Endosomal membrane dynamics underlying cell spreading

Cell migration is an orchestrated and highly coordinated multi-step process that is central to the development and maintenance of multicellular organisms. Dysregulated migration however, is associated with pathological states such as tumor formation and metastasis; thus a clear understanding of the molecular mechanisms that drive this process is critical to the development of counteracting therapeutics. Cell migration and adhesion-dependent cell spreading share a number of features. For example, both processes rely on the activation of mechanisms for the coordinated spatial and temporal assembly/disassembly of focal adhesions, as well as mechanisms controlling actin rearrangements and directed vesicular trafficking. Actin remodeling and vesicular trafficking events are in turn, implicated functions of a variety of small GTPases of the Ras superfamily, which include the Rho and Arf subfamilies. Thus towards efforts of further characterizing the molecular pathways that drive cell spreading, I pursued aims to examine the role of a specific member of the Arf subfamily Arf6, in this process. In contrast to other studies which have primarily used constitutively active or dominant negative mutants of Arf6 to study its cellular function, we employed mouse genetics. In this system, mouse embryonic fibroblasts (MEFs) were derived and immortalized from mice genetically manipulated for the acute deletion of Arf6 using a tamoxifen inducible Cre/loxP recombination system. Acute deletion of Arf6 in these MEFs resulted in a kinetic delay in transferrin recycling as well as in cell spreading. The spreading delay correlated with reduced trafficking of cholera toxin B-labeled intracellular membranes to the plasma membrane. Cholera toxin-B labels the ganglioside GM1, which is enriched in lipid rafts. These specialized membrane domains are thought to serve as signaling hubs bearing many proteins that in turn, mediate trafficking steps required for cell spreading/migration. I further report that the trafficking of these specialized membranes to the plasma membrane involves the retromer complex, a coat-like multi-protein complex primarily known for mediating retrograde transport from endosomes to the trans-Golgi network. Altogether, my studies have confirmed genetically, an involvement of Arf6 in cell spreading and raft trafficking, and established a link between these membrane microdomains and the retromer complex. In separate studies, I have also investigated the role of phospholipase D2 (PLD2) in endocytic trafficking and found that similarly derived cultures exhibit alterations in the expression levels of various trafficking related proteins as well as defects in transferrin and epidermal growth factor receptor trafficking. These results suggest a role for PLD2 and possibly its enzymatic product phosphatidic acid, in these events.