Formation of trans-synaptic complexes at the first visual synapse

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Vision begins in the retina with rod and cone photoreceptor cells. These specialized neurons detect light, then relay signals via synaptic transmission to other downstream neurons called bipolar cells. Proper formation and function of the synapses between photoreceptors and bipolar cells requires accumulation of multiple proteins on both sides of the synapse. The long-term goals of my research program are to understand how proteins are targeted to the photoreceptor-bipolar synapses, how they are functionally and structurally organized at the synapse, and how specificity of synapse formation is controlled. In this proposal, we will focus on trans-synaptic complexes - direct interactions between proteins that form complexes spanning the synaptic cleft. We will take a multipronged approach to identify requirements for synaptic localization and interaction with trans-synaptic partners. In Objective 1, we will examine sequences required for synaptic localization of proteins in both photoreceptors and bipolar cells. For screening of mutants, we will electroporate neonatal mouse retinas with plasmid DNA; this allows cell-type specific expression of tagged proteins in the native environment of the retina tissue. We will test the same mutants for interact with trans-synaptic partners using in vitro pull-down assays. This will help us understand whether trans-synaptic interactions play a role in accumulation of proteins at the synapse. Glycosylations are modifications of protein extracellular domains with sugar molecules, whose roles are still mostly unknown. In Objective 2, we will test the role of glycosylations in synaptic localization and trans-synaptic interactions. We will take two approaches: treatment of proteins with enzymes that remove glycosylations, and electroporation of constructs with mutations that prevent glycosylation. Many synapses contain scaffolding proteins that organize other proteins by binding to repeating structures called PDZ domains. Although photoreceptor-bipolar synapses contain several proteins with PDZ binding motifs, scaffolding protein interactions are still poorly characterized. In objective 3, we will test whether PDZ domain interactions are required for synaptic localization by electroporating constructs whose PDZ binding motifs have been mutated. We will also perform pull-down experiments to test binding to known PDZ-containing proteins at the synapse. My research program addresses gaps in knowledge about synaptic targeting in both photoreceptors and bipolar cells, and assembly of the synapses by which they communicate. Understanding these retinal circuits at the molecular level is crucial for understanding vision. The information gained from these studies will provide new detailed information about the structure and function of proteins at the synapse, and will inform future investigations aimed at understanding synapse development.