Neutrons provide the solution to nanoscale examination of living cell membrane and confirm the existence of lipid rafts.
The cell membrane, a thin bilayer of lipid molecules with embedded proteins, provides the essential function of protecting the cell from its outside environment and controlling the movement of substances in and out of the cell. However, much about this thin bilayer of lipid molecules has remained a mystery despite being extensively studied. This has been due to the difficulty of viewing a living cell membrane; previous methods employed to investigate membrane structure, such as X-rays and electron beams, were not well suited for studying living cells due to their high-energy nature (>5,000 eV) that damage membranes. Using cold neutrons with a low kinetic energy (<0.025 eV), for the first time, researchers performed direct nanoscale examination of a living cell membrane.
Using isotopes to create internal contrast within living cells the membrane structure and thickness of the bacterium, B. subtilis, was determined. In addition, the researchers were able to confirm the existence of the long hypothesized presence of lipid rafts, tightly packed free-floating membrane lipids and proteins thought to be important to cell signaling and facilitating movement of essential biomolecules in and out of the cell, along with a variety of other functions. The methods developed may prove valuable in areas of interest to DOE such as biomass feedstock and biofuel production, in which bacteria have an important role.
Examining a living cellular membrane has remained an unsolved challenge up to this point due to the dynamic, chemically diverse, and fragile nature of living cells. Too small to be seen by a traditional optical microscope, neutrons emerged as the solution to studying a living lipid bilayer at nanoscale without damaging the cell. Neutrons can be used as a probe for characterizing biological materials because a neutron beam scattered by a biological sample creates a pattern that is dependent on the material’s isotopic composition and reflects the material’s structural arrangement. Deuterium is an isotope of a highly abundant atom in biological matter, hydrogen. It contains a neutron and a proton, in contrast to hydrogen, which contains a single proton but no neutron. This seemingly small difference makes substituting deuterium for hydrogen an ideal approach to studying membranes and other nanoscale biological systems. Cells perceive little difference between hydrogen and its isotope, deuterium, while the isotopes appear very differently using the neutron scattering technique. A team of researchers at Oak Ridge National Laboratory (ORNL) was able to introduce enough deuterium into the membrane of the bacterium B. subtilis to differentiate it from other cell components. Further, the team was able to tune the specific proportions of deuterium and hydrogen by introducing into the cell two fatty acid (the molecules that comprise the membrane lipids) types, with unique isotope ratios. The cell incorporated the specific mix of isotope-labeled fatty acids into its membrane and a non-uniform distribution of lipids was observed, confirming the lipid raft hypothesis. These experiments answer some of biology’s longest-standing questions, aligning with the DOE Office of Science mission of providing fundamental science research to address some of the most pressing challenges of our time.
Amy Swain, Ph.D.
Biological Systems Sciences Division
Office of Biological and Environmental Research
Office of Science
U.S. Department of Energy
This research was sponsored by the Laboratory Directed Research and Development Program (grant number 6988) of Oak Ridge National Laboratory (ORNL), managed by UT-Battelle, LLC, for the U. S. Department of Energy (DOE) under Contract No. DE-AC05-00OR22725. Support for J.K. provided by the DOE Office of Basic Energy Sciences, Scientific User Facilities Division and for R.F.S by the DOE Office of Biological and Environmental Research (grant number ERKP-851). This research used resources of the Oak Ridge Leadership Computing Facility at ORNL, supported by the DOE Office of Advanced Scientific Computing Research, Facilities Division. Small-angle neutron scattering was performed at ORNL using the Bio-SANS instrument at the High Flux Isotope Reactor, supported by the DOE Office of Biological and Environmental Research, Biological Systems Science Division, through the ORNL Center for Structural Molecular Biology, and the EQ-SANS instrument at the Spallation Neutron Source, supported by the DOE Office of Basic Energy Sciences, Scientific User Facilities Division (grant number ERKP-SNX). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
J.D. Nickels, S. Chatterjee, C.B. Stanley, S. Qian, X. Cheng, D.A.A. Myles, R.F. Standaert, J.G. Elkins, and J. Katsaras, “The in vivo structure of biological membranes and evidence for lipid domains,” PLoS Biology 5, 15. DOI:10.1371/journal.pbio.2002214. (Reference link)
ORNL Press Release: Neutrons provide the first nanoscale look at a living cell membrane
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