Distribution of lectin binding glycoprotein in osteoclasts

Summary Arachis hypogaea (PNA) lectin, specific for Gal-B-1, 3-GalNac disaccharide and Wheat germ (WGA) lectin, specific for (GlcNac) and terminal neuraminic acid were used to identify histiocytic giant cells, osteoclast like giant cells and osteoclasts. PNA lectin, without neuraminidase predigestion was not bound by the giant cells, while they showed a strong reaction with WGA lectin. Neuraminidase pretreatment decreased WGA lectin binding, which supports that neuraminic acid plays a role in the binding of WGA. On the other hand, neuraminidase digestion liberated large amounts of PNA binding sites in every type of giant cells examined, showing a strong, intracytoplasmic granular staining. This observation is indicative of presence of PNA binding sites masked by neuraminic acid. Instead of the intracytoplasmic PNA binding in some osteoclasts a well defined part of the cytomembrane was havily stained. We suppose that this PNA binding part of cytomembrane equals to the zone of resorption, characterized by the ruffled border of osteoclasts. Our findings indicate that a neuraminic acid substituted PNA binding glycoprotein is synthetized both in osteoclasts and histiocytic giant cells which may indicate a common origin of these cell types.     

Increased synthesis and specific localization of a major lysosomal membrane sialoglycoprotein (LGP107) at the ruffled border membrane of active osteoclasts

The immunocytochemical localization was investigated of a major lysosomal membrane sialoglycoprotein with a molecular mass of 107 kDa, which was designated as LGP107. The study utilized rat osteoclasts with different bone resorbing activity and osteoclast precursors at various stages of differentiation and maturation together with monospecific antibodies to this protein. Despite its localization primarily in lysosomes and endosomes in the other cell types examined, LGP107 was exclusively confined to the apical plasma membrane at the ruffled border of the active osteoclast, where the osteoclast is in contact with the bone surface. The protein was also concentrated in a number of endocytic vacuoles in the vicinity of the ruffled border membrane. However the labeling was not found in the basolateral membranes of the active osteoclast. The ruffled border membrane detached from the bone surface showed a marked decrease in the extent of the immunolabeling. The post-and/or resting osteoclasts, which were located away from the bone surface, were totally devoid of the membraneous localization of LGP107. No definite immunolabeling was found in the immature preosteoclasts. These results indicate that the protein is largely synthesized in the active osteoclast and rapidly translocated to the ruffled border membrane by vectorial vesicle transport. LGP107 is suggested to contribute to the formation and maintenance of the specialized acidic environment for bone resorption.     

Limited and selective localization of the lysosomal membrane glycoproteins LGP85 and LGP96 in rat osteoclasts

 Monospecific antibodies against two major glycoproteins of rat lysosomal membranes with apparent molecular masses of 96 and 85 kDa, termed LGP96 and LGP85, respectively, were used as probes to determine the expression and distribution of lysosomal membranes in rat osteoclasts. At the light microscopic level, the preferential immunoreactivity for both proteins was found at high levels at the side facing bone of actively bone-resorbing osteoclasts. Osteoclasts detached from bone surface were devoid of immunoreactivity for each protein. At the electron microscopic level, both proteins were exclusively confined to the apical plasma membrane at the ruffled border of active osteoclasts with well-developed ruffled border membrane. No immunolabeling for both proteins was observed in the basolateral membrane and the clear zone of bone-resorbing osteoclasts. The plasma membrane of preosteoclasts and post- and/or resting osteoclasts showed little or no reactivity against these two antibodies. The results indicate that lysosomal membrane glycoproteins are actively synthesized in active osteoclasts, rapidly transported to the ruffled border area, and contribute to the formation and maintenance of the acidic resorption lacuna of osteoclasts. Accepted: 9 December 1998     

Membrane modifications in chick osteoclasts revealed by freeze-fracture replicas

Remarkable differences among various membranes of bone cells became evident by examination of freeze-fracture replicas. In osteoclasts, three types of intramembranous particles (IMPs) were identified based on their size and shape: two sizes of isolated globular particles (8 and 12 nm in diameter) and rod-shaped, linear aggregates (8 x 30 nm in dimension). Furthermore, the density and distribution pattern of these IMPs enabled us to distinguish three different domains of membranes of osteoclasts including ruffled border, clear zone, and basolateral regions, as were also observed in thin sections. The highest density of IMPs was 3,500-4,000/microns2 in the ruffled border membrane, and these IMPs included linear aggregates among the usual globular particles. Linear aggregated particles were also observed in the membrane of cytoplasmic vesicles in the vicinity of the ruffled border region, but not in this membrane in other bone cells. In attached osteoclasts, the distribution patterns and densities of IMPs in each ruffled-finger and -plate were extremely variable, from closely to the loosely packed membrane particles. Focal aggregates of membrane particles were also frequently encountered. An important outcome of the present study was the finding that the presence of linear aggregated particles proved to be an additional criterion for distinguishing membrane domains in freeze-replicas of osteoclasts. The surface of the clear zone membrane was not smooth in profile, but revealed a number of eminences that were almost free of particles. Basolateral membranes exhibited a particle density of 2,400/microns2. Globular particles were homogeneously scattered in random fashion on their exposed fracture faces. In some cases, aggregates of IMPs on the basolateral membranes were encountered. In comparison with the ruffled fingers, microprojections from the basolateral surface showed a lesser density of IMPs and were devoid of rod-shaped or linear aggregated particles. Differences between osteoblasts and osteocytes were apparent in the density and the size of IMPs. The membranes of osteoblasts and osteocytes contained the same types of globular particles as seen in osteoclasts. Various sizes of gap junctions were located only on basolateral membranes of the osteoblasts. In contrast, no cellular junctions were observed between osteoclasts and any other type of cells.     

Immunocytochemical localization of cathepsin D in the rat osteoclast.

We performed immunocytochemical localization of cathepsin D in osteoclasts of the proximal growth plate of the rat femurs using both the avidin-biotin-peroxidase complex method for cryo-semi-thin (1 micron) sections and the colloidal gold-labeled IgG method for K4M ultra-thin sections. At the light microscopic level, cathepsin D immunoreactivity in the osteoclasts appeared at the vesicles, granules, and/or small vacuoles. They were distributed throughout the cytoplasm of each cell and were relatively numerous close to the bone surface. This antigen could not be detected at the eroded bone surface. As for other cells, immunoreactivity was seen only in the lysosomes of osteoblast-like cells. Immunoreactivity in the osteoclasts was stronger and greater in the density and number than in osteoblast-like cells. At the electron microscopic level, osteoclasts with well-developed ruffled border possessed numerous cathepsin D-containing lysosomes, vacuoles, and coated vesicle-like structures. Cathepsin D-containing lysosomes fused with cathepsin-negative vacuoles and formed large secondary lysosomes. Osteoclasts with poorly developed ruffled border possessed fewer cathepsin D-containing lysosomes than those with well-developed ruffled border. No immunogold particles were seen in vacuole-like channel expansions of the ruffled borders, between the channels of the ruffled borders, or on the eroded bone surface. These findings demonstrate that osteoclasts contain a large amount of cathepsin D. They suggest that cathepsin D is necessary for osteoclastic bone resorption, that it plays an indirect rather than direct role.     

Cytoplasmic terminus of vacuolar type proton pump accessory subunit Ac45 is required for proper interaction with V(0) domain subunits and efficient osteoclastic bone resorption

Solubilization of mineralized bone by osteoclasts is largely dependent on the acidification of the extracellular resorption lacuna driven by the vacuolar (H+)-ATPases (V-ATPases) polarized within the ruffled border membranes. V-ATPases consist of two functionally and structurally distinct domains, V(1) and V(0). The peripheral cytoplasmically oriented V(1) domain drives ATP hydrolysis, which necessitates the translocation of protons across the integral membrane bound V(0) domain. Here, we demonstrate that an accessory subunit, Ac45, interacts with the V(0) domain and contributes to the vacuolar type proton pump-mediated function in osteoclasts. Consistent with its role in intracellular acidification, Ac45 was found to be localized to the ruffled border region of polarized resorbing osteoclasts and enriched in pH-dependent endosomal compartments that polarized to the ruffled border region of actively resorbing osteoclasts. Interestingly, truncation of the 26-amino acid residue cytoplasmic tail of Ac45, which encodes an autonomous internalization signal, was found to impair bone resorption in vitro. Furthermore, biochemical analysis revealed that although both wild type Ac45 and mutant were capable of associating with subunits a3, c, c', and d, deletion of the cytoplasmic tail altered its binding proximity with a3, c', and d. In all, our data suggest that the cytoplasmic terminus of Ac45 contains elements necessary for its proper interaction with V(0) domain and efficient osteoclastic bone resorption.     

Distribution of lectin binding glycoprotein in osteoclasts

Arachis hypogaea (PNA) lectin, specific for Gal-B-1,3-GalNac disaccharide and Wheat germ (WGA) lectin, specific for (GlcNac) and terminal neuraminic acid were used to identify histiocytic giant cells, osteoclast like giant cells and osteoclasts. PNA lectin, without neuraminidase predigestion was not bound by the giant cells, while they showed a strong reaction with WGA lectin. Neuraminidase pretreatment decreased WGA lectin binding, which supports that neuraminic acid plays a role in the binding of WGA. On the other hand, neuraminidase digestion liberated large amounts of PNA binding sites in every type of giant cells examined, showing a strong, intracytoplasmic granular staining. This observation is indicative of presence of PNA binding sites masked by neuraminic acid. Instead of the intracytoplasmic PNA binding in some osteoclasts a well defined part of the cytomembrane was heavily stained. We suppose that this PNA binding part of cytomembrane equals to the zone of resorption, characterized by the ruffled border of osteoclasts. Our findings indicate that a neuraminic acid substituted PNA binding glycoprotein is synthetized both in osteoclasts and histiocytic giant cells which may indicate a common origin of these cell types.     

Immunocytochemical localization of cathepsin D in the rat osteoclast

Summary We performed immunocytochemical localization of cathepsin D in osteoclasts of the proximal growth plate of the rat femurs using both the avidin-biotin-peroxidase complex method for cryo-semi-thin (1 m) sections and the colloidal gold-labeled IgG method for K4M ultra-thin sections.At the light microscopic level, cathepsin D immunoreactivity in the osteoclasts appeared at the vesicles, granules, and/or small vacuoles. They were distributed throughout the cytoplasm of each cell and were relatively numerous close to the bone surface. This antigen could not be detected at the eroded bone surface. As for other cells, immunoreactivity was seen only in the lysosomes of osteoblast-like cells. Immunoreactivity in the osteoclasts was stronger and greater in the density and number than in osteoblast-like cells. At the electron microscopic level, osteoclasts with well-developed ruffled border possessed numerous cathepsin D-containing lysosomes, vacuoles, and coated vesicle-like structures. Cathepsin D-containing lysosomes fused with cathepsinnegative vacuoles and formed large secondary lysosomes. Osteoclasts with poorly developed ruffled border possessed fewer cathepsin D-containing lysosomes than those with well-developed ruffled border. No immunogold particles were seen in vacuole-like channel expansions of the ruffled borders, between the channels of the ruffled borders, or on the eroded bone surface.These findings demonstrate that osteoclasts contain a large amount of cathepsin D. They suggest that cathepsin D is necessary for osteoclastic bone resorption, that it plays an indirect rather than direct role.     

Atp6v1c1 is an essential component of the osteoclast proton pump and in F-actin ring formation in osteoclasts

Bone resorption relies on the extracellular acidification function of V-ATPase (vacuolar-type proton-translocating ATPase) proton pump(s) present in the plasma membrane of osteoclasts. The exact configuration of the osteoclast-specific ruffled border V-ATPases remains largely unknown. In the present study, we found that the V-ATPase subunit Atp6v1c1 (C1) is highly expressed in osteoclasts, whereas subunits Atp6v1c2a (C2a) and Atp6v1c2b (C2b) are not. The expression level of C1 is highly induced by RANKL [receptor activator for NF-kappaB (nuclear factor kappaB) ligand] during osteoclast differentiation; C1 interacts with Atp6v0a3 (a3) and is mainly localized on the ruffled border of activated osteoclasts. The results of the present study show for the first time that C1-silencing by lentivirus-mediated RNA interference severely impaired osteoclast acidification activity and bone resorption, whereas cell differentiation did not appear to be affected, which is similar to a3 silencing. The F-actin (filamentous actin) ring formation was severely defected in C1-depleted osteoclasts but not in a3-depleted and a3(-/-) osteoclasts. C1 co-localized with microtubules in the plasma membrane and its vicinity in mature osteoclasts. In addition, C1 co-localized with F-actin in the cytoplasm; however, the co-localization chiefly shifted to the cell periphery of mature osteoclasts. The present study demonstrates that Atp6v1c1 is an essential component of the osteoclast proton pump at the osteoclast ruffled border and that it may regulate F-actin ring formation in osteoclast activation.     

Variability of the topography of low-density lipoprotein (LDL) receptors in the plasma membrane of cultured human skin fibroblasts as revealed by gold-LDL conjugates in conjunction with the surface replication technique

In this investigation the membrane-perturbing effect of filipin, a polyene antibiotic which reacts specifically with cholesterol or cholesterol-like compounds in cell membranes, has been exploited to study the distribution of coated pits in cultured human skin fibroblasts. The coated pits, showing no filipin-cholesterol complexes, occurred singly or in clusters without apparent localization of either type to a particular region of the fibroblast membrane. Colloidal gold, conjugated to low-density lipoprotein, has proven to be an excellent marker, allowing the localization of low-density lipoprotein receptors on the surface of cultured cells. A pattern similar to that for the coated pits in the plasma membrane fracture faces was observed in the distribution of gold-low-density lipoprotein conjugates in surface replicas, indicating that the low-density lipoprotein receptors are associated with these coated pits. It was shown that there is an apparent heterogeneity in the distribution of low-density lipoprotein receptors, from cell to cell and even among different areas of the same cell membrane. The binding capacity for gold-low-density lipoprotein complexes, as represented by the extent of surface labeling, was directly related to the cell variety within the culture or to the particular experimental procedure. The observation of differences in the distribution of gold-low-density lipoprotein conjugates, even among adjacent coated pits, provides evidence for various stages of activity of the low-density lipoprotein receptors corresponding to incorporation, mobility, and internalization.     

Apical and basolateral coated pits of MDCK cells differ in their rates of maturation into coated vesicles, but not in the ability to distinguish between mutant hemagglutinin proteins with different internalization signals

In polarized epithelial MDCK cells, all known endogenous endocytic receptors are found on the basolateral domain. The influenza virus hemagglutinin (HA) which is normally sorted to the apical plasma membrane, can be converted to a basolateral protein by specific mutations in its short cytoplasmic domain that also create internalization signals. For some of these mutations, sorting to the basolateral surface is incomplete, allowing internalization of two proteins that differ by a single amino acid of the internalization signal to be compared at both the apical and basolateral surfaces of MDCK cells. The rates of internalization of HA-Y543 and HA-Y543,R546 from the basolateral surface of polarized MDCK cells resembled those in nonpolarized cells, whereas their rates of internalization from the apical cell surface were fivefold slower. However, HA-Y543,R546 was internalized approximately threefold faster than HA-Y543 at both membrane domains, indicating that apical endocytic pits in polarized MDCK cells retained the ability to discriminate between different internalization signals. Slower internalization from the apical surface could not be explained by a limiting number of coated pits: apical membrane contained 0.7 as many coated pits per cell cross-section as did basolateral membranes. 10-14% of HA-Y543 at the apical surface of polarized MDCK cells was found in coated pits, a percentage not significantly different from that observed in apical coated pits of nonpolarized MDCK cells, where internalization was fivefold faster. Thus, there was no lack of binding sites for HA-Y543 in apical coated pits of polarized cells. However, at the apical surface many more shallow pits, and fewer deep, mature pits, were observed than were seen at the basolateral. These results suggest that the slower internalization at the apical surface is due to slower maturation of coated pits, and not to a difference in recognition of internalization signals.     

Electron microscopic and histochemical studies of the mononuclear osteoclast of the mouse.

Osteoclasts collected from the long bones of mice were cultured on dentin slices. To identify osteoclasts, the tartrate-resistant acid phosphatase (TRACPase) activity of cultured cells was histochemically examined by the azo dye method. The TRACPase-positive cells could be distinguished from other cells by light microscopy. The cells were sectioned by alternating semithin and ultrathin sections to observe their ultrastructure and three-dimensional structure. TRACPase activity was detected both in multi-nucleated osteoclasts and in mononuclear cells. Most of the mononuclear TRACPase-positive cells had features similar to preosteoclasts. A mononuclear TRACPase-positive cell was a ruffled border and clear zone was reconstructed three-dimensionally by NIKON COSMOZONE 2SA. The reconstruction showed that this cell possessed a large clear zone and small ruffled border. Under the ruffled border, no lacuna was apparent; but there was disruption of the dentin surface. The results suggest that this cell was a mononuclear osteoclast and that it might have been in the process of making a new lacuna.     

Osteoclast cell-surface changes during the egg-laying cycle in Japanese quail

The medullary bone serves as a source of labile calcium mobilized during calcification of the egg shell in birds. Quantitative histological methods demonstrate that the numbers of medullary bone osteoclasts and nuclei per osteoclast remain unchanged during the egg cycle in the Japanese quail (Coturnix). Therefore, cyclic changes in bone resorption cannot be explained by modulations of osteoclasts from and into other bone cells, a mechanism previously suggested for certain species of birds. Rather, dramatic changes in osteoclast cell-surface features occur during the egg cycle, which might account for cyclic variations in resorptive activity. During egg shell calcification, osteoclasts with ruffled borders are closely apposed to bone surfaces; the cytoplasm is rich in vacuoles that contain mineral crystals and seem to derive from the ruffled border. At the completion of egg shell calcification, the ruffled borders and vacuoles move away from the bone surface, although the osteoclast remains attached to the bone along the filamentous or "clear" zone. Associated with the disappearance of the ruffled borders is the appearance of extensive interdigitated cell processes along the peripheral surface of the osteoclast away from the bone. These unusual structures, which may serve as a reservoir of membrane, largely disappear when ruffled borders and associated structures reappear. Therefore, in these hens, the osteoclasts modulate their cell surface rather than their population during the egg cycle.     

Studies of the mechanism of spleen cell cure for osteopetrosis in ia rats: appearance of osteoclasts with ruffled borders

After ia (osteopetrotic) rats receive whole body radiation and an injection of spleen cells from a normal littermate, the dense, sclerotic skeleton characteristic of osteopetrosis is rapidly remodeled and becomes normal in appearance radiographically and histologically within three weeks. The mechanism of this skeletal transformation has been explored in cured ia rats by light and electron microscopic examination of osteoclasts. In ia rats less than 25 days of age, osteoclasts viewed by electron microscopy lack a ruffled border - the extensive elaboration of plasma membrane next to the bone surface. Cured ia rats have osteoclasts with ruffled borders indistinguishable from those of normal littermates. In ia rats that receive only 600 rads whole body radiation, osteoclasts are still present three weeks later, but appear abnormal by light microscopy, with dense nuclei and lacking cytoplasmic vacuoles next to the bone surface. Cured ia rats have two types of osteoclasts, one type indistinguishable from osteoclasts of normal littermates by light microscopy, the other resembling osteoclasts of ia rats that received radiation only. These data indicate that the mechanism of the spleen cell cure for osteopetrosis in ia rats is rapid remodeling of the skeleton produced by osteoclasts with ruffled borders. Whether normal spleen cells produce these osteoclasts directly by cell division or indirectly by elaboration of some unknown local factor required for formations of ruffled borders by ia osteoclasts is not known.     

Transcytosis of calcium from bone by osteoclast-like cells evidenced by direct visualization of calcium in cells

'Transcytosis' of calcium (Ca) from bone by osteoclasts was identified by using a newly developed method that uses fixed or living osteoclast-like cells previously differentiated in vitro, a Ca-specific cell-membrane-impermeable fluorescent dye, and confocal laser scanning microscopy. This method, called the cell-membrane-impermeable dye method, revealed that in fixed osteoclast-like cells, a large quantity of Ca was confined within vacuoles and transported toward the apical cell membrane in the cells. These Ca-confined vacuoles were co-localized with marker proteins of both ruffled border and lysosome. The vacuoles were disrupted when treated with an inhibitor of ruffled border ATPase. In living osteoclast-like cells, Ca-confined vacuoles were again preferentially located at the central region and near the apical cell membrane. These results suggest actual transcytosis of Ca from bone by osteoclasts, and are the first direct evidence of the significant role of osteoclasts in the entire process of Ca metabolism in bone.     

Specific immunocytochemical localization of cathepsin E at the ruffled border membrane of active osteoclasts

The immunocytochemical localization of cathepsin E, a non-lysosomal aspartic proteinase, was investigated in rat osteoclasts using the monospecific antibody to this protein. At the light-microscopic level, the preferential immunoreactivity for cathepsin E was found at high levels in active osteoclasts in the physiological bone modeling process. Neighboring osteoblastic cells were devoid of its immunoreactivity. At the electron-microscopic level, cathepsin E was exclusively confined to the apical plasma membrane at the ruffled border of active osteoclasts and the eroded bone surface. Cathepsin E was also concentrated in some endocytotic vacuoles of various sizes in the vicinity of the ruffled border membrane, some of which appeared to be secondary lysosomes containing the phagocytosed materials. These results strongly suggest that this enzyme is involved both in the extracellular degradation of the bone organic matrix and in the intracellular breakdown of the ingested substances in osteoclasts.     

The membrane composition of coated pits, microvilli, endosomes, and lysosomes is distinctive in the rat kidney proximal tubule cell

The distribution of a number of membrane proteins on plasmalemmal microdomains (microvilli, coated pits) and in endosomes and lysosomes of the proximal tubule epithelial cell was determined in normal rat kidneys by immunofluorescence and immunoelectron microscopy. Two major brush border proteins, 130 and 94 kD, and gamma-glutamyl transpeptidase were detected on the membranes of the microvilli but were not found on membranes of coated pits. Gp330, the Heymann nephritis antigen, and clathrin were localized in coated pits. The lysosomal membrane glycoprotein, lgp120 (Lewis, V., S. A. Green, M. Marsh, P. Vihko, A. Helenius, and I. Mellman, 1985, J. Cell Biol., 100: 1839-1847) was restricted to lysosomes where it co-localized with beta-glucuronidase. Endosomes, identified by preloading with HRP injected 5-15 min before rats were killed, did not contain detectable amounts of any antigen tested. The distribution of the same proteins was also determined in rats given sodium maleate, which is known to slow or reduce protein absorption by the proximal tubule and to cause vacuolation of the endocytic apparatus. After maleate treatment the distribution of microvillar and lysosomal markers was unchanged, but the coated pit markers were redistributed--gp330 was concentrated in newly formed apical vacuoles, and clathrin was diffusely distributed in the apical cytoplasm or on apical coated vesicles. These findings indicate that the membrane composition of microvilli, coated pits, endosomes, and lysosomes is distinctive in the proximal tubule cell; and that gp330, unlike other known coated pit membrane components, is not transferred to endosomes during endocytosis. After maleate treatment, the coated pits lose their clathrin coats, and the corresponding membrane is internalized.     

Electron microscopic localization of lactate dehydrogenase in osteoclasts of chick embryo tibia

Synopsis Lactate dehydrogenase (LDH) was localized in osteoclasts of fixed and unfixed 19-day chick embryo tibias using a copper ferrocyanide capture reaction and osmiophilic polymer generation. This study revealed that: (1) LDH activity in fixed, briefly rinsed osteoclasts was associated principally with limiting membranes of cytoplasmic vacuoles and vesicles and with the plasma membrane; (2) LDH activity in unfixed osteoclasts was associated only with mitochondria; and (3) some mitochondria were stained in fixed tissue given a long rinse. These results indicate that: cytoplasmic LDH diffused out of unfixed tissue; mitochondrial LDH was inactivated by formaldehyde in fixed tissue; and formaldehyde-inhibited mitochondrial LDH can be reactivated by a long rinse. Although the vesicles that stained for LDH activity were found in all parts of the cell, they were concentrated near the ruffled border, and there is evidence that they contained material from the bone surface. These results suggest that the LDH associated with cytoplasmic vesicles of the osteoclast may be important in processing of material resorbed from the bone surface and that osteoclastic mitochondria may utilize lactate from the bone fluid for energy production.     

Downregulation of small GTPase Rab7 impairs osteoclast polarization and bone resorption

During skeletal growth and remodeling the mineralized bone matrix is resorbed by osteoclasts through the constant secretion of protons and proteases to the bone surface. This relies on the formation of specialized plasma membrane domains, the sealing zone and the ruffled border, and vectorial transportation of intracellular vesicles in bone-resorbing osteoclasts. Here we show that Rab7, a small GTPase that is associated with late endosomes, is highly expressed and is predominantly localized at the ruffled border in bone-resorbing osteoclasts. The decreased expression of Rab7 in cultured osteoclasts by antisense oligodeoxynucleotides disrupted the polarization of the osteoclasts and the targeting of vesicles to the ruffled border. These impairments caused a significant inhibition of bone resorption in vitro. The results indicate that the late endocytotic pathway is involved in the osteoclast polarization and bone resorption and underscore the importance of Rab7 in osteoclast function.     

Localization of cathepsins B,D, and L in the rat osteoclast by immuno-light and -electron microscopy

The localization of cathepsins B, D, and L was studied in rat osteoclasts by immuno-light and-electron microscopy using the avidin-biotin-peroxidase complex (ABC) method. In cryosections prepared for light microscopy, immunoreactivity for cathepsin D was found in numerous vesicles and vacuoles but was not detected along the resorption lacunae of osteoclasts. However, immunoreactivity for cathepsins B and L occurred strongly along the lacunae, and only weak intracellular immunoreactivity was observed in the vesicles and peripheral part of the vacuoles near the ruffled border. In control sections that were not incubated with the antibody, no cathepsins were found in the osteoclasts or along the resorption lacunae of osteoclasts. At the electron microscopic level, strong intracellular reactivity of cathepsin D was found in numerous vacuoles and vesicles, while extracellular cathepsin D was only slightly detected at the base of the ruffled border but was not found in the eroded bone matrix. Most osteoclasts showed strong extracellular deposition of cathepsins B and L on the collagen fibrils and bone matrix under the ruffled border. The extracellular deposition was stronger for cathepsin L than for cathepsin B. Furthermore cathepsins B and L immunolabled some pits and part of the ampullar extracellular spaces, appearing as vacuoles in the sections. Conversely, the intracellular reactivity for cathepsins B and L was weak: cathepsin-containing vesicles and vacuoles as primary and secondary lysosomes occurred only sparsely. These findings suggest that cathepsins B and L, unlike cathepsin D, are rapidly released into the extracellular matrix and participate in the degradation of organic bone matrix containing collagen fibrils near the tip of the ruffled border. Cathepsin L may be more effective in the degradation of bone matrix than cathepsin B.