Experimental Evidence for the Existence of Inter-leaflet Coupled

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Biophysical Chemistry, Biomolecules, and Biomaterials; Surfactants and Membranes

Experimental Evidence for the Existence of Interleaflet Coupled Nanodomains: An MC-FRET study. Ivo Stanislav Vinklárek, Lukáš Ve#as, Petra Riegerova, Kristian Skala, Ilya I. Mikhalyov, Natalia Gretskaya, Martin Hof, and Radek Šachl J. Phys. Chem. Lett., Just Accepted Manuscript • Publication Date (Web): 09 Apr 2019 Downloaded from http://pubs.acs.org on April 9, 2019

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The Journal of Physical Chemistry Letters

Experimental Evidence for the Existence of Inter-leaflet Coupled Nanodomains: An MC-FRET study. Ivo S. Vinklárek,1 Lukáš Vel’as1, Petra Riegerová1, Kristián Skála1,2, Ilya Mikhalyov3, Natalia Gretskaya3, Martin Hof1 and Radek Šachl1 Department of Biophysical Chemistry, J. Heyrovský Institute of Physical Chemistry of the

1

Academy of Sciences of the Czech Republic, 182 23 Prague, Czech Republic; 2

Department of Physical and Macromolecular Chemistry, Faculty of Science, Charles

University, Hlavova 8, CZ-12840 Prague, Czech Republic 3

Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of

Science, Moscow, GSP-7, Russia E-mail: [email protected]

Abstract Plasma membranes of living cells are compartmentalized into small sub-microscopic structures (nanodomains) having potentially relevant biological functions. Despite this, structural features of these nanodomains remain elusive, primarily due to the difficulties in characterizing such small dynamic entities. It is unclear whether nanodomains found in the upper bilayer leaflet are transversally registered with those found in the lower leaflet. Experiments performed on larger microscopic domains indicate that the coupling between the leaflets is strong, forcing the domains to be in perfect registration. But, can the same thing be said about the biologically more relevant nanodomains? This work provides experimental evidence that even small nanodomains of variable sizes between 10 and 160 nm are interleaflet coupled. Importantly, the alternative scenarios of partially registered, independent, or anti-registered nanodomains could be excluded. 1 ACS Paragon Plus Environment

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Considering huge complexity of a cell, it is not surprising that cellular plasma membranes show an inherent heterogeneity on the nanometer scale.1–4 Interestingly, such nanoheterogeneities (called throughout the manuscript lipid nanodomains) were also found in minimalised model systems consisting of only two or three different types of lipids, although the phase diagrams predict them to be homogeneous.5 Such nanodomains contain a few hundreds to a few of thousands of lipid molecules and have a low degree of ordering.6 A review of the literature and theoretical considerations indicate that such nanodomains occur in any fluid lipid bilayer, are dynamic and that the choice of the lipids determine the size of the corresponding heterogeneities.5,7–9 Since only a very few techniques allow their detection, knowledge on these nanodomains is still limited. One of the emerging questions to be answered is whether the nanodomains are inter-leaflet coupled; i.e., whether the nanodomains found in the upper leaflet are transversally aligned with those found in the lower leaflet (Figure 1, Figure 2). Considering the asymmetric composition of plasma membranes, understanding of how the lipids contribute to the inter-leaflet coupling appears not only to be of academic interest, but also biologically relevant.10 Experimental studies on model membranes (i.e. giant unilamellar vesicles; GUVs) have shown that microscopic domains are in registration.11 However, do the interactions that keep the domains coupled even suffice to hold the small nanodomains in registration? Experiments that would answer this question are practically non-existent, mainly because distinguishing registered from inter-leaflet independent nanodomains requires not only high lateral resolution but also resolution along the bilayer’s vertical. FLIM-FRET (fluorescence lifetime imaging of Förster resonance energy transfer) combined with Monte-Carlo simulations (called MC-FRET for short) meets such requirements on high spatial resolution in all three directions. We have developed this method for the determination of sizes and concentrations of the nanodomains in GUVs, and in this work it is used to distinguish registered from inter-leaflet independent



nanodomains.6,12 Principles of MC-FRET remain similar even when resolving registered from inter-leaflet independent nanodomains (Figure 1 and Experimental methods). The method is based on fluorescent probes that possess increased affinity for the nanodomains. Then, formation of nanodomains changes the originally homogeneous distribution of probes into a heterogeneous one, bringing the donors (D) closer to the acceptors (A) and enhancing the efficiency of FRET. By choosing donor/acceptor (D/A) pair whose Förster radius R0 matches the thickness of the lipid bilayer, FRET will occur efficiently from one layer to the other one. This so-called inter-FRET enables to distinguish registered from inter-leaflet independent nanodomains by fitting experimental time-resolved fluorescence decays (TRFDs, Figure 1 and Experimental methods).

Figure 1 depicts basic principles of MC-FRET. (Case 1): D and A are randomly distributed in a homogeneous bilayer. At sufficiently high acceptor concentration, FRET will occur and cause faster fluorescence decay of D (compare the black with the blue TRFD). (Case 2): D and A are preferentially localized in independent nanodomains (orange decay). (Case 3): D and A are preferentially localized in registered nanodomains (green decay). The shape of the decays is influenced by the size and area occupied by the domains, the affinity of the probes 4 ACS Paragon Plus Environment

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for the domains, and inter-leaflet coupling of the nanodomains; the readouts which can be determined by fitting the experimental TRFDs with MC-FRET.

Figure 2: Horizontal projection of the lipid bilayer for six simulated cases: (A): Perfectly registered nanodomains; (B): Partially registered nanodomains, with a relative shift of 50%; (C): Inter-leaflet independent nanodomains; (D): Anti-registered nanodomains; (E): a crowded bilayer with the nanodomains that occupy 50 % of the bilayer and (F): nanodomains 5 ACS Paragon Plus Environment


with a distribution of sizes shown in the insert of panel F. KD(D) = 1000, KD(A) = 1000. The green squares mark the boundaries of a periodic cell.

Prior to experiments, the sensitivity of MC-FRET to inter-leaflet organization of the nanodomains was investigated computationally. We carried out MC-FRET simulations for the two most probable scenarios: membranes either with registered or independent nanodomains (Figure 2). The ability of MC-FRET to distinguish registered from independent nanodomains (characterized by the relative difference in average lifetimes 𝛿(𝜏), for registered⟨𝜏(registered)⟩ vs. independent ⟨𝜏(independent)⟩ nanodomains; 𝛿(𝜏) = [|〈𝜏(independent)〉 − 〈𝜏(registered)〉|]⁄〈𝜏(registered)〉 ) was optimal through a broad range of nanodomain radii 𝑅𝐷 ∈ ⟨5; 200⟩ nm, and fractional areas between 5 and 60% (panel A of Figure 3). Whereas nanodomains with 𝑅𝐷 > 200 nm were beyond of the scope of this study because they are resolvable by microscopes, nanodomains with the total area larger than 70% were not considered further because it was sterically impossible to add more nanodomains into such an already crowded bilayer.


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Figure 3: (A): The relative differences 𝛿(𝜏)(in %) in average lifetimes for registered vs. independent nanodomains are shown for various combinations of the nanodomain radius RD and the fractional area occupied by the nanodomains. 𝛿(𝜏) reports here on the efficiency of MC-FRET to resolve bilayers with registered nanodomains from those with independent ones. The resolution was poor/excellent at Point 1/Point 2. KD(D) = 1000, KD(A) = 1000. (B, C): TRFDs corresponding to Point 1 and Point 2 are displayed here. (D): Dependence of the size of nanodomains on the content of sphingomyelin in DOPC/Chol (25%)/SM bilayers (blue). 10% of DOPC was replaced by either PGPC or POVPC (red or orange).

In our TRFD experiments, we used ganglioside GM1 molecules labelled in the headgroup region by either FL-Bodipy (g-GM1, donor) or 564/670-Bodipy (r-GM1, acceptor) as 7 ACS Paragon Plus Environment


the main D/A pair. This pair was shown to exhibit high affinity for nanodomains, but does not form nanodomains by its own.6,13 It has the Förster radius (R0 = 5.87 nm) very similar to the distance between the layers containing D and A (d = 4.8 nm). Importantly, we confirmed the main conclusions with a chemically different D/A pair, based on 1,2-distearoyl-snglycero- 3-phosphoethanolamine-N-[amino Poly(ethylene glycol) 2000], labelled at the end of the Poly(ethylene glycol) chain with either carboxyfluorescein (CF-PEG-DSPE, donor) or Rhodamine101 (Rh-PEG-DSPE, acceptor, R0 = 6.63 nm, d = 7.3 nm). Both D/A pairs have already been used successfully in the characterization of lipid nanodomains.6,12 To experimentally test the capability of MC-FRET to resolve registered from independent nanodomains for a broad spectrum of nanodomain sizes, we made use of the oxidized phospholipids 1-palmitoyl-2-(5'-oxo-valeroyl)-sn-glycero-3-phosphocholine (POVPC) and 1-palmitoyl-2-glutaryl-sn-glycero-3-phosphocholine (PGPC). Such carboxyor aldehyde-truncated phospholipids were shown to stabilize micron-sized domains in ternary 1-palmitoyl-2-oleolyl-glycero-3-phosphocholine/Cholesterol/Sphingomyelin (POPC/Chol/SM) mixtures by increasing hydrophobic mismatch and the line tension between the domains and the remaining part of the lipid bilayer.14–16 Both POVPC and PGPC are able to induce growth of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)/Chol/SM nanodomains in the range between 2 and 160 nm (Table 1, panel D of Figure 3) while preserving the character of these nanodomains (Figure S4). Specifically, 10 mol% of DOPC was replaced by either POVPC or PGPC, and the content of SM changed from 0 to 12 mol%. Nanodomains found in (DOPC/Chol/SM) (70-65/25/5-10 mol%) bilayers had radii of 2 to 7 nm, and covered between 50 and 58% of the bilayer (Table 1 and 6,13). Fitting TRFDs for a g-GM1/r-GM1 D/A pair to either a model assuming registered or independent nanodomains yielded a similar quality of the fit (compare χ2 values in Table 1). We conclude


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that the MC-FRET approach cannot report on inter-leaflet coupling of the nanodomains smaller than 7 nm (panel A of Figure 3).

Table 1: A summary of output parameters obtained by MC-FRET using a g-GM1/rGM1 D/A pair. Models assuming either registered or independent nanodomains were used for the data fitting. Conclusions about the inter-leaflet coupling of the nanodomains were drawn 2 2 by comparing chi-squared values for either the registered (𝜒𝑟𝑒𝑔 ) or independent (𝜒𝑖𝑛𝑑𝑒𝑝 )

nanodomains.

Lipid mixture

Composition

Registered nanodomains

Independent nanodomains

Radius

Area

Radius

Area

[nm]

[%]

[nm]

[%]

2 𝜒𝑟𝑒𝑔

2 𝜒𝑖𝑛𝑑𝑒𝑝

2 𝜒𝑖𝑛𝑑𝑒𝑝 2 𝜒𝑟𝑒𝑔

DOPC/Chol/SM

70/25/5

2±1

50±5

1.01

1.5±1.0

59±5

1.03

1.02

DOPC/Chol/SM

65/25/10

7±3

58±5

1.59

9±4

65±5

1.63

1.03

DOPC/Chol/PGPC

67.5/25/7.5

10±4

61±5

1.85

17±4

62±5

2.66

1.44

DOPC/Chol/SM/PGPC

63/25/5/7

19±5

63±5

1.89

26±6

60±5

2.46

1.30

DOPC/Chol/SM/PGPC

58.5/25/10/6.5

78±17

63±5

1.94

118±29

47±4

4.90

2.53

DOPC/Chol/SM/PGPC

56.7/25/12/6.3

160±40

58±5

1.67

114±28

42±4

6.41

3.84

DOPC/Chol/POVPC

67.5/25/7.5

6±3

61±5

1.49

8±3

57±5

2.30

1.54

DOPC/Chol/SM/POVPC

63/25/5/7

8±3

58±5

1.90

18±5

62±5

2.24

1.20

DOPC/Chol/SM/POVPC

58.5/25/10/6.5

29±5

65±6

2.29

26±6

59±5

2.97

1.30

DOPC/Chol/SM/POVPC

56.7/25/12/6.3

116±28

61±5

2.01

114±28

45±4

6.30

3.13

Our simulations, however, show that the sensitivity should improve significantly for nanodomains larger than 7 nm in radius (panel A of Figure 3). We tested this prediction by using the g-GM1/r-GM1 pair. For nanodomain radii exceeding 7 nm and ranging up to 160 2 2 nm, the chi-squared values for the registered (𝜒𝑟𝑒𝑔 ) nanodomains ) and independent (𝜒𝑖𝑛𝑑𝑒𝑝

differed from each other by more than 20% in favour of the model that assumed registered 9 ACS Paragon Plus Environment


nanodomains (Table 1, Figure 4). A few representative fits to the TRFD for registered nanodomains are shown in panel F of Figure 4. The quality of the fits improved only slightly if at all by including in the model a distribution of nanodomain sizes (Table S3 and panel F of Figure 1). Importantly, the fits were convincingly better when assuming the model with registered nanodomains also for the second, chemically different D/A pair (Table S4).


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Figure 4: Maps of chi-squared minima obtained by MC-FRET under the assumption of a bilayer with registered (A), independent (B), anti-registered (C), or partially-registered (D-E) nanodomains. The global minima are marked in blue. The lipid mixture contained DOPC/Chol/SM/PGPC (55/25/10/6.5) and g-GM1 and r-GM1 at the probe to lipid ratio of 1:200. Demonstrative fits (black) to experimental TRFDs are shown in panel F. The bilayers contained 2 nm (red), 19 nm (blue) or 160 nm (green) large nanodomains.

To further support this finding, we investigated the remaining two possible scenarios (Figure 2): that the nanodomains can only be in partial registration, the extent of which is characterized by a relative shift of the nanodomains in the upper leaflet in respect to the nanodomains in the lower leaflet; or that they are anti-registered. Here, the nanodomains in the upper leaflet cannot occupy lateral positions that have already been taken by the nanodomains in the lower leaflet. To verify the first scenario we systematically shifted in MC-FRET simulations the nanodomains in one layer with respect to the nanodomains in the other layer, while recording the trends of the χ2 parameter. We carried out this experiment with a g-GM1/r-GM1 D/A pair on adequately large nanodomains (RD = 78 ± 17 nm) found in DOPC/Chol/SM/PGPC (58.5/25/10/6.5) bilayers. Because the quality of the fit gradually decreased when increasing the relative shift, we could exclude this scenario (Figure S5, and panels D-E of Figure 4). Finally, under specific circumstances characterised by a large area (about 50%) occupied by the nanodomains and a strong hydrophobic mismatch between the nanodomains, formation of anti-registered nanodomains may be favoured (panel D of Figure 2).17 We tested that case again on DOPC/Chol/SM/PGPC (58.5/25/10/6.5) bilayers and found that the presence of anti-registered nanodomains is improbable (compare the χ2 values of Figure 4 or Figure S6).



In summary, we provided experimental evidence that lipid nanodomains are in perfect registration over a broad spectrum of nanodomain sizes between 10 and 160 nm. To date, we are only aware of one experimental neutron scattering study, which indicated the presence of registered, 13 nm large nanodomains in 1,2-distearoyl-sn-glycero-3phosphocholine/POPC/Chol (39/39/22) bilayers.18 Interestingly, shear stress experiments performed on microdomains have shown that the threshold shear required to deregister domains increases with decreasing domain size, and is approximately four times higher for those domains 1 µm in radius, compared to domains 11 µm in size.19 Consequently, these results would suggest that also nanodomains need to be registered. However, such extrapolation is not straightforward because the properties of micro- versus nanodomains are in many ways different.5,6 First of all, characterization of nanodomains using global thermodynamic quantities, such as line tension, may not be entirely accurate. In particular, the concept of phase diagrams must be considered with care.5 In this context, it is not surprising that nanodomains were also found outside the phase coexistence region.6,20 In fact, nanodomains appearing due to thermal and compositional fluctuations have been found even in one component systems.9,20 Moreover, nano- in comparison to microdomains are more dynamic with the shortest reported lifetime approaching milli- to microsecond time-scale.6,9,20 In our MC-FRET simulations, we did two a priori assumptions. Firstly, we assumed the nanodomains to be uniform in size. Although this extreme assumption appears unlikely, simulating TRFD with a model comprising a distribution of nanodomain sizes did not give better fits, which supports this assumption (SI and panel F in Figure 2). Secondly, we assumed a circular shape of the nanodomains. Keeping in mind the determined high nanodomain densities (Table 1), the nanodomains have to reside close to each other. We consider such circular nanodomains as basic building blocks, the assembly of which might form larger, non-circularly shaped structures (panel E of Figure 3).


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Mechanisms that lead to the registration of nanodomains are widely debated in the literature.21–23 The observed high density of nanodomains (Table 1) significantly increases the probability that nanodomains residing in the opposite leaflets find each, but does not explain why the nanodomains stay in registration. Originally, cholesterol was suggested as contributing to coupling by its ability to flip-flop between the leaflets. However, a recent study by Thallmair et al. showed that it is not the flip-flop of cholesterol that stabilizes the registered nanodomains, but an intermediate state in which the cholesterol is sandwiched between the leaflets.24,25 In contrast, Galimzyanov et al suggested that domain registration requires neither any special lipid component nor any particular interaction between the leaflets.26,27 Instead, they identified two main mechanisms that drive domain registration: minimization of the line tension that arises along the rim between more ordered and less ordered part of the lipid bilayer; and membrane undulations which distribute stiffer domains into bilayer parts with lower fluctuations in curvature. There is also a mechanism still in play that is associated with dynamic chain interdigitation. This mechanism is thermodynamically favourable if terminal segments of acyl chains penetrate into the opposing leaflet of the lipid bilayer, which might be favoured by registered nanodomains.28 In the here investigated cases of DOPC/Chol/SM/±PGPC or ±POVPC bilayers the acyl chains of SM and DOPC with 18 carbons are long enough to be integrated in the opposing leaflet. Although none of the mechanisms mentioned has yet been shown by an experiment to play a predominant role, nanodomain coupling between the inner and outer leaflet might have considerable implications for biological functions.

Experimental methods General: Details about all of the chemicals, preparation of GUVs, and further experimental details can be found in the supporting information.



MC-FRET:29,30 The entire MC-FRET analysis relies on the assumption that the nanodomains are circular in shape and uniform in size. This allows them to ascribe a certain size characterized by the nanodomain radius RD. Specifically, a defined number of domains was generated on a bilayer of a known thickness. The domains in both leaflets were generated so as to be in 1) perfect or 2) partial registration, 3) inter-leaflet independent, or 4) antiregistered. The probes were distributed according to the distribution constants defined as 𝐾𝐷 (𝑝𝑟𝑜𝑏𝑒) = [𝑝𝑟𝑜𝑏𝑒(𝑖𝑛𝑠𝑖𝑑𝑒)]⁄[𝑝𝑟𝑜𝑏𝑒(𝑜𝑢𝑡𝑖𝑠𝑖𝑑𝑒)]. The number of acceptors corresponded to

the predetermined surface concentration of acceptors (see SI). A donor was randomly excited, and the time at which energy transfer took place was calculated. This process was modulated by the overall energy transfer rate 𝛺𝑖 according to ∆𝑡𝑖 = −𝑙𝑛𝛾⁄𝛺𝑖 , where γ is a randomly generated number between 0-1. The outcome of each simulation step was the time interval Δti, between the excitation and energy transfer event. Each generated configuration was used 100. The total number of all excitation events was 3 x 105. By constructing a histogram of Δti intervals, the total survival probability function 𝐺(𝑡) was obtained, and the simulated decay of D quenched by the acceptors 𝐹𝐷𝐴 (𝑡) calculated 𝐹𝐷𝐴 (𝑡) = 𝐺(𝑡)𝐹𝐷 (𝑡). Here, 𝐹𝐷 (𝑡) is the donor decay in the absence of acceptors. The simulated decay was fitted to the experimental one by varying the input simulation parameters; i.e., the domain radius RD, the area fraction the domains occupied Ar, and KD(D,A). The global minimum was found by scanning the chisquared space of physically acceptable parameters RD, Ar, and KD(D,A). The conclusions about inter-leaflet coupling of the nanodomains were drawn by comparing the global chisquared minima for scenarios 1 to 4.

Acknowledgements RŠ, IV, PR, KS acknowledge financial support from the Czech Science Foundation via grant 18-04871S. KS, IV, and RŠ acknowledge support from Charles University, Faculty of Sciences via GAUK 1072218. MH acknowledges financial support from the Czech Science 14 ACS Paragon Plus Environment

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Foundation via 19-26854X. Computational resources were provided by CESNET LM2015042 and CERIT Scientific Cloud LM2015085 in the framework of the programme ‘Projects of Large Research, Development, and Innovations Infrastructures,’

References (1)

(2)

(3)

(4)

(5) (6)

(7)

(8)

(9) (10)

(11)

(12)

(13)

(14)

(15)

Eggeling, C.; Ringemann, C.; Medda, R.; Schwarzmann, G.; Sandhoff, K.; Polyakova, S.; Belov, V. N.; Hein, B.; von Middendorff, C.; Schönle, A.; et al. Direct Observation of the Nanoscale Dynamics of Membrane Lipids in a Living Cell. Nature 2009, 457 (7233), 1159–1162. Bernardino de la Serna, J.; Schütz, G. J.; Eggeling, C.; Cebecauer, M. There Is No Simple Model of the Plasma Membrane Organization. Front. Cell Dev. Biol. 2016, 4 (September), 1–17. Owen, D. M.; Williamson, D. J.; Magenau, A.; Gaus, K. Sub-Resolution Lipid Domains Exist in the Plasma Membrane and Regulate Protein Diffusion and Distribution. Nat. Commun. 2012, 3, 1256. Ritchie, K.; Iino, R.; Fujiwara, T.; Murase, K.; Kusumi, A. The Fence and Picket Structure of the Plasma Membrane of Live Cells as Revealed by Single Molecule Techniques (Review). Mol. Membr. Biol. 2003, 20 (1), 13–18. Cebecauer, M.; Amaro, M.; Jurkiewicz, P.; Sarmento, M. J.; Šachl, R.; Cwiklik, L.; Hof, M. Membrane Lipid Nanodomains. Chem. Rev. 2018, 118, 11259–11297. Koukalová, A.; Amaro, M.; Aydogan, G.; Gröbner, G.; Williamson, P. T. F.; Mikhalyov, I.; Hof, M.; Šachl, R. Lipid Driven Nanodomains in Giant Lipid Vesicles Are Fluid and Disordered. Sci. Rep. 2017, 7 (1), 5460. Mouritsen, O. G.; Boothroyd, A.; Harris, R.; Jan, N.; Lookman, T.; MacDonald, L.; Pink, D. A.; Zuckermann, M. J. Computer Simulation of the Main Gel-Fluid Phase Transition of Lipid Bilayers. J. Chem. Phys. 1983, 79 (4), 2027–2041. Jørgensen, K.; Sperotto, M. M.; Mouritsen, O. G.; Ipsen, J. H.; Zuckermann, M. J. Phase Equilibria and Local Structure in Binary Lipid Bilayers. BBA - Biomembr. 1993, 1152 (1), 135–145. Mouritsen, O. G.; Jörgensen, K. Dynamic Lipid-Bilyar Heterogeneity: A Mesoscopic Vehicle for Membrane Function? BioEssays 1992, 14 (2), 129–135. Nickels, J. D.; Smith, J. C.; Cheng, X. Lateral Organization, Bilayer Asymmetry, and Inter-Leaflet Coupling of Biological Membranes. Chem. Phys. Lipids 2015, 192, 87– 99. Garg, S.; Rühe, J.; Lüdtke, K.; Jordan, R.; Naumann, C. A. Domain Registration in Raft-Mimicking Lipid Mixtures Studied Using Polymer-Tethered Lipid Bilayers. Biophys. J. 2007, 92 (4), 1263–1270. Amaro, M.; Šachl, R.; Aydogan, G.; Mikhalyov, I. I.; Vácha, R.; Hof, M. GM1 Ganglioside Inhibits B-Amyloid Oligomerization Induced by Sphingomyelin. Angew. Chemie 2016, 55, 9411–9415. Šachl, R.; Amaro, M.; Aydogan, G.; Koukalová, A.; Mikhalyov, I. I.; Boldyrev, I. A.; Humpolíčková, J.; Hof, M. On Multivalent Receptor Activity of GM1 in Cholesterol Containing Membranes. Biochim. Biophys. Acta 2015, 1853 (4), 850–857. Volinsky, R.; Paananen, R.; Kinnunen, P. K. J. Oxidized Phosphatidylcholines Promote Phase Separation of Cholesterol-Sphingomyelin Domains. Biophys. J. 2012, 103 (2), 247–254. Parkkila, P.; Štefl, M.; Olzyńska, A.; Hof, M.; Kinnunen, P. K. J. Phospholipid Lateral 15 ACS Paragon Plus Environment


(16)

(17)

(18)

(19)

(20) (21) (22) (23)

(24)

(25)

(26)

(27) (28)

(29)

(30)

Diffusion in Phosphatidylcholine-Sphingomyelin-Cholesterol Monolayers; Effects of Oxidatively Truncated Phosphatidylcholines. Biochim. Biophys. Acta - Biomembr. 2015, 1848 (1), 167–173. Štefl, M.; Šachl, R.; Olzyńska, A.; Amaro, M.; Savchenko, D.; Deyneka, A.; Hermetter, A.; Cwiklik, L.; Humpolíčková, J.; Hof, M. Comprehensive Portrait of Cholesterol Containing Oxidized Membrane. Biochim. Biophys. Acta - Biomembr. 2014, 1838 (7), 1769–1776. Perlmutter, J. D.; Sachs, J. N. Interleaflet Interaction and Asymmetry in Phase Separated Lipid Bilayers: Molecular Dynamics Simulations. J. Am. Chem. Soc. 2011, 133 (17), 6563–6577. Nickels, J. D.; Cheng, X.; Mostofian, B.; Stanley, C.; Lindner, B.; Heberle, F. A.; Perticaroli, S.; Feygenson, M.; Egami, T.; Standaert, R. F.; et al. Mechanical Properties of Nanoscopic Lipid Domains. J. Am. Chem. Soc. 2015, 137 (50), 15772–15780. Blosser, M. C.; Honerkamp-Smith, A. R.; Han, T.; Haataja, M.; Keller, S. L. Transbilayer Colocalization of Lipid Domains Explained via Measurement of Strong Coupling Parameters. Biophys. J. 2015, 109 (11), 2317–2327. Nielsen, L. K.; Bjørnholm, T.; Mouritsen, O. G. Fluctuations Caught in the Act. Nature 2000, 404 (March), 352. May, S. Trans-Monolayer Coupling of Fluid Domains in Lipid Bilayers. Soft Matter 2009, 5 (17), 3148. Kiessling, V.; Wan, C.; Tamm, L. K. Domain Coupling in Asymmetric Lipid Bilayers. Biochim. Biophys. Acta - Biomembr. 2009, 1788 (1), 64–71. Fujimoto, T.; Parmryd, I. Interleaflet Coupling, Pinning, and Leaflet Asymmetry— Major Players in Plasma Membrane Nanodomain Formation. Front. Cell Dev. Biol. 2017, 4 (January), 1–12. Thallmair, S.; Ingólfsson, H. I.; Marrink, S. J. Cholesterol Flip-Flop Impacts Domain Registration in Plasma Membrane Models. J. Phys. Chem. Lett. 2018, acs.jpclett.8b01877. Marrink, S. J.; De Vries, A. H.; Harroun, T. A.; Katsaras, J.; Wassall, S. R. Cholesterol Shows Preference for the Interior of Polyunsaturated Lipid Membranes. J. Am. Chem. Soc. 2008, 130 (1), 10–11. Galimzyanov, T. R.; Molotkovsky, R. J.; Bozdaganyan, M. E.; Cohen, F. S.; Pohl, P.; Akimov, S. A. Elastic Membrane Deformations Govern Interleaflet Coupling of LipidOrdered Domains. Phys. Rev. Lett. 2015, 115 (8), 1–5. Galimzyanov, T. R.; Kuzmin, P. I.; Pohl, P.; Akimov, S. A. Undulations Drive Domain Registration from the Two Membrane Leaflets. Biophys. J. 2017, 112 (2), 339–345. Raghupathy, R.; Anupama Ambika Anilkumar Polley, A.; Singh, P. P.; Yadav, M.; Johnson, C.; Suryawanshi, S.; Saikam, V.; Sanghapal D. Sawant, A. P.; Guo, Z.; A., V. R.; et al. Transbilayer Lipid Interactions Mediate Nanoclustering of Lipid- Anchored Proteins. Cell 2016, 161 (3), 581–594. Šachl, R.; Humpolíčková, J.; Štefl, M.; Johansson, L. B.-Å.; Hof, M. Limitations of Electronic Energy Transfer in the Determination of Lipid Nanodomain Sizes. Biophys. J. 2011, 101 (11), L60–L62. Šachl, R.; Johansson, L. B.-Å.; Hof, M. Förster Resonance Energy Transfer (FRET) between Heterogeneously Distributed Probes: Application to Lipid Nanodomains and Pores. Int. J. Mol. Sci. 2012, 13 (12), 16141–16156.


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Figure 1 depicts basic principles of MC-FRET. (Case 1): D and A are randomly distributed in a homogeneous bilayer. At sufficiently high acceptor concentration, FRET will occur and cause faster fluorescence decay of D (compare the black with the blue TRFD). (Case 2): D and A are preferentially localized in independent nanodomains (orange decay). (Case 3): D and A are preferentially localized in registered nanodomains (green decay). The shape of the decays is influenced by the size and area occupied by the domains, the affinity of the probes for the domains, and inter-leaflet coupling of the nanodomains; the readouts which can be determined by fitting the experimental TRFDs with MC-FRET.

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Figure 2: Horizontal projection of the lipid bilayer for six simulated cases: (A): Perfectly registered nanodomains; (B): Partially registered nanodomains, with a relative shift of 50%; (C): Inter-leaflet independent nanodomains; (D): Anti-registered nanodomains; (E): a crowded bilayer with the nanodomains that occupy 50 % of the bilayer and (F): nanodomains with a distribution of sizes shown in the insert of panel F. KD(D) = 1000, KD(A) = 1000. The green squares mark the boundaries of a periodic cell.


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Figure 3: (A): The relative differences δ(τ)(in %) in average lifetimes for registered vs. independent nanodomains are shown for various combinations of the nanodomain radius RD and the fractional area occupied by the nanodomains. δ(τ) reports here on the efficiency of MC-FRET to resolve bilayers with registered nanodomains from those with independent ones. The resolution was poor/excellent at Point 1/Point 2. KD(D) = 1000, KD(A) = 1000. (B, C): TRFDs corresponding to Point 1 and Point 2 are displayed here. (D): Dependence of the size of nanodomains on the content of sphingomyelin in DOPC/Chol (25%)/SM bilayers (blue). 10% of DOPC was replaced by either PGPC or POVPC (red or orange).



Figure 4: Maps of chi-squared minima obtained by MC-FRET under the assumption of a bilayer with registered (A), independent (B), anti-registered (C), or partially-registered (D-E) nanodomains. The global minima are marked in blue. The lipid mixture contained DOPC/Chol/SM/PGPC (55/25/10/6.5) and g-GM1 and r-GM1 at the probe to lipid ratio of 1:200. Demonstrative fits (black) to experimental TRFDs are shown in panel F. The bilayers contained 2 nm (red), 19 nm (blue) or 160 nm (green) large nanodomains.


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132x132mm (96 x 96 DPI)


Experimental Evidence for the Existence of Inter-leaflet Coupled

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