Abstract
Efficient intracellular transport is essential for healthy cellular function and structural integrity, and problems in this pathway can lead to neuronal cell death and disease. To spatially and temporally evaluate how transport defects are initiated, we adapted a primary neuronal culture system from Drosophila larval brains to visualize the movement dynamics of several cargos/organelles along a 90 micron axonal neurite over time. All six vesicles/organelles imaged showed robust bi-directional motility at both day 1 and day 2. Reduction of motor proteins decreased the movement of vesicles/organelles with increased numbers of neurite blocks. Neuronal growth was also perturbed with reduction of motor proteins. Strikingly, we found that all blockages were not fixed, permanent blocks that impeded transport of vesicles as previously thought, but that some blocks were dynamic clusters of vesicles that resolved over time. Taken together, our findings suggest that non-resolving blocks may likely initiate deleterious pathways leading to death and degeneration, while resolving blocks may be benign. Therefore evaluating the spatial and temporal characteristics of vesicle transport has important implications for our understanding of how transport defects can affect other pathways to initiate death and degeneration.
Highlights
Within axons the intracellular transport of vesicles, organelles, and biomolecules is essential for the maintenance of its structure and for its function
Our analysis indicates that reduction of motor proteins perturbs vesicle transport both spatially and temporally, and these attributes likely contribute to the defects seen in neuronal growth
Our results led us to two major findings, 1) the long-term temporal changes observed in vesicle motility may reflect functional roles in the developing neuron, and 2) vesicle blockages possess inherent dynamic motility behaviors that change over time
Summary
Within axons the intracellular transport of vesicles, organelles, and biomolecules is essential for the maintenance of its structure and for its function Such a complex pathway utilizes motor proteins, kinesin-1 and dynein, for anterograde and retrograde transport on a vast network of microtubules (MTs) [1,2,3]. Primary neuronal cultures allow for continuous high resolution observation of vesicle motility, long-term, without the confounding effects of anesthetics and larval movements during recordings. Such analysis can lead to new insights on both the spatial and temporal mechanisms of vesicle motility and their role for neuronal growth and maintenance, which are currently unclear
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