Biarsenical-tetracysteine fluorescent protein tagging has been effectively used in a variety of cell types. It has the advantage of requiring a much smaller peptide alteration to existing proteins than fusion to green fluorescent protein (GFP) or monomeric red fluorescent protein (mRFP). However, there are no reports of the tetracysteine tagging system being used in Dictyostelium. In order to establish this tagging system in Dictyostelium, the filamin gene (FLN) was modified to express a C-terminal tetracysteine sequence and then transfected into cells. After addition of either FlAsH-EDT2 or ReAsH-EDT2, the fluorescence intensity of cells increased in a time-dependent manner and reached a plateau after 3 h of incubation. ReAsH had a much stronger and more specifically localized fluorescent signal compared with FlAsH. After removal of the ReAsH-EDT2 reagent, the fluorescence signal remained detectable for at least 24 h. The localization of filamin labelled by ReAsH was similar to that of an FLN-mRFP fusion protein, but the fluorescence signal from the ReAsH-labelled protein was stronger. Our findings suggest that the ReAsH-tetracysteine tagging system can be a useful alternative for in vivo protein tagging in Dictyostelium.
Fluorescent protein tagging allows the visualization of individual protein dynamics in living cells. The cloning of GFP and the development of various colour derivatives have made this approach to cell biological problems generally accessible (Giepmans et al., 2006). However, several studies indicate that the large size of the fluorescent protein tag (25–30 kDa) may, in some cases, affect the function, distribution or maturation of the fusion protein (Andresen et al., 2004). In order to solve this problem, several smaller molecular tags have been developed. One of the most interesting approaches is the tetracysteine (4Cys) tagging system developed by Tsien and colleagues. This method is based on the interaction between small membrane-permeant nonfluorescent biarsenical compounds, 4,5-bis(1,3,2-dithiarsolan-2-yl) fluoresce in (FlAsH-EDT2) or 4,5-bis(1,3,2-dithiarsolan-2-yl) resorufin (ReAsH-EDT2), and a short genetically encoded 4Cys peptide motif (e.g. Cys-Cys-Pro-Gly-Cys-Cys) (Adams et al., 2002). When the biarsenical compound is added to cells expressing 4Cys-tagged proteins, the reagent covalently reacts with the four cysteines, forming an intensely fluorescent complex. Moreover, two different biarsenical compounds that react with the same 4Cys tag are available, which fluoresce green (FlAsH-EDT2) or red (ReAsH-EDT2). The 4Cys tagging system has several advantages over fluorescent protein tagging with GFP and its derivatives. The 4Cys motif adds only 6–9 amino acids to the target protein, and this modification can be done using an oligonucleotide, greatly simplifying vector construction. In addition, the two biarsenical compounds can be added to and removed from cells to transiently label them, or added sequentially to differentially label old and new proteins.
The biarsenical-4Cys system has been used in many different cell types, including mammalian cells (Adams et al., 2002; Gaietta et al., 2002; Martin et al., 2005), Drosophila (Poskanzer et al., 2003), plants (Estevez & Somerville, 2006), Escherichia Coli (Ignatova & Gierasch, 2004) and yeast (Rice et al., 2001; Andresen et al., 2004). However, successful application of this technology in Dictyostelium discoideum (D. discoideum) cells has not been previously described. Dictyostelium is a unicellular organism that has been used as a model system to investigate questions related to cellular, developmental and molecular biology. GFP and RFP have been used extensively in this system to create fluorescent fusion proteins. We sought to develop the 4Cys tagging technique to use in situations in which it appeared that GFP was altering protein function or localization in cells. We have found that the system works, but has unusual limitations that might affect other cell types as well.
Materials and methods
TC-FlAsH(TM) (C24H18As2O5S4) and TC-ReAsH(TM) (C16H13As2NO3S4) II In-Cell Tetracysteine Tag Detection Kits were obtained from Invitrogen, Inc. (Eugene, OR, USA). Oligonucleotides were purchased from MWG (High Point, NC, USA). Enzymes for molecular biology were from New England Biolabs (Ipswich, MA, USA).
To introduce the 4Cys sequence coding for CCPGCCMEP (Martin et al., 2005) into an expression vector, complementary oligonucleotide primers: 5′-TTCCTGAACTGCTGCCCCGGGTGCTGTATGGAGCCCTAAT-3′ and 5′-CTAGATTAGGGCTCCATACAGCACCCGGGGCAGCAGTTCAGGAATGCA-3′ were annealed at a final concentration of 1 pmol μL–1 in T4 DNA ligase buffer (NEB). The vector pDXA-3C (Manstein et al., 1995) was linearized with NsiI and XbaI and then ligated with the annealed sequences overnight. The resulting construct pDXA-4Cys creates an expression cassette under control of an actin-15 promoter, with the 4Cys sequence at the C-terminus with G418 selectivity. To generate a construct containing full-length filamin (ABP-120) fused to the 4Cys tag, pDFLN-GFP (Washington & Knecht, 2008) was digested with Hind III and Bam HI, and the fragment containing the filamin-coding sequence was inserted into pDXA-4Cys so that the 4Cys sequence was at the C-terminus of the fusion protein.
Cell culture and transformation of Dictyostelium cells
Cells of D. discoideum strain GHR (120−) (Rivero et al., 1999) were cultured at 20°C in HL-5 nutrient medium (Sussman & Sussman, 1967). As these cells were already G418-resistant, the pDFLN-4Cys plasmid was co-transformed along with the hygromycin-resistant plasmid pHygTm(plus) (a kind gift of Jeff Williams) by electroporation, as described previously (Pang et al., 1999). After electroporation, the cells were transferred to 35-mm petri dishes and selected with 25 μg mL–1 hygromycin (Calbiochem, Co., San Diego, CA, USA) in HL-5 medium. Once colonies of hygromycin-resistant cells were visible, the medium was changed to LoFlo low fluorescence medium (H. MacWilliams, personal communication, http://dictybase.org/techniques/media/lowflo_medium.html) containing 2 μm ReAsH-EDT2 and fluorescent colonies were picked manually with a micropippetter and put into a new dish to clone the fluorescent cells.
pDFLN-4Cys transfected cells were harvested in HL-5 medium and the concentration was adjusted to 1 × 106 cells mL–1. A 200-μL cell suspension was then added to each well of an eight-well-chambered coverglass slide (Lab-Tek II; Nalge Nunc International, Rochester, NY, USA) and the cells were allowed to adhere for 30 min. The medium was removed and the cells were washed with LoFlo and replaced with 200 μL of LoFlo medium contain 2 μm of FlAsH-EDT2 or ReAsH-EDT2. The cells were incubated in darkness at room temperature for the indicated period of time before imaging.
Under agarose folate chemotaxis assay
The folate chemotaxis assay used in this study was performed as previously described (Woznica & Knecht, 2006). Briefly, transfected cells were labelled by ReAsH-EDT2 for 3 h and then washed twice with SM medium (10 g Bacto® protease peptone no. 2 [Difco, Detroit, MI, USA], 10 g glucose, 1 g yeast extract, 1.9 g KH2PO4, 0.6 g K2HPO4,, 1 g MgSO4· 7H2O, to 1 L, pH 6.5]. The cells were resuspended in the SM medium, adjusted to 1 × 106 cells mL–1, and 100 μL of the suspension was added to a well cut in a 1% agarose gel in a 60-mm glass bottom petri dish (Willco Wells, Amsterdam, The Netherlands). The folic acid chemoattractant was added to the second trough cut 5 mm away from the cell well. Over the next 4–6 h, the cells were imaged as they moved under the agarose towards the folate well.
Imaging was performed with a Leica TCS SP2 confocal microscope system using a 40× (1.25 NA) or 100× (1.40 NA) oil immersion objective. (Leica Microsystems, Heidelberg, Germany). Label intensity was monitored under lower magnification (40× objective), and chemotaxis under higher magnification (100× objective). All images were captured using the same settings so that the intensity of labelling can be compared. All the images were processed using NIH Image J software (Rasband, W.S., ImageJ; National Institutes of Health, Bethesda, MD, USA, http://rsb.info.nih.gov/ij/, 1997–2007) and Canvas (ACD Systems, Inc., Miami, FL, USA).
Results and discussion
In order to test the 4Cys tagging system, we created a 4Cys-labelled version of a probe that has been well characterized as GFP and RFP fusion proteins. Filamin (ABP-120) is a member of the calponin homology (CH) superfamily of actin cross-linking proteins (Popowicz et al., 2006). Filamin cross-links actin filaments into a loose network structure. The cells were transfected with a vector encoding expression of the complete filamin protein with a C-terminal 4Cys motif (pDFLN-4Cys). The same transformed cells were then incubated in LoFlo media containing either 2 μm of FlAsH-EDT2 or 2 μm of ReAsH-EDT2 and imaged over time by fluorescence microscopy (Fig. 1A). The results show that the FlAsH-EDT2-labelled cells had a much weaker fluorescence signal compared with ReAsH-EDT2. The signal was detectable 15 min after adding the label and increased steadily for about 3 h and then levelled off (Fig. 1B).
It was surprising that the FlAsH-EDT2 reagent seemed to label 4Cys-expressing cells poorly compared with ReAsH-EDT2. The manufacturer recommends FlAsH-EDT2 and indicates that ReAsH-EDT2 was more sensitive to photobleaching and had more phenotypic effects on cells (Invitrogen, Inc., product manual). It is possible that in Dictyostelium, the FlAsH-EDT2 reagent has poor membrane permeability and could be complexing specifically with the 4Cys probe, but at a slow rate. To determine if the weak FlAsH-EDT2 signal was specific, wild-type cells and cells expressing FLN-4Cys were labelled with FlAsH-EDT2 or ReAsH-EDT2 and examined by confocal microscopy. The fluorescent signal from FLN-4Cys cells labelled with FlAsH-EDT2 was not significantly different from labelled wild-type cells, and was higher than in unlabelled cells (Fig. 1C; and data not shown). This result indicates that most of the FlAsH-EDT2 signal is coming from nonspecific labelling. The same FLN-4Cys cells labelled with ReAsH-EDT2 produced a bright and localized signal, whereas ReAsH-EDT2-labelled wild-type cells had little detectable background under the same imaging conditions. Similar results have been found with several other 4Cys-tagged proteins in Dictyostelium, indicating these results are not specific to filamin. In order to test for phenotypic effects of the labelling reagents, the motility of cells exposed to FlAsH-EDT2 and ReAsH-EDT2 was measured. The rate of movement of treated cells was not significantly different from untreated cells, indicating that Dictyostelium cells are not affected by these reagents in the same way as mammalian cells (data not shown).
On the basis of the more efficient fluorescence labelling by ReAsH-EDT2, we focused on optimizing the system using this reagent. In order to determine the optimal labelling concentration, transfected cells were incubated with different concentrations of ReAsH-EDT2 for different time intervals (Fig. 2A). The relative labelling intensity increased gradually up to 2 μm. Increasing the concentration to 4 μm produced a small increase in labelling, but given the high cost of the reagent, 2 μm produced a very strong signal and is probably the optimal concentration for most experiments (Fig. 2B). The labelling was also time-dependent, reaching a peak intensity at about 3 h, which is significantly longer than what the company recommends for mammalian cells (30–60 min). We speculate that the increased labelling time and the poor labelling with FlAsH-EDT2 reflect differences in membrane permeability of the reagents into Dictyostelium cells.
The stability of the fluorescent protein complex was also evaluated. The cells were imaged for various times after removal of the ReAsH-EDT2 reagent from the medium. The fluorescent signal is still very strong 3 h after removal of the ReAsH-EDT2 reagent, but after overnight incubation, it has decreased by about 80%. The signal can still be detected 24 h after removal of the ReAsH-EDT2 reagent, but by 48 h, it is mostly gone (Fig. 3A and B). This may be due to the loss of the probe or growth of the cells plus turnover of the protein, but we cannot distinguish between these possibilities.
In order to determine that the probe was not affecting protein function, the localization of the ReAsH-labelled protein was determined. The FLN-4Cys-tagged protein localized to the cellular cortex and macropinosome cups. In a chemotactic gradient of folic acid, the cells become polarized, and the fluorescent protein was found in the cortex at the rear of the cell (Fig. 4). These results are comparable to those obtained with the FLN-GFP and FLN-mRFP probes (Washington & Knecht, 2008).
In order to directly compare the utility of the 4Cys probe with fluorescent protein tags, cells expressing either FLN-GFP or FLN-mRFP were compared with cells expressing FLN-4Cys (Fig. 5). As this experiment was performed on different cells populations, and cells can vary significantly in the expression of transfected probes, it is not possible to make quantitative comparisons, but the results provide a rough guide to the utility of the probes. As expected, the FLN-GFP probe-expressing cells were brighter than the FlAsH-EDT2-treated cells, and the localization was more distinct. This is consistent with the fact that most of the FlAsH-EDT2 signal appears to be background. The signal intensity and localization of the FLN-mRFP and ReAsH-EDT2 probes were similar. Therefore, it is clear that although FlAsH-EDT2 is not useful for live cell imaging in Dictyostelium cells, ReAsH-EDT2 can be a valuable alternative to RFP for fluorescent tagging experiments.
In this paper, we have demonstrated that the versatile 4Cys/biarsenical molecular tagging system can be used in D. discoideum to examine the localization of proteins in live cells. However, this system has limitations that are different from what have been described previously for other systems. ReAsH-EDT2, but not FlAsH-EDT2, specifically labels 4Cys-tagged proteins in a pattern indistinguishable from RFP and GFP fusion proteins. FlAsH-EDT2 labelled 4Cys probe-expressing cells weakly, and the signal was not significantly different from the background labelling of untransfected cells. ReAsH-labelled filamin-4Cys was also compared with filamin-mRFP and both tags show the same cellular localization and similar specific labelling intensity.
We would like to thank Dr. Raymond Washington for providing the cells expressing FLN-GFP and FLN-mRFP. We would also like to thank Michael O'Grady of Invitrogen for providing us with a sample of ReAsH-EDT2 for testing.