Year: 2011
Type: dissertation
Abstract: All eukaryotic cells exhibit a constant turnover of membrane components via trafficking of vesicles between subcellular compartments. A classical example of a dynamic membrane delimited organelle exhibiting a high degree of complexity in terms of structure and function is the synaptic vesicle (SV), which participates in the release of neurotransmitter from neurons. Biochemical analysis of purified SVs was instrumental in the identification and understanding of proteins involved in exocytotic membrane fusion and neurotransmitter uptake. Numerous protocols have been established detailing the isolation of SVs from brain. Protocols resulting in highly purified vesicles often have extremely low yields compared to the starting material required. Here I describe an improved protocol for the small-scale isolation of synaptic vesicles from mouse and rat brain. The procedure relies on standard fractionation techniques, including differential centrifugation, rate-zonal centrifugation and size-exclusion chromatography. The protocol has been optimised to minimize vesicle loss and increase yield during preparation while maintaining a high degree of purity. The protocol can be completed in a very short time compared to classical protocols. Immunoblotting and electron micrographs showed high purity, enrichment profile of one of the most abundant vesicle protein synapotphysin revealed maximum enrichment in the final SV fraction. This opens the possibility to purify SVs from genetically modified mice to further explore the biochemistry of the neurotransmitter release process. Therefore, I purified SVs from Rab-GDI knock-out mice to ensure the applicability of this new protocol. Furthermore, I carried out the biochemical and morphological characterisation of mouse SVs and compared them to rat SVs. To determine any differences of SVs between these two species, physical parameters were analysed like diameter, mass and density. I found that SVs from mouse and rat are relatively similar except for minor differences in their physical p arameters, protein and lipid compositions. To characterise the role of SNARE proteins in membrane fusion and to check for the fusogenic properties of the purified vesicles, in-vitro fusion assays were performed with liposomes containing syntaxin 1 and SNAP-25 and glutamate uptake as a general function was monitored.
DOI: 10.53846/goediss-485
Author: Ahmed, Saheeb
Language: English
Subfield: Cell Biology
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W4213167603