The Role of Phospholipid Headgroup Composition and Trehalose in

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The role of phospholipid headgroup composition and trehalose in the desiccation tolerance of Caenorhabditis elegans Sawsan Abusharkh, Cihan Erkut, Jana Oertel, Teymuras V. Kurzchalia, and Karim Fahmy Langmuir, Just Accepted Manuscript • DOI: 10.1021/la502654j • Publication Date (Web): 07 Oct 2014 Downloaded from http://pubs.acs.org on October 12, 2014

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The role of phospholipid headgroup composition and trehalose in the desiccation tolerance of Caenorhabditis elegans Sawsan E. Abusharkh1,3, Cihan Erkut2, Jana Oertel1, Teymuras V. Kurzchalia2, and Karim Fahmy1* 1

Biophysics Division, Institute of Resource Ecology, Helmholtz-Zentrum Dresden-Rossendorf,

PF 510119, D-01314 Dresden, Germany 2

Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauerstraße 108, D-01307

Dresden, Germany 3

Technische Universität Dresden, Dresden-International Graduate School for Biomedicine and

Bioengineering, D-01062 Dresden, Germany KEYWORDS anhydrobiosis, membrane mechanics, nematode, rapid scan FTIR spectroscopy.

ACS Paragon Plus Environment

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ABSTRACT Anhydrobiotic organisms have the remarkable ability to lose extensive amounts of body water and survive in an ametabolic state. Distributed to various taxa of life, these organisms have developed strategies to efficiently protect their cell membranes and proteins against extreme water loss. Recently, we showed that the dauer larva of the nematode Caenorhabditis elegans is anhydrobiotic and accumulates high amounts of trehalose during preparation to harsh desiccation (preconditioning). Here, we have used this genetic model to study the biophysical manifestations of anhydrobiosis and show that, in addition to trehalose accumulation, dauer larvae dramatically reduce their phosphatidylcholine (PC) content. The chemical composition of the phospholipids (PLs) has key consequences not only for their interaction with trehalose, as we demonstrate with Langmuir-Blodget monolayers. Also the kinetic response of PLs to hydration transients is strongly influenced as evidenced by timeresolved FTIR-spectroscopy. PLs from preconditioned larvae with reduced PC content exhibit a higher trehalose affinity, a stronger hydration-induced gain in acyl chain free volume, and a wider spread of structural relaxation rates of their lyotropic transitions and sub-headgroup Hbond interactions. The different hydration properties of PC and phosphatidylethanolamine (PE) headgroups are crucial for the hydration-dependent rearrangement of the trehalose-mediated Hbond network. As a consequence, the compressibility modulus of PLs from preconditioned larvae is about 2.6 fold smaller than that from non-preconditioned ones. Thus, the biological relevance of reducing the PC:PE ratio by PL headgroup adaptation should be the preservation of plasma membrane integrity by relieving mechanical strain from desiccated trehalose-containing cells during fast rehydration.


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1. INTRODUCTION Anhydrobiotic organisms exhibit the remarkable ability to survive extreme desiccation, during which they can reversibly halt their metabolism and lose most of their body water1. Upon rehydration, these organisms can quickly resume life activities. Anhydrobiosis is a widespread survival strategy observed in diverse taxa of life such as bacteria2, yeast3, tardigrades4, brine shrimps5, plants6, rotifers7 certain insect larvae8 as well as various nematode species9-11. Anhydrobiotes have evolved a number of mechanisms that preserve the structure of their membranes and proteins at extremely diminished water content12,

13

. One mechanism is the

synthesis of disaccharides such as trehalose (α,α-1,1'-glucoside), which has long been correlated with desiccation tolerance in many anhydrobiotic invertebrates14 such as nematodes15,

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,

chironomids8, tardigrades4, rotifers17 as well as prokaryotes2. The decisive role of trehalose in desiccation tolerance came from studies on Caenorhabditis elegans. We introduced this free-living nematode as a true anhydrobiote18 to delineate the molecular mechanisms of anhydrobiosis19. In the metabolically depressed dauer stage, C. elegans tolerates extreme water loss after an initial mild desiccation treatment, called preconditioning. During this process, trehalose increases 4-5 fold, whereas mutants unable to synthesize trehalose are extremely sensitive to harsh desiccation. Light and electron microscopy revealed that upon harsh desiccation, trehalose-deficient worms suffer massive rupture of cell membranes and membranous organelles. Infra-red spectra of living dauer larvae further supported an in vivo role of trehalose for maintaining cell membrane structure20 in agreement with studies on phase transitions in model lipids21. In their natural environment, desiccated anhydrobiotes encounter fast rehydration when rainwater penetrates the habitat after a period of drought. The sudden and steep gradients of


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hydration cause different swelling rates of the diverse biological materials in adjacent compartments. The mechanical response of membranes to this effect is governed by the hydration-induced (lyotropic) phase transitions of their lipids. The coordination of these processes appears to be the critical factor to preserve membrane integrity during the exit from the dry state. We asked whether trehalose plays a role in this coordination. In vitro investigations show that lipid headgroup-sugar interactions might be crucial for this process, because they dramatically lower the phase transition temperature in the dry state22 and preserve phase separation in lipid mixtures21, 23. Molecular dynamics (MD) calculations of pure model lipids confirm the intercalative action of trehalose at the head-group region in general 24-26 but without a clear preference for vitrification, water replacement or hydration force mechanisms27. In contrast, the lyotropic transitions of natural lipid–carbohydrate mixtures from anhydrobiotes have not been investigated. We have surmised that protective mechanisms should be especially effective on the time scale of seconds, during which membrane damage may occur under natural conditions when sudden deviations from equilibrium occur. These processes may not be fully understood by static experiments or simulations with a bulk water phase. Therefore, we have established time-dependent IR-absorption experiments to monitor the structural response of C. elegans phospholipids to transient hydration changes within a few seconds at reduced humidity. The use of IR-spectroscopy for the quantitative evaluation of hydration-dependent states of lipids has been comprehensively reviewed28 and allows the label-free detection of structural changes with chemical group-specific resolution. In this study, we looked for differences at the molecular level in fast lyotropic transitions of phospholipids (PLs) extracted from preconditioned vs. non-preconditioned dauer larvae,


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assuming that during preconditioning, membranes become desiccation-resistant. Our data show that already in the absence of trehalose, PLs extracted from preconditioned larvae (PPLs) respond to hydration transients with a larger increase of acyl chain free volume as compared to PLs of non-preconditioned larvae (NPLs). We found that this property correlates with a dramatic decrease of the PC:PE ratio upon preconditioning. The data further suggest a dynamic and hydration-dependent topology of trehalose in the PL headgroup region. These chemical and physical changes appear to represent an essential molecular adaptation to anhydrobiosis by which PL lateral expansion is adjusted to reduce mechanical strain during hydration transients in cell membranes.

2. EXPERIMENTAL SECTION Caenorhabditis elegans culture conditions. The temperature-sensitive dauer-constitutive daf2(e1370) strain of C. elegans was obtained from CGC (Minneapolis, MN) funded by the NIH Office of Research Infrastructure Programs (P40 OD010440). This strain of C. elegans is a conditional mutant which at 15 °C undergoes a reproductive life cycle similar to the wild type and can thus be propagated. However, eggs grown at 25 ˚C exclusively develop into dauer larvae which at his temperature arrest at this stage. We used the latter regime to obtain large populations of pure dauer larvae whose phenotype can be changed from desiccation-sensitive to tolerant depending on preconditioning (see below). Worms were maintained at 15 °C on nematode growth medium (NGM) agar plates seeded with concentrated Escherichia coli strain NA22 as the food source. Eggs were isolated by alkaline hypochlorite treatment from gravid hermaphrodites. They were then grown at 25 °C in


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complete S medium (liquid culture) supplemented with E. coli for 5 days until they completely arrested as dauer larvae29. For radioactive labeling of dauer lipids, eggs were grown at 25 °C until dauer arrest on NGM agar plates with 10 µCi CH314COONa (Hartmann Analytic, Germany) mixed to the food source. Preconditioning was performed as described previously18. Briefly, dauer larvae from liquid cultures or radioactive labeling plates were collected into 15 ml conical tubes and washed 2–3 times by pelleting and resuspending in distilled water to remove bacteria, salts and debris. Finally, the larvae were resuspended in 2 ml water. One half of the worms were immediately snap-frozen in liquid nitrogen (non-preconditioned group). The remaining was first filtered on TETP membranes (8 µm pore size, Millipore, USA) and then placed on 35 mm plastic Petri dishes into a controlled humidity chamber equilibrated at 98% RH (preconditioned group). After 4 days of preconditioning, they were collected in 1 ml distilled water and snap-frozen. PLs were extracted from homogenized worms using the method of Bligh and Dyer

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as detailed in the

Supporting Information.

Time-resolved ATR-FTIR Spectroscopy. PL films were dried from vesicle suspensions (Suppl. Information) on a diamond 9-reflections attenuated total reflectance (ATR) unit (RESULTEC, Illerkirchberg) and equilibrated overnight at a 75% RH using a saturated salt solution of NaCl. The latter was separated from the sample compartment by a dialysis membrane placed 1 mm above the lipid film on the ATR-crystal. Hydration pulses were generated by an electrical current (0.7 A) sent for 4 s through a heating wire (0.26 Ohm) immersed in the salt solution, which leads to a transient increase of lipid hydration through the gas phase. By calibration of the water ν(OH) absorption of at different RHs, this regime generated an initial


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water uptake by the PL film that corresponded to a RH change of 5 – 8%. IR absorption changes were measured during reversion to the initial hydration state over 40-50 s in ten time slices by rapid scan Fourier transform infrared (FTIR) difference spectroscopy (MCT detector, resolution: 2 cm-1 and 3.2 s; using an IFS/66v/S spectrometer, and OPUS programming software, BRUKER Optics GmbH, Karlsruhe, Germany). PL samples were allowed to fully relax to the initial RH over 8 min before the next hydration pulse was applied (for details see Supporting Information, Fig. S1). Absorption spectra were calculated and co-added for each time slice from 10 hydration pulses in an automated fashion to improve the signal to noise ratio. Difference spectra were calculated relative to the absorption of the PL film before the hydration pulse. Thereby, the difference spectra contain only contributions of those vibrational modes that undergo hydrationinduced absorption changes. Spectral processing was done in an automated fashion as described31 and explained in the Supporting Information.

Langmuir-Blodgett isotherms of lipid monolayers. Lipid monolayers were formed by spreading 7.5 µL of lipid extracts (1 mg/ml in CHCl3: CH3OH (4:1)) on 20 ml of phosphate buffered saline (10 mM, pH = 7.2) in a µ-Trough S (Kibron.Inc, Finland). One hour after spreading, the monolayer was compressed at rate of 0.05 Å2/chain/min to a target pressure of 25 mN/m. Then the monolayer was expanded with the same rate to a pressure of 19 – 20 mN/m. To study the interaction of the lipids with trehalose, 1 ml of 125 mM aqueous trehalose solution was injected into the subphase (20 ml) at constant area giving rise to a pressure increase upon trehalose incorporation into the monolayer. After the pressure has stabilized (~ 1 h) the film was expanded again to 19 – 20 mN/m. The relation


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∆AL/AL = K · exp[-(At · π/RT)]

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(Eq 1)

was used to approximate the molecular area (At) occupied by trehalose in the PL film. It was obtained from the slope of the RT·ln (∆AL/AL) vs. π plot, where AL is the PL surface area at a given pressure of 25 mN/m > π > 19 mN/m in the absence of trehalose and ∆AL the area increase in the presence of trehalose at the same π. K is the partitioning constant of trehalose between the aqueous and the PL phase32. The free enthalpy of trehalose insertion was derived from the y-axis interception of the linear regression line through the RT·ln (∆AL/AL) vs. π plot. The purity of trehalose used in all experiments was verified by FTIR spectroscopy using the IR spectrum of amorphous trehalose as a reference33.

3. RESULTS Dynamics of headgroup hydration and acyl chain disorder in C. elegans phospholipids. The major question in the physiological context of anhydrobiosis in the dauer larvae of C. elegans concerns the structural impact of fast hydration transients on membrane integrity. We used time-resolved Fourier transform infrared (FTIR) difference spectroscopy as a label-free method to monitor fast hydration-induced structural changes in PL films prepared from two populations of larvae (preconditioned and non-preconditioned). The hydration of a PL film was transiently increased in an automated fashion by pulsed evaporation from a hydration reservoir. The infrared (IR) absorption changes of the lyotropic phase transition were then followed by rapid scan FTIR spectroscopy within seconds and were found to show the strongest response at a basal RH of 75% used throughout this study. The chemically and topologically different regions of the PLs contribute to specific IR absorptions such as the antisymmetric stretching vibration of


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Figure 1: Experimental setup for pulsed hydration and infrared spectra acquisition. A) A PL film (sample) on an attenuated-total-reflectance (ATR) crystal is separated by a gas phase and a dialysis membrane (distance < 1mm) from a saturated KCl solution to maintain 75 % RH. After recording an IR reference spectrum, a TTL signal initiates a heating current (4s) in the salt solution to increase the RH by 5-7%. By rapid scan FTIR spectroscopy, the relaxation of the PL film to its initial hydration is monitored in ten time intervals (rectangular trigger levels). B) The first difference spectrum calculated from the first data acquisition interval and the reference spectrum. Absorption increases between 4000 and 3000 cm-1 are caused by water uptake in the PL film. Absorption difference bands of the PL acyl chains (3000-2800 cm-1, water background subtracted for clarity) and headgroups (1800-1000 cm-1) are due to hydration-induced shifts of their vibrational frequencies. C) Evolution of difference spectra in response to a single hydration pulse.


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the phosphates, νas(PO2-) at ~1260 cm-1, the ester carbonyl stretching frequency ν(C=O) at ~1740 cm-1,

and

the

acyl

methylene

modes

ν(CH2)

between

3000

and

2800

cm-1.

The νas( PO2-), and ν(C=O) frequencies are affected directly by hydration-induced Hbonding,whereas the ν(CH2) respond to changes in acyl chain free volume. Thus, the IR signature provides a view on the extent of H-bonding at different sites in the PL headgroups and on the acyl chain order as reviewed28, 34. Figure 1 shows the experimental setup (Fig. 1A) and a representative hydration-induced IR absorption change (Fig. 1B). Data were obtained as a time-dependent sequence of difference spectra (Fig. 1C) with positive hydration-induced absorption bands from which the spectrum of the initial 75% RH state was subtracted (negative peaks). Water uptake causes the increase of the water ν(OH) band between 4000 cm-1 and 2700 cm-1. Frequency shifts of the PL film cause negative (absorption of the initial state) and positive bands (absorption of the hydration-induced state). After a rapid increase of hydration (within 4 s), the PL film reverts to its initial hydration state leading to the successive reduction of the absorption differences over time. The difference spectra reveal the hydration-induced kinetics of H-bond changes and structural transitions at the chemically and topologically different sites of the PLs. Of particular interest is the comparison of the amplitudes of the acyl chain ν(CH2), and the Hbond-dependent νas(PO2-) to the amplitude of the C=O stretching mode. The difference spectra are shown for NPLs and PPLs in Fig. 2A and 2B, respectively. In NPLs, the ν(C=O) shifts down in response to increased H-bonding which gives rise to the negative lobe at 1741 cm-1 (depletion of the initial C=O state). The positive lobe at 1713 cm-1 is caused by the ν(C=O) stretching modes of the more strongly H-bonded ester carbonyl groups formed after hydration. The


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symmetric νs(CH2) and antisymmetric νas(CH2) give rise to difference bands at 2849/2858 cm-1 and 2916/2929 cm-1, respectively. The ν(CH2)/ν(C=O) amplitude ratios reflect the physiologically relevant relation between the gain of acyl chain free volume (up-shift of the ν(CH2) during the lyotropic phase transition35) and the degree of sub-headgroup hydration (down-shift of the ν(C=O)). The decrease of these perturbations over time is exemplified for three difference spectra after the hydration pulse. In addition to the ν(C=O), also the νas(PO2-) becomes down-shifted upon hydration (1204/1204 cm-1 difference band) because at a basal humidity of 75% RH, H-bonding to the PO2- di-ester groups can further increase by hydration. From the water ν(OH) and the νas(PO2-) band in the reference spectrum (not shown) a basal hydration of Γ of 15 and 13 mol H2O/mol P is obtained 36 for NPLs and PPLs, respectively. The hydration-induced increase of the ν(OH) in the first difference spectrum (exemplified in Fig. 1C) corresponds to a water uptake of ∆Γ of 4-5 mol H2O / mol P within 4 s (corresponding to ∆RH of 5-7%). The hydration-induced frequency shifts are almost identical in NPLs and PPLs because they are due to rather general PL properties. The salient difference, however, is the increased amplitude of the ν(CH2) difference bands in PPLs as compared to NPLs, when normalized to the amplitude of the ν(C=O) absorption change. Hence, the spectra reveal a much larger increase of free acyl chain volume in PPLs at the same degree of incremental sub-headgroup hydration. The normalized νs(CH2) amplitudes evidence an approximately three-fold larger gain of acyl chain free volume upon hydration of the sub-headgroup region in PPLs versus NPLs. In contrast, the nearly identical νas(PO2-) / ν(C=O) absorption ratios demonstrate the unaltered relative degree of hydration changes at the phosphate and carbonyl region.


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Figure 2: Hydration-pulse-induced IR absorption changes of PL extracts in the absence of trehalose. A) Representative selection of difference spectra of NPLs recorded at the indicated times after the hydration pulse. The down shift of the ν(C=O) and νas(PO2-) causes the difference bands at 1741/1713 and 1242 /1204 cm-1 due to increased H-bonding to the ester carbonyl and phosphates, respectively. The increase of acyl chain free volume is monitored by the up-shift of the νs(CH2) and νas(CH2) frequencies (2849/2959 and 2917/2949 cm-1, respectively). Spectra are normalized to the amplitude of the ν(C=O) difference band. B) As in (A) obtained with PPLs. C) Relaxation of absorption changes in NPLs. D) as in (C) for PPLs. IR frequencies are assigned to chemical groups as indicated. The loss of water to the gas phase (reduction of ν(OH) amplitude) is shown for comparison (blue). Spectral noise is < 0.1 mAU (Supporting information, Fig. S1)


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The kinetics of selected absorption bands is shown in Figs. 2C and 2D for NPLs and PPLs, respectively. Clearly, the re-equilibration of water with the gas phase (blue traces) precedes all other absorption changes of the PLs. The order of relaxations exhibit a close relation to the topology of the chemical groups: The slower relaxation of the νas(PO2-) and ν(C=O) difference bands is consistent with water being initially released from inter-headgroup hydration sites followed by the synchronous dehydration of the ester-carbonyl and PO2- region, before the acyl chains revert to their more densely packed initial state. The delayed response of the acyl ν(CH2) changes demonstrates that sub-headgroup hydration is not cooperatively coupled to acyl chain order on the time scale of seconds in either PL sample. In summary, hydration transients of NPLs and PPLs induce similar responses by increased Hbonding to PO2- and C=O groups. However, in PPLs, these H-bond networks evoke a much larger increase in acyl chain disorder, as evidenced by the ~3-fold larger ν(CH2) difference band.

Desiccation stress depletes phosphatidylcholine in cell membranes and enhances their interaction with trehalose. The different physical properties of NPLs and PPLs revealed by the IR-spectra suggest underlying chemical differences that are generated during preconditioning. Therefore, the total PL content of dauer larvae was analyzed after incorporation of

14

C into de

novo synthesized lipids by supplementing the food of growing worms with CH314COONa. Extracted PLs were separated by 2-dimensional thin layer chromatography (TLC) according to their hydrophobicity (Fig. 3A). Comparison of the abundance of PL classes shows that preconditioning dramatically decreases the phosphatidylcholine (PC) levels, whereas other PL classes

such

as

phosphatidylethanolamine

(PE),

phosphatidylserine

(PS)

and

phosphatidylinositol (PI) are not much affected.


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Figure 3: Correlation of PL headgroup composition and hydration reponses. A) TLC radiogram of decrease

in

14

C-labeled PLs. Among the major PL classes, only PC displays a dramatic the

preconditioned

sample.

PE:

Phosphatidylethanolamine,

PC:

Phosphatidylcholine, PI: Phosphatidylinositole, PS: Phosphatidylserine, S: Starting point. Dimensions are indicated by arrows. Solvent 1: CHCl3:CH3OH:32%NH3 (65:35:5, v/v/v); solvent

2:

CHCl3:CH3OH:(CH3)2CO:CH3COOH:H2O

(50:10:20:12.5:5,

v/v/v/v/v)).

B)

Hydration-pulse-induced IR absorption changes of an NPL film (black) and a PPL film (green) which was supplemented with bovine PC (PPL:PC=1:3), thereby restoring the ν(CH2) / ν(C=O) amplitude ratio of NPLs.


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Can PC depletion explain the different lyotropic behavior of PPLs? We addressed this question by supplementing PPLs from C. elegans dauer larvae with bovine PC and performing again hydration-induced time-resolved ATR-FTIR difference spectroscopy. Figure 3B shows that the increase of acyl chain disorder in relation to sub-headgroup hydration as measured by the amplitude ratio of the νs(CH2) and ν(C=O) difference bands, is indeed reduced to the value found with NPLs. This confirms the critical role of PC in regulating the lyotropic properties of the cell membranes in the anhydrous state. Our results so far revealed a predominant role of PC-containing lipids in regulating the extent of lyotropic acyl chain transitions. The PLs were devoid of trehalose, because the organic extraction separates PLs and sugars into different phases. However, our previous work clearly showed that the accumulation of large amounts of trehalose during preconditioning of dauer larvae is essential for their desiccation tolerance. Therefore, we asked whether the reduced PC content of PPLs might additionally affect their interaction with trehalose. Trehalose-PL interactions were studied by Langmuir-Blodget monolayers which are suitable models to study the interactions of the sugar with bilayers

25

. Figure 4A shows the expansion

isotherm of a PPL monolayer followed by the injection of trehalose into the subphase at a pressure of ~20 mN/m. Adsorption of the sugar to the monolayer caused a pressure increase to ~23.5 mN/m over one hour. The subsequent film expansion reduced the pressure again. From the area differences between the two branches, we have obtained the fractional area increase ∆AL/AL induced by trehalose at constant pressure for the pressure values covered by both expansion isotherms. The ability of trehalose to increase the monolayer area agrees with previous studies on model lipids37, 38. The slope of the plot RT ln(∆AL/AL) vs. π corresponds to a molar area At = 77 Å2 occupied by trehalose (Fig. 4B). A smaller At = 60 Å2 is obtained for NPLs (Fig. 4C and D).


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Figure 4: Interaction of trehalose with PL monolayers. Expansion of a monolayer of PPLs (A) and NPLs (B) before (I) and after (II) injection of trehalose (TRE) to the subphase at a final concentration of 125 mM. Broken arrows exemplify the area increase induced by trehalose at constant pressure. C) The molecular area At occupied by trehalose in the monolayers is derived from the slope of the linear regressions of the RT· ln(∆AL/AL) plots over a range of pressures. The free enthalpy difference of binding of trehalose to PPLs vs. NPLs is reduced by 1.6 kJ < ∆∆G0 < 5 kJ (determined from the offsets of the linear regressions and allowing an uncertainty of the minimal area per lipid of 45-65 Å2).


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Also the free enthalpy of the binding of the disaccharide to PLs increased from -12.8 kJ/mol in PPLs to -9.5 kJ/mol in NPLs as determined from the zero pressure extrapolation of the two regression lines. These results demonstrate that preconditioning enhances the affinity of the PLs to the disaccharide. Its larger molar area in PPL monolayers suggests that it covers a larger surface in cell membranes in preconditioned worms.

Properties of PLs in the presence of trehalose. The reduction of the PC-content during preconditioning increases the coupling of acyl chain disorder to sub-headgroup hydration on the time scale of seconds, already in the absence of trehalose. Physiologically, this situation is not sufficient to maintain membrane organization upon water loss, because preconditioned but trehalose-deficient worms do not survive desiccation. Motivated also by our finding that trehalose has a higher affinity for PPLs, we have also studied its influence on the structural transitions of NPLs and PPLs by time-resolved ATR-FTIR spectroscopy (Fig. 5). In the presence of trehalose (1:20 trehalose:lipid), the coupling of acyl chain disordering to sub-headgroup hydration, as reflected by the ratio between the ν(CH2) and ν(C=O) difference bands, is again larger in PPLs than in NPLs. However, the νas(PO2-) amplitude relative to the ν(C=O) is reduced to about one third of that measured in both PLs alone (Fig. 2). Clearly, trehalose saturates the majority of H-bond interactions at the PO2- groups which agrees with the water-replacement mechanism22, 39. Since hydration nevertheless increases acyl chain disorder without further PO2H-bonding, the transient hydration of the ester carbonyl region drives the process. Indeed, the shift of the ν(C=O) as compared to trehalose-free PLs evidences a stronger hydration-induced Hbonding at the ester-carbonyls: the negative lobe at 1740 cm-1 is unaffected by the sugar but the


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Figure 5: Hydration-pulse-induced IR absorption changes of PL extracts in the presence of trehalose. A) Selection of difference spectra of NPLs recorded at the indicated times after the hydration pulse. The hydration-induced down shift of the ν(C=O) and νas(PO2-) causes the difference bands at 1741/1700 cm-1 due to increased H-bonding to the ester carbonyl. Little changes are observed for the phosphates at 1249-1200 cm-1. The increase of acyl chain free volume is monitored by the up-shift of the νs(CH2) and νas(CH2) frequencies (2849/2959 and 2917/2949 cm-1, respectively). B) As in (A) obtained with PPLs. C) Relaxation of absorption changes of NPLs. D) as in (C) for PPLs. Infrared frequencies are assigned to chemical groups as indicated. The loss of water to the gas phase (reduction of ν(OH) amplitude) is shown for comparison (blue) as well as the change in H-bonding to trehalose (original spectral data in Supporting Information Fig. S2D).


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positive lobe is down-shifted to ~1700 cm-1 as compared to 1711 cm-1 in the absence of trehalose. The unaffected initial ν(C=O) at 1740 cm-1 shows that stronger carbonyl H bonds are not established by trehalose per se but form transiently. Trehalose thus saturates the PO2- Hbonds and directs further H-bond formation efficiently to the sub-headgroup region in a dynamic hydration-induced process. Likewise, the ν(CH2) at 2850 and 2950 cm-1 are initially unaffected by trehalose. They shift up only after a hydration pulse and their broadening (particularly at ~2950 cm-1) evidences a larger heterogeneity of acyl chain conformations than in the absence of the sugar. The described trehalose PL interactions are exhibited by both PL extracts. A remarkable influence of preconditioning, however, is seen on the relaxation kinetics. The absorption changes of the water ν(OH), ν(C=O) and νas(PO2-) are slowed down by the sugar (Fig. 5C) in accordance with earlier FTIR studies40. They become essentially synchronized with the acyl chain relaxation in NPLs. This is surprisingly not the case for PPLs. The relaxation rates of H-bonding to the different PL chemical groups are clearly more heterogeneous. Particularly, the acyl chain reordering occurs faster than the re-equilibration of water with the gas phase. PPLs are thus unique in allowing trehalose to slow down headgroup hydration changes without rigidifying but actually softening the hydrophobic core of the PPLs as discussed below. In summary, the demonstrated change in lipid composition during preconditioning leads to an increased gain of acyl chain free volume, to accelerated acyl chain dynamics during hydration transients and to a more efficient trehalose insertion into PPLs. These physical parameters correlate with the previously observed occurrence of membrane ruptures in C. elegans dauer larvae after desiccation and are probably of key importance for trehalose-dependent desiccation tolerance.


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4. DISCUSSION Dauer larvae of the nematode C. elegans are capable of adopting an anhydrobiotic state in which they survive desiccation for several weeks. Desiccation tolerance depends on a limited number of strategies that are employed during preconditioning19. One of them is the biosynthesis of trehalose. Trehalose-deficient worms are desiccation sensitive even after preconditioning and exhibit irreversible damage to cell membranes upon rehydration18,

20

. Motivated by these

findings, we studied fast non-equilibrium structural transitions induced by hydration in PLs extracted from dauer larvae. The salient physical feature revealed here is the almost three-fold increase in acyl chain free volume upon fast hydration of PPLs. This behavior is independent of trehalose and originates in a different coupling of the sub-headgroup H-bond network to acyl chain packing. Its chemical basis is the reduction of the PC-content during preconditioning. Previous FTIR work on model lipids at extremely low hydration (Γ of 3-5) demonstrated that PC forms strong water contacts41, whereas PE exhibits weaker water binding upon drying42, 43. Our data suggest that the different intrinsic headgroup hydration patterns contribute to the unexpected differences in coupling headgroup hydration to acyl chain disorder. In concert with other structural preferences, e.g., a tilted acyl chain packing in PC over PE44, the regulation of the PC:PE ratio in vivo could be an essential factor in rendering cell membranes desiccation-resistant. We have asked whether the different physico-chemical properties of the C. elegans PL extracts are important for the protective function of trehalose. The disaccharide saturates the H-bonds to the PO2- groups already at reduced humidity, indicative of water-replacement13,

16

. At reduced humidity,

however, trehalose neither enhances H-bonding nor entraps water in the sub-headgroup region.


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Figure 6: Absorption change ∆A of the symmetric CH2 stretching mode as a function of the chemical potential of water. The time-dependent amplitude of the 2849/2859 cm-1 difference bands (in per cent of the total absorption at 2850 cm-1) is plotted versus the chemical potential of water in the absence (A) and presence of trehalose (B). From the slopes, compressibility modules are derived (see Supporting Information).

Instead, it couples hydration transients very efficiently to the growth of a sub-headgroup H-bond network. This contrasts its action on model lipids in aqueous suspension, where the ν(C=O) gradually decreases with the amount of trehalose45. In agreement with these studies, however, the transiently formed H-bonds are indeed much stronger than in pure PL extracts (positive lobes of the ν(C=O) difference bands at 1711 cm-1 and ~1700 cm-1 in NPLs and PPLs, respectively). The potential of trehalose to organize an unusually large dynamical hydration shell46 may be crucial


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in linking the lipid surface hydration to the increased transient sub-headgroup H-bonding shown here. It was surprising that at 75% RH trehalose did not increase sub-headgroup hydration per se. A dynamic hydration-driven trehalose-mediated H-bond network describes the data much better and appears to be the crucial factor in tailoring PL properties for desiccation tolerance. Since the dynamic properties of PLs also correlate with their mechanical behavior, we have derived estimates of the PL compressibility modulus Kc (Fig. 6), based on the FTIR data in Figs. 2 and 5. Kc relates the area increase per lipid ∆AL to the change of the free enthalpy of water per lipid, i.e., ∆(Γ·ln(aw))47, with aw the water activity at thermodynamic equilibrium (Supporting Information). The frequency change of the acyl chain ν(CH2) (and thus the amplitude ∆A of its difference band) correlates also linearly with ∆AL35. Therefore, under equilibrium conditions, the ν(CH2) amplitude ∆A scales with ∆(Γ·ln(aw)). Of course, an equilibrium is not generally expected for fast hydration transients and a non-linear relation is indeed found for both PL samples in the absence of trehalose (Fig. 6A). However, an almost linear plot is obtained in the presence of trehalose (Fig. 6B), demonstrating that the disaccharide permits a close to equilibrium transition during fast hydration transients. The approximated lateral tensions of 2.6 N/m and 1.0 N/m for NPLs and PPLs correspond to a molar compressibility of 16.0 and 6.4 kJ/Å2, respectively. These estimates agree with recent molecular dynamics (MD) calculations for trehalose-containing bilayers26. Remarkably, PPLs with reduced PC content exhibit a lower compressibility modulus and are thus "softer" than NPLs. In addition to the softened two-dimensional lateral expansion in PPLs, the PL extracts differ in their three-dimensional swelling which includes the contributions from inter-membraneous water. Trehalose increases the fractional gain in volume per added water molecule more


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pronouncedly in PPLs (from 0.86 % to 1.1 %) than in NPLs (from 0.89 % to 0.95%), see Supporting Information (Fig. S2A and B, respectively). In both cases trehalose causes the formation of an additional strongly H-bonded water population (Fig. S2C) and exhibits extensive hydration-dependent H-bond changes (Fig. S2D). Only in the PE rich PPLs, these H-bonds grow into a more voluminous inter-membraneous network. This supports again the crucial role of matching the intrinsic headgroup hydration properties to the trehalose-mediated water structures to produce a favourable lyotropic behavior of the carbohydrate PL composite. Our data show that the PC-reduction is a factor that enhances the effect of trehalose on the lateral and three-dimensional expansion of the native PL mixture. Furthermore, hydration transiently extends the trehalose-mediated H-bond network to the sub-headgroup. This suggests that the sugar slides deeper into the sub-headgroup region upon water uptake. This will further increase the area per lipid particularly in PPLs, which show higher trehalose affinity and molar area requirement. Therefore, PPLs should resist better to strain generated in a cellular compartment that grows during swelling. A correlation between lipid lateral expansion and the number of trehalose to C=O H-bonding has been seen in MD simulations in the fully hydrated state25and recently also in the dehydrated state48. Here, we propose a hydration-driven trehalose insertion into to the ester carbonyl region of PPLs at reduced humidity (Fig. 7). This relocation probably represents an extreme but physiologically relevant case of the trehalose repulsion/attraction mechanism as previously proposed for high and low trehalose:water ratios in hydrated PLs49. Importantly, the bilayer-stabilizing function of trehalose is clearly not only dependent on its crucial α,α-1,1 glycosidic linkage50 but also on PL headgroup composition. MD calculations have focused only on PC and show headgroup fluctuations between parallel and perpendicular orientations with respect to the fully hydrated membrane51. The parallel


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arrangement is populated upon dehydration52, 53 but not influenced by trehalose48. It remains to be elucidated whether such orientational features contribute to PE being the preferred headgroup over PC in the trehalose-dependent desiccation tolerance of the C. elegans dauer larvae.

5. CONCLUSIONS Anhydrobiotes preserve their membranes in the dry state and during quick rehydration. Understanding the physicochemical basis of this behavior can help engineering desiccation tolerance also for practical applications. We have shown that dauer larvae of the nematode C. elegans prepare for anhydrobiosis by reducing the PC headgroup content of their PLs. We think that this is a systemic response to mildly reduced humidity. It results in a higher affinity of the PLs for trehalose. Importantly, lyotropic transitions can then progress close to thermodynamic equilibrium within seconds and with a larger gain in acyl chain free volume as shown schematically in Fig. 7. Hence, trehalose synthesis and PC-reduction act synergistically in promoting desiccation tolerance of cell membranes. Many molecular strategies are shared among anhydrobiotes from different taxa19,

54

. Trehalose is not the only sugar associated with

desiccation tolerance. Plants, for instance, synthesize sucrose as an osmoprotectant55. Coupling hydration transients at biomembrane surfaces efficiently to acyl chain order by matching the PL headgroup chemistry to the carbohydrate structure may be a common mechanism among anhydrobiotes.


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Figure 7: Dynamic interaction of trehalose with lipid headhroups during transient hydration at reduced humidity. Hydration of the trehalose-containing PLs leads to the insertion of the disaccharide into the headgroup/sub-headgroup interface (vertical arrows), allowing direct and water-mediated H-bonding to the ester carbonyls. The stronger water headgroup interaction in PC restricts this motion in NPLs (A), whereas efficient intercalation of the sugar occurs in the PE-dominated PPLs (B). The higher water-induced acyl chain disorder in PPLs further allows a larger gain of molecular area (horizontal arrows) upon fast hydration, leading to a “softer” (lower lateral tension) response of PPLs.


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ASSOCIATED CONTENT Supporting Information. Lipid extraction method, vesicle production, time-resolved FTIR raw data, determination of hydration-induced PL volume changes, derivation of membrane compressibility. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author * Karim Fahmy, Inst. Resource Ecology, Helmholtz-Zentrum Dresden-Rossendorf, PF 510119, D-01314 Dresden, Germany, e-mail: [email protected] . Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Funding Sources Financial support of the Dresden-International-Graduate-School for Biomedicine and Bioengineering (DIGS-BB) to S. Abusharkh is greatfully acknowledged. ACKNOWLEDGMENT We thank Jenny Philipp at the HZDR for biochemical assistance. ABBREVIATIONS MD, Molecular Dynamics; NPLs, phospholipids of non-preconditioned C. elegans dauer larvae; PC, phosphatidyl choline; PE, phosphatidylethanolamine; PI, phosphatidylinositole; PL,


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phospholipid; PPLs, phospholipids of preconditioned C. elegans dauer larvae; PS, phosphatidylserine; RH, relative humidity. REFERENCES 1. 2. 3. 4. 5.

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Bechinger, B.; Seelig, J., Conformational-Changes of the Phosphatidylcholine Headgroup Due to Membrane Dehydration - a H-2-Nmr Study. Chem Phys Lipids 1991, 58, (1-2), 1-5. Ulrich, A. S.; Watts, A., Molecular Response of the Lipid Headgroup to Bilayer Hydration Monitored by H-2-Nmr. Biophys J 1994, 66, (5), 1441-1449. Cruz De Carvalho, R.; Bernardes Da Silva, A.; Soares, R.; Almeida, A. M.; Coelho, A. V.; Marques Da Silva, J.; Branquinho, C., Differential proteomics of dehydration and rehydration in bryophytes: Evidence towards a common desiccation tolerance mechanism. Plant, Cell Environ. 2014, 37, 1499–1515. Martinelli, T., In situ localization of glucose and sucrose in dehydrating leaves of Sporobolus stapfianus. J Plant Physiol 2008, 165, (6), 580-587.


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The Role of Phospholipid Headgroup Composition and Trehalose in

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