Platinum replicas of broken-open osteoclasts imaged by transmission electron microscopy.

BACKGROUND: Preserving the cellular structure at the highest possible resolution is a prerequisite for morphological studies to deepen our understanding of cellular functions. A revival of interest in rapid-freezing methods combined with breaking-open techniques has taken place with the development of effective and informative approaches in platinum replica electron microscopy, thus providing new approaches to address unresolved issues in cell biology. HIGHLIGHT: The images produced with platinum replicas revealed 3D structures of the cell interior: (1) cell membranes associated with highly organized cytoskeletons, including podosomes or geodomes, (2) heterogeneous clathrin assemblies and membrane skeletons on the inner side of the membrane, and (3) organization of the cytoskeleton after detergent extraction. CONCLUSION: In this review, I will focus on the platinum replica method after brokenopen cells have been broken open with mechanical shearing or detergent extraction. Often forgotten nowadays is the use of platinum replicas with stereomicroscopic observations for transmission electron microscopy study; these "old-fashioned" imaging techniques, combined with the breaking-open technique represent a highly informative approach to deepen our understanding of the organization of the cell interior. These are still being pursued to answer outstanding biological questions.

Surface distribution of heterogenous clathrin assemblies in resorbing osteoclasts.

Osteoclasts seeded on either glass coverslips or apatite pellets have at least two morphologically distinct substrate adhesion sites: actin-based adhesion structures including podosome belts and sealing zones, and adjacent clathrin sheets. Clathrin-coated structures are exclusively localized at the podosome belts and sealing zone, in both of which the plasma membrane forms a tight attachment to the substrate surface. When cultured on apatite osteoclasts can degrade the apatite leading to the formation of resorption lacunae. The sealing zone divides the ventral membrane into different domains, outside and inside of the sealing zones. The former facing the smooth-surfaced intact apatite contains relatively solitary or networks of larger flat clathrin structures; and the latter, facing the rough-surfaced degraded apatite in the resorption lacunae contain clathrin in various shapes and sizes. Clathrin assemblies on the membrane domain facing not only a resorption lacuna, or trails but also intact apatite indeed were observed to be heterogeneous in size and intensity, suggesting that they appeared to follow variations in the surface topography of the apatite surface. These results provide a detailed insight into the flat clathrin sheets that have been suggested to be the sites of adhesion and mechanosensing in co-operation with podosomes.

Scattered podosomes and podosomes associated with the sealing zone architecture in cultured osteoclasts revealed by cell shearing, quick freezing, and platinum-replica electron microscopy.

Osteoclasts (OCs) can adhere to a variety of substrate surfaces by highly dynamic actin-based cytoskeletal structures termed podosomes. This tight attachment is established by a sealing zone (SZ), which is made of interconnected individual podosomes. Compared with scattered podosomes in various cell types, the architecture of the SZ is still unclear. Especially, ultrastructural studies on the details of the cytoskeletal structure of an OC have been challenging, because the high density of filaments in their podosomes obscure visualization of individual filaments. Therefore, to study this organization in more exact detail, we employed shearing open combined with replica electron microscopy. The present study provides several new details of the podosome and SZ structure, which were previously unrecognized: (a) the SZ consists of recognizable podosomes with a dense actin network of interpodosomal regions characterized by multiple layers of crossing, branching and anastomosing actin filament networks; (b) the Arp2/3 complex is distributed throughout the actin network of podosomes and SZ, indicating that actin polymerization is concentrated at these regions; (c) a close spatial relationship between the podosome and the dorsal membrane; and (d) a network of membranous organelles in close proximity to the podosomes in the SZ. Taken together, the present study reveals that a more complicated interpodosomal actin network among neighboring individual podosomes, which is more complicated than previously thought, appears to form the SZ. Indeed, individual podosomes are not an isolated structural unit from other organelles; and, in turn, their dynamism might affect the surrounding interpodosomal cytoskeletons, membranous organelles, and plasma membrane.

Ultrastructural analysis of apatite-degrading capability of extended invasive podosomes in resorbing osteoclasts.

Osteoclasts in culture are non-transformed cell types that spontaneously develop specific cell-adhesion devices such as podosomes. An individual podosome is a complex network of filamentous actin (F-actin) unit structure that collectively, with other proteins, self-organizes as the sealing zone. Major matrix degradation on apatite seems to proceed under the ruffled-border domain, which is an enclosed extracellular compartment tightly sealed off by this sealing zone. Presently we found that usually the top of finger-like projections of the ruffled border reached toward the plane of the apatite surface, where a shallow degradation of apatite took place. Simultaneously, we obtained several pieces of structural evidence indicating that a specific protrusion referred to as an invasive podosome (invadopodium), which was continuous with podosomes derived from the sealing zone, invaded deeply into apatite matrix and degraded it. The F-actin architecture of the invasive podosome - an active extracellular matrix-degrading, actin-rich cell protrusion - could be distinguished from that of other punctate F-actin structures including the individual podosome, sealing zone, and ruffled border projection. Invasive podosomes contained 2 different F-actin populations, i.e., an interconnected meshwork and a parallel array of bundles. The morphological variability of these protrusions was apparent, having a single cylindrical to lamella-shaped cytoskeletal organization. Our present observations strongly suggest that the degradation of apatite substrate-resorbing osteoclasts appears to have been preceded by the combined appearance of ruffled border and invasive podosomes, and also occurred simultaneously with cell migration during an alternating cycle of resorption and migration.

Visualization of structural organization of ventral membranes of sheared-open resorbing osteoclasts attached to apatite pellets.

Osteoclasts are highly polarized cells from both morphological and functional points of view. Using quick-freeze, rotary-replication methods combined with cell-shearing, we clarified the variability of cytoplasmic surface of the polarized membranes of osteoclasts seeded on apatite. As to the organization of actin filaments and clathrin sheets, we confirmed almost the same ventral membrane specializations of osteoclasts on apatite as seen on glass plates. The organized actin filaments and membrane-associated particles supported the ruffled border membranes. Inside the actin sealing zone, membrane specializations were not always occupied with the ruffled border but also with other types of membranes. Some osteoclasts formed an actin ring but lacked the ruffled border projections. We report a unique and distinctive membrane modification of apatite-attached osteoclasts, i.e., the presence of dense aggregates of membrane-associated particles and related structures not found in the osteoclasts seeded on glass plates. Actin filament polarity in the podosomes was determined by decoration with myosin S1. The actin filament polarity within podosome appears to be oriented predominantly with its barbed ends toward the core, whereas the interconnecting F-actin appears to be mixed oriented. Two different types of clathrin plaques displayed different distributions: clathrin-dependent endocytosis was observed in the ruffled border regions, whereas flat clathrin sheets were found in the leading edge of lamellipodia and near podosomes. The clathrin sheets adhered to the apatite surface tightly on the ventral membranes overlaying the resorption lacunae. All these membrane specializations as mentioned above may indicate the functional variability of osteoclasts seeded on apatite.

Differential distribution of posttranslationally modified microtubules in osteoclasts.

The differential distribution of microtubules in osteoclasts in culture was examined by using antibodies against acetylated, tyrosinated, or detyrosinated tubulins. Tyrosinated tubulin was found throughout the cytoplasmic microtubules in all cells examined. An expanding protrusion that contained tyrosinated tubulin but none of the detyrosinated or acetylated form was seen in the immature osteoclasts. Detyrosinated or acetylated tubulin was detectable in the peripheral cytoplasm of the mature osteoclasts displaying the loss of the expanding protrusion. Although most of the microtubules were derived from the centrosome, noncentrosomal microtubules were distributed in the expanding protrusion, which was predominantly positive for tyrosinated tubulin. By tracing single microtubules, the authors found that their growing ends were always rich in tyrosinated tubulin subunits. End binding protein 1 bound preferentially to the microtubule ends. Both acetylated and tyrosinated microtubules were shown to be closely associated with podosomes. Microtubules appeared to grow over or into the podosomes; in addition, the growing ends of single microtubules could be observed to target the podosomes. Moreover, a microtubule-associated histone deacetylase 6 was localized in the podosomes of the osteoclast. On the basis of these results, the authors conclude that posttranslational modifications of microtubules may correlate with characteristic changes in podosome dynamics in osteoclasts.

Adhesion structures and their cytoskeleton-membrane interactions at podosomes of osteoclasts in culture.

The organization of the cytoskeleton in the podosomes of osteoclasts was studied by use of cell shearing, rotary replication, and fluorescence cytochemical techniques. After shearing, clathrin plaques and particles associated with the cytoskeleton were left behind on the exposed cytoplasmic side of the membrane. The cytoskeleton of the podosomes was characterized by two types of actin filaments: relatively long filaments in the portion surrounding the podosome core, and highly branched short filaments in the core. Individual actin filaments radiating from the podosomes interacted with several membrane particles along the length of the filaments. Many lateral contacts with the membrane surface by the particles were made along the length of individual actin filaments. The polarity of actin filaments in podosomes became oriented such that their barbed ends were directed toward the core of podosomes. The actin cytoskeletons terminated or branched at the podosomes, where the membrane tightly adhered to the substratum. Microtubules were not usually present in the podosome structures; however, certain microtubules appeared to be morphologically in direct contact with the podosome core. Most of the larger clathrin plaques consisted of flat sheets of clathrin lattices that interconnected neighboring clathrin lattices to form an extensive clathrin area. However, the small deeply invaginated clathrin plaques and the podosomal cytoskeleton were located close together. Thus, the clathrin plaques on the ventral membrane of osteoclasts might be involved in both cell adhesion and the formation of receptor-ligand complexes, i.e., endocytosis.

Possible requirement of intercellular adhesion molecule-1 for invasion of gingival epithelial cells by Treponema medium.

Oral treponemes are members of the spirochete family of bacteria associated with periodontal diseases. In the present study, we demonstrate that intercellular adhesion molecule-1 (ICAM-1) on human gingival epithelial cells (HGEC) contributed to the invasion of Treponema medium, a medium-sized oral Treponema, into those cells. The quantity of T. medium in HGEC was found to peak at 2 h after inoculation and then decreased gradually. Immunofluorescence microscopy findings showed that the bacteria were colocalized with ICAM-1 on HGEC. Furthermore, knockdown of ICAM-1 in HGEC resulted in inhibition of T. medium invasion by RNA interference, whereas that of Toll-like receptor 2 did not. These results suggest that ICAM-1 may be required for the invasion of T. medium into HGEC, and they indicate that the molecule plays a principal role in the primary stages of the development and progression of chronic periodontitis.

The ruffled border and attachment regions of the apposing membrane of resorbing osteoclasts as visualized from the cytoplasmic face of the membrane.

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.

A cytoplasmic xylanase (XynX) of Aeromonas caviae ME-1 is released from the cytoplasm to the periplasm by osmotic downshock.

Aeromonas caviae ME-1 is a multiple xylanase-producing gram-negative bacterium which was isolated from the gut contents of a wild silkworm, Samia cynthia pryeri. One of the xylanases produced by A. caviae ME-1, XynX (38 kDa, family 10 xylanase), hydrolyzes xylan to xylobiose and xylotetraose as final degradation products. Generally, xylanases are extracellular or cell surface enzymes. However, XynX is not exported to the extracellular fluid by A. caviae ME-1 and an Escherichia coli transformant harboring the xynX gene. In this study, we investigated the intracellular localization of XynX in A. caviae ME-1 and an E. coli transformant. XynX was found in the cytoplasm when the cells were grown under normal culture conditions. However, XynX was released from the cytoplasm to the periplasm during osmotic downshock. This release of XynX in the E. coli transformant was blocked in the presence of gadolinium chloride, which has been reported to be an inhibitor of bacterial mechanosensitive channels.

[Cytoskeleton of cultured osteoclasts on bone slices and glass].

The cytoskeletal system in a osteoclast modulates its cell shape and motility. During bone remodeling, osteoclastic adhesion and escapement from bone matrix depend upon the formation and disassembly of podosomes. Highly dynamic adhesion of osteoclasts to the bone matrix is referred to as podosomes which consists of actin cytoskeleton and its associated proteins. The cytoskeleton modulates not only in mechanical support of cell shape and motility but also serves signal transducing function in response to external signals. For the better understanding of osteoclast function, our intention here is to describe a dynamic cytoskeleton of osteoclasts including a specific adhesion termed podosome.

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.