James E. Casanova, PhD
Role of vesicular transport processes in regulating cell motility and invasion
The Casanova laboratory uses the tools of cell biology, molecular biology and biochemistry to understand the mechanisms by which vesicular transport processes are coordinated with cell motility. A major focus of this research is on signaling through small GTPases of the ARF and Rho families. ARFs are best understood as regulators of vesicular transport, where they nucleate the assembly of carrier vesicles and participate in the sorting events that concentrate cargo proteins for delivery to specific cellular destinations. Rho family GTPases are well known regulators of actin cytoskeleton assembly. Recent findings in the laboratory have shown that significant crosstalk exists between these two classes of GTPases. Specifically, activation of one isoform of ARF, ARF6, leads to activation of a Rho family member, Rac1, whose function is necessary for cells to extend their membranes in the direction of movement. In collaboration with Dr. Kodi Ravichandran, it was discovered that ARF6 stimulates recruitment of a Rac1 activator, DOCK180, to the plasma membrane, and it is the presence of this ARF6/DOCK180/Rac1 complex that promotes cytosekeletal reorganization and membrane extension. Moreover, ARF6 stimulates the synthesis of specific phosphoinositide lipids (PI[4,5]P2) that are necessary cofactors for many of the proteins that control actin assembly. This project has made extensive use of the Biomolecular Research Facility for sequencing of mutant DNA constructs and oligonucleotide synthesis.
Each of the ARF and Rho family GTPases is activated by a family of proteins referred to as Guanine Nucleotide Exchange Factors (GEFs). Each ARF and each Rho isoform are regulated by more than one GEF, and the lab is working to understand which GEFs control GTPases action in specific cellular contexts. For example, it was discovered that a specific Arf6 GEF, BRAG2, controls ARF6 activation at the plasma membrane and regulates the internalization of cell surface adhesion molecules. When BRAG2 expression was reduced by RNAi technology, integrins accumulated on the surface of cells, leading to more robust attachment and migration. The laboratory is now working toward understanding the specific mechanisms whereby ARF6 controls integrin internalization, and whether this extends to other cell surface molecules as well. Much of this work was done in collaboration with the Parsons, Horwitz and Schwartz laboratories, and made extensive use of the Flow Cytometry Facility to measure surface levels of adhesion molecules. The overall goal of the research in this laboratory is to understand the mechanisms by which metastatic cells become motile and to identify regulatory molecules in this process that may be targeted for therapeutic intervention.