Vectors for Genetically-Encoded Tags for Electron Microscopy Contrast in Drosophila
© Tsui et al. 2016
Received: 7 July 2015
Accepted: 25 January 2016
Published: 1 February 2016
One of the most notable recent advances in electron microscopy (EM) was the development of genetically-encoded EM tags, including the fluorescent flavoprotein Mini-SOG (Mini-Singlet Oxygen Generator). Mini-SOG generates good EM contrast, thus providing a viable alternative to technically-demanding methods such as immuno-electron microcopy (immuno-EM). Based on the Mini-SOG technology, in this paper, we describe the construction, validation and optimization of a series of vectors which allow expression of Mini-SOG in the Drosophila melanogaster genetic model system.
We constructed a Mini-SOG tag that has been codon-optimized for expression in Drosophila (DMS tag) using PCR-mediated gene assembly. The photo-oxidation reaction triggered by DMS was then tested using these vectors in Drosophila cell lines. DMS tag did not affect the subcellular localization of the proteins we tested. More importantly, we demonstrated the utility of the DMS tag for EM in Drosophila by showing that it can produce robust photo-oxidation reactions in the presence of blue light and the substrate DAB; the resultant electron micrographs contain electron-dense regions corresponding to the protein of interest. The vectors we generated allow protein tagging at both termini, for constitutive and inducible protein expression, as well as the generation of transgenic lines by P-element transformation.
We demonstrated the feasibility of Mini-SOG tagging in Drosophila. The constructed vectors will no doubt be a useful molecular tool for genetic tagging to facilitate high-resolution localization of proteins in Drosophila by electron microscopy.
Materials and Methods
Open reading frame of Drosophila Histone2Av (CG5499) was amplified from cDNA prepared from Drosophila embryos. Mitochondrial-targeting sequence corresponding to the N-terminal 31-amino acid mitochondrial import signal of cytochrome c oxidase subunit VIII (CG7181)  was amplified from adult Drosophila genome. The DNA sequence of Mini-SOG (based on Shu et al., 2011 ), codon-optimized for Drosophila expression, and the associated primer sequences (fourteen 40-mers, named DMS1–DMS14) used for gene assembly, were designed using the online program DNAWorks v3.2.2 (https://hpcwebapps.cit.nih.gov/dnaworks/, National Insitute of Health, USA), with codon frequency threshold set to 20 %. The DMS gene was synthesized by a two-step PCR method [5, 6] . The resultant fragment was subcloned into pUbi as pUbi-C-DMS. All other DMS vectors were also constructed by restriction enzyme cloning. The sequences of the primers used are detailed in Additional file 1: Table S1 and the maps of the vectors are in Additional file 1: Figure S2. The sequences of all constructs were confirmed by Sanger sequencing.
Cell Culture and Transfection
Drosophila cell lines were maintained according to standard protocols. Cells were transfected with 0.1–0.3 ug DNA using Effectene Transfection Reagent (QIAGEN) according to the manufacturer’s protocol. For induction of the metallothionein promoter, 0.2–0.5 mM copper sulphate (final concentration) was added 24 h post-transfection. Two days following transfection, the cells were harvested for confocal microscopy and photo-oxidation.
Cells were incubated with 250 nM of Mitotracker Red (Molecular Probes) in culture medium for 30 min at room temperature, washed once with medium and immediately observed under a microscope.
Transfected cells were plated onto gridded 35 mm glass-bottom dishes (P35G-0-14-C, Matek Corp., USA) for 30 min, and observed under the microscope, either directly or after fixation (as described below). Confocal images were taken using LSM 780 (ZEISS, Germany).
Photo-oxidation by Mini-SOG was performed essentially as described by Shu et al., 2011  with a few modifications. Cells were fixed by 2.5 % glutaldehyde in 0.1 M cacodylate buffer for an initial 5 min at room temperature, followed by a one-hour incubation period on ice. Cells were then washed five times with 0.1 M cacodylate buffer and incubated with blocking buffer (50 mM glycine, 10 mM KCN and 5 mM aminotriazole in 0.1 M cacodylate buffer) for 30 min. Afterwards, the buffer was replaced with oxygen-saturated DAB (0.1 mg/ml DAB in 0.1 M cacodylate buffer). Samples were then illuminated with a 63× oil objective using a high pressure mercury lamp with the CFP filter set (EX436/20. DM455. BA480/40) for 2–6 min, depending on the expression levels of the proteins. After photo-oxidation, cells were washed five times with 0.1 M cacodylate buffer and sent immediately for electron microscope sample preparation.
Samples were prefixed with 2.5 % glutraldehyde in 0.1 M sodium cacodylate buffer, postfixed with 1 % osmium tetroxide (OsO4) in 0.1 M sodium cacodylate buffer for 30 min, and then washed with pure water three times. The samples were subsequently dehydrated by ethanol series. A 1:1 ethanol:epoxy resin mix was then added to the samples and incubated on a mixer for 30 min. Afterwards, the resin mix was replaced by pure resin and incubated overnight, and an extra change of pure resin was performed. The resin was then allowed to polymerize in a 60 ° C oven for two days. The resin block was trimmed to 0.5 mm cubes, which were further sliced to 50 nm sections using a microtome. These sections were mounted onto copper grids (MAXTAFORM HF34, 200 holes) without any stain and observed by a JEM-1230R microscope (JEOL), at an accelerating voltage of 100 keV, using bright field imaging.
Results and Discussion
We engineered the DMS tag at both the N- terminal and C- terminal ends. In addition to the monomeric DMS tag, a tandem DMS dimer was constructed to further enhance the enzymatic efficiency of the tag. There are three choices of promoters: constitutive (ubquitin promoter) and inducible (metallothionein and UAS promoters). The ubiquitin promoter is derived from the Ubqi-p63E gene (CG11624), which drives high level expression in cell lines and tissues in Drosophila flies . We also included a mCherry tag to the DMS vector. Hygromycin gene in pMK33b backbone allows for stable clone generation in cell line while pUAST backbone can be used for the generation of transgenic flies . In addition, the metallothionein promoter in pMK33b allows inducible expression in cell culture. As shown in Fig. 2b, without the addition of copper sulphate, cells transfected with the pMK33B-mCherry-DMS-His2Av constructs showed no red fluorescence and hence no expression of the mCherry-DMS-His2Av fusion protein (Fig. 2c, upper panel). After the addition of 0.5 mM copper sulphate for 48 h, robust expression of mCherry-DMS-His2Av was detected by red nuclear fluorescence (Fig. 2c, lower panel). Altogether, we constructed 14 DMS vectors that should prove to be suitable for a wide variety of needs in EM studies using the Drosophila model system.
We subsequently investigated the time dependence of the photo-oxidation reaction by DMS in Drosophila cells, using histone2Av fusion. We found that the bleaching of mCherry occurs after around 1 min of reaction, while the photo-oxidation reaction continues to be very robust 4 min after the reaction started, as judged by the continuous increase in the darkness of the osmium deposit in the nucleus (Fig. 5, lower panel). This indicates that under correct fixation and photo-oxidation reaction conditions, the DMS fusion can sustain a long period of reaction. This sustained reaction should prove to be particularly useful when the DMS-fusion construct shows lower expression levels.
Over-expression sometimes leads to the mis-localization of the tagged protein. Reducing expression levels may be done in several ways. For the MT promoter, reducing the amount of copper added by titration may be enough to reduce expression levels for correct localization. Another way to get around this is to switch existing promoters with a weaker substitute.
Enough time should be left for a suitable amount of oxidation product to form However, bear in mind that overly extended reactions lead to excessive buildup of free radicals, damaging the internal cellular structure.
Terminal fusion site:
As with any other exogenous protein tagging method, there is a chance that the DMS tags will affect trafficking and localization of the proteins being investigated. Hence, it is advisable to make both N-terminal and C-terminal tagged fusions, then to choose constructs yielding the most natural expression and trafficking. We created vectors for fusion at both ends for this purpose. Typically, the localization of the endogenous protein could be investigated in a separate immunolabelling control experiment under light microscopy to check for proper localization of the fusion protein before starting the EM experiment.
In conclusion, we constructed a set of vectors for optimized Mini-SOG mediated photo-oxidation in Drosophila cells for high resolution EM detection of proteins. These vectors are inducible and can be used for cell culture as well as transgenic preparations. We believe that this vector set will prove to be a useful resource not only to the Drosophila community, but also to anyone interested in using Drosophila as a disease model system in general.
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