Supported bilayers with excess membrane reservoir: A template for reconstituting membrane budding and fission

Thomas J. Pucadyil, Sandra L. Schmid

Research output: Contribution to journalArticle

27 Citations (Scopus)

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.

Original languageEnglish (US)
Pages (from-to)517-525
Number of pages9
JournalBiophysical Journal
Volume99
Issue number2
DOIs
StatePublished - Jul 21 2010

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Membranes
Osmolar Concentration
Liposomes
Fluorescence Microscopy
Silicon Dioxide
Adsorption
Microscopy
Lipids

ASJC Scopus subject areas

  • Biophysics
  • Medicine(all)

Cite this

Supported bilayers with excess membrane reservoir : A template for reconstituting membrane budding and fission. / Pucadyil, Thomas J.; Schmid, Sandra L.

In: Biophysical Journal, Vol. 99, No. 2, 21.07.2010, p. 517-525.

Research output: Contribution to journalArticle

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