is the fluorescence quantum yield. Relative brightness is given as a percentage of the commonly used EGFP. 1 Figures are given for irreversibly activated, kindled, protein.
bleaching techniques [21], the advent of fluorescent proteins that undergo light-induced spectral changes, termed optical highlighters, should facilitate such studies. The first effective highlighter was an avGFP mutant that, following UV or violet illumination, displayed a 100-fold increase in emission upon 488 nm excitation (see Table 1 and Figure 2; [22]). However, the low quantum yield of photoactivatable GFP (PA-GFP) before activation can make targeting difficult. Furthermore, activation with short-wavelength light can cause damage: activation with a single-photon laser of PA-GFP fused to a histone variant prevented nuclear fission in Drosophila embryos [23]. Two-photon excitation reduced chromatin damage, but even so, longer wavelengths and lower energies were required to allow continued nuclear division. To date, PA-GFP has been used in cellular marking [24], and to study inter-organelle [22,25] and membrane-associated protein dynamics [26]. We have used PA-GFP to study plasmodesmatal connections, and plants that have a PAGFP transgene offer an elegant and facile system for studies of symplastic communication within and between tissues (see Figure 3a–c). Since the discovery of PA-GFP, several Anthozoan proteins, including Kaede and Kikume, that fluoresce green before UV or violet illumination and red subsequently have been cloned (see Figure 2; [2,27–29]). Irreversible photoconversion of Kaede results from extension of the chromophore in a reaction that involves the cleavage of the peptide backbone [30]. The utility of these Anthozoan-derived proteins as highlightable fusions is compromised by their oligomerisation, although fusions of Kaede to organelle-targeting sequences have been successfully used to monitor organelle dynamics in plants www.sciencedirect.com
[31]. Mutagenesis of the Anthozoan-derived mRFP has also produced photoactivatable mRFPs [32]. However, like an earlier Anemonia sulcata-derived protein that contains similar amino-acid substitutions and can be reversibly photoactivated with green light (Kindling fluorescent protein [33]), these variants, which are activated by more damaging UV light, are poorly fluorescent after photoactivation. Mutagenesis of the highly fluorescent aceGFP protein, which was derived from a non-fluorescent chromoprotein of the Hydrozoan Aequorea coerulescens, has produced a photoswitchable cyan fluorescent protein (PS-CFP) [34]. Upon violet illumination, the cyan fluorescent form converts to a green fluorescent form (Figure 2). Both forms give approximately 2.5-fold less fluorescence than activated PA-GFP but there is a 1500-fold ratiometric difference between the native and switched forms. PS-CFP has been used to monitor the dynamics of the membraneassociated human dopamine transporter, and there is now a faster-maturing commercial derivative, PS-CFP2 (EvrVgen), which is brighter and gives a ratiometric difference of over 2000-fold. One problem with PSCFP2 is that violet light is used both to image and to photoconvert the cyan fluorescent form, necessitating cautious imaging to prevent premature photoconversion. However, in our hands, PS-CFP2 is a useful protein for studies of the dynamics of different protein pools in plant cells (see Figure 3d,e), and has an advantage over PAGFP in that it is easily detectable pre-activation. The gene for a green-to-red switchable protein, EosFP, which has homology to Kaede, has been cloned from the coral Lobophyllia hemprichii, and dimeric and monomeric forms have been engineered from the tetrameric wildtype proCurrent Opinion in Plant Biology 2005, 8:565–573
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Figure 2
The photoconversion mechanisms for the most useful optical highlighters. The photoconversion sequence for each highlighter is shown running left to right across the page, beginning in each case with the native or inactivated form. Each element shows a leaf epidermal cell containing a nucleus, imaged under different fluorescence excitation wavelengths (in this example under 561, 488 and 405 nm laser light sources). Arrows represent the light used for photoconversion: high-and low-intensity illumination is indicated by large and small arrows respectively, and the colours of the arrows indicate the wavelength used. Emission colours appear in the nucleus and/or cytoplasm of each cell. (a) A cell containing photoactivatable green fluorescent protein (PA-GFP) appears non-fluorescent (grey) prior to photoconversion in the first image. After illuminating the nucleus with 405-nm light (purple arrow), the PA-GFP protein exhibits green fluorescence, which slowly diffuses out into the cytoplasm, as illustrated in subsequent cells. (b) The irradiation of Kaede and Kikume highlighters causes the native, green protein to convert to a red form. The green protein is imaged under 488 nm light and switched to the red form using 405 nm illumination. Once converted, the red form must be imaged using 561 nm illumination. A pool of non-switched protein will still be visible if 488 nm illumination is used. (c) Native PS-CFP protein is cyan fluorescent under violet illumination. Following illumination of the nucleus by high-intensity 405 nm light, cyan fluorescence is quenched and, when excited with 488 nm light, PS-CFP emits green light. The GFP diffuses out of the nucleus and into the surrounding cytoplasm. Non-switched cyan-fluorescent protein is still visible if the cell is imaged using violet excitation. (d) Photoswitching of the green to red form of the monomeric EosFP protein can be achieved, and imaged, using the same methods as described for Kaede and Kikume above. (e) DRONPA exists in two inter-convertible protonated and deprotonated forms. When irradiated at 488 nm, the deprotonated protein pool strongly emits green fluorescence. Following intense illumination with 488 nm light, the protein population is driven towards the almost non-fluorescent protonated form. Subsequent illumination with 405 nm light switches the protein back to the deprotonated, green-fluorescent form.
tein [35]. Like PS-CFP, EosFP is photoconverted by violet light, but the green and red forms are both excited by longer wavelengths (see Figures 2 and 3 Figures 2 and 3f-i). The various forms of EosFP have been used to precisely photoconvert and monitor various fusion proteins in mammalian cells with the sole caveat that the monomeric form is limited to temperatures below 30 8C. A near-ideal monomeric highlighter protein has recently been obtained through the cloning of the gene for a GFP from Pectiniidae coral, and its power demonstrated by Current Opinion in Plant Biology 2005, 8:565–573
monitoring the fast nucleocytoplasmic shuttling of a mitogen-activated protein kinase fusion [36]. Strong illumination of the protein, called DRONPA, with 490 nm light leads to loss of green fluorescence, but this can be repeatedly recovered by illumination with low levels of violet light. Consequently, this is the first protein to allow fluorescence to be switched off and on at will, providing a potentially excellent reporter for dynamic cell processes (see Figures 2 and 3 Figures 2 and 3m,n). DRONPA has a relatively fast bleaching rate under www.sciencedirect.com
New tools for in vivo fluorescence tagging Chapman, Oparka and Roberts 569
Figure 3
Imaging of fluorescent highlighters and bimolecular fluorescence complementation in plants. All images were collected using a Leica SP2 AOBS-spectral confocal laser scanning microscope (CLSM). (a–c) Photoactivation of PA-GFP in a transgenic, PA-GFP-expressing, Arabidopsis apical meristem. (a) Before activation, no green signal is visible. (b) Following the localised photoactivation of a single cell at the tip of the meristem, GFP becomes visible and (c) subsequently moves symplastically into adjacent cells. The scale bars in (a–c) represent 20 mm. (d,e) Photoswitching of a Tobacco mosaic virus (TMV)-expressed fusion of PS-CFP and the viral movement protein (MP) in Nicotiana tabacum epidermal cells. (d) Before switching, the MP fusion is seen as aggregates on the vertices of the endoplasmic reticulum under low-power 405 nm illumination. (e) Following the irradiation of a square region of interest (ROI) with high-power 405 nm light, fluorescence emission changes from blue to green. The scale bars in (d,e) represent 50 mm. (f–i) Imaging and photoswitching of tetrameric EosFP in a Nicotiana benthamiana leaf. If plants are grown in the light, tEosFP undergoes slow photoswitching from the red- to the green-emitting form. This can be seen at the macroscopic scale in (f), in which a TMV vector that expresses tEosFP has unloaded from the veins of a systemically infected leaf. In cells near the veins, where the protein has been present for some time (3 days), the red, switched version of the protein can be seen. Further from the veins, in tissue more recently infected, the protein is detected in the green, un-switched form. The scale bar in (f) represents 2 mm. (g) Under the CLSM, tEosFP is visible in the cytosol and nuclei of infected cells. (h) Following switching of a circular ROI that surrounds the nucleus of one cell, the green emission is reduced throughout the cytosol and bleached in the nucleus, whereas (i) red emission is detected throughout the cytosol. The scale bar in (g) represents 50 mm. (j–l) BiFC detection and localisation of protein–protein interactions in epidermal
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490 nm illumination, but because its brightness is high, multiple images can be obtained using an attenuated excitation light source.
Novel reporters for protein interactions The discovery that avGFP could be circularly permuted led to the development of bimolecular fluorescence complementation (BiFC) assays in which the sites of protein interactions are localised in living cells [37,38]. In these assays, interacting pairs of proteins bring together two fragments of YFP (or other spectral variants [38]) to reconstitute a fluorescent complex. Following its development in mammalian cells, two groups simultaneously adapted the system for use in plants [39,40]. The reporter constructs that were developed by the two groups differ only in minor respects: one amino acid difference in the YFP sequences used, a single residue difference in the permutation site, and differences in the linkers and antibody tags added. These systems have been used to detect homodimeric [39] and heterodimeric interactions [40] in the nucleus and cytoplasm. The two systems should greatly facilitate the detection of protein–protein interactions in planta because they do not require the complex instrumentation and data analysis that are necessary for FRET. However, as the binding of the YFP fragments is essentially irreversible, BiFC cannot be used to monitor dynamic protein interactions. BiFC is a simple method to identify protein interactions in planta, however, producing a fluorescent complex that is easily detected without specialist equipment (see Figure 3j–l).
will reduce the efficiency of energy transfer. Other FRET pairs have been tested in the past, but have generally been flawed in some respect. Mutagenesis of the most commonly used ECFP has produced a variant, Cerulean, that has improved brightness that is reported to have mono-exponential decay kinetics [42], which would facilitate fluorescence lifetime data analysis. More recently a novel intramolecular ECFP–YFP FRET pair has been evolved through DNA shuffling and FACS [43]. The resulting pair of fluorescent proteins, CyPet and YPet, exhibited a 20-fold ratiometric FRET signal change between the on- and off-states; a significant improvement when compared to the three-fold change observed for the parental pair. The advantage in using small fluorophores rather than fluorescent proteins has been highlighted in a study that monitored the activation of the human adenosine A2A receptor using intramolecular FRET [44]. An A2A–ECFP–YFP construct allowed FRET monitoring of the receptor’s conformational change in activation but prevented coupling to adenylyl cyclase. By contrast, an A2A–CCXXCC–YFP construct bound to FlAsH gave a five-fold greater FRET signal upon receptor activation and produced adenylyl cyclase activation equivalent to that of wildtype A2A. A further recent improvement of FRET has been provided by the development of a threeway system, which is advantageous as many biological interactions involve more than two proteins. This novel technique, using ECFP, EYFP and mRFP, has been used successfully to detect and localise a three-way protein interaction in mammalian cells [45].
Protein inactivation For dynamic interactions, FRET, whether measured through intensity-based protocols or by fluorescence decay kinetics, remains a more rigorous method for quantification of in vivo interactions [41]. ECFP and EYFP, the pair of fluorescent proteins most commonly used in FRET studies, are less than ideal. They have suboptimal extinction coefficients and quantum yields, and also have broad excitation and emission spectra with a small Stoke’s shift (the difference between the excitation and emission maxima) for EYFP. Some of the new redshifted spectral variants, such as mRFP and its engineered derivatives [4], might provide better acceptor molecules. These new variants have a disadvantage, however, in that they have reduced overlap between donor emission and acceptor excitation spectra, which
Chromophore- and fluorophore-assisted light inactivation (CALI and FALI) are methods that allow specific protein inactivation with high spatial resolution. Small organic fluorophores that have genetically encoded binding sequences, rather than fluorescent proteins, have been more widely used in such studies [9,46,47]. The reason for this is that these small fluorophores, such as FlAsH and particularly ReAsH, are more efficient than GFP in producing active oxygen species and hence in inactivating the coupled protein [47]. Recently, multiphoton microscopy has been used for CALI of EGFP fusions that are involved in gap-junction function and nuclear division [48]. Significantly, it has been found that a high-power threshold is required for efficient protein inactivation. In wide-field illumination, this would be detrimental to cell
(Figure 3 Legend continued) cells of N. benthamiana plants. (j) Two days after the agro-infiltration of leaves with BiFC vectors that were designed to express the amino- and carboxy-terminal fragments of split YFP as fusions to the tobacco 14-3-3 protein T14-3c, yellow fluorescence was visible in the cytosol of abaxial epidermal cells. (j,k) At the same time point, self-interaction of the Arabidopsis ALY4 exon junction complex protein was detected in the nucleus and nucleolus following co-infiltration of BiFC binary vectors expressing fusions to the ALY4 protein. The scale bars in represent (j) 50 mm, (k) 20 mm, (l) 10 mm and (m) 10 mm. (m,n) Rapid nuclear import of a mitogen-activated protein kinase–DRONPA fusion protein in COS7 cells. In the absence of epidermal growth factor (EGF), ERK (extracellular signal-regulated kinase) is retained in the cytosol (m(i–v)), whereas after stimulation by EGF, ERK moves from the cytosol to the nucleus (n(i–v)). In each panel of images, image (i) was obtained after the erasure of the DRONPA signal to a background level with strong 488 nm light. DRONPA was then selectively activated in a portion of the cytosol using low intensity 405 nm illumination. Images (ii-v) are collected at 1, 11, 21 and 41 seconds after activation, respectively. The scale bar in (m)(i) represents 10 mm for (m–n). C, cytosol; Nu, nucleus. Current Opinion in Plant Biology 2005, 8:565–573
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viability, but this problem is avoided in multiphotonCALI because the excitation is restricted to a very small volume [49]. Multi-photon systems that permit the efficient use of genetically encoded fluorescent fusion proteins should allow more refined analyses in the future because such proteins confer greater specificity than small fluorophores, which may bind to other cellular components.
Conclusions AvGFP-like reporter proteins currently remain the method of choice for localising proteins in vivo. The latest collection of fluorescent proteins [4,5] broadens the spectral range of available reporters, allowing more proteins to be imaged simultaneously. They are likely to make the commonly used mRFP redundant as they offer increased brightness, better tolerance of fusions, faster maturation and greater photostability.
but as yet, there is no method for introducing them into plants other than through the technically challenging procedure of micro-injection. The development of BiFC systems for detecting protein– protein interactions in plants is likely to be the most important recent breakthrough. Although BiFC cannot monitor dynamic interactions, the regeneration of a fluorescent complex requires that the two parts of YFP are brought into a proximity similar to that required for FRET between two fluorescent proteins. Thus, BiFC systems offer a far more readily accessible method than FRET for the detection of proteins interactions in planta. Perhaps of greatest advantage of BiFC over FRET is that whereas FRET is not amenable to high-throughput studies, BiFC offers the prospect of screening for proteins interactions in planta rather than in a heterologous system such as yeast.
Although it is over three years since PA-GFP was discovered, there have been no published reports of its use in plants to date. Such highlighter proteins should be an invaluable tool for high-resolution, subcellular studies; in particular for monitoring protein trafficking and possibly, through the use of the fusions to ribonucleic acid binding proteins such as the MS2 bacteriophage coat protein, for monitoring RNA trafficking. The latest three highlighters (PS-CFP, EosFP and DRONPA) [34,35,36] should be easier to use than PA-GFP as they can be imaged easily before photoconversion, obviating the possible need to mark target sites with a second fluorescent label. Like PAGFP, these three highlighters are photoswitched by violet light, which can have deleterious side effects [23], although dual photon microscopy might alleviate such problems while additionally permitting photoswitching with higher spatial resolution. Given the rapid recent development of fluorescent proteins, it is probably just a matter of time before there are highlighters that can be switched by less damaging, longer wavelength light.
Acknowledgements
Although invaluable as reporters and in FRET studies, the size of avGFP-like proteins can lead to artefacts and can disrupt protein function [10,44]. Small peptides that bind fluorescent ligands are a possible alternative, but cannot yet confer the same absolute specificity of the entirely genetically encoded fluorescent proteins. There is no means to engineer smaller reporters from current fluorescent proteins because their b-barrel structure is intrinsic to their function. Thus, if smaller fluorescent proteins are to be developed, a novel source will have to be identified. It might be that technological advances in other areas of science will have an impact on the future of fluorescent-tagging methods; recent advances in light microscopy have provided resolutions that were previously thought unattainable [50] and the imaging of single molecules is now feasible. The extreme brightness of quantum dots makes them attractive for such studies, www.sciencedirect.com
We would like to acknowledge funding from the Scottish Executive for the Environment and Rural Affairs Development (grants SCR/0611 and SCR/0612). The authors would like to thank Atsushi Miyawaki, Petra Boevink, Kath Wright, Sabrina Da¨hne and Christian Lauterbach for providing some of the images in Figure 3, and Stuart MacFarlane and Joachim Uhrig for providing ALY4 clones.
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Congratulations to the winners of the Current Opinion in Plant Biology poster prizes at the 2nd Tri-national Arabidopsis meeting A jury of the conference committee awarded prizes for the best poster in each of four sessions: Photosynthesis and metabolism Nu´ria Sa´nchez Coll, Antoine Danon and Klaus Apel Characterization of the signal transduction pathway activated by singlet oxygen. Epigenetics and gene silencing Claudia Kerzendorfer, Svetlana Akimcheva, Dieter Schweizer and Peter Schlo¨gelhofer Characterisation of ATFANCD2 in Arabidopsis thaliana. Development and evolution Patricia Torres-Galea and Cordelia Bolle The role of GRAS proteins in light signaling Biotic and abiotic stress Khaoula Belhaj, Baiqing Lin and Felix Mauch RPH1, a novel Arabidopsis gene involved in resistance to Phytophthora brassicae.
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