Coordination of microtubules and the actin cytoskeleton is important in osteoclast function, but calcitonin disrupts sealing zones without affecting microtubule networks

Bone-resorbing osteoclasts form sealing zones and ruffled borders toward the bone surface. The sealing zone consists of a ring-like alignment of F-actin dots and surrounds the ruffled border, from which protons are secreted into the bone surface. Vacuolar-type proton ATPase (V-ATPase) in osteoclasts is a ruffled border-associated enzyme responsible for the proton secretion. We studied the interaction between microtubules and the actin cytoskeleton in osteoclasts. Confocal microscopic observation revealed that osteoclasts on glass coverslips, dentine slices and Osteologic™ discs formed the ring-like structure of F-actin dots, and microtubules overlapped the top of the F-actin dots. Osteoclasts cultured on dentine formed resorption pits within 48 h. The treatment of osteoclasts with cytochalasin D, an F-actin-depolymerizing reagent, induced perturbation of the microtubules in osteoclasts on glass and inhibited their pit-forming activity on dentine in a dose-dependent and reversible manner. Conversely, nocodazole, a microtubule-depolymerizing reagent, disrupted sealing zones and inhibited pit-forming activity of osteoclasts in a dose-dependent and reversible manner. V-ATPase showed a tendency to be localized inside sealing zones in osteoclasts. Treatment of osteoclasts with calcitonin induced both disruption of sealing zones and dispersion of V-ATPase to the whole area of the cytoplasm within 60 min. The microtubule networks in osteoclasts remained unchanged for 60 min even in the presence of calcitonin. These results suggest that coordination of the actin cytoskeleton and microtubules is important in the function of osteoclasts, but calcitonin selectively affects the actin cytoskeleton and induces the dispersion of V-ATPase without causing significant changes in the microtubules.