Microtubule Plus-End Tracking Protein and Polymerase, XMAP215, affects the Neuronal Microtubule and Actin Cytoskeletons to control Axon Outgrowth and Guidance Mechanisms

Abstract

While XMAP215 (CKAP5 / ch-TOG) has been best characterized for its microtubule polymerase function, recent studies have highlighted a novel role for XMAP215 in facilitating an interaction between microtubules and F-actin in the embryonic neuronal growth cone, a critical structure involved in neuronal outgrowth and guidance mechanisms. Microtubule and F-actin cytoskeletal cross talk and reorganization are important aspects of axonal guidance mechanisms, but how associated proteins facilitate this function largely remains a mystery. In addition, it has long been established that neuronal growth cone navigation depends on changes in microtubule (MT) and F-actin architecture downstream of guidance cues. However, the mechanisms by which MTs and F-actin are dually coordinated remain a fundamentally unresolved question. Here, I report that the well-characterized MT polymerase, XMAP215 (also known as ch-TOG / CKAP5), plays an important role in mediating MT–F-actin interactions within the growth cone. I demonstrate that XMAP215 regulates MT–F-actin alignment through its N-terminal TOG 1–5 domains. Additionally, I show that XMAP215 directly binds to F-actin in vitro and co-localizes with F-actin in the growth cone periphery. By working with lab colleagues, we also find that XMAP215 is required for regulation of growth cone morphology and response to the guidance cue, Ephrin A5. Our findings provide the first strong evidence that XMAP215 coordinates MT and F-actin interaction in vivo. It is here that I suggest a model in which XMAP215 regulates MT extension along F-actin bundles into the growth cone periphery and that these interactions may be important to control cytoskeletal dynamics downstream of guidance cues. Furthermore, I then go on to study this dual microtubule and F-actin role, diving deeper into the mechanism behind this novel ability of XMAP215. Here, I report that XMAP215 is capable of spatially localizing populations of microtubules into distinct domains in the growth cone through its less well-characterized microtubule-lattice binding activity. In addition, through the use of purified proteins and biochemical assays, I show that XMAP215 is capable of binding directly to F-actin, facilitated by its unique TOG5 domain. Finally, through biochemical means and super resolution imaging, I show that this novel function of XMAP215 is mediated by polymerase-incompetent mutants of XMAP215. Taken together, my findings show strong evidence of a non-microtubule-polymerase function of XMAP215, providing mechanistic insights into how microtubule populations can be guided by interaction with the F-actin cytoskeleton. In conclusion, I explore a novel and functionally important role for XMAP215 in facilitating interactions between microtubule and actin cytoskeletons, bridging the two structural components of the cell together. In this way, XMAP215 is now known as a distinct microtubule/F-actin regulator that governs microtubule exploration through the help of actin in neurons, in addition to its previously characterized function as a microtubule polymerase. While this thesis explores the very groundwork of XMAP215’s new novel function, there is still a great deal more to learn about the overall mechanism occurring, as well as an understanding of its role in various cell types.