COMPUTATIONAL AND FUNCIONAL CHARACTERIZATION OF THE MOLECULAR STEPS IN MEMBRANE FUSION

膜融合分子步骤的计算和功能表征

• 批准号: 9064856
• 负责人: MARIA BYKHOVSKAIA
• 金额: $ 30.54万
• 依托单位: WAYNE STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-04-21 至 2018-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9064856
• 关键词: Address; Alzheimer' s Disease; Binding; Binding Proteins; Biochemical; Biochemistry; Biological Models; Biomedical Engineering; Biomedical Research; Biophysics; Cell membrane; Cereals; Chimeric Proteins; Complex; Computer Simulation; Coupled; Docking; Doctor of Medicine; Doctor of Philosophy; Drosophila genus; Educational Curriculum; Educational process of instructing; Electrophysiology (science); Employment Opportunities; Enrollment; Epilepsy; Equilibrium; Funding; Goals; Hispanic-serving Institution; Institution; Instruction; Ions; Kinetics; Lead; Learning; Link; Mechanics; Mediating; Membrane; Membrane Fusion; Membrane Lipids; Membrane Proteins; Memory; Memory impairment; Microscopy; Modeling; Molecular; Molecular Biology; Molecular Models; Mutate; Mutation; Neurobiology; Neuromuscular Junction; Neurons; Neurosciences; Neurosciences Research; Neurotransmitters; Optics; Parkinson Disease; Physiology; Point Mutation; Process; Proteins; Puerto Rico; Reaction; Regulation; Research; Research Training; SNAP receptor; Schizophrenia; Series; Site; Stimulus; Students; Synapses; Synaptic Cleft; Synaptic Membranes; Synaptic Transmission; Synaptic Vesicles; Testing; Training; Transgenic Animals; Underrepresented Minority; United States National Institutes of Health; Universities; Vesicle; genetic manipulation; graduate student; in vivo; membrane model; molecular modeling; multidisciplinary; nervous system disorder; neurotransmitter release; programs; protein complex; research study; synaptotagmin; tool; training opportunity; undergraduate student

DESCRIPTION (provided by applicant): Intellectual merit: At the cellular level, learning and memory are governed by changes in the efficacy of synaptic transmission and in particular, by the dynamic regulation of neuronal transmitter release. Neurotransmitters are packaged into synaptic vesicles that dock at the synaptic membrane, undergo a series of preparatory steps, open a pore, and fuse with the synaptic membrane, resulting in neurotransmitter release into the synaptic cleft. This process is very dynamic, plastic, and highly regulated. Although molecular components of docking and fusion have been identified, it is not yet understood how they interact to regulate the dynamics of docking, pore opening, and fusion. In particular, little is known about the detailed mechanics of protein interactions that regulate synaptic vesicle fusion. The present application will focus on this critical question by combining modeling and experimentation to investigate the molecular machinery that regulates synaptic vesicle docking and fusion. Vesicles tightly dock at the plasma membrane via a specialized protein complex (SNARE), which is thought to provide the necessary force to overcome inter-membrane repulsion and thus mediate vesicle fusion. Stimulus evoked fusion is triggered by an influx of Ca2+ ions that interact with a vesicle protein, synaptotagmin (Syt), which is tightly coupled with the SNARE complex. Fusion pore opening is thought to be controlled by the interaction of Syt and a small protein complexin (Cpx) with the SNARE complex. Although molecular interactions of these proteins have been studied with biochemical and molecular biology tools, there is still a lack of understanding of how these proteins interact dynamically and how the forces of the protein fusion machinery counterbalance forces generated within the synaptic and vesicle membranes. To elucidate these mechanisms, we propose to build a molecular model of the fusion machinery and to perform computer simulations of the dynamics of the fusion complex. To understand the interactions between the vesicle, synaptic membrane and the protein fusion machinery, we will develop a coarse grain model of membrane/vesicle dynamics and integrate it with the atomic model of the fusion protein complex. To validate the model, we will simulate the effect of single point mutations in the fusion complex on the release kinetics and test our predictions experimentally. The experiments will be performed at Drosophila neuromuscular junctions (NMJ), a model system ideally amendable to genetic manipulations. To test the predictions of the model, we will combine electrophysiology and optical fluorescent microscopy to assess release kinetics in NMJs where the fusion machinery is modified by point mutations with computationally predicted effects on membrane fusion. This research will be performed by a multidisciplinary team that includes experts in molecular modeling (Dr. Jagota), membrane mechanics and dynamics (Dr. Hui), synaptic physiology (Dr. Bykhovskaia) and Drosophila neurobiology (Dr. Littleton). An attack on this problem by a collaborative team with balanced representation of all its aspects will lead to new, detailed and quantitative, understanding of the regulated synaptic vesicle fusion process. Broader impact: Universidad Central del Caribe (UCC) is a Hispanic serving institution in Puerto Rico (U.S. Commonwealth). The proposed project will allow the UCC to develop tight links with highly regarded mainland institutions and will thus create training and employment opportunities for students with diverse backgrounds. The PI, Dr. Bykhovskaia, directs the Specialized Neuroscience Research Program (SNRP) at UCC (funded by NIH), that has a goal of raising research standards in institutions with a predominant enrollment of underrepresented minorities. Thus, the proposed project will involve underrepresented B.S., M.S., Ph.D., and M.D. students in biomedical research. Furthermore, Dr. Jagota directs the undergraduate and graduate Bioengineering programs at Lehigh University, an institution with a balanced emphasis on research, teaching and training. The proposed research will be performed by graduate students, and will actively involve undergraduate students through research for credit and summer opportunities. This research will be incorporated into the undergraduate bioengineering curriculum through a course on Biomolecular and Cellular Mechanics, developed by Dr. Jagota at Lehigh University. It will be tightly integrated with other activities, including student exchange and transdisciplinary seminars, and thus will promote integration of research and training across diverse intellectual and ethnic backgrounds.

描述(申请人提供)：智力优势：在细胞水平上，学习和记忆是由突触传递效率的变化，特别是神经元递质释放的动态调节决定的。神经递质被包装成突触小泡，停靠在突触膜上，经过一系列准备步骤，打开一个孔，与突触膜融合，导致神经递质释放到突触裂隙中。这一过程是非常动态的、可塑性的，并且受到高度监管。虽然对接和融合的分子成分已经确定，但还不清楚它们是如何相互作用来调节对接、孔道开放和融合的动力学的。特别是，对调节突触小泡融合的蛋白质相互作用的详细机制知之甚少。目前的应用将集中在这一关键问题上，通过建模和实验相结合的方式来研究调节突触小泡对接和融合的分子机制。囊泡通过一种特殊的蛋白质复合体(SNARE)紧密地停靠在质膜上，被认为提供了克服膜间斥力的必要力量，从而介导了囊泡融合。刺激引起的融合是由钙离子的涌入触发的，它与与SNARE复合体紧密耦合的囊泡蛋白突触素(SYT)相互作用。融合孔的打开被认为是由SYT和一个小蛋白复合体(CPX)与SNARE复合体的相互作用控制的。尽管已经用生化和分子生物学工具研究了这些蛋白质的分子相互作用，但仍然缺乏对这些蛋白质如何动态相互作用以及蛋白质融合机制的力如何抵消突触和囊泡膜内产生的力的了解。为了阐明这些机制，我们建议建立融合机制的分子模型，并对融合复合体的动力学进行计算机模拟。为了了解囊泡、突触膜和蛋白质融合机制之间的相互作用，我们将建立膜/囊泡动力学的粗粒度模型，并将其与融合蛋白复合体的原子模型相结合。为了验证模型，我们将模拟融合复合体中单点突变对释放动力学的影响，并通过实验验证我们的预测。这些实验将在果蝇神经肌肉接头(NMJ)上进行，这是一个理想的可用于遗传操作的模型系统。为了验证模型的预测，我们将结合电生理学和光学荧光显微镜来评估NMJ中的释放动力学，其中融合机制通过点突变进行修改，并通过计算预测对膜融合的影响。这项研究将由一个多学科团队进行，其中包括分子建模专家(贾戈塔博士)、膜力学和动力学专家(许博士)、突触生理学专家(Bykhovskaia博士)和果蝇神经生物学专家(Littleton博士)。由一个平衡地代表所有方面的协作团队对这个问题进行攻击，将导致对 调节突触小泡融合过程。更广泛的影响：中央加勒比大学(UCC)是波多黎各(美国联邦)的一家拉美裔服务机构。拟议的项目将使UCC能够与备受推崇的内地机构发展紧密联系，从而为不同背景的学生创造培训和就业机会。这位名叫Bykhovskaia的PI是UCC专门神经科学研究项目(SNRP)的负责人(由NIH资助)，该项目的目标是提高招生人数占多数的少数族裔院校的研究标准。因此，拟议的项目将涉及生物医学研究中代表性不足的理工科、理工科、博士和医学博士学生。此外，贾戈塔博士还指导利哈伊大学的本科生和研究生生物工程项目，这是一所平衡强调研究、教学和培训的机构。拟议的研究将由研究生进行，并将通过研究学分和暑期机会积极吸引本科生参与。这项研究将通过利哈伊大学的贾戈塔博士开发的生物分子和细胞力学课程纳入本科生物工程课程。它将与其他活动紧密结合在一起，包括学生交流和跨学科研讨会，从而促进不同智力和种族背景的研究和培训的整合。

项目成果

Hydrodynamics govern the pre-fusion docking time of synaptic vesicles.

流体动力学控制突触小泡的融合前对接时间。

• DOI: 10.1098/rsif.2017.0818
• 发表时间: 2018
• 期刊: Journal of the Royal Society, Interface
• 作者: Singh,Pankaj;Hui,Chung-Yuen
• 通讯作者: Hui,Chung-Yuen

Interaction of the Complexin Accessory Helix with Synaptobrevin Regulates Spontaneous Fusion.

• DOI: 10.1016/j.bpj.2016.09.017
• 发表时间: 2016-11
• 期刊: Biophysical journal
• 影响因子: 3.4
• 作者: Alexander Vasin;Dina Volfson;J. Littleton;M. Bykhovskaia

Complexin Mutants Reveal Partial Segregation between Recycling Pathways That Drive Evoked and Spontaneous Neurotransmission.

复合蛋白突变体揭示了驱动诱发神经传递和自发神经传递的循环途径之间的部分分离。

• DOI: 10.1523/jneurosci.1854-16.2016
• 发表时间: 2017
• 期刊: The Journal of neuroscience : the official journal of the Society for Neuroscience
• 作者: Sabeva,Nadezhda;Cho,RichardW;Vasin,Alexander;Gonzalez,Agustin;Littleton,JTroy;Bykhovskaia,Maria
• 通讯作者: Bykhovskaia,Maria

A continuum model of docking of synaptic vesicle to plasma membrane.

突触小泡与质膜对接的连续体模型。

• DOI: 10.1098/rsif.2014.1119
• 发表时间: 2015
• 期刊: Journal of the Royal Society, Interface
• 作者: Liu,Tianshu;Singh,Pankaj;Jenkins,JamesT;Jagota,Anand;Bykhovskaia,Maria;Hui,Chung-Yuen
• 通讯作者: Hui,Chung-Yuen

Phosphatidylinositol (4, 5)-bisphosphate targets double C2 domain protein B to the plasma membrane.

• DOI: 10.1111/tra.12528
• 发表时间: 2017-12
• 期刊: Traffic (Copenhagen, Denmark)
• 作者: Michaeli L;Gottfried I;Bykhovskaia M;Ashery U
• 通讯作者: Ashery U