Lipid Driven Nanodomains in Giant Lipid Vesicles are Fluid and Disordered

It is a fundamental question in cell biology and biophysics whether sphingomyelin (SM)- and cholesterol (Chol)- driven nanodomains exist in living cells and in model membranes. Biophysical studies on model membranes revealed SM and Chol driven micrometer-sized liquid-ordered domains. Although the existence of such microdomains has not been proven for the plasma membrane, such lipid mixtures have been often used as a model system for ‘rafts’. On the other hand, recent super resolution and single molecule results indicate that the plasma membrane might organize into nanocompartments. However, due to the limited resolution of those techniques their unambiguous characterization is still missing. In this work, a novel combination of Förster resonance energy transfer and Monte Carlo simulations (MC-FRET) identifies directly 10 nm large nanodomains in liquid-disordered model membranes composed of lipid mixtures containing SM and Chol. Combining MC-FRET with solid-state wide-line and high resolution magic angle spinning NMR as well as with fluorescence correlation spectroscopy we demonstrate that these nanodomains containing hundreds of lipid molecules are fluid and disordered. In terms of their size, fluidity, order and lifetime these nanodomains may represent a relevant model system for cellular membranes and are closely related to nanocompartments suggested to exist in cellular membranes.

concerning properties of biological membranes question the biological relevance of the L o phase microdomains found in model membranes. The assumption that experiments on model membranes can reveal biologically relevant information leads us to two central questions: firstly, can lipid driven domains in model membranes be smaller than 40 nm and secondly if so, do such nanodomains have a L o character?
In this work we used MC-FRET in combination with novel monosialoganglioside GM 1 fluorescent probes to uncover the existence of nanodomains in lipid bilayers that should be in a homogeneous liquid disordered (L d ) phase according to published phase diagrams 14,15 . FRET has been frequently used in the past to reveal microto nano-scale heterogeneities in lipid membranes [16][17][18] but mostly on a qualitative level. Combination of FRET with MC simulations enabled us quantifying the sizes of domains down to a few nanometers and the fractional area occupied by these domains. To assess the fluidity and phase of the nanodomains we employed solid state wide-line and high resolution MAS (magic angle spinning) NMR spectroscopy, two-color z-scan fluorescence correlation spectroscopy (FCS) 19 and FRET.

Determination of nanodomain size and fractional bilayer area by MC-FRET.
Description of the MC-FRET approach. FRET between a single donor and a single acceptor occurs at distances between 1 to 10 nm and can be used as a molecular ruler within this accessible range. The situation is different when FRET occurs in a lipid bilayer that contains nanodomains and an ensemble of heterogeneously distributed donors and acceptors. Here, the formation of nanodomain structures forces a homogeneous distribution of donors and acceptors (Fig. 1, case 1) into a heterogeneous one (Fig. 1, cases 2 and 3) when using appropriate fluorescent probes that possess either an increased or decreased affinity for such nanodomains. This causes a change in FRET efficiency that can be seen in the recorded fluorescence decays (Fig. 1, bottom right corner). In these cases, the range of accessible distances (domain radii) that can be determined is significantly broader (2-50 nm) 20 and lies exactly in the region where other techniques become less efficient. The remarkably broad range of accessible distances is a consequence of FRET that occurs at the boundary of the nanodomains and of the fact that the length of that boundary depends on the nanodomain radius R D . The entire process of energy transfer can be modeled using MC simulations under certain assumptions (see Materials and Methods for details). The simulated decay curves were fitted to the experimental data by varying the radius of the nanodomains R D , the fractional bilayer area occupied by the nanodomains Ar (which is proportional to nanodomain concentration c D by c D = Ar/(πR D )) and the distribution constants of donors K D (D) and acceptors MC-FRET can detect various kinds of membrane heterogeneities, such as domains or pores 21,22 . However, its resolution significantly depends on both K D (D) and K D (A). If the probes possess equal affinity for the nanodomains and the remaining bilayer, then the formation of heterogeneities in a bilayer will not induce a heterogeneous probe distribution and therefore no change in FRET efficiency will occur. Consequently, such heterogeneities will not be 'seen' by FRET and the selected probes. Thus, Donor/Acceptor (D/A) pairs with suitable K D have to be chosen. Based on literature 23 and our previous work 24, 25 we used two different D/A pairs for the detection of lipid driven nanodomains. The first one consisted of ganglioside GM 1 molecules labeled at the headgroup with either FL-BODIPY (g-GM 1 ) or 564/570-BODIPY (r-GM 1 ). Both g-GM 1 and r-GM 1 show increased affinity for the L o microdomains but also to less ordered fluid nanodomains (refs 24 and 25 and as shown on Table 1 and Table 2). Importantly, these GM 1 probes do not intrinsically self-aggregate at the concentrations used in the FRET experiments (see SI and ref. 26). The second D/A pair consisted of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino (Polyethyleneglycol) 2000] labeled at the end of the Polyethyleneglycol chain with either carboxyfluorescein (CF-PEG-DSPE) or Rhodamine101 (Rh-PEG-DSPE). PEG-DSPE lipids were shown to have increased affinity for the L o phase 23 , which is confirmed by this work (Table 2). In addition, here we demonstrate that these probes preferentially partition to the nanodomains that are rich in Chol and SM but still maintain their liquid-disordered character.
Nanodomains outside the L d /gel phase coexistence region in DOPC/SM. These mixtures phase-separate at 23 mol% of SM (L d + gel phase coexistence) and are fully converted into the gel phase at 81 mol% of SM at room ; ** as compared to FRET obtained in a homogeneous bilayer; *** this conclusion is drawn based on K D s given in Table 1.
temperature ( Fig. 2) 27,28 . To detect and characterize nanodomains in this binary system we performed MC-FRET experiments in the range 0-15 mol% of SM with g-GM 1 /r-GM 1 and CF-PEG-DSPE/Rh-PEG-DSPE D/A pairs. All bilayers appeared homogeneous in confocal images (see an example in Fig. 2). MC-FRET results obtained using g-GM 1 /r-GM 1 D/A pair indicated that the bilayers were homogeneous at DOPC/SM (100-92/0-8), while at (90-85/10-15) we were able to detect nanodomains ( Table 1). The presence of nanodomains is reflected in an enhanced relative FRET efficiency (see Table 1 for results and Materials and Methods for definition) and a faster  Scientific RepoRts | 7: 5460 | DOI:10.1038/s41598-017-05539-y fluorescence decay of the donor g-GM 1 in the presence of r-GM 1 (compare the two decays in the top left panel of Fig. 3). Determination of average domain radius yielded two global minima at R D = (8 ± 1) nm, Ar = (37 ± 10) % and R D = (12 ± 3) nm, Ar = (55 ± 10) %. The best fit was obtained for K D = 10, showing a high affinity of the GM 1 probes for the domains. In contrast, the distribution of the CF-PEG-DSPE/Rh-PEG-DSPE D/A pair in the bilayer was not affected by the presence of nanodomains (K D (D,A) = 1), which did not allow for the detection of nanodomains by means of this D/A pair (see Table 1 and overlapping decays in the bottom left panel of Fig. 3). It is worth noting that binary DOPC/Chol mixtures exhibit different behavior. We showed previously that lipid mixtures of DOPC/Chol (65/35) were homogeneous as determined by FRET 24 . Transient nanodomains were found for this binary mixture only close to the phase separation boundary by other methods 29,30 where miscibility of Chol with DOPC is low 28 and Chol starts to phase-separate into anhydrous and monohydrate crystals 31 .
Nanodomains outside the L d /L o phase coexistence region in DOPC/Chol/SM. Addition of 25 mol% of Chol to the DOPC/SM bilayers promoted the formation of nanodomains. Here nanodomains were detected at DOPC/ Chol/SM (70-65/25/5-10) by g-GM 1 /r-GM 1 D/A pair. The enhanced relative FRET efficiency as compared to the homogeneous DOPC/Chol/SM (75/25/0) bilayer and the time resolved fluorescence decays of the donor g-GM 1 can be seen in Table 1 and Fig. 3, respectively. Determination of domain sizes by MC-FRET yielded an average R D (9 ± 1) nm and Ar = (45 ± 5) %. Deep chi-squared minima were only reached when K D (D) and K D (A) were at least 20, demonstrating that the GM 1 probes were highly localized in the nanodomains (see Fig. SI4). Moreover, the nanodomains were also detected by the CF-PEG-DSPE/Rh-PEG-DSPE DA pair at DOPC/Chol/ SM (65/25/10) and (63/25/12) ( Table 1 and Fig. 3). The affinity of the PEG-DSPE probes for the domains was lower (K D (D) and K D (A) ≈ 5), but sufficient to cause a change in the relative FRET efficiency and enable the determination of domain sizes at the higher SM amounts. The determined average R D = (8 ± 1) nm and Ar = (55 ± 5) % are in good agreement with the parameters determined using the g-GM 1 /r-GM 1 pair.

Supportive evidence for nanodomain existence by z-scan FCS. Our FRET measurements indi-
cate that the nanodomains occupy up to 55% of the entire bilayer area in binary DOPC/SM as well as ternary DOPC/Chol/SM lipid mixtures and exhibit an average radius of approximately 10 nm. According to our previous work focusing on MC simulations of molecular probe diffusion in a lipid bilayer 32 , the presence of stable (ca. >10 ms) nanodomains at such high domain concentration slows down the diffusion of fluorescently labeled lipids (=probes, Fig. 4). The extent to which the diffusion of the probes is slowed down depends in particular on their K D , the size of the nanodomains, the diffusion coefficient of the nanodomains themselves D (nanodomain), and the diffusion coefficient of the probes within those nanodomains D (probe). The strongest impact on probe diffusion occurs when the nanodomains are immobile and the probes have high affinity for them. When using classical FCS (where the focal waist is much larger than the nanodomains, about 300 nm vs. 10 nm) the presence of nanodomains is reflected in a slower diffusion behavior of the probe that can still be described by the free diffusion model (for example at K D (probe) = 25, domain radius = 50 nm and diffusion coefficient of nanodomains = 0.8 μm 2 /s probe diffusion is 5 times slower) 32 . However, considering the small size of the nanodomains described in this manuscript, it can be expected that they are mobile. In such case their impact on probe diffusion is less pronounced but still significant in most cases (for details see ref. 32). When probes avoid entering the nanodomains (K D < 1, panel A of Fig. 4) their diffusion is slowed down as well and does not exhibit any deviations from free diffusion as seen by classical FCS. In general, their sensitivity to the presence of nanodomains is smaller compared to probes that partition mostly towards nanodomains.
Considering the high affinity of g-GM 1 for nanodomains (Table 1), we used it as a probe to detect nanodomains by FCS. For comparison, we also used the DiD probe. It can be inferred from FRET experiments using g-GM 1 and DiD (Fig. SI3), that DiD is homogeneously distributed between the nanodomains and the remaining bilayer. Therefore, the expected impact of nanodomains on its diffusion will be smaller.
In DOPC/SM lipid mixtures (panel B of Fig. 4) the dependence of the diffusion of g-GM 1 on SM content in the lipid bilayer could be divided into two regimes: In the first regime, at DOPC/SM (100-92/0-8), the diffusion coefficients were constant within the error of the FCS measurement. In this regime, our FRET experiments showed a homogeneous bilayer. At DOPC/SM (90-85/10-15), where FRET detected nanodomains, the g-GM 1 diffusion coefficient decreased. A similar trend but with slightly less distinct differences between the two regimes was obtained for DiD. According to panel B of Fig. 4, the diffusion of g-GM 1 was on average about 5% slower in bilayers with nanodomains than in homogeneous bilayers, whereas the diffusion of DiD slowed down on average about 3%. In order to judge the significance of the decrease in the diffusion coefficient D a t-test was performed (see Table SI2 and SI3 in SI). P-values lower than 0.1 determine a significant difference between two sets of data. In case of g-GM 1 the change in D in respect to the composition DOPC/SM (100/0), which contains no nanodomains, was significant for the compositions (90/10) and (85/15) and insignificant for (88/12). In case of DiD the drop was significant only for (85/15).
The impact of nanodomains on the diffusion of g-GM 1 was much stronger in DOPC/Chol/SM mixtures (panel C of Fig. 4), where g-GM 1 partitioned into the nanodomains more efficiently (see Table 1 for K D s), compared to the DOPC/SM bilayers. A significant drop in the diffusion of g-GM 1 (see Table SI3 in SI) occurred already at (70/25/5), where nanodomains were detected by FRET. The diffusion slowed down further as more SM was accumulated into the nanodomains (note that the average domain radius and area coverage remain the same (Table 1) as SM content is increased). Based on the similarity of the hydrophobic molecular regions of SM and g-GM 1 , and the results of the MC simulation of probe diffusion in the presence of nanodomains 32 this can be explained by more efficient entrapment (longer dwell-time) of g-GM 1 in the SM-rich nanodomains. The abrupt increase in the diffusion coefficient at (63/25/12) occurred due to formation of microscopically phase-separated domains and concentration of Chol and SM into such domains. The bilayer was very close to the L d /L o phase separation boundary at this composition. Here, there is an increased risk of measurements being unintentionally Scientific RepoRts | 7: 5460 | DOI:10.1038/s41598-017-05539-y performed on phase-separated GUVs, where the diffusion in the L d phase is fast 33 . This is also reflected in the large standard deviation error bar associated with this data point (panel C of Fig. 4). A similar pattern of diffusion coefficients was obtained for DiD. For comparison, the diffusion coefficient of g-GM 1 decreased on average about 14% in bilayers containing nanodomains, whereas DiD diffusion was slowed down on average about 5%. Of note, the decrease in D of DiD in (67/25/8) and (65/25/10) bilayers was determined to be significant by the t-test (Table SI3). Nanodomain fluidity. According to the published phase diagrams 27, 28 , the investigated bilayers (Table 1) should be homogeneous and in a neat liquid-disordered state. However, we observed nanodomains under these conditions, thus, we questioned to which extent these nanodomains were fluid and disordered. We   Table 2, Atto-488-DOPE, Atto-633-DOPE and DiD partition preferably into the L d phase and are efficiently expelled from microscopic L o domains in DOPC/ Chol/SM (55/25/20) bilayers. This composition is close in the phase diagram (Fig. 2) to those where nanodomains are observed (e.g. DOPC/Chol/SM 65/25/10). Based on these results, one expects that these probes should be driven out of nanodomains with L o character. This would change the relative FRET efficiency for both D/A pairs (see Table 2 for implications in FRET in the presence of L d or L o nanodomains). On the other hand, formation of L d nanodomains would not change either the distribution of the probes or the relative FRET efficiency for both DA pairs. As Figs SI2 and SI3 show, the kinetics of fluorescent relaxation of the donors remained the same for all the cases. This indicates that the relative FRET efficiency for both D/A pairs remained constant in all investigated bilayer compositions, with and without nanodomains. This finding supports our hypothesis that the nanodomains have a L d character.
To obtain further insight at a molecular level into the organization and dynamics of the lipid bilayers studied here, additional solid-state wide-line and high-resolution MAS 31 P NMR experiments were performed 34,35 . Wide-line NMR spectra obtained for pure DOPC bilayers (Fig. 5) exhibited a "powder-like" lineshape at 298 K, which is typical for a lamellar PC bilayer in its liquid-crystalline L d -phase 36,37 . Under these conditions, the individual lipid molecules in the bilayer undergo fast rotational dynamics, which causes the typical shape and reduced width of the obtained NMR spectra. Analysis of the lineshapes revealed a chemical shift anisotropy, Δσ, where Δσ = σ ∥ − σ ⊥ is the width of NMR spectrum, of approximately 45.3 ppm, which is typical for this phase. Addition of 25 mol% of cholesterol to the DOPC bilayers generated a spectrum representative for a lamellar bilayer system at 298 K (Fig. 5B) with a hint of a second subspectrum, i.e. showing a homogeneous bilayer with perhaps a small fraction of a second, slightly more ordered subdomain. In contrast, the sample composed of DOPC/SM (90/10) resulted in significant changes in the corresponding NMR lineshape (Fig. 5A). The NMR spectrum is clearly composed of two sub-spectra, the main component with an intense 90° edge at −14.6 ppm and a second at −18.3 ppm. As SM is a minor component (10%) in the lipid bilayer, we attribute these two sub-spectra to DOPC in two different dynamic environments. Fitting the lineshape to two axially symmetric powder patterns reveals that the first sub-spectrum, characterized by a chemical shift anisotropy of 38.4 ppm, contributes approximately 47% of the total intensity. This component reflects a lipid environment with a slightly increased disorder in the headgroup region of the DOPC lipids. The second sub-spectrum with an intense 90° edge at 16 ppm, is characterized by a larger chemical shift anisotropy of approximately 49.0 ppm. This increase indicates a different membrane environment with the lipid headgroup regions (and presumably the whole lipid molecules) undergoing reduced dynamics (with less motional averaging of the chemical shift anisotropy) presumably due to the close proximity of stiff SM molecules. In summary, the DOPC/SM (90/10) membranes appear to consist of two different environments: 47% of the bilayer was presumably SM free, fluid and disordered whereas the rest of the bilayer was richer in SM with DOPC lipids in direct contact with SM molecules. Such estimation is in agreement with our MC-FRET results, according to which the SM rich domains occupied 37% or 55% (two global chi-squared minima) of the entire bilayer area (Table 1). Also in agreement with FRET data, 5 mol% SM was not able to induce nanodomains as seen in Fig. 5A, which would have been visible as a second NMR sub-spectrum characterized by a larger chemical shielding anisotropy.
For the ternary systems composed of DOPC/Chol/SM (70,65/25/5,10) lipid mixtures, static NMR spectra were clearly composed of two sub-spectra with different widths (Fig. 5B). Although multiple components (subspectra) are present in the system the spectral properties of each are inconsistent with the broader spectra that would be expected of lipids in their gel phase. The broader of these two components has an intense 90° edge at −14 ppm, whilst the remaining component has a smaller chemical shift anisotropy similar to that of DOPC/Chol bilayers in the absence of SM. Fitting the lineshape to two axially symmetric powder patterns indicates that the SM rich contributes approximately 44% of the entire spectral intensity in good agreement with MC-FRET results.
Our MAS 31 P NMR results further confirm the L d character of the nanodomains found in the studied bilayers. As seen in Fig. 6, the isotropic NMR signal occurred at −0.71 ppm, which is the isotropic chemical shift value expected for DOPC bilayers. Upon addition of SM, or Chol and SM, the variations in this value were minor (upfield to −0.74 ppm) but the NMR linewidth increased dramatically from 15 Hz to 45 Hz. Despite the increase in the linewidth, the resonances remained relatively narrow, supporting the disordered character of the lipid headgroups within the domains. The lipids are undergoing fast dynamics and the isotropic lineshapes of the MAS spectra are influenced by exchange processes. Such behaviour is also typical for L d phase. The isotropic linewidths in the MAS 31 P NMR spectra of lipids are largely dominated by the spin-spin/transverse relaxation times which are sensitive to motion on the ms to µs timescale 38 . The similarity of the linewidths obtained for DOPC in the presence of SM, Chol or both SM and Chol indicates that both species are likely to interact with DOPC headgroups, slowing down the motions on the ms to µs timescale and resulting in reduction of the T 2 and an increase in the corresponding linewidth. The absence of more significant changes in the 31 P powder lineshape reflects that the exchange processes occurring do so from populations of lipids exhibiting similar isotropic and anisotropic chemical shielding suggesting that both populations exhibit similar dynamic properties.
The fact that the nanodomains were detected both by NMR and FCS helps to restrict the range of possible nanodomain lifetimes. The readouts of both techniques are influenced by processes that occur on the micro-to millisecond time scale. Therefore, the lifetimes of nanodomains should be roughly in this range. The broadening that is present in the SM/DOPC MAS 31 P NMR spectra (Fig. 6, red colour) together with the underlying broad subspectrum (ranging from 0.3 to −1.5 ppm) indicates that the DOPC is in exchange between different environments on the NMR timescale (ms/µs) in a manner analogous to that reported by e.g. by Bonev et al. 39 . This observation is consistent with SM lipids forming a dynamic complex with multiple DOPC molecules over the timescale of the NMR experiment (ms/µs). Interestingly, g-GM 1 , r-GM 1 , CF-DSPE-PEG and Rh-PEG-DSPE probes seem to exhibit increased affinity for this dynamic complex (see Table 1 for K D s). Preferable localization of these probes in the nanodomains originates presumably from structural similarity of hydrophobic regions of these probes with SM and allows for determination of nanodomain sizes by MC-FRET.
Of note, transient and dynamic heterogeneities of much shorter lifetime, about 100 ns, and size of 10 nm were revealed by neutron scattering 29,40 and MARTINI simulations 41 . These were reported to exist in DPPC/Chol and DMPC/Chol bilayers close to the phase separation boundary where Chol crystals start to form. Moreover, longer lived fluctuations in composition of about 0.8 ms were observed in ternary mixtures of high and low melting Scientific RepoRts | 7: 5460 | DOI:10.1038/s41598-017-05539-y temperature lipids near miscibility critical points 42 . In this work, on the contrary to the above mentioned cases, the nanodomains are found further away from the phase separation boundaries (Fig. 2).
Although the mechanism of how these nanodomains are formed is not yet well understood, we expect that the process is facilitated by the following factors. First, geometrical factors result in different packing preferences of DOPC and SM. Consequently, SM tends to be surrounded by other SM rather than DOPC molecules. Moreover, it has been documented by a variety of experimental approaches that SM and Chol preferably interact with each other (for a review see ref. 43). It is also known that Chol promotes segregation of different PC components at low Chol contents whereas it suppresses the segregation at higher concentrations (above 50 mol%) 44 . All these interactions seem to be re-enforced by hydrogen bonding between -NH group of SM and hydroxyl group of Chol and between SM and DOPC 43,[45][46][47] . In addition, temporal thermal fluctuations and fluctuations in concentration may perhaps be involved in the formation of nanodomains 41 . Groupings of three to five molecules have been found even in ideal binary mixtures with only nearest neighbor interactions 48 . Thus, hypothetically, it is possible that these temporal fluctuations function as seeds for the liquid disordered nanodomains in a similar way as nanodomains work as formation platforms for microscopic L o phase domains.
Implications for the raft theory. So far, scientists have frequently used L o microdomains as a model system for rafts despite insufficient experimental evidence for the existence of such domains in cells. The size and physical properties of such microdomains are extreme in the context of the plasma membrane, but they are still used as a model system for putative nano-scale rafts in cellular membranes. In respect to the complex composition of a cellular plasma membrane where sharp and well-defined phase transitions can hardly be expected, differences between various local environments are presumably more subtle than the differences between the L d and L o phases encountered in model systems. To bridge the gap between the extremes of L d or L o phase bilayers in artificial model systems and the plasma membrane of living cells scientists started using giant plasma membrane vesicles (GPMVs) as an intermediate model system that more or less preserve biological complexity of native plasma membranes 5,49 . Similarly to the synthetic model membranes of GUVs, GPMVs may phase separate into two distinct microscopic phases. However, the differences between the L d and L o phase are significantly smaller to those observed in GUVs, presumably better corresponding to what is encountered in plasma membranes of living cells. A disadvantage might be a worse control of the GPMVs' composition 6 and phase behaviour which is dependent on the detergent used 49 .
The L d nanoscale domains that have been found and characterized in this work have in analogy to the L o microdomains encountered on GUVs a very simplified composition. However, in terms of domain sizes and their physical properties the nanodomains seem to represent a good model system for cellular rafts. Moreover, the plasma membrane of a living cell is permanently changing. Thus, rafts can be expected to form and disappear or change their properties during their lifetime. In this context, the transient nature of the L d nanodomains may also correspond better to the properties of cellular rafts.

Summary.
In this work, we discovered and characterized nanodomains in binary DOPC/SM and ternary DOPC/Chol/SM bilayers of compositions that should result in homogeneous bilayers according to published phase diagrams. The results of our MC-FRET, solid-state NMR and z-scan FCS experiments are summarized in the phase diagram of Fig. 2 Table 3. Briefly, all three methods indicate that binary mixtures DOPC/SM (100-92/0-8) are homogeneous and that DOPC/SM (90-85/10-15) exhibit nanodomains. In the ternary lipid mixtures containing Chol, nanodomains were revealed at DOPC/Chol/SM (70-65/25/5-10).

and in
The nanodomains of approximately 10 nm can be estimated to consist of roughly 400 to 500 molecules, are enriched in SM but still contain a high amount of DOPC molecules, which is sufficient to maintain the nanodomains fluid and disordered. Despite their L d character the nanodomains exhibit subtle differences on average environment and dynamics as compared to the surrounding. The nanodomains appear long-lived with a lifetime in the range of microseconds to several milliseconds. In terms of their size, fluidity, order and lifetime these nanodomains may represent a relevant model system for cellular membranes and perhaps be more closely related to heterogeneities, e.g. nanocompartments, observed in cellular plasma membranes.

Methods
GUV preparation. GUVs were prepared by the electroformation method as described previously by Angelova et al. 50 . All lipid mixtures were made from stock solutions in chloroform. The lipid mixture (100 nmol in approximately 200 μL of chloroform) containing the additional labelled lipids was spread onto two hollowed titanium plates. These were placed on a heating plate at approximately 47 °C to facilitate solvent evaporation. The plates were subsequently put under vacuum for at least 1 h to evaporate remaining solvent traces. The lipid-coated plates were assembled using one layer of Parafilm as an insulating material. The electroswelling chamber was filled with 1 ml of preheated sucrose solution (with the osmolarity of 103 mOsm/kg) and sealed with Parafilm. An alternating electrical field of 10 Hz rising from 0.02 V to 1.1 V (peak-to-peak voltage) during the first 45 min was applied and kept at 1.1 V and 47 °C for additional 1.5 h. This sequence was followed by a so-called detaching phase at 4 Hz and 1.3 V for 30 min. Finally, the GUVs were added to a microscope chamber containing glucose buffer (~80 mM glucose, 10 mM HEPES and 10 mM NaCl, pH 7.2) at the osmolarity of 103 mOsm/kg. All lipid mixtures contained 2 mol% of biotinyl-PE to immobilize the GUVs on the bottom of a chamber coated with BSA-biotin/streptavidin. For the FCS experiments, the probe-to-lipid ratio was 1:100000 whereas for the FRET experiments, the donor (acceptor)-to-lipid ratio was 1:1000 (1:200) in case of g-GM 1 /DiD pair and 1:200 (1:200) in case of g-GM 1 /r-GM 1 pair, respectively.
Sample preparation for NMR experiments. The lipid mixtures were prepared by dissolving the appropriate lipids in a 2/1 vol/vol HCCl 3 /MeOH solution, followed by evaporation, resuspending in water and freeze-drying, as described previously 51 . To produce multilamellar vesicles, appropriate amounts (around 20 mg) of dry lipid powder was then rehydrated using in the same buffer as used above (except D 2 O was used here instead) at a one-to-one weight ratio, followed by several freeze-thaw cycles and vortexing. Finally the membrane suspensions were pelleted into 4 mm MAS NMR rotors (Bruker, Germany) and measured immediately or kept at −20 °C prior NMR experiments.

FCS and FLIM-FRET measurements.
Both types of measurements were performed on a home-built confocal microscope consisting of an inverted confocal microscope body IX71 (Olympus, Hamburg, Germany) and pulsed diode lasers (LDH-P-C-470, 470 nm, and LDH-D-C-635, 635 nm PicoQuant, Berlin, Germany) operated at 10 MHz repetition rate. The lasers were pulsing alternatively to avoid artifacts caused by signal bleed-through. The laser light was coupled to a polarization maintaining single mode optical fiber and re-collimated at the output with an air space objective (UPLSAPO 4X, Olympus). The light was up-reflected to a water immersion objective (UPLSAPO 60x, Olympus) with a 470/635 dichroic mirror. The signal was split between two single photon avalanche diodes using 515/50 and 697/58 band pass filters (Chroma Rockingham, VT) for green and red channel, respectively. z-scan measurements were conducted on the top of selected GUVs. First, a membrane was placed to the waist of a laser, moved 1.5 µm below the waist afterwards and finally, scanned vertically in 20 steps (150 nm spaced). A 60 second long measurement was performed at each step. The laser intensity at the back aperture of the objective was around 6 µW for each laser line. To obtain the average diffusion coefficients presented in Fig.4 z-scan FCS measurements on 5-10 different GUVs were performed. Further details of the data analysis are given elsewhere 19 .
FLIM-FRET measurements were done by acquiring an image (512 × 512 pixels, 0.6 ms/pixel) of a GUV at its cross-section. The experimental fluorescence decay of the donor that was taken for further analysis was obtained by summing up the measured fluorescence decays from at least five different GUVs. However, variability between fluorescence decays obtained from individual GUVs was negligible (see Fig. SI6). Laser intensity of 1 µW for the 470 nm laser was chosen low enough to avoid pile-up effect for the FLIM-FRET measurements. The experiments were performed at 25 °C. NMR experiments. All 31 P NMR experiments were acquired using a 500 MHz Avance III spectrometer (Bruker, Switzerland). Static wideline NMR spectra of multilamellar vesicles were acquired at 298 K using a Hahn echo pulse sequence with a single π/2 pulse of 7.8 µs pulse length, an inter-pulse delay of 50 µs and a recycle rate of 4 s. During acquisition, TPPM proton decoupling 52 was used (40 W) and ca. 10000 scans were accumulated. For high-resolution MAS NMR spectra, the samples were spun at 5 kHz and a single pulse excitation followed by proton decoupling (parameters as for static NMR experiments) was used. Between 200 to 600 scans were accumulated.
NMR data was processed in matNMR 53 , with all spectra zero-filled to 4096 pts and 30 Hz line-broadening added prior to Fourier transform. Powder lineshapes were analyzed by fitting to one or two axially symmetric powder patterns, using the fitting routines within matNMR.
Analysis of FLIM-FRET data. Förster resonance energy transfer (FRET) was analyzed from fluorescence lifetime images (FLIM). Each pixel contains information on the arrival times of individual photons. These times are used to construct the fluorescence decay, whose shape can be modified by FRET. Analysis of the decay by an appropriate mathematical model yields further information. In this work, the so-called Baumann-Fayer (BF) model was used (see SI) to (i) determine the experimental surface concentration of the acceptors, which was required as one of the input parameters for the MC simulations; (ii) obtain information about how donors and acceptors were distributed in the lipid bilayer. Relative FRET efficiency E rel used in the manuscript is defined as the ratio between FRET efficiency 54 for a heterogeneous bilayer E hetero (with nanodomains) and the FRET efficiency for a homogeneous bilayer E homo (without nanodomains). Homogeneous bilayers were selected to contain 0% of SM and the same amount of Chol as the heterogeneous bilayers.
The determination of nanodomain sizes was performed by analyzing the experimental fluorescence decay with Monte Carlo simulations. Two sets of GUVs are always prepared: the first set contains GUVs with homogeneous bilayers and is used to calculate the number of acceptors in the GUVs by the Baumann-Fayer model (see SI). The number of acceptors is assumed to be the same in the other set of GUVs, where nanodomains with unknown dimensions might exist. This is done in order to reduce the number of optimized parameters. The entire fitting procedure was described in detail elsewhere 24 and is shortly summarized in what follows. A defined number of donors, acceptors and circular domains with a given radius R D was generated in the lipid bilayer. Whereas the number of donors was kept at a sufficiently high value for statistical reasons, the number of acceptors had to be determined by the BF model (SI) to correspond to the actual experimental conditions. First, the donors and acceptors were distributed according to the distribution constants defined as K D (D) = [D inside ]/[D outside ], K D (A) = [A inside ]/[A outside ]. In the next step, a donor was randomly excited and the time at which an energy transfer event took place calculated. This process was random and modulated by the overall energy transfer rate Ω i according to Δt i = −lnγ/Ω i where γ is a randomly generated number between 0-1. The outcome of each simulation step was the time interval Δt i between the excitation and the energy transfer event. To achieve good statistics, each generated configuration was used 100 times before a new configuration was generated. The total number of all excitation events was 3 × 10 5 . By constructing a histogram of Δt i intervals the total survival probability function G(t) was obtained and the simulated decay of donors quenched by the acceptors calculated. The simulated decay was fitted to the experimental one by varying the input simulation parameters, i.e. the domain radius R D , the area fraction the domains occupied Ar and K D (D,A). The global minimum was found by scanning the chi-squared space of physically acceptable parameters R D , Ar, and K D (D,A). Because of structural similarity between donors and acceptors and a weak dependence of R D and Ar on the actual values of K D , K D (D) was kept identical to K D (A).
Analysis of z-scan FCS data has been described many times before 19,55 and is briefly summarized in SI.