MOLECULAR MECHANISMS OF ANTEROGRADE TRANSPORT

顺行运输的分子机制

• 批准号: 7955686
• 负责人: ELAINE L BEARER
• 金额: $ 0.68万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-08-01 至 2010-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7955686
• 关键词: Actins; Affect; Alzheimer' s Disease; Amino Acid Sequence; Amyloid beta-Protein Precursor; Axon; Axonal Transport; Biological Assay; Cells; Computer Retrieval of Information on Scientific Projects Database; Confocal Microscopy; Cultured Cells; Dynein ATPase; Funding; Grant; Herpesvirus 1; Human; Image; Institution; Kinesin; Label; Length; Life Cycle Stages; Location; Maintenance; Mediating; Microtubules; Modeling; Molecular; Molecular Motors; Motor; Myosin ATPase; Neurons; Pathogenesis; Peptide Fragments; Peptides; Physiology; Process; Proteolysis; Research; Research Personnel; Resources; Role; Simplexvirus; Source; Squid; Synapses; Time; Travel; United States National Institutes of Health; Virus; Virus Receptors; anterograde transport; base; cell motility; computational anatomy; protein aminoacid sequence; receptor; reconstitution; retrograde transport; tool

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Anterograde transport is crucial for maintenance of the synapse in neurons. Anterograde transport over long distances in neuronal processes is mediated by a combination of actin and microtubule tracks and molecular motors, kinesin, dynein and myosin. How cargo attaches to the correct motor to be transported to specific locations within the cell is a major question in neuronal physiology. Numerous studies suggest a role for amyloid precursor protein (APP), which when proteolyzed produces the toxic AB fragment of Alzheimer's disease. We have developed human herpes simplex virus 1, HSV, as a tool to discover molecular mechanisms of cargo-transport interactions. At different times in its life cycle, HSV travels in either the retrograde or the anterograde direction in neuronal processes. We reconstituted retrograde transport of HSV in the giant axon of the squid by injecting GFP-VP16 labeled HSV into axon and imaged transport by confocal microscopy. In this project, anterograde transport of GFP- and mRFP1- labeled HSV will be reconstituted in the giant axon. This assay will be used to discover the molecular basis for cargo-motor interactions. In Preliminary Results, we show that GFP-VP16-labeled HSV is also transported in the anterograde direction in the giant axon. Motile virus co-purifies with full length APP -- and extraction of APP removes motility. By coating fluorescent beads with peptide fragments of APP and injecting then into the axon, we discovered a 15 amino acid sequence sufficient to mediate transport of the beads. In this application, we will: (1) analyze the direction and velocity of HSV anterograde transport and identify the motor receptor(s) that the virus uses, (2) determine the peptide sequence of APP sufficient for anterograde motility using peptide-coated fluorescent beads injected into the giant axon, (3) determine how HSV acquires the motor receptor during its synthesis in cultured cells, and (4) discover whether HSV affects APP proteolysis into toxic fragments. Results from these studies will definitively identify the mechanism of HSV transport and the role of APP. Thus, these results will yield answers to universal questions on axonal transport, specific facts about herpes pathogenesis, and fundamental new information about the role of APP in Alzheimer's disease.

该副本是利用众多研究子项目之一 由NIH/NCRR资助的中心赠款提供的资源。子弹和 调查员（PI）可能已经从其他NIH来源获得了主要资金， 因此可以在其他清晰的条目中代表。列出的机构是 对于中心，这不一定是调查员的机构。 顺行转运对于维持神经元突触至关重要。肌动蛋白和微管轨道和分子电机，驱动蛋白，动力蛋白和肌球蛋白的组合介导了神经元过程中长距离的顺行传输。货物如何连接到正确的电动机，以运输到细胞内的特定位置是神经元生理学的主要问题。大量研究表明，淀粉样蛋白前体蛋白（APP）的作用，当蛋白水解产生阿尔茨海默氏病的有毒AB片段时。我们已经开发了人类单纯疱疹病毒1，HSV，是发现货物交通相互作用的分子机制的工具。在其生命周期的不同时间，HSV在神经元过程中的逆行或顺行向行进。我们通过将GFP-VP16标记为轴突标记为轴突，并通过共聚焦显微镜将HSV注入GFP-VP16，重新组建了HSV的逆行运输。在这个项目中，将在巨型轴突中重构GFP和MRFP1标记的HSV的顺行运输。该测定法将用于发现货物运动相互作用的分子基础。在初步结果中，我们表明GFP-VP16标记的HSV也沿巨型轴突的顺行向运输。 Motile病毒与全长应用共同绘制 - 提取应用程序可以消除运动性。通过用App的肽片段涂层荧光珠并将其注射到轴突中，我们发现了一个足以介导珠子转运的15个氨基酸序列。在此应用中，我们将：（1）分析病毒使用的HSV前进运输的方向和速度，并确定病毒使用的运动受体，（2）确定APP的肽序列足以使用肽涂层的荧光磁珠注射到巨型轴突中，（3）确定HSV的conts and cann and in Crantirors and cann cann and cann cann and interodrorade sotility and cann and in Cyn cann and cann and and cant and cant and cant and cant and cants and（3） HSV会影响APP蛋白水解成有毒片段。这些研究的结果将确定确定HSV转运的机制和APP的作用。因此，这些结果将为有关轴突运输的普遍问题，有关疱疹发病机理的具体事实以及有关APP在阿尔茨海默氏病中作用的基本新信息的答案。