The dynein motor complexes responsible for the beating of motile cilia are among the largest molecular machines in the cell.
In recent years about 10 'assembly factors' have been identified for the cytoplasmic assembly of dynein motor complexes prior to their trafficking to the cilium. These are hypothesised to be part of a highly-conserved chaperone assembly pathway. In humans, mutations in these factors causes the disease, Primary Ciliary Dyskinesia. To understand this assembly pathway there is a need to (1) discover further assembly factors to complete the 'tool kit' (2) carry out detailed analysis of their function, preferably in an organismal context capitalising on recent advances for genetic and microscopy analysis. Our recent research has firmly established Drosophila as a powerful model for these two aims. In Drosophila, motile cilia are confined to sensory neurons and sperm, which are readily accessible for analysis, and yet the entire known dynein assembly pathway is conserved. Our recent functional genomic screen in Drosophila led to the identification of new dynein assembly factors (Zmynd10 and Heatr2). In collaboration with human geneticists, both these genes were subsequently found to cause PCD in humans.
In this project, the student will analyse CRISPR-generated mutations in several new candidate assembly factors identified in our Drosophila screen. These will be examined for immotile cilia and dynein assembly defects using behavioural assays, immunofluorescence, confocal microscopy and electron microscopy. Interactions with other proteins (co-chaperones and dynein clients) will be determined by immunoprecipitation of tagged proteins from testes and identification of interactors by mass spectrometry. New tools for the analysis of dynein subunit dynamics (localisation and turnover) will be generated using CRISPR technology to tag endogenous genes, thereby allowing sophisticated microscopy analysis in an in vivo context. The student will work in parallel with work conducted on mouse models by co-supervisor Pleasantine Mill, and there will be opportunity for interaction with human geneticists specialising in PCD.
Prof Andrew Jarman
Dr Barry Denholm
Dr Pleasantine Mill