Investigating the role of R2TP-like co-chaperone complexes during axonemal dynein assembly
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Date
02/09/2022Author
Lennon, Jennifer
Metadata
Abstract
Motile cilia are specialised cell-types which in humans have important roles in
the linings of the airways, the reproductive system and the brain. The
movement, required for this type of cilia to function, is facilitated by structures
called axonemal dynein motor complexes. These are large, multi-subunit
structures, and so it is crucial that they are assembled correctly. In humans, if
the motility of these is defective, it can lead to a disorder called Primary Ciliary
Dyskinesia, or PCD. This is a heterogeneous, autosomal recessive disorder –
symptoms of which include abnormally positioned organs, chronic respiratory
infections and infertility. Therefore, the development and structure of the motile
cilia is tightly regulated by multiple proteins including chaperones, dynein
axonemal assembly factors (DNAAFs), microtubule inner proteins (MIPs), the
outer arm docking complex (ODA-DC) and the nexin-dynein regulatory
complex (N-DRC). Chaperones work with co-chaperones to regulate their
many functions within the cell. One of these co-chaperones is the R2TP
complex, which was originally discovered in yeast but is conserved in higher
organisms. This multi-protein co-chaperone is involved in the assembly of
multi-subunit complexes such as the axonemal dynein motors. Two of the
R2TP subunits, Pontin and Reptin, are involved in many cellular functions both
in this co-chaperone complex and independently. It is thought that as some
DNAAFs share similar protein domains to the components of the R2TP
complex, they may form R2TP-like complexes. However, the specific details
surrounding the roles of these complexes during the assembly process
remains unclear. The structure of motile cilia is highly conserved throughout
evolution and Drosophila melanogaster has been shown previously to be an
excellent model for furthering understanding into the development and function
of these structures as only two cell types in the fly contain axonemal dynein
motor complexes. These are the chordotonal neuron, which has a motile
ciliated dendrite essential for its mechanosensory function, and the sperm
flagellum. In this thesis, I use the Drosophila model to further characterise
putative ciliary genes (Wdr16 and Dpcd) identified by a transcriptome analysis
previously carried out in the lab. RNAi knockdown experiments as well as
expression analysis supported motile cilia functions. The diversity which has
been identified regarding the roles of these two putative ciliary genes highlights
how proteins can be involved in motile cilia in different ways. I also use this
genetically tractable model to further understand the roles of the individual
proteins of a previously identified R2TP-like complex (R2DP3). Electron
microscopy, proteomics and investigation into how the localisation of dynein
subsets was affected in null mutants (generated using CRISPR/Cas9) allowed
for the role of this R2TP-like complex in the dynein assembly process to be
further specified. Using co-immunoprecipitation and affinity purification, we
identified an additional protein complex featuring Pontin and Reptin of the
R2TP complex, alongside the DNAAF Heatr2 and the putative DNAAF Dpcd.
As well as a role in dynein assembly, both DNAAFs are additionally expressed
in the neuroblasts of the CNS, and disruption to their function results in a late
larval lethality. Therefore, we have found these genes to not be specific to the
dynein assembly process and hypothesise that Dpcd may have an additional
function (working with Pontin, Reptin and potentially Heatr2) in the regulation
of AKT signalling and therefore impact cell proliferation.