Using supported bilayers to study the spatiotemporal organization of membrane-bound proteins
Nguyen, Phuong A.
Field, Christine M.
Groen, Aaron C.
Mitchison, Timothy J.
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KeywordSupported bilayer; Cell division; In vitro reconstitution; Cytokinesis signaling; E. coli; Xenopus; Lipids; Microtubules; FtsA; FtsZ
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 (TIRF) 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.
© The Author(s), 2015. This is the author's version of the work and is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Methods in Cell Biology 128 (2015): 223-241, doi:10.1016/bs.mcb.2015.01.007.
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