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

<div><p>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 <i>Bacillus subtilis</i> 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 <i>B</i>. <i>subtilis</i> 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.</p></div

Contrast variation small-angle neutron scattering (SANS) reveals the structure of the <i>B</i>. <i>subtilis</i> membrane.

<p>(a) Schematic of the hydrogen (H)-labeled membrane as seen by neutrons. The cell wall and cytoplasmic contents are contrast-matched at 85% D₂O, hence invisible to neutrons, while the membrane with incorporated H-fatty acids (FAs) stands out in strong contrast. (b) SANS data plotted as scattered intensity, <i>I(q)</i>, as a function of scattering wavevector, <i>q</i> (Å⁻¹), from the <i>B</i>. <i>subtilis</i> membrane. Error bars correspond to ± <i>σ</i>. The experimental data are shown superimposed with the best-fit (red solid line) using a lamellar form factor,[<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002214#pbio.2002214.ref045" target="_blank">45</a>] revealing an average hydrophobic thickness of 24.3 ± 0.9 Å, consistent with a fluid-phase lipid bilayer (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002214#pbio.2002214.s015" target="_blank">S3 Data</a>). Gas chromatography/mass spectrometry (GC/MS) analysis of the membrane FAs is shown in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002214#pbio.2002214.g002" target="_blank">Fig 2c</a> with peak integrals in Table E in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002214#pbio.2002214.s012" target="_blank">S1 Text</a>.</p

Detecting lateral lipid organization in <i>B</i>. <i>subtilis</i>.

<p>(a) Schematic of the experiment. Neutron contrast in the membrane was controlled using different blends of hydrogen (H)- and deuterium (D)-fatty acids (FAs). The control mixture consisted of anteiso-pentadecanoic acid (a15:0) and normal-hexadecanoic acid (n16:0), each 30% H and 70% D (inset to <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002214#pbio.2002214.g003" target="_blank">Fig 3b</a>, red curve). This mixture is contrast-matched to 85% D₂O and produces only background scattering whether or not the lipids are uniformly distributed (lower left and lower center panels). The experimental mixture contained the same FAs, but with a different isotopic distribution, i.e., n16:0 being 100% D and a15:0 being 40/60 H/D. In <i>B</i>. <i>subtilis</i>, this mixture produces an overall ratio of 30/70 H/D because a15:0 is more abundant (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002214#pbio.2002214.g002" target="_blank">Fig 2c</a>, bottom). If the lipids are uniformly distributed the membrane will be contrast-matched and produces no net scattering above background (indistinguishable from the control, lower center panel). However, if the lipids are organized in a manner that the high-melting n16:0 and low-melting a15:0 in membrane lipids are nonuniformly distributed, neutron contrast and a scattering signal will arise—illustrated in the lower right panel of (a) and in (c), which have patches enriched in n16:0. Just as these patches create contrast visible to the eye in the illustration, they create contrast visible to neutrons in the experiment in the form of excess scattering. (b) Small-angle neutron scattering (SANS) spectra of cerulenin-treated <i>B</i>. <i>subtilis</i> Δ<i>yusL</i>, incorporating experimental and control mixtures. The experimental mixture displays clear excess scattering (inset) that can result only from the nonuniform distribution of lipids with different contrast. From the relation ℓ = 2π/<i>q</i>, it can be inferred that the excess scattering in the range <i>q</i> = 0.01–0.15 Å⁻¹ corresponds to structures with length scales of 3–40 nm. Error bars correspond to ± <i>σ</i>. See <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002214#pbio.2002214.s016" target="_blank">S4 Data</a>. Gas chromatography/mass spectrometry (GC/MS) analyses of membrane FAs are provided in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002214#pbio.2002214.s010" target="_blank">S10 Fig</a>. (c) Representation of the membrane having visible lipid patches (of arbitrary structure), with the buffer, cell wall, and cytoplasmic contents all being contrast-matched.</p

Envelope structure and scattering properties of <i>B</i>. <i>subtilis</i>.

<p>(a) Representation of the cell wall, the membrane and a portion of the cytoplasm. (b) Neutron scattering length densities, <i>ρ</i>, of the different unlabeled biomolecules as a function of percent D₂O in the aqueous medium. Line colors correspond to (a). Sloped lines are the result of partial deuteration via exchange of NH, OH, and SH hydrogens with increasingly deuterated water. Where the line for each type of biomolecule crosses the black water line, e.g., at approximately 42% D₂O for protein (orange line), the class of biomolecule is contrast-matched and is effectively invisible to neutrons. (c) Total neutron scattering from cells grown in ordinary hydrogen (H) medium and resuspended in phosphate buffered saline (PBS) at different D₂O concentrations (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002214#pbio.2002214.s013" target="_blank">S1 Data</a>). The estimated intensity <i>I</i>(0) is shown with the observed intensity, expressed as the Porod invariant <i>Q</i>*, from small-angle neutron scattering (SANS) experiments. <i>I</i>(0) was calculated using the <i>ρ</i> values in (b) and the biomolecular composition of <i>B</i>. <i>subtilis</i> (Tables A-E in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002214#pbio.2002214.s012" target="_blank">S1 Text</a>). The contribution of individual species is shown using the same color scheme as in (b). Intensity scales with Δ<i>ρ</i>² and is plotted as its square root to maintain proportionality in the ordinates between (b) and (c).</p

Growth conditions to control membrane composition and neutron contrast.

<p>(a) Scattering length densities, <i>ρ</i>, for the different classes of biomolecules in <i>B</i>. <i>subtilis</i> cultured in 90% D₂O with hydrogen (H)-glucose. Compared to <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002214#pbio.2002214.g001" target="_blank">Fig 1b</a>, there is a clear shift of <i>ρ</i> to higher values for all classes of biomolecules as a result of deuteration, converging in the range 90%–100% D₂O. Incorporation of exogenous H-fatty acids (FAs; solid blue line) generates strong neutron contrast (blue arrow down). (b) Total neutron scattering from cells grown in deuterated medium and resuspended in phosphate buffered saline (PBS) at different D₂O concentrations. The observed scattered intensity <i>Q</i>* is shown along with the calculated intensities <i>I</i>(0) for Δ<i>yusL</i> cells grown in the absence (open circles) or presence (open squares) of cerulenin and H-FAs. The difference at 85% D₂O (blue up arrow) results from the strong contrast between H-FAs and the rest of the cell and buffer. (c) Cellular lipids were extracted, then methanolyzed to liberate FAs as methyl esters for analysis by gas chromatography/mass spectrometry (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002214#pbio.2002214.s003" target="_blank">S3 Fig</a>). (top) <i>B</i>. <i>subtilis</i> membranes have a mixture of 7 linear and branched saturated FAs.[<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002214#pbio.2002214.ref034" target="_blank">34</a>] (middle) Growth of cells in deuterated medium does not significantly alter FA composition (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002214#pbio.2002214.s004" target="_blank">S4 Fig</a> and Table B in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002214#pbio.2002214.s012" target="_blank">S1 Text</a>). Peaks shift because deuteration reduces retention on the column and broaden due to the presence of multiple isotopomers, averaging approximately 70% deuterium (D). (bottom) Cerulenin-treated Δ<i>yusL</i> cells rescued by the addition of 2 native FAs, H-anteiso-pentadecanoic acid (a15:0) and H-normal-hexadecanoic acid (n16:0), incorporate only these 2 FAs in their membranes. See <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002214#pbio.2002214.s014" target="_blank">S2 Data</a>. (d) FA degradation via β-oxidation was suppressed by deletion of the <i>yusL</i> (<i>fadN</i>) gene (locus BSU32840), and FA biosynthesis was blocked conditionally by the addition of cerulenin to the medium. Exogenous FAs rescue growth and become incorporated into membrane phospholipids.</p

<i>Bacillus subtilis</i> Lipid Extract, A Branched-Chain Fatty Acid Model Membrane

Lipid extracts are an excellent choice of model biomembrane; however at present, there are no commercially available lipid extracts or computational models that mimic microbial membranes containing the branched-chain fatty acids found in many pathogenic and industrially relevant bacteria. We advance the extract of <i>Bacillus subtilis</i> as a standard model for these diverse systems, providing a detailed experimental description and equilibrated atomistic bilayer model included as Supporting Information to this Letter and at (http://cmb.ornl.gov/members/cheng). The development and validation of this model represents an advance that enables more realistic simulations and experiments on bacterial membranes and reconstituted bacterial membrane proteins