The Influence of Binding and Crowding on Synaptic Protein Mobility

描述（申请人提供）：突触的蛋白质组成受到精细调节，以维持健康的大脑功能。突触包含多种重要的跨膜蛋白，包括神经递质受体、细胞粘附分子和离子通道。这些成分的精确结构组织建立突触后功能。本研究的主要目标是了解这些关键的突触后蛋白如何在兴奋性突触内集中，以及它们的数量和位置的调节如何影响健康和疾病中的神经可塑性机制。特别重要的是ampa型谷氨酸受体（AMPARs）的调节，因为激活AMPARs的数量在多种形式的神经可塑性中受到控制和调节。ampar在神经元表面膜上自由扩散，并通过该机制进入和退出突触后密度（PSD）。为了维持突触的强度，突触减缓受体的移动以保留它们。不幸的是，尽管深入研究，控制突触内受体移动的机制仍然不清楚。包括受体横向运动的高分辨率成像在内的令人信服的数据表明，与PSD内的伙伴结合，特别是支架PSD-95，对受体积累至关重要。然而，数值模拟和间接的实验证据表明了另一种可能性，即突触中充满了如此密集的蛋白质，以至于这个障碍场阻止了受体的逃逸。然而，这一机制（突触内受体的空间位阻）了解甚少，尚未系统地解决。基于这些观察结果，我推测PSD中的位阻和生化结合共同调节突触跨膜蛋白如受体的迁移。我结合了两种单分子成像方法，以必要的纳米级分辨率分辨突触内部和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位阻的影响。如果突触环境对进入PSD的蛋白质施加立体影响，这种影响应该取决于蛋白质的大小。第一组实验通过改变另一种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验证这一预测。然后，通过改变膜两侧相同蛋白质的体积大小并跟踪其在活突触中的运动，我评估突触中细胞外和细胞内环境中的位阻是否会对受体的迁移产生不同的影响。接下来的实验通过跟踪携带可与PSD- 95结合的配体的小的、通用的跨膜蛋白的运动来测试突触结合对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。

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.