S-acylation dependent regulation of fast axonal transport in neurons

神经元是一种巨大的、形态复杂的细胞，它通过位于树突上的突触或神经元之间的连接接收输入。然后，信息通过被称为轴突的长输出投射传递到下游神经元。神经元蛋白和细胞器到特定亚细胞位置的有效运输和递送对神经元功能至关重要，但在长轴突距离（人类长达一米）上可能具有挑战性。尽管贩运神经元很重要，但人们对其控制机制知之甚少。快速轴突运输（Fast axonal transport， FAT）是指携带神经递质和神经营养因子、核内体和溶酶体、mRNA颗粒、自噬体和促退行性信号复合物的囊泡通过动力蛋白和动力蛋白马达沿微管进行连续、快速的运动。为了实现FAT，电机必须在长距离上连续移动货物，这需要恒定的能量来源。有趣的是，最近在FAT囊泡上发现了糖酵解酶，它们为发动机提供“机载”持续的能量供应。然而，在FAT过程中分子马达是如何起作用的，以及胞质糖酵解酶是如何与囊泡相连的，目前尚不清楚。将蛋白质导向特定膜的一种机制是将脂肪酸共价添加到蛋白质半胱氨酸残基上，这一过程被称为s -酰化。最近对s -酰基蛋白质组学的研究表明，糖酵解酶以及许多运动蛋白和动力蛋白运动亚基及其激活剂都是s -酰基化的。因此，我假设s -酰化将多种运动蛋白、它们的激活剂和糖酵解酶拴在囊泡上，以提供脂肪所需的“机载”能量和持续运动。本提案旨在建立一个研究项目，专注于了解轴突中蛋白质和细胞器运输的分子基础。我这个研究项目的短期目标将集中在阐明s -酰化在FAT中的作用，通过以下具体目标：(1)定义脂肪转运机制的s -酰化的酶和分子调控，(2)确定脂肪转运机制是否需要s -酰化，(3)表征体内脂肪对s -酰化的依赖性。这项工作将使用一系列先进的生化和细胞生物学技术进行，包括常规和微流体神经元细胞培养，病毒介导的敲除/拯救，神经元和视神经外植体的活细胞共聚焦成像，以及专门的s -酰化测定。总的来说，这个多方面的项目将使我的团队处于研究FAT与神经发育、突触功能和轴突退化相关的分子机制的前沿。

Neurons are large, morphologically complex cells that receive input at synapses or neuron-neuron connections located on short projections called dendrites. Information is then transmitted to downstream neurons via long output projections known as axons. The efficient trafficking and delivery of neuronal proteins and organelles to specific subcellular locations is critical for neuronal function but can be challenging over long axonal distances, up to a metre in humans. Despite the importance of trafficking in neurons, the governing mechanisms are poorly understood. Fast axonal transport (FAT) is the continuous, fast movement of vesicles carrying neurotransmitters and neurotrophins, endosomes and lysosomes, mRNA granules, autophagosomes, and pro-degenerative signaling complexes along microtubules by dynein and kinesin motors. To achieve FAT, motors must move cargo continuously over long distances, which requires a constant source of energy. Interestingly, glycolytic enzymes were recently found on FAT vesicles where they provide motors with an `on-board' constant supply of energy. However, how molecular motors function during FAT and how the cytosolic glycolytic enzymes are tethered to vesicles is not known. One mechanism to direct proteins to specific membranes is the covalent addition of fatty acids to protein cysteines residues, a process known as S-acylation. Recent curation of S-acyl proteomic studies reveals that glycolytic enzymes as well as a number of kinesin and dynein motor subunits and their activators are S-acylated. I therefore hypothesize that S-acylation tethers multiple motor proteins, their activators, and glycolytic enzymes to vesicles to provide `on-board' energy and continuous movement required for FAT. This proposal seeks to establish a research program focused on understanding the molecular basis of protein and organelle transport in axons. My short-term goals for this research project will focus on elucidating the role of S-acylation in FAT through the following specific objectives: (1) define the enzymatic and molecular regulation of S-acylation of FAT transport machinery, (2) determine if S-acylation of transport machinery is required for FAT, and (3) characterize the dependence of FAT on S-acylation in vivo. This work will be carried out using a range of advanced biochemical and cell biological techniques including conventional and microfluidic neuronal cell culture, viral mediated knockdown/rescue, live cell confocal imaging in neurons and optic nerve explants, and specialized S-acylation assays. Overall, this multifaceted project will position my group at the forefront of research into the molecular mechanisms that govern FAT with implications for neurodevelopment, synaptic function, and axon degeneration.