Jill C. Wildonger - US grants

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.