Clathrin sheets on the protoplasmic surface of ventral membranes of osteoclasts in culture

Physical cell-shearing resulted in various degrees of disruption of the basolateral (upper) membranes, cytoskeletons or cell organelles and exposed the protoplasmic surface of ventral (adhesion) membranes of osteoclasts that were attached to the underlying substratum, such as coverslips, mica or synthetic apatite plates. Freeze-dried replicas of the ventral membranes left behind on the substratum after cell-shearing provided three-dimensional information on the ultrastructure of the protoplasmic membrane surface of cultured osteoclasts. An extensive area of the protoplasmic surface and various amounts of cytoskeletal structures attached to the adherent ventral surface of the plasma membrane were visible. In particular, the most characteristic finding of the present study is that numerous clathrin sheets displaying various sizes, shapes and curvature were revealed on the ventral membrane. The polygon substructures of the clathrin lattices appeared to be composed of hexagons with a few pentagons interspersed. They were seen at the peripheral membranes where they were situated at the sites of close contact with the underlying substratum. In addition, clathrin lattices were never observed on the basolateral (upper) membranes. In favourable stereo views, most cytoskeletons were not in direct contact with the clathrin sheets. However, a few observations indicated possible remnants of cytoskeletons attached to clathrin lattices. Podosomes did not have a direct structural relationship to clathrin lattices. Although it is generally accepted that cytoskeletal podosomes in motile cells, such as osteoclasts, play a major role in cell adhesion, the present study indicates that membrane-associated clathrin might also function during attachment to the substrate. In this regard, clathrin is thought to be required for receptor-mediated endocytosis, but whether it might also function in cell attachment is still a matter for debate. This type of clathrin-related adhesion appears to be a previously unrecognized site of cell/substrate adhesion in osteoclasts. To assess this possible function, we focused on clathrin and related cytoskeletal elements on the ventral membranes of cultured osteoclasts.     

Ultrastructural study of the effects of cytochalasin D administration on the structure and acid trimetaphosphatase activity of osteoclast.

To determine the role of the cytoskeleton consisting of the microfilaments in osteoclasts, cytochalasin D (CD) conjugated with PBS was administered intravenously to rats at a dose of 1 mg/100 g b.w. Control rats were given only PBS. Then the osteoclasts in the metaphyses of the humeri were examined ultrastructurally, as well as acid trimetaphosphatase (TMPase) cytochemistry. The plasma calcium (Ca) concentrations before and after CD and calcitonin administration were also measured. CD administration first caused a prominent reduction of ruffled borders and spreading of clear zones associated with the occurrence of large pale vacuoles in adjacent cytoplasm. At 1 hr after CD administration, the osteoclasts mostly lacked both ruffled borders and clear zones, but still maintained a normal intracellular organization of cytoplasmic organelles. Morphometric analysis confirmed that CD administration resulted in about 70% reduction of both ruffled border and clear zone areas. TMPase secretion from osteoclasts towards the resorbing bone surfaces was strongly inhibited by CD administration. At 1 hr after administration, although CD caused a decrease of plasma Ca concentrations in 4 of 7 examined rats similar to that caused by calcitonin treatment, there was a slight increase of plasma Ca concentrations in the other 3 rats. These results suggest that the structure and bone-resorbing function of the ruffled border-clear zone complex of the osteoclast is highly regulated by cytoskeleton consisting of microfilaments containing F-actin.     

Alkaline phosphatase activity in the osteoclasts induced by experimental tooth movement.

The localization of alkaline phosphatase (Alpase) activity in the osteoclasts was examined cytochemically. Alpase activity was located in the basolateral membrane in mature osteoclasts having ruffled borders and clear zones, and also in the basolateral membrane in the osteoclasts lacking a ruffled border or a clear zone on the bone surface. But in the preosteoclasts situated away from the bone surface the enzyme activity was noted in the whole plasma membrane. These results suggest that the localization of Alpase activity may be altered in relation to the changes in morphology associated with the functional activity of the osteoclasts.     

Osteoclast differentiation at growth plate cartilage-trabecular bone junction in newborn rat femur

Using 3-day-old newborn rats, we examined the differentiation processes of osteoclasts associated with the destruction of the femoral growth plate cartilage and primary trabecular bone. In the growth plate cartilage, thin mineralized areas were detected solely in the longitudinal septal cartilage matrix in the hypertrophic zone, but the transverse septal cartilage matrix between adjacent chondrocytic lacunae within a row of chondrocytes remained unmineralized. The longitudinal septal cartilage between adjacent rows of chondrocytes appeared to persist, forming the walls of opened lacunar canals. Consistent with the removal of the transverse septal cartilage matrix, the longitudinal canals of opened chondrocytic lacunae were deeply invaded by capillary vessels, mononuclear cells and multinucleated pre-osteoclasts lacking a ruffled border. CD34-positive endothelial cells of capillary vessels deeply penetrated into the transverse septal cartilage matrix facing the medullary cavity and the opened chondrocytic lacunae. ED1-positive monocytes/macrophages were distributed at the chondro-osseous junction, but they were distant from the erosive front of the transverse septa. Tartrate-resistant acid phosphatase-positive multinucleated pre-osteoclasts lacking a ruffled border and differentiated osteoclasts with a ruffled border were localized mainly at two locations: the chondro-osseous junction and the growth front of primary bone trabeculae. Osteoclasts were located on the type-I collagen-positive bone trabeculae close to the growth plate, but they appeared to be distant from the type-II collagen-positive cartilage matrix. Even within opened chondrocytic lacunae, when osteoclasts were distant from the cartilage and bone matrix, they lacked polarized cytoplasmic organization and a ruffled border. The osteoclasts located in the remaining septal cartilage also exhibited neither a ruffled border nor a clear zone. Osteoclasts with a prominent ruffled border and clear zone were located in bone matrix covering the remaining septal cartilage. These results suggest that osteoclasts require hydroxyapatite crystals and bone matrix constituents for ruffled border formation and are not involved in resorption of the unmineralized transverse and mineralized longitudinal septal cartilage without covering bone matrix at the chondro-osseous junction.     

Antibody-decorated dystrophin molecule of murine skeletal myofiber as seen by freeze-etching electron microscopy

Dystrophin is the protein product of Duchenne muscular dystrophy gene which is defective in this genetic disorder. Here we identified ultrastructurally the dystrophin molecule from the various cytoskeletons at the cytoplasmic surface of murine myofiber plasma membrane by using quick-freeze, deep-etch, rotary-shadow replica of anti-dystrophin antibody-decorated muscle samples. The molecule was really cytoskeleton and incorporated in the meshwork of the plasma membrane-associated cytoskeletons. The molecule appeared to connect directly and/or through another cytoskeletal molecule with actin filament.     

The three-dimensional structure of the clear zone of a cultured osteoclast

Osteoclasts collected from long bones of newborn mice were cultured on dentine slices. Then, osteoclasts were sectioned by alternating semithin and ultrathin sections, and the three-dimensional reconstruction was made by the serial semithin sections. By this method, the present study showed first the three-dimensional structure of an osteoclast, especially the clear zone. A reconstructed osteoclast with complicated contours shifted from the lacuna, and its clear zone was a ring-shaped structure. By TEM, a new small lacuna was formed under the ruffled border, and the clear zone could be further classified into three types. The present study suggested that the synthetic observations of both three-dimensional structure and ultrastructure by transmission electron microscopy were necessary to determine whether the osteoclast was resorbing or migrating. According to these observations, the reconstructed osteoclast seemed to be migrating.     

Effects of osteoprotegerin administration on osteoclast differentiation and trabecular bone structure in osteoprotegerin-deficient mice

Osteoprotegerin (OPG)-deficient mice exhibit severe bone loss including the destruction of growth plate cartilage. Using OPG-deficient mice, we attempted to clarify the differentiation and ultrastructure of osteoclasts located on the destroyed growth plate cartilage and trabecular bone matrix in long bones. In (-/-) homozygous OPG knockout mice, adjacent to the growth plate cartilage, the formation of bone trabeculae without a calcified cartilaginous core resulted in an irregular chondrocyte distribution in the growth plate cartilage. At the metaphyseal ossification center, TRAP-positive osteoclasts showed unusual localization on both type-II collagen-positive cartilage and type-I collagen-positive bone matrix. Osteoclasts located on cartilage matrix lacked a typical ruffled border structure, but formed resorption lacunae. During growth plate cartilage destruction, osteoclasts formed ruffled border structures on bone matrix deposited on the remaining cartilage surfaces. These findings suggest that, in OPG (-/-) mice, osteoclast structure differs, depending on the matrix of either cartilage or bone. Then, we examined the effects of OPG administration on the internal trabecular bone structure and osteoclast differentiation in OPG (-/-) mice. OPG administration to OPG (-/-) mice significantly inhibited trabecular bone loss and maintained the internal trabecular bone structure, but did not reduce the osteoclast number on bone trabeculae. For most osteoclasts, OPG administration caused disappearance or reduction of the ruffled border, but induced neither necrotic nor apoptotic damages. These results suggest that OPG administration is an effective means of maintaining the internal structure and volume of trabecular bone in metabolic bone diseases by inhibition of osteoclastic bone resorption.     

Thiamine pyrophosphatase activity in the Golgi apparatus of calcitonin-treated osteoclasts.

The thiamine pyrophosphatase (TPPase) activity described by Novikoff and Goldfisher was examined in osteoclasts affected by calcitonin in order to elucidate whether the morphological and functional changes of the osteoclasts have an influence over the secretion function of their Golgi apparatus. The Golgi apparatus of osteoclasts of which the ruffled border had disappeared and bone resorption discontinued as the result of treatment with calcitonin showed a slight TPPase activity. The reaction products of the enzyme in these inactive osteoclasts were distinctly fewer than that of control osteoclasts, which were not affected by calcitonin. From these results, it is suggested that there may be a connection between the morphological and functional changes of osteoclasts and the secretion function of the Golgi apparatus.     

Osteoclast differentiation and characteristic trabecular bone formation during growth plate destruction in osteoprotegerin-deficient mice

Osteoprotegerin (OPG) is an osteoblast-derived secreted member of the tumour necrosis factor receptor superfamily that inhibits osteoclastogenesis. Mice that are OPG-deficient have severe bone loss, including growth plate cartilage destruction. Using OPG-deficient mice as a useful animal model, we attempted to clarify differentiation and ultrastructural features of osteoclasts located on destructed growth plate cartilage and trabecular bone matrix. In the humerus and femur of OPG homozygous (-/-) mice, adjacent to the growth plate cartilage, bone trabeculae without a calcified cartilage core were characteristically formed at the metaphyseal side of the medullary cavity, which resulted in an irregular chondrocyte distribution and arrangement in growth plate cartilage. During growth plate cartilage destruction, osteoclasts positive for tartrate-resistant acid phosphatase showed unusual localization on both type-II collagen-positive cartilage and type-I collagen-positive trabecular bone matrix at the ossification centre of the epiphyseal/metaphyseal border. Although multinucleated osteoclasts were distributed within open lacunar canals in the growth plate, those on uncalcified cartilage matrix lacked a ruffled border. Facing the calcified cartilage matrix within lacunar canals, osteoclasts showed irregularly formed ruffled borders. After growth plate destruction, a thin bone layer was deposited on the remaining cartilage surfaces by invading osteoblasts. Osteoclasts formed prominent ruffled border structures on bone matrix, deposited on the remaining growth plate cartilage. These results suggest that, in OPG (-/-) mice, terminal osteoclast differentiation requires the presence of newly produced bone matrix, as the coupled phenomenon of bone formation and resorption, as well as osteoblast-derived cytokines.     

Differentiation and function of osteoclasts cultured on bone and cartilage

We examined the differentiation and resorptive function of osteoclasts (OC) cultured on the slices of calcified bone, decalcified bone and hyaline cartilage, and found that OC differentiation depends on the co-cultured substratum, as well as osteoblast-derived factors. Bone marrow-derived macrophages (BMM) were formed from marrow cells of 5 week old ddY mice and cultured for 3 days on freeze-dried slices of calcified bone, decalcified bone or cartilage, all prepared from rabbit costal bone. BMM cultured on calcified bone slices exhibited tartrate-resistant acid phosphatase (TRAP) activity and were structurally characterized by multinucleation and ruffled border development. However, on decalcified bone slices, BMM seldom became multinucleated and exhibited weak TRAP activity. BMM cultured on cartilage slices were mononuclear, devoid of TRAP activity and structurally resembled mononuclear phagocytes. In SEM observations of co-cultured slices, resorption lacunae were formed only on calcified bone slices, and not on slices of decalcified bone and cartilage. Our results, therefore, indicated that BMM could differentiate into functional OC only on calcified bone slices, suggesting a key role of calcified components in the bone matrix for the terminal OC differentiation. Then, we cultured BMM on the same slices with yeast particles. In cultures with yeast particles, BMM exhibited intense TRAP activity, developed a ruffled border-like structure and formed resorption lacunae even on decalcified bone and cartilage slices. Vacuolar-type H+-ATPase was strongly expressed along the ruffled border membranes of these OC. Only the BMM that had not incorporated yeast particles developed a ruffled border, whereas the BMM that had incorporated yeast particles did not become multinucleated and lacked a ruffled border structure. Thus, our results further suggest that, even on uncalcified substrata, the terminal differentiation of BMM into functional OC is induced by an unidentified external stimulus, which may be contained in the cell membrane of yeast particles.     

Localization of receptor activator of NFkappaB ligand, RANKL, in periodontal tissues during experimental movement of rat molars

The receptor activator of NFkappaB ligand, RANKL, is one of the key regulatory molecules in osteoclast formation and function. We examined RANKL localization in the periodontal tissues during experimental movement of rat molars. To produce orthodontic force, an elastic band was inserted between the upper first and second molars for 4 days, and the dissected maxillae were subjected to light and electron microscopic immunocytochemical examination for RANKL. Expression of RANKL protein was detected in osteoblasts, osteocytes, fibroblasts, and osteoclasts mostly located in resorption lacunae. In osteoblasts, osteocytes, and fibroblasts, RANKL localization was mainly observed in the cytoplasm, the cisternae of rough endoplasmic reticulum and along plasma membranes. In osteoclasts, RANKL was expressed along the ruffled, border membranes and in the cytoplasm, including the clear zone. These results suggest that during tooth movement, osteoclast differentiation and activation are regulated, at least in part, by RANKL, possibly produced by osteoblasts/stromal cells and osteoclasts themselves in the periodontal tissues.     

Osteoclastic features of multinucleated giant cells responding to synthetic hydroxyapatite implanted in rat jaw bone.

Multinucleated giant cells (MGCs) that responded to synthetic hydroxyapatite (HAP) implanted in rat mandibles were studied with electron microscopy. HAP used in this study sintered at 200 degrees C (HAP200) and at 125 degrees C (HAP1250) after the synthesis by a wet method. One to three weeks after the intraosseous implantation of HAP, MGCs responding to HAP200 had not only well-developed ruffled border and the clear zone but well-developed perinuclear Golgi complex, many mitochondria and vesicles in their cytoplasms. MGCs responding to HAP1250 had the clear zone, but not the ruffled border although they showed similar cytoplasmic features to those of MGCs responding to HAP200. They merely extended short slender cytoplasmic processes to HAP1250. These results suggest that although osteoclast-like MGCs respond to HAP implanted in the bone, the development of the ruffled border-clear zone system depends on physicochemical properties of HAP.     

Filament Nucleation Tunes Mechanical Memory in Active Polymer Networks

Incorporating growth into contemporary material functionality presents a grand challenge in materials design. The F‐actin cytoskeleton is an active polymer network that serves as the mechanical scaffolding for eukaryotic cells, growing and remodeling in order to determine changes in cell shape. Nucleated from the membrane, filaments polymerize and grow into a dense network whose dynamics of assembly and disassembly, or “turnover,” coordinates both fluidity and rigidity. Here, the extent of F‐actin nucleation is varied from a membrane surface in a biomimetic model of the cytoskeleton constructed from purified protein. It is found that nucleation of F‐actin mediates the accumulation and dissipation of polymerization‐induced F‐actin bending energy. At high and low nucleation, bending energies are low and easily relaxed yielding an isotropic material. However, at an intermediate critical nucleation, stresses are not relaxed by turnover and the internal energy accumulates 100‐fold. In this case, high filament curvatures template further assembly of F‐actin, driving the formation and stabilization of vortex‐like topological defects. Thus, nucleation coordinates mechanical and chemical timescales to encode shape memory into active materials.     

'秦冠'苹果花粉培养及微丝骨架的变化

以'秦冠'苹果花粉为试材,采用花粉离体培养法,研究了不同培养基组分以及温度对苹果花粉萌发的影响.结果表明:(1)蔗糖和硼酸在一定质量浓度范围内,对苹果花粉萌发及花粉管生长起促进作用,但超过一定质量浓度时则起抑制作用.秦冠苹果花粉最适的培养基为20%蔗糖和0.01%硼酸,花粉的萌发率可以达到60%.在此基础上,以Alexa Fluor 488phalloidin作为肌动蛋白微丝的探针,运用激光共聚焦显微镜技术,观察了苹果花粉萌发过程中微丝骨架的分布变化动态;(2)在未水合的苹果花粉粒中,均匀分布着短小弯曲、颗粒环形微丝.在水合而未萌发的的花粉中,微丝呈网状分布在胞质中,萌发沟部分肌动蛋白束网络较致密,中央胞质部分较疏松.花粉萌发后,微丝转变为平行排列,向花粉管伸长的方向延伸聚集,与花粉管纵向平行排列;(3)生长异常的花粉管内其微丝状态也异常.     

A comparative atomic force microscopy study on living skin fibroblasts and liver endothelial cells

Atomic force microscopy (AFM) has been used to image a wide variety of cells and has proven to be successful in cellular imaging, by comparing results obtained by AFM with SEM or TEM. The aim of the present study was to investigate further the conditions for AFM imaging of living cells and compare the results with those obtained by SEM. We chose to image skin fibroblast and liver sinusoidal endothelial cells of two different sources, because these cells have been well described and characterized in earlier studies. AFM imaging of living cells mainly reveals submembranous structures, which could not be observed by SEM. This concerns the visualization of the overall cytoskeletal architecture and organelles, without the necessity of any preparative steps. The AFM study of living cells allows a time lapse study of dynamic changes of the actin cytoskeleton under the influence of the cytoskeleton-disturbing drug cytochalasin B in cells that can be followed individually during the process. However, softer samples, such as the fenestrated parts of living rat liver sinusoidal endothelial cells in culture could not be visualized. Apparently, these cell parts are disrupted due to tip-sample interaction in contact mode. To avoid the lateral forces and smearing artefacts of contact mode AFM, non-contact imaging was applied, resulting in images of higher quality. Still, endothelial fenestrae could not be visualized. In contrast, contact imaging of immortomouse liver sinusoidal endothelial cells, which are devoid of fenestrae, could easily be performed and revealed a detailed filamentous cytoskeleton.     

Crystal structure of troponin and the molecular mechanism of muscle regulation

Troponin plays a central role in the regulation of skeletal and cardiac muscle contraction. The protein consists of three polypeptide chains (TnT, TnI and TnC) and is located on polymerized actin together with tropomyosin, forming muscle thin filament. We have determined the molecular structures of the core domains (relative molecular mass of 46,000 and 52,000) of human cardiac troponin in the Ca2+-saturated form by X-ray crystallography. Analysis of the four structures derived from the two crystal forms reveals that the core domain is further divided into sub-domains, connected by linkers, making the entire molecule highly flexible. The structures of the troponin ternary complex suggests that the Ca2+-binding to the regulatory TnC site displaces the carboxyl-terminal portion of TnI from actin/tropomyosin, thereby altering mobility and/or flexibility of the troponin/tropomyosin strand on the actin filament. These Ca2+-dependent changes in the properties of the tropomyosin strand on the actin filament may in turn alter accessibility of myosin heads (motor protein) to the actin filament.     

Ultrastructural and cytochemical study of the odontoclasts in physiologic root resorption of human deciduous teeth.

Using extracted human deciduous teeth undergoing physiologic root resorption, this author studied the ultrastructural and cytochemical features of odontoclasts. The scanning electron microscopic observation of trypsin-treated dentin and cementum surfaces of resorption lacunae showed the exposure of collagen fibrils and prominent loss of the peritubular matrices around the dentinal tubules. In the resorption lacunae formed in enamel, there was dissolution of either the rod or the interrod regions. The odontoclasts developed extensive ruffled borders apposed to these resorbing matrices and had round phagosomes containing tannic acid-stainable fine amorphous inclusions, which were identical to those in the extracellular canals of the ruffled borders. The odontoclasts did not phagocytose the collagen fibrils. The odontoclasts showed the enzymatic activities of the acid trimetaphosphatase and acid p-nitrophenyl phosphatase (p-NPPase) in the Golgi-lysosome system, the ruffled border region, and along the resorbing dentin surfaces. The p-NPPase activity was inhibited by sodium tartrate. Also, the odontoclasts showed H(+)-K(+)-ATPase activity in the cytoplasm along the plasma membranes including those of ruffled border and the limiting membranes of the lysosomes. These results suggest that: 1) the odontoclasts are associated with resorption of non-collagenous organic matrices and/or extracellularly-degraded collagenous fragments rather than the incorporation of intact collagen fibrils; 2) the odontoclasts release the hydrolytic enzymes onto the lacunal surfaces and/or the lysosomes for the extra/intracellular degradation of the organic matrices; and 3) they also have H(+)-K(+)-ATPase for extracellular demineralization of the inorganic crystals.     

Improved method for visualizing coated pits,microfilaments, and microtubules in cryofixed and freeze-substituted plant cells

We have optimized the conditions for visualizing microfilaments, microtubules, and coated pits in the cortical cytoplasm of high-pressure frozen and freeze-substituted plant cells, in both tobacco root tips and onion cotyledons, individual microfilaments and the supramolecular structure of coated pits can be seen clearly in freeze-substituted samples treated with OsO4 at 40 degrees C followed by 5% uranyl acetate. Treatment with uranyl acetate alone resulted in poorly stained cytoplasmic organelles, whereas microfilaments were difficult to discern in specimen treated with OsO4 alone. The combination of a 40 degrees C OsO4 staining step followed by staining with uranyl acetate at 4 degrees C should prove useful for more detailed plant cytoskeletal/membrane studies in the future.     

A wideband radar for high-resolution mapping of near-surface internal layers in glacial ice

Snow accumulation rate is an important parameter in determining the mass balance of polar ice sheets. Accumulation rate is currently determined by analyzing ice cores and snow pits. Inadequate sampling of the spatial variations in the ice sheet accumulation has resulted in accumulation rate uncertainties as large as 24%. We designed and developed a 600-900-MHz airborne radar system for high-resolution mapping of the near-surface internal layers for estimating the accumulation rate of polar ice sheets. Our radar system can provide improved spatial and temporal coverage by mapping a continuous profile of the isochronous layers in the ice sheet. During the 2002 field season in Greenland, we successfully mapped the near-surface layers to a depth of 200 m in the dry-snow zone, 120 m in the percolation zone, and 20 m in the melt zone. We determined the water equivalent accumulation rate at the NASA-U/spl I.bar/1 site to be 34.9/spl plusmn/5.1 cm/year from 1964 to 1992. This is in close agreement with the ice-core derived accumulation rate of 34.6 cm/year for the same period.