Organization of cytoskeletal F-actin, G-actin, and gelsolin in the adhesion structures in cultured osteoclast

Immunofluorescence using Cc protein (group-specific component or vitamin D binding protein [DBP]) as a marker of G-actin showed that nonfilamentous, m onomeric G-actin is a component of the podosomes of: osteoclasts cultured o n glass plates or bone slices. Typical individual podosomes of the well-spr ead cells on glass plates were rosette in form. When viewed from the basola teral surface, the core portion of the dotlike podosomes was associated wit h packed P-actin filaments surrounded by G-actin organized in a ringlike st ructure. 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. Wi th cell spreading on glass plates, the clustering of the podosomes formed a continuous belt of tightly packed podosomes as an adhesion structure at th e paramarginal area. In addition, these structures were seen on the ventral cell surface. Similar changes in cell shape were seen in the osteoclasts w hen 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 bec ame evident then that F- and G-actin dissociated from each other in the pod osomes. The staining patterns of G-actin varied from a discrete dot to a di ffuse one. Toward the nonresorption phase, the osteoclasts lost their conti nuous F-actin band but dotlike podosomes appeared in the leading and the tr ailing edges. In such a cell undergoing translational movements, G-actin wa s located diffusely in the cytoplasm behind the lamellipodia and along some segments of the leading edge. Cytochalasin B treatment caused cells to dis organize the actin cytoskeletal architecture, which indicated the disassemb ling of F-actin into G-actin in podosomes and disappearance of actin-ring o f cultured osteoclasts. Staining with polyclonal actin antibody or monoclon al p-actin was overlapped with the distribution pattern of G- and F-actin. Gelsolin was detected in the region of the adhesion area corresponding to t he podosome. The observation that F-actin, G-actin, and gelsolin were detec ted in the osteoclastic adhesion structures suggests that the podosomes may represent sites where a rapid polymerization/depolymerization of actin occ urs. These dynamic changes in cytoskeletal organization and reorganization of G-actin may reflect changes in the functional polarization of the osteoc last during the bone resorption cycle and suggest the important role of G-a ctin in the regulation of osteoclast adhesion.