Methods to Investigate the Role of Rho GTPases in Osteoclast Function.

The actin cytoskeleton is essential for the biology of osteoclasts, in particular during bone resorption. As key regulators of actin dynamics, the small GTPases of the Rho family are very important in the control of osteoclast activity. The study of Rho GTPase signaling pathways is essential to uncover the mechanisms of bone resorption and can have interesting applications for the treatment of osteolytic diseases. In this chapter, we describe various techniques to obtain primary osteoclasts from murine bone marrow cells, to measure Rho GTPase activation levels, to monitor bone resorption activity of osteoclasts and to introduce the expression of proteins of interest using a retroviral approach. We illustrate the different methods with experimental examples of the effect of Rac1 activation by the exchange factor Dock5 on bone resorption by osteoclasts.