Dynamic regulation of the actin cytoskeleton is required for synapses to form and maintain their shape. The actin cytoskeleton is regulated by Rho GTPases in response to genetic and extracellular signals. Rho GTPases are regulated by guanine nucleotide exchange factors and GTPase activating proteins (GAPs). Syd-1 is a protein that has been identified as necessary for synapse formation in worms, with similar proteins in flies, and mice. Little is known about the molecular mechanism by which Syd-1 is acting. While genetic techniques are great tools for examining synapse development, they are limited by their inability to consider the molecular nature of the protein product. By studying the biochemical nature of synaptic proteins, we can begin to understand their function with a new level of clarity. Syd-1 has a predicted Rho GAP domain; however it is thought to be inactive. The activity of the fly protein, Dsyd-1, has never been examined although it has been speculated that it is inactive in all invertebrates. Recently the mouse version was reported to have Rho GAP activity. By performing GTPase activity assays on purified proteins, I found the GAP domain of Dsyd-1 increased the GTPase activity of Rac-1 and Cdc42 but not RhoA. Members of our lab found the activity of Dsyd-1 is necessary for proper synapse formation both at the Drosophila neuromuscular junction as well as in R7 neurons. In Caenorhabditis elegans, Syd-1 was found to interact with presynaptic protein RSY-1. Since RSY-1 is evolutionarily conserved, I tested whether or not RSY-1 has a similar effect on R neurons in Drosophila. I also isolated mRNA from R neurons and evaluated the possibility of analyzing mutant neurons using comparative transcriptomics. This dissertation includes previously unpublished coauthored material.

Synapse development is a stepwise process that requires the recruitment of key synaptic components to active zones, followed by continual maintenance of these structures to maintain connectivity and stability throughout the life of the organisms. Early synapse development requires the recruitment of early scaffolding proteins to establish stable connectivity as well as provide sites of recruitment of other vital synaptic proteins. One of the earliest proteins to be localized to the synapse is the conserved protein Syd-1. Syd-1 proteins contain a Rho GTPase activating protein (GAP)-like domain of unclear significance. Here I show that Drosophila Syd-1 interacts with all six fly Rhos and has GAP activity towards RAC1. I then show that lacks GAP activity localizes normally to presynaptic sites and is sufficient to recruit Nrx-1 but fails to cluster Brp normally and genetically interacts with RAC1 in vivo. I conclude that contrary to previous models, the GAP domain of fly Syd-1 is active and required for presynaptic development. Additionally, I’ve identified a previously uncharacterized protein, Vezl, as being critical for retrograde axonal transport and synaptic maintenance. I found that Vezl required for normal neuronal growth and that vezl loss resulted in decreased neuron size and the formation of swollen neuronal terminals that accumulated membrane markers and axonal transport cargo. I found that vezl mutants specifically retrograde transport of cargo and particularly affected signaling endosomes. The signaling endosomes were unable to initiate retrograde transport in vezl mutants and remained stuck within the distal boutons unable to relay their signaling peptides back to the nucleus. I conclude that Vezl is serving a role in attaching retrograde cargo to dynein and the microtubules specifically at neuron tips so that they can undergo retrograde axonal transport. This dissertation includes previously published and unpublished co-authored material. / 2020-09-06

Neurons in the central nervous system are typically not replaced and must therefore maintain their choice of fate and their synaptic connections throughout the life of an organism. I have used Drosophila genetics to analyze genes that prevent neurons from switching fates and allow them to form synapses onto target neurons. The Drosophila fly eye is composed of approximately 750 ommatidia, each comprising eight photoreceptor neurons (R1-R8) surrounded by non-neuronal accessory cells. These photoreceptor neurons undergo a well-defined developmental specification process and form synapses at defined locations in the brain. I have taken advantage of this system to investigate two questions: 1) how do neurons maintain their fate after specification? and 2) how do neurons form stable synapses? For the first half of my dissertation, I have focused my research on a gene, Sce, that I have shown is essential to prevent R7 neurons from undergoing a late switch in cell fate. Sce is an integral component of the Polycomb Group (PcG) complex that is essential for maintaining repression of multiple genes throughout the genome. I found that PcGs are required to prevent R7s from derepression of the R8-specific transcription factor Senseless. For the second half of my dissertation, I focused on the gene syd-1 that was identified to be required for proper presynaptic formation of R7 neurons. Previous studies in Caenorhabditis elegans suggested that Syd-1 acts upstream of Liprin-α and that Liprin-α promotes presynaptic development by binding the kinesin Kif1a to promote axon transport. I used live image analysis to show that, unlike Liprin-α, Syd-1 is not necessary to promote axon transport. Instead, we show that in R7s, Syd-1 acts upstream of Trio, and our results suggest that Syd-1's function is to promote Trio activity. This dissertation includes both my previously published and co-authored materials. / 10000-01-01