Force Pathway to Synaptic Vesicle Clustering in Embryonic Fruit Fly Neuro Muscular Junctions

动物的记忆和学习是通过神经元-神经元或神经-肌肉连接处（称为突触）的神经传递来实现的。这些突触形成于被称为轴突的神经元长索状延伸的末端。连接的轴突部分称为突触前末端。它拥有小的（~50纳米）囊泡，其中含有神经递质。一些囊泡位于靠近突触壁的活性部位，准备释放它们的内容物。其他的则聚集在突触内作为储备池。当神经元放电时，一个被称为动作电位的电信号就会到达突触。活性部位的一些囊泡释放神经递质并刺激突触后末端。这样，就传送了信号。来自储备池的新囊泡加入活性位点。显然，为了实现神经传递，神经元必须在突触上聚集储备池囊泡，以防止扩散，并为它们提供定向的流动性，以取代活性部位释放的囊泡。尽管经过数十年的研究，这种二元性的机制仍然难以捉摸。本项目试图通过将轴突的机械特性（即其收缩性或机械张力）与囊泡聚集、动力学和释放联系起来，来解决这一悖论。PI先前在胚胎果蝇（果蝇）上的工作表明，神经肌肉突触前末端的囊泡聚集取决于轴突的机械张力。这项研究的发现将通过与一名戏剧老师合作，与高中生一起制作一部短剧来传播给更广泛的观众，以表现神经传递——角色模仿囊泡、离子、肌动蛋白和突触素- 1。这项研究还将通过让来自代表性不足群体的本科生参与研究、在当地儿童博物馆举办展览、向高中教师教授生物物理学等方式，与教育相结合。该项目基于一种假设，即在胚胎果蝇中形成神经-肌肉连接的运动神经元轴突在其整个长度上都有一个可收缩的肌动蛋白网络，包括突触。这种力的连续性导致了突触中稳定的f -肌动蛋白结构。离子敏感粘附蛋白，如：突触蛋白I将（胶）囊泡附着在突触f -肌动蛋白上，从而聚集并固定它们以防止扩散。在动作电位期间，突触钙离子浓度增加，粘附蛋白释放囊泡。然后它们被运动蛋白沿着f -肌动蛋白纤维移动到或离开活性位点。因此，突触f -肌动蛋白结构作为囊泡聚集的支架，以及它们定向移动的双车道高速公路。这一假设将通过研究胚胎果蝇的神经-肌肉连接三步来验证。首先，测试肌动蛋白机制是否参与包括突触在内的整个轴突的收缩力产生。其次，测试在突触中是否存在由轴突收缩力稳定的f -肌动蛋白结构，以及它是否作为囊泡粘附的支架，以及它们运输的高速公路。最后，测试轴突力是否调节神经传递。新型纳米机械力传感器、微流体、高分辨率显微镜（随机光学重建显微镜，STORM）、纳米探针和循环伏安法将被用来检验这一假设。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

Memory and learning in animals are achieved by neurotransmission at neuron-neuron or neuro-muscular junctions called synapses. These synapses are formed at the end of long cable-like extensions of neurons, called axons. The axonal part of the junction is called the pre-synaptic terminal. It hosts small (~50 nm) vesicles containing neurotransmitters. Some of the vesicles are at the active site close to the wall of the synapse, ready to release their contents. Others are clustered within the synapse as a reserve pool. When the neuron fires, an electric signal, known as the action potential, arrives at the synapse. Some of the vesicles at the active site release their neurotransmitters and stimulate the post synaptic terminal. Thus, a signal is transmitted. New vesicle from the reserve pool join the active site. Clearly, to achieve neurotransmission, neurons must cluster reserve-pool vesicles at the synapse against diffusion, and yet provide them directed mobility to replace the released ones at the active site. In spite of decades of research, the mechanism of this duality remains elusive. This project attempts to resolve this paradox by linking a mechanical property of the axon, namely its contractility or mechanical tension, with vesicle clustering, dynamics and release. Prior work of the PI on embryonic Drosophila (fruit fly) revealed that vesicle clustering at the neuromuscular presynaptic terminal depends on mechanical tension of the axons. The findings of this research will be disseminated to the broader audience by developing a short drama with high school students, in collaboration with a drama teacher, to represent neurotransmission -- with characters mimicking vesicles, ions, actin and synapsin-I. The research will also be integrated with education through involvement of undergraduate students from underrepresented groups in research, exhibition modules at the local Children's Museum, and teaching biophysics to high school teachers. The project is based on the hypothesis that axons of motor neurons forming neuro-muscular junctions in embryonic flies have an contractile acto-myosin network along their entire length, including the synapse. This force continuity results in a stable F-actin architecture at the synapse. Ion sensitive adhesion proteins, e,g., synapsin I, attach (glue) vesicles to synaptic F-actin, thus clustering and immobilizing them against diffusion. During an action potential, synaptic Calcium ion concentration increases, and the adhesion proteins release the vesicles. They are then moved by motor proteins to and away from the active sites along the F-actin fibers. Thus, synaptic F-actin architecture serves as a scaffold for vesicles to cluster, as well as a double-lane highway for their directed mobility. This hypothesis will be tested by studying the neuro-muscular junction of embryonic Drosophila in three steps. First, to test whether acto-myosin machinery is involved in contractile force generation along the entire length of the axon including synapse. Second, to test whether there exists an F-actin architecture at the synapse stabilized by the axonal contractile force, and whether it serves as a scaffold for the vesicles to adhere, as well as a highway for their transport. Finally, to test whether axonal force modulates neurotransmission. Novel nano-mechanical force sensors, micro-fluidics, high resolution microscopy (Stochastic Optical Reconstruction Microscopy, STORM), nano-probe and cyclic voltammetry will be used to test the hypothesis.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.