CRCNS: Receptor mobility and scaffold dynamics at single glutamatergic synapses

描述（由申请人提供）：智力优势：突触强度的活动调节变化是学习和神经发育分子理论的核心。在大脑的谷氨酸突触中，受体数量的调节是突触强度快速变化的核心机制。突触后密度（PSD）的多结构域蛋白结合受体并调节其运输，这导致了一个模型，即这些蛋白质充当“槽”，其占用决定突触强度。然而，缺乏对该模型的直接测试，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观点，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。在如此拥挤的空间中，扩散的基本特征被改变，以至于受体可以被限制在非常小（纳米大小）的区域内，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，以及突触蛋白的光学和电子显微镜成像支持这一观点。基于这些观察结果，我们假设PSD中的大分子拥挤与生化相互作用一致，决定了突触受体的数量。拥挤空间中扩散和反应的计算模型和最先进的活细胞成像技术将使我们能够验证这一假设。为此，我们将：具体目标1：在PSD中开发核心支架组织的结构模型。一组相互连接的支架蛋白构成了PSD的核心，是控制受体数量的核心，但核心内蛋白质的分布，特别是在活突触中的分布，仍然没有记载。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和生化文献，我们将详细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运动和寿命的模型。为了扩展该模型，使其能够解释受体的迁移，我们将使用高通量单分子跟踪PALM和突触亚域的高分辨率光漂白和光激活来测量突触内的蛋白质迁移。测量核心支架的突触内移动性，以及跨膜膜蛋白或居住在膜外或膜内小叶中的膜蛋白的移动性将提供模型约束。我们将从Aim 1扩展模型，以允许使用用于模拟物理系统（如胶体）的技术来测量特征。特异性目的3：测试支架密度、间距和迁移率的改变是否影响PSD内受体迁移率和受体寿命。为了测试Aim 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核心支架的特征，并测量它们对细胞中受体迁移的影响。为了改变拥挤，我们将通过改变支架连接结构域的工程蛋白来改变支架内的间距。为了控制支架的移动性，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。使用上述导出的约束条件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。特异性目的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。利用膜片钳电生理学和谷氨酸光解，我们将测试突触效能是否随着PSD拥挤和支架移动的改变而协同变化。模拟谷氨酸释放将用于测试重建受体的迁移和分布是否导致实验观察到的突触反应。

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.