AbstractEvolutionary cell biology is a broad subject that involves resolution of molecular mechanisms based on differences and similarities at a cellular level between taxa. In this PhD the focus was on two areas of evolutionary cell biology. The first area revolved around three classes of protein that contain tubulin cofactor c (TBCC) domains. The archetypal member of the family is TBCC. It acts as a chaperone for α/β tubulin heterodimerisation in the tubulin folding pathway from which microtubules are formed. The second class of TBCC domain-containing protein, Retinitis Pigmentosa 2 (RP2) is required for eukaryotic flagellum assembly. The third class is the little-studied tubulin binding cofactor C domain 1 (TBCCD1) protein which appears involved in Golgi-centrosome interactions. Phylogenetic study into TBCC domain-containing proteins found that they are generally conserved across eukaryotic evolution with secondary loss of TBCCD1 early in fungal evolution and canonical TBCC occasionally during the evolution of the Taphiromycota. Occasional losses of TBCCD1 and TBCC were seen in diverse protist lineages. RP2 was found only in flagellate eukaryotes with a strong correlation observed between presence of an RP2 and the ability to assemble a ciliary gate at the transition zone near the base of the flagellum. Following the phylogenetics plasmids for localisation studies of HA-epitope tagged TBCCD1 and RP2 in the diplomonad Spironucleus vortens were prepared.
The second area focused on the dynamic formation by some metabolic enzymes in yeast of filaments and puncta. Such aggregation, dependent on the enzyme concerned, has been considered a regulatory response to changes in the concentration(s) of substrate, product or other metabolic intermediates or as response to environmental stress. Phylogenetics revealed possible conservation of filament and fociforming enzymes in three diverse eukaryotes. The results of the analysis were the basis for heterologous expression in Leishmania tarentolae of CTP synthetase from Crithidia fasciculata and asparagine synthetase from Naegleria gruberi – the former is a prime example of a filament-forming enzyme in
prokaryotes, mammals, yeast and an apicomplexan protist. Homologous episomal expression of the transaldolase Tal1 in Capsaspora owczarzaki was also conducted using a recently documented reverse genetics approach. Collectively, the results obtained from expressing fluorescent protein fusions indicated a tendency for filament and foci-forming may be conserved in diverse eukaryotes.
|Date of Award||9 Mar 2023|
|Supervisor||Michael Ginger (Main Supervisor) & Jane Harmer (Co-Supervisor)|