Using supported bilayers to study the spatiotemporal organization of membrane-bound proteins

Methods Cell Biol. 2015;128:223-241. doi: 10.1016/bs.mcb.2015.01.007. Epub 2015 Apr 8.

Abstract

Cell division in prokaryotes and eukaryotes is commonly initiated by the well-controlled binding of proteins to the cytoplasmic side of the cell membrane. However, a precise characterization of the spatiotemporal dynamics of membrane-bound proteins is often difficult to achieve in vivo. Here, we present protocols for the use of supported lipid bilayers to rebuild the cytokinetic machineries of cells with greatly different dimensions: the bacterium Escherichia coli and eggs of the vertebrate Xenopus laevis. Combined with total internal reflection fluorescence microscopy, these experimental setups allow for precise quantitative analyses of membrane-bound proteins. The protocols described to obtain glass-supported membranes from bacterial and vertebrate lipids can be used as starting points for other reconstitution experiments. We believe that similar biochemical assays will be instrumental to study the biochemistry and biophysics underlying a variety of complex cellular tasks, such as signaling, vesicle trafficking, and cell motility.

Keywords: Cell division; Cytokinesis signaling; E. coli; FtsA; FtsZ; In vitro reconstitution; Lipids; Microtubules; Supported bilayer; Xenopus.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Aurora Kinase A / metabolism
  • Aurora Kinase B / metabolism
  • Bacterial Proteins / metabolism
  • Carrier Proteins / metabolism
  • Cell Cycle Proteins / metabolism
  • Cell Division
  • Cell Membrane / metabolism*
  • Cell Movement / physiology
  • Cytoskeletal Proteins / chemistry
  • Cytoskeletal Proteins / metabolism
  • Escherichia coli / metabolism*
  • Escherichia coli Proteins / metabolism
  • Lipid Bilayers / metabolism*
  • Membrane Proteins / metabolism*
  • Microscopy, Fluorescence / methods
  • Ovum / metabolism
  • Protein Binding / physiology
  • Tissue Extracts / metabolism
  • Xenopus Proteins / metabolism
  • Xenopus laevis / metabolism*

Substances

  • Bacterial Proteins
  • Carrier Proteins
  • Cell Cycle Proteins
  • Cytoskeletal Proteins
  • Escherichia coli Proteins
  • FtsA protein, E coli
  • FtsZ protein, Bacteria
  • Lipid Bilayers
  • Membrane Proteins
  • Tissue Extracts
  • Xenopus Proteins
  • ZipA protein, E coli
  • Aurora Kinase A
  • Aurora Kinase B
  • aurkb protein, Xenopus