Insights into the function and protein network of TALPID3
Fraser, Amy Margaret
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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.