Computational and Functional Characterization of the Molecular Steps in Membran*

膜分子步骤的计算和功能表征*

• 批准号: 8511841
• 负责人: MARIA BYKHOVSKAIA
• 金额: $ 28.39万
• 依托单位: UNIVERSIDAD CENTRAL DEL CARIBE
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-07-16 至 2017-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8511841
• 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; Hispanics; 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; Plastics; Point Mutation; Process; Proteins; Puerto Rico; Reaction; Regulation; Research; Research Training; SNAP receptor; Schizophrenia; Series; Simulate; 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; 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（由申请人提供）： 智力优点：在细胞水平上，学习和记忆受突触传递功效的变化，特别是神经元递质释放的动态调节的控制。神经递质被包装到突触小泡中，突触小泡停靠在突触膜上，经历一系列准备步骤，打开一个孔，并与突触膜融合，导致神经递质释放到突触间隙中。这个过程是非常动态的、可塑的并且受到高度监管。尽管对接和融合的分子成分已被识别，但尚不清楚它们如何相互作用以调节对接、孔开放和融合的动力学。特别是，人们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。本申请将通过结合建模和实验来研究调节突触小泡对接和融合的分子机制来关注这一关键问题。囊泡通过特殊的蛋白质复合物（SNARE）紧密地停靠在质膜上，这被认为提供了克服膜间排斥所需的力，从而介导囊泡融合。刺激诱发的融合是由 Ca2+ 离子的流入触发的，Ca2+ 离子与囊泡蛋白突触结合蛋白 (Syt) 相互作用，而突触结合蛋白与 SNARE 复合体紧密耦合。融合孔的开放被认为是由 Syt 和小蛋白复合物 (Cpx) 与 SNARE 复合物的相互作用控制的。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如何平衡突触和囊泡膜内产生的力的了解。为了阐明这些机制，我们建议建立聚变机制的分子模型并对聚变复合物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，我们将开发膜/囊泡动力学的粗粒模型，并将其与融合蛋白复合物的原子模型集成。为了验证该模型，我们将模拟融合复合物中单点突变对释放动力学的影响，并通过实验测试我们的预测。这些实验将在果蝇神经肌肉接头（NMJ）上进行，这是一个非常适合基因操作的模型系统。为了测试模型的预测，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。这项研究将由多学科团队进行，其中包括分子建模（Jagota 博士）、膜力学和动力学（Hui 博士）、突触生理学（Bykhovskaia 博士）和果蝇神经生物学（Littleton 博士）方面的专家。一个平衡地代表各个方面的协作团队对这个问题的攻击将带来对这个问题的新的、详细的和定量的理解。 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）的一所西班牙裔服务机构。拟议的项目将使UCC能够与备受推崇的内地院校建立紧密联系，从而为具有不同背景的学生创造培训和就业机会。 PI Bykhovskaia 博士负责指导 UCC 的专业神经科学研究计划 (SNRP)（由 NIH 资助），该计划的目标是提高以代表性不足的少数族裔为主的机构的研究标准。因此，拟议的项目将涉及生物医学研究中代表性不足的学士、硕士、博士和医学博士生。此外，Jagota 博士还负责理海大学 (Lehigh University) 的本科生和研究生生物工程课程，该大学是一家均衡重视研究、教学和培训的机构。拟议的研究将由研究生进行，并将积极让本科生通过研究获得学分和暑期机会。这项研究将通过理海大学 Jagota 博士开发的生物分子和细胞力学课程纳入本科生生物工程课程。它将与其他活动紧密结合，包括学生交流和跨学科研讨会，从而促进不同知识和种族背景的研究和培训的融合。