ARTICLE |
Correspondence to: Andrzej J. Janecki, Div. of Gastroenterology, Hepatology and Nutrition, U. of Texas Medical School at Houston, 6431 Fannin, 4.234 MSB, Houston, TX 77030. E-mail: ajaneck@heart.med.uth.tmc.edu
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Summary |
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We developed a confocal morphometric analysis to quantitate the relative plasma membrane (PM) expression of the Na/H exchanger NHE3 in living PS120 fibroblasts. NHE3 is a membrane transport protein that is acutely regulated by changes in the number of molecules expressed at the PM. To quantitate the PM expression of NHE3 under various experimental conditions, we stably expressed a chimera of rabbit NHE3 and green fluorescent protein (NHE3GFP) in PS120 fibroblasts. A three-dimensional (3D) map of the intracellular distribution of NHE3GFP was obtained by confocal laser scanning microscopy (CLSM) of cells superfused with a styryl dye, FM 4-64. This fluorophore rapidly and reversibly labeled the outer lipid layer of the PM, which allowed generation of a digital mask of the PM and calculation of the fraction of a total cellular NHE3GFP expressed at the PM. This analysis was successfully used to quantitate the relative PM expression of NHE3GFP in control cells (25%) and a decrease in the expression caused by subsequent exposure of cells to wortmannin (5.1%). Reliability of the method was confirmed by cell surface biotinylation, which yielded very similar results. Confocal morphometric analysis is fast and reproducible and could potentially be used for investigations on regulation of expression of other membrane proteins. (J Histochem Cytochem 48:14791491, 2000)
Key Words: NHE3, confocal microscopy, green fluorescent protein, morphometric analysis
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Introduction |
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The sodium/hydrogen exchanger NHE3 is a transmembrane protein expressed at the apical membrane domain of Na+-absorbing epithelia, predominantly in the kidney and in the small and large intestine. The protein performs Na/H exchange with stoichiometry 1:1 and is driven by the Na+ concentration gradient across the plasma membrane (reviewed in
The steadily growing number of membrane proteins shown to be regulated by changes in their recycling kinetics has stimulated investigations into the mechanisms responsible for controlling intracellular trafficking of the PM proteins. One of the important tasks in these investigations was an accurate quantitation of the protein of interest within various intracellular compartments. Three major approaches have commonly been used to address this problem: cell fractionation, surface biotinylation, and direct or indirect immunolabeling. In conjunction with use of specific markers for various intracellular compartments, these methods have provided a reasonably good characterization of the pathways and kinetics of recycling of several membrane transporters. However, none of these techniques can easily be applied to dynamic quantitation of PM proteins in living cells. This limitation could be at least partially circumvented by using reporter molecules readily detectable in living cells. One such molecule is a recently cloned and characterized green fluorescent protein (GFP) from a jellyfish, Aequorea victoria (
In this report we describe a novel morphometric method that we developed to dynamically quantitate expression of NHE3 in PM of living cells. For the purpose of these studies, NHE3 was stably expressed in PS120 fibroblasts as a fusion molecule with a green fluorescent protein (NHE3GFP). The total cytoplasmic and PM-associated fluorescence intensity of NHE3GFP was measured in serial optical sections of examined cells obtained by confocal laser scanning microscopy (CLSM), and was subsequently digitally integrated for the entire cell. To accurately map the PM, we labeled the living cells with a lipophilic, membrane-impermeable fluorophore, FM 4-64, which exhibits excellent binding and fluorescent properties. Here we describe details of the procedure itself, and details and rationale for the important control experiments. Although confocal morphometric analysis was used for studies on regulation of PM expression of NHE3, it could potentially be used for similar studies on other membrane transport proteins and receptors.
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Materials and Methods |
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Cell Culture and Transfection
Chinese hamster lung fibroblasts (PS120 cells; a gift from Dr. J. Poussegur) were cultured in DMEM supplemented with 0.1 mM nonessential amino acids, 1 mM pyruvate, penicillin (50 IU/ml), streptomycin (50 µg/ml), and 10% FBS, in a 10% CO2 humidified incubator at 37C. PS120 cells are deficient in endogenous Na/H exchangers and are unable to excrete H+ ion in the absence of external bicarbonates (
Examination of Fluorescent Properties of NHE3GFP
To test the relationship between NHE3GFP concentration and its fluorescence intensity, we measured the fluorescence intensity of serial dilutions of whole-cell lysates prepared from PS120E3G cells. Briefly, cells cultured in 100-mm Petri dishes were rinsed with PBS, scraped with a rubber policeman, and sonicated on ice in lysis buffer (150 mM NaCl, 1% Triton X-100, 10 µg/ml aprotinin, 1 mM PMSF, 5 mM iodoacetamide, 10 µg/ml leupeptin, 50 mM Tris, pH 7.4). The lysate was rocked for 30 min at 4C and spun at 14,000 x g for 10 min. The supernatant was collected and serially diluted with lysis buffer. Aliquots of 45 µl were placed between two glass coverslips spaced by 0.2 mm. Images were collected using a Zeiss LSM410 laser confocal microscope equipped with a Zeiss x 40 water immersion lens (NA 1.2). This microscopic setup was used in all experiments described here unless otherwise indicated. Excitation was set at 488 nm and emission was collected using a 510-nm dichroic mirror and a 505530-nm bandpass barrier filter. Images (8-bit) were recorded at eight-times frame averaging, and each sample was measured in 10 randomly chosen areas of 40 x 40 pixels. For calculation of the emission intensity, images were background-corrected (by subtracting signal from the lysate obtained from wild-type PS120 cells) and the integrated pixel intensity was examined using MetaMorph software (Universal Imaging; West Chester, PA)
To test the degree of photobleaching of NHE3GFP under the experimental conditions used during confocal morphometric analysis, PS120EG3 cells cultured on glass coverslips were lightly fixed in paraformaldehyde (2% in PBS, 10 min at 4C). The fixation step was necessary to prevent lateral diffusion of NHE3GFP molecules in the optical section examined. Images were collected every 3 sec at four-times frame averaging using a confocal microscope, with laser power set at 100 µW. Up to 80 serial images were obtained from the same field (total of 320 single frame scans). For analysis, 30 x 30-pixel regions with an initial average fluorescence intensity of approximately 220 (on a scale of 255) were chosen on a random basis. After background correction, the integrated pixel values in each region (the average pixel value times the number of pixels in the area) were measured in all images in the series using MetaMorph software.
Confocal Morphometric Analysis
Because we were specifically interested in quantitation of NHE3GFP at the plasma membrane (PM), we developed a method that enabled us to create a mask of the PM in living PS120E3G, which was subsequently used to precisely map the PM in optical sections of the cells. PS120E3G cells grown on coverslips were mounted in a superfusion chamber, placed on the confocal microscope stage, and initially superfused with control medium (130 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgSO4, 0.8 mM Na2H(PO4), 0.2 mM NaH2(PO4), 25 mM glucose, 20 mM HEPES, pH 7.4). The temperature of the medium was set so that the actual temperature at the cell surface was 46C. After a short equilibration period, a styryl dye FM 4-64 (20 µM; Molecular Probes, Eugene, OR) was added to the control medium. Kinetics of binding of FM 4-64 to and dissociation from the PM of PS120E3G cells were evaluated in a separate set of experiments. Once the binding of FM 4-64 to the PM was saturated (60 sec), serial optical sections were obtained along the Z-axis at 0.4-µm steps, and series of images spanning entire cells were stored on the magneto-optical disk. Excitation wavelength was set at 488 nm and emission signals were collected by separate photomultipliers using 505530-nm and 620650-nm bandpass filters, respectively. Gain and offset were set manually for each measured cell to ensure that the fluorescence intensities of eGFP and FM 4-64 were contained within the 15235 intensity range on the 8-bit gray density scale. Correction factor for a distance-related attenuation of fluorescence in the Z-axis was obtained by measuring the fluorescence intensity of 2-µm fluorescent beads (Molecular Probes) placed on the cell surface at various distances from the coverslip. Fluorescent beads were also used to set the optimal pinhole aperture and the optimal value of the Z step. Quantitation of the relative PM content of NHE3GFP was performed using MetaMorph software, as follows. The overlay images were color-decoded, background subtracted, and thresholded. Next, images of the PM (FM 4-64 signal) were binarized and reversed so that the signal from FM 4-64 was ascribed the value of "0" and the remainder of the image was ascribed the value of "1." The Boolean logical operation "AND" was then performed on the corresponding images, representing signals from eGFP (distribution of NHE3GFP) and from FM 4-64 (reversed binary mask). This resulted in generation of a new image in which the eGFP fluorescent signal corresponding to the PM was digitally subtracted from the image. The intensity of eGFP fluorescence corresponding to the PM was calculated by subtracting the value of integrated fluorescence intensity of eGFP within the cytoplasm from the total cellular fluorescence intensity (cytoplasm plus PM). Quantitation of the PM content of NHE3GFP in the entire cell (expressed as percent of total cellular content) was performed using the formula
in which n stands for the number of optical sections required to scan the entire cell, and IFIt and IFIc stand for integrated fluorescence intensity of the entire cell and of the cytoplasm within a given optical section, respectively.
In some experiments, cells on coverslips were lightly fixed with 2% paraformaldehyde in PBS (10 min at 4C) and processed further as described above for living cells.
In a separate set of experiments, PS120E3G cells were incubated with a phosphatidylinositol 3-kinase (PI 3-K) inhibitor wortmannin (100 nM; 30 min at 37C), a procedure known to significantly decrease the PM content of NHE3 (
Cell Surface Biotinylation
To complement the confocal morphometric analysis, we also evaluated the relative PM content of NHE3GFP by cell surface biotinylation, as described in details elsewhere (
Measurement of Na/H Exchange Rate
To evaluate the NHE3 activity, we used a method based on SNARF-1 as an intracellular pH (pHi) indicator and confocal microscopy, as described in detail previously (5.9, cells were superfused with medium containing 131 mM Na+. This allowed rapid recovery of pHi, monitored by sequential images taken every 6 sec. The ratio of SNARF-1 emissions at 640 nm and 580 nm was semiautomatically calculated in the serial pairs of respective images using MetaMorph software. Final pHi values were calculated using a calibration curve and a nigericin equilibration method as previously described (
In some experiments, cells were incubated with wortmannin (100 nM; 30 min at 37C) before measurement of pHi recovery.
Statistical Analysis
Numerical data are expressed as means ± SEM, and the significance of difference between experimental groups was analyzed by the two-tailed Student's t-test.
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Results |
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NHE3GFP Expression and Fluorescent Properties
In cells transiently transfected with NHE3GFP, approximately 25% of all cells exhibited diffuse cytoplasmic expression of NHE3GFP, suggesting overexpression and lack of membrane targeting of fusion protein. In contrast, in the stably transfected clonal cells NHE3GFP was characteristically distributed among three compartments: the PM, a juxtanuclear vesicular compartment, and a population of small particles dispersed throughout the cytoplasm and most probably representing recycling vesicles (Fig 1A). High-magnification images revealed that some of the particles were closely associated with the PM (Fig 1B). Because the diameter of these particles was approximately 0.10.4 µm, they might represent recycling vesicles at initial stages of endocytosis and/or during exocytic fusion with PM. Indeed, time-lapse images of living cells revealed that some of the particles initially located within a distance of 0.51.0 µm from the PM moved towards the cell surface and then rapidly disappeared, most likely fusing with the PM (not shown). The general pattern of intracellular distribution of NHE3GFP closely resembled that of NHE3 fused with VSVG but lacking the GFP tag (PS120E3V cells; Fig 1C).
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The NHE3 moiety of the fusion protein expressed at the PM was functionally active, as revealed by measurement of pHi recovery rate in the presence of 131 mM Na+. The average recovery rate was 0.052 ± 0.004 (pHi/sec; at pHi 6.7; mean ± SEM from 180 cells in 12 experiments), and it was very similar to the average rate of pHi recovery in PS120E3V cells (0.040 ± 0.006; mean ± SEM from 40 cells in three experiments). These data indicate that fusion of the carboxy terminus of NHE3 with eGFP did not significantly compromise the protein targeting and activity.
Variants of wild-type GFP with the S65T mutation have been reported to be 2035-fold more resistant to photobleaching (12% of the initial value. These results suggest that the effect of photobleaching on the obtained results was negligible when a cell was examined only once (approximately 20 scans). However, in experiments in which the same cell had to be examined more than once, the degree of photobleaching should be taken into account during quantitative analysis.
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Quantitation of any fluorophore critically depends on the linearity of correlation between the fluorophore concentration and its fluorescent intensity. We examined this correlation using serial dilutions of NHE3GFP in the lysates obtained from PS120E3G cells. As shown in Fig 3, the intensity of NHE3GFP fluorescence changed linearly within two logs of magnitude of the protein concentration. This finding supported the rationale for the confocal morphometric analysis, because the actual range of fluorescence intensity of eGFP measured in living PS120GFP cells (20230 gray density levels in 8-bit images) was well contained within the range of linear correlation found in the experiments with cell lysates.
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We also investigated the possibility that the hypothetical pHi gradient within the narrow zone of cytoplasm immediately adjacent to the PM might result in higher concentrations of H+ ions in that zone and, consequently, in a decrease in fluorescence intensity of the PM-bound NHE3GFP ( 7.4; Fig 4A). Under these conditions, the average pHi in a 0.2-µm zone of cytoplasm adjacent to the PM was not significantly different from that measured randomly in the cell interior (7.38 ± 0.08 vs 7.41 ± 0.06, respectively; means ± SE from 120 areas in 30 cells).
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Cellular Distribution of NHE3GFP
To quantitate the PM expression of NHE3GFP in living cells, we developed a morphometric analysis based on CLSM. The principle of this method was to create a 3 D digital map of the PM and then to quantitate the relative amount of NHE3GFP within this area. To accomplish this, we used a styryl fluorescent dye, FM 4-64. Preliminary experiments revealed that FM 4-64 binding to the PM of PS120E3G cells was saturated at concentrations of 1015 µM (Fig 5A). Therefore, a 20 µM concentration was used in all subsequent experiments. Kinetics of binding and washout of FM 4-64 to the PM at 4C are shown in Fig 5B. The fluorophore saturated the PM binding sites very rapidly, with t1/2 20 sec and with 95% saturation achieved within
60 sec. Once saturation was achieved, the fluorescence intensity of FM 4-64 bound to the PM remained constant, as long as the dye was present in the superfusate at saturating concentration (not shown). The washout phase observed after removal of the dye from solution was also very rapid, with 80% of the fluorescence dissociated from the PM within
70 sec (Fig 5B).
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An example of a series of images obtained from scanning of a PS120E3G cell superfused with FM 4-64 is shown in Fig 6. Qualitatively similar images were obtained when cells were lightly fixed with paraformaldehyde (not shown). A graphic representation of principles of the confocal morphometric analysis is shown in Fig 7. Each optical section of the cell (Fig 7A) was color-decoded and the fluorescent signal from FM 4-64 was converted into a binary mask. The image was then reversed (signal from PM was ascribed a value of 0, whereas the rest of image had a value of 1; Fig 7B) and used for mapping eGFP pixels corresponding to PM within the same optical section. As a result of a logical operation "AND" performed on the images of the binary mask and the respective optical section of the cell (Fig 7B and Fig 7C), the intensity of pixels corresponding to the PM in the new image equaled 0, whereas the remainder of the image retained its original intensity pattern (Fig 7D). Most of the steps of confocal analysis were performed semiautomatically on the series (stacks) of 2225 images spanning the entire cell. However, mapping the portion of PM facing the culture substratum (the "basal" membrane) was complicated by the fact that access of FM 4-64 to this portion of the PM was limited, and therefore the pixel intensity in some areas was below the set threshold level. To solve this problem, we defined the location of the membrane/cytoplasm interface manually. The principle of this procedure is illustrated in Fig 8. First, images of the PM were digitally reconstructed in the XZ plane using a stack of serial images of FM 4-64 obtained from scanning of the examined cell. The PM/cytoplasm interface (Fig 8B, arrow) was ascribed to an optical section (approximately four horizontal pixel rows) in which the average pixel intensity decreased by more than 60% compared to the immediately preceding section. This procedure let us map the interface layer with axial (Z) accuracy of 0.4 µm (thickness of the optical section) and the maximal resolution of 0.1 µm (dimension of a single pixel).
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Results of confocal analysis of the intracellular distribution of NHE3GFP in PS120E3G cells are shown in Fig 9. Under basal conditions, 25.0 ± 2.5% of the total cellular NHE3GFP was located at the PM. This value did not change significantly among several batches of PS120E3G cells, regardless of the variability in cell size and the absolute expression of NHE3GFP among the cells examined (data not shown). To test the ability of confocal morphometric analysis to detect changes in PM expression of NHE3GFP in the same living cell, we exposed PS120E3G cells to a PI 3-K inhibitor, wortmannin. Inhibition of PI 3-K has been previously reported to decrease the PM expression of NHE3 and, consequently, to significantly inhibit NHE3 activity in AP-1 cells (pHi/sec); mean ± SE from 56 cells in three separate experiments). The degree of the inhibitory effect of wortmannin on the NHE3GFP activity roughly corresponded to the degree of decrease of expression of the protein at the PM, which suggested that most if not all regulation of NHE3 activity by wortmannin occurred via removal of NHE3GFP molecules from the PM.
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To verify the results of confocal morphometric analysis, we evaluated the PM expression of NHE3 in PS120E3G cells using quantitative cell surface biotinylation (Fig 11). This method yielded the values of relative PM content of NHE3GFP in PS120E3G cells of 22 ± 1% and 5 + 1% for control and wortmannin-treated cells, respectively, thus very similar to the results provided by confocal analysis (Fig 9). Under both experimental conditions, values obtained from biotinylation were slightly lower than those provided by confocal analysis. This difference, however, was not statistically significant.
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Discussion |
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In this report we describe details of a novel morphometric method developed to quantitate NHE3GFP expression at the PM of living PS120 fibroblasts. The method, based on CLSM and digital image processing, is reproducible, fast, and the measurement can be performed in the same cell more than once, thus enabling direct comparison of pre- and post-treatment expression of NHE3GFP at the PM.
In the majority of applications, immunofluorescent or immunohistochemical methods are used primarily as qualitative tools and only rarely have been applied for semiquantitation of the antigen of interest. The reasons for this are the limitations related to the very principles of these methods. These include a difficult to define stoichiometry of antigenantibody binding, problems with reproducibility and sensitivity, and the interference of out-of-focus fluorescence when epifluorescent illumination is used. The latter problem could be relatively easily solved by using confocal microscopy or digital deconvolution algorithms. The other difficulties are not easy to overcome. A major advance in the area of quantitative fluorescence microscopy was the introduction of GFP as a fluorescent tag for the protein of interest. The initially used wild-type GFP cloned from the jellyfish Aequorea victoria (-helix region critical for the fluorophore formation (F64L/S65T) (
In the first step during development of the confocal morphometric analysis, we evaluated the susceptibility of NHE3GFP to photobleaching. Although eGFP was reported to be very resistant to photobleaching, photobleaching-related loss of fluorescence might be a problem because of the large number of scans needed to complete the analysis. The bleaching control experiments were performed using PS120E3G cells that were lightly fixed to prevent lateral diffusion of NHE3GFP during examination. After approximately 80 single-frame scans (needed to image an entire living cell) performed with the laser power set at the level used in actual experiments, the photobleaching-related loss of fluorescence was only 5% of the average initial level. Moreover, the major parameter measured in our experiments (PM expression of NHE3GFP relative to the total cell content of the fusion protein) was relatively insensitive to the bleaching-related proportional drop of fluorescence intensity within an optical section of the examined cell. Therefore, we did not correct the results for the degree of photobleaching when cells were scanned only once. However, we did correct for photobleaching when the same cell was scanned twice (e.g., in the cell shown in Fig 10).
The second parameter of critical importance was the linearity of the protein concentration/fluorescence intensity relationship for NHE3GFP. We examined this parameter using serial dilutions of NHE3GFP obtained from lysed PS120E3G cells. As demonstrated in Fig 3, the doseresponse curve was linear over two logs of magnitude, and it became nonlinear only with very low concentrations of NHE3GFP (not shown on the graph). This high degree of linearity of the doseresponse curve provides an important justification for relative quantitation of the proteins of interest fused with GFP, and also for recently described calculations of the absolute amounts of the protein of interest in given intracellular compartments (6.8 (
100 nm (linear dimension of a single pixel) cytoplasmic zone adjacent to the PM in the resting cells, in which NHE3 operated at the very low steady-state level. In theory, such a gradient might still exist in a zone smaller than the resolution of the system. It should still be detectable at the level of a single pixel, however, considering that the SNARF-1 method detects pHi differences as low as 0.02 pH units (
The major goal during development of confocal morphometric analysis was a reproducible quantitation of distribution of the total cellular pool of NHE3 between the PM and the cell interior under various experimental conditions. To achieve this goal, we had to be able to reliably distinguish between NHE3 molecules located at the PM and those associated with intracellular structures adjacent to the PM, including particles most likely representing recycling vesicles (Fig 1B). In one recent report, a GFP fluorescence associated with PM was quantitated using a visual subjective definition of the PM boundaries (
Once FM 4-64 was removed from the perfusate, it dissociated from the PM very rapidly, a property important in experiments in which the PM content of NHE3 was evaluated twice, i.e., before and after experimental treatment that affected the PM expression of the exchanger. An example of such an experiment is shown in Fig 10. A PI 3-K inhibitor, wortmannin, significantly inhibited the Na/H exchange rate in PS120E3G cells, which was accompanied by a visible decrease in the PM expression of NHE3GFP. Confocal morphometric analysis performed in the same living cell before and after wortmannin treatment provided evidence that the decrease in the Na/H exchange rate resulted from wortmannin-induced translocation of NHE3 molecules from the PM into the juxtanuclear cytoplasmic compartment. To prevent endocytosis of PM-bound FM 4-64 during optical scanning, superfusion with the fluorophore was performed at 46C. At these temperatures, the vesicle trafficking in eucariotic cells is, for all practical purposes, arrested (
One important aspect of the confocal morphometric analysis requires additional comments. Although FM 4-64 maximally saturated the PM during measurements, the measured fluorescence intensity depended on the local concentration of the fluorophore within the thickness of the optical section. This concentration was highest in optical sections in which the PM was oriented perpendicularly to the XY plane, and was decreasing sharply with the angle between the PM plane and the section plane approaching 0o. Binary transformation of the FM 4-64 images eliminated the problem of varying fluorescence intensity, because any pixel value above the set threshold was ascribed the constant value of 1. This approach also facilitated automation of the analysis in areas of the PM directly contacting the perfusate, where the fluorescence intensity of FM 4-64 was significantly higher than threshold level. However, an automatic algorithm could not be applied to the PM facing the culture substrate, where the fluorescence intensity of FM 4-64 was often below the threshold level, because of impaired access of the fluorophore. In these areas, the PM/cytoplasm interface was defined manually using orthogonal sections of the cells digitally reconstructed in the XZ plane. In this approach, scattered pixels of intensity higher than threshold level were used to define the PM/cytoplasm interface (Fig 8). To verify the results obtained we calculated the relative PM content of PS120E3G cells cultured on a permeable support, which significantly improved basal penetration of FM 4-64. The results of these experiments were not significantly different from those obtained from cells cultured on glass coverslips (data not shown).
Results of confocal morphometric analysis were confirmed by experiments in which the relative PM expression of NHE3GFP was evaluated by cell surface biotinylation. The latter method, however, may suffer from its own set of potential pitfalls. These include a difficult-to-estimate efficiency of biotinylation, a possible contamination of the "surface" fraction with biotinylated intracellular proteins, and impaired penetration of biotin to the PM facing the culture substratum (
In conclusion, we describe a novel morphometric method to reliably and reproducibly estimate the relative PM content of NHE3 in living cells under various experimental conditions. The method is fast, and all major calculations are performed automatically by the computer software, thus minimizing the effect of investigator bias. Use of FM 4-64 as a intravital marker for PM resulted in a precise definition of the PM/cytoplasm interface and, consequently, in exclusion from calculations of the submembrane particles containing NHE3GFP. We believe that this method has potential applicability in experiments on expression and regulation of a growing number of transport proteins and receptors shown to be regulated by rapid changes in the expression level at the PM.
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Acknowledgments |
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Supported by NIDDK grants K08DK02557, RO1DK26523, PO1DK44484, R29DK43778, T32DK0763205, and by the Meyerhoff Digestive Diseases Center for Epithelial Disorders.
We thank Dr Shaoyou Chu and Mr Greg Martin for expert advice and help with the confocal microscopy and immunostaining. We also express our thanks to Mr David SzentGyorgyi and Dr Neal R. Glicksman from Universal Imaging Corp. for invaluable advice with MetaMorph software.
Received for publication May 9, 2000; accepted May 10, 2000.
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Literature Cited |
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![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Akhter S, Cavet ME, Tse CM, Donowitz M (2000) C-Terminal domains of Na(+)/H(+) exchanger isoform 3 are involved in the basal and serum-stimulated membrane trafficking of the exchanger. Biochemistry 39:1990-2000[Medline]
Beron J, Forster I, Beguin P, Geering K, Verrey F (1997) Phorbol 12-myristate 13-acetate down-regulates Na,K-ATPase independent of its protein kinase C site: decrease in basolateral cell surface area. Mol Biol Cell 8:387-398[Abstract]
Betz WJ, Mao F, Smith CB (1996) Imaging exocytosis and endocytosis. Curr Opin Neurobiol 6:365-371[Medline]
Chalfie M, Tu Y, Euskirchen G, Ward WW, Prasher DC (1994) Green fluorescent protein as a marker for gene expression. Science 263:802-805[Medline]
Cormack BP, Valdivia RH, Falkow S (1996) FACS-optimized mutants of the green fluorescent protein (GFP). Gene 173:33-38[Medline]
Cubitt AB, Woollenweber LA, Heim R (1999) Understanding structure-function relationships in the Aequorea victoria green fluorescent protein. San Diego, London, Boston, New York, Sydney, Tokyo, Toronto, Academic Press, pp. 1930
Delagrave S, Hawtin RE, Silva CM, Yang MM, Youvan DC (1995) Red-shifted excitation mutants of the green fluorescent protein. Biotechnology 13:151-154[Medline]
Donowitz M, Janecki A, Akhtar S, Cavet ME, Sanchez F, Lamprecht G, Khurana S, LeeKwon W, Yun CHC, Tse CM (1999) Short-term regulation of NHE3 by growth factors/protein kinases involves vesicle trafficking in epithelial cells and fibroblasts. In Domschke W, Stoll R, Brasitus TA, Kagnoff MF, eds. Intestinal Mucosa and Its Diseases. Pathophysiology and Clinics. Dordrecht, Boston, London, Kluwer Academic Publisher, 99-115
Donowitz M, Levine S, Yun C, Brant S, Nath S, Yip J, Hoogerwerf S, Pouyssegur J, Tse C (1996) Molecular studies of members of the mammalian Na/H exchanger gene family. In Schultz SG, Andreoli TE, Brown AM, Fambrough DM, Hoffman AJ, Welsh MJ, eds. Molecular Biology of Membrane Transport Disorders. New York, London, Plenum Press, 259-275
D'Souza S, GarciaCabado A, Yu F, Teter K, Lukacs G, Skorecki K, Moore HP, Orlowski J, Grinstein S (1998) The epithelial sodium-hydrogen antiporter Na+/H+ exchanger 3 accumulates and is functional in recycling endosomes. J Biol Chem 273:2035-2043
Forte TM, Machen TE, Forte JG (1977) Ultrastructural changes in oxyntic cells associated with secretory function: a membrane recycling hypothesis. Gastroenterology 73:941-955[Medline]
Franchi A, Cragoe E, Jr, Pouyssegur J (1986) Isolation and properties of fibroblast mutants overexpressing an altered Na+/H+ antiporter. J Biol Chem 261:14614-14620
Gerdes HH, Kaether C (1996) Green fluorescent protein: applications in cell biology. FEBS Lett 389:44-47[Medline]
Gottardi CJ, Dunbar LA, Caplan MJ (1995) Biotinylation and assessment of membrane polarity: caveats and methodological concerns. Am J Physiol 268:F285-295
Heim R, Tsien RY (1996) Engineering green fluorescent protein for improved brightness, longer wavelengths and fluorescence resonance energy transfer. Curr Biol 6:178-182[Medline]
Hirschberg K, Miller CM, Ellenberg J, Presley JF, Siggia ED, Phair RD, LippincottSchwartz J (1998) Kinetic analysis of secretory protein traffic and characterization of Golgi to plasma membrane transport intermediates in living cells. J Cell Biol 143:1485-1503
Janecki AJ, Janecki M, Akhter S, Donowitz M (2000) Basic fibroblast growth factor stimulates surface expression and activity of Na+/H+ exchanger NHE3 via mechanism involving phosphatidylinositol 3-kinase. J Biol Chem 275:8133-8142
Janecki AJ, Montrose MH, Zimniak P, Zweibaum A, Tse CM, Khurana S, Donowitz M (1998) Subcellular redistribution is involved in acute regulation of the brush border Na+/H+ exchanger isoform 3 in human colon adenocarcinoma cell line Caco-2. Protein kinase C-mediated inhibition of the exchanger. J Biol Chem 273:8790-8798
Katsura T, Verbavatz JM, Farinas J, Ma T, Ausiello DA, Verkman AS, Brown D (1995) Constitutive and regulated membrane expression of aquaporin 1 and aquaporin 2 water channels in stably transfected LLC-PK1 epithelial cells. Proc Natl Acad Sci USA 92:7212-7216[Abstract]
Kempson SA, Lotscher M, Kaissling B, Biber J, Murer H, Levi M (1995) Parathyroid hormone action on phosphate transporter mRNA and protein in rat renal proximal tubules. Am J Physiol 268:F784-791
Kneen M, Farinas J, Li Y, Verkman AS (1998) Green fluorescent protein as a noninvasive intracellular pH indicator. Biophys J 74:1591-1599
Kuismanen E, Saraste J (1989) Low temperature-induced transport blocks as tools to manipulate membrane traffic. Methods Cell Biol 32:257-274[Medline]
Kurashima K, Szabo EZ, Lukacs G, Orlowski J, Grinstein S (1998) Endosomal recycling of the Na+/H+ exchanger NHE3 isoform is regulated by the phosphatidylinositol 3-kinase pathway. J Biol Chem 273:20828-20836
Lostao MP, Hirayama BA, PanayotovaHeiermann M, Sampogna SL, Bok D, Wright EM (1995) Arginine-427 in the Na+/glucose cotransporter (SGLT1) is involved in trafficking to the plasma membrane. FEBS Lett 377:181-184[Medline]
Mellman I (1996) Endocytosis and molecular sorting. Annu Rev Cell Dev Biol 12:575-625[Medline]
Ogawa H, Inouye S, Tsuji FI, Yasuda K, Umesono K (1995) Localization, trafficking, and temperature-dependence of the Aequorea green fluorescent protein in cultured vertebrate cells. Proc Natl Acad Sci USA 92:11899-11903[Abstract]
Ormo M, Cubitt AB, Kallio K, Gross LA, Tsien RY, Remington SJ (1996) Crystal structure of the Aequorea victoria green fluorescent protein. Science 273:1392-1395[Abstract]
Paillard M (1997) Na+/H+ exchanger subtypes in the renal tubule: function and regulation in physiology and disease. Exp Nephrol 5:277-284[Medline]
Patterson GH, Knobel SM, Sharif WD, Kain SR, Piston DW (1997) Use of the green fluorescent protein and its mutants in quantitative fluorescence microscopy. Biophys J 73:2782-2790[Abstract]
Piston DW, Patterson GH, Knobel SM (1999) Quantitative imaging of the green fluorescent protein. San Diego, London, Boston, New York, Sydney, Tokyo, Toronto, Academic Press, pp. 3148
Pouyssegur J, Sardet C, Franchi A, Paris S (1984) A specific mutation abolishing Na+/H+ antiport activity in hamster fibroblasts precludes growth at neutral and acidic pH. Proc Natl Acad Sci USA 81:4833-4837[Abstract]
Powell KA, Campbell LC, Tavare JM, Leader DP, Wakefield JA, Gould GW (1999) Trafficking of glut4-green fluorescent protein chimaeras in 3T3-L1 adipocytes suggests distinct internalization mechanisms regulating cell surface glut4 levels. Biochem J 344:535-543[Medline]
Prasher DC, Eckenrode VK, Ward WW, Prendergast FG, Cormier MJ (1992) Primary structure of the Aequorea victoria green-fluorescent protein. Gene 111:229-233[Medline]
Prince LS, Workman RB, Jr, Marchase RB (1994) Rapid endocytosis of the cystic fibrosis transmembrane conductance regulator chloride channel. Proc Natl Acad Sci USA 91:5192-5196[Abstract]
Robart FD, Ward WW (1990) Solvent perturbations of Aequarea green fluorescent protein. Photochem Photobiol 51:92s
Robey RB, Ruiz O, Santos AV, Ma J, Kear F, Wang LJ, Li CJ, Bernardo AA, Arruda JA (1998) pH-dependent fluorescence of a heterologously expressed Aequorea green fluorescent protein mutant: in situ spectral characteristics and applicability to intracellular pH estimation. Biochemistry 37:9894-9901[Medline]
Thomas JA, Buchsbaum RN, Zimniak A, Racker E (1979) Intracellular pH measurements in Ehrlich ascites tumor cells utilizing spectroscopic probes generated in situ. Biochemistry 18:2210-2218[Medline]
Ward WW, Prentice HJ, Roth AF, Coddy CW, Reeves SC (1982) Spectral perturbations of the Aequorea green fluorescent protein. Photochem Photobiol 35:803-808
Yang F, Moss LG, Phillips GN (1996a) The molecular structure of green fluorescent protein. Nature Biotechnol 14:1246-1251[Medline]
Yang J, Holman GD (1993) Comparison of GLUT4 and GLUT1 subcellular trafficking in basal and insulin-stimulated 3T3-L1 cells. J Biol Chem 268:4600-4603
Yang TT, Cheng L, Kain SR (1996b) Optimized codon usage and chromophore mutations provide enhanced sensitivity with the green fluorescent protein. Nucleic Acids Res 24:4592-4593
Yip KP, Tse CM, McDonough AA, Marsh DJ (1998) Redistribution of Na+/H+ exchanger isoform NHE3 in proximal tubules induced by acute and chronic hypertension. Am J Physiol 275:F565-575