Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.662056
Title: The regulation of dynamin I interactions in synaptic vesicle endocytosis
Author: Smillie, K. J.
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2005
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Abstract:
Dynamin 1 is a 96kDa GTPase and is one of eight proteins known to undergo coordinated dephosphorylation upon nerve terminal stimulation and subsequent rephosphorylation. They are collectively referred to as the dephosphins and are all dephosphorylated by calcineurin and at least three are rephosphorylated by cyclin-dependent kinase 5. Phosphomemetic peptides were used to investigate the specific role of dynamin 1 phosphorylation in synaptic vesicle endocytosis. The peptide mimicking dephosphorylated dynamin 1 was nearly four times more effective at inhibiting synaptic vesicle endocytosis than the peptide mimicking phosphorylated dynamin 1, indicating that there is an essential role for these phosphorylation sites in endocytosis. Experiments to probe the function of the phosphorylation sites identified syndapin 1 as a stimulation dependent binding partner to this region of dynamin 1. The interaction was further characterised by immunoprecipitation and GST-pulldown assays. Dynamin 1 can also be regulated by Ca2+ either directly or indirectly through Ca2+-dependent binding partners. Dynamin 1 is published to bind to the cytosolic C-terminus of the integral vesicle protein synaptophysin in a Ca2+-dependent manner. This thesis shows that the over-expression of the C-terminus of synaptophysin-eGFP in cerebellar granule neurites inhibits vesicle recycling and that a peptide designed from this region of synaptophysin also inhibits vesicle recycling in isolated nerve terminals. GST-pulldowns with the C-terminus of synaptophysin extracted dynamin 1, in the presence of Ca2+, but also a more prominent band shown by western blot to be amphiphysin 1. Amphiphysin 1 was confirmed to bind to the C-terminus of synaptophysin independently from dynamin 1. Amaphiphysin 1 may interact with synaptophysin, in nerve terminals, linking synaptophysin to a potential role in synaptic vesicle endocytosis.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.662056  DOI: Not available
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