CRCNS: Receptor mobility and scaffold dynamics at single glutamatergic synapses

CRCNS：单个谷氨酸突触的受体移动性和支架动力学

• 批准号: 8451516
• 负责人: Sridhar Raghavachari
• 金额: $ 30.2万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-07-01 至 2015-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8451516
• 关键词: Accounting; Affect; Alzheimer' s Disease; Autistic Disorder; Baltimore; Binding; Biochemical; Biological Assay; Brain; Cells; Characteristics; Colloids; Computer Simulation; Computing Methodologies; Core Protein; Crowding; Diffusion; Disease; Electrons; Electrophysiology (science); Environment; Epilepsy; Excitatory Synapse; Functional disorder; Glutamates; Heart; Image; Lead; Learning; Life; Light; Link; Literature; Maps; Maryland; Measurement; Measures; Membrane; Membrane Proteins; Microscopic; Modeling; Molecular; Neurobiology; Neurodegenerative Disorders; Photobleaching; Protein Engineering; Proteins; Publishing; Reaction; Receptor Activation; Regulation; Research; Resolution; Scaffolding Protein; Schizophrenia; Science; Spatial Distribution; Structural Models; Synapses; Synaptic Receptors; System; Techniques; Testing; Training; Work; base; cellular imaging; crosslink; density; high school; inhibitor/antagonist; nanoscale; neurodevelopment; neuropsychiatry; neurotransmission; novel; outreach; patch clamp; photoactivation; photolysis; physical model; postsynaptic; postsynaptic density protein; presynaptic density protein 95; protein distribution; public health relevance; receptor; receptor binding; response; scaffold; simulation; single molecule; synaptic function; theories; tool; trafficking; undergraduate student

DESCRIPTION (provided by applicant): Intellectual merit: Activity-regulated changes in synapse strength lie at the heart of molecular theories of learning and neural development. At glutamatergic synapses of the brain, regulation of receptor number is a core mechanism for rapidly changing synaptic strength. Multi-domain proteins of the postsynaptic density (PSD) bind receptors and regulate their trafficking, and this has lead to a model that these proteins serve as "slots" whose occupancy determines synaptic strength. However, direct tests of this model are lacking, and recent work suggests that it is incomplete. We have recently developed computational models to test the idea that the spatial distribution and mobility of proteins that make up the PSD may result in a phenomenon called macromolecular crowding. In such crowded spaces, the fundamental character of diffusion is altered such that receptors can be confined within very small (nanometer-sized) regions even without any need for binding to PSD-95 and similar scaffold molecules. High-resolution imaging studies of receptor diffusion in synapses, as well as light and electron microscopic imaging of synaptic proteins support such a view. Motivated by these observations, we hypothesize that macromolecular crowding in the PSD acts in concert with biochemical interactions to determine the number of synaptic receptors. Emerging computational models of diffusion and reaction in crowded spaces and state-of-the-art live-cell imaging will now allow us to test this hypothesis. To do so, we will: Specific Aim 1: Develop a structural model of core scaffold organization in the PSD. An interconnected set of scaffolding proteins forms the core of the PSD and is central to control of receptor numbers, but the distribution of proteins within the core, particularly in living synapses, remains undocumented. We will use super-resolution live-cell imaging to map the distribution of core proteins in the PSD. Then, incorporating structural, EM, and biochemical literature, we will elaborate and refine our published models to reproduce these measurements of PSD organization. Specific Aim 2: Develop a model of receptor mobility and lifetime in excitatory synapses. To extend this model so that it can account for receptor mobility, we will use high-throughput single-molecule tracking PALM and high-resolution photobleaching and photoactivation in synaptic subdomains to measure protein mobility within the synapse. Measurements of the intrasynaptic mobility of core scaffolds, and mobility of transmembrane membrane proteins or those resident in the external or internal membrane leaflets will provide model constraints. We will extend the model from Aim 1 to allow for measured characteristics using techniques developed to model physical systems such as colloids. Specific Aim 3: Test whether alterations in scaffold density, spacing, and mobility affect receptor mobility and receptor lifetime within the PSD. To test predictions of the model generated in Aim 2, we will use several molecular strategies to alter characteristics of the PSD core scaffold, and measure their influence on receptor mobility in cells. To alter crowding, we will alter spacing within the scaffold by engineering proteins with altered scaffold-linking domains. To control scaffold mobility, we will acutely cross-link targeted PSD constituents and use cytoskeletal inhibitors to arrest the internal dynamics of the PSD. Computer simulations of receptor diffusion using constraints derived above will be performed to extract mobility and lifetimes and compared to the measurements. Specific Aim 4: Test whether alterations of scaffold distribution and mobility in the PSD affect synaptic strength. The crowding effect can regulate the number or spatial arrangement of receptors, which are expected to affect receptor activation during neurotransmission. Using patch-clamp electrophysiology and glutamate photolysis, we will test whether synaptic efficacy changes in coordination with alterations of PSD crowding and scaffold mobility. Simulations of glutamate release will be used to test whether the reconstructed receptor mobility and distribution results in experimentally observed synaptic responses. Broader Impact: This work will greatly advance our fundamental understanding of synapse function and plasticity, thus also aiding research into synaptic dysfunction that underlies neuropsychiatric and neurodegenerative diseases. Second, this project, based on the synergy between theoretical sciences, novel computational methods, and new techniques in neurobiology, will provide a unique crossdisciplinary environment for training of young neuroscientists at Duke and Maryland. Finally, the project will be integrated into ongoing outreach efforts to expose local underrepresented high school and undergraduate students in Durham and Baltimore to advanced math and science.

描述（由申请人提供）：智力优点：突触强度的活性调节变化是学习和神经发育的分子理论的核心。在大脑的神经元突触中，受体数量的调节是快速改变突触强度的核心机制。突触后密度（PSD）的多结构域蛋白结合受体并调节它们的运输，这导致了一个模型，这些蛋白质作为“插槽”，其占用决定突触强度。然而，这个模型的直接测试是缺乏的，最近的工作表明，它是不完整的。我们最近开发了计算模型来测试这样一种想法，即构成PSD的蛋白质的空间分布和流动性可能导致一种称为大分子拥挤的现象。在这种拥挤的空间中，扩散的基本特征被改变，使得受体可以被限制在非常小的（纳米尺寸）区域内，甚至不需要与PSD-95和类似的支架分子结合。突触中受体扩散的高分辨率成像研究，以及突触蛋白质的光镜和电镜成像都支持这一观点。出于这些观察结果，我们假设，大分子拥挤的PSD行为与生化相互作用，以确定突触受体的数量。新兴的计算模型的扩散和反应在拥挤的空间和国家的最先进的活细胞成像现在将允许我们测试这一假设。具体目标1：开发PSD中核心支架组织的结构模型。一组相互连接的支架蛋白形成了PSD的核心，并且是控制受体数量的核心，但是核心内蛋白质的分布，特别是在活突触中，仍然没有文献记载。我们将使用超分辨率活细胞成像来绘制PSD中核心蛋白的分布。然后，结合结构，EM和生物化学文献，我们将阐述和完善我们发表的模型，以重现PSD组织的这些测量。具体目标2：建立兴奋性突触中受体迁移率和寿命的模型。为了扩展该模型，使其能够解释受体的迁移率，我们将使用高通量单分子跟踪PALM和高分辨率的光漂白和突触子域中的光活化来测量突触内的蛋白质迁移率。核心支架的突触内流动性的测量和跨膜蛋白或那些驻留在外部或内部膜小叶的流动性将提供模型约束。我们将扩展目标1的模型，以允许使用开发的物理系统（如胶体）建模技术测量特性。具体目标3：测试支架密度、间距和迁移率的改变是否影响PSD内的受体迁移率和受体寿命。为了测试目标2中生成的模型的预测，我们将使用几种分子策略来改变PSD核心支架的特性，并测量它们对细胞中受体迁移率的影响。为了改变拥挤，我们将通过改造具有改变的支架连接结构域的蛋白质来改变支架内的间距。为了控制支架的流动性，我们将急性交联靶向PSD成分，并使用细胞骨架抑制剂来阻止PSD的内部动力学。将使用以上导出的约束进行受体扩散的计算机模拟，以提取迁移率和寿命，并与测量结果进行比较。具体目标4：测试PSD中支架分布和移动性的改变是否影响突触强度。拥挤效应可以调节受体的数量或空间排列，从而影响神经传递过程中受体的激活。使用膜片钳电生理学和谷氨酸光解，我们将测试突触功效是否与PSD拥挤和支架移动性的改变协调变化。谷氨酸释放的模拟将用于测试重建的受体迁移率和分布是否导致实验观察到的突触反应。 更广泛的影响：这项工作将极大地推进我们对突触功能和可塑性的基本理解，从而也有助于研究神经精神和神经退行性疾病的基础突触功能障碍。第二，这个项目，基于理论科学之间的协同作用，新的计算方法，和神经生物学的新技术，将提供一个独特的跨学科的环境，在杜克和马里兰州的年轻神经科学家的培训。最后，该项目将被纳入正在进行的外联工作，使达勒姆和巴尔的摩当地代表性不足的高中和本科生接触高等数学和科学。