Single Molecule Imaging of RIM1 Scaffold in Photoreceptor Neurotransmission

项目002(079)：光感受器神经传递中RIM1支架的单分子成像，Choi PL 项目摘要/摘要 视网膜中的光感受器通过调节释放，通过它们的突触传递对光的反应 活动区内含有谷氨酸的突触小泡。光感受器突触使用带状突触 由多结构域支架蛋白组成的活性区域促进神经递质的快速和持续释放 这是正常视力所必需的。参与突触传递的支架蛋白调节失调 与导致失明的视网膜疾病有关。然而，我们对这种分子的了解有限。 这些病理基础的机制，这对于理解和治疗这些疾病至关重要。我们的 长期目标是确定在活动区和突触小泡释放的分子机制 视网膜疾病中细胞结构是如何失调导致视力障碍的。总体目标 这一应用的目的是确定Rab3相互作用分子1(RIM1)中的常染色体显性突变 导致致盲疾病，7型视锥-杆状营养不良(CORD7)。我们对这一提议的中心假设是 RIM1的结构动力学调节支架活性，而支架活性被CORD7疾病破坏 突变(目标1)，通过改变相分离和小泡而导致功能活动区受损 融合(目标2)。 RIM1是通过直接或间接相互作用调节活动区功能的中心成分 与所有其他活性区带蛋白结合。RIM1由高度结构化的域串联阵列组成，通过以下方式连接 本质上无序的连接子，这排除了用传统方法进行结构分析的可能性。要克服这一点 限制，我们将使用最先进的单分子荧光共振能量转移(SmFRET)来 确定RIM1的动态结构。我们已经建立了一个体外重组系统 活动区的突触几何结构，允许在生物学上观察蛋白质的动力学和功能 相关背景。我们的新测试将提供RIM1的第一个结构细节以及CORD7突变是如何 通过改变支架活性来影响RIM1的功能。 细胞机制是如何组织起来允许释放突触小泡的，目前还不完全清楚。 明白了。RIM1经历液-液相分离(LLP)，浓缩成无膜 “细胞器”，当与其他活性区带蛋白混合时。这个脚手架组织建议有限责任合伙公司 对活跃区的囊泡释放至关重要。确定RIM1 LLP在囊泡中的功能意义 为了进行融合，我们将进行我们开发的新型单囊泡融合试验。这些方法将提供 LLLP过程中RIM1的第一个结构细节以及LLPS如何驱动囊泡融合。实现单分子 在与失明相关的生物学背景下解析蛋白质动力学及其功能将发现新的 隐藏在系综测量中的RIM1的机制，并为分子分析提供了一个平台 疾病突变，如CORD7。

Project 002 (079): Single Molecule Imaging of RIM1 Scaffold in Photoreceptor Neurotransmission, Choi PL PROJECT SUMMARY/ABSTRACT Photoreceptors in the retina transmit a response to light across their synapses by regulating the release of glutamate-containing synaptic vesicles at the active zone. The photoreceptor synapse uses a ribbon-type active zone composed of multi-domain scaffold proteins to promote fast and continuous neurotransmitter release that is needed for normal vision. Dysregulation of the scaffold proteins involved in synaptic transmission is associated with retinal diseases leading to blindness. However, we have limited knowledge of the molecular mechanisms underlying these pathologies, which is critical for understanding and treating these diseases. Our long-term goal is to determine the molecular mechanisms of synaptic vesicle release at the active zone and how the cellular machinery is dysregulated in retinal disease leading to visual disabilities. The overall objective of this application is to determine how an autosomal dominant mutation in Rab3-interacting molecule 1 (RIM1) causes the blinding disease, cone-rod dystrophy type 7 (CORD7). Our central hypothesis for this proposal is that the structural dynamics of RIM1 regulates the scaffolding activity, which is disrupted by the CORD7 disease mutation (Aim 1), leading to impairment of a functional active zone by altering phase separation and vesicle fusion (Aim 2). RIM1 is the central component regulating the function of the active zone by directly or indirectly interacting with all other active zone proteins. RIM1 consists of tandem arrays of highly structured domains connected by intrinsically disordered linkers, which precludes structural analysis by traditional methods. To overcome this limitation, we will use the state-of-the-art single-molecule fluorescence resonance energy transfer (smFRET) to determine the dynamic structure of RIM1. We have established an in vitro reconstituted system recapitulating the synaptic geometry of the active zone, allowing protein dynamics and function to be observed in a biologically relevant context. Our novel assay will provide the first structural details of RIM1 and how the CORD7 mutation affects the function of RIM1 by altering the scaffolding activity. How the cellular machineries are organized to allow the release of synaptic vesicles is incompletely understood. RIM1 undergoes liquid-liquid phase separation (LLPS), condensing into a membrane-less “organelle”, when mixed with other active zone proteins. This scaffold organization suggests that LLPS is essential for vesicle release at the active zone. To determine the functional significance of RIM1 LLPS in vesicle fusion, we will conduct a novel single-vesicle fusion assay that we developed. These approaches will provide the first structural details of RIM1 during LLPS and how LLPS drives vesicle fusion. Achieving single-molecule resolution of protein dynamics and its function in a biological context related to blindness will uncover novel mechanisms of RIM1 that are hidden in ensemble measurements, and set a platform for molecular analysis of disease mutations such as CORD7.