From the
Division of Biological Sciences, Institute of Scientific and Industrial Research, Osaka University, and Core Research for Evolutional Science and Technology of the Japan Science and Technology Corporation, Osaka 567-0047,
Department of Physiology, Kansai Medical University, Moriguchi, Osaka 570-8506, Japan
Received for publication, March 10, 2003
, and in revised form, April 2, 2003.
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ABSTRACT |
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INTRODUCTION |
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V-ATPase is a multisubunit complex formed from a catalytic V1 sector and a membrane-spanning VO sector (713). The VO sector may have a pertinent role in localizing V-ATPases to various cellular membranes. The yeast VO sector is composed of five subunits (a, c, c', c'', and d) and has two subunit a isoforms, Stv1p and Vph1p. The nematode Caenorhabditis elegans has two c (1416) and four a (17) isoforms with different localizations. On the other hand, a single gene for the c subunit has been detected in mammals (18, 19), and no subunit c' gene has been found so far. Only one gene for the c'' subunit was found in three organisms (14, 2022). Mouse embryos lacking the c subunit die shortly after the blastocyst stage during early development (23). In contrast to the c subunit, three subunit a isoforms (a1, a2, and a3) have been identified in mouse (24, 25), and a fourth isoform, a4, was recently detected in mouse (26, 27) and humans (28).
V-ATPase with the a3 isoform was localized to the osteoclast plasma membrane (25). In addition, disruption of the Atp6i gene for mouse a3 causes severe osteopetrosis due to the loss of osteoclast-mediated acidification of resorption lacunae (29). A spontaneous mouse osteopetrotic mutation (30) has been mapped to the same gene (31). These results established that the a3 isoform is an essential subunit for the osteoclast plasma membrane V-ATPase. However, the a3 isoform is expressed in all tissues so far examined, and it is not restricted to osteoclasts (25). Therefore, the subcellular localization of a3 in other cell types remains to be determined.
In this study, we found unexpectedly that the V-ATPase with the a3 isoforms was localized to late endosomes and lysosomes in NIH3T3 and RAW 264.7 cells. RAW 264.7 can form multinuclear cells when cultured with the extracellular domain of RANKL (receptor activator of nuclear factor B ligand) (32). The differentiated cells express osteoclast markers such as tartrate-resistant acid phosphatase, calcitonin receptor, c-src, and cathepsin K. After RANKL stimulation, the a3 isoform together with lysosome markers are localized on dot-like organelles associated with the filamentous structures of microtubules extending to the cell surface and reside on the plasma membrane of mature multinuclear osteoclast-like cells. These findings suggest that lysosomal V-ATPase is targeted to the ruffled border upon osteoclast differentiation.
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EXPERIMENTAL PROCEDURES |
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Cell CultureRAW 264.7 and NIH3T3 cells were grown in DMEM containing 10% fetal bovine serum, non-essential amino acids, 100 units/ml penicillin, and 100 µg/ml streptomycin, and maintained at 37 °C under 5% CO2. For osteoclast differentiation, RAW 264.7 cells were harvested with a 0.05% trypsin solution containing 1 mM EDTA (pH 8.0) and then starved for 5 h in serum-free MEM containing non-essential amino acids. They were then cultured on a plastic dish for 7 days in MEM containing 2% fetal bovine serum, non-essential amino acids, 100 ng/ml sRANKL, 10 ng/ml M-CSF, 100 units/ml penicillin G, and 100 µg/ml streptomycin and then maintained by replacing the medium every 2 days. Histochemical staining for tartrate-resistant acid phosphatase was performed as described previously (33).
HistologyTo stain the osteoclasts attached to the bone surface, mice (C57Bl/6J male; age, 6 weeks) were anesthetized and then perfused with 4% paraformaldehyde in PBS (pH 7.4). Their tibiae and femora were dissected out and immersed in the same solution overnight at 4 °C. After washing with PBS, the tibiae were decalcified with an EDTA solution (9% EDTA-2Na-2H2O, 10% EDTA-4Na-4H2O) at 4 °C for 1 week. They were successively infiltrated with 30% sucrose in PBS, embedded in OCT compound (Miles), and stored frozen. They were then sectioned at 6-µm thickness, mounted on gelatin-coated slides, and stained with hematoxylin. Sections were stained immunochemically as described previously (34).
ImmunochemistryCells were washed with phosphate-buffered saline (PBS) and fixed at room temperature for 25 min in 0.1 M sodium phosphate buffer (pH 7.0) containing 2% paraformaldehyde. After rinsing with PBS containing 0.1 M glycine, and incubation for 15 min at room temperature in PBS containing 0.4% saponin, 1% bovine serum albumin, and 2% normal goat serum, the cells were incubated overnight at 4 °C with the primary antibodies. They were washed with the same solution, and incubated with the secondary antibodies for 1 h at room temperature. They were then washed again and finally mounted on a glass slide with Vectashield (Vector Laboratories).
For observation of acidic organelles, osteoclast-like cells were incubated with DMEM containing 2% fetal bovine serum, 1 µM LysoTracker Red DND-99 for 30 min at 37 °C under 5% CO2. They were then fixed at room temperature for 60 min with 0.1 M sodium phosphate buffer (pH 7.0) containing 4% paraformaldehyde, washed with 0.1 M glycine in PBS, and permeabilized with 0.2% saponin, 1% bovine serum albumin, and 2% normal goat serum in PBS. The cells were incubated with anti-a3 polyclonal antibody at room temperature for 60 min and then stained with the Alexa 488-labeled antibodies for 60 min. They were finally mounted with PermaFluor (Shandon). Fluorescence images were acquired with a confocal microscope, LSM 510 (Carl Zeiss).
Electron MicroscopyThe pre-embedding silver enhancement immunogold method was used as described previously (35). Osteoclast-like cells were washed with PBS and fixed with 4% paraformaldehyde at room temperature for 1 h (36). Cryosections (1 µm) were reacted with 10 µg/ml anti-a3 antibodies overnight, followed by incubation with colloidal gold-conjugated antibodies. For double-labeling immunoelectron microscopy, sections were reacted with anti-a3 and -lamp2 antibodies. They were then incubated with goat anti-rabbit IgG gold conjugate (5-nm diameter) and goat anti-rat IgG gold conjugate (10-nm diameter), followed by observation under an Hitachi H-7000 electron microscope.
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RESULTS |
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Localization of the a3 Isoform in Late Endosomes and Lysosomes of RAW 264.7 Cells before DifferentiationWe addressed the question of whether the V-ATPase localization is similar in the progenitor RAW 264.7 cells, which can differentiate into osteoclast-like multinuclear cells (32). In contrast to those in osteoclasts, anti-a3 antibodies stained dot-like structures around the nuclei, and no positive staining was observed on the plasma membrane or its vicinity (Fig. 2A). As shown in Fig. 2B, the a3 signal was colocalized with lamp2, a marker for late endosomes and lysosomes (37, 38). Essentially the same observation was made for mouse NIH3T3 cells (Fig. 2B). These results suggest that V-ATPase with the a3 isoform is localized to the late endosomes and lysosomes in the osteoclast progenitor cells and fibroblasts. Signals of both the a1 and a2 were not superimposed with that of lamp2 as shown in Fig. 2C, indicating that V-ATPases with a1 or a2 are not present in lysosome/late endosomes. The signal for GM130, a cis-Golgi marker (39), was not superimposed with those of a3 (Fig. 2D). GM130 overlapped with a1 and a2, suggesting that these isoforms are localized to the Golgi complex in RAW 264.7 cells. Furthermore, a1 was also localized to organelles other than lysosomes/late endosomes or the Golgi apparatus.
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Localization of V-ATPase with the a3 Isoform in Lysosomes during DifferentiationIt became of interest to determine the cellular localization or organization of lysosomes during differentiation, because the a3 isoform was localized to lysosomes before stimulation (Fig. 2B). RAW 264.7 cells were cultured in the presence of RANKL and M-CSF, and osteoclast-like multinuclear cells were identified by staining for TRAP (Fig. 3A). The number of TRAP-positive cells reached a plateau level after stimulation for 7 days (data not shown). The localization of the dot-like organelles stained with anti-a3 antibodies dramatically changed upon differentiation: at 13 days after stimulation, the organelles were distributed evenly in the cytoplasm; at 5 days, they were found around the nuclei and on the filaments extending to the plasma membrane (Fig. 3B). After 7 days, a3 mostly localized to the plasma membrane and its close vicinity, similar to the osteoclasts derived from bone marrow cells (25).
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We found that lamp2 was also localized on the filaments extending to the plasma membrane at 5 days after stimulation and mostly on the plasma membrane and its close vicinity after 7 days (Fig. 3B). The lamp2 staining was merged with that of a3 in x-y and x-z sections (Fig. 3, B and C, respectively).
Consistent with the immunofluorescence staining, electron microscopic observation revealed that both the a3 isoform and lamp2 were localized to lysosome-like multivesicular bodies (Fig. 4). Interestingly, lamp2 signal was observed on both limiting membranes and vesicular or tubular structures inside the multivesicular bodies (10-nm gold particles in Fig. 4, arrowheads); however, the a3 localization was restricted to the limiting membranes (5-nm particles in Fig. 4, arrows).
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LysoTracker, an indicator of luminal acidic organelles, could stain the lumens of the organelles (about 1-µm diameter) carrying a3 (Fig. 5). The signals of LysoTracker decreased during the permeabilization of immunofluorescence, even though we still observed a significant fraction of a3 staining positively for LysoTracker. A lack of LysoTracker staining in the presence of bafilomycin A1, a V-ATPase inhibitor, confirmed that the organellar lumens were acidified by V-ATPase. These results suggest that V-ATPase with the a3 isoform in lysosomes/late endosomes membranes was transported to the periphery, and part of it was fused with the plasma membrane to yield the cell surface proton pump.
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Judging from the results of immunoblotting, the amount of a3 increased about 5-fold upon stimulation together with V1 subunit A, indicating that V-ATPase with the a3 isoform had increased (Fig. 6). Based on this observation, the amount applied to the gel was standardized as to the culture, where a significant population had differentiated into osteoclast-like cells. On the other hand, no increase in a1 or a2 was detected, consistent with previous results for osteoclasts derived from bone marrow cells (25). The a4 isoform, which is expressed specifically in kidney (26, 27), was not detectable.
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Microtubule Association of Organelles Carrying V-ATPase with the a3 IsoformThe dot-like organelles stained with anti-a3 and lamp2 antibodies (a3/lamp2-positive compartments) were colocalized with microtubules but not with actin filaments in osteoclast-like cells (Fig. 7A), as shown previously for osteoclasts derived from bone marrow cells (25). Nocodazole and cytochalasin D are known to depolymerize microtubules (40, 41), and actin filaments (42), respectively. No filamentous structures with the a3 isoform and lamp2 were visible after nocodazole treatment, although the signals for the two proteins showed similar localizations (Fig. 7B). The a3 localization was not affected by cytochalasin D, suggesting that actin filaments were not involved in the filamentous distribution of a3. These results suggest that the a3/lamp2-positive compartments are localized on the microtubules extending to the plasma membrane.
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Polarized Distribution of V-ATPase with the a3 Isoform Although V-ATPases with the a3 isoform were found in the plasma membrane region (Fig. 3), the results for vertical (x-z) sections indicate that it is localized to the region facing both the medium and the culture dish surface (Fig. 3C). It is also noteworthy that the multinuclear cells did not form podosomes (Fig. 7A), a discrete ring of actin filaments found in osteoclasts (43, 44). The even distribution may be a result of the cells being cultured on a plastic surface. Thus, we transferred RAW 264.7 cells stimulated for 6 days to a bone surface and cultured them for an additional day. The osteoclast-like cells on the bone showed a significantly polarized distribution of the a3 isoform surrounded by actin rings, a structure similar to podosomes (Fig. 8A). A lateral section (x-z) also indicated that podosomes are formed on the bone side, and a3 is concentrated inside the ring (Fig. 8B). The a3 was localized to the plasma membrane, and it was near the bone surface, whereas syntaxin 4, a plasma membrane marker, merged with a3 in areas facing the bone surface but did not merge in membranes facing the medium. Immunoelectron microscopy confirmed that the a3 signal was highly concentrated in the cell periphery and plasma membrane facing the bone matrix (Fig. 9, A and B). All these results indicate that V-ATPase with the a3 isoform is targeted from multivesicular compartments (Fig. 9B, upper right inset) to the ruffled border membrane forming resorption lacunae.
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DISCUSSION |
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We were interested in membrane-embedded VO subunits, because their unique isoforms may play significant roles in the localization of V-ATPases to specific membranes. The four mouse VO subunit a isoforms, a1, a2, and a3 are expressed ubiquitously (25). However, a3 was found specifically in the plasma membrane and near the osteoclasts derived from bone marrow cells (25, 28). Consistent with this localization, a3 mutations caused osteopetrosis in humans and mouse (29, 31, 45, 46). Recently, we also identified isoforms of mouse V1 and VO subunits C, G, E, and d (4750). In each case, one isoform is ubiquitously expressed and one or more additional ones are in unique cells or tissues. The isoforms of the V1 sector may also be involved in determining the activity and/or localization of V-ATPase within the cell through interaction with a specific a isoform. In this regard, all isoforms except B1 and B2 for the B subunit are localized in the stalk region connecting the catalytic domain with the VO sector (7, 9, 11, 13).
Osteoclasts differentiate from hematopoietic progenitor cells through interaction of their plasma membrane receptor (RANK) with RANKL of osteoblasts (1, 2, 51). A murine macrophage line, RAW 264.7, can differentiate into osteoclast-like multinuclear cells, when cultured with the extracellular domain of RANKL (32, 52). The a3 is localized to the lysosomes/late endosomes of unstimulated RAW 264.7 cells, whereas most of the a2 and a1 isoforms were in the Golgi complex and not colocalized with a late endosome/lysosome marker lamp2. The kidney-specific a4 subunit was not found in RAW 264.7 cells. These results indicate that no a subunit and, accordingly, no V-ATPase were localized to the plasma membrane before stimulation.
Upon differentiation into multinuclear cells, a3 was found on the surface of inside-acidic organelles stained with lamp2, which were on the filamentous microtubules extending to the plasma membrane. These results indicate that V-ATPase with the a3 isoform is localized to the lysosomes/late endosomes of progenitor cells and that these organelles are transported near the plasma membrane of osteoclast-like cells. Electron microscopy confirmed that the V-ATPase with the a3 isoform was finally targeted to the plasma membrane. Thus, an exciting possibility is that lysosomes or late endosomes, transported to the vicinity of the cell surface, directly fuse with the plasma membrane. Our results indicate that such a fusion event is essential for the formation of a functional proton pump on the cell surface. The molecular machinery underlying the lysosome fusion with plasma membrane in osteoclasts remains unclear. Several genetic diseases, which are characterized by varying degrees of hypopigmentation, prolonged bleeding, and immunological deficiency, are the result of impairment of lysosome exocytosis in cell types, including cytotoxic T cells and melanocytes (53, 54). However, there is no evidence concerning any defects in bone metabolism in patients and mutant mice.
The assembly of a3 into V-ATPase was confirmed by immunoprecipitation: anti-a3 antibodies precipitated subunit A of the V1 sector and the c subunit of VO from the solubilized fraction (with 2% octylglucoside) or RAW 264.7 membranes.2 Furthermore, the amounts of the a3 isoform and the A subunit of V1 increased to the same degree after differentiation, suggesting that expression of V-ATPase with the a3 isoform was induced during osteoclast differentiation. Our results showed the re-routing of the lysosome pool of V-ATPase to the plasma membrane, but it is still difficult to rule out the possibility that newly synthesized enzyme with the a3 isoform was deposited to plasma membrane. We have tried to monitor the a3-green fluorescent protein fusion protein transferring to the plasma membrane in living cells. However, the green fluorescent protein-tagged protein as well as other fusions, including hemagglutinin-tagged and myctagged a isoforms appeared to be degraded readily or did not exhibit the corresponding localization as the authentic protein. In this regard, there is still no information on any amino acid determinants specific to a3 isoform that may mediate proper targeting.
In eukaryotic cells, organellar transport to a destination is accomplished by microtubule-associated motor proteins, including kinesin and cytoplasmic dynein (55). It has been shown that c-src, which plays a pivotal role in osteoclast differentiation, is co-localized with intracellular microtubules through a direct interaction with tubulin (56). The V-ATPase is colocalized with microtubules in osteoclasts (25, 56), suggesting that the organelles containing molecules involved in bone resorption are transported to the bone-apposed cell surface through microtubules. Consistently, the depolymerization of microtubules disturbed the a3 localization with the filamentous structures. It has also been shown that the B subunit of V-ATPase contains a filamentous actinbinding site (57). The luminal acidic organelles (vesicles) delivered to the vicinity of the plasma membrane may be concentrated to form an active resorption domain through an interaction with microfilaments.
It should be emphasized finally that lysosomes/late endosomes and, eventually, V-ATPases change their intracellular localization during the expression of differentiated functions. For changes in the final destination of lysosomal V-ATPase, the subunit a3 isoform should play a pertinent role, because other isoforms (a1 and a2) are not assembled into the plasma membrane. Thus, apparently ubiquitous lysosomal V-ATPase becomes a specific plasma membrane pump in highly differentiated osteoclasts.
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FOOTNOTES |
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¶ To whom correspondence should be addressed. Tel.: 81-6-6879-8480; Fax: 81-6-6875-5724; E-mail: m-futai{at}sanken.osaka-u.ac.jp.
1 The abbreviations used are: V-ATPase, vacuolar H+-ATPase; NF-B, nuclear factor kappa B; RANK, receptor activator of NF-
B; RANKL, RANK ligand; sRANKL, extracellular domain of RANKL; MCSF, macrophage colony-stimulating factor; DMEM, Dulbecco's modified Eagle's medium; MEM, minimal essential medium; PBS, phosphate-buffered saline; lamp2, lysosome-associated membrane protein 2; TRAP, tartrate-resistant acid phosphatase.
2 T. Toyomura, Y. Murata, A. Yamamoto, T. Oka, G.-H. Sun-Wada, Y. Wada, and M. Futai, unpublished data.
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ACKNOWLEDGMENTS |
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REFERENCES |
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