Analysis of axonal transport mutants in Drosophila

Members of the dynein and kinesin superfamilies are the main motor proteins that are responsible for axonal transport. To better understand the mechanism of the transport process, and to learn how it is regulated and what proteins are involved, we conducted a genetic screen in Drosophila. A number of axonal transport defective mutants were identified. These mutants had two characteristics in common. They all had a unique tail flip phenotype and showed abnormal accumulations of synaptic proteins in their segmental nerves. Two such mutants are the focus of my work. I mapped the first mutant, redtape, to the left arm of the second chromosome at cytological location 23E. I was able to narrow the region in which redtape is located to 30kb. There are a number of interesting genes in this region that are currently being studied.; The second mutant, gridlock, was mapped by recombination and complementation mapping to the third chromosome, to cytological location 87C. I showed that gridlock is a missense mutation in the arp1 (actin related protein) gene. Arp1 is the most abundant subunit of the dynactin complex, which associates with the dynein motor. Further analysis of the arp1 mutant showed that in this mutant, the dynactin complex is disrupted. This allowed me to compare the phenotype of the arp1 mutant, which lacks dynactin, to dynein heavy chain (dhc) mutants. I showed that both dynein and dynactin are required for many aspects of the cell cycle and oogenesis, and that lack of dynein or dynactin leads to programmed cell death in the larval brain. I did not observe a significant phenotypic difference between arpl and dhc. Thus, I used biochemical assays to further probe dynactin's function. Using a microtubule-binding assay, I showed that in the absence of dynactin, less dynein binds to microtubules. This finding is consistent with the proposal that dynactin is required for dynein's processivity and microtubule attachment. I also tested the role of the dynactin complex in binding dynein to membranous cargo. I performed a membrane fractionation assay and showed that with or without dynactin, dynein binds efficiently to membranes. In vivo analysis of the arpl mutant showed profound inhibition in rate and number of APP containing vesicles moving in either direction.