Abstract
Cytokinesis is the final stage of mammalian cell division, which leads to membrane abscission separating the two daughter cells. Proteins with roles in executing and controlling cytokinesis are recruited to the cleavage furrow of dividing cells, however current knowledge is limited regarding lipid-dependent recruitment. Two complementary approaches have been taken to address this problem. In the first, an RNAi screen of lipid biosynthetic enzymes was undertaken and analysed for their impact on cytokinesis. Whilst limited insight into how lipids influence protein recruitment was obtained, issues associated with such RNAi screens are addressed. In the second approach, attention was focused on the kinase, PKCε. PKCε is recruited to the cleavage furrow and, as a complex with 14-3-3, is essential for cytokinesis completion when the abscission checkpoint is engaged.Catalytic activity is required for release of PKCε from the cleavage furrow, but the mechanism by which recruitment/retention and release occurs is not known. Manipulation of the C1 domains is consistent with a requirement for diacylglycerol in the activity-dependent recruitment of PKCε. While not required for diacylglyceroldependent membrane binding, the “inter-C1 domain” was found to be necessary for retention. Mutations in this domain weaken PKCε localisation at the cleavage furrow, and interfere with cells successfully completing cytokinesis. Genetic code expansion enabling crosslinking of PKCε identified an interactor, whose association with PKCε weakens upon mutations at this site, correlating with loss of PKCε at the cleavage furrow. It was found that PKCε-14-3-3 complex formation was not required for recruitment/retention, despite the requirement for cytokinesis, and that the requirement for this complex lies downstream, associated with the phosphorylation of Aurora B. The mechanisms of this complex assembly were addressed through in vitro and in vivo studies, revealing the previously unrecognised requirement for dephosphorylation of PKCε Ser350.
The metabolically-derived lipidome is challenging in linking to biological functions. However, the analysis of specific protein recruitment, as here for PKCε, provides a directly tractable problem, and understanding the behaviour of PKCε has enabled specific insight into its requirement during cytokinesis.
| Date of Award | 1 Dec 2020 |
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| Original language | English |
| Awarding Institution |
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| Supervisor | Ulrike Eggert (Supervisor) & Peter Parker (Supervisor) |