High molecular weight tropomyosins regulate osteoclast cytoskeletal morphology

Abstract

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.

Tropomyosin – master regulator of actin filament function in the cytoskeleton

“…Tpm1.6 and Tpm3.1 display diffuse localization throughout the interior of the cell, whereas Tpm4.2, Tpm1.8 and Tpm1.9 localize to actin-rich adhesion structures, such as the podosomes and the sealing zone (McMichael et al, 2006). RNA interference studies further demonstrated that Tpm1.6 contributes to osteoclast morphogenesis and motility, whereas Tpm4.2 is involved in assembly and/or maintenance of adhesion structures Kotadiya et al, 2008 Fig. 3.…”

Regulated Proteolysis of Nonmuscle Myosin IIA Stimulates Osteoclast Fusion

“…Mature osteoclasts, as evidenced by a peripheral belt of podosomes (35), were present by days 5-7 of culture. Resorption and motility assays were performed as previously described (34,36).…”

“…Cells were fixed and permeabilized, as previously described (28,33,34,36). Primary antibodies were added in a standard polyethylene glycol blocking buffer and were detected using Alexacoupled secondary antibodies (Invitrogen).…”

“…RAW264.7 cells were transfected with Lipofectamine 2000 (Invitrogen), and marrow-derived osteoclasts were transfected via electroporation, as previously described. Osteoclasts were transfected by these methods with Ͼ95% efficiency (34,36). In other experiments, a human myosin IIA cDNA in the pEGFP-C3 vector (40) or the empty vector (Clontech) was transiently transfected into RAW264.7 cells on day 3 of RANKL treatment and assayed for cell perimeter and nuclear number on day 5.…”

Tropomyosin isoforms and reagents

“…16,21,[77][78][79][80] In addition, overexpression studies complemented with targeted gene siRNA knockdown experiments have demonstrated the importance of specific Tm isoforms in regulating the structure and dynamic properties of focal adhesions. 76,81 Finally, recent siRNA knockdown experiments have played an important role in establishing the nonredundant roles of Tm isoforms in stress fiber assembly.…”