Postsynaptic Rapsyn Dynamics at the Neuromuscular Junction of Living Animals

神经冲动传递到肌细胞的效率取决于肌膜上乙酰胆碱受体蛋白的数量和密度。这些受体定位于称为突触的特殊结构，以突触后膜中的折叠为特征。受体被一系列特殊的支架蛋白牢牢地固定在折叠的顶端。这项研究的目的是研究当突触活动水平被操纵时，这种支架的关键成分Rapsyn的动力学。为了解决Rapsyn在突触处的动态变化，PI将水母绿色荧光蛋白(GFP)与Rapsyn融合，并将该结构引入小鼠的颈部(胸锁乳突肌)。这使得在活着的动物身上监测和成像几天过程中特定的突触成为可能。受体蛋白将被荧光银环蛇毒素标记，银环蛇毒素是一种与受体高度亲和力结合的蛇毒。PI将调查以下问题：第一，Rapsyn在体内单个突触的寿命是多少。第二，Rapsyn过表达对受体体内代谢稳定性有何影响。最后，PI将在活体内研究突触活动对单个突触中缝合蛋白动力学的影响。国际学生联合会将促进对研究生、本科生和高中生的培训，包括那些代表性不足的群体。PI在培养本科生方面有丰富的经验。此外，这项提议将加强神经科学领域的教学业绩和学习。结果将通过出版物和在会议上介绍研究结果的方式向科学界传播。最后，PI的实验室将参与社区的外展工作。

The efficiency of nerve impulse transmission to muscle cells depends on the number and density of acetylcholine receptor proteins on the muscle membrane. These receptors are localized to specialized structures known as synapses, characterized by folds in the postsynaptic membrane. Receptors are firmly held in the crest of the folds by a host of specialized scaffold proteins. The goal of this study is to investigate the dynamics of one critical component of this scaffold, rapsyn, when levels of synaptic activity are manipulated. To address the dynamics of rapsyn at the synapse, the PI will fuse jellyfish green fluorescent protein (GFP) to rapsyn and introduce the construct into the neck of the mouse (sternomastoid muscle). This makes it possible to monitor and image a particular synapse over the course of several days in the living animal. Receptor proteins will be labeled by fluorescent bungarotoxin, a snake venom that binds with high affinity to the receptor. The PI will investigate the following questions: first, what is the lifetime of rapsyn at individual synapse in vivo. Second what is the effect of rapsyn overexpression on the metabolic stability of receptors in vivo. Finally, the PI will investigate the effect of synaptic activity on the dynamics of rapsyn at individual synapses in vivo. The PI will promote the training of graduate, undergraduate and high school students, including those of underrepresented groups. The PI has extensive experience in training undergraduate students. In addition this proposal will enhance teaching performance and learning in the areas of neuroscience. Results will be disseminated to the scientific community through publications and by presenting findings in meetings. Finally the PI's lab will be involved in outreach to the community.