Thesis advisor: Laura Anne Lowery / Thesis advisor: David Burgess / 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. / Thesis (PhD) — Boston College, 2020. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

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Purification, chemistry and application are three very important aspects of current research on carbon nanotubes (CNTs). In the dissertation, the purification of nitric acid treated single-walled carbon nanotubes (SWNTs), the dissolution and dichlorocarbene addition of SWNTs, and the effects of chemically functionalized CNTs on neuronal growth are discussed.The nitric acid treated SWNTs were purified by chemical treatment, cross-flow filtration, and centrifugation methods. The effects of nitric acid treatment on the SWNTs and the efficiency of different purification methods was evaluated by the measurement of purify of SWNTs via solution phase NIR. Nitric acid reflux followed with controlled pH centrifugation can produce SWNTs with high purity. This purification mechanism was explained by the relationship of the concentration of the acidic sites on SWNTs and the zeta potential of SWNTs.The dissolution of SWNTs was achieved via chemical functionalization of SWNTs with octadecylamine (ODA). Dichlorocarbene addition to the sidewall of both ODA functionalized and as-prepared SWNTs was investigated. ODA functionalized HiPco-SWNTs were found to have the highest functionality of dichlorocarbene. Vis-NIR spectra of the dichlorocarbene functionalized SWNTs showed a significant decrease in the interband transitions of the semiconducting SWNTs, which indicated that the chemical functionalization of the sidewall of SWNTs changes the electronic properties of SWNTs. Far-IR spectra of the dichlorocarbene functionalized SWNTs showed a dramatic decrease in the electronic transitions at the Fermi level of metallic SWNTs, which was opposite to the effect of ionic doping by bromine. This difference in the far-IR spectroscopy can be used to distinguish covalent chemical functionalization and ionic doping effects of SWNTs.Chemically functionalized multi-walled carbon nanotubes (MWNTs) were applied as substrates for neuronal growth. By manipulating the charge carried by functionalized MWNTs we are able to control the outgrowth and branching pattern of neuronal processes. Chemically functionalized water soluble SWNTs graft copolymers were used in the modulation of outgrowth of neuronal processes. The graft copolymers were prepared by the functionalization of SWNTs with poly-m-aminobenzene sulphonic acid and poly-ethylene glycol. These functionalized water soluble SWNTs were able to increase the length of selected neuronal processes after their addition to the culturing medium.

The debilitating effects of spinal cord injury can be attributed to a lack of regeneration in the central nervous system. Identification of growth-promoting pathways, particularly ones that can be controlled by small molecules, could provide significant advancements in regenerative science and lead to potential treatments for spinal cord injury. The biological investigations of neuroregenerative small molecules, specifically the natural products clovanemagnolol and vinaxanthone, have been expanded to a whole organism context using the nematode Caenorhabditis elegans (C. elegans) as a tool for these studies. A straightforward assay using C. elegans was developed to screen for compounds that promote neuronal outgrowth in vivo. This outgrowth assay was then used to guide the design of chemically edited analogs of clovanemagnolol that maintained biological activity while possessing structures amenable to further modification for mechanism of action studies. Pull-down experiments using affinity reagents synthesized from a neuroactive structural derivative, clovanebisphenol, and the C. elegans proteome combined with mass spectrometry-based protein identification and genetic recapitulation using mutant C. elegans identified the putative protein target of the small molecule as a kinesin light chain, KLC-1. Furthermore, the small molecule-promoted regeneration of injured neurons in vivo was studied using laser microsurgery to cut specific axons in C. elegans followed by treatment with a library of analogs of the growth-promoting natural product vinaxanthone. Enhanced axonal regeneration was observed following small molecule treatment and the results were used to determine the structure-activity relationship of vinaxanthone, which may guide future development of potential drug candidates for the treatment of spinal cord injury. / text