Molecular mechanisms of anterograde transport

描述（由申请人提供）： 顺行运输对于神经元中突触的维持至关重要。神经元过程中的长距离顺行运输由肌动蛋白和微管轨道以及分子马达、驱动蛋白、动力蛋白和肌球蛋白的组合介导。货物如何附着到正确的马达上并被运送到细胞内的特定位置是神经生理学中的一个主要问题。许多研究表明淀粉样前体蛋白（APP）的作用，当蛋白水解时产生阿尔茨海默病的有毒AB片段。我们已经开发了人类单纯疱疹病毒1，HSV，作为一种工具，发现货物运输相互作用的分子机制。在其生命周期的不同时期，HSV在神经元过程中以逆行或顺行方向行进。我们通过将GFP-VP 16标记的HSV注射到鱿鱼的巨大轴突中重建HSV的逆行运输，并通过共聚焦显微镜成像运输。在这个项目中，GFP和mRFP 1标记的HSV的顺行运输将在巨大的轴突中重建。该试验将用于发现货物-马达相互作用的分子基础。在初步结果中，我们发现GFP-VP 16标记的HSV也在巨大轴突中以顺行方向运输。运动病毒与全长APP共同纯化-APP的提取去除了运动性。通过用APP的肽片段包被荧光珠，然后注射到轴突中，我们发现了足以介导珠转运的15个氨基酸序列。在此应用程序中，我们将：（1）分析HSV顺行运输的方向和速度并鉴定病毒使用的运动受体，（2）使用注射到巨轴突中的肽包被的荧光珠确定足以进行顺行运动的APP的肽序列，（3）确定HSV在其在培养细胞中的合成期间如何获得运动受体，以及（4）发现HSV是否影响APP蛋白水解成毒性片段。这些研究的结果将明确确定HSV运输的机制和APP的作用。因此，这些结果将产生关于轴突运输的普遍问题的答案，关于疱疹发病机制的具体事实，以及关于APP在阿尔茨海默病中的作用的基本新信息。

DESCRIPTION (provided by applicant): 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.