Surface modifications at the periosseous region of chick osteoclast as revealed by freeze-substitution.код для вставкиСкачать
THE ANATOMICAL RECORD 222:323-332 (1988) Surface Modifications at the Periosseous Region of Chick Osteoclast as Revealed by Freeze-Substitution TOSHITAKA AKISAKA, GUS PERMANA SUBITA, AND YOSHIO SHIGENAGA Department of Anatomy, School of Dentistry, Hiroshima University, Kasumi 1-2-3, Minami-ku, Hiroshima 734, Japan ABSTRACT Improved preservation of osteoclast fine structure can be achieved by quick freezing, freeze-substitution, or detergent extraction. With such techniques the ruffled border mainly contains a disorganized, interconnected meshwork of microfilaments (5-7 nm in diameter), whereas in the clear zone a few ordered arrays of intermediate-type filaments (10-12 nm in diameter) are detectable among the network of microfilaments. In well-frozen samples, well-preserved matrix may have occluded the cytoskeleton; detergent extraction permits visualization of the cytoskeletal components. In fresh-frozen cells an extracellular fuzzy coat overlays the ruffled border. At the site of attachment of the clear zone to the bone surface, extracellular cementing material is detected only after quick freezing. The superiority of quick freezing to preserve ultrastructure is shown in various cytoplasmic organelles. Most vesicles and vacuoles found close to the ruffled border seemed not to make contact with the extracellular matrix. Anhydrous procedures using quick freezing and freezesubstitution stabilize bone mineral in some vacuoles and in the channels of the ruffled border. Osteoclasts (bone-destroying cells) are highly motile and show morphological changes in response to calcium-regulating hormones and various substrates (Hancox and Boothroyd, 1961; Cameron et al., 1967; Kallio et al., 1972; Lucht, 1973; Lucht and Maunsbach, 1973; Holtrop et al., 1974; Weisbrode et al., 1974; Miller, 1977; Chambers et al., 1984). On the surface next to the calcified matrix, two distinct areas become identifiable: the attaching, organelle-free clear zone and the complicated membrane infoldings of the ruffled border, both of which appear to be active features of osteoclasts involved in bone resorption (Gothlin and Ericsson, 1976; Holtrop and King, 1977). These two membrane modifications seem t o endow the osteoclast with its functional and morphological characteristics. It is apparent that the osteoclast cytoskeleton may play an important role in regulating cell shape, motility, and adherence to bone. Despite the considerable attention paid t o the surface modifications of osteoclasts, their cytoskeletal organization has yet to be confirmed. Most information about the osteoclast cytoskeleton has come from in vitro experiments (King and Holtvop, 1975; ZamboninZallone et al., 1983; Marchisio et al., 1984). However, it is questionable whether the in vitro state always reflects the in vivo state. Morphological differences between osteoclasts in vitro and in vivo have been suggested. The inability to preserve the cytoplasmic structure of in vivo osteoclasts may in part be ascribed to the deleterious effects of conventional processing. Chemical fixatives cannot penetrate rapidly and evenly into the deeper portion of calcified tissues, since the 0 1988 ALAN R. LISS, INC. calcified matrix prevents a smooth and even penetration of fixatives. Such a delay in fixation causes various postmortem changes, leading to distortion, destruction, or loss of cytoplasmic structures. Probably, the high concentration of hydrolytic enzymes or organic acids both inside and outside of osteoclasts can accelerate the postmortem changes. Also, soluble components may be extracted or precipitated during aqueous treatment, again resulting in an artifactual situation. The main purpose of the present study was to elucidate the cytoplasmic structure of in vivo osteoclasts, especially focusing on their ruffled border and clear zone. To minimize the various disadvantages of conventional methods, the present study has employed quick freezing, freeze-substitution, or detergent extraction, by which the organization of cytoplasmic structure has been successfully and recently clarified on various types of cells (Small, 1981; Schnapp and Reese, 1982; Bridgman and Reese, 1984; Bridgman et al., 1986). As expected, these methods have contributed toward the improved preservation of the ultrastructure of in vivo osteoclasts. MATERIALS AND METHODS Forty white leghorn chicks (1-2 weeks old) were used for the present study. After cervical dislocation without anesthesia, tibial metaphyses were dissected out quickly. Received September 21, 1987; accepted February 15, 1988. Gus Permana Subita’s permanent address is Department of Oral Medicine, Faculty of Dentistry, Indonesia University, JL Salemba Raya 4, Jakarta, Indonesia. 324 T.AKISAKA ET AL. Fig. 1 . A low-power electron micrograph of a quick-frozen (QF) and freeze-substituted(FS) osteoclast attaching to the metaphyseal bone. The d e d border (RB)and clear zone (CZ) are identifiable. Bar pm. x 7,800. = 1 325 FREEZE-SUBSTITUTEDOSTEOCLAST Direct Quick Freezing Followed by Freeze-Substitution Slabs of tissue were frozen by the metal contact method introduced by Van Harreveld and Crowell (1964). Immediately after tissue dissection, within 30 sec, samples without any cryoprotectant were firmly contacted by hand against a polished copper block precooled by liquid nitrogen. Frozen samples were stored in a vial containing 1%tannic acid (Merk, West Germany) in absolute acetone for 1-2 days a t -78°C and then transferred into another vial containing 2%osmium tetroxide (OsO,) (Merk) in absolute acetone and left there 1-2 days at - 78°C. The vials were gradually brought to room temperature. Subsequently, the samples were washed in absolute acetone and embedded in Epon-Araldite. Chemical Fixation Followed by Freeze-Substitution Tissue blocks were immersed in a fixative containing 1% tannic acid, 1%acrolein (Merk), and 2% glutaraldehyde (GA) (Taab, U.K.) in 0.1 M cacodylate buffer (pH7.2) at 4°C for 2 hr. After fixation, the samples were frozen by the metal contact method as described above or by submersion into Freon cooled by liquid nitrogen. Followingfreezing,the samples were processed for freezesubstitution as mentioned above. Detergent Extraction To extract the soluble components from the cytoplasm, tissues were immersed in buffered fixative containing 2%paraformaldehyde (Taab), and 0.2% saponin (Katayama Chemicals, Japan) for 1-2 hr. Some tissues were soaked for 20-60 min in a buffer (100 mM KC1; 30 mM HEPES, pH 7.2; 5 mM MgC1,; and 2 mM EGTA) containing 0.2% saponin without chemical fixation for 20-60 min. After detergent extraction, tissues were frozen by the Freon or metal contact method. Freezesubstitution and embedding were the same as described above. Conventional Processing For a control, tissues were first fmed for 2 hrs in 1% tannic acid, 2% paraformaldehyde (PA), and 2.5% GA buffered with 0.1 M cacodylate (pH 7.2) and then postfured in buffered 2% OsO,. After dehydration through a graded series of acetones, the samples were embedded. Ultrathin sections were doubly stained with uranyl acetate and lead citrate or left unstained. They were examined with a Hitachi 500 electron microscope at an accelerating voltage of 100 kV. RESULTS General Features The degree of ultrastructural preservation of rapidly frozen osteoclasts was dependent on the size of ice crystals that caused the deformation of various structures. The essential ultrastructural features observed after quick freezing were similar to those seen after conventional furation (Fig. 1).However, variable improvements for morphological preservation could be achieved by using quick freezing (QF) and freeze-substituion(FS). The nuclear membrane displayed an apparently smoother contour after quick freezing than after conventional processing. Various spherical vacuoles close to the ruffled border, which were often coalesced with each other or the ruffled border, seemed t o be true vac- uoles. All the membrane components were well preserved, showing a trilaminar substructure without signs of rupture (Fig. 2). Calcified matrix was also retained well in situ (Figs. 1, 2). Mineral crystals from the dissolved bone were often observed in the vacuoles and in the extracellular channels of the ruffled border (Fig. 2). At low magnification, the cytoplasmic matrix of the osteoclast was uniformly well preserved (Fig. 1). Regarding the reproducibility of the method utilizing direct quick freezing without cryoprotectant, this method imposed restrictions. On the other hand, chemical furation before freezing remarkably decreased the ice crystal damage in the sample. However, several differences between the morphology of fresh-frozen and fmed-frozen materials became evident. At low magnification, the cytoplasmic matrix of chemically fixed specimens exhibited a granular appearance (Fig. 3). The contents of vacuoles seemed to have a condensed or aggregated form. Flocculent materials adhering to the ruffled border membrane, in the quick frozen sample (Figs. 2, see also Fig. 5), were absent from the ruffled border after chemical fixation and freeze-substitution (see Fig. 8). Following saponin extraction, a finely disorganized meshwork consisting of microfilaments was observed beneath the ruffled border, whereas a loose network of thicker filaments showing 10-12 nm in diameter was visible in the deeper cytoplasm (Fig. 4). Ruffled Border The methods employed in the present study gave different ultrastructural pictures of the ruffled border (Figs. 5-7). In directly fresh-frozen and freeze-substituted samples, a uniform appearance caused by a densely interconnected, disorganized microfilament meshwork was revealed (Fig. 5). In some places, protuberances of electron-dense material projected from the inside of the plasma membrane into the cytoplasm. Fuzzy material appeared to coat the outside of the ruffled border membrane. In well-preserved cytoplasm it was difficult to identify the cytoskeletal structure. In the sample treated by saponin before quick freezing, a clearer picture of the cytoskeletal organization and undercoat structure was obtained (Fig. 6). Complex, disorganized, interconnected microfilaments (5-7 nm in diameter) were still preserved. Neither microtubules nor intermediate filaments could be detected. In the conventionally processed samples, cytoplasmic structure in the ruffled border fingers was poorly preserved (Fig. 7). A partial membrane rupturing was frequently encountered. Extracellular fuzzy material was also undetectable. Freeze-substitution after aqueous chemical fixation (CF) also provided better ultrastructural preservation than did conventional processing (Fig. 8).The interfibrillar substance seen in directly frozen, freeze-substituted samples (Fig. 5) was not observed in samples subjected to an initial aqueous chemical fixation. Distortion of the filamentous network was also recognizable. Along the inner side of the ruffled border membrane, small protuberances and undercoat material were observed (Fig. 8).Casual contacts among the outer leaflets of the ruffled fingers were also often encountered. After freeze-substitution, the asymmetry of the ruffled border membrane became apparent, since the inner leaflet was more obvious than the outer one (Figs. 5, 8). FREEZE-SUBSTITUTED OSTEOCLAST Within the cytoplasm adjacent to the ruffled border, the contents and limiting membrane in various vesicles and vacuoles seemed to be well preserved. The fusion of the d e d border and vesicular membranes was rarely observed. Most vesicles and vacuoles seemed not to make contact with the extracellur space (Fig. 1). Clear Zone The boundary between the clear zone and the ruffled border was obscure in some cases. The clear zone was characterized by an area that was organelle free except for a few vesicular structures. After quick freezing, the typical clear zone exhibited a dense, interconnected meshwork similar to that in the ruffled border. In addition, electron-dense amorphous material and a few parallel arrays of intermediate filaments (10-12 nm in diameter) were recognizable (Figs. 9-13). The typical clear zone in longitudinal sections exhibited bundles of filamentous structures associated with electron-dense dark bands (Fig. 9). On the contrary, in cross sections through the clear zone, the amorphous material was observed to have a uniform cloudy appearance (Fig. 10). At high magnification, intermediate filaments were seen interwoven among the disorganized microfilments with amorphous, electron-dense material (Fig. 11). The contour of the clear zone membrane followed that of the bone surface. In most cases. the clear zone showed a smooth contour (Fig. 12). Even after quick freezing, a constant narrow extracellular space in the contact area where the clear zone was closest to the bone surface was observed. Within the space, structures connecting the cell membrane to the bone surface were revealed only after quick freezing (Fig. 12). Along the inner side of the clear zone membrane, an intimate relationship was suggested between the microfilaments and the membrane via the undercoat material. Saponin treatment clarified the cytoskeletal organization of the clear zone: Microfilaments formed a network, while intermediate filaments tended to make a few ordered arrays (Fig. 13). DISCUSSION Although a large amount of information on osteoclast fine structure has been accumulated using conventional processing (Gothlin and Ericsson, 1976; Holtrop and King, 1977), recent methods utilizing quick freezing followed by freeze-substitution have been demonstrated to be superior to the conventional method for preser- Fig. 2. The ruffled fingers aRer QF and FS show a homogenous appearance, which reflects filamentous and interfibrillar materials, while the clear zone contains a few thicker filaments. Arrowheads indicate bone crystals in the channels of the ruffled border. Fuzzy material (asterisks) adhem to the rutned border membrane.Bar = 0.5 )~m.x 38,000. Fig. 3. This section was treated with chemical furation (CF) before freezing followed by freeze-substitution.A granularity of cytoplasmic matrix becomes evident in comparison with Figure 2. The fuzzy coat has almost disappeared from the ruffled border (RB).The content of vacuoles (V) appears to be condensed. CZ, clear zone. Bar = 0.5 pm. x 33,600. Fig. 4. The sample was extracted with saponin before CF, freezing, and freeze-substitution. Cytoskeletal components become more clearly visualized. The ruffled border (RB)contains a fine filamentous network, while the remaining cytoplasm exhibits a loose network of thicker fdaments. Bar = 0.5 pm. ~ 3 2 , 0 0 0 . 327 vation of the ultrastructure of calcifyingtissue (Akisaka and Shigenaga, 1983; Goldberg and Escaig, 1984; Akisaka et al., 1987). It can be concluded from these results that quick freezing can minimize the disadvantage of chemical fixation. In this study, a great improvement on the preservation of osteoclast ultrastructure was achieved by quick freezing and freeze-substitution. The combination of detergent extraction with freeze-substitution also allowed a good visualization of cytoskeletal structures. The application of the metal contact freezing technique to mineralized tissue seems to be an extremely difficult approach because 1) the hardness of the calcified matrix prevents firm contact between the sample and the cooled copper block, which could lead to uneven heat transfer; and 2) the calcified matrix may have areas of different thermal conductivity. In fact, during this study, only a few osteoclasts were successfully preserved at the electron microscopic level because of severe ice crystal damage. On the other hand, chemical fixation with aqueous aldehydes before freezing provided more reproducible results, but made it difficult to retain some labile materials and delicate cytoskeletal structures (Bridgman and Reese, 1984). However, it is apparent that freezesubstitution even after an initial aqueous chemical fixation exhibited better morphological preservation than did the conventional processing alone. The finding of the microfilament meshwork in the ruffled border of the osteoclast was previously undescribed. These filaments may be actin, as judged from their diameter of 5-7 nm. It is impossible, however, with further study using the antibody localization technique, to determine the exact nature of the filaments. Apparently, the d e d border of the osteolcast is filled with a disorganized,interconnected microfilament meshwork and shows extremely variable forms. It is most likely that these differences in cytoskeletal organization may reflect the different functional roles. Probably, the shape of the ruffled border reflects its degree of involvement in bone resorption. It is well known that calcitonin induces the detachment of the cell body of the osteoclast from the bone surface and the shortening of the ruMed fingers (Kallio et al., 1972), while parathyroid hormone may function to develop the ruffled border (Holtrop et al., 1974). Consequently, one may speculate that the disorganized meshwork structure would be a more adaptable or flexible form to allow changes in the ruffled fingers than would the highly ordered arrays of filaments. Marchisio e t al. (1984) showed multiple actincontaining dots in cultured osteoclasts that seemed ultrastructurally to correspond to the clear zone of in vivo osteoclasts. In the clear zone of osteoclasts, the microfilaments were decorated by heavy meromyosin, which identified actin filaments (King and Holtrop, 1975). The present study demonstrated that the clear zone was composed of a disorganized microfilament meshwork, parallel arrays of intermediate filaments, and electrondense amorphous material. This amorphous material showed parallel dark bands in the longitudinal section, while the clear zone had a uniform cloudy appearance in cross section. Morphological differences in the clear zone may be dependent on the environment in vivo or in vitro (Malkani et al., 1973; Marchisio et al., 1984). The adhesive properties of isolated osteoclasts may be ascribed to the focal contact between the cell and the Fig,5.The ruffled fingers aRer QF and FS. Small protuberances are visible on the inner leaflet (arrowheads).The inner leaflet is thicker than the outer leaflet. The fuzzy coat (asterisks) is well preserved. Bar = 0.5 pm. ~85,000. Fig. 6.Saponin treatment before QF followed by FS. The filamentous structures and undercoat material (arrowheads)become evident in the ruffled hgers. Bar = 0.5 pm. x 55,100. Fig. 7.Conventional processing (CP). Filamentous structures in the ruffled border are indistinguishable.Bar = 0.5 pm. x 59,000. Fig. 8. A cross sectioon of the d e d fingers after chemical furation (CF) and FS. Small protuberances are visible along the inner side of the plasma membrane (arrowheads), but other fragile cytoskeletal structures appear to be deformed because of the aqueous chemical furation. A casual contact between the ruffled fmgers shows a gap junction-like appearance (arrows). Bar = 0.1 pm. x 135,200. Fig. 9. A osteoclast containing a typical clear zone (CZ). Parallel dark bands (arrows) with the bundle of filaments are visible in the longitudinal section of the clear zone. QF and FS. Bar = 1pm. x 12,600. Inset: A higher magnification of the parallel dark bands. Two types of filaments were identificable: microfilament (mf) and intermediate filament (if).Bar = 0.5 pm. ~46,200. 331 FREEZE-SUBSTITUTEDOSTEOCLAST substratum (Marchisio et al., 1984). Similar focal contact between cells and substratum has been reported for various types of motile cells (Izzard and Lochner, 1976; Wehland et al., 1979; Chen and Singer, 1982). A similar observation was also made in the clear zone of the in vivo osteoclast at sites of attachment. At the contact site, the dense filamentous network can stabilize the clear zone membrane, which may strengthen the adhesion between the cell and the bone surface. The present study showed the presence of cementing material within the narrow extracellular space between the clear zone membrane and bone. However, the chemical nature of the cementing material detected by freezesubstitution in the osteoclast is completely unknown. Cytochemical differences between the ruMed border and clear zone membrane have been suggested (Doty and Schofield, 1976; Akisaka and Gay, 1986). They differ morphologically as well. Kallio et al. (1971) showed fine bristle-like structures along the inside of the ruffled border membrane. The present freeze-substitution work exhibited a difference in undercoat structure: small protuberances associated with electron-densematerial were seen in the undercoat of the ruffled border membrane but not in that of the clear zone membrane. Different preparative procedures have led to various interpretations of the ultrastructure of undercoat structures. Undercoat structures may be composed of various enzymes or cytoskeletal components. The superiority of the quick freezing technique for the preservation of osteoclast ultrastructure was also demonstrated in the fuzzy coat along the extracellular aspect of the ruffled border. Within the enclosed microenvironment of the ruffled border, its membrane is exposed to the extracellular fluid which contains various hydrolytic enzymes and organic acids (Vaes, 1968; Doty and Schofield, 1972). Such an environment of the ruffled border region is destructive for the integrity of osteclasts. It is, therefore, likely that the fuzzy coat may play a protective role against the extracellular resorbing fluid. In conclusion, the present method-quick freezing followed by freeze-substitution-seems to provide a more accurate picture of the ultrastructural details of in vivo osteoclasts, especially of the ruffled border and clear zone. Quick freezing can stabilize fragile structures, while freeze-substitution allows anhydrous treatment of the tissue. By use of these techniques several preFig. 10.A cross-sectional view of the QF and FS clear zone. The amorphous material appears to be a continous form within the network of filaments. Bar = 1 Fm. X 14,400. Fig. 11. A high-power of cross-sectioned clear zone after QF and FS. Within the amorphous material, a filamentous network appears to be composed of microfilaments (mfl and intermediate filaments (If).Bar = 0.5 pm. x 57,200. Fig. 12.A longitudinal section of the clear zone adjacent to the calcified matrix. The filamentous network appears to be in direct wntact with the clear zone membrane. Within a narrow extracellular space, bridging structures connect the cell membrane with the surface of calcified matrix. QF and FS. Bar = 0.1 pm. x 132,000. Fig. 13. A few microprojections from the clear zone into the bone. Microfilaments and intermediate filaments seem to be interwoven. This CF-fixed sample was treated with saponin followed by freeze-substitution. Bar = 0.5 pm. ~ 4 0 , 0 0 0 . viously unreported observations have been made regarding osteoclast ultrastructure. ACKNOWLEDGMENTS The authors are indebted to Mrs. Yoshi Shirana for typing the manuscript. This investigation was supported in part by Grant-in-Aid for Scientific Research No. 62570808 from the Japanese Ministry of Education, Science, and Culture to T.A. LITERATURE CITED Akisaka, T., and C.V. Gay 1986 An ultracytochemical investigation of ouabain-sensitive pnitrophenyl p h o s p h a h in chick osteoclasts. Cell Tissue Res., 244:57-62. Akisaka, T., and Y.. Shigenaga 1983 Ultrastructure of growing epiphyseal cartilage processed by rapid freezing and freeze-substitution. J . Electron Mimsc., 32:305-320. Akisaka,T., G.P. Subita, and Y. Shigenaga 1987 Ultrastructural observations on chick bone processed by quick-freezing and freezesubstitution. Cell Tissue Res.,247:469-475. Bridgman, P.C., B. Kachar, and T.S. 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