Organization of Cytoskeletal F-Actin, G-Actin, and Gelsolin in the Adhesion Structures in Cultured Osteoclast

Abstract

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. Cytochalasin B treatment caused cells to disorganize the actin cytoskeletal architecture, which indicated the disassembling of F-actin into G-actin in podosomes and disappearance of actin-ring of cultured osteoclasts. Staining with polyclonal actin antibody or monoclonal β-actin was overlapped with the distribution pattern of G- and F-actin. Gelsolin was detected in the region of the adhesion area corresponding to the podosome. The observation that F-actin, G-actin, and gelsolin were detected in the osteoclastic adhesion structures suggests that the podosomes may represent sites where a rapid polymerization/depolymerization of actin occurs. These dynamic changes in cytoskeletal organization and reorganization of G-actin may reflect changes in the functional polarization of the osteoclast during the bone resorption cycle and suggest the important role of G-actin in the regulation of osteoclast adhesion.