Recombination Between 13 C and 2 H to Form Acetylide ( 13 C 2 2 H -) Probes Nanoscale Interactions in Lipid Bilayers Via Dynamic Secondary Ion Mass Spectrometry: Cholesterol and GM 1 Clustering
Although it is thought that there is lateral heterogeneity of lipid and protein components within biological membranes, probing this heterogeneity has proven challenging. The difficulty in such experiments is due to both the small length scale over which such heterogeneity can occur, and the significant perturbation resulting from fluorescent or spin labeling on the delicate interactions within bilayers. Atomic recombination during dynamic nanoscale secondary ion imaging mass spectrometry (NanoSIMS) is a non-perturbative method for examining nanoscale bilayer interactions. Atomic recombination is a variation on conventional NanoSIMS imaging whereby an isotope on one molecule combines with a different isotope on another molecule during the ionization process, forming an isotopically enriched polyatomic ion in a distance-dependent manner. We show that the recombinant ion, 13 C2 2 H -, is formed in high yield from 13 C-and 2 H-labeled lipids. The low natural abundance of triply labeled acetylide also makes it an ideal ion to probe GM1 clusters in model membranes and the effects of cholesterol on lipid-lipid interactions. We find evidence supporting the cholesterol condensation effect as well as the presence of nanoscale GM1 clusters in model membranes.
INTRODUCTION<!>MATERIALS AND METHODS<!>RESULTS AND DISCUSSION<!>CONCLUSIONS<!>ASSOCIATED CONTENT
<p>Biological membranes are composed of a diverse collection of proteins and lipids. Some of the most studied mammalian lipids are glycerophospholipids, sphingolipids and sterols. Cholesterol is of particular interest because it is thought to mediate the formation of nanoscale ordered and disordered domains composed of saturated lipids and unsaturated lipids, respectively. 1,2 This nanoscale separation has implications for biological functions such as signal transduction 3 , trafficking 4 and viral binding. 5 Furthermore, cholesterol is thought to have a condensing effect on lipid bilayers, whereby the average area per lipid decreases as a function of increasing cholesterol concentration. This has been attributed to cholesterol having an ordering effect on lipid tail groups; however, much of the evidence for this condensing effect is limited to simulations [6][7][8] or compression of monolayers. [9][10][11] The glycolipid GM1, found widely in brain tissue, has also been suggested to engage in nanoscale clustering, [12][13][14] with downstream effects on amyloid fibril formation 15,16 and growth factor function. 17,18 It is challenging to study lipid clustering on a molecular length-scale, and direct, non-perturbative experimental evidence for such clustering remains elusive. Many studies examine domains via detergent extraction, 19-21 fluorescence [22][23][24][25][26][27] or single particle tracking, 2,28,29 to name a few. 23,30,31 These methods all involve significant perturbations to the lipid system, 32,33 which makes it difficult to determine if the evidence for domain existence and size is due to the colocalization of the natural components or the perturbations caused by the analytical method. [32][33][34][35][36] Additionally, there is evidence that nanodomains may be smaller than 10 nm, placing them well below the diffraction limit of conventional microscopy. 2 Even the most sophisticated super-resolution methods, 37,38 which require perturbative dye labeling, 33 are challenged by these dimensions, further complicating how such interactions are investigated. Therefore, if clustering on the nanometer scale is to be studied, novel analytical methods need to be developed.</p><p>Nanoscale secondary ion mass spectrometry (NanoSIMS) can probe the organization of bilayers on the nanometer scale using isotopically labeled lipids. The NanoSIMS 50L functions by rastering a freeze-dried bilayer sample with a Cs + primary ion beam. Sputtered secondary ions from the sample are then separated by a mass analyzer, allowing for up to seven unique masses to be detected simultaneously. The advantage of this technique is that it can provide compositional information, via the calculation of high-precision isotope ratios. [39][40][41] This can be done on length scales ranging from a few microns to as little as 1 nm using secondary ion recombination, discussed in detail in the following. 42 This makes NanoSIMS an appealing method to examine nanoscale organization. Furthermore, the isotopic labeling required to identify unique lipid species causes minimal perturbation to the delicate intermolecular interactions within a bilayer.</p><p>Our group has developed methods to examine the macroscale organization of lipid bilayer patches using the 50 nm lateral resolution (determined by the primary ion beam diameter) of the NanoSIMS. 41,43 However, we recently demonstrated that atomic recombination can be applied to examine membrane organization below this lateral resolution limit. 42 This is particularly useful as colocalization of membrane components could take place on the nm length scale. 44,45 Atomic recombination is observed following sample ionization by the primary cesium beam. For example, if the sample is labeled with 13 C and 15 N on different molecules, then collisional sputtering of the sample results in the formation of 13 C 15 N -, 46 possibly through the gas-phase reaction of unstable species. Although the mechanism behind the process of recombination is not fully understood, it has been established that recombination depends not only on the chemical connectivity of labels (i.e. what the labels are covalently attached to), but also on the average distance between the two differently labeled molecules. 42,46,47 Critically, this distance dependence has been shown to be sensitive within 1-3 nm. This makes recombination a promising method to examine nanoscale colocalization of lipids within bilayers.</p><p>Here, recombination between 13 C and 2 H to form the triply labeled acetylide ion, 13 C2 2 His applied to examine nanoscale lipid interactions. Figure 1A shows schematically that as the average distance between a 13 C-labeled lipid and a 2 H-labeled lipid decreases, the formation of triply labeled acetylide is expected to increase. While both recombinant 13 C2 2 Hand 13 C 15 Nare well-separated from any interfering ions (Figure 1B), the advantage of recombinant 13 C2 2 His that it has much lower natural abundance (20 ppb) than 13 C 15 N -(44,000 ppb). This means that recombinant acetylide has improved signal-to-background. Furthermore, since there are more labeling sites for 2 H relative to 15 N on lipids, more 2 H labels can be incorporated into a lipid of interest, improving sensitivity. Additionally, 2 H labeling opens up the possibility of labeling lipid tail groups, where interactions critical to lateral heterogeneity are presumed to take place. Finally, although the acetylide ion is a triatomic species, it forms in higher yield compared to other CxHyions. Given these advantages, 13 C and 2 H recombination to form acetylide should be an ideal method to investigate the subtle effects of cholesterol on lipid organization as well as interactions between lipids that are present in lower concentrations in biological membranes, such as the important glycolipid GM1. Using the acetylide ion, the condensing effect of cholesterol is demonstrated via recombination. Additionally, 13 C and 2 H recombination to form acetylide is used to demonstrate nanoscale GM1 clustering in model membranes. Finally, nanoscale interaction between saturated lipids is demonstrated in brain lipid extracts.</p><!><p>Unlabeled phospholipids, cholesterol (CHOL), 2 H31-POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine), and 2 H70-DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine) were purchased from Avanti Polar Lipids. Unlabeled GM1 was purchased from Biosynth-Carbosynth. Texas Red 1,2dihexadecanoyl-sn-glycero-3-phosphoethanolamine (TR-DHPE) was purchased from Thermo Fisher Scientific. All solvents were purchased from Fisher. Four-inch <100> ptype silicon wafers with a 9.5 nm SiO2 layer were purchased from Silicon Quest International and were diced to 5x5 mm to fit in the NanoSIMS sample holder. This thickness of SiO2 has previously been found previously to provide a compromise between charge dissipation during the NanoSIMS measurements and bilayer stability. 43,48 A grid (25, 50 or 100 μm 2 ) patterned with chrome (5 nm height and 5 μm width) was imprinted on the substrates via photolithography to facilitate correlative imaging.</p><p>Labeled Lipid Synthesis. 13 C18-POPC, 13 C18-DSPC, 13 C18-GM1 and 2 H35-GM1 were synthesized by adapting previously described methods 41,49,50 to attach isotopically labeled fatty acids to the appropriate lysolipid. Detailed synthetic protocols and methods can be found in Sections 1 and 2 (and in Figures S1 and S2) of the Supporting Information. Structures for these as well as all other labeled lipids used in this study are shown in Figure 2.</p><p>NanoSIMS sample preparation. Supported lipid bilayer (SLB) patches were formed via fusion of giant unilamellar vesicles (GUVs) to oxidized silicon substrates. GUVs were produced by the method of gentle hydration. 51,52 Briefly, lipid mixtures containing 13 C-and 2 H-labeled lipids, as well as 0.1 mole percent TR-DHPE, were mixed from stock solutions before being dried down under a stream of argon and then further desiccated for at least 4 hours under house vacuum. The concentrations of lipid stock solutions were regularly calibrated via ESI-MS with lipid standards. The mixtures were then re-hydrated in a submicron filtered 500mM sucrose solution and heated to 65 ˚C, above the melting point of the high melting point lipid (DSPC 54 ˚C) for 15 hours. Silicon substrates were plasma cleaned for 10 minutes. Following plasma cleaning, GUVs were cooled to room temperature and deposited onto silicon substrates in phosphate buffer (240 mM NaCl, 10 mM NaH2PO4 at pH 7.4). The deposition of GUVs was monitored via fluorescence The formation of the triply labeled acetylide ion ( 13 C2 2 H -) depends on the average distance between 13 C and 2 H labels on different lipids in a supported lipid bilayer during Cs + bombardment (typical beam diameter is 50 nm, not to scale, and limiting the lateral resolution for conventional imaging). When the differently labeled lipids are closer together, more triply labeled acetylide is detected. In this figure and all subsequent figures, blue colored lipids will correspond to 13 C-labeled lipids, while the purple lipids correspond to 2 H-labeled lipids. (B) The acetylide ion mass is well-separated from any interfering isobars. Panel B is adapted from reference 42. microscopy using trace amounts (0.1 mole percent) of TR-DHPE, until 30-40% of the substrate was covered in SLB patches. The substrates and bilayer were then washed extensively with ultra-pure water, flash-frozen in liquid nitrogen and subjected to low pressure for 12 hours to sublime vitreous ice. This procedure has previously been shown to preserve bilayer morphology at high resolution. 43 The substrates were then examined by fluorescence microscopy. The chrome pattern on the substrate further assists in finding regions of the substrate where individual bilayers were formed from the rupture of single GUVs.</p><p>Asymmetric bilayers were prepared via Langmuir-Blodgett (LB) -Langmuir-Schaffer (LS) methods. First, LB monolayers were compressed and deposited onto diced silicon wafers using a KSV NIMA KN 2002 (Biolin Scientific, Stockholm, Sweden) Langmuir trough (273 cm 2 ). Whatmann filter paper was used as a Whilhelmy plate to monitor surface pressure. The lipid mixture for the lower leaflet dissolved in chloroform was spread on water (>18 MΩ from Milli-Q system) placed within the clean trough. The chloroform was left to evaporate for 10 minutes before compressing the barriers at 10 mm/min until the surface pressure reached 32 mN/m. Plasma cleaned NanoSIMS substrates were glued with E6000 adhesive (Eclectic Products) to a glass slide. The glass slide and attached substrate were then pulled through the air-water interface at a rate of 1 mm/min while the surface pressure was maintained at 32 mN/m, creating a monolayer on the surface (such monolayers are stable in air). 42 Then, a new monolayer with the upper leaflet lipid mixture was formed on the trough. The substrate with the deposited lower leaflet monolayer was then removed from the glass slide before the glue dried and passed through the air-water interface perpendicular to the surface to deposit the upper leaflet. Since SLBs are not stable in air 53 , the substrate was removed such that a drop of water remained on the surface with the asymmetric bilayer. The substrate was then rapidly flash frozen in liquid nitrogen and subjected to low pressure as previously described.</p><p>NanoSIMS Analysis. Analysis was performed on the Cameca NanoSIMS 50L at Stanford University. Images were collected with a 2 pA 133 Cs + primary beam. Ten 25 μm x 25 μm scans of 256 x 256 pixels were collected (enough to remove all the deposited lipid from the surface) with a dwell time of 1 ms/pixel. The ion detectors were set to 2 H -, 12 C -, 13 C -, 12 C2 1 H -, 12 C2 2 H -, 13 C2 1 H -, and 13 C2 2 H -. Secondary electron images were collected simultaneously. Standard samples were regularly analyzed to ensure that isotope ratios were reproducible from day to day and session to session.</p><p>Data Analysis. Images were analyzed using ImageJ (National Institutes of Health, USA) with the OpenMIMS plugin (National Resource for Mass Spectrometry, Harvard University USA). Planes were summed and regions of interest were manually selected to exclude debris on the sample. Total counts within each region of interest were determined via the "Tomography" tab. These counts were then used to determine the ratio of interest, ℜ( 13 C2 2 H -) ≡ 13 C2 2 H -/ ( 13 C2 2 H -+ 13 C2 1 H -+ 12 C2 2 H -), to control for the size of the analyzed bilayer (formed from the rupture of a single GUV) and for variations in ionization efficiency.</p><!><p>Recombination to form Acetylide is Distance-Dependent. To demonstrate that the formation of triply labeled acetylide depends on the average distance between 13 C and 2 H labels on different molecules, GUV patches (formed from the rupture of individual GUVs) with microscale coexisting phases were prepared, as was done in earlier work with 13 C and 15 N recombination to form 13 C 15 N -. 42 A mixture of 50:50 POPC:DSPC was examined. This mixture has microscale phase separation between DSPC and POPC that is visible by conventional NanoSIMS imaging (50 nm lateral resolution). Furthermore, mixtures of these lipids are well-studied in GUVs. 44,45 . In this composition, the saturated lipids cluster together into gel domains, while the unsaturated lipids cluster into disordered domains. The effect of altering the average distance between isotopic labels was examined by comparing recombination in bilayers containing 13 C18-DSPC and 2 H70-DSPC to bilayers containing 13 C18-POPC and 2 H70-DSPC. If the formation of 13 C2 2 His a distance-dependent process, then recombination between 13 C and 2 H to form 13 C2 2 Hshould be high in bilayers where both the 13 C and 2 H lipids localize to the gel domain. Conversely, the formation of 13 C2 2 Hshould be low in bilayers where the 13 C-labeled lipid localizes to the disordered domain and the 2 H-labeled lipid localizes to the gel domain. As can be seen in Figure 3A, when both labels are in the same domains ( 2 H70-DSPC -13 C18-DSPC) there are high standardized counts (ratio shown above the images in Figure 3A) of the recombinant ion within the DSPC-rich domains. When the labels are placed on lipids that localize to different domains ( 2 H70-DSPC -13 C18-POPC), such high signal is not seen visually. Quantification of the recombination ratio, ℜ( 13 C2 2 H -) ≡ 13 C2 2 H -/ ( 13 C2 2 H -+ 13 C2 1 H -+ 12 C2 2 H -), within the gel domains of both samples is shown in Figure 3B. This quantification recapitulates the visual results from Figure 3A. Only in bilayers where the 13 C-and 2 H-labeled lipids colocalize to the same region is high ℜ( 13 C2 2 H -) seen. If the 13 C-and 2 H-labeled lipids localize to different domains, and are consequently farther apart on average, there is a substantial drop in ℜ( 13 C2 2 H -). Having established that recombination to form 13 C2 2 His distancedependent, much like 13 C 15 N -, 42 atomic recombination was then used to examine the effects of cholesterol on nanoscale lipid-lipid interactions. Effects of Cholesterol on Lipid-Lipid Interactions. The advantage of atomic recombination is that it circumvents the lateral resolution limit of the NanoSIMS 42 (approximately 50 nm). This makes it possible to use 13 C2 2 Hformation to probe subtle effects of cholesterol in mixtures with nanoscale, rather than microscale, phase separation or clustering. To probe the effects of cholesterol on the average distance between lipids, bilayers composed of POPC/DSPC/CHOL were studied, as this mixture exhibits nanoscale heterogeneity at certain compositions. 44,45 As can be seen in Figure 4A, conventional NanoSIMS imaging cannot resolve any heterogeneity in the displayed bilayer, in contrast to the bilayers shown in Figure 3A. However, atomic recombination can still distinguish between samples where labeled lipids are placed in the same versus different nanoscale clusters in similar samples (Figure S3).</p><p>To examine the effects of cholesterol on lipid-lipid interactions, bilayers containing 15, 20, 25 and 30 mole percent cholesterol with a constant 1:1 POPC:DSPC lipid ratio were prepared. These samples contained 13 C-and 2 Hlabeled lipids in either the same ordered ( 13 C18-DSPC and 2 H70-DSPC), disordered ( 13 C18-POPC and 2 H31-POPC) or different ( 13 C18-DSPC and 2 H31-POPC) nanoscale domains. This allowed the effects of cholesterol on lipids within and between domains to be systematically probed by tracking how ℜ( 13 C2 2 H -) varies with increasing cholesterol concentration. Detailed compositional information can be found in Table S1 and Figure S4. To maximize ℜ( 13 C2 2 H -), a 1:1 mole ratio of 13 C-labeled lipid to 2 H-labeled lipid was used for all samples (Figure S5).</p><p>The effects of cholesterol concentration on ℜ( 13 C2 2 H -) can be seen in Figure 4B-D. As cholesterol concentration is increased, there is a statistically significant increase in ℜ( 13 C2 2 H -) for bilayers containing 13 C18-DSPC and 2 H70-DSPC, as well as bilayers containing 13 C18-POPC and 2 H31-POPC. Conversely, there is a significant decrease in ℜ( 13 C2 2 H -) for bilayers containing 13 C18-DSPC and 2 H31-POPC. It can therefore be concluded that for the first two cases ( 13 C18-DSPC -2 H70-DSPC and 13 C18-POPC -2 H31-POPC), the average distance between the 13 C-and 2 H-labeled lipids decreases as cholesterol concentration increases. For the third case ( 13 C18-DSPC -2 H31-POPC) the average distance between the 13 C-and 2 H-labeled lipids increases as cholesterol concentration increases.</p><p>The trend observed for 13 C18-DSPC -2 H70-DSPC and 13 C18-POPC -2 H31-POPC bilayers can be attributed to the cholesterol condensation effect, which should closely pack lipids within a nanoscale domain 7,[54][55][56] . This close packing decreases the average distance between 13 C-and 2 H-labeled lipids resulting in a larger ℜ( 13 C2 2 H -). Interestingly, the measured ℜ( 13 C2 2 H -) in 13 C18-DSPC and 2 H70-DSPC containing bilayers also decreases when transitioning from macroscale gel phase (Figure 3B) to nanoscale gel plus Lo phase (Figure 4B). This result means that cholesterol initially decreases average distances between lipids within the same domain during the gel to Lo phase transition. The decrease in ℜ( 13 C2 2 H -) upon transition likely results from a combination of the change in domain size and the decreased lipid order in the Lo phase relative to the gel phase. 44 The upper row displays a bilayer where the 2 H-labeled lipid ( 2 H70-DSPC) and the 13 Clabeled lipid ( 13 C18-DSPC) both localize to the DSPC-rich gel domains. High recombination is observed in these domains since the 13 C and 2 H labels are close. The lower row of NanoSIMS images displays the second bilayer, where the 2 H-labeled lipid ( 2 H70-DSPC) localizes to the gel domains and 13 C-labeled lipid ( 13 C18-POPC) localizes to the disordered domains. Less recombination is observed as the 13 C and 2 H labels are farther apart on average. (B) Quantification of recombination between 13 C and 2 H to form 13 C2 2 Hin the gel domains. Samples where labeled lipids localize to the same ( 2 H70-DSPC -13 C18-DSPC) or different ( 2 H70-DSPC -13 C18-POPC) domains are compared. The recombination ratio ℜ( 13 C2 2 H -) ≡ 13 C2 2 H -/ ( 13 C2 2 H -+ 13 C2 1 H -+ 12 C2 2 H -) is higher when both labeled lipids are in the DSPC-rich gel domains and are closer together on average. Error bars in this and all subsequent figures display the 95% confidence interval. Seven bilayer patches were examined for each sample.</p><p>The ℜ( 13 C2 2 H -) measured in bilayers containing 13 C18-DSPC and 2 H31-POPC displays the opposite trend. As cholesterol concentration is increased, ℜ( 13 C2 2 H -) decreases. This corresponds to an increase in the average distance between the 13 C-and 2 H-labeled lipids. This could be due to cholesterol localizing at domain interfaces, as has been suggested by simulations. [56][57][58] Interfacial partitioning may also provide an explanation for why cholesterol does not dilute the labeled lipids in ordered domains as its concentration increases (if this were a dominating factor, bilayers containing 13 C18-DSPC and 2 H70-DSPC would display decreasing ℜ( 13 C2 2 H -) as cholesterol concentration increases). There are, however, potential effects due to the labeling strategy used which may influence the observed ℜ( 13 C2 2 H -) trend. In particular, the decreasing label concentration may increase the average distance between labeled lipids, decreasing ℜ( 13 C2 2 H -) (See Figures S6-7, Table S1, and the discussion in Section 7 in the Supporting Information).</p><p>GM1 Clustering in Model Membranes. Previous work, largely simulations, 12,13 suggests that GM1 can engage in nanoscale or macroscale clustering. 16,59 A truly nonperturbative confirmation of such clustering would help further understanding of membrane interactions as well as protein-lipid interactions, as GM1 clustering may have downstream implications for amyloid fibril formation 60,61 and growth factor function. 62,63 Atomic recombination was further applied to probe this clustering.</p><p>Since GM1 is present at low concentration in the plasma membrane of neurons, the extent of labeling necessary to identify nanoscale clustering was investigated. The result from this investigation can be seen in Figure S3, where ℜ( 13 C2 2 H -) in bilayers containing 2 H70-DSPC and 13 C18-DSPC (same nanoscale domain) is compared to ℜ( 13 C2 2 H -) in bilayers containing 2 H70-DSPC and 13 C18-POPC (different nanoscale domain). Due to nanoscale phase separation between DSPC and POPC lipids, higher ℜ( 13 C2 2 H -) is seen in bilayers containing 2 H70-DSPC and 13 C18-DSPC even when using as little as 4 mole percent total isotopically labeled lipid (2 mole percent each of 13 C-and 2 H-labeled lipids). Atomic recombination is therefore a plausible method for investigating nanoscale heterogeneity of components present in low concentrations, such as GM1.</p><p>To examine whether GM1 engages in local clustering, isotopically labeled GM1 was synthesized as shown in Figure S2. This enables ℜ( 13 C2 2 H -) in bilayers containing 13 C18-GM1 and 2 H35-GM1 could be compared to ℜ( 13 C2 2 H -) in bilayers containing 2 H35-GM1 and either 13 C18-POPC or 13 C18-DSPC. Previous work has suggested that GM1 may localize to the upper leaflet in supported lipid bilayers. 64 Therefore, in SLBs formed from GUV rupture, the average distance between 13 C18-and 2 H35-GM1 may be artificially lower due to these lipids preferentially localizing to the upper leaflet. This in turn would lead to artificially higher ℜ( 13 C2 2 H -) relative to samples containing 13 C18-POPC or 13 C18-DSPC, as neither of these lipids localize to a particular leaflet. As a result, the conventional approach using GUV deposition of symmetric bilayers is not applicable to this experiment. Instead, asymmetric bilayers were formed via Langmuir-Blodgett (LB) -Langmuir-Schaffer (LS) methods. A lower leaflet of POPC was deposited as a monolayer onto a patterned silicon substrate, after which it was plunged through a second monolayer containing 2 H35-GM1, and either 13 C18-GM1, 13 C18-POPC or 13 C18-DSPC, before being  S1 for detailed compositional information. For each data point, a minimum of ten individual bilayer patches were examined. rapidly freeze-dried. All lipid mixtures were compressed to 32 mN/m, which corresponds to approximately 60 A 2 for each molecule. The resulting bilayers have asymmetric leaflets as shown schematically in Figure 5A, with isotopically labeled lipids in only the upper leaflet.</p><p>The measured ℜ( 13 C2 2 H -) for bilayers with each labeled lipid pair ( 2 H35-GM1 -13 C18-GM1, 2 H35-GM1 -13 C18-DSPC and 2 H35-GM1 -13 C18-POPC) is shown in Figure 5B. The displayed ratios demonstrate highest ℜ( 13 C2 2 H -) in bilayers containing 2 H35-GM1 and 13 C18-GM1. ℜ( 13 C2 2 H -) in bilayers containing 2 H35-GM1 and either 13 C18-DSPC or 13 C18-POPC are significantly lower. Bilayers containing 2 H35-GM1 and 13 C18-DSPC do not display a significant difference in ℜ( 13 C2 2 H -) relative to those containing 2 H35-GM1 and 13 C18-POPC (p = 0.078). Therefore, there is not sufficient evidence to suggest a preference for GM1 interactions between saturated or unsaturated lipids. It is possible that such a preference exists, given that a saturated lipid may pack better with GM1 relative to an unsaturated lipid; however, within the limits of this experiment, there is no detectable difference. Prior simulations suggest that GM1-GM1 interactions are mediated by specific interactions between cholesterol and GM1. This, in addition to hydrogen bonding between GM1 headgroups, may mediate the formation of clusters. 12,61,65 The experiments described here provide direct and labelfree evidence for GM1 clustering, consistent with previous simulations.</p><p>Recombination in Lipid Extracts. While the model systems examined thus far contain 3-4 components, biological membranes are considerably more complex. Therefore, a more complex mixture was examined to test if greater lipid diversity abolishes the nanoscale heterogeneity demonstrated above. A first approximation for this diversity can be achieved using natural lipid extracts. Although commercially available extracts obviously do not recapitulate the effect that peripheral and transmembrane proteins have on bilayer organization, they at least serve as a model for a more complex system with greater variety in terms of lipid headgroups and unsaturation levels. To probe heterogeneity in this complex mixture, 3nmol of 13 C18-DSPC and 2 H70-DSPC (same nanoscale domain) or 13 C18-POPC and 2 H70-DSPC (different nanoscale domains) were mixed into brain lipid extracts (Figure 6A). ℜ( 13 C2 2 H -) was compared between bilayer patches using the two labeling schemes. If nanoscale separation between saturated and unsaturated lipids is present in this more complex bilayer, then ℜ( 13 C2 2 H -) should be greater in bilayers containing 13 C18-DSPC and 2 H70-DSPC relative to those with 13 C18-POPC and 2 H70-DSPC. The resulting recombination ratios can be seen in Figure 6B. The measured ℜ( 13 C2 2 H -) for bilayers containing 13 C18-DSPC and 2 H70-DSPC is significantly greater than that measured for bilayers containing 13 C18-POPC and 2 H70-DSPC. This demonstrates that the increased lipid diversity of a comparatively more biological system does not abolish nanoscale clustering of saturated lipids. It is possible that these interactions are mediated by cholesterol or result from favorable packing between saturated lipids relative to packing between saturated and unsaturated lipids.</p><!><p>The paucity of analytical methods to interrogate the lateral organization of biological membranes on the nanometer scale has proven to be a significant barrier to improving our understanding of these complex systems. This is largely due to the small length scale over which lipidlipid interactions can occur and the significant perturbations conventional labeling strategies can have on the delicate and transient interactions between bilayer components. In this work we build on previously established atomic recombination methods and take For each labeling scheme, 10 mol% of labeled lipid (5 mol% 13 C18-GM1, 13 C18-POPC or 13 C18-DSPC and 5 mol% 2 H35-GM1) replaced the respective neutral lipid(s). Twenty independent bilayers were examined for each sample. The ℜ( 13 C2 2 H -) in bilayers containing 2 H35-GM1 and 13 C18-GM1 is significantly different from the ℜ( 13 C2 2 H -) in bilayers containing 2 H35-GM1 and 13 C18-POPC or 13 C18-DSPC. advantage of a previously unused ion ( 13 C2 2 H -) with improved sensitivity. We apply this ion to examine fundamental questions regarding the effects of cholesterol on lipid interactions as well as the nanoscale interactions of GM1. Recombination to form acetylide has also been leveraged to examine more complex systems that may serve as a better approximation of compositional heterogeneity present in a biological membrane.</p><p>There has been considerable debate over the past few decades over the role of cholesterol in lipid bilayers; much of the evidence for the condensation effect of cholesterol relies on simulation. 7,66 Recombination to form acetylide is a novel method for examining the subtle effects that cholesterol can have on bilayer organization. A particular advantage of atomic recombination is its non-perturbative nature, as it does not rely on fluorescent or spin labels, 30,67 commonly used for investigating membrane organization. Although a significant advantage of these conventional labels is that they can be present in low concentration and still attain high signal, the work presented here demonstrates that atomic recombination is also quite sensitive. The large number of 13 C and 2 H labels that can be placed on lipids with relative ease leads to reasonable signal even at relatively low (a few mole percent) labeled lipid concentration.</p><p>Using this high sensitivity, we were able to demonstrate that there is evidence of preferential interaction between GM1 lipids relative to other bilayer components, providing evidence for nanoscale GM1 clustering. This emphasizes that recombination can be applied to study interactions between bilayer components that are present in low concentrations in biological membranes. Recombination methods can also be applied to more complex systems, such as lipid extracts, to detect nanoscale clustering. To obtain an even clearer picture of bilayer organization, atomic recombination could be used to examine more than lipid-lipid interactions such as protein-lipid or peptide-lipid interactions, and these applications are being pursued.</p><!><p>Synthesis of 13 C18-POPC and 13 C18-DSPC; synthesis of 13 C18 NHS stearate and 2 H35 NHS stearate; synthesis of 13 C18-GM1 and 2 H35-GM1; examining lower limits of recombination; lipid compositions; optimization of recombination signal; concentration dependence of recombination Figure 6. Recombination in brain lipid extracts. (A) 13 C18-DSPC and 2 H70-DSPC or 13 C18-POPC and 2 H70-DSPC are mixed into brain lipid extracts and the mixture is then used to form supported lipid bilayers via rupture of single GUVs. Once mixed in, isotopically labeled POPC and DSPC may be incorporated into different nanoscale domains in bilayers, highlighted schematically by the red dashed lines. For both 13 C18-POPC -2 H70-DSPC and 13 C18-DSPC -2 H70-DSPC samples, 3 nmol of 13 C lipid (either 13 C18-DSPC or 13 C18-POPC) and 3 nmol of 2 H70-DSPC were mixed into brain lipid extracts (approximately 10 percent of the total mass of the deposited bilayer). Unlabeled lipids were added to each mixture so that the lipid mixture added to the brain lipid extracts was the same between the two samples (9 nmol of DSPC and 3 nmol POPC). (B) The measured ℜ( 13 C2 2 H -) is higher in bilayers containing the saturated 13 C18-and 2 H70-DSPC lipids than those containing unsaturated 13 C18-POPC and saturated 2 H70-DSPC. Ten bilayer patches were examined for each sample.</p>