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神经元神经元或神经肌肉连接的神经传递来实现的，称为突触。这些突触在神经元的长电缆状延伸末端（称为轴突）形成。连接的轴突部分称为突触前末端。它含有含有神经递质的小（约50 nm）囊泡。一些囊泡位于靠近突触壁的活跃部位，准备释放其内容物。其他人则被当作储备池聚集在突触中。当神经元发射时，一个称为动作电位的电信号到达突触。活性部位的某些囊泡释放其神经递质并刺激突触后末端。因此，信号传输。储备池的新囊泡加入了活动地点。显然，为了获得神经传递，神经元必须在突触中群集储备囊泡反对扩散，但仍可以定向移动性来代替活跃部位的释放的移动性。尽管进行了数十年的研究，但这种双重性的机制仍然难以捉摸。该项目试图通过将轴突的机械性能（即其收缩性或机械张力）与囊泡聚类，动力学和释放联系起来来解决此悖论。 PI在胚胎果蝇（果蝇）上的先前工作表明，神经肌肉突触前末端的囊泡聚类取决于轴突的机械张力。这项研究的发现将通过与戏剧老师合作制作一部简短的戏剧来代表神经传递，以模仿囊泡，离子，肌动蛋白和突触素1，将其传播给更广泛的受众。这项研究还将与教育融合，通过参与来自人数不足的小组的研究，当地儿童博物馆的展览模块以及向高中教师教生物物理学的研究。该项目基于以下假设：在胚胎蝇中形成神经肌肉连接的运动神经元的轴突沿其整个长度（包括突触）具有收缩的肌动蛋白网络。这种力连续性导致突触处的F-肌动蛋白结构稳定。离子敏感的粘附蛋白，e，g。，突触素I，附着（胶）囊泡与突触F-肌动蛋白，从而将它们聚集并固定在扩散中。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，它们被运动蛋白移动到沿F-肌动蛋白纤维的活性位点并远离活性位点。因此，突触F-肌动蛋白结构是囊泡聚集的脚手架，以及针对其定向迁移率的双车道高速公路。该假设将通过研究三个步骤的胚胎果蝇的神经肌肉连接来检验。首先，要测试Acto-Myosin机械是否沿轴突的整个长度参与收缩力的产生，包括突触。 其次，要测试是否存在轴突收缩力稳定的突触处的F-肌动蛋白结构，以及它是否是囊泡粘附的脚手架，以及其运输的高速公路。 最后，测试轴突力是否调节神经传递。新型的纳米机械力传感器，微氟化物，高分辨率显微镜（随机光学重建显微镜，风暴），纳米探针和环状伏安法将用于测试假设。该奖项反映了NSF的法定任务，并通过评估了CRAM的知识群体，并通过基金会的范围进行了评估。

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.