Jill C. Wildonger - US grants

[unreadable] DESCRIPTION (provided by applicant): Project Summary: Normal human cognition relies on functional neuronal circuits that are assembled during development. Neuronal polarity and morphology are integral to forming proper neuronal connections, although the question of how neuronal polarity and morphology are established in vivo remains largely unanswered. Using the fruit fly as a model system, the aims of this proposal are to (1) conduct an in vivo forward genetic screen to uncover novel neuronal polarity and morphology regulators, (2) characterize a polarity mutant identified in this screen and (3) identify the mammalian homologs of fly neuronal polarity/morphology genes and analyze their function in cultured mammalian neurons. The genetic screen uses a GFP construct to visualize a class of multipolar neurons in living fly embryos and larvae, facilitating the screening process. The screen has uncovered a polarity mutant, which will be characterized by first identifying the gene corresponding to the mutation and then determining the mechanism by which it functions. Lastly, to better understand the conserved mechanisms regulating neuronal polarity and morphology, genes identified in the genetic screen will be analyzed in cultured mammalian neurons. Specifically, mammalian homologs of the fly mutants will be identified and their role in neuronal polarity/morphology will be assessed using cultured hippocampal neurons. In conclusion, the goal of this proposal is identify novel regulators of neuronal polarity and morphology and to characterize their function in both flies and cultured mammalian neurons. Therefore, the results of these studies will likely shed light on conserved mechanisms regulating neuronal polarity and morphology. Public Health Relevance: Multiple human developmental disorders, including lissencephaly and mental retardation, are associated with abnormal neuronal polarity and/or morphology. This goal of this proposal is understand how neuronal polarity and morphology are established in vivo, which will contribute to our understanding of how neural circuits assemble. Such knowledge can be applied to treating human disorders. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Shortly after differentiating, neurons establish distinct axonal and dendritic compartments that are specialized to send and receive signals, respectively. Polarity is essential for neurons to function in a neuronal circuit, yet how neurons polarize within a developing organism remains virtually unknown. My long-term goal as an independent biomedical researcher is to identify the mechanisms that create distinct axonal and dendritic compartments within neurons and to understand how this contributes to normal neuronal function in vivo. This proposal is based on our finding in fruit flies that the microtubule-based molecular motor dynein is necessary for two key features of neuronal polarity: the polarized localization of dendritic proteins and organelles and the uniform plus-end distal orientation of axonal microtubules. Two outstanding questions I will address in this proposal are: (1) How is dynein's function in neurons controlled by its interactions with different cofactors? and (2) How is dynein's activity regulated by its interaction with microtubules;more specifically, do microtubule modifications (such as acetylation, detyrosination, and polyglutamylation) provide spatial cues that influence dynein's activity and thereby shape neuronal polarity? The mentored phase of this award will be carried out at the University of California, San Francisco (UCSF), under the guidance of Dr. Yuh Nung Jan. During the mentored phase, I will use a genetic approach to characterize the cofactors that provide functional specificity to dynein developing fruit fly nervous system (Aim 1). Next, I will extend my studies in vitro and develop a new dynein motor construct to determine how dynein motor activity is affected by microtubule modifications (Aim 2). To do so, I will collaborate with Dr. Ronald Vale (UCSF) to learn in vitro techniques to analyze motor- microtubule interactions, including single molecule motility assays. To address how microtubule modifications affect neuronal polarization in vivo (Aim 3), I will use new knock-in technique called "genomic engineering" to build reagents for my independent phase. Dr. Yang Hong (University of Pittsburgh), who pioneered the genomic engineering technique, will serve as a consultant, as will Dr. Anthony Wynshaw-Boris (UCSF), a leader in the study of genes linked to human neurodevelopmental disorders such as classical lissencephaly. During the independent phase, I will address the following questions: Are microtubule modifications necessary for neurons to form distinct axonal and dendritic compartments in vivo? Is any one modification particularly important, or are there combinations of modifications that specify axon or dendrite formation? How do microtubule modifications regulate polarized transport in developing neurons in vivo? To answer these questions, I will use genomic engineering to knock-in multiple tubulin alleles with targeted mutations that block different microtubule modifications, both singly and in combination. Using currently available reagents and new polarity markers that I will generate, I will then characterize the effect of these mutations on neuronal polarity and dynein-mediated polarized transport within developing fruit fly nervous system. Through this combination of in vitro and in vivo approaches, these studies will provide significant new insight into microtubule-based mechanisms that shape neuronal polarity in a developing organism. PUBLIC HEALTH RELEVANCE: Project narrative: Neuronal polarity is essential for developing neurons to properly integrate into functional neuronal circuits. Loss of polarity during early stages of development has been associated with several human developmental disorders, including classical lissencephaly. By characterizing the microtubule-based mechanisms that govern neuronal polarization in vivo, this project will provide important new insight into how neurons normally polarize and how disrupting this process leads to human disease.

The microtubule cytoskeleton is essential to neuronal activity. Microtubules have an intrinsic polarity that motors read out to localize cargo, and differences in microtubule orientation between axons and dendrites are a defining feature of a polarized neuron (microtubule polarity is uniform in axons and mixed in dendrites). Despite the importance of microtubule organization to neuronal function, the mechanisms that create and maintain polarized microtubule arrays in axons and dendrites are poorly understood. While existing models focus largely on the motor-mediated translocation of microtubules into and out of neurites, there is now strong evidence from both vertebrates and invertebrates that local, non-centrosomal microtubule nucleation affects microtubule polarity in axons and dendrites, signaling the need for new models and a better understanding of local nucleation mechanisms. Our proposal addresses three fundamental, outstanding questions. How is nucleation machinery localized by molecular motors to specific compartments (Aim 1)? How is local ?-tubulin- mediated microtubule nucleation regulated to maintain the unique polarities of axonal and dendritic cytoskeletons, and how does local microtubule growth affect intracellular transport (Aim 2)? Using a fly model, we exploit cutting-edge genome engineering and live imaging approaches to dissect novel mechanisms of motor-based transport and local nucleation in vivo. There are two known platforms for ?-tubulin-mediated microtubule nucleation in neurons: Golgi outposts (dendrites only) and augmin (dendrites and axons). In Aim 1, we delineate a novel mechanism of polarized transport in which the coordinated and spatially regulated activities of kinesin-1 and dynein localize Golgi outposts to dendrites. In Aim 2, we determine how the localization of ?-tubulin to Golgi outposts or augmin (or novel nucleation centers) regulates microtubule polarity in axons and dendrites, and the effects of local microtubule growth on the transport of vesicles and organelles. To identify novel regulators of microtubule nucleation and microtubule polarity, we are leveraging our in vivo system in a forward genetic screen to gain new insights into the poorly understood mechanisms controlling local nucleation and microtubule polarity. Our studies will create a new mechanistic framework for understanding how polarized transport of Golgi outposts and local microtubule nucleation maintains neuronal polarity and supports intracellular trafficking. Multiple human disorders are associated with deficits in microtubule-based trafficking, and with mutations in kinesin-1 and dynein, and our investigations may shed light on the pathology of these diseases.

Microtubules are essential to virtually all aspects of neuronal activity, and post-translational modifications are thought to be key regulators of microtubule function. Microtubule acetylation is a post-translational modification that plays an important role in basic cellular activities, such as intracellular trafficking, that underlie normal neuronal function. However, fundamental questions remain: first, how do stable, but not dynamic, neuronal microtubules accumulate acetylation? Second, how does microtubule acetylation affect neuronal microtubule networks and neuronal structure? In this work, we will use biophysical and cellular experiments to elucidate whether the ?-tubulin lysine 40 acetyltransferase ?TAT1 preferentially acetylates stable microtubules, or whether ?TAT1 does not preferentially acetylate stable microtubules, but rather acts to select against dynamic microtubules. Then, we will develop a multi-scale, mechanistic computational model to integrate, interpret, and extend our experimental results. We will then leverage this framework to investigate previously uncharacterized ?-tubulin acetylation sites. These experiments and computer simulations will provide insights into how a post-translational modification that is enriched on neuronal microtubules affects neuronal morphogenesis, which has broad implications for a range of human disorders that are linked to dysfunction of the microtubule cytoskeleton, including Huntington's and Parkinson's diseases.

PROJECT SUMMARY/ABSTRACT Communication between neurons and their targets depends on proper synaptic growth and activity. The microtubule cytoskeleton plays a central role in synaptic terminal development, and microtubule dysfunction is associated with many neurological disorders. Neurons contain stable and dynamic microtubules, and these two populations must be properly balanced for synapses to grow and form stable connections. In this proposal, we use a synergistic combination of in vivo genetic analyses and cell-free in vitro biophysical approaches to elucidate the mechanisms by which microtubule dynamics and stability are balanced. We leverage a novel ?- tubulin mutant that alters the normal microtubule balance and perturbs synaptic growth. This tubulin mutation disrupts a highly conserved, essential ?-tubulin site that is acetylated. Post-translational modifications (PTMs), such as acetylation, have the potential to directly and specifically regulate microtubule stability and dynamics to shape synaptic morphogenesis, yet relatively few microtubule PTMs have been studied. Our preliminary data implicate this previously uncharacterized ?-tubulin site in regulating the addition of tubulin dimers to growing microtubule ends, which suggests a novel acetylation-based mechanism to control microtubule dynamics. Based on our preliminary findings, we will test the hypothesis that microtubule dynamics and stability are balanced by ?-tubulin acetylation and other known regulators to shape synaptic terminal morphogenesis (Aim 1). We will use the Drosophila neuromuscular junction as a model and investigate the effects of manipulating microtubule dynamics and stability on two different motor neuron types, called type Ib and type Is, whose synaptic terminals have distinct morphologies and transmission properties. Our preliminary data indicate that altering the microtubule cytoskeleton has strikingly different effects on the growth of type Ib and Is synaptic terminals. We will test the hypothesis that stable and dynamic microtubules are uniquely balanced in different neuron types to establish distinct neuron-specific synaptic structures and activities (Aim 2). Combined, our studies will reveal novel mechanisms that regulate synaptic microtubule networks and provide fundamental new insight into the central role that microtubules play in creating diverse synaptic morphologies and functions.