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


Author(s): Nguyen, Phuong A; Field, Christine M; Groen, Aaron C; Mitchison, Timothy J; Loose, Martin
Article/Chapter Title: Using supported bilayers to study the spatiotemporal organization of membrane-bound proteins
Affiliation IST Austria
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: Xenopus; cell division; In vitro reconstitution; Cytokinesis signaling; E. coli; FtsA; FtsZ; Lipids; Microtubules; Supported bilayer
Book Title: Building a Cell from its Components Parts
Volume: 128
ISBN: 0091-679X
Publisher: Academic Press  
Date Published: 2015-01-01
Start Page: 223
End Page: 241
Sponsor: P.A.N., C.M.F., and A.C.G. were supported by NIH grant GM39565 awarded to T.J.M.; MBL fellowships from the Evans Foundation, MBL Associates, and the Colwin Fund (T.J.M. and C.M.F.); HFSP fellowship LT000466/2012-L (M.L.).
URL:
DOI: 10.1016/bs.mcb.2015.01.007
Notes: We thank M. Hanley for comments on the manuscript. R. Ohi, D. Burgess, D. Miyamoto, and E. Tan for reagents; H. Basu for preliminary work; A. Bridges for sharing the RCA cleaning protocol, the Nikon Imaging Center at Harvard Medical School and Nikon at the Woods Hole Marine Biological Laboratory (MBL) for microscopy support; and the National Xenopus Resource at MBL for Xenopus animals and care.
Open access: yes (repository)