Formation of trans-synaptic complexes at the first visual synapse

视觉始于视网膜的视杆细胞和视锥细胞。这些专门的神经元探测光线，然后通过突触传输将信号传递给其他下游神经元，称为双极细胞。光感受器和双极细胞之间的突触的正确形成和功能需要在突触两侧积累多种蛋白质。我的研究计划的长期目标是了解蛋白质是如何定位于光感受器-双极突触的，它们在突触的功能和结构上是如何组织的，以及突触形成的特异性是如何控制的。在这个提案中，我们将重点放在跨突触复合体--蛋白质之间形成跨越突触间隙的复合体的直接相互作用上。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互动的要求。在目标1中，我们将研究在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电穿孔新生小鼠的视网膜；这允许标记蛋白在视网膜组织的自然环境中特定细胞类型的表达。我们将使用体外下拉试验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的积累中发挥作用。糖基化是蛋白质胞外结构域与糖分子的修饰，其作用大多尚不清楚。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，以及用阻止糖基化的突变对构建物进行电穿孔。许多突触含有支架蛋白，通过与被称为PDZ结构域的重复结构结合来组织其他蛋白。尽管光感受器-双极突触含有几种带有PDZ结合基序的蛋白质，但支架蛋白相互作用的特征仍然很差。在目标3中，我们将通过电穿孔结构来测试突触定位是否需要PDZ结构域的相互作用，这些结构的PDZ结合基序已经发生突变。我们还将进行下拉实验，以测试与突触上已知的含有PDZ的蛋白质的结合。我的研究项目解决了关于光感受器和双极细胞中突触靶向的知识空白，以及它们通信所用的突触的组装。在分子水平上了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提供有关突触蛋白质结构和功能的新的详细信息，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.