Formation of trans-synaptic complexes at the first visual synapse

视觉开始于视网膜的杆状和锥状感光细胞。这些特殊的神经元检测光线，然后通过突触传递信号给其他下游的神经元，即双极细胞。光感受器和双极细胞之间的突触的正常形成和功能需要突触两侧多种蛋白质的积累。我的研究计划的长期目标是了解蛋白质如何靶向光感受器双极突触，它们如何在突触上进行功能和结构组织，以及如何控制突触形成的特异性。在这个提议中，我们将重点关注跨突触复合物-形成跨越突触间隙复合物的蛋白质之间的直接相互作用。我们将采取多管齐下的方法来确定突触定位和与跨突触伙伴相互作用的需求。在目标1中，我们将检查光感受器和双极细胞中蛋白质突触定位所需的序列。为了筛选突变体，我们将用质粒DNA电穿孔新生小鼠视网膜；这允许在视网膜组织的自然环境中标记蛋白的细胞类型特异性表达。我们将使用体外下拉法测试相同的突变体与跨突触伙伴的相互作用。这将有助于我们了解跨突触相互作用是否在突触蛋白质积累中发挥作用。糖基化是糖分子修饰蛋白质胞外结构域的过程，其作用目前尚不清楚。在目标2中，我们将测试糖基化在突触定位和跨突触相互作用中的作用。我们将采取两种方法：用去除糖基化的酶处理蛋白质，以及用防止糖基化的突变对结构体进行电穿孔。许多突触含有支架蛋白，通过结合被称为PDZ结构域的重复结构来组织其他蛋白质。尽管光感受器-双极突触包含几种具有PDZ结合基序的蛋白质，但脚手架蛋白相互作用的特征仍然很差。在目标3中，我们将通过PDZ结合基序发生突变的电穿孔结构体来测试PDZ结构域的相互作用是否需要突触定位。我们还将进行下拉实验，以测试与突触中已知含有pdz的蛋白质的结合。我的研究计划解决了关于光感受器和双极细胞的突触靶向知识的空白，以及它们通过突触进行交流的突触组装。在分子水平上理解这些视网膜回路对于理解视觉至关重要。从这些研究中获得的信息将为突触蛋白质的结构和功能提供新的详细信息，并将为未来旨在了解突触发育的研究提供信息。

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.