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The in vivo structure of biological membranes and evidence for lipid domains

Examining the fundamental structure and processes of living cells at the nanoscale poses a unique analytical challenge, as cells are dynamic, chemically diverse, and fragile. A case in point is the cell membrane, which is too small to be seen directly with optical microscopy and provides little obse...

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Bibliographic Details
Published in:	PLoS biology 2017-05, Vol.15 (5), p.e2002214-e2002214
Main Authors:	Nickels, Jonathan D, Chatterjee, Sneha, Stanley, Christopher B, Qian, Shuo, Cheng, Xiaolin, Myles, Dean A A, Standaert, Robert F, Elkins, James G, Katsaras, John
Format:	Article
Language:	English
Subjects:	Acids Algorithms Bacillus subtilis - chemistry Bacillus subtilis - drug effects Bacillus subtilis - growth & development Bacillus subtilis - metabolism Bacterial Proteins - genetics Bacterial Proteins - metabolism BASIC BIOLOGICAL SCIENCES Biochemistry Biological membranes Biology Biology and Life Sciences Cell Membrane - chemistry Cell Membrane - drug effects Cell Membrane - metabolism Cells (biology) Cerulenin - pharmacology Chemical composition Deuterium Editing Enoyl-CoA Hydratase - genetics Enoyl-CoA Hydratase - metabolism Fatty Acid Synthesis Inhibitors - pharmacology Fatty acids Fatty Acids - chemistry Fatty Acids - metabolism Funding Gene Deletion Hydrogen Hydrophobic and Hydrophilic Interactions Hydrophobicity In vivo methods and tests Isotopes Isotopic labeling Laboratories Layoffs Lipid Bilayers - chemistry Lipid Bilayers - metabolism Lipid rafts Lipids Lymphocytes B Mathematical analysis Medicine and Health Sciences Membrane Microdomains - chemistry Membrane Microdomains - drug effects Membrane Microdomains - metabolism Membrane structure Membranes Methods Microbial Viability - drug effects Microscopy Models, Biological Molecular biology Nanostructure Neutron Diffraction Neutron scattering Neutrons Optical microscopy Palmitic Acids - chemistry Palmitic Acids - metabolism Physical Sciences Radioactive labeling Rafts Research and Analysis Methods Scattering Scattering, Small Angle Spectra Stereoisomerism Studies Writing
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Summary:	Examining the fundamental structure and processes of living cells at the nanoscale poses a unique analytical challenge, as cells are dynamic, chemically diverse, and fragile. A case in point is the cell membrane, which is too small to be seen directly with optical microscopy and provides little observational contrast for other methods. As a consequence, nanoscale characterization of the membrane has been performed ex vivo or in the presence of exogenous labels used to enhance contrast and impart specificity. Here, we introduce an isotopic labeling strategy in the gram-positive bacterium Bacillus subtilis to investigate the nanoscale structure and organization of its plasma membrane in vivo. Through genetic and chemical manipulation of the organism, we labeled the cell and its membrane independently with specific amounts of hydrogen (H) and deuterium (D). These isotopes have different neutron scattering properties without altering the chemical composition of the cells. From neutron scattering spectra, we confirmed that the B. subtilis cell membrane is lamellar and determined that its average hydrophobic thickness is 24.3 ± 0.9 Ångstroms (Å). Furthermore, by creating neutron contrast within the plane of the membrane using a mixture of H- and D-fatty acids, we detected lateral features smaller than 40 nm that are consistent with the notion of lipid rafts. These experiments-performed under biologically relevant conditions-answer long-standing questions in membrane biology and illustrate a fundamentally new approach for systematic in vivo investigations of cell membrane structure.
ISSN:	1545-7885 1544-9173 1545-7885
DOI:	10.1371/journal.pbio.2002214