Photoconvertible Fluorescent Protein Research Articles

Monitoring cellular movementin vivowith photoconvertible fluorescence protein “Kaede” transgenic mice

Kaede is a photoconvertible fluorescence protein that changes from green to red upon exposure to violet light. The photoconversion of intracellular Kaede has no effect on cellular function. Using transgenic mice expressing the Kaede protein, we demonstrated that movement of cells with the photoconverted Kaede protein could be monitored from lymphoid organs to other tissues as well as from skin to the draining lymph node. Analysis of the kinetics of cellular movement revealed that each subset of cells in the lymph node, such as CD4(+) T, CD8(+) T, B, and dendritic cells, has a distinct migration pattern in vivo. Thus, the Kaede transgenic mouse system would be an ideal tool to monitor precise cellular movement in vivo at different stages of immune response to pathogens as well as in autoimmune diseases.

Live visualization of protein synthesis in axonal growth cones by microinjection of photoconvertible Kaede into Xenopus embryos

Photoconvertible fluorescent proteins, such as Kaede, can be switched irreversibly from their native color to a new one. This property can be exploited to visualize de novo mRNA translation, because newly synthesized proteins can be distinguished from preexisting ones by their color. In this protocol, Kaede cDNA linked to the 3' untranslated region (UTR) of beta-actin is delivered into cells fated to become the retina by injection into Xenopus blastomeres. Brief exposure (6-10 s) to UV light (350-410 nm) of Kaede-positive retinal axons/growth cones efficiently converts Kaede from its native green fluorescence to red. The reappearance of the green signal reports the synthesis of new Kaede protein. This approach can be used to investigate the spatiotemporal control of translation of specific mRNAs in response to external stimuli and to test the efficiency of full-length versus mutant UTRs. The 3-d protocol can be adapted for broad use with other photoactivatable fluorescent proteins.

Direct measurement of protein dynamics inside cells using a rationally designed photoconvertible protein

All biological reactions depend on the diffusion and re-localization of biomolecules. Our understanding of biological processes requires accurate measurement of biomolecule mobility in living cells. Currently, approaches for investigating the mobility of biomolecules are generally restricted to measuring either fast or slow diffusion kinetics. We describe the development and application of a photoconvertible fluorescent protein, Phamret, that can be highlighted by UV light stimulation inducing a change in fluorescence emission from cyan fluorescent protein (CFP) to photoactivated GFP (PA-GFP). Phamret can be monitored by single excitation-dual emission mode for visualization of molecular dynamics for a broad range of kinetics. We also devised a microscopy-based method to measure the diffusion coefficient from the fluorescence decay after photostimulation of Phamret, enabling analysis of diffusion kinetics ranging from less than 0.1 microm2/s up to approximately 100 microm2/s, and found significant changes in free protein movement during cell-cycle progression.

Synthesis and properties of the red chromophore of the green-to-red photoconvertible fluorescent protein Kaede and its analogs

Green fluorescent protein (GFP) and homologous proteins possess a unique pathway of chromophore formation based on autocatalytic modification of their own amino acid residues. Green-to-red photoconvertible fluorescent protein Kaede carries His–Tyr–Gly chromophore-forming triad. Here, we describe synthesis of Kaede red chromophore (2-[(1 E)-2-(5-imidazolyl)ethenyl]-4-( p-hydroxybenzylidene)-5-imidazolone) and its analogs that can be potentially formed by natural amino acid residues. Chromophores corresponding to the following tripeptides were obtained: His–Tyr–Gly, Trp–Tyr–Gly, Phe–Trp–Gly, Tyr–Trp–Gly, Asn–Tyr–Gly, Phe–Tyr–Gly, and Tyr–Tyr–Gly. In basic conditions they fluoresced red with relatively high quantum yield (up to 0.017 for Trp-derived compounds). The most red-shifted absorption peak at 595 nm was found for the chromophore Trp–Tyr–Gly in basic DMSO. Surprisingly, in basic DMF non-aromatic Asn-derived chromophore Asn–Tyr–Gly demonstrated the most red-shifted emission maximum at 642 nm. Thus, Asn residue may be a promising substituent, which can potentially diversify posttranslational chemistry in GFP-like proteins.

In vivo manipulation of fluorescently labeled organelles in living cells by multiphoton excitation

Femtosecond laser pulses in the near-infrared region have potential applications in the imaging and manipulation of intracellular organelles. We report on the manipulation of intracellular organelles by two-photon excitation. The dynamics of green fluorescent protein (GFP)-histone are investigated by two-photon fluorescence recovery after photobleaching (FRAP). Intracellular ablation of fluorescently labeled organelles in living cells is performed by focusing femtosecond laser pulses. We report on the selective marking of individual organelles by using two-photon conversion of a photoconvertible fluorescent protein.

Using Photoactivatable Fluorescent Protein Dendra2 to Track Protein Movement

Photoactivatable fluorescent proteins are capable of dramatic changes in fluorescent properties in response to specific light irradiation. For example, they can be converted from cyan to green, or from green to red, or from nonfluorescent to a brightly fluorescent state. Several types of such proteins were developed recently, and some of them are already becoming popular tools to study protein mobility. Here we provide detailed recommendations on application of the monomeric green-to-red photoconvertible fluorescent protein Dendra2 for protein tracking in living cultured cells.

Method for Real-Time Monitoring of Protein Degradation at the Single Cell Level

Protein degradation is important in practically all aspects of cellular physi-ology (1). Together with transcription and translation, proteolysis ensures maintenance and rapid regulation of individual protein concentration in living cells. The half-life for different proteins varies between a few minutes and days. Moreover, decay rate for many proteins change drastically throughout the cell cycle or in response to external stimuli.Two methods are commonly used to determine a protein’s half-life, namely radioactive pulse-chase analysis and cycloheximide chase (2). Pulse-chase analysis provides minimal distortion of normal cell physiology. The main disadvantages of this method are its laboriousness and necessity for radio-labeling. In contrast to pulse-chase analysis, cycloheximide chase strongly affects cellular metabolism that should be considered a serious disadvantage for this approach. Importantly, both methods do not allow real-time measurements at the single cell level.The use of green fluorescent protein (GFP) provided an opportunity to apply fluorescence microscopy and flow cytometry to protein degradation analysis (3–6). However, to extract information regarding protein degra-dation using GFP, one should block the synthesis of new GFP molecules (e.g., by cycloheximide) (3,4). Even in presence of cycloheximide, residual GFP chromophore maturation affects estimation of degradation rate, especially when maturation and degra-dation half-times are comparable.Since 2002, a number of so-called photoactivatable fluorescent proteins (PAFPs) have been developed (7). Most commonly, local PAFP activation is used to visualize protein movement within cells. Here we propose to use PAFP activation within a whole cell to monitor protein degradation (Figure 1). Indeed, while steady-state fluorescence intensity of an FP-tagged protein depends on both synthesis and degradation rates, photo-activation creates a fluorescent signal that depends only on protein degra-dation (fluorescence bleaching during observation should be taken into account and made allowance for). Thus, time-lapse imaging of activated PAFP allows quantification of the tagged protein degradation process. Also, this protocol can be potentially adapted for PAFP photoactivation in large cell culture samples and their further analysis by flow cytometry or microplate readers.To demonstrate the usability of the method proposed, we used the green-to-red photoconvertible fluorescent protein Dendra2 (Evrogen, Moscow, Russia), which is a commercially available improved version of Dendra (8). In the dark, Dendra2 matures up to the green fluorescent state with excitation-emission maxima at 490 and 507 nm, respectively. Upon maturation, it can be converted into a red-emitting protein (excitation-emission at 553/573 nm) by irradiation with violet (e.g., 405 nm) or blue (e.g., 488 nm) light. Due to its monomeric state, Dendra2 can be safely used for protein labeling. In contrast to other PAFPs, Dendra2 can be activated with blue light, which is less damaging compared with ultraviolet (UV) or violet light. Using a well-established assay based on dithionite reduction of chromo-phore of urea-denatured fluorescent protein followed by dilution of the sample and maturation of the fluorescent protein starting from the native polypeptide (9), we measured Dendra2 maturation half-time as 90 min at 37°C.Similar to GFP, Dendra2 was found to be a long-lived protein. In HeLa and HEK 293 cells, we observed no decrease in Dendra2 green fluorescence for several hours after the addition of cycloheximide (not shown). To photoconvert Dendra2 throughout the experiments, we applied 10–20 s of irradiation with blue light from a 100 W Hg-lamp using the GFP filter set. As a result, the green signal decreased while clearly detectable red fluores-cence appeared. Then, we monitored red fluorescence at 37°C in the confocal mode (Leica DMIRE2 TCS SP2 micro-scope; Leica Microsystems GmbH, Wetzlar, Germany) using the 543-nm laser line. Practically no decay of red fluorescence after Dendra2 photocon-version was observed (Figure 2A), demonstrating very high stability of this protein in living cells. Next we tested the influence of peptides or proteins known to determine fast degra-

Single-organelle tracking by two-photon conversion

Spatial and temporal information about intracellular objects and their dynamics within a living cell are essential for dynamic analysis of such objects in cell biology. A specific intracellular object can be discriminated by photoactivatable fluorescent proteins that exhibit pronounced light-induced spectral changes. Here, we report on selective labeling and tracking of a single organelle by using two-photon conversion of a photoconvertible fluorescent protein with near-infrared femtosecond laser pulses. We performed selective labeling of a single mitochondrion in a living tobacco BY-2 cell using two-photon photoconversion of Kaede. Using this technique, we demonstrated that, in plants, the directed movement of individual mitochondria along the cytoskeletons was mediated by actin filaments, whereas microtubules were not required for the movement of mitochondria. This single-organelle labeling technique enabled us to track the dynamics of a single organelle, revealing the mechanisms involved in organelle dynamics. The technique has potential application in direct tracking of selective cellular and intracellular structures.

Contributions of host and symbiont pigments to the coloration of reef corals

For a variety of coral species, we have studied the molecular origin of their coloration to assess the contributions of host and symbiont pigments. For the corals Catalaphyllia jardinei and an orange-emitting color morph of Lobophyllia hemprichii, the pigments belong to a particular class of green fluorescent protein-like proteins that change their color from green to red upon irradiation with approximately 400 nm light. The optical absorption and emission properties of these proteins were characterized in detail. Their spectra were found to be similar to those of phycoerythrin from cyanobacterial symbionts. To unambiguously determine the molecular origin of the coloration, we performed immunochemical studies using double diffusion in gel analysis on tissue extracts, including also a third coral species, Montastrea cavernosa, which allowed us to attribute the red fluorescent coloration to green-to-red photoconvertible fluorescent proteins. The red fluorescent proteins are localized mainly in the ectodermal tissue and contribute up to 7.0% of the total soluble cellular proteins in these species. Distinct spatial distributions of green and cyan fluorescent proteins were observed for the tissues of M. cavernosa. This observation may suggest that differently colored green fluorescent protein-like proteins have different, specific functions. In addition to green fluorescent protein-like proteins, the pigments of zooxanthellae have a strong effect on the visual appearance of the latter species.

Activity- and mTOR-Dependent Suppression of Kv1.1 Channel mRNA Translation in Dendrites

Mammalian target of rapamycin (mTOR) is implicated in synaptic plasticity and local translation in dendrites. We found that the mTOR inhibitor, rapamycin, increased the Kv1.1 voltage-gated potassium channel protein in hippocampal neurons and promoted Kv1.1 surface expression on dendrites without altering its axonal expression. Moreover, endogenous Kv1.1 mRNA was detected in dendrites. Using Kv1.1 fused to the photoconvertible fluorescence protein Kaede as a reporter for local synthesis, we observed Kv1.1 synthesis in dendrites upon inhibition of mTOR or the N-methyl-d-aspartate (NMDA) glutamate receptor. Thus, synaptic excitation may cause local suppression of dendritic Kv1 channels by reducing their local synthesis.

Semi‐rational engineering of a coral fluorescent protein into an efficient highlighter

Kaede is a natural photoconvertible fluorescent protein found in the coral Trachyphyllia geoffroyi. It contains a tripeptide, His 62-Tyr 63-Gly 64, which acts as a green chromophore that is photoconvertible to red following (ultra-) violet irradiation. Here, we report the molecular cloning and crystal structure determination of a new fluorescent protein, KikG, from the coral Favia favus, and its in vitro evolution conferring green-to-red photoconvertibility. Substitution of the His 62-Tyr 63-Gly 64 sequence into the native protein provided only negligible photoconversion. On the basis of the crystal structure, semi-rational mutagenesis of the amino acids surrounding the chromophore was performed, leading to the generation of an efficient highlighter, KikGR. Within mammalian cells, KikGR is more efficiently photoconverted and is several-fold brighter in both the green and red states than Kaede. In addition, KikGR was successfully photoconverted using two-photon excitation microscopy at 760 nm, ensuring optical cell labelling with better spatial discrimination in thick and highly scattering tissues.

Plant mitochondrial fission and fusion

Single plant cells have hundreds of mitochondria that move around and change their shape through the processes of fission and fusion. The Arabidopsis genome has genes for two dynamin-related proteins, DRP3A and DRP3B, that are similar to genes involved in mitochondrial fission in yeasts. DRP3A and DRP3B were localized to mitochondrial constricted sites and ends. Over-expression of DRP3A or DRP3B with point mutations caused severe elongation of mitochondria. These results show that DRP3A and DRP3B are functional homologues of the dynamin-related protein for mitochondrial fission in yeasts. On the other hand, Arabidopsis appears to have no genes similar to those involved in yeast mitochondrial fusion. Little has been written about mitochondrial fusion in plants. Using a novel photoconvertible fluorescent protein, we have recently shown that mitochondria in live plant cells rapidly undergo fusion and fission on a time scale of seconds. In this mini-review, we focus on plant mitochondrial fission and fusion, and compare them with mitochondrial fission and fusion in yeasts and animals, about which much has been learned in recent years.

Frequent fusion and fission of plant mitochondria with unequal nucleoid distribution.

The balance between mitochondrial fusion and fission influences the reticular shape of mitochondria in yeasts. Little is known about whether mitochondria fusion occurs in plants. Plant mitochondria are usually more numerous and more grain-shaped than animal mitochondria. BLAST searches of the nuclear and mitochondrial genome sequences of Arabidopsis thaliana did not find any obvious homologue of mitochondrial fusion genes found in animals and yeasts. To determine whether mitochondrial fusion actually occurs in plants, we labeled mitochondria in onion epidermal cells with a mitochondria-targeted, photoconvertible fluorescent protein Kaede and then altered the fluorescence of some of the mitochondria within a cell from green to red. Frequent and transient fusion of red and green mitochondria was demonstrated by the appearance of yellow mitochondria that subsequently redivided. We also show that mitochondrial fission occasionally occurs without an equal distribution of the nucleoid (DNA-protein complex in mitochondria), resulting in the coexistence of mitochondria containing various amounts of DNA within a single cell. The heterogeneity of DNA contents in mitochondria may be overcome by the frequent and transient fusion of mitochondria.

Anencephalus and spina bifida.

Rhodopsin is a cilia-specific GPCR essential for vision. Rhodopsin mislocalization is associated with blinding diseases called retinal ciliopathies. The mechanism by which rhodopsin mislocalizes in rod photoreceptor neurons is not well understood. Therefore, we investigated the roles of trafficking signals in rhodopsin mislocalization. Rhodopsin and its truncation mutants were fused to a photoconvertible fluorescent protein, Dendra2, and expressed in <i>Xenopus laevis</i> rod photoreceptors. Photoconversion of Dendra2 causes a color change from green to red, enabling visualization of the dynamic events associated with rhodopsin trafficking and renewal. We found that rhodopsin mislocalization is a facilitated process for which a signal located within 322–326 aa (CCGKN) is essential. An additional signal within 327–336 aa further facilitated the mislocalization. This collective mistrafficking signal confers toxicity to rhodopsin and causes mislocalization when the VXPX cilia-targeting motif is absent. We also determined that the VXPX motif neutralizes this mistrafficking signal, enhances ciliary targeting at least 10-fold, and accelerates trafficking of post-Golgi vesicular structures. In the absence of the VXPX motif, mislocalized rhodopsin is actively cleared through secretion of vesicles into the extracellular milieu. Therefore, this study unveiled the multiple roles of trafficking signals in rhodopsin localization and renewal.