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Chapter 22 Postembedding Detection of Acidic Compartments
Chapter 22
Postembedding Detection of Acidic Compartments RICHARD G. W. ANDERSON Department of Cell Biology and Anatomy The University of Texas Southwestern Medical Center Dallas, Texas 7S235
I. Introduction 11. Materials and Methods A. Materials B. Indirect Immunofluorescence Microscopy C . Indirect Immunoelectron Microscopy 111. Results and Discussion A. Light Microscopy B. Electron Microscopy IV. Conclusions References
I. Introduction Acidic membrane-bound compartments are stable elements of all eukaryotic cells. Endocytic vesicles (Anderson et al., 1984; Galloway et af., 1983; Maxfield, 1982; Tycko and Maxfield, 1982; Yamashiro et af., 1983), lysosomes (Ohkuma and Poole, 1978; Poole and Ohkuma, 1981), portions of the trans-Golgi apparatus (Anderson and Pathak, 1985; Orci et af.,1985, 1986), certain secretory vesicles (Fishkes and Rudnick, 1982; 463 METHODS IN CELL BIOLOGY, VOL. 31
Copyright 0 1989 by Academic Press, Inc. All rights of reproduction in any form reserved.
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Hutton, 1982; Johnson and Scarpa, 1984; Orci et al., 1986; Russell, 1984), and plant tonoplasts (Boller and Wiemken, 1986) are listed among the known acidic compartments. The function of low pH in each compartment is not entirely understood. In some compartments the low pH helps to maintain ionic gradients across the vacuole membrane, whereas in others the high H+ concentration unfolds proteins, permitting the release of bound ions such as iron or exposing a hydrophobic site that facilitates the passage of the molecule across the membrane. The low-pH environment can activate the proteolytic enzymes of both lyosomes and trans-Golgi vesicles or control the proper sorting of molecules that travel either the endocytic or the exocytic pathway. (These and other functions are reviewed in Mellman et al., 1986, and Anderson and Orci, 1988.) The identification of acidic compartments in living cells has long depended on vital staining techniques (Metchnikoff, 1968). Vital dyes that accumulate in acidic compartments are weak bases. At neutral pH they are hydrophobic and readily cross membranes; however, in an acidic environment they acquire a charge (an absorbed H') and leave the compartment slowly. As an example, acridine orange is a fluorescent weak base that has been used extensively to study, by light microscopy, the dynamic distribution of acidic compartments in living cells. The proton gradient across the membrane of each acidic vacuole is generated by an ATP-dependent proton pump (Mellman et al., 1986; Al-Awqati, 1985; Rudnick, 1986). The pump must be active for vital dyes to accumulate. When the pump is inactivated, the H+ gradient dissipates and any vital indicator that had accumulated leaves the compartment. Thus, vital indicators have been severely limited in their use because of the requirement that cells remain alive during the experimental procedure. To overcome these limitations, weak bases have been found that accumulate in living cells but are retained in acidic compartments following fixation with aldehyde fixatives (Anderson et al., 1984; Anderson and Pathak, 1985; Orci et al., 1986, 1987a,b; Schwartz et al., 1985). The reagent can then be localized by immunocytochemical techniques after the cells or tissues have been embedded in plastic. In this way, the H+ gradient can be captured as it existed in the living cell and made visible with either the light or electron microscope. The postembedding detection of acidic compartments (PEDAC) has proved to be a valuable adjunct to other techniques for studying this interesting aspect of organelle physiology.
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11. Materials and Methods
A. Materials 3-(2,4-Dinitroanilino)-3’-amino-N-methyldipropylamine (DAMP) and monoclonal antidinitrophenol (anti-DNP) IgG (Oxford Biomedical Research, Oxford, MI) Affinity-purified anti-mouse IgG or anti-mouse IgG coupled to tetramethylrhodamine isothiocyanate (Zymed, San Francisco, CA) Saponin, ovalbumin, and 3’-diaminobenzidine tetrahydrochloride (Sigma) Anti-mouse IgG coupled to horseradish peroxidase (HRP) (Cappell, Westchester, PA) Protein A-gold or goat anti-rabbit IgG-gold conjugates ( Janssen, Olen, Belgium) Lowicryl K4M (Polysciences, Warringtin, PA) Epon (Fluka, Hauppauge, NY) Monesin (Calbiochem, San Diego, CA)
B . Indirect Immunofluorescence Microscopy Cultured cells are easily labeled with the weak base, DAMP, by first growing the monolayers on coverslips and incubating the coverslips with 30-50 pM DAMP in normal medium for 30 minutes at 37°C. Following the incubation, cells are washed with culture medium and then fixed for 15 minutes at room temperature with 3% (w/v) paraformaldehyde in buffer A (10 mM sodium phosphate, 150 mM NaCl, 2 mM MgC12, pH 7.4). To control for specificity, a set of cells is subsequently incubated with 25 p M monensin for 5 minutes at 37°C before fixation, which dissipates the proton gradient and causes DAMP to leave the compartment. Cells are then fixed and the coverslips are washed with 2 ml of 15 mil4 NH4Cl and twice with buffer A. Each monolayer is then permeabilized by overlaying the coverslip with 2 ml of 0.1% (v/v) Triton X-100 in buffer A for 5 minutes at - 10°C. Each coverslip is then placed in a Petri dish, cell-side up, covered with 60 pl of monoclonal mouse anti-DNP IgG (15 pg/ml) and incubated for 60 minutes at 37°C in a moist chamber. Following four washes with buffer A (15 minutes each) the cells are incubated with 50 pI of tetramethylrhodamine isothiocyanate-labeled rabbit anti-mouse IgG (40 pglml) for 60 minutes at 37°C. Coverslips are then washed once more, mounted on glass slides, and viewed with a fluorescence microscope.
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Tissue samples can also be labeled with DAMP. There are two general methods of labeling: tissue perfusion with DAMP or incubation of tissue slices with the DAMP. Although 30-50 p M DAMP is still a workable concentration, penetration is a problem and thin tissue slices are recommended. Regardless of the method of administering the DAMP, to localize sites of DAMP accumulation, tissues are fixed for 2 hours at room temperature with 1% glutaraldehyde in 0.1 M sodium phosphate buffer (pH 7.3), dehydrated, and embedded in Epon. Thick sections (0.5-1.0 pm) are prepared of the Epon-embedded material, collected on glass slides, and processed to remove Epon (Orci et al., 1986). Sections are then incubated with monoclonal anti-DNP IgG (5 pg/ml) for 1 hour at 37"C, washed with phosphate buffer (2X, 5 minutes each), and then incubated with rhodamine-labeled anti-mouse IgG for 1 hour at 37°C. Sections can then be mounted and viewed by either epifluorescence or transmitted-fluorescence microscopy (Orci et al., 1986).
C. Indirect Immunoelectron Microscopy I . IMMUNOPEROXIDASE For cultured cells, cells are incubated with DAMP as just described, and then fixed with either 2% paraformaldehyde in buffer B (10 mM sodium periodate, 0.75 M lysine, 37.5 mhl sodium phosphate, pH 6.2) or with a 3% paraformaldehyde in buffer C [ 100 mM sodium phosphate (pH 7.8), 3 mM KC1, 3 mM MgC12, and 3 mM 2,4,6-trinitrophenol]. The cells are then processed while attached to the dish for indirect immunoperoxidase localization of DAMP (Anderson et al., 1984), using 50 pg/ml of anti-DNP IgG, 0.5 mg/ml of HRP-conjugated goat anti-mouse IgG. Saponin concentration for permeabilization must be adjusted for the cell type. To reveal the HRP sites, cells are incubated at room temperature for 10 minutes with 0.2% (w/v) diaminobenzidine and 0.01% (v/v) H202.The cells are then fixed in 2% (w/v) osmium tetroxide, and 1% (w/v) potassium ferrocyanide in 0.1 M sodium cacodylate (pH 7.3), dehydrated, released from the dish in propylene oxide, pelleted, and embedded in Epon (Anderson et al., 1981). The same method can be used to localize DAMP in tissues.
LOCALIZATION 2. IMMUNOGOLD Incubation of either cultured cells or tissue samples with DAMP is carried out as described earlier. Cells or tissues are then fixed with I% glutaraldehyde in 0.1 M sodium phosphate buffer (pH 7.3) for 1 hour at
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room temperature. Cultured cells are pelleted in the fixative. Pellets or tissue samples are washed with 0.1 M sodium phosphate buffer and incubated in 0.5 M NHdCl in phosphate buffer for 30 minutes at room temperature. Samples are washed once again with phosphate buffer and embedded in Lowicryl K4M at -20°C as described (Anderson and Pathak, 1985; Orci et al., 1986). Thin sections are prepared and mounted on formvar-carbon-coated nickel grids. The grids are floated face-down on buffer C [ O S M NaC1, 0.1% NaN3, 1% (w/v) ovalbumin, 0.01 M Tris-HC1 buffer, pH 7.21 for 30 minutes at room temperature. Grids are then edge-dried on filter paper (Whatman, no. 50) and transferred to a drop (70 p1) of buffer C containing 5 pg/ml of anti-DNP IgG for 16 hours at 4°C. Grids are then rinsed thoroughly in buffer D (0.1 M Tris-HC1, 0.15 M NaCl, pH 7.2) for 10 minutes, quickly dried on filter paper, and immediately floated face-down on buffer E (buffer D + 0.02% PEG-20, 0.1% NaN3) in a porcelain spot plate. Grids are then incubated for 2 hours at 37°C with 5 pg/ml of rat anti-mouse IgG in buffer E followed by 1 hour incubation at room temperature with 1 : 70 dilution of protein A-gold (10 r 2 nm diameter) in buffer E. All antibody solutions are centrifuged at 100,000 g for 30 minutes prior to use. After the protein A-gold incubation, grids are washed with a stream of distilled water for 20 seconds and air-dried. Labeled sections are double-stained at room temperature with 5% aqueous uranyl acetate (10 minutes) and lead citrate (3 minutes). Tissue samples or cells incubated in the presence of DAMP can also be embedded in Epon. Epon-embedded samples are then labeled by the immunogold procedure according to the protocol just outlined, with the following modifications. Before any labeling, the sections are etched by floating the grids, section-side down on saturated solution of sodium metaperiodate for 1 hour at room temperature, followed by thorough rinsing with distilled water, before processing for immunolabeling.
3. QUANTIFICATION OF GOLDLABEL The number of gold particles per square micron of compartment can be evaluated directly in electron micrographs by standard procedures (Orci et al., 1986). The density of gold particles due to anti-DNP IgG binding can be used to calculate pH if the number of gold particles is proportional to the proton concentration (Orci et al., 1986). Apparently DAMP accumulates in proportion to the proton concentrations in a compartment; however, the quantitative retention of DAMP is related to the number of available crosslinking sites in the compartment. For protein-rich secretory vacu-
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oles, most likely all of the accumulated DAMP becomes crossolinked during fixation; however, for a protein-poor compartment such as the tonoplast, most likely all of the DAMP would be released after fixation. Compartments such as transitional vesicles of the Golgi apparatus, endosomes, and lysosomes seem to have ample crosslinking sites. The pH of a compartment can be estimated using the formula: pH = 7.0 - log D 1 / D 2where , D 1 = density of DAMP-specific gold particles in the compartment of interest, and D2 = density of gold particles in a pH 7.0 compartment such as the nucleus.
111. Results and Discussion
A. Light Microscopy Although the acidic compartments of tissue culture cells can be easily identified with reagents such as acridine orange, PEDAC offers a broader range of experimental options because the cells have been fixed. Figure 1 shows a typical micrograph of cultured fibroblasts that have been incubated with DAMP. Acidic compartments appear as brightly fluo-
FIG. I . Light-microscopic visualization of acidic compartments in cultured human fibroblasts. The bright dots represent sites of DAMP accumulation. 50 pLM DAMP, 30 minutes at 37°C. ~ 2 5 0 0 .
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rescent vacuoles that are often clustered around the nucleus of the cell. A similarly prepared sample can be used to colocalize antigens that are suspected of residing, either permanently or transiently, in acidic compartments. Moreover, with a suitably labeled ligand, the dynamics of acidification during endocytosis can be studied. There is very little known about the distribution of acidic compartments in the various tissues. The PEDAC technique provides a convenient way to survey by light microscopy various tissues for the presence of acidic compartments. The antigenicity of the DNP group on DAMP survives fixation and embedding in plastic; therefore, immunolocalization can be performed directly on thick (0.5-1.0 pm) sections. The option is available to do colocalization studies if there is an interest in identifying molecules that might reside in the acidic compartments. The major technical difficulty with applying PEDAC to tissue samples is obtaining thorough penetration of DAMP into cells. Perfusion of tissues with DAMP is recommended. Tissue slices can be incubated with DAMP, although we have found that DAMP may not reach the inner cells of the slice. In this case, caution is recommended: only the outer cell layer should be used for analysis.
B . Electron Microscopy The reason for developing the PEDAC procedure was to be able to visualize acidic compartments with the resolution of the electron microscope. Two basic formats are available: immunoperoxidase, which gives qualitative information about the distribution of these compartments, and immunogold, which can be used to quantify the distribution of acidic compartments. The choice of techniques depends on the type of study being performed. Generally, immunogold gives better resolution than immunoperoxidase . The PEDAC procedure can provide high-resolution morphological information about the distribution of acidic compartments in tissues. We already know that lysosomes, endosomes, certain secretory vacuoles, and portions of the Golgi apparatus are acidic. However, little information is available as to whether certain tissues utilize special acidic compartments for specific functional needs. There is also the possibility that certain disease processes cause abnormal regulation of intracellular vacuolar pH. Electron-microscopic PEDAC has a major application in mapping the function of low-pH compartments. Double-labeling experiments allow, for example, the colocalization of acidic compartments and the movement of endocytic markers (Fig. 2). Likewise, double-labeling with antibodies to a specific antigen can give valuable information about the traffic pattern
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FIG. 2. Colocalization of LDL-gold (15-nm particles) and DAMP (5-nm particles) in endosomes of human fibroblasts. Cultured human fibroblasts grown according to standard conditions (Anderson et a / . , 1984), were incubated in the presence of LDL-gold (20 pg/ml) plus 50 pM DAMP for 30 minutes at 37°C. Cells were washed, fixed, and embedded in Lowicryl K4M. Thin sections were then processed to localize DAMP. Notice that the endosome on the right contains both LDL-gold and DAMP, whereas the endosome on the left contains tittle DAMP-specific labeling, indicating that the pH of the former is quite a bit lower than the latter. X55,OOO.
of antigens relative to the regulation of acidification. An extreme refinement of this application is to utilize conformation-specificmonoclonal antibodies (mAb) to detect the relationship between the shape of a molecule and the pH of the compartment in which it resides (Orci et al., 1986). For example, there is good biochemical evidence that transferrin loses its iron in an acidic endosomal compartment (Mellman ef al., 1986). With mAb that can distinguish iron-loaded transferrin from apo-
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transferrin, one could obtain important information about the relationship between the delivery of iron and the acidification of the endosome. Aside from being able to identify compartments that are acidic, the PEDAC procedure also offers the opportunity to estimate the pH of intracellular compartments that are inaccessible to measurement with fluoresceinated molecules (Tycko and Maxfield, 1982). This method relies on a simple numerical relationship between the pH and the density of immunogold labeling due to DAMP accumulation in a particular compartment. As with any technique, these calculations are dependent on several assumptions. Critical among these is that DAMP accumulates proportionally to the proton concentration in the compartment and that fixatives quantitatively retain DAMP at sites of accumulation. For some tissues these conditions appear to hold true (Orci et al., 1986); however, caution should be applied in approaching any new system. A highly acidic compartment that does not have sufficient crosslinking sites for DAMP to bind to during fixation will not retain the marker after fixation even though it may have accumulated to a high concentration in the living cell. Another area of investigation where PEDAC could be valuable is studying the regulation of pH in these compartments. There is considerable evidence that the magnitude of the H+ gradient within vacuoles of a continuous exocytic or endocytic pathway are quite different (see Fig. 2). For example, whereas trans-Golgi vesicles of certain exocrine cells are slightly acidic (pH 5 . 9 , the mature secretory vesicles derived from these vesicles have a neutral pH. There are several ways that the pH might be regulated. The pH may be regulated by controlling the number of proton pumps in the membrane (Al-Awqati, 1985). Alternatively, the leakiness of the membrane to protons may be adjusted at various stages in the life cycle of the vesicle. Because proton pumps are electrogenic (Al-Awqati, 1985), the modulation of permeant anion fluxes (e.g., C1-) could also be an important way to control the movement of protons in response to the activity of the pump. Whereas there will be an important application of biochemical techniques to determining the mechanism of pH regulation, the PEDAC technique can play a central role in distinguishing among various hypotheses.
IV. Conclusions Postembedding detection of acidic compartments (PEDAC) is a technique that has the potential for unraveling some of the mysteries surrounding the function of low-pH compartments in health and disease.
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There are many applications that have not been touched on in this brief review. When combined with cell fractionation, biochemical, and physiological techniques, PEDAC offers the interested investigator a method for understanding the dynamic function of low-pH compartments in living cells.
ACKNOWLEDGMENTS I would like to thank Dr. Ravindra Pathak for preparing the light and electron micrographs. Some of these techniques were developed in collaboration with Drs. Lelio Orci, Michael Brown, and Joseph Goldstein. I greatly appreciate the help of Ms. Mary Surovik in preparing this manuscript.
REFERENCES Al-Awqati, Q. (1985). Annu. Rev. Cell Biol. 2, 179. Anderson, R. G. W., and Orci, L. (1988). J. Cell Biol. 106, 539. Anderson, R. G. W., and Pathak, R. K. (1985). Cell (Cambridge, Mass.) 40, 635. Anderson, R. G. W., Brown, M. S., and Goldstein, J. L. (1981). J . Cell Biol. 88, 441. Anderson,R. G. W., Falck, J. R., Goldstein, J. L., and Brown, M. S. (1984). Proc. Natl. Acad. Sci. U.S.A. 81, 4838. Boller, T., and Wiemken, A. (1986). Annu. Rev. Plant Physiol. 37, 137. Fishkes, H., and Rudnick, G. (1982). J. Biol. Chem. 257, 5671. Balloway, C. J., Dean, G. E., Marsh, M., Rudnick, G., and Mellrnan, I. (1983). Proc. Nut/. Acad. Sci. U.S.A. 80,3334. Hutton, J. C . (1982). Biochem. J. 204, 171. Johnson, R. G., and Scarpa, A. (1984). In “Electrogenic Transport: Fundamental Principles and Physiological Implications” (M. P. Blaustein and M. Lieberman, eds.), pp. 71-91. Raven Press, New York. Maxfield, F. R. (1982). J . Cell Biol. 95, 676. Mellman, I., Fuchs, R., and Helenius, A. (1986). Annu. Rev. Biochem. 55, 663. Metchnikoff, E. (1968). “Lectures on the Comparative Pathology of Inflammation.” Dover, New York. Ohkuma, S., and Poole, B. (1978). Proc. Natl. Acad. Sci. U.S.A. 75, 3327. Orci, L., Ravazzolla, M., Amherdt, M., Madsen, O., Vassalli, J.-D., and Perrelet, A. (1985). Cell (Cambridge, Mass.) 42, 671. Orci, L., Ravazzola, M., Amherdt, M., Madsen, O., Perrelet, A., Vassalli, J.-D., and Anderson, R. G. W. (1986). J. Cell Biol. 103, 2273. Orci, L., Ravazzola, M., and Anderson, R. G. W. (1987a). Nature (London) 326, 77. Orci, L., Ravazzola, M., Storch, M A . , Anderson, R. G. W., Vassalli, J.-D., and Perrelet, A. (1987b). Cell (Cambridge, Mass.) 49, 865. Poole, B., and Ohkuma, S. (1981). J . Cell Biol. 90, 665. Rudnick, G. (1986). Annu. Reu. Physiol. 48, 403. Russell, J. T. (1984). J . Biol. Chem. 259, 9496. Schwartz, A. L., Strous, G. J. A. M., Slot, J. W., and Geuze, H. J. (1985). EMBO J . 4,899. Tycko, B., and Maxfield, F. R. (1982). Cell (Cambridge, Muss.) 26, 643. Yamashiro, D. J., Fluss, S. R., and Maxfield, F. R. (1983). J. Cell Biol. 97, 929.
Postembedding Detection of Acidic Compartments RICHARD G. W. ANDERSON Department of Cell Biology and Anatomy The University of Texas Southwestern Medical Center Dallas, Texas 7S235
I. Introduction 11. Materials and Methods A. Materials B. Indirect Immunofluorescence Microscopy C . Indirect Immunoelectron Microscopy 111. Results and Discussion A. Light Microscopy B. Electron Microscopy IV. Conclusions References
I. Introduction Acidic membrane-bound compartments are stable elements of all eukaryotic cells. Endocytic vesicles (Anderson et al., 1984; Galloway et af., 1983; Maxfield, 1982; Tycko and Maxfield, 1982; Yamashiro et af., 1983), lysosomes (Ohkuma and Poole, 1978; Poole and Ohkuma, 1981), portions of the trans-Golgi apparatus (Anderson and Pathak, 1985; Orci et af.,1985, 1986), certain secretory vesicles (Fishkes and Rudnick, 1982; 463 METHODS IN CELL BIOLOGY, VOL. 31
Copyright 0 1989 by Academic Press, Inc. All rights of reproduction in any form reserved.
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Hutton, 1982; Johnson and Scarpa, 1984; Orci et al., 1986; Russell, 1984), and plant tonoplasts (Boller and Wiemken, 1986) are listed among the known acidic compartments. The function of low pH in each compartment is not entirely understood. In some compartments the low pH helps to maintain ionic gradients across the vacuole membrane, whereas in others the high H+ concentration unfolds proteins, permitting the release of bound ions such as iron or exposing a hydrophobic site that facilitates the passage of the molecule across the membrane. The low-pH environment can activate the proteolytic enzymes of both lyosomes and trans-Golgi vesicles or control the proper sorting of molecules that travel either the endocytic or the exocytic pathway. (These and other functions are reviewed in Mellman et al., 1986, and Anderson and Orci, 1988.) The identification of acidic compartments in living cells has long depended on vital staining techniques (Metchnikoff, 1968). Vital dyes that accumulate in acidic compartments are weak bases. At neutral pH they are hydrophobic and readily cross membranes; however, in an acidic environment they acquire a charge (an absorbed H') and leave the compartment slowly. As an example, acridine orange is a fluorescent weak base that has been used extensively to study, by light microscopy, the dynamic distribution of acidic compartments in living cells. The proton gradient across the membrane of each acidic vacuole is generated by an ATP-dependent proton pump (Mellman et al., 1986; Al-Awqati, 1985; Rudnick, 1986). The pump must be active for vital dyes to accumulate. When the pump is inactivated, the H+ gradient dissipates and any vital indicator that had accumulated leaves the compartment. Thus, vital indicators have been severely limited in their use because of the requirement that cells remain alive during the experimental procedure. To overcome these limitations, weak bases have been found that accumulate in living cells but are retained in acidic compartments following fixation with aldehyde fixatives (Anderson et al., 1984; Anderson and Pathak, 1985; Orci et al., 1986, 1987a,b; Schwartz et al., 1985). The reagent can then be localized by immunocytochemical techniques after the cells or tissues have been embedded in plastic. In this way, the H+ gradient can be captured as it existed in the living cell and made visible with either the light or electron microscope. The postembedding detection of acidic compartments (PEDAC) has proved to be a valuable adjunct to other techniques for studying this interesting aspect of organelle physiology.
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11. Materials and Methods
A. Materials 3-(2,4-Dinitroanilino)-3’-amino-N-methyldipropylamine (DAMP) and monoclonal antidinitrophenol (anti-DNP) IgG (Oxford Biomedical Research, Oxford, MI) Affinity-purified anti-mouse IgG or anti-mouse IgG coupled to tetramethylrhodamine isothiocyanate (Zymed, San Francisco, CA) Saponin, ovalbumin, and 3’-diaminobenzidine tetrahydrochloride (Sigma) Anti-mouse IgG coupled to horseradish peroxidase (HRP) (Cappell, Westchester, PA) Protein A-gold or goat anti-rabbit IgG-gold conjugates ( Janssen, Olen, Belgium) Lowicryl K4M (Polysciences, Warringtin, PA) Epon (Fluka, Hauppauge, NY) Monesin (Calbiochem, San Diego, CA)
B . Indirect Immunofluorescence Microscopy Cultured cells are easily labeled with the weak base, DAMP, by first growing the monolayers on coverslips and incubating the coverslips with 30-50 pM DAMP in normal medium for 30 minutes at 37°C. Following the incubation, cells are washed with culture medium and then fixed for 15 minutes at room temperature with 3% (w/v) paraformaldehyde in buffer A (10 mM sodium phosphate, 150 mM NaCl, 2 mM MgC12, pH 7.4). To control for specificity, a set of cells is subsequently incubated with 25 p M monensin for 5 minutes at 37°C before fixation, which dissipates the proton gradient and causes DAMP to leave the compartment. Cells are then fixed and the coverslips are washed with 2 ml of 15 mil4 NH4Cl and twice with buffer A. Each monolayer is then permeabilized by overlaying the coverslip with 2 ml of 0.1% (v/v) Triton X-100 in buffer A for 5 minutes at - 10°C. Each coverslip is then placed in a Petri dish, cell-side up, covered with 60 pl of monoclonal mouse anti-DNP IgG (15 pg/ml) and incubated for 60 minutes at 37°C in a moist chamber. Following four washes with buffer A (15 minutes each) the cells are incubated with 50 pI of tetramethylrhodamine isothiocyanate-labeled rabbit anti-mouse IgG (40 pglml) for 60 minutes at 37°C. Coverslips are then washed once more, mounted on glass slides, and viewed with a fluorescence microscope.
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Tissue samples can also be labeled with DAMP. There are two general methods of labeling: tissue perfusion with DAMP or incubation of tissue slices with the DAMP. Although 30-50 p M DAMP is still a workable concentration, penetration is a problem and thin tissue slices are recommended. Regardless of the method of administering the DAMP, to localize sites of DAMP accumulation, tissues are fixed for 2 hours at room temperature with 1% glutaraldehyde in 0.1 M sodium phosphate buffer (pH 7.3), dehydrated, and embedded in Epon. Thick sections (0.5-1.0 pm) are prepared of the Epon-embedded material, collected on glass slides, and processed to remove Epon (Orci et al., 1986). Sections are then incubated with monoclonal anti-DNP IgG (5 pg/ml) for 1 hour at 37"C, washed with phosphate buffer (2X, 5 minutes each), and then incubated with rhodamine-labeled anti-mouse IgG for 1 hour at 37°C. Sections can then be mounted and viewed by either epifluorescence or transmitted-fluorescence microscopy (Orci et al., 1986).
C. Indirect Immunoelectron Microscopy I . IMMUNOPEROXIDASE For cultured cells, cells are incubated with DAMP as just described, and then fixed with either 2% paraformaldehyde in buffer B (10 mM sodium periodate, 0.75 M lysine, 37.5 mhl sodium phosphate, pH 6.2) or with a 3% paraformaldehyde in buffer C [ 100 mM sodium phosphate (pH 7.8), 3 mM KC1, 3 mM MgC12, and 3 mM 2,4,6-trinitrophenol]. The cells are then processed while attached to the dish for indirect immunoperoxidase localization of DAMP (Anderson et al., 1984), using 50 pg/ml of anti-DNP IgG, 0.5 mg/ml of HRP-conjugated goat anti-mouse IgG. Saponin concentration for permeabilization must be adjusted for the cell type. To reveal the HRP sites, cells are incubated at room temperature for 10 minutes with 0.2% (w/v) diaminobenzidine and 0.01% (v/v) H202.The cells are then fixed in 2% (w/v) osmium tetroxide, and 1% (w/v) potassium ferrocyanide in 0.1 M sodium cacodylate (pH 7.3), dehydrated, released from the dish in propylene oxide, pelleted, and embedded in Epon (Anderson et al., 1981). The same method can be used to localize DAMP in tissues.
LOCALIZATION 2. IMMUNOGOLD Incubation of either cultured cells or tissue samples with DAMP is carried out as described earlier. Cells or tissues are then fixed with I% glutaraldehyde in 0.1 M sodium phosphate buffer (pH 7.3) for 1 hour at
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room temperature. Cultured cells are pelleted in the fixative. Pellets or tissue samples are washed with 0.1 M sodium phosphate buffer and incubated in 0.5 M NHdCl in phosphate buffer for 30 minutes at room temperature. Samples are washed once again with phosphate buffer and embedded in Lowicryl K4M at -20°C as described (Anderson and Pathak, 1985; Orci et al., 1986). Thin sections are prepared and mounted on formvar-carbon-coated nickel grids. The grids are floated face-down on buffer C [ O S M NaC1, 0.1% NaN3, 1% (w/v) ovalbumin, 0.01 M Tris-HC1 buffer, pH 7.21 for 30 minutes at room temperature. Grids are then edge-dried on filter paper (Whatman, no. 50) and transferred to a drop (70 p1) of buffer C containing 5 pg/ml of anti-DNP IgG for 16 hours at 4°C. Grids are then rinsed thoroughly in buffer D (0.1 M Tris-HC1, 0.15 M NaCl, pH 7.2) for 10 minutes, quickly dried on filter paper, and immediately floated face-down on buffer E (buffer D + 0.02% PEG-20, 0.1% NaN3) in a porcelain spot plate. Grids are then incubated for 2 hours at 37°C with 5 pg/ml of rat anti-mouse IgG in buffer E followed by 1 hour incubation at room temperature with 1 : 70 dilution of protein A-gold (10 r 2 nm diameter) in buffer E. All antibody solutions are centrifuged at 100,000 g for 30 minutes prior to use. After the protein A-gold incubation, grids are washed with a stream of distilled water for 20 seconds and air-dried. Labeled sections are double-stained at room temperature with 5% aqueous uranyl acetate (10 minutes) and lead citrate (3 minutes). Tissue samples or cells incubated in the presence of DAMP can also be embedded in Epon. Epon-embedded samples are then labeled by the immunogold procedure according to the protocol just outlined, with the following modifications. Before any labeling, the sections are etched by floating the grids, section-side down on saturated solution of sodium metaperiodate for 1 hour at room temperature, followed by thorough rinsing with distilled water, before processing for immunolabeling.
3. QUANTIFICATION OF GOLDLABEL The number of gold particles per square micron of compartment can be evaluated directly in electron micrographs by standard procedures (Orci et al., 1986). The density of gold particles due to anti-DNP IgG binding can be used to calculate pH if the number of gold particles is proportional to the proton concentration (Orci et al., 1986). Apparently DAMP accumulates in proportion to the proton concentrations in a compartment; however, the quantitative retention of DAMP is related to the number of available crosslinking sites in the compartment. For protein-rich secretory vacu-
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oles, most likely all of the accumulated DAMP becomes crossolinked during fixation; however, for a protein-poor compartment such as the tonoplast, most likely all of the DAMP would be released after fixation. Compartments such as transitional vesicles of the Golgi apparatus, endosomes, and lysosomes seem to have ample crosslinking sites. The pH of a compartment can be estimated using the formula: pH = 7.0 - log D 1 / D 2where , D 1 = density of DAMP-specific gold particles in the compartment of interest, and D2 = density of gold particles in a pH 7.0 compartment such as the nucleus.
111. Results and Discussion
A. Light Microscopy Although the acidic compartments of tissue culture cells can be easily identified with reagents such as acridine orange, PEDAC offers a broader range of experimental options because the cells have been fixed. Figure 1 shows a typical micrograph of cultured fibroblasts that have been incubated with DAMP. Acidic compartments appear as brightly fluo-
FIG. I . Light-microscopic visualization of acidic compartments in cultured human fibroblasts. The bright dots represent sites of DAMP accumulation. 50 pLM DAMP, 30 minutes at 37°C. ~ 2 5 0 0 .
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rescent vacuoles that are often clustered around the nucleus of the cell. A similarly prepared sample can be used to colocalize antigens that are suspected of residing, either permanently or transiently, in acidic compartments. Moreover, with a suitably labeled ligand, the dynamics of acidification during endocytosis can be studied. There is very little known about the distribution of acidic compartments in the various tissues. The PEDAC technique provides a convenient way to survey by light microscopy various tissues for the presence of acidic compartments. The antigenicity of the DNP group on DAMP survives fixation and embedding in plastic; therefore, immunolocalization can be performed directly on thick (0.5-1.0 pm) sections. The option is available to do colocalization studies if there is an interest in identifying molecules that might reside in the acidic compartments. The major technical difficulty with applying PEDAC to tissue samples is obtaining thorough penetration of DAMP into cells. Perfusion of tissues with DAMP is recommended. Tissue slices can be incubated with DAMP, although we have found that DAMP may not reach the inner cells of the slice. In this case, caution is recommended: only the outer cell layer should be used for analysis.
B . Electron Microscopy The reason for developing the PEDAC procedure was to be able to visualize acidic compartments with the resolution of the electron microscope. Two basic formats are available: immunoperoxidase, which gives qualitative information about the distribution of these compartments, and immunogold, which can be used to quantify the distribution of acidic compartments. The choice of techniques depends on the type of study being performed. Generally, immunogold gives better resolution than immunoperoxidase . The PEDAC procedure can provide high-resolution morphological information about the distribution of acidic compartments in tissues. We already know that lysosomes, endosomes, certain secretory vacuoles, and portions of the Golgi apparatus are acidic. However, little information is available as to whether certain tissues utilize special acidic compartments for specific functional needs. There is also the possibility that certain disease processes cause abnormal regulation of intracellular vacuolar pH. Electron-microscopic PEDAC has a major application in mapping the function of low-pH compartments. Double-labeling experiments allow, for example, the colocalization of acidic compartments and the movement of endocytic markers (Fig. 2). Likewise, double-labeling with antibodies to a specific antigen can give valuable information about the traffic pattern
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FIG. 2. Colocalization of LDL-gold (15-nm particles) and DAMP (5-nm particles) in endosomes of human fibroblasts. Cultured human fibroblasts grown according to standard conditions (Anderson et a / . , 1984), were incubated in the presence of LDL-gold (20 pg/ml) plus 50 pM DAMP for 30 minutes at 37°C. Cells were washed, fixed, and embedded in Lowicryl K4M. Thin sections were then processed to localize DAMP. Notice that the endosome on the right contains both LDL-gold and DAMP, whereas the endosome on the left contains tittle DAMP-specific labeling, indicating that the pH of the former is quite a bit lower than the latter. X55,OOO.
of antigens relative to the regulation of acidification. An extreme refinement of this application is to utilize conformation-specificmonoclonal antibodies (mAb) to detect the relationship between the shape of a molecule and the pH of the compartment in which it resides (Orci et al., 1986). For example, there is good biochemical evidence that transferrin loses its iron in an acidic endosomal compartment (Mellman ef al., 1986). With mAb that can distinguish iron-loaded transferrin from apo-
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transferrin, one could obtain important information about the relationship between the delivery of iron and the acidification of the endosome. Aside from being able to identify compartments that are acidic, the PEDAC procedure also offers the opportunity to estimate the pH of intracellular compartments that are inaccessible to measurement with fluoresceinated molecules (Tycko and Maxfield, 1982). This method relies on a simple numerical relationship between the pH and the density of immunogold labeling due to DAMP accumulation in a particular compartment. As with any technique, these calculations are dependent on several assumptions. Critical among these is that DAMP accumulates proportionally to the proton concentration in the compartment and that fixatives quantitatively retain DAMP at sites of accumulation. For some tissues these conditions appear to hold true (Orci et al., 1986); however, caution should be applied in approaching any new system. A highly acidic compartment that does not have sufficient crosslinking sites for DAMP to bind to during fixation will not retain the marker after fixation even though it may have accumulated to a high concentration in the living cell. Another area of investigation where PEDAC could be valuable is studying the regulation of pH in these compartments. There is considerable evidence that the magnitude of the H+ gradient within vacuoles of a continuous exocytic or endocytic pathway are quite different (see Fig. 2). For example, whereas trans-Golgi vesicles of certain exocrine cells are slightly acidic (pH 5 . 9 , the mature secretory vesicles derived from these vesicles have a neutral pH. There are several ways that the pH might be regulated. The pH may be regulated by controlling the number of proton pumps in the membrane (Al-Awqati, 1985). Alternatively, the leakiness of the membrane to protons may be adjusted at various stages in the life cycle of the vesicle. Because proton pumps are electrogenic (Al-Awqati, 1985), the modulation of permeant anion fluxes (e.g., C1-) could also be an important way to control the movement of protons in response to the activity of the pump. Whereas there will be an important application of biochemical techniques to determining the mechanism of pH regulation, the PEDAC technique can play a central role in distinguishing among various hypotheses.
IV. Conclusions Postembedding detection of acidic compartments (PEDAC) is a technique that has the potential for unraveling some of the mysteries surrounding the function of low-pH compartments in health and disease.
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There are many applications that have not been touched on in this brief review. When combined with cell fractionation, biochemical, and physiological techniques, PEDAC offers the interested investigator a method for understanding the dynamic function of low-pH compartments in living cells.
ACKNOWLEDGMENTS I would like to thank Dr. Ravindra Pathak for preparing the light and electron micrographs. Some of these techniques were developed in collaboration with Drs. Lelio Orci, Michael Brown, and Joseph Goldstein. I greatly appreciate the help of Ms. Mary Surovik in preparing this manuscript.
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