Jan 29, 1985 - A.L.S. was supported in part by a John A. and George Hart- ... Geuze,H., Slot,J.W., Strous,G.J.A.M., Peppard,J., von Figura,K., Hasilik,A.
The EMBO Journal vol.4 no.4 pp.899-904, 1985
Immunoelectron microscopic localization of acidic intracellular compartments in hepatoma cells
Alan L.Schwartz, Ger J.A.M.Strous, Jan Willem Slot and Hans J.Geuze Division of Pediatric Hematology/Oncology, Children's Hospital, DanaFarber Cancer Institute and Department of Pediatrics, Harvard Medical School, Boston, MA 02115, USA, and Center for Electron Microscopy and Laboratory for Histology and Cell Biology, Medical Faculty, University of Utrecht, The Netherlands Communicated by P.A.Peterson
Using protein A-colloidal gold immunoelectron microscopy and monospecific antibodies to the weak base primaquine, we have delineated acidic intracellular compartments in the human hepatoma cell line, HepG2. Primaquine specifically accumulated within endocytotic compartments (including CURL vesicles, multivesicular bodies and lysosomes). In addition, the Golgi cisternae were positive. However, the CURL tubules, which contain recycling asialoglycoprotein receptor, did not accumulate primaquine. Thus, there may be a gradient of acidification within the endocytotic pathway. Key words: receptor-mediated endocytosis/immunocytochemistry/acidification/primaquine Introduction The mechanisms governing the intracellular trafficking and sorting of proteins during biosynthesis, secretion, exocytosis and endocytosis remain largely unknown. Recent evidence has suggested a role for acidification in many of these processes including the synthesis and secretion of adenocorticotrophic hormone (ACTH) in pituitary tumor cells (Moore et al., 1983), exocytosis of granule storage products (reviewed in Ives and Rector, 1984), and receptor-mediated endocytosis and cellular entry of viruses (Marsh et al., 1983) and macromolecular ligands such as lowdensity lipoprotein (reviewed in Dean et al., 1984). Acidic intracellular compartments were first detected at the light microscopic level in intact cells with the use of acridine orange (Allison and Young, 1964). More recent studies have directly demonstrated acidification of coated vesicles (Stone et al., 1983; Forgac et al., 1983), the endosomal compartment (Tycko and Maxfield, 1982; Maxfield, 1983; Galloway et al., 1983; Merion et al., 1983) as well as lysosomes (Ohkuma and Poole, 1978). Many of these observations used intact cells and fluorescent probes and employed ionophores (e.g., monensin) and the socalled 'lysosomotropic' weak bases (e.g., NH4CI, chloroquine). This latter class of compounds (deDuve et al., 1974) neutralizes the pH of acidic intracellular compartments as a result of protonation of the base (Ohkuma and Poole, 1978). Osmotic swelling of acidic intracellular compartments and subsequent cytoplasmic vacuolization often result (Ohkuma and Poole, 1981). These weak bases are taken up by both passive diffusion as well as active transport processes and accumulate substantially within cells against a concentration gradient (Dean et al., 1984). However, the precise intracellular localization and distribution of the lysosomotropic bases and hence the localization of acidic intracellular compartments is not known. IRL Press Limited, Oxford, England.
To address this question we have defined the intracellular localization of primaquine, an 8-amino-quinolone similar to chloroquine, but containing a free-NH2, which allows cross-linking and quantitative fixation. Both primaquine and chloroquine are weak bases and inhibit receptor-mediated endocytosis and recycling of the asialoglycoprotein receptor (ASGP-R) in hepatoma cells (Schwartz et al., 1984). Using immunoelectron microscopy with monospecific antibodies to primaquine we have delineated the acidic intracellular compartments which accumulate primaquine endocytotic compartments including CURL vesicles and multivesicular bodies (Geuze et al., 1983), lysosomes and Golgi. -
Results Table I demonstrates the specificity of the affinity-purified antiprimaquine antibody. Of note is that this antibody recognizes the antigen following cross-linking with glutaraldehyde. Furthermore, there is no cross-reactivity with hepatoma HepG2 cell extracts. Primaquine enters cells in the uncharged form and concentrates within acidic compartments following protonation. We have previously shown that incubation of HepG2 cells with primaquine inhibits ASGP-R recycling and ligand degradation (Schwartz et al., 1984). Initial experiments were designed to maximize primaquine cross-linking in extracytoplasmic acidic compartments while minimizing the amount of primaquine within the cytosol. The concentration of primaquine, time of incubation, time of wash, type and time of fixation were varied as described in Materials and methods. The optimal conditions included incubation of cells for 30 min with 300 ktM primaquine, wash for 15 min in media without added primaquine (pH 7.3), 3-4 h fixation in 2% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) and wash overnight in phosphate buffer prior to immersion in sucrose and freezing. Use of milder fixation conditions was Table I. Specificity of anti-primaquine antibody
Coating of well
Contents of well Primaquine
HSA HSA HSA HSA
+ +
HepG2 HepG2 HepG2 HepG2
+ +
-
-
Glutaraldehyde
A410
+
1.085 0.018 0.021 0.016 1.125 0.028 0.017 0.021
+ +
+
Microtiter wells were coated with either HSA or HepG2 cell extract in 1% Triton X-100 as described in the text. Some wells were then incubated with primaquine and/or glutaraldehyde. All wells were blocked by incubation of 10% fetal bovine serum. 1 ug affinity-purified anti-primaquine antibody in 50 Al of 10% FBS in PBS was incubated for 2 h at 37°C. Following rinsing, 50 kd of 10% FBS in PBS containing 0.1 tl goat anti-rabbit antibody conjugated with alkaline phosphatase was added. After rinsing, 50 Al containing 250 Ag p-nitrophenyl phosphate in 25 mM borate (pH 9.3), 10 mM MgCI2 was incubated for 15 min at 37°C. Following dilution the absorbance was determined at 410 nm.
899
A.L.Schwartz et al.
Fig. 1. Ultrathin cryosection of HepG2 cell showing immunogold (5 nm) labelling for primaquine in the lumena of the swollen cisternae of a Golgi complex (G). 118 000 x.
%;
Fig. 2. As in Figure 1, except that in addition primaquine labelling can be seen in flattened portions of the Golgi cisternae (G). The coated vesicles (arrowheads) show only a few particles. N, nucleus. 115 000 x.
associated with a marked loss of specific primaquine labelling. HepG2 cells incubated with primaquine tended to be more rounded and less polygonal than control cells. In addition, they demonstrated more numerous as well as swollen lysosomes and multivesicular bodies. Occasionally there was swelling within the 900
Golgi cisternae. CURL tubules, however, were not swollen (see below, Figures 3-5). The immunochemical localization of primaquine was limited to the lysosomes, multivesicular bodies, CURL vesicles and Golgi cisternae. The most dense labelling reaction occurred in lyso-
Localization of acidic intracellular compartments
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Fig. 4. Section immuno double-labelled for the demonstration of primaquine (6 nm gold) and ASGP-R (10 nm gold). Primaquine is confined to the CURL vesicle (V) which is slightly obliquely sectioned, whereas ASGP-R is present in both the vesicle and the CURL tubule (T). 103 000 x.
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3 Fig. 3. CURL vesicle showing primaquine labelling (10 nm gold). At the arrowhead the membrane of the lower vesicle is continuous with the membrane of a CURL tubule without label. 25 000 x. somes (Figures 5, 6) and multivesicular bodies (Figure 5). Lysosomes were identified by positive reactivity with anti-cathepsin D, whereas multivesicular bodies were negative (not shown). Primaquine was localized to the swollen Golgi cisternae (Figures
1, 2). In cells fixed under milder conditions (see Materials and methods), Golgi complexes were negative. This may reflect difficulty in cross-linking the drug in swollen Golgi cisternae. In addition, there is a paucity of label over trans-Golgi elements including coated vesicles (Figure 2). Primaquine labelling was absent from CURL tubules (Figures 3-5), which showed significant ASGP-R labelling (Figures 4, 5). Since the diameter of CURL tubules ( 20 nm) falls within the thickness of the section (- 100 nm), membrane continuities between vesicles and tubules were only seldomly seen (see Figure 3). Labelling was present in CURL vesicles without internal vesicles and multivesicular bodies with numerous internal vesicles. Nuclei, cytosol and rough endoplasmic reticulum were negative (Figures 2, 3, 6). There was variable primaquine reactivity in mitochondria (compare Figure 5 with 6), however, this appeared to be independent of acidification (see below). The specificity of primaquine localization to acid compartments was addressed with three independent controls. First, pre-
L
.-
I
'.. ' i *
We
T
5
Fig. 5. Section double-labelled for the demonstration of ASGP-R (6 nm gold) and primaquine (10 nm gold). Primaquine is present in a multivesicular body (MVB) and mitochondrion (M), but was undetectable in CURL tubules (T) which are identified by the presence of ASGP-R. 130 000 x.
adsorption of the anti-primaquine antibody with primaquine prior yielded no labelling reaction in any compartment. Second, use of either anti-rat pancreas amylase in place of anti-primaquine or use of protein A-gold without prior incubation with antibody yielded no labelling reaction. Third, pre-incubation of HepG2 cells at 37°C for 15 min with 25 mM NH4C1 prior to the standard 30-min incubation with primaquine markedly reduced the anti-primaquine labelling. However, some reaction was still found in lysosomes. Of importance, the mitochondria reactivity remained.
to incubation with the sections
Discussion Hepatoma HepG2 cells incubated with primaquine specifically accumulate this weak base within endocytotic structures, especially the vesicles of the CURL compartment and lysosomes, as well 901
A.L.Schwartz et al.
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Fig. 6. Section double-labelled for the demonstration of ASGP-R (6 nm gold) and primaquine (10 nm gold). Only a few particles representing ASGP-R are present in and at the periphery of the large lysosomes. Anti-primaquine strongly labels the lysosome but is unreactive with the nucleus (N), rough endoplasmic reticulum (R) and cytosol. In contrast to Figure 5, the mitochondria (M) of this preparation were almost negative for primaquine. 80 000 x.
as the Golgi complex and mitochondria. The accumulation within lysosomes and multivesicular bodies was the most prominent. The specificity of primaquine localization within these compartments could have resulted from either specific binding sites for the amine, or from accumulation of the amine via protonation within acidic compartments. Primaquine does not specifically bind to the hepatoma cells or cell extracts (Table I). The localization of primaquine to acidic intracellular organelles was demonstrated by the loss of reactivity in CURL vesicles, multivesicular bodies, lysosomes and Golgi in cells pre-treated with NH4Cl. The variable reactivity of the mitochondria, which is insensitive to NH4Cl pretreatment, most probably represents binding of the quinolone to mitochondrial protoporphin IX (Chou et al., 1979; McChesney and Fitch, 1984). This must represent a very small amount of reactivity of the whole cell (Table I). Thus, it appears that the amine accumulates within these specific compartments as a result of protonation. The contents of the lysosome have a pH of 4.5 -5.0 (Ohkuma and Poole, 1978) as does the endosomal compartment (Tycko and Maxfield, 1982). The low pH of the lysosomes appears to be a requirement for optimal activity of many acidic hydrolases, whereas the acid pH of the endocytotic structures probably functions in dissociating internalized ligand from receptor (e.g., iron from transferrin, Dautry et al., 1983; ASGP from ASGP-R, Wolkoff et al., 1984). In addition the acid milieu of this compartment appears to be required for viral and toxin entry into the cytoplasm (Marsh et al., 1983) as well as for receptor recycling (Schwartz et al., 1984). Of note, CURL tubules were unreactive for primaquine,
902
whereas CURL vesicles and especially multivesicular bodies were positive. This may reflect a lower H+ concentration within the tubules. In agreement with the present findings are the observations of acidification of isolated endocytotic vesicles (Merion et al., 1983; Galloway et al., 1983) and Golgi (Glickman et al., 1983). However, organelle isolation via cellular fractionation can result in contamination which can bias the interpretation of the results. Similar changes in the overall morphology of these organelles have been made in cells treated with monensin (Strous et al., 1983) or the lysosomotropic amines (Ohkuma and Poole, 1981; Conconi and Walker, 1984). Thus, the present observations provide direct immunocytochemical evidence for accumulation of the weak base primaquine within lysosomes, multivesicular bodies, CURL vesicles, and the Golgi complex. Recently, Anderson et al. (1984) localized the intracellular sites of accumulation of a basic congener of dinitrophenol in fibroblasts via anti-DNP antibodies and either fluorescence microscopy or peroxidase staining combined with electron microscopy. They demonstrated localization in coated vesicles and intracellular uncoated vesicular and tubular structures as well as classical lysosomes. Using an independent approach we have also demonstrated localization of the weak base primaquine to lysosomes and uncoated vesicular structures. In addition, utilizing different fixation techniques we have noted abundant labelling in multivesicular bodies as well as Golgi cisternae. We have shown previously that ASGP-R/ligand complexes are transferred to CURL (Geuze et al., 1983). Receptor-ligand uncoupling takes place and vesicles containing free ligand prob-
Localization of acidic intracellular compartments
ably detach from the tubules (Geuze et al., 1983, 1984). Our observation that CURL vesicles are acidic whereas CURL tubules are apparently not deserves comment as a difference in acidity between vesicles and tubules may be important for the uncoupling mechanism of CURL. Consistent with our findings are the recent observations of Yamashiro et al. (1984), who demonstrated that the para-Golgi tubular structures in CHO cells were mildly acidic (pH 6.5) in contrast to the more acidic endocytic vesicles. These para-Golgi tubules within CHO cells appear to be the equivalent of CURL tubules in liver cells (Geuze et al., 1983). In addition, even in the absence of ligand, ASGP-R molecules recycle from the cell surface into the cell. These ASGP-R molecules are sequestered within an intracellular compartment in cells treated with primaquine or other weak bases (Schwartz et al., 1984). Thus, it will be important to define the intracellular site of receptor sequestration in cells treated with these agents. In summary, then, we have directly demonstrated the accumulation of the weak base, primaquine, and hence the acidic nature of the lysosomes, endocytotic compartment including CURL vesicles and multivesicular bodies and the Golgi complex.
Materials and methods Primaquine was obtained as the biphosphate from Sigma Chemical Company. Keyhole limpet hemocyanin (KLH) and bovine gamma globulins (BGG) were obtained from Calbiochem. Goat anti-rabbit IgG antibody conjugated with alkaline phosphatase was obtained from Zymed laboratories. Preparation of primaquine-protein conjugates Primaquine was cross-linked to KLH or BGG in the following manner: 10 mg of KLH or BGG were mixed with 8 mg primaquine in 10 ml PBS at 40C. The solution was made 1 % with fresh glutaraldehyde and mixed for 1 h. The protein solutions turned mildly cloudy. Protein solutions were dialyzed overnight against PBS at 4'C and stored at -200C. The degree of cross-linking of primaquine to protein was determined by removing an aliquot of the protein/primaquine solu,tion prior to and following the addition of glutaraldehyde and adding an equal volume of ice-cold 20% trichloracetic acid, precipitating the protein-primaquine complex. Addition of trichloracetic acid to primaquine results in a major absorbance peak at 330 nm which can be quantitated in the supernatant directly. Approximately 45 % of the primaquine was conjugated to either KLH or BGG, resulting in a primaquine to KLH ratio of 3950 mol/mol and primaquine to BGG ratio of 126 mol/mol. Immunization of rabbits New Zealand white rabbits were immunized by s.c. injection of 1 mg of proteinprimaquine conjugates following emulsification 1 to 1 in Freund's adjuvant. On days 5, 6 and 7 following booster immunization the rabbits were bled and serum prepared. Affinity purification of anti-primaquine antibodies Primaquine was covalently coupled to cyanogen bromide-activated Sepharose 4B in the standard manner using 3 mg of primaquine per 5 ml of swelled Sepharose 4B. More than 95 % of the primaquine was covalently coupled. Affinity purified anti-primaquine antibodies were prepared by absorbing the rabbit antisera to the primaquine-Sepharose 4B column following by extensive washing with PBS and elution with 3 M KSCN. The affinity purified antibody was dialyzed against PBS at 4°C and stored in PBS with 0.01% sodium azide at 4°C. Determination of specificity of the anti-primaquine antibody Polyvinyl chloride microtiter wells (Dynatech) were coated with either 1 mg human serum albumin (HSA) in 1% Triton X-100 in PBS or a cell extract of HepG2 cells (5 x 106 clone a16 cells per 2 mlI1% Triton X-100 in PBS) for 2 h at room temperature. Following rinsing in PBS, some wells received 50 1l of 1 mg primaquine/mI PBS and/or glutaraldehyde 0.013% in PBS for 1 h at room temperature. Following rinsing in PBS, all remaining sites on the wells were blocked by incubation for 2 h at room temperature with 10% fetal bovine serum in PBS. Following a final rinse in PBS, the wells were stored at 4°C. In order to assay anti-primaquine antibody specificity, appropriate wells were incubated with 50 yl of 10% fetal bovine serum containing 1 tig affinity-purified anti-primaquine antibody for 2 h at 37°C. Following rinsing in PBS, all wells were then incubated for 2 h at 37°C with 50 pAl of 10% fetal bovine serum in PBS containing 0.1 Il goat anti-rabbit conjugated alkaline phosphatase. Following rinsing in PBS, each well was incubated for 15 min at 37°C with 50 pI containing 250 Ag p-nitrophenol phosphate, 25 mM sodium borate (pH 9.3), 10 mM
The optical density was determined at 410 nm following dilution of the MgCl2. 20-fold
sample
(Table I).
Cell processing for immunocytochemistry Cells were incubated with 300 pM primaquine for 30 min or with 12.5 AM or 50 pM primaquine for 2 h at 37°C. The cells were then washed in situ in medium without primaquine for 10, 20 s, 5, 10, 15 min or 1 h and thereafter fixed. The following fixatives were used: 4% formaldehyde, 1 % acroleine + 0.2% glutaraldehyde, 2 % glutaraldehyde, all for periods ranging from 1 to 7 h. All fixatives were prepared in 0.1 M sodium phosphate buffer (PB), pH 7.4. Following fixation the cells were rinsed in PB, harvested and embedded in 10% gelatin as detailed previously (Geuze and Slot, 1980). Control cells were pre-incubated with 25 mM NH4C1 at 37°C prior to treatment with primaquine as detailed above. Washing and fixation were the same as that ultimately used for the experimental cells (see Results). Immunoelectron microscopy Gelatin blocks with cells were immersed in 2.3 M sucrose and frozen in liquid nitrogen. Ultrathin cryosections were prepared according to the Tokuyasu method (Tokuyasu and Singer, 1976) and indirectly labelled with protein A-colloidal gold probes as described previously (Geuze et al., 1981). 5 nm gold particles were prepared and sized as detailed previously (Slot and Geuze, 1981). The 6 and 10 nm particles were made by a new tannic acid/citrate reduction method (to be described elsewhere). There is no overlap in size between 6 and 10 nm particles. Doubleet al., labelling of ASGP-R and primaquine was as previously described (Geuzecellulose 1981). Sections were stained with uranyl acetate and embedded in methyl
according to Tokuyasu (1978).
of the Immunocytochemical control preparations included: (i) pre-absorption with the sections; anti-primaquine antibody with primaquine prior to incubation (ii) use of affinity-purified rabbit anti-rat pancreas amylase in place of anti-ASGP-R or anti-primaquine; (iii) use of protein A-gold probe without prior incubation with antibody.
Acknowledgements The authors are indebted to all of their colleagues in the respective laboratories. We thank especially Janice M.Griffith for her excellent tissue preparations, Tom van Rijn for printing the photographs, and Ireta Ashby for secretarial assistance. The study was supported in part by grants from the Koningin Wilhelmina Fonds, The Netherlands, the National Foundation (Basil O'Connor Research Award), the National Institutes of Health, USA (GM 32477), NATO (818/83), NSF, USA (INT-8317418). A.L.S. was supported in part by a John A. and George Hartford Foundation Fellowship.
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Received on 6 November 1984; revised on 29 January 1985
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