Visualization of intracellular structures using fluorescent proteins intobacco BY-2 cells
We have established several intracellular structure-visualized cell lines of tobacco
BY-2 using fluorescent proteins. GFP-tubulin, GFP-AtVAM3, GFP-ABD2 and histone
H2B-RFP visualized microtubules, vacuolar membrane, actin microfilaments andchromosomes/ cell nuclei, respectively (Kumagai et al. 2001, Kutsuna and Hasezawa
2002, Sano et al. 2005). In this protocol, we will introduce the procedure used toestablish these cell lines.
I. Construction of fusion genes
Plasmid construction was carried out in a modified pUC18 vector which contained
the CaMV 35S promoter and the nopaline synthase terminator sequence from pBI101.
The constructed region was subsequently subcloned into a binary vector. Molecularcloning was carried out by the standard method and only the sequence information isshown below.
I-1. Fluorescent proteins
GFP: sGFP(S65T), Niwa et al. 1999
RFP: tandem-dimer Tomato (tdTomato), Shaner et al. 2004, R Tsien
http://www.tsienlab.ucsd.edu/
Although we used sGFP(S65T) as GFP for every construct, EGFP (BD Biosciences
Clontech, http://www.clontech.com/) can be used as well.
Because the commercially available DsReds of DsRed (now off the market), DsRed2
and DsRed-Express form tetramer that often aggregates in living cells when fused withinterested proteins, monomeric RFPs are recommended (Campbell et al. 2001).
Thanks to the energetic efforts of R. Tsien’s lab in California, monomeric forms of RFP
were developed and improved. The first, mRFP1, was improved from the DsRed(Campbell et al. 2001). Although we succeeded in visualizing some of the intracellular
structures of microtubules and actin microfilaments in tobacco BY-2 cells, because of
the low fluorescent quantum yield of 0.25 they were unsuitable for time-sequential
observations over a long period or for obtaining images with a very short exposure time.
Thanks to the improvement of the monomeric RFPs, monomeric red, orange and yellow
fluorescent proteins have been developed (Shaner et al. 2004). We used tandem-dimer
Tomato (tdTomato), whichforms a dimmer intra-molecularly, and succeeded invisualizing chromosomes in tobacco BY-2 cells by preparing fusion proteins withhistone H2B (manuscript submitted). However, with some proteins, such as membraneproteins, the fusion proteins with tdTomato did not show proper intracellular
localization, possibly because of problems with the three-dimensional structures of thefusion proteins or because of the intra-molecular dimerization of tdTomato.
I-2. Intracellular proteinsTobacco α-tubulin: Accession number AB052822 (Kumagai et al. 2001)
Arabidopsis AtVam3: Accession number TU88045 (Sato et al. 1997)
Arabidopsis AtFim1: Accession number U66424 (McCurdy and Kim et al. 1998)
Tobacco histone H2B: clone number: 35066r1 in the TAB (Transcriptome Analysis ofBY-2) database (http://mrg.psc.riken.jp/strc/index.htm).
I-3. Choice of N- or C-terminus tags and linkers
Tobacco α-tubulin, AtVAM3 and the actin binding domain (ABD) of AtFim1
visualized the intracellular structures with N-terminus tags of fluorescent proteins (ex.
GFP-tubulin). In contrast, tobacco histone H2B visualized chromosomes with aC-terminus tag of the fluorescent protein (histone H2B-RFP). In the reported literature,membrane proteins often worked with C-terminus fluorescent tags.
Between the fluorescent protein and the protein of interest, some liker sequence ofseveral amino acids is often required to keep the three-dimensional conformation ofboth the fluorescent protein and the protein of interest. We inserted two and elevenamino acids of GA (glycine-alanine) or GAGAGAGAGAG to prepare GFP-tubulin andGFP-ABD2, respectively.
I-4. Binary vectors
pBI series (pBI101, pBI121): Clontech (out of the market)
pCAMBIA series: CAMBIA (http://www.cambia.org/daisy/cambia/home.html)
pGWB series: Nakagawa et al. (unpublished,http://bio2.ipc.shimane-u.ac.jp/nakagawalab.htm)
pCAMBIA vectors were developed to overcome the difficulties of handling theexisting binary vectors. pCAMBIA vectors are relatively small (about 9 kbp), have ahigh copy number in E.coli and several selectable markers for bacteria and plants.
pGWB vectors are designed to enable gene fusion by using Gateway cloning
technology and cloning efficiency has been dramatically improved (Nakagawa et al.

unpublished result). We believe the pGWB vectors in combination with the Gateway
technology are the most efficient available at this moment, though there are still somepoints to be attended to. The pGWB vectors provided 14 (pGWB5) or 9 amino acids(pGWB6) between the GFP and proteins of interest by recombination and the relatively
long linker sequences may sometimes affect GFP fluorescence. Since the original
pGWB vectors have two selective markers against kanamycin and hygromycin forplants, dual transformation will be difficult when the pGWB vectors are chosen for thefirst transformation. Although we have not yet tried them, new pGWB vectors havebeen developed to overcome these disadvantages (Nakagawa et al. in press).
II. Transformation into BY-2 cells
The fusion protein-construct in a binary vector was transformed into Agrobacterium
tumefaciens strain, LBA4404. A 5-ml aliquot of 2-d old BY-2 cells was incubated with
100 µl of an overnight culture of the transformed A. tumefaciens. After 2-d incubation at27°C, the BY-2 cells were washed 4 times in a 10 ml liquid medium and then plated
onto solid medium containing 30 to 50 mg liter-1 of kanmycin or hygromycin to selecttransformants and 250 to 500 mg liter-1 carbenicillin or claforan to remove bacteria.
Calluses, which appeared after 3 to 4 weeks (2-5 mm in diameter), were transferred onto
new solid plates and cultured independently until they reached approximately 1 cm indiameter. To prepare liquid cultures, pore 0.1 to 1 g callus (about 0.5 to 1 cm indiameter) in a small amount of fresh liquid medium (5-10 ml);when they’re fullygrown , add fresh medium (10-20 ml). The repetition of this cycle increases the culture
volume. After 2-3 days, transfer the calluses into liquid medium.Gentle pipetting of thecalluses will accelerate their growth.
To dual-visualize intracellular structures, two binary-vector constructs weresequentially transformed into tobacco BY-2 cells. For example, to dual-visualize
microtubules and chromosomes, the binary-vector construct of histone H2B-RFP wastransformed into the established BY-2 cell line that visualized microtubules with
GFP-tubulin.
References
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