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Title: Discovery and characterisation of membrane-associated proteins involved in cell division
Author: Dona, Federico
ISNI:       0000 0004 8500 2688
Awarding Body: King's College London
Current Institution: King's College London (University of London)
Date of Award: 2019
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Cytokinesis is the final step of cell division that results in the segregation of chromosomes and the division of the cytoplasm into two identical daughter cells. Cytokinesis is an important step during the cell cycle, and although a fair amount is known about this essential process, it is not completely understood which proteins and pathways are involved and there are many outstanding questions about how these are linked and regulated. We used an RNAi based screen as a tool to study this process and to discover new possible contributors to cytokinesis. Using this screen, we discovered and characterised interesting proteins, including Stomatin and Glypican 5. Stomatin is a monotopic plasma membrane protein that belongs to a family of four other members and it was not known to be involved in cell division. I show that Stomatin is involved in cell division in human cells. Depletion of Stomatin by RNA interference or CRISPR/Cas9 results in an increase of binucleated cells. The cell division failure is characterised by a mitotic delay followed by abscission failure. This phenotype can be rescued using RNAi-resistant GFP-Stomatin, which, like wild-type, localises at the plasma membrane throughout cell division. Stomatin has two palmitoylation sites and RNAi-resistant point mutants in these sites localise to the plasma membrane but cannot rescue RNAi-induced binucleation. In contrast, an RNAi-resistant C-terminal mutant lacking the intramembrane domain does rescue binucleation. These data suggest that plasma membrane binding is necessary but not sufficient for Stomatin's function during cell division. A lipidomic analysis in Stomatin depleted cells showed significant changes to several lipid species relative to control, suggesting that this protein and lipid metabolism are intimately connected. This project has identified a new peripheral plasma membrane protein involved in cell division helping us to understand better the role of the plasma membrane in the cell division process. Glypican 5 belongs to a family of heparan sulphate proteoglycans that are most conspicuously found bound to the plasma membrane through GPI anchors. These proteins have roles in cellular signaling and as tumour suppressors, but none has been associated with cytokinesis prior to our group's work. I show that depletion of Glypican 5 leads to binucleated cells due to abscission failure and to defective and slower migration. In conclusion, the work presented here illustrates the identification and characterisation of two different proteins in cell division, with a primary focus on Stomatin.
Supervisor: Eggert, Ulrike Sophie ; Ridley, Anne Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available