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

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

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

Description of Hydration Water in Protein (Green Fluorescent Protein) Solution

The structurally and dynamically perturbed hydration shells that surround proteins and biomolecules have a substantial influence upon their function and stability. This makes the extent and degree of water perturbation of practical interest for general biological study and industrial formulation. We present an experimental description of the dynamical perturbation of hydration water around green fluorescent protein in solution. Less than two shells (∼5.5 Å) were perturbed, with dynamics a factor of 2–10 times slower than bulk water, depending on their distance from the protein surface and the probe length of the measurement. This dependence on probe length demonstrates that hydration water undergoes subdiffusive motions (τ ∝ <i>q</i>^–2.5 for the first hydration shell, τ ∝ <i>q</i>^–2.3 for perturbed water in the second shell), an important difference with neat water, which demonstrates diffusive behavior (τ ∝ <i>q</i>^–2). These results help clarify the seemingly conflicting range of values reported for hydration water retardation as a logical consequence of the different length scales probed by the analytical techniques used

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

Subnanometer Structure of an Asymmetric Model Membrane: Interleaflet Coupling Influences Domain Properties

Cell membranes possess a complex three-dimensional architecture, including nonrandom lipid lateral organization within the plane of a bilayer leaflet, and compositional asymmetry between the two leaflets. As a result, delineating the membrane structure–function relationship has been a highly challenging task. Even in simplified model systems, the interactions between bilayer leaflets are poorly understood, due in part to the difficulty of preparing asymmetric model membranes that are free from the effects of residual organic solvent or osmotic stress. To address these problems, we have modified a technique for preparing asymmetric large unilamellar vesicles (aLUVs) via cyclodextrin-mediated lipid exchange in order to produce tensionless, solvent-free aLUVs suitable for a range of biophysical studies. Leaflet composition and structure were characterized using isotopic labeling strategies, which allowed us to avoid the use of bulky labels. NMR and gas chromatography provided precise quantification of the extent of lipid exchange and bilayer asymmetry, while small-angle neutron scattering (SANS) was used to resolve bilayer structural features with subnanometer resolution. Isotopically asymmetric POPC vesicles were found to have the same bilayer thickness and area per lipid as symmetric POPC vesicles, demonstrating that the modified exchange protocol preserves native bilayer structure. Partial exchange of DPPC into the outer leaflet of POPC vesicles produced chemically asymmetric vesicles with a gel/fluid phase-separated outer leaflet and a uniform, POPC-rich inner leaflet. SANS was able to separately resolve the thicknesses and areas per lipid of coexisting domains, revealing reduced lipid packing density of the outer leaflet DPPC-rich phase compared to typical gel phases. Our finding that a disordered inner leaflet can partially fluidize ordered outer leaflet domains indicates some degree of interleaflet coupling, and invites speculation on a role for bilayer asymmetry in modulating membrane lateral organization

<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

Mechanical Properties of Nanoscopic Lipid Domains

The lipid raft hypothesis presents insights into how the cell membrane organizes proteins and lipids to accomplish its many vital functions. Yet basic questions remain about the physical mechanisms that lead to the formation, stability, and size of lipid rafts. As a result, much interest has been generated in the study of systems that contain similar lateral heterogeneities, or domains. In the current work we present an experimental approach that is capable of isolating the bending moduli of lipid domains. This is accomplished using neutron scattering and its unique sensitivity to the isotopes of hydrogen. Combining contrast matching approaches with inelastic neutron scattering, we isolate the bending modulus of ∼13 nm diameter domains residing in 60 nm unilamellar vesicles, whose lipid composition mimics the mammalian plasma membrane outer leaflet. Importantly, the bending modulus of the nanoscopic domains differs from the modulus of the continuous phase surrounding them. From additional structural measurements and all-atom simulations, we also determine that nanoscopic domains are in-register across the bilayer leaflets. Taken together, these results inform a number of theoretical models of domain/raft formation and highlight the fact that mismatches in bending modulus must be accounted for when explaining the emergence of lateral heterogeneities in lipid systems and biological membranes