In vitro reconstitution of ARF-regulated cytoskeletal dynamics on Golgi membranes

Function of the secretory pathway is intimately connected to the cytoskeleton. Cytoskeletal dynamics and molecular motors are involved in organelle morphology and positioning, as well as the formation and translocation of trafficking intermediates such as vesicles. At least three classes of small GTPases, the ADP-ribosylation factor (ARF), Rho, and Rab families, have been implicated in regulating cytoskeleletal dynamics and molecular motor function within the secretory pathway. We have used the reconstitution of transport vesicle formation on isolated Golgi membranes to characterize mechanisms of ARF1 regulated actin polymerization. ARF1 affects cytoskeletal function in part by recruiting a complex between the vesicle-coat protein, coatomer, and the Rho-related GTPase, Cdc42, to the Golgi apparatus. Cdc42 can activate actin polymerization on Golgi membranes through an Arp2/3-dependent mechanism. Coatomer-bound Cdc42 plays a further role in regulating vesicle motility via the motor protein, dynein. Future studies elucidating the molecular mechanisms connecting vesicular transport with actin dynamics will provide important clues to the overall contribution of the cytoskeleton and molecular motors to protein transport. This article describes methods and reagents for characterizing cytoskeletal regulation at the Golgi apparatus through the cell-free reconstitution of vesicle formation.