Abstract
Cholesterol helps to stabilize membrane fluidity and many membrane proteins interact with cholesterol and are functionally clustered in cholesterol rich “rafts.” Synaptic vesicle (SV) membranes are enriched in cholesterol in comparison to other organelles. Attempts to study the function of this high cholesterol content have been hampered by the inability to extract cholesterol from SVs in live presynaptic terminals. Here, we describe a method to extract vesicular cholesterol using a temperature-sensitive Drosophila dynamin mutant, shibire-ts1 (shi), to trap SVs on the plasma membrane. Trapped SVs are more accessible to cholesterol extraction by the cholesterol chelator, methyl-β-cyclodextrin (MβCD). This method can likely be extended to extract other lipids from SVs and could also be used to add lipids. We speculate that this method could be used on mammalian preparations in conjunction with dynamin inhibitors.
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Acknowledgements
This work was supported by a grant from CIHR, Canada MOP-82827 (M.P.C.).
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Dason, J.S., Charlton, M.P. (2014). A Novel Extraction Protocol to Probe the Role of Cholesterol in Synaptic Vesicle Recycling. In: Ivanov, A. (eds) Exocytosis and Endocytosis. Methods in Molecular Biology, vol 1174. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-0944-5_25
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DOI: https://doi.org/10.1007/978-1-4939-0944-5_25
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