S-acylation dependent regulation of fast axonal transport in neurons

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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.