Search

Osteoclasts perform bone resorption vital to the maintaining of healthy bone and blood calcium levels. Osteoclasts function by forming unique actin attachment structures for migration (podosomes) and bone resorption (actin rings). For these studies, the role of two actin binding protein classes, tropomyosins and myosins, are studied to reveal their function in osteoclast differentiation and podosome/actin ring dynamics.Our research initially demonstrated the presence of nine tropomyosin isoforms in distinct locations in osteoclasts. Tm4 co-localized with the interior faces of actin rings and podosomes. RNAi-mediated suppression led to reductions in actin ring thickness, bone resorption, and motility. Overexpression of exogenous Tm4 resulted in abnormal, thicker podosomes that were unusually distributed along with reduced cell motility and abolished bone resorption. We hypothesize that the main role of Tm4 is regulation of the adhesion structures of osteoclasts by stabilizing filamentous actin in podosomes and actin rings and thus affecting migration and bone resorption.Myosins are a class of molecular motors that provide the ATP-dependent force to generate movement such as vesicle transport, cell migration, and cell division. Our previous studies showed MyoIIA to be distributed within podosomes and the actin ring of polarized osteoclasts. During differentiation, MyoIIA protein levels temporarily diminished corresponding to fusion initiation. Cathepsin B regulated the temporary protein decrease. RNAi of MyoIIA was used during the latter stages of osteoclastogenesis. This suppression generated very large, less motile osteoclasts that were a result of increased fusion (elevated numbers of nuclei per cell) and cell spreading (perimeter per nuclear number). While the large cells, which formed large actin rings, were capable of resorbing bone, their diminished motility resulted in minimal changes in resorption. These data suggest MyoIIA plays an inhibitory role during osteoclastogenesis to limit the extent of precursor fusion and to promote osteoclast motility.In mature osteoclasts, podosomes are arranged in belt-like formations at the cell periphery from a microtubule-dependent process. The multifaceted Myo10 has been shown to bind multiple podosome regulatory proteins including microtubules. Initial analysis showed Myo10 localized immediately adjacent to podosome belts, suggesting a potential role in podosome positioning. RNAi suppression of Myo10 resulted in diminished spreading of osteoclasts and a microtubule-based podosome patterning defect. Dominant negative overexpression of the MyTH4 (microtubule binding) tail domain similarly inhibited podosome positioning. Overexpression of full-length Myo10 led to increased podosome belt formation in both osteoclasts and macrophages. Analysis of osteoclasts during podosome positioning shows Myo10 localized between podosomes and the surrounding microtubule system. These results suggest Myo10 links the microtubule network to podosomes through its MyTH4 tail domain.Together Tm4, MyoIIA, and Myo10 seem to be playing various roles in regulating osteoclast attachment and function.