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- Results and Discussion
- Materials and Methods
- Supporting Information
Fluorescent reporter proteins that allow repeated switching between a fluorescent and a non-fluorescent state are novel tools for monitoring intracellular protein trafficking. A codon-optimized variant of the reversibly photoswitchable fluorescent protein DRONPA was designed for the use in transgenic Arabidopsis plants. Its codon usage is also well adapted to the mammalian codon usage. The synthetic protein, DRONPA-s, shows photochemical properties and switching behavior comparable to that of the original DRONPA from Pectiniidae both in vitro and in vivo. DRONPA-s fused to the RNA-binding protein AtGRP7 (Arabidopsis thaliana glycine-rich RNA-binding protein 7) under control of the endogenous AtGRP7 promoter localizes to cytoplasm, nucleoplasm and nucleolus of transgenic Arabidopsis plants. To monitor the intracellular transport dynamics of AtGRP7-DRONPA-s, we set up a single-molecule sensitive confocal fluorescence microscope. Fluorescence recovery after selective photoswitching experiments revealed that AtGRP7-DRONPA-s reaches the nucleus by carrier-mediated transport. Furthermore, photoactivation experiments showed that AtGRP7-DRONPA-s is exported from the nucleus. Thus, AtGRP7 is a nucleocytoplasmic shuttling protein. Our results show that the fluorescent marker DRONPA-s is a versatile tool to track protein transport dynamics in stably transformed plants.
Upon transcription and throughout their life, mRNAs are bound by a suite of proteins. Their dynamic association with RNA defines pre-mRNA processing, lifetime, export from the nucleus and the rate at which a specific mRNA is translated (1). AtGRP7 (Arabidopsis thaliana glycine-rich RNA-binding protein 7) is a representative of a class of small glycine-rich RNA-binding proteins with a single RNA recognition motif (RRM) at its N-terminus. AtGRP7 is part of the endogenous timing system that controls the daily life of the plant. Further, it promotes the transition to flowering and participates in pathogen defense in Arabidopsis presumably via the regulation of target transcripts at the post-transcriptional level (2–5). The AtGRP7 transcript itself undergoes so-called circadian, i.e. 24-h, oscillations in steady-state abundance with a peak at the end of the daily light phase. AtGRP7 impacts the oscillations of its own transcript by negative autoregulation at the post-transcriptional level. It binds to its own pre-mRNA, causing the production of an alternative splice form with a premature termination codon that is rapidly degraded through nonsense-mediated decay (6–8). The influence of AtGRP7 on splice-site selection of its pre-mRNA obviously occurs in the nucleus. Presently, it is not known whether AtGRP7 impacts additional processing steps such as RNA degradation or translation. However, its effect on the steady-state abundance of a suite of transcripts suggests it also has a cytoplasmic function (4,6).
Immunogold labeling has first shown the presence of the orthologous protein SaGRP (Sinapis alba glycine-rich protein) in the nucleus of mustard plants (9). To reach the nuclear compartment, proteins translated on ribosomes in the cytoplasm need to cross the nuclear envelope. This occurs through the nuclear pore complexes (NPCs) embedded in the nuclear envelope (10,11). NPCs contain a central aqueous channel with an estimated radius of about 2.6 nm, as measured in HeLa cells (12). Proteins with a molecular mass above around 40 kDa require facilitated, energy-dependent transport, whereas smaller proteins can passively diffuse through the channel (11,13). However, simple diffusion does not allow proteins to accumulate against a concentration gradient and some smaller proteins including histones pass the nuclear envelope also by carrier-mediated transport. One advantage of such a carrier-dependent passage is the possibility to restrict the access as an additional layer to control, e.g. gene expression.
Nuclear import is mediated by peptide motifs in the cargo proteins that are recognized by receptor proteins (11,13). Conventional nuclear localization signals (NLSs) consist of one or two stretches of basic amino acid residues, in particular lysines. AtGRP7 has a different nuclear import signal, an M9 domain originally found in some mammalian heterogeneous nuclear ribonucleoproteins (hnRNPs) implicated in many aspects of RNA processing (14). M9 domains have been reported to mediate transport both into and out of the nucleus (15). The AtGRP7 M9 domain is recognized by AtTRN1, the Arabidopsis orthologue of the import receptor transportin (also known as karyopherin β2). AtGRP7 is imported into the nucleus of tobacco BY2 cells (16). Notably, it can also be imported into the nuclei of permeabilized HeLa cells by both Arabidopsis AtTRN1 and human transportin (16). In Arabidopsis, an AtGRP7-GFP (green fluorescent protein) fusion protein driven by the strong constitutive Cauliflower Mosaic Virus (CaMV) promoter is found in both the nucleus and the cytoplasm (5,17). The molecular mechanisms of the nuclear uptake have not been investigated and it is not known whether AtGRP7 is re-exported from the nucleus to the cytoplasm.
Fluorescence recovery after photobleaching (FRAP) and photoactivation of fluorescent reporter proteins provide insights into carrier-mediated, directed transport and diffusion in the cell. However, photobleaching and photoactivation are irreversible processes in conventional fluorescent markers such as eGFP (enhanced GFP) or pa-GFP (photoactivatable GFP).
To overcome these limitations, Ando et al. have cloned a fluorescent protein from a Pectiniidae that can be reversibly photoswitched between a bright and a dark state, and thus allows repeated measurements or even different kinetic measurements on a single cell (18). It has been named ‘DRONPA’, after ‘dron’, a ninja designation for ‘vanishing’, and ‘pa’ for ‘photoactivation’. When irradiated with 488-nm light, DRONPA emits bright green fluorescence. With increasing laser power, DRONPA is photoswitched to a non-fluorescent off-state. Irradiation at 400 nm, however, recovers the fluorescent on-state. Moreover, photoswitching of DRONPA to the off-state requires many fewer photons (quantum yield φ = 3.2 × 10−4) than photobleaching of eGFP (quantum yield φ = 8 × 10−6), allowing fluorescence recovery after selective photoswitching measurements to be performed at much lower excitation intensities with less cell damage (18,19).
Recently, Martin et al. have used DRONPA as a reporter in a tobacco (Nicotiana benthamiana) transient expression system (20). In another study, a DRONPA fusion to secretory carrier membrane protein 2 (SCAMP2) monitored the movement of secretory vesicles through the endomembrane system in transiently transformed tobacco BY-2 cells (21). Often, however, intact plants are preferred systems because faithful monitoring of intracellular movement requires the macromolecule's native environment to allow for specific interactions or post-translational modifications to occur. This requires the use of endogenous promoters, whereas transient expression commonly relies on the strong constitutive CaMV promoter. In the case of some GFP-tagged RNA-binding proteins, this has been found to alter their dynamic properties (22–25).
To avoid potential incongruence of the Pectiniidae sequence with the mRNA processing and translation apparatus in plants, we designed a synthetic gene, DRONPA-s (synthetic DRONPA), by adapting the codons to the codon bias of the model plant A. thaliana. This approach was prompted by the previous observation that an 84-nucleotide sequence within GFP was mistaken as a cryptic intron in planta, preventing proper GFP expression in transgenic plants (26). This was remedied by modifications of the GFP codons that removed the sequences erroneously taken as splice sites (26). The newly designed DRONPA variant DRONPA-s is also better adapted to the codon usage of mammals than the original DRONPA sequence cloned from the coral Pectiniidae. High-level expression of DRONPA-s was achieved in transgenic Arabidopsis plants, in a tobacco (N. benthamiana) transient expression system as well as in transfected HeLa and COS-7 cells.
To monitor intracellular transport dynamics, we set up a confocal fluorescence microscope with enhanced sensitivity that allows the use of a low laser power and thus reduces off-switching of DRONPA-s fusion proteins during imaging.
Fluorescence recovery after selective photoswitching experiments in transgenic Arabidopsis plants expressing the AtGRP7-DRONPA-s fusion protein revealed that AtGRP7 is imported into the nucleus via carrier-mediated transport. Furthermore, photoactivation experiments in the nucleus provided evidence that AtGRP7-DRONPA-s is exported out of the nucleus. This shows that AtGRP7 undergoes bidirectional nucleocytoplasmic trafficking.
- Top of page
- Results and Discussion
- Materials and Methods
- Supporting Information
Additional Supporting Information may be found in the online version of this article:
Figure S1: Alignment of the DRONPA nucleotide sequence of the commercial vector DRONPA-GREEN pDG-S1 (MLB Group Company) and the codon-optimized DRONPA-s. Changes in DRONPA-s are highlighted in black. The deduced amino acid sequence is shown below the nucleotide sequences.
Figure S2: Photoswitching performance of DRONPA-s. A) CAI for the original DRONPA and the codon-optimized DRONPA-s, determined by GeneArt using their in-house proprietary software GENEOPTIMIZER®. The histograms show the percentage of sequence codons, which fall into a quality class (defined by the frequency of usage). The quality of the most often used codon for a given amino acid is set to 100, the remaining codons are scaled accordingly (42). B) Details on sequence motifs removed during the optimization process.
Figure S3: Photoswitching performance of DRONPA-s. A) Absorption spectrum (blue, broken line) and emission spectrum (green, solid line) of the synthetic DRONPA-s in solution. B) Decay in fluorescence intensity after irradiation with 488 nm (20 mW/cm2). Each data point corresponds to the integral of the emission spectrum between 500 and 580 nm.
Figure S4: Functional complementation of the atgrp7-1 T-DNA insertion line with the AtGRP7-DRONPA-s fusion protein. A) An immunoblot of total protein extracts from atgrp7-1, Col-0 wt and two independent transformants of AtGRP7-DRONPA-s in atgrp7-1 was probed with an antibody against DRONPA to detect the fusion protein (the asterisk indicates a degradation product) (top) and reprobed with a light-harvesting chlorophyll-binding protein (LHCP) antibody to confirm equal loading. In parallel, a blot was probed with an antibody against AtGRP7 (middle). The part corresponding to endogenous AtGRP7 protein is shown. The blot was reprobed with the LHCP antibody to confirm equal loading. On the blot probed with an antibody against AtGRP8 (bottom), unspecific cross-reaction with the large subunit of ribulose-1,5-bisphosphate carboxylase (RbcL) confirms equal loading. B) The flowering time of Col-0 wt, atgrp7-1 and AtGRP7-DRONPA-s in atgrp7-1 grown in short days was compared by determining total leaf number. Data are based on two independent experiments with n ≥ 20 each. The leaf number in atgrp7-1 and the complemented line is significantly different (p value = 0.000385).
Figure S5: Switching behavior of immobilized DRONPA-s. A) A 20 × 20-µm region of recombinant DRONPA-s embedded in a Mowiol matrix is switched off by exposure to 488 nm. Subsequently, a small spot in the region of the laser focus is switched back to the fluorescent state by irradiation with 404-nm light. Fluorescence intensity is indicated by the quantitative color code: yellow corresponds to the highest fluorescence intensity. B) Calculation of the spot size with the full width at half maximum (FWHM) convention. Shown are the fluorescence intensities along the indicated line in (A).
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