Motile cilia are microtubule based projections that assist in the movement of fluid
over the surface of cells, such as in the respiratory epithelium, or of cells through
a fluid, such as in sperm. Ciliary movement is driven by axonemal dyneins (ADs),
large molecular complexes which contain long heavy chain ATPase motor
subunits. The stability of ADs has been shown to be dependent on multiple
cytoplasmically localised proteins, which are involved in their assembly and
trafficking to the cilia. The heavy chain subunits have been suggested to be
particularly reliant on specialised chaperoning pathways in order to fold into the
correct tertiary structure. Hereditary defects in genes encoding the proteins of the
ADs or proteins involved in their assembly result in an incurable human disease,
primary ciliary dyskinesia (PCD). PCD results in neonatal respiratory distress with
lifelong respiratory complications and is also highly heterogenous with mutations
in 40 genes associated with it so far. Despite the identification of many putative
assembly factors, where and how they interact with AD proteins remains unknown.
In order to investigate AD complexes, from the translation of their subunits to their
degradation, in greater spatial and temporal detail a heavy chain outer dynein arm
subunit (ODA), Dnah5, was tagged with the adaptable SNAP tag in mice. Dnah5
is the largest AD heavy chain and the most commonly mutated gene in PCD. When
developing novel therapeutics the SNAP-Dnah5 mouse could be used as a
reporter for functional rescue in PCD mouse models which exhibit loss of these
complexes from the cilia. The effectiveness of the therapy could then be graded
on the restoration of SNAP-DNAH5 fluorescence in the motile cilia. As a secondary
aim this project also sought to improve the efficiency of CRISPR/Cas9 induced
gene correction, via a novel linkage method, to develop a genome editing therapy
for PCD, which could be tested using SNAP-Dnah5 mice.
Using the SNAP-Dnah5 mouse tracheal epithelial cells I have directly imaged
DNAH5’s docking onto the motile axoneme from the distal end and have
demonstrated that there is a very low level of ODA turnover in mature cilia. I have
also shown that the Dnah5 transcript localises to large apical clusters in ciliated
tracheal epithelial cells and via preliminary pulldown experiments that SNAPDNAH5
might interact with RNA regulatory proteins in maturing motile ciliated cells
suggestive of translational regulation. This project demonstrates the utility of this
mouse model for future studies
