| Organelle transport in eukaryotic cells is regulated by a precisely coordinated activity of phosphoinositide lipids, small GTPases, and molecular motors. Despite the extensive study of functional activities of individual regulators, how these activities promote precise deliveries of particular membrane proteins to specific cellular locations remained unclear. Ankyrin-B, which is previously well recognized as a plasma membrane adaptor that assembles diverse specialized plasma membrane domains, exhibited an unexpected role in intracellular transport. This thesis establishes ankyrin-B as a master integrator of the polarized long range organelle transport via direct interactions with Rab GTPase Activating Protein 1 Like (RabGAP1L), phosphatidylinositol 3-phosphate (PI3P) and dynactin 4. In Chapter 2, I identified an ankyrin-B death domain binding partner, RabGAP1L, that specifically interacts with ankyrin-B on intracellular organelles and requires ankyrin-B for its proper localization. In Chapter 3, I demonstrated that ankyrin-B is a PI3P-effector in mouse embryonic fibroblasts (MEFs) and promotes the polarized transport of associated organelles in migrating cells in a RabGAP1L-dependent manner. I continued to investigate what membranes/membrane-associated proteins utilize the ankyrin-B/RabGAP1L pathway in Chapter 4 and identified alpha5beta1-integrin as a cargo whose polarized transport and recycling are ankyrin-B-dependent. I further presented that ankyrin-B, through recruiting RabGAP1L to PI3P-positive/Rab22A-associated endosomes containing alpha5beta1-integrin, promotes polarized recycling of alpha5beta1-integrin in migrating mouse embryonic fibroblasts. In collaboration with James Bear (UNC Chapel Hill), we further demonstrated that this ankyrin-B/RabGAP1L-mediated pathway is required for haptotaxis along fibronectin gradients. In Chapter 5, I elucidated the in vivo interaction between ankyrin-B and RabGAP1L. I demonstrated that ankyrin-B specifically interacts with RabGAP1L at long axon tracts in the brain and at costameres in the skeletal muscle. I also show the phenotypic analysis of ankyrin-B floxDD mice as an initial attempt to determine the physiological function of the ankyrin-B death domain in vivo. Together, this thesis dissects an ankyrin-B-mediated molecular mechanism for polarized endosomal trafficking and alpha5beta1-integrin recycling during directional cell migration, and provides new insights into how phosphoinositide lipids, Rab GTPases, and molecular motor activities are coordinated to control the directional transport of specialized membrane cargos. |
