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Am J Physiol Cell Physiol 292: C1519-C1522, 2007. First published December 13, 2006; doi:10.1152/ajpcell.00435.2006
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METHODS IN CELL PHYSIOLOGY

Xenopus oocyte plasma membrane sheets for FRET analysis

Michela Ottolia,1 Kenneth D. Philipson,1,2 and Scott John2

Departments of 1Physiology and 2Medicine and Cardiovascular Research Laboratories, David Geffen School of Medicine at University of California, Los Angeles, California

Submitted 14 August 2006 ; accepted in final form 7 December 2006


   ABSTRACT
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 ABSTRACT
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 GRANTS
 REFERENCES
 
Plasma membrane sheets from Xenopus oocytes have been isolated for use in fluorescence resonance energy transfer (FRET) measurements. This system has the following advantages: 1) fluorescent recordings from a large surface area to maximize the signal-to-noise ratio, 2) reduction in background fluorescence from proteins retained in intracellular compartments, and 3) access to the cytoplasmic surface of the plasma membrane for rapid solution changes. To demonstrate the utility of this approach, we have examined a previously published FRET-based Ca2+ sensor, namely, the Cameleon-PM. This construct targets to the plasma membrane and, upon various Ca2+ additions to the cytoplasmic face of the membrane, shows ratiometric FRET changes. From the ratiometric changes recorded, an apparent Ca2+ affinity of 1.65 µM was determined. Thus preparation of Xenopus oocyte plasma membrane sheets and FRET measurements demonstrates all three of the advantages outlined above.

fluorescence resonance energy transfer

FLUORESCENCE RESONANCE ENERGY TRANSFER (FRET) is a noninvasive optical technique used to measure distances between molecules or changes in conformation occurring within an individual molecule (10). For FRET to occur, fluorescent moieties must be in close proximity (<10 nm) so that energy can be transferred from the donor to the acceptor (10).

Numerous studies have detected FRET within the cytoplasm of living cells; e.g., detection of conformational changes of a protein in response to changes in intracellular Ca2+ (4) or protein-protein interactions. However, these studies become more challenging when phenomena occur at the plasma membrane: it is difficult to optically isolate the plasma membrane using an epifluorescent microscope, since a large portion of the cell is excited. The background fluorescence originating from fluorescent proteins outside the region of interest (e.g., proteins retained within intracellular membrane compartments such as the endoplasmic reticulum) can decrease the signal-to-noise ratio and may mask small changes in FRET magnitude occurring at the plasma membrane. Further limitations include the difficulty in accessing the cytoplasmic surface of the plasma membrane for fast solution changes. Although the use of either a confocal or TIRF (total internal reflection fluorescent) microscope improves the isolation of the signal from the plasma membrane, this does not provide a solution for improving accessibility to the cytoplasmic surface of the membrane.

To circumvent these obstacles, we adapted and improved a method originally developed for immunostaining experiments using plasma membranes from Xenopus oocytes (9). We isolated sheets of plasma membrane from Xenopus oocytes expressing fluorescent proteins on coverslips suitable for optical measurements. This new technique simultaneously eliminates background fluorescence from intracellular membranes, allowing fluorescent recordings from a large surface area, and permits rapid access to the cytoplasmic side of the membrane.


   MATERIALS AND METHODS
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 MATERIALS AND METHODS
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cDNA for Cameleon-PM (Cam-PM) was generously provided by Dr. Atsushi Miyawaki. cDNA for a pair of fluorescent proteins, cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP), targeted to the intracellular membranes of the endoplasmic reticulum (CFP-ER) and Golgi (CFP-Golgi) and the plasma membrane (YFP-PM) of Xenopus oocytes were purchased from Clontech (Mountain View, CA). All constructs were subcloned into an RNA expression vector. RNA was synthesized using mMessage mMachine (Ambion, Austin, TX) and injected into Xenopus oocytes as described previously (7). Thin sections of the oocytes were prepared as described previously (7).

Fluorescent plasma membrane sheets were prepared by first manually removing the vitelline membrane of the oocyte under isotonic solutions. To partially remove invaginations of the oocyte plasma membrane, the devitellinized oocyte can be incubated for 1–2 min in water to osmotically swell the oocyte. The oocyte is placed on a dry polylysine-treated coverslip embedded in a plastic petri dish. A 5-mm coverslip is then placed on top of the oocyte. The solution surrounding the oocytes is removed, leaving sufficient fluid to keep the oocyte wet. The oocyte flattens between the two coverslips, and a large portion of the surface of the oocyte is in direct contact with the polylysine-treated coverslip. Slight pressure favors adhesion of the membrane to the coverslip. After ~10 min, the top coverslip and the oocyte are removed by suction, leaving a plasma membrane sheet attached to the coverslip with the cytoplasmic surface exposed. The coverslip is rinsed several times and placed on an inverted epifluorescent microscope stage for optical studies. These sheets are visible using the excitation light of the chromophore; no signal is observed in bright field.

Images (16-bit) were acquired using a Nikon Eclipse TE300 microscope fitted with a x40 (NA 1.2) oil-immersion lens (Nikon) and equipped with the following filters/dichroic sets (nm): 1) CFP cube: excitation 436/20b (band pass), emission 480/40b, dichroic 455 (long pass; DCLP); and 2) YFP cube: excitation 500/20b, emission 535/30b, DCLP 515 (Chroma Technology, Rockingham, VT). Light-emitting diodes (LEDs; Lumileds, San Jose, CA) were used as light sources: one emitting at 455 ± 20 nm (royal blue) and the other emitting at 505 ± 15 nm (cyan). LEDs and camera exposure were controlled by MetaFluor Imaging 6.1 software (Molecular Devices, Sunnyvale, CA).

Ratiometric FRET measurements were performed by simultaneously monitoring CFP and YFP emissions of the sample when excited at the wavelengths for CFP (royal blue LED) (3–5, 12). The ratio between YFP and CFP emission was measured online in real time using the MetaFluor Imaging software. YFP and CFP images were acquired simultaneously using the Dual View (Optical Insights, Tucson, AZ) image splitter equipped with a 505-nm long-pass dichroic filter to separate the CFP and YFP signals, a CFP emission filter (480/30), and a YFP emission filter (535/40). Images were captured with a Cascade 512B digital camera (Photometrics, Tucson, AZ). Exposure times were optimized for each sheet but varied between 300 and 600 ms, and were recorded at a constant rate for each sheet but varied between 0.2 and 0.33 Hz.

Defined Ca2+ solutions contained (mM) 100 CsCl, 10 HEPES, and 10 EGTA, pH 7. CaCl2 was added to obtained a free [Ca2+] of 30, 10, 3, 1, 0.5, 0.3, and 0.1 µM (calculated with MAXc software). The Ca2+ solutions were perfused directly over the sheet, consisting of 20-ml syringes connected to an eight-way perfusion device (Warner Instruments, Hamden, CT) by electrically controlled solenoids (Lee, Westbrook, CT). Input and output of solution volumes to the recording chamber (glass-bottomed petri dish) were equilibrated to maintain constant flow rates and pressures within the recording chamber.

To obtain the apparent Ca2+ affinity, we examined five sheets. The complete range of defined Ca2+ solutions (30–0.1 µM) were tested on each sheet. Ca2+-containing solutions were applied in random order. Between each Ca2+ application, a Ca2+-free buffer was perfused over the sheet. Values of FRET ratio for each Ca2+ concentration were recorded. The FRET ratio was corrected by subtracting the background noise (measured from areas absent plasma membrane sheets), the amount of CFP bleeding through into the YFP channel, and the amount of direct YFP excitation. The values reported represent the corrected FRET ratios from the five experimental determinations along with the standard errors. A graph was plotted of Ca2+ concentration against normalized, corrected FRET ratio. Data points were fitted to a Hill function to extrapolate an apparent Ca2+ affinity of 1.65 µM.


   RESULTS AND DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
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Characterization of fluorescent plasma membrane sheets. Fluorescent plasma membrane sheets were prepared from Xenopus oocytes injected with cRNAs encoding for several fluorescence-tagged proteins. To test our system, we used the established FRET pair of fluorescent proteins CFP (donor) and YFP (acceptor) (4, 8, 11). Specific targeting of these constructs to the ER (CFP-ER), Golgi (CFP-Golgi), and plasma membrane (YFP-PM) was achieved by fusing the fluorescent proteins to the ER targeting sequence of calreticulin (Clontech) (1), the sequence encoding the NH2-terminal 81 amino acids of human beta1,4-galactosyltransferase (Clontech) (13), and the membrane anchor sequence of Ki-Ras (6), respectively. Two to three days after cRNA injection, oocytes showing strong fluorescence were selected using an epifluorescent microscope equipped with filters to visualize both CFP and YFP fluorescence. Some oocytes were used for preparation of plasma membrane sheets, whereas others were prepared for conventional fixation and thin sections (see MATERIALS AND METHODS). Figure 1A shows epifluorescent pictures of intact oocytes (top) and thin sections (8 µm; middle) of oocytes expressing the indicated constructs. Figure 1A, bottom, shows images of the fluorescent plasma membrane sheets expressing the indicated construct.


Figure 1
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  		Fig. 1. Expression patterns of targeting constructs in Xenopus oocytes. A: fluorescent images of selected areas of intact oocytes (top), 8-µm oocyte sections (middle), and fluorescent plasma membrane sheets (bottom) expressing the indicated constructs. Endoplasmic reticulum (ER) images were obtained from the animal pole (see MATERIALS AND METHODS). B: fluorescent plasma membrane sheets obtained from the animal (left) and vegetal poles (right) of oocytes coexpressing YFP-PM and CFP-ER. Patches were imaged using either the yellow fluorescent protein (YFP; left) or the cyan fluorescent protein (CFP; right) filters to differentiate the plasma membrane (PM) fluorescent signal from ER staining. CFP images at higher gray scale also are shown. Gray scale bars are shown at right of the images. PM-Cam, Cameleon-PM.

 
As shown in Fig. 1A, we occasionally observed a fluorescence signal from fluorescent plasma membrane sheets expressing CFP-ER, indicating the presence of ER membranes in the plasma membrane sheets. Since the distribution of the Xenopus oocyte ER is asymmetric, being more prominent in the animal pole (2), isolation of fluorescent plasma membrane sheets from the vegetal pole drastically decreases CFP-ER (see Fig. 1B). These date indicate that the membrane isolated from Xenopus oocyte onto coverslips is mainly composed of plasma membrane and not intracellular membranes such as ER or Golgi.

Fluorescent plasma membrane sheets are useful for FRET investigations. To determine whether the fluorescent plasma membrane sheets could be utilized for FRET studies, we expressed a fluorescent indicator for Ca2+ that is targeted to the plasma membrane (6). This construct, Cam-PM, consists of CFP-calmodulin-calmodulin binding peptide-cpYFP (circularly permutated YFP) fused to the membrane anchor sequence of Ki-Ras (6). Binding of Ca2+ to calmodulin shifts the protein from an extended to a more compact conformation, increasing FRET efficiency. Fluorescent plasma membrane sheets were prepared from oocytes injected with Cam-PM to monitor Ca2+-dependent changes in FRET ratio (see MATERIALS AND METHODS). Changes in FRET ratio were measured as changes in the YFP/CFP ratio (4, 8). As shown in Fig. 2A, rapid switches between solutions with different Ca2+ concentrations caused fast changes in the YFP/CFP ratio, indicating that the Cam-PM is readably accessible to the perfusion solution. [Occasionally, a slower and smaller response to Ca2+ was observed. In these cases, a very mild treatment of the fluorescent plasma membrane sheet with Triton X-100 (0.01%) recovered the response of Cam-PM to Ca2+.] A dose-response curve fitted to a Hill function to extrapolate the apparent Ca2+ affinity is shown in Fig. 2B. An apparent Ca2+ affinity of ~1.65 µM and a Hill coefficient of 1.2 were determined. There are no previous studies reporting the affinity of Cam-PM for Ca2+. The soluble Cameleon has an apparent Ca2+ affinity of 250 nM (6). Possibly, anchoring of Cameleon to the plasma membrane modifies structure and alters apparent affinity for Ca2+. To test specificity, we applied the same solutions containing different Ca2+ concentrations to fluorescent plasma membrane sheets from oocytes expressing equal amounts of CFP and YFP targeted to the plasma membrane (Fig. 2C). In this case, the YFP/CFP ratio did not change with changes in Ca2+ concentration, corroborating our results.


Figure 2
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  		Fig. 2. Fluorescent plasma membrane sheets can be utilized for fluorescence resonance energy transfer (FRET) experiments. A: representative recording of changes in corrected YFP and CFP emission (see MATERIALS AND METHODS) of Cam-PM on the fluorescent plasma membrane sheets during the perfusion of solutions with different Ca2+ concentrations. The corresponding YFP/CFP ratio also is shown. In this fluorescent plasma membrane sheet, Ca2+ was perfused at the following concentrations: 30, 10, 3, 1, 0.5, and 0.1 µM. B: Ca2+ dose-response curve for Cam-PM. Points are the averages of 5 experiments; error bars are standard errors. The apparent affinity for Ca2+ was calculated by fitting the data points to a Hill function. The value determined was 1.65 µM. C: changes in corrected YFP/CFP ratio recorded from a fluorescent plasma membrane sheet isolated from oocytes coexpressing CFP and YFP targeted to the plasma membrane during perfusion of the solutions with the different Ca2+ content (indicated by bars).

 
We tried to extend this type of study to mammalian expression systems, namely, plasma membrane sheets from HEK-293 and HeLa cells. The plasma membrane sheets isolated from mammalian cells were much smaller (on the order of ~1–2 µm compared with the 50–100 µm from Xenopus oocytes), drastically increasing the noise signal. Because of these observations, we have paused in pursuing this line of investigation further.

Because the Xenopus oocyte expression system has been the foundation for many ion channel and transporter biophysical characterization studies, we expect that this technically simple approach will be not only useful for others to monitor conformation changes of ion channel and transporter proteins but also more widely applicable to other plasma membrane proteins, using FRET-based approaches.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by Beginning Grant-In-Aid 0365104Y from the American Heart Association Western States Affiliate (to M. Ottolia), National Heart, Lung, and Blood Institute Grant HL-49101 (to K. D. Philipson), and the Laubisch Fund (to S. A. John).


   ACKNOWLEDGMENTS
 
We thank John Parker for technical assistance and Dr. A. Miyawaki for sharing the Cam-PM construct.


   FOOTNOTES
 

Address for reprint requests and other correspondence: S. John, Cardiovascular Research Laboratories, MRL 3-645, David Geffen School of Medicine at UCLA, Los Angeles, CA 90095-1760 (e-mail: sjohn{at}mednet.ucla.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
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 RESULTS AND DISCUSSION
 GRANTS
 REFERENCES
 
1. Fliegel L, Burns K, MacLennan DH, Reithmeier RA, Michalak M. Molecular cloning of the high affinity calcium-binding protein (calreticulin) of skeletal muscle sarcoplasmic reticulum. J Biol Chem 264: 21522–21528, 1989.[Abstract/Free Full Text]

2. Marchant JS, Parker I. Xenopus tropicalis oocytes as an advantageous model system for the study of intracellular Ca2+ signalling. Br J Pharmacol 132: 1396–1410, 2001.[CrossRef][Web of Science][Medline]

3. Miyawaki A, Griesbeck O, Heim R, Tsien RY. Dynamic and quantitative Ca2+ measurements using improved cameleons. Proc Natl Acad Sci USA 96: 2135–2140, 1999.[Abstract/Free Full Text]

4. Miyawaki A, Llopis J, Heim R, McCaffery JM, Adams JA, Ikura M, Tsien RY. Fluorescent indicators for Ca2+ based on green fluorescent proteins and calmodulin. Nature 388: 882–887, 1997.[CrossRef][Medline]

5. Miyawaki A, Tsien RY. Monitoring protein conformations and interactions by fluorescence resonance energy transfer between mutants of green fluorescent protein. Methods Enzymol 327: 472–500, 2000.[Web of Science][Medline]

6. Nagai T, Yamada S, Tominaga T, Ichikawa M, Miyawaki A. Expanded dynamic range of fluorescent indicators for Ca2+ by circularly permuted yellow fluorescent proteins. Proc Natl Acad Sci USA 101: 10554–10559, 2004.[Abstract/Free Full Text]

7. Ottolia M, John S, Qiu Z, Philipson KD. Split Na+-Ca2+ exchangers. Implications for function and expression. J Biol Chem 276: 19603–19609, 2001.[Abstract/Free Full Text]

8. Ottolia M, Philipson KD, John S. Conformational changes of the Ca2+ regulatory site of the Na+-Ca2+ exchanger detected by FRET. Biophys J 87: 899–906, 2004.[CrossRef][Web of Science][Medline]

9. Singer-Lahat D, Dascal N, Mittelman L, Peleg S, Lotan I. Imaging plasma membrane proteins in large membrane patches of Xenopus oocytes. Pflügers Arch 440: 627–633, 2000.[CrossRef][Web of Science][Medline]

10. Stryer L. Fluorescence energy transfer as a spectroscopic ruler. Annu Rev Biochem 47: 819–846, 1978.[CrossRef][Web of Science][Medline]

11. Tsien RY. The green fluorescent protein. Annu Rev Biochem 67: 509–544, 1998.[CrossRef][Web of Science][Medline]

12. Wang Y, Botvinick EL, Zhao Y, Berns MW, Usami S, Tsien RY, Chien S. Visualizing the mechanical activation of Src. Nature 434: 1040–1045, 2005.[CrossRef][Medline]

13. Watzele G, Berger EG. Near identity of HeLa cell galactosyltransferase with the human placental enzyme. Nucleic Acids Res 18: 7174, 1990.[Free Full Text]




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