Abstract
Trafficking of intracellular cargoes along the neuronal axon microtubule tracks is a motor-protein-dependent process. Here we use a targeted genetic approach to knockdown candidate kinesin genes involved in trafficking organelles in male and female Drosophila melanogaster. Live-imaging experiments revealed intracellular trafficking changes, and kinesins 1 and 3 were identified as critical regulators. Disruptions in either gene product reduce rates of axonal trafficking in motor neurons, and lead to the formation of large intracellular aggregates. Kinesin disruptions led to significant changes in neuropeptide abundance at boutons, and changes in synaptic morphology. Confocal imaging revealed fewer neuropeptides trafficking through, or getting captured by synapses in kinesin knockdown experiments, and a dramatic reduction in neuropeptide release at motor neuron terminals. A profound reduction in neuromuscular transduction, and excitation-contraction coupling in kinesin 1 knockdowns, but not for kinesin 3 was observed. Collectively, the targeted genetic screen of kinesin proteins revealed disruptions in kinesin 1 and 3 greatly impact intracellular axonal trafficking. Taken together, several kinesins were identified which critically regulate organelle trafficking, and genetic disruptions in key kinesins also revealed critical disruptions in cellular morphology, function, physiology, and behavior.
Significance Statement Intracellular trafficking of cargo is a vital process critical to the survival of most cells. The functional cells within the nervous system, in neurons are particularly dependent on trafficking given their profound energy consumption, and need to transport materials over long distances, often exceeding one meter. Here we conduct a targeted genetic screen to identify motor proteins involved in trafficking cargo in motor neurons. We reveal two genes previously identified in kinesin 1 and 3, critically involved in cargo transport. Our data show profound reductions in intracellular trafficking, and genetic disruptions lead to massive intracellular aggregations. Using cutting-edge approaches, our investigations uniquely show associated downstream impairments in neuromuscular junction structure, function, physiology, development, and behavior.
Footnotes
Research supported by the National Institute of General Medical Sciences of the National Institutes of Health under Award Number R15GM155985 to KGO. We also thank generous financial support from Middle Tennessee State University to KGO.
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