Monitoring RhoGTPase activity in lymphocytes

L-selectin is a cell adhesion molecule (CAM) that is essential for the tethering and subsequent rolling of naïve lymphocytes along the luminal wall of postcapillary venules entering lymph nodes. As with many CAMs, L-selectin has the capacity to transduce intracellular signals in response to ligand binding. This implicates CAMs involved in tethering and rolling as contributors to intracellular signals that lead to the transition from rolling to arrest. In addition, studies in L: -selectin-null mice have also revealed a role for L: -selectin in chemokine-directed cell migration of leucocytes in tissues. The Ras homology (Rho) family of small GTPases are intracellular proteins that respond to signals received from the surrounding environment. The RhoGTPases typically activate downstream targets involved in the remodelling of the actin cytoskeleton, which is essential for continued progression through the multi-step adhesion cascade. This chapter will focus on how to prepare, perform and monitor RhoGTPase activation assays in response to L: -selectin stimulation. Although this section focuses on L: -selectin stimulation, the methods outlined here can be applied to analysing RhoGTPase activity in response to stimulating other receptors involved in tethering/rolling such as CD44, P-selectin glycoprotein ligand-1 and E-selectin ligand-1.