Understanding neuronal vesicle transport : a study of the complex interplay of molecular motors and effects of anesthetics

Abstract

Neurons are a unique cell type with many distinct membrane domains. Two of these domains, the dendrites and the axon, perform distinct functions in neuronal signaling and require specific complements of membrane proteins. These proteins are delivered by vesicle transport that is mediated by molecular motors. Kinesins and dynein mediate long-range transport along microtubules, and dimeric myosins mediate short-range transport near the cellular membrane. Nearly all eukaryotic cells utilize vesicle transport for survival, but a neuron’s large size and polarity make it particularly reliant on intracellular transport. Correspondingly, deficits in vesicle transport in neurons can contribute to neurodegenerative conditions. This document contains two separate studies examining elements of myosins and kinesins in vesicle trafficking and protein accumulation. Myosin V motor proteins are dimeric and transport to the barbed end of actin filaments. To systematically characterize the vesicle populations bound by myosin V, I developed a novel labeling strategy to visualize myosin Va- and Vb-labeled vesicles in cultured hippocampal neurons. Both myosin Vs bound vesicles that were polarized to the somatodendritic domain where they underwent bidirectional long-range transport. Through a series of two-color imaging experiments, it became clear that myosin Vs specifically colocalized with two different dendrite-selective vesicle populations. Additionally, myosin V bound vesicles concurrently with two Kinesin-3 family members, KIF13A and KIF13B. These results show that coregulation of kinesin and myosin V on vesicles is likely to play an important role in neuronal vesicle transport. This new assay will be applicable in a broad range of cell types to determine the function of myosin V motor proteins. My second project was directed to ask whether the most widely used general anesthetic propofol (2,6-diisopropylphenol) alters the transport properties of neuronal vesicles whose transport is mediated by Kinesin-1 family member KIF5C and Kinesin-3 family member KIF1A. Propofol is known to disrupt the processivity of three kinesins including Kinesin-1 in single molecule motility experiments by shortening the run length without disrupting the velocity. I assessed the effect of propofol on vesicles transported by KIF5C and KIF1A in cultured hippocampal neurons. The experiments showed that propofol reduced the run length and velocity of vesicles transported by KIF5C. This decrease in processivity resulted in a reduction in cargo accumulation at the axon terminal suggesting the potential for propofol to impact neuronal vesicle transport during anesthesia for surgery. Furthermore, vesicles moved by Kinesin-3 family member KIF1A, the primary transporter of presynaptic vesicles, also displayed reduced run length and velocity in response to propofol treatment. These results illustrate that propofol has physiologically relevant effects on at least two different neuronal vesicle populations. Future experiments will address whether propofol also alters fusion of synaptic vesicles at the axonal terminal. In summary, I developed a novel labeling strategy to characterize myosin V-labeled vesicles and I studied the impact of propofol on Kinesin-1- and Kinesin-3-mediated vesicle transport. These results provide new insights into the complexities of the intracellular trafficking machinery that ensures that the vesicle proteins and membrane reach their specific destinations.