Monitoring of in vitro and in vivo translation of green fluorescent protein and its fusion proteins by fluorescence correlation spectroscopy
2001; Wiley; Volume: 44; Issue: 1 Linguagem: Inglês
10.1002/1097-0320(20010501)44
ISSN1097-0320
AutoresYasutomo Nomura, Hirotoshi Tanaka, Lorenz Poellinger, Fumihiro Higashino, Masataka Kinjo,
Tópico(s)Molecular Biology Techniques and Applications
ResumoCytometryVolume 44, Issue 1 p. 1-6 Original ArticleFree Access Monitoring of in vitro and in vivo translation of green fluorescent protein and its fusion proteins by fluorescence correlation spectroscopy Yasutomo Nomura, Corresponding Author Yasutomo Nomura ynomura@imd.es.hokudai.ac.jp Laboratory of Supramolecular Biophysics, Research Institute for Electronic Science, Hokkaido University, Sapporo, JapanLaboratory of Supramolecular Biophysics, Research Institute for Electronic Science, Hokkaido University, Sapporo 060-0812, JapanSearch for more papers by this authorHirotoshi Tanaka, Hirotoshi Tanaka Department of Clinical Immunology and AIDS Research Center, Institute of Medical Science, University of Tokyo, Tokyo, JapanSearch for more papers by this authorLorenz Poellinger, Lorenz Poellinger Department of Cell and Molecular Biology, Medical Nobel Institute, Karolinska Institute, Stockholm, SwedenSearch for more papers by this authorFumihiro Higashino, Fumihiro Higashino Department of Oral Pathology, Hokkaido University School of Dentistry, Sapporo, JapanSearch for more papers by this authorMasataka Kinjo, Masataka Kinjo Department of Clinical Immunology and AIDS Research Center, Institute of Medical Science, University of Tokyo, Tokyo, JapanSearch for more papers by this author Yasutomo Nomura, Corresponding Author Yasutomo Nomura ynomura@imd.es.hokudai.ac.jp Laboratory of Supramolecular Biophysics, Research Institute for Electronic Science, Hokkaido University, Sapporo, JapanLaboratory of Supramolecular Biophysics, Research Institute for Electronic Science, Hokkaido University, Sapporo 060-0812, JapanSearch for more papers by this authorHirotoshi Tanaka, Hirotoshi Tanaka Department of Clinical Immunology and AIDS Research Center, Institute of Medical Science, University of Tokyo, Tokyo, JapanSearch for more papers by this authorLorenz Poellinger, Lorenz Poellinger Department of Cell and Molecular Biology, Medical Nobel Institute, Karolinska Institute, Stockholm, SwedenSearch for more papers by this authorFumihiro Higashino, Fumihiro Higashino Department of Oral Pathology, Hokkaido University School of Dentistry, Sapporo, JapanSearch for more papers by this authorMasataka Kinjo, Masataka Kinjo Department of Clinical Immunology and AIDS Research Center, Institute of Medical Science, University of Tokyo, Tokyo, JapanSearch for more papers by this author First published: 12 April 2001 https://doi.org/10.1002/1097-0320(20010501)44:1 3.0.CO;2-0Citations: 22AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Abstract Background Because the process of protein translation is an event of sparse molecules, the measurement requires high sensitivity. One of the candidates for studying the molecules is fluorescence correlation spectroscopy (FCS), which gleans quantitative information from fluctuating fluorescence signals in a diluted solution. Methods Using FCS, the translation products of expression plasmid for green fluorescent protein (GFP) and its fusion proteins were measured in vitro and in vivo. Results In in vitro translation, the number of products increased linearly for 90 min upon concentration of the plasmid. The autocorrelation function for GFP was fitted with a one-component model with a diffusion time of 0.18 ms, which was identical to the value expected from the molecular weight. In the cases of GFP- tagged hypoxia-inducible factor-1α and glucocorticoid receptor, each fitting result was significantly improved with a two-component model. The slow component with a diffusion time of 6 ms appeared to be related to the ribosome or polysome. In response to the addition of dexamethasone, the nuclear translocation from cytosol clearly induced the decrease in number of molecules in the focal point. Conclusions FCS permits monitoring of the number of molecules translated in vitro and in vivo, the translation rate, and the molecular weight. Cytometry 44:1–6, 2001. © 2001 Wiley-Liss, Inc. Green fluorescent protein (GFP) from Aequorea victoria is a powerful fluorophore formed autocatalytically by internal cyclization and oxidation of the Ser-Tyr-Gly sequence at positions 65–67 within the 238 amino acid (1). Following the cloning of the cDNA for GFP, several investigators took a great interest in the expression of GFP and GFP-fusion proteins as a unique fluorophore to monitor a range of intracellular processes, especially the localization of proteins (2, 3). Fluorescence correlation spectroscopy (FCS) measures sensitive fluctuations in fluorescence intensity due to only a few fluorescent molecules diffusing in and out of a small volume element in solution (4-6). The frequency of fluctuations is determined by the diffusion time of fluorescent molecules, which depends on the molecular weight of fluctuations. The width of the fluctuations is related to the number of fluorescent molecules (7-12). The technique requires several microliters of sample volume, which can be reused after the experiments have been performed because the measurement causes little damage (13). Monitoring GFP fluorescence in living cells requires a sensitive technique that detects low expression of GFP-fusion protein and measures molecular changes in intracellular compartments. FCS is ideal for this purpose (14, 15). It permits observation of the translation process of GFP-fusion proteins when cells are transfected with the expression vector. In the present study, FCS was used to compare the translation of GFP and GFP-fusion protein in vitro and in vivo because few studies had made the in vitro versus in vivo comparison. MATERIALS AND METHODS Sample Preparation Three expression plasmids were used in this study as described previously (16, 17): pCMX-GFP, pCMX-GFP-hypoxia-inducible factor (HIF)1α, and pCMX-GFP-glucocorticoid receptor (hGR). Briefly, pCMX-GFP has a modified and highly chromophoric form of GFP cDNA under the control of CMV immediate-early and T7 promoters. This mutant GFP contains an S65A mutation, which confers a wavelength shift and temperature resistance to the protein as well as a Y145F substitution, increasing the intracellular stability of GFP (18). pCMX-GFP-HIF1α (17) and pCMX-GFP-hGR (16) are the expression plasmids that encode GFP and full-length cDNA for HIF1α and hGR, respectively. The proteins were synthesized using the TNT® in vitro transcription/translation system (Promega, Madison, WI), which contains either wheat germ extract or reticulocyte lysate and T7 RNA polymerase, according to the manufacturer's instruction. Before use, wheat germ extract was centrifuged 10,000 × g for 20 min at 4°C. Cos7 cells (American Type Culture Collection, Rockville, MD) were routinely maintained in Dulbecco's minimal essential medium supplemented with 10% fetal calf serum plus penicillin and streptomycin at 37°C. The cells were cultured on 6-cm diameter plastic dishes and the medium was changed to OPTI-MEM medium that lacked phenol red (Life Technologies, Rockville, MD) before transfection. A plasmid cocktail containing 6 μg in total of the expression plasmids of pCMX-GFP, pCMX-GFP-HIF1α, or pCMX-GFP-hGR was mixed with 12 μl of TransIT LT-1 (Panvera, Madison, WI) and added to the culture. FCS Measurement FCS measurement was performed using a ConfoCor fluorescence correlation measurement system (Carl Zeiss, Jena, Germany) with an objective (C-Apocromat 40× 1.2 NA; Carl Zeiss). A pinhole (30 μm) was set in front of the photodiode. Sample cells or solution were excited by a 488 nm line of the laser (about 4 kW/cm2). The sample droplet (20 μl) on the eight-well–chambered glass was set on the objective. The fluctuation of fluorescence intensity was analyzed using an autocorrelation function, which provided the average number of molecules and the translational diffusion time of fluorescent particles in the focused field in which the molecules moved in and out due to Brownian motion. The fluorescence autocorrelation function (G(t)) was fitted to a simple two-component model with the average number of fluorescent molecules (N) and the translational diffusion time of the free fast-moving product (Dfree) and slow-moving product (Dslow), according to Equation 1, with Dfree, slow = wo2/4Dfree, slow and S = wo/zo, (1) where y is the fraction of the slow-moving component, wo is the radius of the detection field (volume element), 2zo is the field length, and Dfree and Dslow are the translational diffusion constants of free fast-moving product and slow-moving product, respectively. S is the structure parameter (19). For the one-component model, y = 0. The data analysis was performed using the nonlinear least-squares method with the FCS ACCESS computer program (EVOTEC Biosystems, Hamburg, Germany). The structure parameter was 0.192 and the diffusion time of rhodamine 6G in pure water was 0.049 ms. In the case of in vitro translation, unless otherwise stated, measurements at 15-min intervals after the addition of the plasmid were performed at 23°C. Because the fluorescence intensity was very weak just after the addition, FCS measurements were not carried out. Transiently expressed GFP/GFP-fusion proteins in Cos 7 cells were detectable between 24 and 72 h after transfection. Cytosol within the cells was observed at room temperature at 24 h after transfection. FCS measurement for cultured cells was carried out according to previous reports (20, 21). Cells were cultured in LabTek eight-well chambered coverglass (Nalge Nunc International, Naperville, IL) under normal conditions. Before the measurement, cells were washed several times with phenol red-free Iscove's modified Dulbecco's medium (IMDM) or Hank's balanced salt solution to remove phenol red dye. Fluorescent cells were targeted under a normal microscope and the measuring point (laser-focused point) fixed in cytosolic space, excluding the nucleus. Next, the emission intensity profile along the ordinate axis (z-axis) from the coverglass was determined by moving the stage up and down slowly. The focal point was fixed at the maximum fluorescence intensity for FCS measurement. RESULTS AND DISCUSSION In Vitro In the reticulocyte lysate, increases in fluorescence intensity and autocorrelation functions were observed after the addition of the plasmid under the reaction temperature of 30°C (data not shown). However, to avoid the optical interference due to the coexistence of hemoglobin, we used the wheat germ extract system. Although the manufacturer-supplied wheat germ extract was opaque, after centrifugation, the clear supernatant had a significant translational activity at 23°C. Therefore, the data obtained from the supernatant were analyzed. Figure 1 shows the typical autocorrelation function of in vitro translation of GFP at the various times after the addition of pCMX-GFP. The sequential decrease in peak values of autocorrelation indicates that fluorescent molecules increased with time. The autocorrelation functions could be fitted well using the one-component model and the diffusion time did not change. Then, it can be explained that single fluorescent species increased during the reaction. As shown in Figure 2, the number of molecules and the fluorescent intensity increased with time. In the range from 10 ng to 1 μg of pCMX-GFP, the increase in number of molecules reached plateau at the end of the reaction time (Fig. 2A). The plateau effect may result from the decrease in enzyme activity rather than from the shortage of substrates such as amino acid and ATP. The number of fluorescent products was linearly dependent on the concentration of pCMX-GFP until 100 ng (Fig. 2B). At 1 μg of pCMX-GFP, the change in number of molecules showed the plateau phase. When pCMX-GFP above 100 ng was transcribed, a part of abundant mRNA was not translated. The diffusion time of the product for pCMX-GFP was constant and independent of the concentration of the plasmid (Table 1). The molecular weight expected from the diffusion time was approximately 20,000 Da, which agreed well with the value of 27,000 Da reported in the literature (15). From the volume element (0.4 fl) and the number of molecules (80 molecules by 1 μg plasmid), GFP production was calculated as 0.3 μM in the in vitro translation system. According to the manufacturer's data sheets, this system can synthesize the maximum level, 1 μM luciferase. Judging from these values, FCS estimated successfully the protein translation in this system. Figure 1Open in figure viewerPowerPoint Autocorrelation function of in vitro translation products of pCMX-GFP. Curves show each autocorrelation function of 15-min intervals after the addition of the plasmid. The concentration of pCMX-GFP was 60 ng. Insertion is the time-course of diffusion time. According to Equation (1), each function fitted the one-component model and the diffusion time was calculated. Figure 2Open in figure viewerPowerPoint A: Number of molecules in different concentrations of pCMX-GFP. The concentration is shown on the right. Insertion is the time-course of fluorescent intensity of the same sample. B: Effect of pCMX-GFP concentration on the number of molecules after 150 min of the addition of plasmid. Table 1. Diffusion Time of Three Proteins in Translation System In Vitro* Plasmid pCMX-GFP pCMX-GFP-HIF1α pCMX-GFP-hGR Condition One component fitting plasmid concentration Two component fitting Two component fitting 1–60 ng 100 ng–1 μg Fast Slow Fast Slow Diffusion time (ms) Mean 0.16 0.18 0.33 (fixed) 6.0 0.31 (fixed) 6.3 SD 0.01 0.02 — 1.7 — 1.5 Fraction (%) Mean — — 89 11 84 16 SD — — 2.0 2.0 2.0 2.0 Sample 6 3 2 2 Data points 30 15 10 10 * Individual sample solution was measured five times during the plateau phase of the increase in number of molecules. As shown in Figure 3, autocorrelation functions of pCMX-GFP-HIF1α and pCMX-GFP-hGR products were different from that of the pCMX-GFP product. The results indicate that these products were larger than the pCMX-GFP product. Furthermore, the autocorrelation function of the pCMX-GFP-HIF1α product was slightly shifted to the left than that of the pCMX-GFP-hGR product. The molecular weight for GFP-HIF1α was approximately 147,000 Da (16) and the diffusion time was as expected, 0.326 ms. When we analyzed the data with the two-component model, one diffusion time was fixed to the value. The fraction with the diffusion time of 0.326 ms slightly increased with reaction time and then remained constant. Inversely, the diffusion time of the slow-moving molecule decreased and remained stable. The second diffusion time decreased with reaction time and stabilized at approximately 6 ms. We observed 20% of fluorescent molecules moved slowly (slow-moving molecule). Because this component reflected polysomes that contained GFPs as described below, we believe that the relative efficiency is higher than that of the GFP monomer. Therefore, the equation for various species with different efficiencies (22) should be used. However, the relative efficiencies of polysome-containing GFPs are unknown and the fraction of the slow component is less than 20%. We applied a simple analysis as the first step of this study. A detailed analysis of the slow component will be provided in the near future. The diffusion times for the products of GFP-fusion proteins are summarized in Table 1. Figure 3Open in figure viewerPowerPoint A: Normalized autocorrelation functions of pCMX-GFP, pCMX-GFP-HIF1α, and pCMX-GFP-hGR products. Insertion is the time-course of number of molecules of products of pCMX-GFP-HIF1α and pCMX-GFP-hGR. In the early stage, fluorescence was weak and the number of molecules changed irregularly. The number of molecules was less than that of pCMX-GFP but increased sequentially. B: Fraction of two components and diffusion time of slow component in pCMX-GFP-HIF1α products by fitting the two-component model. The diffusion time of the fast component was fixed to 0.326 ms (see text). The number of fluorescent pCMX-GFP products increased dependently on the concentration of its template, i.e., pCMX-GFP. The diffusion time of the product was almost the same as that expected from the molecular weight of GFP as reported in the literature. This is consistent with the explanation that GFP mRNA was transcribed from pCMX-GFP, which was dependent on the concentration of plasmid, and that GFP was stoichiometrically translated from the mRNA before the plateau phase. We may conclude that the translation process of GFP within the small sample volume of 20 μl was able to monitor in real time. Several investigators reported that protonation of the hydroxy group of Tyr-66, which is part of the chromophore in GFP, induces the nonfluorescent state (23). However, the in vitro translation system used in the present study is buffered by 20 mM HEPES-KOH at pH 7.5 (24), enabling maintenance of pH. Our analytical model, therefore, did not contain the protonation state. The number of GFP-fusion proteins, GFP-HIF1α, and GFP-hGR was calculated to be less than that of GFP. Correspondingly, in the Cos7 cells, which were transiently transfected with pCMX-GFP-HIF1α and pCMX-GFP-hGR, the fluorescence from whole cells was weaker than that from the cells with pCMX-GFP (data not shown). Using FCS, if the transfection efficiency is known or constant, the difference of translation efficiency among plasmids in cultured cells may be predicted even in in vitro translation systems. The autocorrelation function of GFP can be fitted well with the one-component model. For the GFP-fusion proteins, the two-component model was more suitable. The slow-moving component had a molecular weight of 109 Da, which was calculated from the diffusion time of 6 ms. Compared with GFP that contains 238 amino acids, the calculated molecular weight of GFP-HIF1α is only fourfold that of GFP. Because the molecular weight of a complex of GFP-HIF1α mRNA and ribosome was expected to be 6 × 106 Da, it is likely that formation of polysomes could be detected (25). The decrease in the fraction of the slow component agreed with the explanation that GFP-HIF1α free from polysome increases. However, in the presence of high biological complexity, the simple globular shape-model might have a limitation in FCS measurement. A more detailed analysis model with a higher-order term will be the subject of future investigations. In Vivo As shown in Figure 4, the autocorrelation function of GFP in cell was different from that in PBS. The results indicate that GFP in cells fluctuated slower than that in solution. Taking account of an identical translated product, this difference suggests that the viscosity in cytosol is higher than that in solution. Figure 4Open in figure viewerPowerPoint Autocorrelation function of GFP in cell and solution (0.1 mg/ml, 25 mM Tris-HCl, pH 7.8, 1 mM dithiothreitol, 1 mM EDTA). Both HIF-1α and hGR are the transcription regulatory proteins and translocate from the cytoplasm to the nucleus by different stimuli, such as hypoxia and the glucocorticoid hormone, respectively (16, 17). Although the detailed mechanism for nuclear transport remains unknown, hGR forms the homodimer in contrast to HIF-1α (16, 17). Because the diffusion time for GFP-hGR in vitro was slightly longer than that for GFP-HIF1α (Fig. 3) and unchanged even after addition of dexamethasone (data not shown), GFP-hGR might form homodimer just after the translation in the absence of hormonal ligand. Interestingly, the diffusion time for GFP-hGR in cells was also unchanged, although the nuclear transport occurred in response to the addition of dexamethasone (Figs. 5, 6). Furthermore, the nuclear translocation from cytosol clearly induced the decrease in number of molecules in volume element. We also noted a changing of fluorescence intensity per particle (count per particle) after stimulation with dexamethasone (Fig.6). The value of the parameter increased about twice that of the initial state, although it is difficult to distinguish between monomer and dimer by the diffusion time. This result may suggest that GFP-hGR forms a homodimer in the cytosol by the stimuli. However, more detailed examination could help to shed light on the phenomenon. Figure 5Open in figure viewerPowerPoint GFP-hGR shows dexamethasone-induced nuclear translocation. Cos7 cells were transiently transfected with pCMX-GFP-hGR. After 24-h expression, they were incubated for 30 min with 100 nM dexamethasone. Localization of GFP-hGR was observed with a conventional fluorescence microscope (Zeiss, Axiotron, objective 20×). Figure 6Open in figure viewerPowerPoint Diffusion time, number of molecules, and intensity for GFP-hGR molecule after the addition of 1 μM dexamethasone. CONCLUSIONS We may conclude that, using an extremely small sample volume, FCS enables us to determine the number of translated GFP/GFP-fusion protein in vitro and in vivo. Finally, we would mention that a sample measured by FCS is noninvaded by any physical condition so that translation products can be used for other biochemical analysis or application without dilution, degradation, or contamination. FCS permits monitoring of the in vitro and in vivo translation of GFP/GFP-fusion protein. Acknowledgements We thank Dr. Hidesato Ogawa (Kyoto University) for providing pCMX-GFP and pCMX-GFP-hGR. LITERATURE CITED 1 Cubitt AB, Heim R, Adams SR, Boyd AE, Gross LA, Tsien RY. Understanding, improving and using green fluorescent proteins. Trends Biochem Sci 1995; 20: 448– 455. 2 Llopis J, McCaffery JM, Miyawaki A, Farquhar MG, Tsien RY. Measurement of cytosolic, mitochondrial, and Golgi pH in single living cells with green fluorescent proteins. 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