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1 Role of the Intermediate Filament Protein Nestin in Modulating 1 Role of the Intermediate Filament Protein Nestin in Modulating Growth Cone Morphology and Behavior Christopher Joseph Bott Rock Hill, South Carolina B.S., Clemson University, 2009 A Dissertation presented to the Graduate Faculty of the University of Virginia in Candidacy for the Degree of Doctor of Philosophy Department of Cell Biology University of Virginia November, 2019 2 Abstract Axonal patterning in the neocortex requires that responses to an individual guidance cue vary among neurons in the same location, and within the same neuron over time, to establish the varied and diverse sets of connections seen in the developed brain. The growth cone is the structure at the tip of a growing axon that interprets these guidance cues and translates them into morphological rearrangements. Different brain regions often reuse the same individual guidance cue molecules. Consequently, the sensitivity or type of response the growth cone has to various guidance cues varies over the course of maturation of the neuron, as the growth cone weaves its way into and out of different brain regions in order to reach its target. In addition there are different responses between different parts of the same neuron: the dendritic growth cones respond very differently compared to axonal growth cones to the same guidance cue using the same basic cytoskeletal proteins. How a growth cone responds to a guidance cue is thus a complex cell biological question and the molecular mechanism are not well understood. I propose that the atypical intermediate filament protein nestin, in its role as a kinase modulator, functions in this manner to change responsiveness to the same guidance cue in time and space. Nestin is expressed highly in neural progenitors and is thus used widely as a progenitor marker. In my work, I discovered a subpopulation of mouse embryonic cortical neurons which transiently express nestin in their axons, but not in their dendrites. Since the prevailing view contends that intermediate filaments are not present in growth cones, intermediate filaments are understudied in developing axons, with very few studies looking into the role of intermediate filaments in axon guidance. Many studies, on the other hand, investigate the role of actin and microtubule dynamics in growth cones. I used high resolution imaging to demonstrate that nestin containing filaments are present in the distal axon and the growth cone and even extend into peripheral regions of the growth cone including filopodia. Nestin expression is thus not restricted to neural progenitors but persists for 2-3 days at lower levels in newborn neurons. Since protein expression level in progenitors is far greater than in axons, without careful and technically challenging analysis axonal nestin may be easily (although incorrectly) dismissed as background staining- particularly in vivo. This early period of neuronal maturation, in which nestin protein can be detected, is a critical period that establishes the orientation of the axon and its future trajectory. I initially found that nestin-expressing neurons have smaller growth cones, suggesting that nestin affects cytoskeletal dynamics. Nestin, unlike other intermediate filament subtypes, regulates Cdk5 kinase via binding the Cdk5 activator p35. Cdk5 is a critical kinase that operates during early neuronal development, and is part of the downstream signaling machinery of the repulsive axon guidance cue, Sema3a. Sema3a is important for initial axon positioning and organization within the intermediate zone of the developing cortex. I find that nestin selectively facilitates the phosphorylation of the lissencephaly-linked protein doublecortin (DCX) by Cdk5/p35, but surprisingly the phosphorylation of other Cdk5 substrates is not affected by nestin. DCX is a microtubule binding protein, which stabilizes microtubules. Phosphorylation of DCX by Cdk5 decreases microtubule binding and thus induces microtubule instability. DCX and DCX phosphorylation have previously been shown to be important for axon guidance. I uncover that the Cdk5 substrate selectivity imparted by nestin is based on the ability of 3 nestin to specifically interact with DCX, but not with other Cdk5 substrates. In addition, I find that other intermediate filaments do not interact with DCX. Nestin thus creates a selective scaffold for DCX with activated Cdk5/p35 to facilitate robust phosphorylation. Neurons that express nestin are more sensitive to the repulsive effects of Sema3a, and nestin siRNA reduces growth cone sensitivity to Sema3a. A nestin mutant that does not interact with DCX or p35 and does not affect DCX phosphorylation, does not have the same effects on growth cone morphology as WT nestin. Lastly, I use cortical cultures derived from DCX knockout mice to show that the effects of nestin on growth cone morphology and on Sema3a sensitivity are DCX-dependent, thus suggesting a functional role for the specific DCX-nestin complex in neurons. DCX null neurons are more sensitive to Sema3a in a nestin-independent manner. This observation suggests that DCX is operating as a brake or buffer to resist the cytoskeleton destabilizing effects induced by Sema3a. Nestin-enhanced Cdk5 phosphorylation of DCX removes the DCX brake on microtubules and thus DCX dissociates from microtubules in WT neurons. I propose that nestin changes growth cone behavior by regulating subsets of substrates that are phosphorylated by intracellular kinase signaling downstream of guidance cues in developing neurons. Thus, the transient expression of nestin could allow for temporal and/or spatial modulation of a neuron's response to Sema3a, particularly during early axon guidance. Temporally, at 2-3 days, when nestin expression can no longer be detected in vitro, is the same time period in vivo in which cortico-cortical axons are growing into a Sema3a rich region- and these are insensitive to the repulsive effects of Sema3a. This is consistent with the loss of nestin during that time period. Spatially, nestin’s absence from dendrites may play a role in the lack of sensitivity of dendrites to the repulsive effects of Sema3a. This is one example of how generalized signaling pathways can have variable signaling outcomes depending on which signaling modifying proteins are present. For example, the absence or presence of signaling modifying proteins, such as nestin, which can alter the subsets of substrates that are phosphorylated, may provide a mechanism how Cdk5 can be involved in both the attractive response of dendrites to Sema3a, and the repulsive response of axons to the same guidance molecule. This raises the question if other intermediate filaments may be playing similar roles. The exquisite developmental and cell type variability of intermediate filament expression could in principle provide unique signaling chemistries depending on which intermediate filaments are expressed. Intermediate filaments may thus have functions beyond mechanically providing a structure or skeleton to cells, but modify signaling networks. 4 Acknowledgments I would first like to thank my advisor Bettina Winkler. Her enthusiasm inspired me, and her pragmatism kept me grounded. Bettina has provided me with an ideal amount of support, independence, and at times, patience. I truly appreciate her permitting me to pursue ideas and concepts wherever they led, and never bounding me by the labs expertise. I will always be grateful to her for allowing me to pursue this degree in her lab. I want to thank all the members of the Winckler lab for so much help along the way. First of all, Chan Choo is one of the most knowledgeable scientists that I have ever worked with, and I am thankful that she was able to remind and inform me of the limitations of our techniques, as well as best practices. I want to thank my fellow graduate student lab mate Kelly Barford, who started in this lab with me and finished her degree well ahead of me, and in doing so provided me inspiration to keep going. Laura Digilio was just so very helpful with so many day to day tasks, and helped me keep my mouse colony down to reasonable numbers. Lloyd McMahon was a fantastic bench mate, whose knowledge of the finer points of the chemistries of our techniques provided frequent and often heated conversation. I think we both helped each other optimize our experimental techniques. Finally, I want to thank our undergraduates in the lab, Meheret Kinfe and Isabelle Witteveen, for their help in processing data, and more importantly, providing a fresh dose of enthusiasm in the lab. I am thankful to the members of my committee, Noelle Dwyer, Xiaowei Lu, Doug DeSimone, and the frequently added member Judy White. Other members of the cell biology department that both inspired, encouraged, or helped me is various ways are David Castle, Jim Casanova, and Mary Hall. Finally I want to thank my encouraging family and supportive Wife. My parents have always been an inspiration for doing hard work and the importance of completing a task. Their support gave me the confidence to pursue the goals that I was passionate about, even though it was not always a linear path. My sister provided me much needed encouragement along the way. And finally, I want to thank my wife Emily, who has faced the challenges of us both pursuing degree’s in higher education, and I think it has strengthened our bond. I have been truly inspired by her accomplishments, and I could not have done this without her. 5 Table of Contents Abstract Acknowledgements Table of Contents List of Figures Chapter 1: Introduction intermediate filaments, the Doublecortin, and the growth cone 1.1 A brief introduction to intermediate filaments 1.1.1 IF proteins possess conserved structural features which underlie their ability to assemble into filaments. 1.1.2 IF proteins are encoded by a large multi-gene family subdivided into assembly classes. 1.1.3 IF proteins are expressed in a highly cell-lineage restricted manner. 1.1.4 Human diseases are linked to IFs.
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