Bar Domains and Neuronal Membrane Structure

Bar 结构域和神经元膜结构

• 批准号: 8470247
• 负责人: TOBIAS MEYER
• 金额: $ 26.37万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-01 至 2015-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8470247
• 关键词: 3-Dimensional; Algorithms; Architecture; Binding; Binding Proteins; Biological Assay; Cell membrane; Cell physiology; Cells; Cellular Neurobiology; Characteristics; Complementary DNA; Complex; Computer Vision Systems; Defect; Dendrites; Development; Disease; Family; Filopodia; Generations; Goals; Hippocampus (Brain); Image Analysis; Impairment; In Vitro; Individual; Intracellular Membranes; Kinetics; Length; Life; Measurement; Measures; Medical; Membrane; Membrane Lipids; Membrane Proteins; Methodology; Microscopy; Molecular; Monitor; Nanostructures; Natural regeneration; Neck; Nerve Degeneration; Neurites; Neurodegenerative Disorders; Neurons; Pharmaceutical Preparations; Process; Property; Protein Binding; Protein Binding Domain; Proteins; Psyche structure; Radial; Role; Set protein; Shapes; Small Interfering RNA; Specificity; Structural Protein; Structure; Synapses; System; Tertiary Protein Structure; Vertebral column; Work; base; cell type; cellular imaging; drug development; in vitro Assay; new technology; novel; overexpression; preference; programs; response; self assembly

DESCRIPTION (provided by applicant): The goal of the proposed work is to systematically explore whether and how proteins that sense and shape the curvature of plasma membranes are responsible for building the intricate dendritic and axonal arbors that distinguish neurons from other cell types. The formation of complex 3-dimensional branched membrane structures is one of the most fundamental properties of neurons that enable them to transmit information between neurons and from neurons to other cell types. The ability of selected proteins to sense membrane curvature during this differentiation process is important as defects in proteins, such as Oligophrenin and srGAP2, that can bind to and shape lipid membranes cause neurodegenerative diseases. Our proposal aims to develop and execute a scalable experimental strategy to understand the process of arbor formation by focusing on a family of Bar domain containing proteins that are known from in vitro studies to be able to bind to and shape curved membranes. We will systematically investigate their function in generating the branched extended plasma membrane architecture of neurons. Currently available in vitro assays and structural studies of proteins with membrane binding domains can determine the radius of the membrane curvature that results from the formation of oligomers by curvature sensing proteins. Using this approach, proteins have been identified that sense and shape membranes with positive and negative curvatures. Nevertheless, it is difficult from these assays to know to which curved intracellular membranes these proteins may bind, and if or how they act dynamically to generate distinct types of curved plasma membranes in a living cell. We developed a novel assay to investigate curvature dependent processes that is based on fabricated nanostructures that trigger plasma membrane curvature in living cells. Specifically, our project will deliver a new scalable assay based on these nanostructures that allows one to measure in living cells the intracellular membrane localization and the curvature preference as well as the dynamic assembly, disassembly and exchange rate of curvature sensing membrane binding proteins. Our initial studies already identified and characterized a key regulator that binds to positively curved plasma membranes and is critically involved in controlling neuronal architecture. We have combined this approach with parallel high-throughput live-cell imaging and automated image analysis of cultured hippocampal neurons that enables us to systematically analyze the cellular roles of these same Bar domain binding proteins in controlling the neuronal architecture. At the center of our work is the development of this synergistic dual experimental approach that can ultimately be used as an unbiased and systematic platform to investigate the neuronal roles of a large number of putative neuronal membrane binding proteins. Together, our project will provide a molecular framework to understand the program used by neurons to create the vast repertoire of different neuronal architectures.

描述（由申请人提供）：拟议工作的目标是系统地探索感知和塑造质膜曲率的蛋白质是否以及如何负责构建将神经元与其他细胞类型区分开来的复杂树突和轴突乔木。复杂的三维分支膜结构的形成是神经元最基本的特性之一，使它们能够在神经元之间以及从神经元到其他细胞类型传递信息。选定的蛋白质在分化过程中感知膜曲率的能力非常重要，因为寡糖蛋白和 srGAP2 等蛋白质的缺陷可以与脂质膜结合并塑造脂质膜，从而导致神经退行性疾病。我们的提案旨在开发和执行可扩展的实验策略，通过关注含有 Bar 结构域的蛋白质家族来了解乔木形成的过程，这些蛋白质从体外研究中已知能够结合并塑造弯曲的膜。我们将系统地研究它们在生成神经元分支延伸质膜结构中的功能。 目前可用的具有膜结合域的蛋白质的体外测定和结构研究可以确定由曲率传感蛋白形成寡聚物而产生的膜曲率半径。使用这种方法，已经鉴定出能够感知和塑造具有正曲率和负曲率的膜的蛋白质。然而，从这些测定中很难知道这些蛋白质可能结合哪些弯曲的细胞内膜，以及它们是否或如何动态地作用以在活细胞中产生不同类型的弯曲质膜。 我们开发了一种新颖的测定方法来研究曲率相关过程，该方法基于触发活细胞质膜曲率的制造纳米结构。具体来说，我们的项目将提供一种基于这些纳米结构的新的可扩展测定方法，允许人们测量活细胞中的细胞内膜定位和曲率偏好，以及曲率传感膜结合蛋白的动态组装、拆卸和交换率。我们的初步研究已经确定并表征了一个关键的调节因子，该调节因子与正弯曲的质膜结合，并且在控制神经元结构中发挥着至关重要的作用。我们将这种方法与培养海马神经元的并行高通量活细胞成像和自动图像分析相结合，使我们能够系统地分析这些相同的 Bar 结构域结合蛋白在控制神经元结构中的细胞作用。我们工作的核心是开发这种协同双重实验方法，该方法最终可以用作一个公正的系统平台来研究大量假定的神经元膜结合蛋白的神经元作用。总之，我们的项目将提供一个分子框架来理解神经元使用的程序来创建不同神经元结构的庞大库。