Immunofluorescence using Gc protein (group-specific component or vitamin D binding protein [DBP]) as a marker of G-actin showed that nonfilamentous, monomeric G-actin is a component of the podosomes of osteoclasts cultured on glass plates or bone slices. Typical individual podosomes of the well-spread cells on glass plates were rosette in form. When viewed from the basolateral surface, the core portion of the dotlike podosomes was associated with packed F-actin filaments surrounded by G-actin organized in a ringlike structure. The podosomes, when viewed perpendicular to the substrate, showed a conical shape as a bundle of short F-actin core and a ring of G-actin. With cell spreading on glass plates, the clustering of the podosomes formed a continuous belt of tightly packed podosomes as an adhesion structure at the paramarginal area. In addition, these structures were seen on the ventral cell surface. Similar changes in cell shape were seen in the osteoclasts when they were plated on bone slices. With the loss of dotlike podosomes, a continuous band of F-actin was formed around the resorption lacunae. It became evident then that F-and G-actin dissociated from each other in the podosomes. The staining patterns of G-actin varied from a discrete dot to a diffuse one. Toward the nonresorption phase, the osteoclasts lost their continuous F-actin band but dotlike podosomes appeared in the leading and the trailing edges. In such a cell undergoing translational movements, G-actin was located diffusely in the cytoplasm behind the lamellipodia and along some segments of the leading edge.

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.

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.

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.