A screen for novel interactions within the vertebrate centriole
| Autor: | Barnabas, Deepak David |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2019 |
| Předmět: | |
| DOI: | 10.17863/cam.44114 |
| Popis: | The centrioles organize the centrosomes and cilia which play a role in cell division, motility, sensing, polarity, trafficking and signalling. There has been an increase in the understanding of centriole composition and function recently. However, the exact assembly mechanism of the centriole at the molecular level is only beginning to be understood. Knowledge of the canonical assembly mechanism is important to understand how centriole dysfunction materialises in the case of some human diseases. The identification of protein-protein interactions (PPIs) can progress our understanding of the assembly process. In this thesis, I follow two approaches towards this objective. First, I conduct a large-scale combinatorial screen of 3025 binary test pairs amongst a subset of 55 vertebrate centriole/centriole associated proteins. The screen detected 28% of the interactions identified already in the literature and further identified 67 novel binary PPIs within the vertebrate centriole. Of these, 13 are likely to be physiologically relevant due to colocalization of the components within the same centriolar sublocation. I then identify the minimal interaction regions (MIRs) of the proteins involved in these binary interactions and validate such interactions in the mammalian cell context. I perform diagnostic pull-down assays of these putative full-length binary protein complexes recombinantly expressed in insect cells with the aim of subjecting the complexes to structural/biophysical studies. Only 1 novel interaction eluted (PLK1-KIF24) robustly as a binary complex. I validate this interaction in-vitro using SEC-MALS and map the binding region further using NMR. In the second approach towards identifying novel PPIs, I attempt to purify literature-predicted putative multi-protein sub-complexes of the centriole. In summary, the main objective of this thesis has been achieved. The novel PPIs identified in this study, especially those with potential physiological relevance present a good starting point for future investigation. Further, the approach taken in this thesis could serve as a framework to explore other subcellular structures. University of Cambridge, Medical Research Council (MRC), Cambridge Cancer Centre |
| Databáze: | OpenAIRE |
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