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Title: Spatial organisation of the SNARE proteins : the role of munc18-1
Author: Medine, C.
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2009
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In this study we show using in vitro and in vivo approaches that in neuroendocrine cells munc18-1 has two modes of binding to syntaxin 1a. I show that munc 18-1 plays a vital role in binding to a closed-form of syntaxin 1a, keeping syntaxin 1a inactive and permitting its trafficking to the plasma membrane. Munc18-1 is also able to bind to an active open form of syntaxin 1a, allowing it to bind to other SNAREs. I demonstrate that in the absence of munc 18-1, syntaxin 1a and SNAP-25 can readily interact in the Golgi complex, forming reactive SNARE complexes that fail to traffic to plasma membrane. The exocytotic SNARE proteins are highly promiscuous in their interactions with other SNAREs, and thus it is essential to traffic the exocytotic SNARE proteins through intracellular compartments while avoiding ectopic interactions between non-cognate SNARE proteins. Munc 18-1 has a vital regulatory role in preventing the formation of the binary complex between syntaxin 1a and SNAP-25 before the proteins reach their target destination of the plasma membrane. Upon delivery to the plasma membrane that t-SNAREs have been shown to form cholesterol dependent clusters in several cell types of between 60-7509 nm. Recently, it was shown that syntaxin 1a forms dynamic clusters by virtue of self association. Here I present evidence to show that once at the plasma membrane syntaxin 1a and SNAP-25 are concentrated in clusters of high local concentration that co-localise in neuroendocrine cells. Interestingly, despite reducing the affinity of SNAP-25 for syntaxin 1a using mutagenesis, these proteins still co-cluster and interact on the plasma membrane. The newly delivered t-SNAREs, less than 48 hours old, can form interaction-heterogeneous clusters with interactions modulated by elevated Ca2+ levels. Furthermore, quantitative changes in the lipid microenvironment by cholesterol depletion play a role in cluster integrity.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available