Abstract:
:A complete mechanistic understanding of membrane-localized processes in vesicular transport, such as membrane budding and fission, requires their reconstitution with biochemically-defined components from a biochemically-defined substrate. Supported bilayers formed by vesicle fusion represent an attractive substrate for this purpose. However, conventional supported bilayers lack a sufficient membrane reservoir to recreate membrane budding and fission events. We describe the formation of supported bilayers with excess membrane reservoir (SUPER) templates from the fusion of liposomes containing negatively charged lipids on silica beads under high-ionic-strength conditions. Using a fluorescence microscopy-based assay to monitor early and late stages of supported bilayer formation, we show that an increase in ionic strength leads to an increase in the rates of liposome adsorption and subsequent fusion during formation of supported bilayers. The two rates, however, increase disproportionally, leading to accumulation of excess reservoir with an increase in ionic strength. SUPER templates allow the seamless application of microscopy-based assays to analyze membrane-localized processes together with sedimentation-based assays to isolate vesicular and nonvesicular products released from the membrane. The results presented here emphasize the general utility of these templates for analyzing vesicular and nonvesicular transport processes.
journal_name
Biophys Jjournal_title
Biophysical journalauthors
Pucadyil TJ,Schmid SLdoi
10.1016/j.bpj.2010.04.036subject
Has Abstractpub_date
2010-07-21 00:00:00pages
517-25issue
2eissn
0006-3495issn
1542-0086pii
S0006-3495(10)00534-5journal_volume
99pub_type
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