Cytoskeleton rearrangements during calcitonin-induced changes in osteoclast motility in vitro

Abstract

Although relatively little is known about osteoclastic degradation of bone, observations both in vivo and in tissue culture in vitro suggest that osteoclast motility is required. Osteoclasts with abundant motile activity and responsiveness to a bone-regulating hormone, calcitonin, have recently been isolated from rat femur and maintained in culture for short periods of time. Using indirect immunofluorescence with antibodies to tubulin and myosin and NBD-phallacidin staining for F-actin, we have studied cytoskeleton distributions in such osteoclasts, either untreated or treated with calcitonin. Untreated and unresponsive cells were well spread on the substratum and displayed striking motility in time-lapse cinematography. In these cells a fine network of microfilaments but no stress fibers could be seen. Ruffles, both at the cell periphery and on the dorsal cell surface, stained intensely for F-actin. In contrast, myosin staining appeared in a relatively diffuse dotty pattern that diminished toward the cell periphery and was absent from the ruffled borders. Microtubules labeled in a pattern similar to that typically seen in a variety of mononuclear cells with microtubules radiating from the perinuclear region toward the cell periphery. In cells that responded to calcitonin by ceasing motile activity and retracting cytoplasm, microtubules were not detectably altered. Both myosin and actin labeling, however, changed dramatically, with retraction fibers labeled brightly for actin but not for myosin. Early in cytoplasmic retraction, myosin stained most intensely as a ring at the cell periphery at the base of retraction fibers. These data indicate that specific components of the cytoskeleton are altered in response to stimuli, such as calcitonin, that an actomyosin interaction may be involved in the early stages of cytoplasmic retraction, and that these components may play an active role in the molecular mechanisms by which cells respond to this hormone.