The National Science Foundation's (NSF) Tokyo Office periodically receives and disseminates reports on research developments in Japan that are related to the Foundation's mission. NSF-sponsored researchers currently working in Japan prepare many of these reports. These reports present information for use by NSF program managers and policy makers; they are not statements of NSF policy.
Special Scientific Report #00-09 (October 11, 2000)
Ms. Eunice Park, a graduate student in Chemistry, Stanford University, prepared the following report. Ms. Park was a participant in the 2000 Summer Institute sponsored in the United States by NSF/NIH/USDA and the Science and Technology Agency and Japan Science and Technology Corporation in Japan. Dr. Atsushi Miyawaki of the Brain Science Institute at RIKEN (Institute of Physical and Chemical Research) in suburban Tokyo, hosted Ms. Park. Ms. Park can be reached via email at: epark@stanford.edu.
In the summer of 2000, I was fortunate enough to work in the laboratory of Dr. Atsushi Miyawaki at the Brain Science Institute, RIKEN, Japan. The main focus of his laboratory is ‘cruising inside cells’, that is, visualizing intracellular functions in a living cell mostly using a fluorescent probe. This work is very different from what I have done at Stanford as a graduate student and I hoped to broaden my science horizon to molecular biology and cell biology in addition to learning cell imaging techniques by conducting research in his lab during the summer. Luckily and with the kind help of people in the lab, I was not only able to achieve my initial goal, but also to obtain some results. With thanks to everybody in the lab, here is the report on the work that I did during the summer.
1. Introduction
Green fluorescent protein (GFP) has recently attracted the attention of many people by its fluorescent nature.1 GFP is an excellent fluorescence label in live-cell imaging for various reasons. It can be easily incorporated into proteins by genetic fusion and it is easy to target to specific intracellular locations. No cofactor is needed to generate fluorescence. It is non-destructive and less photobleaching than most chemical fluorescent molecules such as Fura-2.
Recently, Miyawaki et al, have developed a Ca2+ indicator, “cameleon”, consisting of cyan and yellow mutants of GFP, calmodulin (CaM), and the CaM-binding domain of myosin light chain kinase (M13).2 Cameleon uses fluorescence energy transfer (FRET) between two GFP mutants upon the structure change in the presence or absence of Ca2+. Despite of its big success, it has some room to be improved. The biggest problem of the cameleon, more precisely GFP variants, is its thermostability of folding and pH sensitivity. The thermostability of GFP folding can be especially problematic when it is used in mammalian cells. The inevitable incubation at 37 °C when expressed in mammalian cells lowers its expression level.1 The pH sensitivity of the fluorescence of GFP variants can yield misleading results when it is used to indicate Ca2+. The pKa’s of some GFP variants like YFP are close to the pH of the cell (pH ~ 7).3 Thus, slight changes of pH in the cell can affect its fluorescence spectrum dramatically. In addition, the rather big size of the camelon (90 kDa) limits its use to the cytosol. Due to this problem, Takeharu Nagai in the Miyawaki lab recently developed another Ca2+ indicator, “pericam”, using circularly permuted GFP. The original C and N terminus are connected with a linker and the new C and N terminals are generated in the middle of the original sequence of GFP. CaM and M13 are connected with the new C and N terminal, respectively. (Fig. 1) Since it uses only one GFP, the new pericam is much smaller than cameleon (12 kDa) and thus it can monitor the Ca2+ dynamics in the nucleus and the cytosol simultaneously. Unlike cameleon, it uses the change of the fluorescence intensity upon the structural change of CaM and M13 in the presence and absence of Ca2+.
In what follows, I report the improvement in thermostability of GFP folding including a pericam by introducing a mutation near the chromophore analogous to other reported GFP variants. Then, the Ca2+ dynamics in HeLa cells measured using this newly improved pericam will be presented. Finally, the preliminary result of imaging Ca2+ in Xenopus embryo using this new pericam will be also presented.
Figure 1. The Construction of Pericam
2. Results
2.0 Improvement on the folding efficiency: Figure 2 shows the fluorescence spectra of E. coli cultures transformed with a plasmid containing various GFP mutant genes. Cultures were grown in 3 mL of LB medium at 37 °C for 15 hours, harvested, and resuspended in PBS(-). The spectra were normalized to OD = 0.5 at 600 nm. The fluorescence spectra were taken with excitation at 485 nm. The dotted lines represent the spectra of the conventionally available GFP mutants known to have much better folding and pH properties than wild type GFP (red: EYFP; black: SEYFP). The solid lines represent the spectra of mutants which contain a single mutation near the chromophore in EYFP or SEYFP. It is clearly shown that this single mutation increases the folding efficiency dramatically when incubated at 37 °C. The molecular extinction coefficients and the quantum yields were also measured (data not shown here). It was confirmed that this dramatic increase in the fluorescence intensity is mostly from the increase of the folding efficiency of the proteins.
The same mutation was also introduced in the pericam variants. Figure 3 shows their fluorescence spectra. The experimental condition was the same as described above. The blue and green lines represent earlier versions of pericam developed in the Miyawaki lab. The black and red lines represent the improved versions of pericam. Both the black and red spectra represent variants containing the same mutation that was confirmed to increase the folding efficiency for GFP variants. The variant represented by the black spectrum differs from the blue variant by only one amino acid change, whereas the green variant contains additional changes. Even though this single amino acid change affects the folding efficiency of the protein tremendously, it was observed to have little effect on the Ca2+ dynamic range, pH sensitivity, molecular extinction coefficient and quantum yield. The Miyawaki lab is currently preparing these results on GFP and pericam variants for the patent and the paper submission.
2.1
Imaging
Ca2+ dynamics in HeLa cells using the improved pericam:
The newly improved pericam was used to image Ca2+ dynamics in HeLa
cell. Figure 4 shows the images of
cells transfected with the improved pericam.
The left panel shows cells before stimulation, while the right panel
shows the same cells after elevation of Ca2+ by 0.1 mM
histamine. It is shown that the
fluorescence intensity increases as the concentration of Ca2+ in the
cell increases.
Figure 4. The Fluorescence Images of HeLa Cells Transfected With The Improved Pericam.
2.2 Detecting Ca2+ in Xenopus embryo using the improved pericam: The improved folding efficiency of the improved pericam enables us to detect Ca2+ in early stages of development in Xenopus embryos. Figure 5 shows images of a Xenopus embryo two and a half days after the injection of pericam mRNA into the animal side of a Xenopus embryo in the 4-cell stage. Left is the bright field image and right is the fluorescence image of the embryo. Strong fluorescence is emitted at the location of the brain and eye as the expressed, properly folded pericam binds to Ca2+.
 |
 |
3. Conclusion
In this work, it was found that thermostability of GFP folding including a pericam is improved significantly by introducing a mutation near the chromophore. The pericam containing this mutation is shown to image Ca2+ dynamics in HeLa cells and a Xenopus embryo. This result suggests that this new mutation may overcome the difficulty of using GFP in mammalian cells and enable study of the role of Ca2+ in early stages of development in Xenopus.
4. References
(1) Tsien, R. Y. Ann. Rev. Biochem. 67, 509 (1998)
(2) Miyawaki, A., Llopis, J., Heim, R., McCaffery, J.M., Adams, J.A., Ikura, M. & Tsien, R.Y. Nature (London) 388, 882 (1997)
(3) Miyawaki, A., Griesbeck, O., Heim, R., & Tsien, R.Y. Proc. Natl. Acad. Sci. USA 96, 2135 (1999)
5. Acknowledgement
This work was supported by the National Science Foundation (FAA GC-1) and the Science and Technology Agency of Japan. I am grateful to Dr. Miyawaki for hosting me in his lab at RIKEN and other members in his lab for their help during my stay. I would especially like to thank Dr. Nagai and Chikako Hara for their advice and helpful discussions.