Rho GTPases and cell migration: Measurement of macrophage chemotaxis

Abstract

Publisher Summary The chapter discusses Rho guanosine 5'-triphosphates (GTPases) and cell migration as a measurement of macrophage chemotaxis. The Boyden chamber is the most widely used method for assaying chemotaxis and is based on scoring cells that have migrated into or through a filter membrane toward a source of putative chemotactic factor. A combination of direct cell observation and rigorous statistical analysis makes the use of the Dunn chamber that is ideal in cases where small numbers of cells are examined for their migratory phenotype following experimental manipulation. To determine the way in which Rho, Rac, and Cdc42 regulate cell migration and chemotaxis, the chapter presents a mouse macrophage cell line, BAC1.2F5, that resembles primary macrophages in being dependent on colony stimulating factor-1 (CSF-1) for survival and proliferation, as well as exhibiting many of the markers of normal activated macrophages. The most significant finding of the study presented in the chapter is that Cdc42 is required for BAC1.2F5 cell chemotaxis, but not for migration. Constitutively active (V12Cdc42) protein stimulates the production of filopodia around the cell margin, which correlates with a failure of the cells to polarize. Cells microinjected with the dominant inhibitory mutant of Cdc42 (N17Cdc42) showed a significant increase in migration speed when compared with control microinjected cells (33 versus 15μm/hr, respectively). Cdc42 is therefore not required for migration and actually exerts a restraint on the speed of locomotion, but it is essential for detection of the cytokine gradient.