The Influence of Binding and Crowding on Synaptic Protein Mobility

结合和拥挤对突触蛋白迁移性的影响

• 批准号: 8649954
• 负责人: Tuo Peter Li
• 金额: $ 3.68万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-09-26 至 2018-09-25
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8649954
• 关键词: Address; Affect; Animal Model; Autistic Disorder; Binding; Biochemical; Biological Assay; Brain; Cell Adhesion Molecules; Characteristics; Conflict (Psychology); Crowding; Cytoplasmic Tail; Data; Diffuse; Diffusion; Discrimination; Disease; Environment; Excitatory Synapse; Extracellular Domain; Figs - dietary; Functional disorder; G-Protein-Coupled Receptors; Generic Drugs; Glutamate Receptor; Goals; Health; Image; Integral Membrane Protein; Investigation; Ion Channel; Knowledge; Lateral; Left; Life; Ligands; Measures; Membrane; Mental disorders; Microscopy; Modeling; Molecular; Monitor; Motion; Movement; Neuronal Plasticity; Neurons; Neurotransmitter Receptor; Play; Positioning Attribute; Prevention approach; Proteins; Receptor Cell; Regulation; Relative (related person); Resolution; Role; Schizophrenia; Shapes; Side; Surface; Synapses; Synaptic Cleft; Tail; Testing; Time; Work; cognitive function; density; design; extracellular; improved; nanometer; nanoscale; neuropsychiatry; postsynaptic; presynaptic density protein 95; public health relevance; receptor; receptor binding; research study; scaffold; single molecule; stargazin; synaptic function

DESCRIPTION (provided by applicant): The protein composition of synapses is exquisitely regulated to maintain healthy brain function. The synapse contains diverse set of centrally important transmembrane proteins, including neurotransmitter receptors, cell adhesion molecules, and ion channels. The precise architectural organization of these components establish postsynaptic function. The broad goal of this proposal is to understand how these critical postsynaptic proteins concentrate within excitatory synapses, and how regulation of their number and position contribute to mechanisms of neural plasticity in health and disease. Of particular importance is regulation of the AMPA-type glutamate receptors (AMPARs), because the number of activated AMPARs is controlled and modulated during many forms of neural plasticity. AMPARs diffuse freely on the neuronal surface membrane, and enter and exit the postsynaptic density (PSD) via this mechanism. In order to sustain synaptic strength, the synapse slows mobility of receptors to retain them. Unfortunately, despite intensive investigation, the mechanisms governing intrasynaptic receptor mobility remain unclear. Compelling data including high resolution imaging of receptor lateral movement suggest that binding to partners within the PSD, notably the scaffold PSD-95, is essential for receptor accumulation. However, numerical modeling and indirect experimental evidence suggest an alternative possibility, i.e. the synapse is so dense with proteins that this obstacle field prevens receptors from escaping. Nevertheless, this mechanism (intrasynaptic steric hindrance of receptors) is poorly understood and has not been systematically addressed. Motivated by these observations, I hypothesize that steric hindrance and biochemical binding in the PSD act in combination to regulate the mobility of synaptic transmembrane proteins like receptors. I have combined two single-molecule imaging approaches that permit discrimination of protein mobility within and around synapses with the necessary, nanometer-scale resolution. Using these approaches to monitor motion of a uniquely designed set of transmembrane protein probes will allow me to examine the influence of binding and steric hindrance separately. If the synaptic environment exerts steric influence on proteins entering the PSD, the effect should depend on protein size. The first set of experiments tests this prediction by altering the extracellular sizeof an otherwise identical binding-deficient transmembrane protein, and tracking their movements in the living synapse. I then assess whether steric hindrance in the extracellular and intracellular environment in the synapse could have different effects on receptor mobility, by altering the bulk size of the same protein on either side of the membrane and tracking their movements in the living synapse. The next experiments test the sufficiency of synaptic binding to slow transmembrane protein by following the movements of a small, generic transmembrane protein carrying a ligand which can bind to PSD- 95. These results greatly clarify important mechanisms controlling key proteins of the synapse.

描述(由申请者提供)：突触的蛋白质组成受到微妙的调节，以维持健康的大脑功能。突触含有多种中枢重要的跨膜蛋白，包括神经递质受体、细胞黏附分子和离子通道。这些组件的精确结构组织建立了突触后功能。这一提议的广泛目标是了解这些关键的突触后蛋白是如何集中在兴奋性突触中的，以及对它们的数量和位置的调节如何有助于健康和疾病中的神经可塑性机制。尤其重要的是对AMPA型谷氨酸受体(AMPAR)的调节，因为在许多形式的神经可塑性过程中，激活的AMPAR的数量受到控制和调节。AMPAR在神经元表面膜上自由扩散，通过这种机制进入和退出突触后密度(PSD)。为了维持突触的力量，突触减慢受体的移动速度以保持它们。不幸的是，尽管进行了深入的研究，但控制突触内受体移动性的机制仍然不清楚。令人信服的数据，包括受体横向运动的高分辨率成像表明，与PSD内的伙伴结合，特别是支架PSD-95，对于受体的积累是必不可少的。然而，数值模拟和间接实验证据表明了另一种可能性，即突触含有如此密集的蛋白质，以至于这个障碍场阻碍了受体的逃逸。然而，这种机制(突触内受体的空间位阻)还知之甚少，也没有被系统地解决。在这些观察的启发下，我假设PSD中的空间障碍和生化结合共同作用，调节突触跨膜蛋白类受体的流动性。我结合了两种单分子成像方法，允许以必要的纳米级分辨率区分突触内和突触周围的蛋白质流动性。使用这些方法来监测一组独特设计的跨膜蛋白探针的运动，将使我能够分别检查结合和空间位阻的影响。如果突触环境对进入PSD的蛋白质施加空间位阻影响，其影响应取决于蛋白质大小。第一组实验通过改变一种完全相同的结合缺陷跨膜蛋白的细胞外大小，并跟踪它们在活突触中的运动来检验这一预测。然后，我评估了突触细胞外和细胞内环境中的空间位阻是否会通过改变膜两边相同蛋白质的体积大小并跟踪它们在活突触中的运动来对受体的流动性产生不同的影响。接下来的实验通过跟踪携带可以与PSD-95结合的配体的小型通用跨膜蛋白的运动来测试突触结合减慢跨膜蛋白的充分性。这些结果极大地阐明了控制突触关键蛋白的重要机制。