S-acylation dependent regulation of fast axonal transport in neurons

神经元是大型的、形态复杂的细胞，其在位于称为树突的短突起上的突触或神经元-神经元连接处接收输入。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。将神经元蛋白和细胞器有效运输和递送到特定的亚细胞位置对于神经元功能至关重要，但在人类长达一米的长轴突距离上可能具有挑战性。尽管神经元运输的重要性，但对其管理机制知之甚少。快速轴突运输（FAT）是通过动力蛋白和驱动蛋白马达沿着微管携带神经递质和神经营养因子、内体和溶酶体、mRNA颗粒、自噬体和促变性信号复合物的囊泡的连续快速运动。为了实现FAT，电机必须长距离连续移动货物，这需要恒定的能源。有趣的是，糖酵解酶最近被发现在脂肪囊泡，他们提供了一个'板上'恒定的能量供应电机。然而，分子马达如何在FAT期间起作用以及胞质糖酵解酶如何拴系到囊泡上尚不清楚。将蛋白质引导至特定膜的一种机制是脂肪酸与蛋白质半胱氨酸残基的共价加成，该过程称为S-酰化。最近的S-酰基蛋白质组学研究表明，糖酵解酶以及一些驱动蛋白和动力蛋白运动亚基及其激活剂是S-酰化的。因此，我推测，S-酰化拴多种马达蛋白，其激活剂，糖酵解酶囊泡提供“船上”的能量和脂肪所需的连续运动。该提案旨在建立一个研究计划，重点是了解轴突中蛋白质和细胞器运输的分子基础。本研究项目的短期目标将集中于阐明S-酰化在FAT中的作用，具体目标如下：（1）定义FAT转运机制的S-酰化的酶和分子调控，（2）确定转运机制的S-酰化是否是FAT所需的，以及（3）表征FAT对体内S-酰化的依赖性。这项工作将使用一系列先进的生物化学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中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的分子机制的最前沿，这些机制对神经发育，突触功能和轴突变性有影响。

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.