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

胚胎果蝇神经肌肉接头突触小泡聚集的力通路

• 批准号: 1935181
• 负责人: Taher Saif
• 金额: $ 73.72万
• 依托单位: University of Illinois at Urbana-Champaign
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-10-01 至 2023-09-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1935181&HistoricalAwards=false
• 关键词: Force; Pathway; Synaptic; Vesicle; Clustering

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.

动物的记忆和学习是通过称为突触的神经元-神经元或神经-肌肉接头的神经传递来实现的。这些突触是在神经元的长索状延伸的末端形成的，称为轴突。连接的轴突部分被称为突触前终末。它含有含有神经递质的小泡(~50 nm)。一些囊泡位于靠近突触壁的活性部位，准备释放其内容物。其他的则聚集在突触内作为一个储备池。当神经元放电时，一个被称为动作电位的电信号到达突触。活动部位的一些小泡释放它们的神经递质，刺激突触后终末。因此，发送信号。来自储备池的新水泡加入活动站点。显然，为了实现神经传递，神经元必须在突触聚集储备池小泡以防止扩散，同时为它们提供定向移动性，以取代活动部位释放的小泡。尽管进行了数十年的研究，但这种二元性的机制仍然难以捉摸。这个项目试图通过将轴突的机械属性，即其收缩或机械张力，与囊泡聚集、动力学和释放联系起来，来解决这一悖论。在果蝇胚胎上的PI研究表明，神经肌肉突触前末端的囊泡聚集依赖于轴突的机械张力。这项研究的发现将通过与一名戏剧老师合作，与高中生合作制作一部短剧来传播给更广泛的观众，以代表神经传递--人物模仿囊泡、离子、肌动蛋白和突触素-I。这项研究还将与教育相结合，让来自代表性不足群体的本科生参与研究，在当地儿童博物馆展示模块，以及向高中教师教授生物物理学。该项目基于这样的假设，即在胚胎果蝇中，形成神经肌肉连接的运动神经元的轴突在包括突触在内的整个长度上都有一个收缩的肌球蛋白网络。这种力的连续性导致了突触处稳定的F-肌动蛋白结构。离子敏感的黏附蛋白，例如，突触素I，将囊泡附着(粘合)到突触F-肌动蛋白上，从而聚集和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，黏附蛋白释放小泡。然后，它们被马达蛋白沿着F-肌动蛋白纤维移动到活性部位或离开活性部位。因此，突触F-肌动蛋白构筑既是囊泡聚集的支架，也是其定向移动的双车道高速公路。这一假说将通过分三步研究果蝇胚胎的神经-肌肉连接来验证。首先，为了测试acto-myosin机制是否参与了包括突触在内的整个轴突的收缩力量的产生。第二，测试突触是否存在由轴突收缩力稳定的F-肌动蛋白构筑，以及它是否作为囊泡附着的支架，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)、纳米探头和循环伏安法将用于验证假设。该奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

项目成果

Synapses without tension fail to fire in an in vitro network of hippocampal neurons

• DOI: 10.1073/pnas.2311995120
• 发表时间: 2023-12-26
• 期刊: PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
• 影响因子: 11.1
• 作者: Joy，Md Saddam Hossain;Nall，Duncan L.;Saif，M. Taher A.
• 通讯作者: Saif，M. Taher A.