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Characterization of the Behavior and Regulation of Pioneer Microtubules at the Larval Drosophila Neuromuscular Junction


Here live imaging and genetic manipulations are combined to visualize and quantitatively characterize a population of dynamic pioneer microtubules within the Drosophila neuromuscular junction (NMJ). First, a GFP-tagged microtubule plus-end binding protein, EB1-GFP, was used to qualitatively and quantitatively characterize the growth and invasion of dynamic axonal and synaptic microtubule plus-ends in vivo. Axonal microtubules were found to be uniformly oriented. EB1-GFP movement in the axons was processive and constrained to a straight path. Interestingly, plus-end growth was found to be significantly slower in sensory (3.02 µm/minute ± 0.12) compared to motor (4.12 µm/min ± 0.038) axons, and is significantly faster within the NMJ (4.66 µm/min ± 0.10) compared to the motor axon. Unlike axonal movement, plus-end movement at the synapse was of mixed orientation. Genetic and imaging analysis revealed the movement of dynamic plus-ends to be largely independent of the Map1b-like protein Futsch, though the dynein/dynactin complex was shown to be required for normal plus-end growth. EB1-GFP labeled puncta are often observed exploring nascent and newly formed synaptic regions, an exploratory behavior that may play a role in synapse development. Supporting movies are presented as supplemental data. This tool was next applied to the study of the formin protein, diaphanous (dia), a gene identified in a screen for mutations effecting growth at the NMJ. Diaphanous is present both pre- and postsynaptically however, genetic rescue and interaction experiments support the conclusion that dia is necessary presynaptically for normal NMJ growth. Formin family proteins are potent regulators of the cytoskeleton and defects in cytoskeletal organization or regulation could correlate with the synaptic growth defect. Live imaging of dynamic microtubules and actin revealed defects in both cytoskeletal elements at the synapse. Finally, genetic evidence is presented demonstrating Dia functions downstream of the presynaptic receptor tyrosine phosphatase Dlar and the Rho-type GEF trio to control NMJ growth. Based upon the established function of DRFs as Rho-GTPase dependent regulators of the cell cytoskeleton, a model is proposed in which Diaphanous links receptor tyrosine phosphatase signaling at the plasma membrane to growth-dependent modulation of the synaptic actin and microtubule cytoskeletons.

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