Axonal transport of proteins and organelles between the neuronal cell body and axon terminals is essential for axon outgrowth, formation of functional synapses, and neuronal survival. While anterograde transport (cell body to axon terminal) relies on the large family of kinesin motor proteins, retrograde cargo transport (from axon terminals towards the cell body) primarily utilizes one motor complex, cytoplasmic dynein. How cargo binds to this single motor selectively and is transported to the proper location is largely unknown. It has been postulated that this process involves adaptor proteins, which bind cargo to either the core dynein motor complex or its accessory complex, dynactin. My long-term goals are to identify mediators of specific retrograde cargo transport, define their function and determine how disruption of this process impacts circuit formation and activity. Because of their unique genetic tools and imaging accessibility, zebrafish are the ideal system to study retrograde axonal transport and the functional consequences of its disruption in an intact vertebrate. Importantly, most cellular processes that regulate axonal transport are highly conserved between mammals and zebrafish. To begin addressing my goals, I used a forward genetic screen to identify four mutant strains that display phenotypes indicative of interrupted retrograde cargo transport, including axon terminal swellings. One of these strains carries a mutation in JNK-interacting protein 3 (Jip3). Preliminary analyses revealed that jip3 mutants exhibit truncation of long axons and accumulation of activated Ret (GDNF responsive receptor tyrosine kinase) in mutant axon growth cones.
In Aim 1, I will address the hypothesis that Jip3 serves as an adaptor protein required for retrograde transport of Ret signaling endosomes, which is necessary for axon extension. The second mutant identified in my screen displays accumulation of mitochondria in axon terminal swellings due to interrupted retrograde transport of this organelle. Anterograde mitochondrial transport and retrograde transport of other cargos are normal. The phenotype in this mutant is due to loss of Actr10, a known member of the dynein accessory complex, dynactin.
In Aim 2, I will determine whether Actr10 functions as an adaptor mediating retrograde transport of mitochondria using in vivo imaging and biochemical dissection of interaction domains in the Actr10 protein.
In Aim 3, I will use my established protocols and new techniques to determine if retrograde transport of specific cargos is disrupted in my additional novel mutants and how these defects affect function of the circuit. My preliminary data show that these strains have mutations in known dynein interactors, all with unknown functions in axonal transport. Finally, in Aim 4, I will engineer transgenic zebrafish strains which will be used to identify the Actr10 interactome and further dissect the molecular mechanisms that govern retrograde axonal transport of specific cargos. With the data, skill sets, and tools acquired from the proposed experiments, I will be poised to decipher the modulation of retrograde axonal transport of various cargos as an independent investigator.
Disruption of retrograde axonal transport contributes significantly to diseases of the nervous system. Strikingly little is known about how selective retrograde cargo transport is accomplished, though understanding this process is critical to designing therapeutic strategies. The work proposed here will advance our understanding of selective retrograde cargo transport, shedding light on basic mechanisms corrupted in disease states.
|Drerup, Catherine M; Lusk, Sarah; Nechiporuk, Alex (2016) Kif1B Interacts with KBP to Promote Axon Elongation by Localizing a Microtubule Regulator to Growth Cones. J Neurosci 36:7014-26|
|Drerup, Catherine M; Nechiporuk, Alex V (2016) In vivo analysis of axonal transport in zebrafish. Methods Cell Biol 131:311-29|