Biochimie (1991) 73, 1311-1316 © SociEt6 franqaise de biochimie et biologie mol6culaire / Elsevier, Paris

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Influence of p h o s p h o l i p i d u n s a t u r a t i o n on the cholesterol d i s t r i b u t i o n in m e m b r a n e s M Pasenkiewicz-Giepda 1,3, WK Subczynskil,3*, A Kusumi 2,3 *Biophysics Department, Institute of Molecular Biology, Jagiellonian University, 31-120 Krakow, Poland; 2Department of Pure and Applied Sciences, College of Arts and Sciences The University of Tokyo, Meguro-ku, Tokyo 153, Japan; 3National Biomedical ESR Center, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, Wisconsin 53226. USA (Received 24 May 1991; accepted 20 August 1991)

Summary - - Over the last half decade, we have studied saturated and unsaturated phosphatidylcholine (PC)-cholesterol membranes, with special attention paid to fluid-phase immiscibility in cis-unsaturated PC-cholesterol membranes. The investigations were carried out with fatty acid and sterol analogue spin labels for which reorientational diffusion of the nitroxide was measured using conventional ESR technique. We also used saturation recovery ESR technique where dual probes were utilized. Bimolecular collision rates between a membrane-soluble square-planar copper complex,3-ethoxy-2-oxobutyraldehyde bis(N4,N4-dimethylthiosemicarbazonato)copper(II) (CuKTMS2) and one of several nitroxide radical lipid-type spin labels were determined by measuring the nitroxide spin-lattice relaxation time (TO. The results obtained in all these studies can be explained if the following model is assumed: 1) at physiological temperatures, fluid-phase micro-immiscibility takes place in cis-unsaturated PC-cholesterol membranes, which induces cholesterol-rich domains in the membrane due to the steric nonconformability between the rigid fused-ring structure of cholesterol and the 30 ° bend at the ci~ double bond of the alkyl chains of unsaturated PC. 2) The cholesterol-rich domains are small and/or of short lifetime (10 - 9 S tO < 10- .7 s). Our results also suggest that the extra space that is available for conformational disorder and accommodation of small molecules is created in the central part of the bilayer by intercalation of cholesterol in cis-unsaturated PC membrane due to the mismatch in the hydrophobic length and nonconformability between cis-unsaturated PC alkyl chains and the bulky tetracyclic ring of cholesterol. phospholipid unsaturation / cholesterol / phase separation / model membrane

Introduction In recent years, the d o m a i n structure o f cellular p l a s m a m e m b r a n e s has b e e n e x t e n s i v e l y studied [1]. The m o l e c u l a r m e c h a n i s m s for f o r m a t i o n o f various specialized d o m a i n s in the p l a s m a m e m b r a n e s are o f particular interest b e c a u s e o f their functional significance [2]. S i n c e the p l a s m a m e m b r a n e s o f eukaryotic cells c o n t a i n various a m o u n t s o f cholesterol and unsaturated p h o s p h o l i p i d s , fluid-phase *Correspondence and reprints (to the American address) Abbreviations: PC, phosphatidylcholine; DMPC, L-0c-dimyristoylphosphatidylcholine; DSPC, L-0t-distearoylphosphatidylcholine; DOPC, L-o~-dioleoylphosphatidylcholine; POPC, L-Otpalmitoyl-2-oleoylphosphatidylcholine; EYPC, egg yolk phosphatidylcholine; 5-SASL, 5-doxyl stearic acid spin label; 16-SASL, 16-doxyl stearic acid spin label; Tempo, 2,2,6,6tetramethy l-piperidine-N-oxyl; T-PC, tempocholine diplamitoylphosphatidic acid ester; CSL, cholestance spin label; ASL, androstane spin label; CuKTMS2, 3-ethoxy-2-oxobutyraldehyde bis(N4,Na-dimethylthiosemicarbazonato)copper(II); TPX, methylpentene polymer; T~, spin-lattice relaxation time

separation o f cholesterol-rich d o m a i n s m a y play an i m p o r t a n t role in f o r m a t i o n o f these specialized d o m a i n s and structures in the m e m b r a n e s [3-5]. For r e v i e w s on cholesterol effects on m e m b r a n e s see

[6-1o]. M a t e r i a l s and m e t h o d s Object of investigation The membranes used in these studies were multilamellar dispersions of phospholipids (liposomes) with and without cholesterol.

Conventional ESR In spin-label studies of membrane structure and dynamics, two classes of labels have been used: spin label lipid analogues, phospholipid spin labels and stearic acid spin labels (SASL) to study alkyl chain motion and spin-labeled sterol analogues; cholestane spin label (CSL) and androstane spin label (ASL) to study cholesterol motion. Due to the large intramolecular mobility of SASL (trans-gauch isomerization of alkyl chains),

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the spin label reports on the motion at the site of doxyl group attachment to the alkyl chains, whereas CSL and ASL, which have a fairly rigid structure, report on motion of the molecule as a whole. "h must be remembered that the experiments carded out with the probe molecules necessitate due caution in interpretation of the results and that the labeled molecules cannot be expected to mimic all properties of phospholipid alkyl chains and cholesterol. Nevertheless, the interaction of CSL, ASL or SASL with phospholipid and cholesterol should, to a certain degree, approximate cholesterol-PC and cholesterol-cholesterol interactions in the membrane because of the overall similarity of the molecular structure and similarity of the phase behavior of membranes with and without spin labels [ 11-13]. From experimental ESR spectra of SASL, the reorientational correlation time of the nitroxide attached to the lipid as well as apparent order parameter of SASL in the membrane above the main phase transition temperature can be immediately obtained. Obtaining dynamical information from ESR spectra of CSL and ASL needs computer simulations. In our analysis, we treated CSL and ASL motion in the membrane as Brownian rotational diffusion of a rigid rod within the confines of a cone imposed by the membrane environment. It allowed us to obtain the wobbling (rotational diffusion about the perpendicular axis of the spin label) diffusion constant (D,) of the long axis of CSL and ASL, its activation energy and the cone angle (0 c) of the confines for various membranes in the liquid-crystalline phase. These experiments suggested that in PC-cholesterol membranes, cholesterol-rich domains may be formed.

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Saturation recovery ESR In order to gain more knowledge about the domain structure and dynamics of PC-cholesterol membranes, we used an observable sensitive to longer time-scale processes than the reorientational diffusion of the lipid-type spin labels. Instead of the latter, translational diffusion of a small membrane-soluble copper complex, CuKTSM 2 (Mw = 393.5, 8 x 8/~ square plane) was indirectly examined. Since CuKTSM2 is paramagnetic, has a short spin-lattice relaxation time (T= < 10°8 s) and is hydrophobic, it acts as an effective relaxing agent for the nitroxide in the membrane 1141. llae transiationaa diffusion of CuKTSM 2 in the membrane was estimated from the rate of bimolecular collision between CuKTSM2 and the nitroxide radical on the basis of T='s measured in the presence and absence of CuKTSM 2. Since the T~ of spin label (10 -6 - 10 -5 s) is much longer than its reorientational correlation time (10 -i° - 1009 s), membrane dynamics in longer time-space scales can be observed, and thereby, it is possible to obtain more information about the size and lifetime of lipid domains formed in the membrane. We have measured CuKTSM2 transport (= diffusion) at various "depths" in the membrane using tempocholine phosphatidic acid ester (T-PC) to probe the head-group region, 5-doxyl stearic acid spin label (5-SASL) to probe the hydrocarbon region near the membrane surface, and 16-do×yl stearic acid spin label (16-SASL) to probe the central region of the membrane, which together enable us to study three-dimensional organization of the membrane. We also used steroid-type spin labels, CSL and ASL. They are likely to partition into the cholesterol-rich domains, with the nitroxide radical of CSL in the membrane surface region and that of ASL in the central part of the bilayer. Molecular structures of spin labels and CuKTSM2 are shown in figure 1. Utilization of all these labels was important because we were interested in the mismatch between the fused-ring backbone of cholesterol and PC alkyl chains both in length and conformation.

~s~ CuKTSM2 Fig 1. Structures of C u K T S M 2, T-PC, 5-SASL, 16-SASL, C S L and ASL.

Necessity of deoxygenation Presence of oxygen in the ESR sample can lead to an increase in the linewidth of the conventional ESR spectra [15] and shortening of the spin-lattice relaxation time of spin labels in saturation recovery experiments [16]. To overcome that problem, all samples were deoxygenated by placement in a gas-permeable capillary made from a methylpentene polymer called TPX I17]. This plastic is permeable to oxygen and nitrogen, and is substantially impermeable to water. Samples were equilibrated in the spectrometer's resonators with nitrogen gas used for temperature control. Discussion

Microimmiscibility of phosphatidylcholine-cholesterol membranes We explained our studies on the rotational diffusion of C S L and A S L , cholesterol-analog spin labels and the

Microimmiscibility of PC-cholesterol membranes motional freedom of SASL, lipid-analogue spin labels in a group of papers [ 13, 18-20]. From comparison of the results for both groups of spin labels in saturated and unsaturated PC membranes, the following three key observations were made: 1), in cis-unsaturated membranes (DOPC, EYPC) the cholesterol effect is much greater on ASL and CSL than on SASL; 2) the cholesterol effects of ASL (CSL) motion are less in cis-unsaturated PC membranes than in saturated PC membranes. An increase in cholesterol mol fraction from 0 to 30%, decreases Dw by a factor of about 2.5 and 0c by a factor of about 1.3 in DOPC membranes as compared with decreases of a factor of 9-15 for Dw and 2 for 0c in DMPC membranes; 3) in saturated PC membranes, both ASL (CSL) and SASL are strongly influenced by the presence of cholesterol. To explain these results, we propose that the key feature of cis-unsaturated PC-cholesterol interaction is the considerable nonconformability of molecular shapes of cis-unsaturated PC and cholesterol in the membrane. The cholesterol backbone is the rigid planar transfused tetracyclic ring structure of a steroid, which reaches up to 9 or 10 carbons of extended alkyl chain and to a somewhat deeper level in the hydrophobic region of the membrane in the liquidcrystalline phase [10, 12, 21]. As is shown in figure 2A, the rigid skeleton of cholesterol (and ASL and CSL) and the bend structure of DOPC may not conform with each other when they are in direct contact in the membrane. Although the effect of this sharp bend is somewhat reduced by the simultaneous occurrence of kinks in the oleoyl chain [12, 21], the cis-double bond would certainly creaie serious problems in the packing of cholesterol and oleoyl chains in the membrane. This nonconformability leads to two major differences in the PC-cholesterol interaction between saturated and unsaturated PC-cholesterol membranes: A) cholesterol molecules tend to be excluded from DOPCdomains and segregated out as shown in figure 2D. Thus, it is likely that the cholesterol mol fraction in the cholesterol-rich phase is higher in DOPC (cis-unsaturated PC membranes) than in saturated PC membranes. (The formation of cholesterolrich domains (or cholesterol o!igomers) in DOPCcholesterol membranes is supposedly driven by the differences in enthalpy of PC-cholesterol, PC-PC, and cholesterol-cholesterol interactions. The PC-cholesterol interaction is the weakest. The balance between these interaction enthalpies and mixing entropy determines the equilibrium between the two phases. A similar delicate balance between interaction enthalpies and mixing entropy in membranes was found in the molecular association of rhodopsin molecules in rod outer segment membranes and in reconstituted membranes [22].) B) the ordering effect of cholesterol

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0 Fig 2. Schematic drawings oh,,,,~,o th,~ i n t e r ~ o t ; . o n of cholesterol with saturated and unsaturated PC molecules in the membrane. A. A static view of the nonconformability of the rigid structure of cholesterol (rectangles) and the rigid bend at the C9-C10 cis-double bond in an unsaturated DOPC alkyl chain (bent rods), which would induce vacant pockets (packing defects) in the membrane. Snapshot drawings of DMPC-cholesterol (B); DSPC-cholesterol (C) and DOPC-cholesterol (D) membranes with CuKTSM v The drawings on the right show the top view models for saturated PC-cholesterol (top) and for unsaturated PCcholesterol (bottom) membranes. The open circles represent the alkyl chains of the phospholipids, and the solid structures indicate cholesterol molecules (after Martin and Yeagle [31 ]). o|t,,..,

,,

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~

. . . . . . . . . . . . . . .

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(a contact interaction of cholesterol with saturated PC, which promotes trans conformation in PC alkyl chains) is much weaker in cis-unsaturated PC membranes (DOPC) compared with that in saturated PC membranes. (In these arguments, only a shortrange effect of cholesterol is considered; ie, choles-

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terol affects the motion of lipids in direct contact, and it minimally influences the lipid in the second annular ring around the cholesterol molecule in the liquidcrystalline phase. In contrast, the long-range effect of cholesterol was observed below the phase transition temperature [23].) Size and lifetime o f cholesterol-rich domains

The most important observation in the study of the rotational diffusion of spin labels that showed anomaly in mixing of unsaturated PC and cholesterol is that, in unsaturated PC-cholesterol membranes, incorporation of cholesterol decreases mobilities of cholesterol-analogue spin labels, but causes little effect on PC analogue spin labels. These results indicate that cholesterol-rich and unsaturated PC-rich domains coexist in the membrane, the former being detected with cholesterol-type spin labels and the latter with phospholipid-type spin labels, and that the reorientational mobility of cholesterol is more restricted in cholesterol-rich domains. Unfortunately, the studies provide little information about the stability and size of cholesterol-rich domains formed in unsaturated PC-cholesterol membranes. To gain knowledge about the size and lifetime of the cholesterol-rich domain, we performed dual-probe saturation recovery experiments [24], and obtained the following results. In the liquid-crystalline phase of saturated PC membranes, incorporation of cholesterol decreases the collision rate between the nitroxide and CuKTSM 2 at all dephts in the membrane, and the effect of cholesterol is smallest in u,~ ,uuua~ of the bilayer. In cis-unsaturated r'c membranes, virtually no effect of cholesterol on the collision between the nitroxide and CuKTSM2 was observed, either with cholesterol-type or phospholipid-type spin labels. This result is in clear contrast with our orevious observation summarized above. ff we assume, on the basis of our above observation, that the product of the local concentration and the local translational diffusion coefficient of CuKTSM2 is smaller in the cholesterol-rich domains than in cis-unsaturated PC-rich domains, these contrasting results suggest that the lifetime of the cholesterol-rich domains (or cholesterol oligomers) is shorter than 10-6 s (T~ of CSL and ASL in PCcholesterol membrane in the liquid-crystalline phase), and/or the size of the domain is so small that all molecules in the domain are in contact with the bulk phase (see the model displayed in fig 2B, C and D). Since the effect of cholesterol on the reorientational mobility of the cholesterol-type spin labels has been detected, the lifetime of the spin label in the cholesterol-rich domain should be longer than 10 -9 s. In conclusion [ 19, 20, 24, 26, 27], we propose a model in ...! ~ ! I

which the fluid-phase immiscibility is prevalent in cisunsaturated PC-cholesterol membranes, but where cholesterol-rich (cholesterol-oligomeric) domains are small (several lipids) and/or of short lifetime ( 1 0 --9 - 10-7 S). Relevant studies have been made by using 1-palmitoyl-2-oleoyl PC (POPC) membranes [28-30]. Shin and Freed [28] and Shin et al [29] reported a cholesterol-induced increase in both order parameter and wobbling rotation diffusion coefficient (Rp~rp) of 16doxyl-PC spin label. They concluded that acyl chain unsaturation leads to poorer mixing of cholesterol in POPC membranes in the liquid-crystalline phase. Hyslop et al [30] examined POPC membranes with a fluorescent analogue of cholesterol and reported that 'sterol molecules in the membrane matrix were not associated to any greater degree'. This conclusion is not necessarily different from that by Freed's group; however, Hyslop et al proposed a model for the sterol/PC phase at l:l mol/mol as each sterol having four PC molecules as nearest neighbors and sterol molecules being 1.06 nm away from each other, which is in marked contrast with the conclusion by Freed's group. Our results indicate that the structures of DOPC-cholesterol and EYPC-cholesterol membranes are different from the model proposed for POPC-cholesterol membranes by Hyslop et al. Since we have not studied POPC membranes, and, in addition, the phase diagram of POPC-cholesterol membrane has not been known, we would refrain from making further comments on POPC-cholesterol membranes. Free volume created in the central part 03"the bilayer in phosphatidylcholine-cholesterol membranes

In saturated PC membranes above the phase transition temperature of the host lipids, the presence of cholesterol decreases the collision rate between the nitroxide and CuKTSM2 at any depth in the membrane. However, the effect of cholesterol in the center of the bilayer is much larger in DMPC membranes than in DSPC membranes [24]. These results can be explained by the mismatch in the hydrophobic length between cholesterol (the bulky tetracyclic ring, in particular) and PC in DSPCcholesterol membranes, which create more space in the central region of the membrane. The cholesterol molecule contains three well-distinguished regions: small polar hydroxyl group, rigid plate-like steroid ring system, and an iso-octyl chain tail. When cholesterol intercalates into the membrane, its polar hydroxyl group is positioned near the middle of the glycerol backbone region of the PC molecule. It separates the PC head groups and decreases the interaction between them. Water molecules come into the

Microimmiscibility of PC-cholesterol membranes

free space between the separated head groups [13]. The rigid steroid ring intercalates between hydrocarbon chains, interacts with them, and promotes the trans conformation in PC alkyl chains (if there is no double bond) from the membrane surface to a depth of about the 7th and 10th carbon [25]. The rest of the PC alkyl-chain tails stay flexible. The presence of cholesterol increases the free space in the central part of the bilayer because the cross-section of the steroid ring is larger than that of its hydrocarbon tail. This 'fluid' region, created in the center of the bilayer by the presence of cholesterol, is much wider in DSPC membranes than in DMPC membranes, thereby accommodating CuKTSM2 molecules more easily in DSPC membranes (fig 2B and C). In cis-unsaturated PC membranes, cholesterol has almost no effect on the collision rate between the nitroxide and CuKTSM2 at any depth in the membrane [24]. Structural nonconformity between the rigid tetracyclic ring of cholesterol and the rigid bend at the C 9 - C I 0 cis-double bond creates even more space available for conformational disorder and diffusion of small molecules in the bilayer (fig 2D) than the simple mismatch in length, as in the case of DSPC-cholesterol membranes. Conclusions In the studies of cholesterol-containing membranes, we think it is important to pay attention to the following three factors: 1), the conformational mismatch between the rigid ring structure of cholesterol and the bend at the cis-doubie bonds in unsaturated aikyi chains; 2), the mismatch in hydrophobic length between cholesterol and PC alkyl chains; and 3), the time-space scales of the lipid domains (cholesterol oligomers). Since the plasma membranes of eukaryotic cells contain various amounts of cholesterol and a variety of phosphelipid alkyl chains, the delicate balance among PC-PC, PC-cholesterol, and cholesterol-cholesterol interactions may play important roles in formation of specialized domain structures in the plasma membranes [ 1-5, 22].

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Acknowledgment This work was supported in part by US Public Health Service Grants GM22923, GM27665, and RR01008; and grants-in-aid from the Japanese Ministry of Education, Science and Culture.

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