Many tasks within the cell are accomplished by highly organized multi-component protein complexes, the biogenesis of which requires carefully guided assembly by molecular chaperones. A particularly striking example is the molecular machinery that drives movement of motile cilia. Motile cilia are ancient eukaryotic organelles required for movement through fluid (e.g. the flagellum of sperm and protozoa) or movement of fluid (e.g. cerebrospinal fluid, mucociliary clearance, left-right patterning in the embryonic node). The dynein motor complexes that drive ciliary beating are massive (over 1 MDa) and have high subunit complexity (with some huge subunits up to 550 kDa). Defective dynein motors result in ciliary immotility, and in the case of humans mutations of ciliary motility genes causes the disease Primary Ciliary Dyskinesia (PCD). The key question of this proposal is how are these large multicomponent complexes built during ciliogenesis. Analysis in a range of organisms, including characterisation of human PCD mutations, suggests motor complexes are pre-assembly in the cytoplasm before being transported into the cilium, entailing steps of protein folding/stabilization, macromolecular assembly, and transport. PCD mutations identify about 10 novel conserved proteins that are required for motor pre-assembly (‘dynein assembly factors’). They are thought to be molecular chaperones linked to Hsp90, but currently their molecular functions are poorly understood. In this project, the student will use Drosophila as a powerful model for a structural and functional analysis of motor complex assembly.
Amelia Shoemark is an expert in using electron tomography to characterise the molecular-scale defects in cilium structure in PCD patients, for both diagnostic and research purposes . Andrew Jarman has pioneered the use of Drosophila as a model organism for analysing motile cilium defects and motor function, including the use of genetics, whole organism imaging, and proteomics . In this project, the student will receive training that combines these different expertises, based in both Edinburgh and Dundee. The student will initially build a molecular model of Drosophila motile cilium structure using electron tomography (the first time this will be attempted). This will provide the baseline for tomography analysis of ciliary defects in mutants of a range of Drosophila genes orthologous to human PCD genes (recently characterised and assembled in the Jarman lab). Characterisation of the ultrastructural defects will allow the student to propose mechanisms of protein complex assembly that can be explored by analysis of data on proteome changes in these mutants, as well as immunofluorescent microscopy analysis of motor assembly. In particular, the student will build on recent bioimaging work in the Jarman lab that uses SNAP-tagged motor protein subunits to follow real-time dynein assembly processes in live tissues by fluorescence microscopy. Overall, the student will gain a training in complementary bioimaging techniques, cellular and whole-organism analysis, as well as the manipulation and analysis of extensive proteomic datasets.
1. Burgoyne et al. Characterizing the ultrastructure of primary ciliary dyskinesia transposition defect using electron tomography. Cytoskeleton 2014 (https://doi.org/10.1002/cm.21171)
2. zur Lage et al. Ciliary dynein motor preassembly is regulated by Wdr92 in association with HSP90 co-chaperone, R2TP. J. Cell Biol. 2018 (https://doi.org/10.1083/jcb.201709026)