The Influence of Binding and Crowding on Synaptic Protein Mobility

描述（由申请人提供）：突触的蛋白质组成被精细地调节以维持健康的脑功能。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了突触后功能。该提案的广泛目标是了解这些关键的突触后蛋白如何集中在兴奋性突触内，以及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。特别重要的是AMPA型谷氨酸受体（AMPAR）的调节，因为激活的AMPAR的数量在许多形式的神经可塑性过程中受到控制和调节。AMPAR在神经元表面膜上自由扩散，并通过这种机制进入和退出突触后致密区（PSD）。为了维持突触强度，突触减慢受体的移动以保留它们。不幸的是，尽管深入的研究，突触内受体的流动性的机制仍然不清楚。令人信服的数据，包括高分辨率成像的受体横向运动表明，结合到合作伙伴内的PSD，特别是支架PSD-95，是必不可少的受体积累。然而，数值模拟和间接的实验证据表明了另一种可能性，即突触是如此密集的蛋白质，这一障碍领域防止受体逃逸。然而，这种机制（突触内受体的空间位阻）知之甚少，并没有得到系统的解决。出于这些观察结果，我假设空间位阻和生化结合在PSD的行为相结合，以调节突触跨膜蛋白的流动性，如受体。我结合了两种单分子成像方法，可以在必要的纳米级分辨率下区分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的蛋白质施加空间影响，则该影响应取决于蛋白质大小。第一组实验通过改变细胞外大小来测试这一预测，否则相同的结合缺陷跨膜蛋白，并跟踪它们在活突触中的运动。然后，我评估是否空间位阻在细胞外和细胞内的环境中的突触可能有不同的影响受体的流动性，通过改变体积大小的相同的蛋白质在膜的两侧，并跟踪他们的运动在活突触。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跨膜蛋白的运动来测试突触与慢跨膜蛋白结合的充分性。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.