| dc.description.abstract | More than 1500 proteins have been identified as centrosome or cilia
proteins, however it is still unknown how these proteins form networks to
control functions such as centrosome orientation, maturation and
ciliogenesis. TALPID3 is a centrosomal protein that plays a role in
centrosome orientation and migration, ciliogenesis and Hedgehog pathway
signal transduction. Although several proteins that interact with TALPID3
have been identified, the TALPID3 protein interacting network has not been
established. Loss of TALPID3 leads to a loss of ciliogenesis, as seen in the
embryonic lethal talpid3 chicken. Unlike the talpid3 chicken however, human
ciliopathy patients with mutations in KIAA0586 (human TALPID3 orthologue)
have a range of defects that often do not result in lethality.
Human KIAA0586 was modelled in the chicken, in order to understand the
functional domains that are affected by mutations that are predicted to be
hypomorphic. A construct containing human KIAA0586 was able to rescue
Hedgehog-dependent expression patterns, when electroporated into the
developing neural tube of talpid3 chicken embryos. An attempt to introduce
a fluorescent tag at the endogenous TALPID3 locus to look at specific
subcellular localisation, proved unsuccessful; however a super resolution
imaging approach confirmed localisation of TALPID3 to the centrosome.
Structured Illumination Microscopy showed that TALPID3 localises to the
distal end of centrioles with more TALPID3 on one centriole than the other
centriole, but no specific regions of localisation elsewhere in the cell.
A proteomic approach on isolated centrosomes was undertaken to
investigate the TALPID3 protein network. Proteomic studies in the
centrosome have previously been carried out in KE37 cells, however in order
to establish alternative cell lines, specifically chicken Primordial Germ Cells
(PGCs), as models for studying the centrosome and cilia, this study was
completed in human Jurkat cells and chicken PGCs. PGCs can be derived
from the talpid3 flock, therefore by including chicken PGCs the aim was to
construct the centrosomal proteome of talpid3 vs wildtype centrosomes
from talpid3 PGCs. Through this study, I have shown that Jurkat cells have a
low frequency of ciliogenesis with modifications of the centrioles that
correlate with their low ciliogenesis frequency; however PGCS are capable
of forming cilia and have modifications of the centrioles that correlate with
their ability to form cilia, highlighting PGCs as a suitable cell model for
carrying out centrosome and cilia studies. A mass spectrometry screen on
centrosomes isolated from Jurkat cells and PGCs failed to identify TALPID3;
however, the screen identified CCDC77 and CCDC127 as novel centrosome
proteins, which was confirmed by immunofluorescence. CRISPR/Cas9
editing, demonstrated that mutations in CCDC127 resulted in a significant
reduction of ciliogenesis and altered centrosome protein localisation
patterns in RPE1 cells.
Isolation of centrosomes from PGCs did not prove effective for constructing
the proteome of talpid3, therefore an alternative approach was taken using
protein extracted from whole cell lysate of talpid3 and wildtype PGCs. A
quantitative proteomic study was undertaken in talpid3 PGCs with the aim
of understanding how protein networks are altered in talpid3. Following a
Tandem Mass Tag (TMT) mass spectrometry approach, pathway analysis in
Ingenuity Pathway Analysis software (IPA) identified down-regulation of
protein pathways linked to regulation of the actin cytoskeleton and showed
an overall change to protein pathways associated with cholesterol
biosynthesis. Immunofluorescence in talpid3 embryo sections was used to
examine changes to proteins involved in actin regulation, including F-actin,
Profilin, Cofilin, Twinfilin and RhoC.
The main findings of this thesis include evidence that talpid3 embryos can be
used to model human ciliopathy mutations, demonstrate that PGCs are a
primary cell model that can be used to study the centrosome and identify
CCDC127 as a novel centrosome protein that is necessary for ciliogenesis in
human RPE1 cells. Additionally, the findings show that protein pathways
associated with the regulation of the actin cytoskeleton are downregulated
in talpid3. Together the results produced in this thesis provide insight into
the centrosome from the perspective of chicken PGCs as well as a better
understanding of protein pathways altered in talpid3. | en |
