Our long-standing goal is to understand the molecular events that underlie homing of cells during the process of inflammation and its subsequent resolution. These events require that key participating cells migrate to areas of inflammation. At the molecular level, at least three major intracellular events must be coordinated for cell migration to occur. First, specific cell surface proteins must undergo repeated cycles of binding and release from their sites of attachment for regulated cell adhesion. Second, adhesion molecules are internalized at the retracting edges to be recycled to the leading edges through endocytic transport pathways, so that directional movement occurs. Third, actin filaments that provide a cellular cytoskeleton must re-organize to form membrane protrusions during cell migration. These three major cellular events are coordinated by crosstalk among mechanistic pathways regulated by small GTPases, in order to accomplish the complicated task of cell migration. The family of small GTPases named ADP-ribosylation factor (ARF) regulates intracellular transport pathways. Recently, ARF6 has been shown to regulate endocytosis and actin cytoskeletal organization. Significantly, these two cellular events are also regulated by the Rho family of small GTPases that plays a critical regulatory role in the formation of membrane protrusions. These findings provide the basis for this grant proposal. First, we have recently identified a novel coat protein complex that is predicted to mediate the ability of ARF6 to regulate the recycling pathway of endocytosis. Thus, we propose to purify this novel coat protein complex, in order to understand ultimately how it mediates the ability of ARF6 to regulate endocytosis. Second, as the Rho family of GTPases has been shown also to regulate endocytosis, we will determine whether their regulation is mediated by the novel coat protein complex that we have identified. The result of these studies will likely provide a better molecular understanding of how membrane protrusions are formed during cell migration. This understanding will have wide-spread applications, as cell migration is required for the many different types of cells that participate in initiating and resolving areas of inflammation.
