High molecular weight tropomyosins regulate osteoclast cytoskeletal morphology.

Tropomyosins are coiled-coil dimers that bind to the major groove of F-actin and regulate its accessibility to actin-modifying proteins. Although approximately 40 tropomyosin isoforms have been identified in mammals, they can broadly be classified into two groups based on protein size, that is, high molecular weight and low molecular weight isoforms. Osteoclasts, which undergo rounds of polarization and depolarization as they progress through the resorptive cycle, possess an unusual and highly dynamic actin cytoskeleton. To further define some of the actin regulatory proteins involved in osteoclast activity, we previously performed a survey of tropomyosin isoforms in resting and resorbing osteoclasts. Osteoclasts were found to express two closely related tropomyosins of the high molecular weight type, which are not expressed in monocytic and macrophage precursors. These isoforms, Tm-2 and Tm-3, are not strongly associated with actin-rich adhesion structures, but are instead distributed diffusely throughout the cell. In this study, we found that Tm-2/3 expression occurs late in osteoclastogenesis and continues to increase as cells mature. Knockdown of these isoforms via RNA interference results in flattening and increased spreading of osteoclasts, accompanied by diminished motility and altered resorptive capacity. In contrast, overexpression of Tm-2, but not Tm-3, caused morphological changes that include decreased spreading of the cells and induction of actin patches or stress fiber-like actin filaments, also with effects on motility and resorption. Suppression of Tm-2/3 or overexpression of Tm-2 resulted in altered distribution of gelsolin and microfilament barbed ends. These data suggest that high molecular weight tropomyosins are expressed in fusing osteoclasts to regulate the cytoskeletal scaffolding of these large cells, due at least in part by moderating accessibility of gelsolin to these microfilaments.