Afzal, Khoula
(2023)
Rescuing the paralysed phenotype of unc-18 (e81) null mutant C. elegans.
PhD thesis, University of Liverpool.
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Abstract
Munc-18 (also known as STXBP1) functions as a core component of the fusion machinery at the presynapse, with a role in several stages of the vesicle cycle. In humans, heterozygous de novo mutations in Munc18-1 are associated with infantile epileptic encephalopathy, however, there is currently no cure for the disorder. This investigation was interested in whether the function of Munc18-1 could be bypassed if it was completely dysfunctional. As null mutations in Munc18-1 and its homologues in several organisms such as yeast and mammals are unviable, the C. elegans nematode was chosen as the model organism. C. elegans remain viable yet demonstrate a severely paralysed phenotype. Prior to this work, ethyl methanesulfonate mutagenesis (EMS) of unc-18 (e81) null mutants led to the discovery of a mutant, termed unc-18 rescue, in which locomotion appeared indifferent from wild-type. Whole genome SoLiD sequencing confirmed the presence of the unc-18 (e81) mutation in the unc-18 rescue mutant, as well as two novel mutations in dgk-1 (ulv1) and sorf-2 (ulv2), and a third mutation in sorf-2 (ulv3), which were all hypothesised to be putatively involved in the unc-18 rescue phenotype. In this investigation, behavioural analysis confirmed that locomotion of unc-18 rescue mutants was successfully restored to wild-type levels when on a surface, but not when in solution. Expression of wild-type dgk-1 in unc-18 rescue mutants reversed improvements in locomotion, confirming the necessity of the dgk-1 (ulv1) for the unc-18 rescue phenotype. However, diacylglycerol kinase (DGK) inhibition or elevated diacylglycerol (DAG) in unc-18 (e81) null mutants did not improve locomotion suggesting the necessity of another gene. Transgenic expression of wild-type sorf-2 reduced the rate of locomotion and confirmed the necessity of sorf-2. Together, sorf-2 RNAi and dgk-1 inhibition successfully improved locomotion in unc-18 (e81) null mutants, but not in unc-18 (e81) null mutants lacking the sorf-2 (ulv3) mutations, or in alternative unc-18 null mutants, suggesting unc-18 (e81) null specificity for the rescue phenotype. Further, 1H nuclear magnetic resonance (NMR) spectroscopy was able to successfully identify differences in lipid and polar metabolite abundance between wild-type, unc-18 (e81) null, and unc-18 rescue worms, highlighting the importance of lipid pathways in physiological processes. Together these findings confirmed the necessity of the dgk-1 (ulv1) and sorf-2 (ulv2 and ulv3) mutations in the unc-18 rescue phenotype and suggest that the unc-18 rescue phenotype is produced by the combined efforts of proteins and lipids which bypass the function of unc-18 in synaptic vesicle fusion.
| Item Type: | Thesis (PhD) |
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| Divisions: | Faculty of Health and Life Sciences |
| Depositing User: | Symplectic Admin |
| Date Deposited: | 30 Nov 2023 10:42 |
| Last Modified: | 30 Nov 2023 10:43 |
| DOI: | 10.17638/03173000 |
| Supervisors: |
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| URI: | https://livrepository.liverpool.ac.uk/id/eprint/3173000 |
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