Genetic analysis of dynein function in the nervous system and in mitosis
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Cytoplasmic dynein-1 is an essential microtubule-based molecular motor. Aided by several adaptors and co-factors, dynein orchestrates retrograde transport of diverse cargoes in interphase cells and plays multiple roles in mitosis. The importance of this motor is underlined by the fact that mutations in Dynein heavy chain, the motor-containing subunit of dynein, cause neurological disorders in humans. How dynein exerts its diverse roles in healthy cells, and how defective motor function causes disease, are not well understood. In this thesis, I describe a genetic dissection of dynein function by using disease-associated, as well as novel, missense mutations in the gene encoding Dynein heavy chain. These mutations had been introduced into the Drosophila melanogaster gene (Dhc) using CRISPR-Cas9 mutagenesis. I document a broad phenotypic characterisation of these mutant alleles, which revealed a subset that causes different degrees of lethality and neurological defects, such as focal protein accumulations along nerves. I show that the severity of the phenotypes observed in flies correlates with the severity of defects observed in previous in vitro studies of motor complex function, indicating that the fly is a valuable model for functional studies of the mutations. I subsequently focussed on two of these mutations: Loa and S3372C. The Loa mutation is located in dynein’s tail domain and was previously shown to compromise processivity of the motor. Flies carrying this allele display severe neurological defects, consistent with previous observations in a mouse model for neurodegenerative disease. I also show, using the translucent wing nerve, that this mutation impairs retrograde transport of mitochondria while not affecting their anterograde transport, suggesting an uncoupling of opposite-polarity molecular motors. The second mutation, S3372C, is a novel, missense change in a highly conserved residue within dynein’s microtubule-binding domain, at the interface with the microtubule. Remarkably, this mutation arrests nuclear divisions in the early embryo but has no discernible effect on neuronal cargo trafficking in flies or motility of dynein complexes along mammalian brain-derived microtubules in vitro. Besides the marked metaphase arrest, mitotic divisions in S3372C mutant embryos display centrosomal and spindle aberrations, as well as a paucity of astral microtubules, correlating with diminished recruitment of γ-tubulin at centrosomes. In addition, dynein shows dramatic accumulations at the metaphase plate in these mutant spindles, presumably contributing to the mitotic defects. This work suggests a novel mechanism for γ-tubulin localisation mediated by dynein, and shows a unique dynein accumulation at the metaphase plate never reported before. Several hypotheses are presented and tested to explain the selective phenotype observed in S3372C mutant embryos, related to mechanochemical properties of single dynein molecules, analysis of phosphorylation regulatory mechanisms, ectopic disulphide bond formation or dynein’s interaction with maternal tubulin isotypes. In general, this work pinpoints features of dynein that are important for its discrete functions in vivo, and illustrate the utility of combining Drosophila and in vitro systems to investigate motor biology. More specifically, these studies provide further insight into how dynein mutations affect cargo trafficking in neurons and suggest unappreciated complexity in how interactions of the motor with microtubules govern its diverse functions.