Motor protein disruption critically alters organelle trafficking, NMJ formation, and excitation contraction coupling.

Abstract

Trafficking of intracellular cargoes along the neuronal axon microtubule tracks is a motor-protein-dependent process. It is well-established that the motor protein kinesin is responsible for anterograde trafficking of axonal cargo, while the dynein/dynactin complex regulates retrograde trafficking. However, there is still much to uncover regarding the various isoforms of these motor proteins as well as the adapter and cargo-associated proteins involved in the precise trafficking dynamics. Here we use a targeted genetic approach to knockdown candidate kinesin genes involved in trafficking organelles like synaptic vesicles, mitochondria, and dense core vesicles in motor neurons. Using fluorescently tagged cargo proteins; live-imaging experiments were conducted to quantify intracellular trafficking changes, and 2 genes, 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 in somas and axons. Downstream, disruptions in both kinesin 1 and 3 expression led to significant changes in neuropeptide (NP) abundance at boutons, and changes in synaptic morphology, including innervation length, bouton number, and active zone composition. Spinning disc confocal imaging revealed fewer NP trafficking through, or getting captured in kinesin knockdown experiments, and a dramatic reduction in NP release at motor neuron terminals. We go on to show profound reductions in neuromuscular transduction, and excitation-contraction coupling in kinesin 1 knockdowns, but not for kinesin 3. Changes in larval crawling as well as development were observed for kinesin 1 knockdowns. Taken together we have not only identified which kinesins are critically involved in organelle trafficking, but also revealed critical disruptions in cellular morphology, function, physiology, and behavior in genetically disrupted animals.