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Published in final edited form as: Chem Phys Lipids. 2021 May 7;238:105090. doi: 10.1016/j.chemphyslip.2021.105090

Ceramide-Mediation of Diffusion in Supported Lipid Bilayers

Masroor Hossain 1, G J Blanchard 1,*
PMCID: PMC8222156  NIHMSID: NIHMS1701805  PMID: 33971138

Abstract

The fluidity and compositional heterogeneity of the mammalian plasma membrane play deterministic roles in a variety of membrane functions. Designing model bilayer systems allows for compositional control over these properties. Ceramide is a phospholipid capable of extensive headgroup-region hydrogen bonding, and we report here on the role of ceramide in planar model bilayers. We use fluorescence recovery after photobleaching (FRAP) to obtain translational diffusion constants of two chromophores in supported model bilayers composed of cholesterol, 1,2-dioleoyl-sn-phosphatidylcholine (DOPC), sphingomyelin, and ceramide. FRAP data for perylene report on the acyl chain region of the model bilayer and FRAP data for 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) sense diffusional dynamics in the bilayer headgroup region. Dynamics in the headgroup region exhibit anomalous diffusion behavior that is characteristic of spatially heterogeneous media.

Keywords: ceramide, supported lipid bilayer, phospholipid, translational diffusion, fluorescence recovery after photobleaching, anomalous diffusion, heterogeneity

Introduction

A biological cell is the smallest fundamental unit of life, and the selectively permeable plasma membrane surrounding it protects the cell from extracellular species, aids in cell signaling, and mediates cellular processes. It is composed primarily of phospholipids and contains a range of other molecular species, including cholesterol, proteins, and carbohydrates. The fluidity of the membrane plays a key role in the function of certain enzymes and transmembrane proteins. Control over the molecular interactions that mediate membrane fluidity can be established through experiments on model lipid bilayers because of their comparative compositional simplicity. A variety of artificial membranes have been demonstrated,(Castellana and Cremer 2006, Richter, Bérat et al. 2006) including black lipid membranes (BLM),(Mueller, Rudin et al. 1962, Mueller, Rudin et al. 1963, Tien, Carbone et al. 1966, Tien and Ottova-Leitmannova 2000, Beerlink, Mell et al. 2009) polymer-cushioned lipid bilayers,(Elender, Kühner et al. 1996, Sackmann 1996, Wong, Park et al. 1999, Shen, Boxer et al. 2000, Zhang, Longo et al. 2000, Baumgart and Offenhäusser 2002, Tanaka and Sackmann 2005) hybrid bilayers,(Plant 1993, Plant, Brigham-Burke et al. 1995, Rao, Plant et al. 1997, Hubbard, Silin et al. 1998, Plant 1999, Kastl, Ross et al. 2002, Rao, Tutumluer et al. 2002) tethered lipid bilayers,(Beyer, Elender et al. 1996, Cornell, Braach-Maksvytis et al. 1997, Seitz, Wong et al. 1998, Hausch, Zentel et al. 1999, Naumann, Schmidt et al. 1999, Seitz, Ter-Ovanesyan et al. 2000, Cornell, Krishna et al. 2001, Naumann, Prucker et al. 2002) suspended lipid bilayers,(Römer, Lam et al. 2004, Römer and Steinem 2004) supported vesicular layers,(Svedhem, Pfeiffer et al. 2003, Yoshina-Ishii and Boxer 2003) supported lipid bilayer patches,(Miller, Stubbington et al. 2019) and supported lipid bilayers (SLB).(Dominska, Mazur et al. 2007, Domińska, Krysiński et al. 2008, Greiner, Pillman et al. 2009, Oberts and Blanchard 2009, Setiawan and Blanchard 2014, Setiawan and Blanchard 2014, Mize and Blanchard 2016) Each type of model membrane has advantages for particular types of experiments. For example, BLMs are well suited for electrical conductivity characterization but typically have limited lifetimes of less than an hour.(Khan, Dosoky et al. 2013) We have chosen to use SLBs for the experiments reported here because they are stable for extended periods of time, are amenable to characterization by multiple means, and there is an established body of knowledge about SLBs.(Dominska, Mazur et al. 2007, Domińska, Krysiński et al. 2008, Greiner, Pillman et al. 2009, Oberts and Blanchard 2009, Setiawan and Blanchard 2014, Setiawan and Blanchard 2014, Mize and Blanchard 2016) Of particular interest is measuring the fluidity of SLBs, and noteworthy techniques include electron spin resonance (ESR) spectroscopy, deuterium nuclear magnetic resonance (2H-NMR) spectroscopy, and fluorescence microscopy.(Dzikovski and Freed 2013) The behavior of spin probes is studied in ESR, and associated spectroscopic parameters may be connected to the degree of membrane fluidity.(Yawata, Sugihara et al. 1984, Man, Olchawa et al. 2010, Man and Olchawa 2017) In 2H-NMR spectroscopy, specific carbon-deuterium bond orientations in hydrophobic regions or arrangement of amphiphile molecules at certain temperatures may elucidate details of membrane phase separations, thereby characterizing the membrane’s fluidity.(Filippelli, Rossi et al. 2011, Gomez-Murcia, Torrecillas et al. 2016, Dazzoni, Grelard et al. 2020) Of particular interest in the Blanchard research group is fluorescence microscopy. The rotational diffusion behavior and fluorescence lifetime of a fluorescent probe, obtained via fluorescence anisotropy decay imaging (FADI) and/or time-correlated single-photon counting (TCSPC) experiments, may be correlated to the fluidity of a bilayer.(Dominska, Mazur et al. 2007, Lapinski and Blanchard 2007, Domińska, Krysiński et al. 2008, Lapinski and Blanchard 2008, Greiner, Pillman et al. 2009, Pillman and Blanchard 2010, Setiawan and Blanchard 2014, Setiawan and Blanchard 2014, Mize and Blanchard 2016) In this work, our primary focus is on the measurement of translational diffusion of chromophores in selected SLBs.

The composition of SLBs is known to have an effect on bilayer morphology and fluidity. Certain phospholipids are known to exert significant influence on the resulting bilayer properties, in many cases because of their propensity to hydrogen bond with other bilayer constituents. Ceramide in particular, has been used as a model bilayer constituent because of its ability to form hydrogen-bonded networks, resulting in the modification of bilayer permeability, morphology and fluidity.(Notman, den Otter et al. 2007, Sullan, Li et al. 2009, Guo, Moore et al. 2013, Jiménez-Rojo, García-Arribas et al. 2014, Moore, Hartkamp et al. 2018) Our motivation for the inclusion of ceramide in SLBs comes from a recent study that focused on mitigating ceramide levels in mouse circulating angiogenic cell (CAC) membranes.(Chakravarthy, Navitskaya et al. 2016) That work examined potential treatment options for people afflicted with diabetic retinopathy (DR).(Fong, Aiello et al. 2004, Chew, Ambrosius et al. 2010, https://www.diabetes.org/2020) CACs are bone marrow-derived reparative cells that promote angiogenesis (development of new blood vessels). The membrane fluidity of CACs is compromised in dyslipidemia,(Kady, Yan et al. 2017) due to the presence of high levels of ceramide.(Chakravarthy, Navitskaya et al. 2016, Jiang, Huang et al. 2019)

We have synthesized supported lipid bilayers with controlled lipid composition to study the effects of ceramide on the fluidity of a planar membrane. Bilayer fluidity was measured using FRAP. The data reported here demonstrate the role of ceramide in decreasing membrane fluidity, and the spot size-dependence of the FRAP data demonstrates the role of compositional heterogeneity in mediating membrane fluidity.

Experimental

Materials.

Lipids and rhodamine-tethered probe were purchased from Avanti® Polar Lipids and used without further purification. Perylene, calcium chloride, Trizma® hydrochloride, and Trizma® base were purchased from Sigma-Aldrich. A 10 mM Tris® buffer (Trizma® HCl and Trizma® base, pH ~7.5) was prepared with Milli-Q® water (18 MΩ•cm). The solid substrate was high grade mica (Ted Pella, Inc.).

Vesicle Formation and Extrusion.

The control system consisted of 10 mole % cholesterol (ovine wool), 40 mole % sphingomyelin (chicken egg), 47-49 mole % DOPC, and 1-3 mole % perylene. The ceramide system consisted of 10 mole % cholesterol, 20 mole % sphingomyelin, 20 mole % ceramide, 47 mole % DOPC, and 3 mole % perylene. The rhodamine-containing system consisted of 10 mole % cholesterol (ovine wool), 40 mole % sphingomyelin (chicken egg), 49 mole % DOPC, and 1 mole % 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine sulfonyl rhodamine B). Appropriate amounts of constituents, present in chloroform, were dried under N2 (g) and Tris® buffer was added to result in 1 mg/mL lipid concentration. The mixture underwent five freeze-thaw-vortex cycles of 5, 5, and 2 minutes, respectively; liquid nitrogen was used for freezing and a 60 °C water bath for heating.(Lapinski and Blanchard 2007, Setiawan and Blanchard 2014, Setiawan and Blanchard 2014, Chakravarthy, Navitskaya et al. 2016) The vesicle solution of the control was extruded 11 times through a 50-nm polycarbonate membrane disc, ceramide system through a 400-nm filter followed by 200-nm, and rhodamine system through a 100 nm filter. The particle size distribution was characterized by Dynamic Light Scattering measurements, which showed average diameters of the control, ceramide, and rhodamine-based solutions of 133, 145, and 131 nm, respectively.

Planar Bilayer Formation via Vesicle Deposition Fusion.

A hole on a plastic petri dish was cut, and a mica slide was glued to the dish for support. Thereafter, a mica sheet was meticulously peeled, followed by addition of 7 μL of CaCl2 and 60 μL of extruded vesicle solution, which sit for 15 minutes before adding 60 μL of Tris® buffer. The dish assembly was covered tightly with Parafilm®, and one hour elapsed before performing fluorescence recovery after photobleaching measurements. For the rhodamine-containing system, the surface was rinsed with a few milliliters of Tris® buffer after the one-hour period.

Translational Diffusion Measurements.

The fluorescence recovery after photobleaching microscopy technique was utilized to measure the mobility of the fluorescent probe perylene. The fluorescing molecules in a region of interest (ROI) on a plasma membrane are irreversibly photobleached with high laser intensity (405 nm herein).(Axelrod, E. et al. 1976, Ishikawa-Ankerhold, Ankerhold et al. 2014, Meddens, Keijzer et al. 2014) Thereafter, some surrounding fluorophores may diffuse translationally into the ROI, while bleached ones exit. The fluorescence recovery is monitored over time and the fitting of appropriate models to the data are used to extract a recovery time constant (τ), which are converted to translational diffusion constants (D). The instrumental setup was described elsewhere.(Kijewska and Blanchard 2017) Briefly, the system consists of an inverted confocal microscope (Nikon C2+), moveable stage, lasers (405, 488, 561, and 640 nm), an LED illuminator, and standard objectives (Nikon). Data were analyzed with a pure diffusion model (discussed below) using MATLAB software (MathWorks).

Results and Discussion

The goal of this work was to synthesize model lipid bilayers that could mimic a cell membrane and to perform fluidity measurements of those systems. On the macroscopic level, samples containing ceramide behaved differently than those not containing ceramide. There was substantial resistance to extrusion of ceramide-containing vesicles compared to non-ceramide-containing vesicles through membranes with pore diameters of 50 and 100 nm. The ceramide amino and hydroxyl groups participate in hydrogen bonding interactions with those of the lipids, rigidizing the system (Figure 1). This effect was supported by data from fluorescence recovery after photobleaching (FRAP) experiments. A pure diffusion model was fitted to fluorescence recovery curves (of perylene), from which fluorescence recovery time constants, τD, were obtained(Soumpasis 1983, Baumler 2017)

IFRAP(t)=[I0(τD2t)+I1(τD2t)]exp(τD∕2t) (1)
τD=(ω24DT) (2)

where I0 and I1 are modified Bessel functions of the first kind and ω is the ROI radius. The τ value was converted to the translational diffusion constant, DT, of perylene. DT, in turn, is related directly to the fluidity of the synthetic bilayer and is inversely related to the viscosity, η, of the medium - seen from the Stokes-Einstein equation (Eq. 3),

DT=kBT6πηr (3)

where r is the radius of the diffusing molecule.(Soumpasis 1983, Brilliantov, Denisov et al. 1991, Kijewska and Blanchard 2017)

Figure 1.

Figure 1.

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Molecular structures of lipids and fluorescent probes. From left to right: cholesterol, 1,2-dioleoyl-sn-phosphatidylcholine (DOPC), sphingomyelin, ceramide, perylene, and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine sulfonyl rhodamine B).

Mammalian plasma membranes consist of a large number of distinct lipids and proteins, along with carbohydrates.(Heimburg 2003) Recent advances in characterization techniques, such as mass spectrometry, enable the elucidation of some of the complexity.(Harkewicz and Dennis 2011, Schey, Grey et al. 2013, Frick, Hofmann et al. 2018, Gupta, Li et al. 2018, Bolla, Agasid et al. 2019) We use a simplified model system containing DOPC, sphingomyelin, cholesterol, and perylene (composition given above), and this is the same control system used previously by our group. Upon introduction of ceramide, the fluorescence images appear the same by visual inspection (Figure 2). However, the control has an average value within one standard deviation of 4.2 ± 0.8 μm2/s (n = 56) while the ceramide containing system yields a value of 2.8 ± 0.8 μm2/s (n = 113), which is consistent with ceramide rigidizing the membrane. Although the average values are different, the distribution of values are clearly overlapped (Figure 2). It is important to note that the widths of these distributions is substantially larger than the uncertainties in the individual determinations of DT. We view this result as being consistent with there being a distribution of environments in the multi-component bilayers we report on here.

Figure 2.

Figure 2.

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(left) Histograms of translational diffusion constants and (right) fluorescence images of supported lipid bilayers. Top: control system representing 56 measurements; middle: ceramide-based system with 113 measurements; bottom: rhodamine system with 21 measurements. Perylene was excited at 405 nm, and emission was collected at ca. 480 nm. For the fluorescence image on the bottom right, dark regions are cholesterol domains and bright spots are mostly phospholipids; the rhodamine chromophore was excited at 561 nm and emits at ca. 630 nm.(Setiawan and Blanchard 2014)

Structural heterogeneity has been observed even in single-component lipid bilayers by Nojimata and Iwata who utilized picosecond time-resolved fluorescence spectroscopy to study rotational dynamics of trans-stilbene in their model systems.(Nojima and Iwata 2014) In that work, two distinct environments with very different local viscosities were found to co-exist. Our ternary control system (DOPC, sphingomyelin, cholesterol) and the ceramide-containing system (DOPC, sphingomyelin, cholesterol, ceramide) both exhibit heterogeneity as is manifested in the distribution of diffusion constant values. This result has also been observed in similar systems and reported by González-Ramírez, who varied the amounts of N-palmitoyl derivatives of sphingomyelin, ceramide, cholesterol, and dipalmitoylphosphatidylcholine. (González-Ramírez, Artetxe et al. 2019) In artificial biomembranes rich in cholesterol and sphingomyelin, it was shown that liquid-ordered (Lo) and liquid-disordered (Ld) domains co-exist, and freely floating lipid rafts may be present in Lo states.(Veiga, Arrondo et al. 2001, Simons and Ehehalt 2002, Quinn and Wolf 2009, Lingwood and Simons 2010) Moreover, a phase diagram by Aufderhorst-Roberts et al. with their lipid system of 10 %, 45 %, and 45 % of cholesterol, sphingomyelin, and DOPC, respectively, that most closely matches our control system indicates the existence of three-phases consisting of the Lo, Ld, and solid, highly viscous Lβ one, providing more support to compositional heterogeneity.(Aufderhorst-Roberts, Chandra et al. 2017) While the domains have certainly been observed, the elusive nature of the postulated rafts as well as their nanoscopic dimensions(Pralle, Keller et al. 2000, Eggeling, Ringemann et al. 2008, Lajoie, Goetz et al. 2009) make them difficult to detect by FRAP due to its diffraction-limited optical resolution and the size of the sampled region relative to raft dimensions.(Kenworthy, Nichols et al. 2004, Lagerholm, Weinreb et al. 2005, Meddens, Keijzer et al. 2014) Nonetheless, similar values of the diffusion constants were obtained by Chiantia et al. where diffusion coefficients of fluorophores ranged from about 2.5 - 4.5 μm2/s based on the ceramide content in their planar bilayer system on mica at room temperature, composed of DOPC/cholesterol:(SM+Cer) 1:1:1 (molar ratio), and assumed to be in liquid-ordered domains.(Chiantia, Kahya et al. 2006) They utilized an approach of combined atomic force microscopy, fluorescence correlation spectroscopy, and confocal fluorescence imaging. Although our ceramide system had greater DOPC content, specifically 1:2:2:4.7:0.3 molar ratio of cholesterol:sphingomyelin:ceramide:DOPC:perylene, compared to theirs, the trend is similar of decreased diffusion coefficients as a function of ceramide content. Furthermore, it is possible that increased levels of ceramide may lead to gel-like and liquid-disordered phases.

In an effort to probe head group mobility as well, the optical probe perylene was replaced by a headgroup-tagged chromophore. For these experiments we used one mole percent of the sulforhodamine tagged lipid 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) (18:1 Liss Rhod PE) (Figure 1). This probe differs not only in the location of the chromophore but also because the chromophore is tethered to the acyl chains, where perylene was not.

Fluorescence images obtained using the rhodamine probe were similar to those reported earlier, where the dark regions indicate cholesterol-rich domains and the rhodamine-PE probe resides preferentially in the DOPC-rich regions (Figure 2).(Setiawan and Blanchard 2014) It is challenging to measure fluidity in such systems because the dark regions of varying dimensions translate within the bilayer and interfere with ROIs, frequently precluding data collection. Nonetheless, the varying diameters and distributions of spots in the fluorescence images provide some insight into the compositional heterogeneity of the film. Despite this complication, we sought to understand the heterogeneous nature of these synthetic bilayers by conducting size-dependence FRAP measurements on the ceramide- and rhodamine-containing bilayers.

One of the hallmarks of diffusion in a heterogeneous system is the apparent dependence of the recovered diffusion constant on the size of the photobleached region. While the actual diffusion is constant, the presence of non-uniformities in the medium serves to alter the motion of the diffusing molecule, with the details of how the motion is altered being reflected in an apparent time-dependence of the diffusion constant. For a two-dimensional system where the diffusing entities exhibit Brownian motion, Gaussian diffusion may be modeled by Eq. 4,(Ratto and Longo 2003)

〈r2〉=4DTt (4)

where ⟨r2⟩ represents the mean square displacement, DT is the translational diffusion constant, and t is time. For a homogeneous medium, the recovered diffusion constant, DT, should be invariant with spot size, leading to a linear relationship between <r2> and the time constant of the bleach recovery. This is not seen for both perylene and rhodamine chromophores in the systems examined here (Fig. 3). Ratto and Longo had noticed via Monte Carlo simulations of lipid bilayers that diffusion is normal, given a long period of time, but appears to be anomalous at short times.(Ratto and Longo 2003) Proteins and other obstructions in the bilayer can give rise to anomalous diffusion.(Duncan, Reddy et al. 2017) The observation of anomalous diffusion in our ceramide-containing supported lipid bilayers indicates that different constituents within a multi-component bilayer also produces anomalous diffusion.

Figure 3.

Figure 3.

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Plot of DT for perylene (open circles) and tethered rhodamine (solid circled) in ceramide-containing supported lipid bilayer as a function of photobleached spot radius squared.

There are several interesting features contained in the data shown in Fig. 3. The first is that the diffusion constant, DT, appears to increase with increasing spot size. Anomalous diffusion has been considered in detail before, with the starting point being the modeling of data in the context of a random walk.(Ott, Bouchard et al. 1990, Balescu 1995, Bychuk 1995, Stapf, Kimmich et al. 1995, Bodurka, Seitter et al. 1997, Weeks and Swinney 1998, Luedtke and Landman 1999, Schaufler, Schleich et al. 1999) Anomalous sub-diffusion (i.e. DT appearing to decrease with increasing spot size) has been explained in the context of random-walk diffusion being interrupted and molecular motion redirected by heterogeneities in the (fluid) medium. Anomalous super-diffusion, (i.e. DT appearing to increase with increasing spot size) has been somewhat more challenging to explain. Typically, random walk motion is considered where the characteristic distance of each diffusive “step” is described by a Gaussian distribution. For anomalous super-diffusion, so-called Levy flights have been invoked,(Ott, Bouchard et al. 1990, Stapf, Kimmich et al. 1995, Bodurka, Seitter et al. 1997, Weeks and Swinney 1998, Luedtke and Landman 1999, Schaufler, Schleich et al. 1999) where the distribution of diffusional step lengths can occasionally be much longer than the average. The physical explanation for such events may involve diffusional motion along a phase boundary, or other structural feature that could allow for uninterrupted motion in a given direction. Unfortunately, it is not possible to resolve the physical basis for the anomalous super-diffusion behavior found in the bilayers we have examined here.

Another interesting feature contained in Figure 3 is that the perylene diffusion constant is consistently smaller than that seen for the tethered rhodamine chromophore. The perylene is confined in the bilayer acyl chain region while the rhodamine chromophore is located in the polar bilayer headgroup region. More important is the fact that the rhodamine chromophore is tethered to acyl chains that are incorporated into the bilayer. Perylene is thus sensing the effective viscosity of the acyl chain region while the rhodamine chromophore motion should be constrained by translation of the acyl chains within the bilayer. Intuition would suggest that both chromophores should experience the same viscosity in a homogeneous medium, with the tethered rhodamine chromophore exhibiting slower diffusional motion due to its larger size. This is not seen experimentally, suggesting that the perylene is sensing a different, more viscous environment within the nonpolar region of the bilayer than the tethered rhodamine chromophore. The tethered rhodamine chromophore should partition into the phosphocholine-rich regions of the bilayer. The data in Fig. 3 point to structural heterogeneity in the bilayer, and the fluorescence images shown in Fig. 2 demonstrate that the tethered rhodamine chromophore does not partition into cholesterol-rich regions; no corresponding partitioning is seen for perylene in the bilayer. While partitioning into unresolved domains within the bilayer is possible for both chromophores, any such effect would be spatially averaged by virtue of the characteristic length scale of the FRAP measurements. The DT data for perylene would thus represent the average of diffusion in the cholesterol-rich and phosphocholine-rich regions of the bilayer while the tethered rhodamine DT data would reflect diffusion in the phosphocholine-rich region, which would be expected to be more fluid (i.e. less viscous) than the cholesterol-rich regions.

Conclusions

We have studied three different lipid bilayer systems supported on mica. The control system consisted of cholesterol, sphingomyelin, DOPC, and perylene and a histogram of the translational diffusion values, obtained from FRAP experiments, was indicative of a heterogeneous distribution. The ceramide-containing system was similar to the control but also included ceramide, which drastically rigidified the vesicle solution, noticed from resistance in extrusion. Additionally, the histogram of diffusion constants was skewed toward lower values, which is sensible since ceramide may incorporate into gel domains within the bilayer. The third batch was similar to the control, but perylene was replaced with a rhodamine-tethered lipid probe and exhibited dark spots of cholesterol and bright spots of mainly phosphocholines. Thereafter, we probed the ceramide- and rhodamine-containing system further by changing the spot size and noticed that the diffusion constant increased with spot radius, indicative of anomalous diffusion.

Even by simplifying our model of the plasma membrane by excluding proteins, complexities are present in lipid bilayers due to possible lipid and dye aggregates, phase segregation, and/or lipid rafts; it is important to understand how those variables are involved in giving the membrane its heterogeneous nature.

The bright spots in the rhodamine system were unexplored in our work, but it is known from prior work in the Blanchard group that these bright spots increased in size upon laser irradiation. It is possible that the system was being heated, then annealing occurred, resulting in microcrystalline lipid domains. It would be worthwhile to investigate this concept further with controlled experiments.

When preparing the supported lipid bilayers, the method of vesicle and bilayer preparation can impact results significantly. This area has been advanced using techniques such solvent-assisted lipid bilayer (SALB) methods.(Ferhan, Yoon et al. 2019, Jackman and Cho 2020) Clearly a deeper understanding of these systems will require further exploration of compositional parameter space, and the effects of other variables such as pH, temperature, and ionic strength may prove useful in understanding the range of spatial heterogeneity achievable in such model bilayers.

Highlights.

Ceramide controls the rigidity of model supported lipid bilayers.

Ttranslational diffusion reveals the effect of ceramide head-group hydrogen bonding.

Directly relevant to supported bilayer organization

Explains why acid sphingomyelinase causes plasma membrane rigidization

Acknowledgements

We are grateful to the National Institutes of Health for funding this project through grants 2R01EY016077-08A1 and 5R01EY025383-02 R01.

Footnotes

The Authors have no conflicts of interest to declare.

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