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Building a presynaptic specialization [electronic resource] : from neutral activity to endocytosis

The nervous system is comprised of a complex network of neurons that are connected by specialized structures called synapses. Each synapse contains a myriad of proteins that fulfill different functions, ranging from the release and reception of neurotransmitters to the maintenance and strengthening of the signals between neurons. Given the multitude of proteins present at the synapse, one question is how do they arrive and remain there? In my thesis, I use Caenorhabditis elegans to explore the cellular processes that contribute to the proper localization of important presynaptic proteins. In the first part of my thesis, I explored how presynaptic proteins are properly localized to the signal-sending process, called the axon, and excluded from the signalreceiving process, called the dendrite. In the motor neuron DA9, synaptic vesicles localize in a stereotyped region of the axon, but in cdk-5 mutants, 40% of the vesicle material is mislocalized to the dendrite. Chan-Yen Ou, a postdoctoral fellow in the lab, isolated a mutant that suppressed cdk-5, suggesting that the gene acts downstream or parallel to cdk-5. I mapped this mutant to the unc-101 locus, which encodes the [Mu]-subunit of the AP1 complex. AP complexes are players in clathrin-mediated endocytosis, and the [Mu]-subunit is the cargo recognition molecule within the complex. The AP1 complex plays a well-established role at the trans-golgi network in the cell body, but we present three results that suggest UNC-101 also acts at presynapses. The first result is the strong localization of UNC-101 at the synapse. The second result is that disrupting synaptic vesicle endocytosis (SVE) using genetic mutations causes a v similar phenotype as unc-101 mutations; animals mutant for unc-57/endophilin, unc- 26/synaptojanin, or dyn-1/dynamin 1 also suppress the cdk-5 dendritic phenotype. The third result is that the transport of synaptic vesicles from the synaptic region towards the dendrite decreases in an unc-101; cdk-5 double mutant compared to the cdk-5 single mutant, suggesting that UNC-101 is preventing retrograde flow from the synapses. While these results suggest a synaptic role for UNC-101, they do not exclude the possibility that UNC-101 also acts at the cell body. Indeed, I also show that UNC-101 affects the localization of postsynaptic proteins, which may occur by sorting proteins at the cell body. Additionally, postsynaptic proteins are unaffected by unc-57, suggesting an SVE-independent role for unc-101. Thus, I provide evidence that the AP1 subunit UNC-101 acts at presynapses and contributes to the molecular polarity of the DA9 motor neuron. The second part of my thesis contains my findings regarding a new system that I established to study synapse formation: the AFD thermosensory neuron. I found that the synaptic pattern in AFD is highly stereotyped, and I also isolated a mutant from a forward genetic screen that I mapped to the tax-4 locus. tax-4 and tax-2 encode two subunits of a cyclic nucleotide-gated channel that is necessary for sensory activity in AFD. When the genes are mutated, the localization of multiple presynaptic proteins is disrupted. Interestingly, they are not all similarly affected. Clusters of synaptic vesicles and the active zone protein SYD-2/liprin-[Alpha] are dimmer and more numerous in tax-4 and tax-2 than wild-type animals. While SAD-1/SAD kinase clusters are also dimmer, there are fewer in tax-4 and tax-2 than wild-type animals. These results suggest that sensory activity can have different effects depending on the presynaptic vi protein. Thus, for the second part of my thesis, I describe the establishment of a new system to study synapse development, the results of a screen, and a link between neural activity and the localization of presynaptic proteins.