TY - JOUR
AU - Suzuki,, Reiko
AB - Abstract The aim of our present research was to visualize how the plasma membrane is modified and how the cytoskeleton interacts with the attachment and ruffled border regions of resorbing osteoclasts. In order to view the surface modification of membranes and associated cytoskeleton, we employed the method of cell-shearing combined with quick-freezing and rotary replication to expose and replicate an extensive area of the cytoplasmic face of the surface membrane of osteoclasts in contact with synthetic apatite as a substratum. The membrane apposed to the apatite was composed of three different domains: the attachment zone, ruffled border and the remainder. In the attachment zone, a highly organized actin filament network formed dot-shaped, F-actin rich adhesion sites, so-called podosomes, and the actin ring. The cytoskeletal filament of podosomes and actin ring appeared to be in direct contact with the cytoplasmic surface of the underlying membrane. Within the actin ring, individually recognizable podosomes were well preserved, which indicates that the actin ring was probably derived from the fusion of podosomes. After shearing at the ruffled border region, the ruffled border projections and membrane regions among the projections were left behind. These ruffled border projections contained the cytoskeletal network. These actin networks also appeared to be in direct contact with the inner side of the ruffled border membrane or in contact with it via membrane-associated particles. At the basal portion of the ruffled border, numerous clathrin-coated patches or pits were well preserved. Deeper clathrin-coated pits and vesicles were also found, which indicates an active site for receptor-mediated endocytotic events. Clathrin sheets were also observed in the cell periphery outside of the actin ring. This type of clathrin sheets adhered to the apatite substrate, but was not anchored to the actin microfilaments. Our study thus clearly visualized the interaction between the cytoskeletal filaments and the underlying membrane at the ruffled border, attachment zone and podosome in osteoclasts cultured on apatitepellets. osteoclast, actin ring, podosome, ruffled border, cell-shearing, quick-freezing Introduction Osteoclasts are large, multinucleated cells specialized for the degradation of mineralized matrix. In the harmony with osteoblastic bone formation, osteoclastic bone resorption is essential for the dynamic equilibrium of bone homeostasis [1]. The resorbing osteoclast is a highly polarized cell with a surface membrane that makes contact with the mineralized matrix via different three membrane domains, i.e. an attachment zone which faces the bone and is formed by cytoskeletal F-actin organization, a ruffled border which is formed by a highly convoluted membrane structure and the remaining domain at the cell periphery. A tight attachment to the mineralized substrate is established surrounding the ruffled border region [2,3]. Within this closed microenvironment between bone and osteoclast, bone destruction and removal of degraded matrix occur. At the resorption front, bone resorption involves the endocytosis of bone material by the invagination and pinching-off of the membrane of the ruffled border, transcytosis of the endocytotic vesicle though the cytoplasm and expulsion of the vesicular contents at the side opposite to the basolateral membrane surface [4–8]. Osteoclasts produced hydrogen ions, which acidify the closed resorption lacuna below the ruffled border and maintain an acidic pH within the resorption lacuna induces the bone mineral [9,10]. Thus, this acidification provides an optimal environment for the action of acid hydrolases and other various enzymes for the destruction of the organic material of the bone. So, the ruffled border region is a site of vigorous site for endocytosis and exocytosis [7,11,12]. Isolated osteoclasts cultured in vitro have provided valuable information for better understanding of the mechanism of bone resorption by osteoclasts. Osteoclasts cultured on glass and on a mineralized substratum show clear differences in their morphology and function [13]. Osteoclasts are capable of adhering to various substrates including glass, plastic, bone, dentin and crystals of various chemical compositions. Nevertheless, they can only resorb mineralized matrices because it has been shown that demineralized bone cannot be resorbed [14–16]. Data are still lacking on the membrane ultrastructure of cultured osteoclasts cultured on artificial synthetic hydroxyapatite in terms of the attachment and ruffled border regions of the membranes. Osteoclasts are characterized by unique cell adhesion structures, the so-called podosomes [3]. Multiple rows of podosomes are localized in the area corresponding to the attachment zone. Podosomes consist of F-actin and associated actin binding proteins [17–19]. Furthermore, the ruffled border serves as an exchange site for various materials from or to the cell. Both the attachment zone and riffled border membrane of osteoclasts have been a matter of much discussion. Here, we cultured osteoclasts on the artificial synthetic hydroxyapatite and examined the cytoplasmic surface for membrane modifications at the resorption front. Cell-shearing combined with quick-freezing microscopy [20,21] was used as we considered that this method would give more detailed information on the cytoplasmic surface of membrane in the attachment zone and ruffled border region. Methods Cultures Osteoclasts were prepared from newborn rabbits by a mechanical desegregation of bone marrow tissues from long bones, and the cells were maintained in Medium 199 containing 10% bovine serum, 1% fungizone and 1% penicillin/streptomycin. Cells were plated onto synthetic hydroxyapatite (HA) pellet (Pentax, Tokyo, Japan) or HA-coated glass cover slips (BD Bioscience, MA, USA). After 48 h in culture, cell morphology was observed with an inverted microscope. All procedures were approved by the Animal Care and Use Committees of Asahi University School of Dentistry. Cell shearing A modified method using ZnCl2 according to Avnur and Geiger [22] was employed to expose the cytoplasmic face of the attachment membrane of adherent osteoclasts on the HA pellets or HA-coated glass cover slips. Apatite pellets were rinsed briefly with a 10 mM PIPES (piperazine-N,N′-bis [2-ethanesulfonic acid]) buffer, pH 7.3 and then incubated for 3–5 min at room temperature in PIPES buffer, pH 6.0 consisting of 3 mM ZnCl2, 60 mM NaCl and 0.67 mM KCl. To shear cells open, we sprayed ice cold 50 mM PIPES buffer (containing 10 mM MgCl2, pH 6.0) onto the entire surface of pellets by using a Pasteur pipette or syringe. For some experiments, cell-shearing was performed by exposure to a jet of tiny cavitation bubbles for ∼20 s from a 6.5 mm diameter microtip probe with a disruptor horn, which when coupled to converter, increases the amplitude of the ultrasonic vibrations and transits them to the solution. An ultrasonic cell disruptor (Branson Sonifier model 250D) operated at a very gentle 30 W of power was used to apply an ultrasonic burst straight down from the microtip probe tip held 2–3 cm above and at a 45° angle to the apatite. Fluorescent microscopy Some apatite specimens were fixed for 30 min with 2% paraformaldehyde buffered with 0.1 M PBS buffer (pH 7.3) prior to staining. Non-sheared open cells were permeabilized with 0.1% Triton X-100 in PBS buffer for 10 min and blocked with 1% BSA in PBS. Osteoclasts were identified by F-actin staining with rhodamine-conjugated phalloidin (Molecular Probe, USA) for 60 min at 37°C. Confocal images were obtained by using a confocal laser scanning microscope (BioRad MRC 2045). Quick-freezing and rotary replication for electron microscopy After the cell-shearing step, the coverslips were quickly rinsed in PIPES buffer and finally in double distilled water. The samples were placed in the freezing device (Eiko FD-5A, Japan) and were quick-frozen by contact with a copper block at liquid nitrogen temperature. For rotary-shadowed replica images, quick-frozen cells were freeze-dried at −90°C for 2 h below a vacuum better than 10−5 Pa in a VFD-300 vacuum device (Vacuum Device, Japan). Freeze-dried specimens were rotary shadowed with platinum (to a 2 nm thickness) at 45° angle and with carbon (to a 20 nm thickness) at a 90° angle with an Eiko FD-5A freeze etching device (Eiko, Japan). Replicas were examined and stereo pairs of electron micrographs were taken with a ±15° tilt angle on a goniometer in a Hitachi H-7100 transmission electron microscope operated at 100 kV. All images of replicas were photographically reversed to aid interpretation; thus, platinum deposits looked white on a dark background. This procedure enhanced the 3D appearance of the replica images. For the 3D visualization, we used the well-known stereo pair and anaglyph stereo electron micrograph. The anaglyph stereoscopic approach enables us to provide valuable visual information available to various types of microscopic images. The anaglyph stereoscopic image pairs were obtained by commercially available image processing software [23,24]. Each original black/white image was converted to green/blue for the right eye and to red for the left eye. By use of commercially available software Adobe Photoshop Element, the two images were superimposed into one image. Anaglyph images of the ultrastructure (Figs 3–12) were obtained by viewing through eyeglasses with blue/red filters. Scanning electron microscopy To examine the substrate surface of cultured osteoclasts we chemically fixed the cells for scanning electron microscopy. Cultures were fixed for 1 h with 2% glutaraldehyde buffered with PIPES, pH 7.3. The samples were dehydrated with a graded series of ethanols, followed by critical drying and examined in a Hitachi S-4500 scanning electron microscope. Results Osteoclasts grown on the apatite pellets were easily identified, as they were significantly larger and abundant filopodia periphery than any of the contaminating cells and had an abundance of filopodia in their periphery (Fig. 1). After 48 h of having been cultured on apatite pellets, the osteoclasts adhered, migrated, spread and started to resorb the substratum. Resorbing osteoclasts could be identified easily, as signs indicating destructing of the apatite surface around such cells were evident. Many resorption lacunae were observed on the surface of apatite pellets. Typical resorbing osteoclasts cultured on apatite were and stained for F-actin by using rhodamine phalloidin. These cells exhibited dot-shaped F-actin rich podosomes, a large band of actin ring and F-actin positive structure inside the actin ring which corresponded to the ruffled border (Fig. 2). The podosome showed a diameter of up to 0.1–0.5 μm, a height of ∼0.5 μm. Fig. 1 Open in new tabDownload slide Scanning electron micrograph of an osteoclast creating resorption lacunae on apatite pellet. (a) Resorption pit on the surface of apatite is clearly demarcated from the sound apatite surface. (b) Numerous filopodia adjacent to the resorption pit project into retracting the rear part of the cell. Bar = 10 μm. Fig. 1 Open in new tabDownload slide Scanning electron micrograph of an osteoclast creating resorption lacunae on apatite pellet. (a) Resorption pit on the surface of apatite is clearly demarcated from the sound apatite surface. (b) Numerous filopodia adjacent to the resorption pit project into retracting the rear part of the cell. Bar = 10 μm. Fig. 2 Open in new tabDownload slide Confocal images of F-actin staining in resorbing osteoclast cultured on the apatite pellets. (a) Actin ring and dot-shaped podosomes are detected by F-actin staining. Some fluorescent inside of the actin ring is responsible for the ruffled border. (b) White line in the upper panel indicates the plane of the corresponding vertical section of the same cell showing actin ring (arrows), ruffled border and podosomes. Arrowheads indicate the basolateral side of the osteoclast. Bar = 10 μm. Fig. 2 Open in new tabDownload slide Confocal images of F-actin staining in resorbing osteoclast cultured on the apatite pellets. (a) Actin ring and dot-shaped podosomes are detected by F-actin staining. Some fluorescent inside of the actin ring is responsible for the ruffled border. (b) White line in the upper panel indicates the plane of the corresponding vertical section of the same cell showing actin ring (arrows), ruffled border and podosomes. Arrowheads indicate the basolateral side of the osteoclast. Bar = 10 μm. In actively resorbing osteoclasts, three different domains of the ventral membrane apposed to the apatite were identified: the attachment zone, which is formed by the actin ring for the cellular attachment, the ruffled border which is the active site for exchange of materials and the remaining of domain at the periphery (Fig. 3). Then, we expose the cytoplasmic face of the apposing membrane in a way that would preserve its ultrastructure. The cell-shearing employed in the present study removed the upper portion of the osteoclasts containing most of the basolateral membrane together with the cytoplasmic matrix, organelles and nucleus. Aggregates of membrane-associated particles were distributed over the cytoplasmic face of the membrane attached to the substratum. These aggregates were clearly seen when the cytoskeletal filaments were removed by shearing. Almost similar results were obtained after either the ZnCl2 method or ultrasonication was used for cell-shearing. The exposed cytoplasmic face of the membrane in the attachment and ruffled border regions remained bound to the culture substratum. Fig. 3 Open in new tabDownload slide Expansive view of the apposed membrane of the cytoplasmic surface of the resorbing osteoclasts. Actin ring (AR) and ruffled border (RB) are easily identifiable. Arrows indicate the cell margin of the osteoclast on apatite pellets. All the anaglyph stereo images (Figs 3–12) can be viewed using red/green stereo viewers. ×4600. Fig. 4 Individually recognizable podosomes (arrows) are fused by dense networks of actin filaments and form a continuous belt of podosomes. Arrows indicate the cell margin of the osteoclast on apatite pellets, where the basolateral membrane remained after shearing ×6900. Fig. 5 Podosomes (P) appear to be a bundle of actin filaments radiating from its core that seem to be composed of tightly packed actin filaments and aggregated materials. The core portion of podosome stands out prominently when viewing in this anaglyph image. Actin filaments seem to terminate to the inner membrane surface. A microtubule (arrowheads) is occasionally observed crossing in the vicinity of the podosome. ×21000. Fig. 6 Interaction of actin filaments with the membrane particles on the inner membrane surface. Remaining actin filaments after shearing appear to directly contact the membrane particles (arrowheads). Membrane-associated particles can be seen the area in which most of actin filaments were sheared away. ×32000. Fig. 7 The area of clathrin lattices on the apposed membrane outside of the actin ring. All the clathrin patches are interconnected with each other. These clathrin lattices are of flat type, which are variable in shapes and sizes. There is no sign of invagination from the clathrin lattice of the membrane. ×30000. Fig. 3 Open in new tabDownload slide Expansive view of the apposed membrane of the cytoplasmic surface of the resorbing osteoclasts. Actin ring (AR) and ruffled border (RB) are easily identifiable. Arrows indicate the cell margin of the osteoclast on apatite pellets. All the anaglyph stereo images (Figs 3–12) can be viewed using red/green stereo viewers. ×4600. Fig. 4 Individually recognizable podosomes (arrows) are fused by dense networks of actin filaments and form a continuous belt of podosomes. Arrows indicate the cell margin of the osteoclast on apatite pellets, where the basolateral membrane remained after shearing ×6900. Fig. 5 Podosomes (P) appear to be a bundle of actin filaments radiating from its core that seem to be composed of tightly packed actin filaments and aggregated materials. The core portion of podosome stands out prominently when viewing in this anaglyph image. Actin filaments seem to terminate to the inner membrane surface. A microtubule (arrowheads) is occasionally observed crossing in the vicinity of the podosome. ×21000. Fig. 6 Interaction of actin filaments with the membrane particles on the inner membrane surface. Remaining actin filaments after shearing appear to directly contact the membrane particles (arrowheads). Membrane-associated particles can be seen the area in which most of actin filaments were sheared away. ×32000. Fig. 7 The area of clathrin lattices on the apposed membrane outside of the actin ring. All the clathrin patches are interconnected with each other. These clathrin lattices are of flat type, which are variable in shapes and sizes. There is no sign of invagination from the clathrin lattice of the membrane. ×30000. In resorbing osteoclasts, the attachment region appeared as an actin ring, which was composed of highly organized actin filaments (Fig. 4). The actin cytoskeleton formed a dense network of the actin ring and surrounded the ruffled border regions, thereby sealing off the resorption lacunae. Although most of the cytoskeletal elements of the cytoplasmic matrix had been removed, the apposing membrane, the actin ring, podosomes and other membrane modifications were left behind after the shearing (Figs 4 and 5). Within the network of the actin ring, we often identified still individually recognizable podosomes that raised above the background of membrane level. These podosomes were characterized by radial filament coming from their core, which was composed of tightly packed short actin filaments and unknown materials that survived the shearing procedure (Fig. 5). These microfilaments appeared to be ∼8 nm in diameter (corrected for the 2 nm platinum coat) and showed a cross striation with a repeating of 5–6 nm. These ultrastructural appearances identified the filament unambiguously as actin microfilaments. Most of the remaining actin filaments were variable length and usually showed no preferred orientation relative to the podosomes or actin rings. Microtubules and intermediate filaments were recognized by their larger size and appearance. The podosomes was characterized by actin rich complex, but microtubules and intermediate filaments were not integral components of the podosomes. Careful observation of the components of podosome relative to that of other cytoskeletal elements such as microtubules and intermediate filaments revealed a close association of microtubules with podosomes (Fig. 5). Tightly packed filaments oriented perpendicularly to the apatite surface. The interaction of radially oriented filaments of the podosomes with apposing membrane surface appeared to occur along the length of the individual filaments. Solitary podosomes were also observed outside of the actin ring. The protoplasmic surface of the membrane, together with portions of the cytoskeletal actin ring or podosomes was resistant to the physical or chemical shearing. Most of the actin filaments appeared to terminate in the membrane-associated particles, whereas a few of them ended directly on the attachment membrane (Fig. 6). The remaining actin filaments after shearing appeared to interact with the membrane via membrane-associated particles on the inner membrane surface. In areas of the membrane that lacked actin filaments, membrane particles were sometimes arranged in a linear array, suggesting that they had been in contact with actin filaments before the filaments were sheared away. The membrane-associated particles were ranging 4–9 nm in diameter. At the periphery of the membrane apposed to the substratum, short filaments forming an irregular network adhered to the cytoplasmic face of the membrane. In this region, clathrin sheets, but not clathrin pits, were often encountered (Fig. 7). Sheet-like (planar) clathrin were connected with each other making an extensive area which appeared to be forming at area devoid of actin filaments (to compare with Fig. 12). The larger clathrin lattice showed the pleomorphic shape, which indicates that newly formed polygons seem to assemble into the margin of the existing, lattices and grow in the size. As is well known, the ruffled border was characterized by numerous surface projections and cytoplasmic infoldings. In favorable views, a ruffled border projection was filled with the cytoskeletal network, which was organized by short actin filaments (Figs 8 and 9). These actin filaments appeared to be in direct contact with the inner side of the ruffled border membrane or make contact with it via the membrane-associated particles. A morphological similarity between the ruffled projections and filopodia has been suggested. Judging from the images of sheared opened cells, however, they appeared to be quiet different structures. A dense actin meshwork formed the core of the ruffled border projections, whereas the actin bundles forming the core of filopodia were distinct from the parallel oriented of individual bundles of filaments. One of the disadvantages of the imaging of sheared-open cells is the limitation that only the cell membrane at the base of the ruffled border could be exposed. Also at the deeper portions of ruffled border projections, the actin filaments were not coated sufficiently by the rotary shadowing metals. However, on the exposed protoplasmic surface at the basal portion of the ruffled border, small non-coated vesicular structures or invaginations were distributed on the surface of the ruffled border membranes (Fig. 10). Careful observation indicates that this type of structure is not aggregate of some protein but small vesicles or invaginations, suggesting the sequence of structural changes occurring in the vesicular membrane during exocytosis. This type of vesicle or invagination had a wide range of sizes (0.1–0.5 μm). In addition abundant clathrin-coated patches or pits could be clearly imaged (Fig. 11). These clathrin coats appeared as extended arrays of polygons in pentagonal or hexagonal fashion showing variable degrees of curvature. Actin microfilament network overlying these clathrin coats were absent. In a well-preserved sample, clathrin lattices and other cytoskeletons were left behind after the shearing (Fig. 12). Clathrin lattices appeared in area of the membrane that lacked overlying actin microfilaments. Usually actin microfilaments attached the edges of clathrin lattice or pits. The majority of clathrin coats consisted of planar or slightly curved lattices. Membrane aggregations showing various sizes and shapes distributed on the cytoplasmic membrane surface linked by short actin filaments. The membrane in this area was principally rich in cytoskeleton without clathrin lattices or vesicular structures. Fig. 8 Open in new tabDownload slide Cytoplasmic membrane surface of the ruffled border region. The inner surface of the ruffled fingers (RF) seems to contain actin cytoskeletons, while the membrane surface among them has no associated the cytoskeleton, presumably almost filaments removed by shearing. ×26000. Fig. 9 A high magnification view of the RB. Interconnected actin filaments are left behind after shearing on the inner surface of membranes. The relationships between the actin filament network and the inner surface membrane can be seen. Some of the actin filaments of the ruffled border appear to contact directly with the membranes (arrows). ×34500. Fig. 10 Smooth-surfaced vesicles or invaginations showing various sizes are observed at the basal region of the ruffled border. This type of structure is clearly distinguishable from the clathrin-coated vesicles as shown in Fig. 11. ×28000. Fig. 11 Clathrin-coated pits are observed on the surface membrane at the basal region of the ruffled border. Note the clathrin lattice showing various degrees of curvature from flat to deeply invaginated one that protrude above the plane of the membrane. ×50000. Fig. 12 In well-preserved region of ruffled border membrane, clathrin lattices (arrows), membranous components (asterisk) and other cytoskeletons scattered. Cytoskeletal elements often approach the edge of clathrin lattices or pits, but do not contact with them. The cytoplasmic surface was covered by a loose irregular network of actin filaments, clathrin lattices and the membrane-associated particles. ×30000. Fig. 8 Open in new tabDownload slide Cytoplasmic membrane surface of the ruffled border region. The inner surface of the ruffled fingers (RF) seems to contain actin cytoskeletons, while the membrane surface among them has no associated the cytoskeleton, presumably almost filaments removed by shearing. ×26000. Fig. 9 A high magnification view of the RB. Interconnected actin filaments are left behind after shearing on the inner surface of membranes. The relationships between the actin filament network and the inner surface membrane can be seen. Some of the actin filaments of the ruffled border appear to contact directly with the membranes (arrows). ×34500. Fig. 10 Smooth-surfaced vesicles or invaginations showing various sizes are observed at the basal region of the ruffled border. This type of structure is clearly distinguishable from the clathrin-coated vesicles as shown in Fig. 11. ×28000. Fig. 11 Clathrin-coated pits are observed on the surface membrane at the basal region of the ruffled border. Note the clathrin lattice showing various degrees of curvature from flat to deeply invaginated one that protrude above the plane of the membrane. ×50000. Fig. 12 In well-preserved region of ruffled border membrane, clathrin lattices (arrows), membranous components (asterisk) and other cytoskeletons scattered. Cytoskeletal elements often approach the edge of clathrin lattices or pits, but do not contact with them. The cytoplasmic surface was covered by a loose irregular network of actin filaments, clathrin lattices and the membrane-associated particles. ×30000. Discussion We isolated the cytoplasmic face of the membrane of cultured osteoclasts that was in apposition to the synthetic apatite pellets and employed quick-freeze, freeze-dry, rotary replication to visualize the interface between the cytoskeletal filaments and the cytoplasmic surface of the underlying membrane apposing to the substratum. The apposing membrane and cytoskeletal structures including podosomes could survive to various degrees after the shearing. These observations indicate that the cytoskeletal filaments in the actin ring or podosomes were in tight contact with the membrane. The upper layer of the cytoskeletal filaments on the membrane had been sheared away. The apposing membrane of resorbing osteoclasts exhibited three distinguishable domains: the attachment zone, the ruffled border and the remainder of this membrane. The attachment zone was composed of a highly organized actin network, which seems to have been derived from the fusion of podosomes. This type of adhesion structure is found in a variety of cells such as osteoclasts, macrophages and transformed fibroblasts [25–27]. Podosomes are highly dynamic adhesion sites that turn over rapidly, suggesting a mechanism for coupling assembly with disassembly [2,13,28]. As is well known, the podosomes appear to function in adhesion and migration. Moreover, podosomes have been implicated in matrix degradation by the finding that the membrane-type matrix metalloproteinase is localized in the plasma membrane of the podosomes [29]. Cytoskeletal organization of osteoclasts is thought to be linked to different functional phases of the bone resorption cycle. It is generally believed that the actin ring might be derived from the fusion of podosomes [2,17,18,30,31]. However, different findings suggest that the attaching actin ring and podosome belts are formed independently. Furthermore, podosomes do not fuse together to form the actin ring onto the apatite [13], and podosome belts are observed only in spread osteoclast adhering to glass. In this study, we found still individually recognizable podosomes in the network of the actin ring, and solitary podosome outside the actin ring at the ultrastructural level. The interaction between the podosomal cytoskeletal filaments and the membrane was clearly seen by exposing the cytoplasmic face of the membrane. Actin filaments of attaching actin ring or podosomes were in direct contact with the membrane via membrane-associated particles, which could represent integral membrane particles. The relationship between the underlying membrane and cytoskeletal filaments still remained intact. Although the role of the actin cytoskeleton in regulating osteoclasts is complex, a direct binding of vacuolar H+-ATPase to actin filament might be involved in controlling the transport of vacuolar H+-ATPase to the ruffled border membrane [32]. Osteoclasts can adhere to various substrates such as glass, mineralized bone or various synthetic materials. However only when the cells are cultured on a mineralized matrix can they create resorption pits [13]. During the destruction of mineralized matrix, the region apposing membrane surrounded by the actin ring formed complicated membrane infoldings and enlarged into the ruffled border projections. Only the basal portion of the ruffled border projections was exposed by shearing. On the surface of their membranes, unevenly distributed membrane particles were observed, which seems to be responsible for the heterogeneous origin of membranes. In addition, recent data suggest that the site of ruffled border region has distinct subdomains for secretion of cytoplasmic products and uptake of degraded matrix [7]. For the destruction of mineralized bones, the osteoclasts secrete protons, lysosomal proteases and other enzymes into the close microenvironment of the resorption lacuna [11]. These exportable materials are carried by vesicular transports via a biosynthetic pathway. Consequently, their vesicular membranes are destined to become incorporated into the ruffled border membrane [7,12,33,34]. The cytoskeletal filaments appeared to end at the ruffled border membrane. These filaments in the ruffled border projections appeared as a highly interwoven meshwork of filaments, similar to that seen in lamellipodia. Actin bundles forming the core of filopodia were quite different from those at the ruffled border [35,36]. The ruffled border is a site for not only the exocytosis but also endocytosis. Degraded organic or inorganic materials are taken up and transcytosed through the entire cytoplasm of osteoclasts. Finally, the degraded materials are destined to be discharged from the basolateral membrane into the extracellular space [4,5,8]. Such a dynamic equilibrium of the ruffled border membranes is maintained by exocytotic events of biosynthetic transport and endocytotic events of transcytotic transport. On the membrane at the basal region of the ruffled border clathrin sheets, pits and vesicles were observed in this study. The polygonal clathrin lattice of coated pits undergoes a molecular rearrangement that converts the sheet-like coated pits into invaginated ones, which subsequently bud off from the membrane [37]. The sheet-like coated pits may correspond to newly formed lattice that increased in size by the formation of polygons. Altogether, the clathrin lattice is a dynamic structure capable of assuming a variety of shapes in response to various stimuli [38]. The pleomorphic shape of sheet-like clathrin lattices may indicate the functional heterogeneity. The presence of clathrin in osteoclasts has been demonstrated in the previous studies [7,39,40]. The present study clearly showed the presence of a clathrin lattice inside and outside of the actin ring resorbing osteoclasts. In the ruffled border region inside of the actin ring, the clathrin lattice exhibited a planar to vesicle form. In contrast, mostly the planar type of clathrin lattice was seen outside of the actin ring. Similar type of clathrin sheets was found on the apposing membranes of osteoclasts cultured on glass plates [39]. Receptor independent mediated endocytosis has still not been excluded in the case of osteoclasts. One role of the clathrin sheets in the cell periphery is to maintain stable attachments of the osteoclast to the substrate. Similar clathrin sheet on the membrane structure has been found at the sites of cell-substratum contact in other cell types [41–45]. These observations suggest a role for clathrin not only in receptor-mediated endocytosis but also in cell attachment and spreading. Conclusion We used the method of cell-shearing combined with quick-freezing and rotary replication to identify an extensive area of the cytoplasmic face of the surface membrane of osteoclasts in contact with synthetic apatite as a substratum. 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TI - The ruffled border and attachment regions of the apposing membrane of resorbing osteoclasts as visualized from the cytoplasmic face of the membrane
JF - Journal of Electron Microscopy
DO - 10.1093/jmicro/dfl012
DA - 2006-04-01
UR - https://www.deepdyve.com/lp/oxford-university-press/the-ruffled-border-and-attachment-regions-of-the-apposing-membrane-of-IVDg2IXC09
SP - 53
EP - 61
VL - 55
IS - 2
DP - DeepDyve
ER -