Langmuir 2008, 24, 8443-8447
8443
Evidence That Phosphatidylinositol Promotes Curved Membrane Interfaces Xavier Mulet,*,†,§ Richard H. Templer,† Rudiger Woscholski,‡ and Oscar Ces† The Chemical Biology Centre, Department of Chemistry, and DiVision of Cell and Molecular Biology, Imperial College London, South Kensington Campus, London SW7 2AZ, U.K. ReceiVed April 9, 2008. ReVised Manuscript ReceiVed June 13, 2008 We have identified the phase behavior of phosphoinositol (PI) lipid extracts from bovine liver and wheat in dioleoylphosphatidylcholine (DOPC) model membranes under physiological conditions (pH 7.4) and show, for the first time, that the physicochemical properties of phosphatidylinositol lipids are capable of driving changes in membrane curvature. Ten mole percent phosphoinositol (PI) extract in DOPC is sufficient to induce the formation of the inverse hexagonal (HII) and inverse micellar cubic (Fd3m) phases at 37 °C. The phase behavior of several hydrated lipid samples was analyzed using small-angle X-ray scattering, and their lattice parameters were calculated.
Introduction The inositol phospholipid family is involved in a range of cellular processes where the fact that the headgroup can be reversibly phosphorylated into one of seven possible phosphoinositol moieties (Supporting Information Figure S1) plays the central role. Altering the headgroup phosphorylation of inositol phospholipids in cells is known to drive signal transduction, the regulation of membrane traffic, the cytoskeleton, nuclear events, and the permeability and transport functions of membranes.1 In turn, phosphoinositide metabolism is tightly regulated by cellular signaling cascades and compartmentalization, which creates a dynamic flux (steady state) of the phosphorylated inositol lipids as well as their major precursor phosphatidylinositol (PI). Whereas the former lipids are present in small amounts, the latter is by far the most abundant inositol lipid in eukaryotic cells. There is ample evidence that phosphoinositides present in membrane compartments are participating in biological membrane fusion and fission processes such as endocytosis and secretion,2 which are ultimately changing the curvature of their corresponding membrane compartments.3–6 We hypothesize that these biological functions are sensitive to the curvature elastic state of the membrane and that this in turn is being regulated by phosphorylation and dephosphorylation of the inositol ring. This links the chemical activity of phosphatases and kinases on inositol headgroups to gross alterations in membrane structure and their mechanical properties. Given the degree of compartmentalization of lipids from the phosphatidylinositol family and their presence around regions where membrane structure is being altered, it is * Corresponding author. E-mail:
[email protected]. † The Chemical Biology Centre and Department of Chemistry. ‡ The Chemical Biology Centre and Division of Cell and Molecular Biology. § Present address: CSIRO Molecular Health and Technologies, Bag 10, Clayton South VIC 3169, Australia.
(1) Di Paolo, G.; De Camilli, P. Nature 2006, 443, 651–7. (2) Ford, M. G.; Pearse, B. M.; Higgins, M. K.; Vallis, Y.; Owen, D. J.; Gibson, A.; Hopkins, C. R.; Evans, P. R.; McMahon, H. T. Science 2001, 291, 1051–5. (3) Botelho, R. J.; Teruel, M.; Dierckman, R.; Anderson, R.; Wells, A.; York, J. D.; Meyer, T.; Grinstein, S. J. Cell Biol. 2000, 151, 1353–68. (4) Hill, M. M.; Feng, J.; Hemmings, B. A. Curr. Biol. 2002, 12, 1251–5. (5) DeWolf, C.; Leporatti, S.; Kirsch, C.; Klinger, R.; Brezesinski, G. Chemistry and Physics of Lipids 1999, 97, 129–138. (6) Wang, Y. J.; Wang, J.; Sun, H. Q.; Martinez, M.; Sun, Y. X.; Macia, E.; Kirchhausen, T.; Albanesi, J. P.; Roth, M. G.; Yin, H. L. Cell 2003, 114, 299–310.
clear that the phosphoinositides are at the right place at the right time for membrane transformation processes. Studies of the phase behavior of phosphoinositol lipids, in particular, with respect to their curvature-inducing properties, have been limited.7–13 Experimental studies of the curvature elastic effect of PI lipids on model and cell membrane systems have not matched the pace of discovery with respect to their role in regulating key cellular processes. Recently, the role of curvature in membranes in vivo has been reassessed in the light of increasing evidence demonstrating that regions of differing membrane curvature may specify the location of effector proteins such as actin filaments or scaffold proteins.14 Membrane curvature sensing, generating, and stabilizing molecules play an important role in maintaining these regions of distinct membrane morphology and function.15–18 In addition, the lipids themselves are capable of promoting the formation of curved interfaces19 with the mixture of bilayer to nonbilayer lipids also being responsible for regulating the function and activity of numerous membraneassociated proteins by compositional control of the bending stress stored in the membrane.14,20 Consequently, one could assume that lipid phase behavior may have an influence on membrane curvature, which in turn affects morphology and ultimately its function. The lamellar structure (Figure 1a) provides the basic building block of all biological membranes, but nonlamellar phases also (7) Hammond, K.; Reboiras, M. D.; Lyle, I. G.; Jones, M. N. Biochim. Biophys. Acta 1984, 774, 19–25. (8) Litman, B. J. Biochemistry 1973, 12, 2545–54. (9) Low, M. G.; Zilversmit, D. B. Biochim. Biophys. Acta 1980, 596, 223–34. (10) Ohki, K.; Sekiya, T.; Yamauchi, T.; Nozawa, Y. Biochim. Biophys. Acta 1981, 644(2), 165–74. (11) Tarahovsky, Y. S.; Koynova, R.; MacDonald, R. C. Biophys. J. 2004, 87, 1054–64. (12) Ter-Minassian-Saraga, L.; Madelmont, G. FEBS Lett. 1982, 137, 137– 40. (13) Waninge, R.; Nylander, T.; Paulsson, M.; Bergenstahl, B. Chem Phys Lipids 2003, 125, 59–68. (14) Ces, O.; Mulet, X. Signal Transduction 2006, 6, 112–132. (15) Bigay, J.; Gounon, P.; Robineau, S.; Antonny, B. Nature 2003, 426, 563–6. (16) Ford, M. G.; Mills, I. G.; Peter, B. J.; Vallis, Y.; Praefcke, G. J.; Evans, P. R.; McMahon, H. T. Nature 2002, 419, 361–6. (17) Gallop, J. L.; McMahon, H. T. Biochem Soc Symp 2005, 223–31. (18) Hubner, S.; Couvillon, A. D.; Kas, J. A.; Bankaitis, V. A.; Vegners, R.; Carpenter, C. L.; Janmey, P. A. Eur. J. Biochem. 1998, 258, 846–53. (19) Zimmerberg, J.; Kozlov, M. M. Nat ReV Mol Cell Biol 2005, 7, 9–19. (20) van den Brink-van der Laan, E.; Killian, J. A.; de Kruijff, B. Biochim. Biophys. Acta 2004, 1666, 275–88.
10.1021/la801114n CCC: $40.75 2008 American Chemical Society Published on Web 07/23/2008
8444 Langmuir, Vol. 24, No. 16, 2008
Letters
Figure 1. Schematic of (a) the fluid lamellar phase, (b) the inverse hexagonal phase, and (c) the micellar cubic (Fd3m) phase with individual micelle packing shown. The packing of the two types of inverse micelles is shown for each site. In this instance, the size of the polyhedra has been reduced for the sake of clarity, giving the appearance that they are not touching.
play key biological roles. Structural elements of both the hexagonal (Figure 1b) and inverse micellar cubic phase (Figure 1c) are postulated to act as transient intermediates in biological phenomena that require topological rearrangements of lipid bilayers such as membrane fusion/fission21,22 and the trans-bilayer transport of lipids and polar solutes.23 The inverted cubic phases include the inverse micellar cubic phases24 (Figure 1c) that are made up of a cubic packing of inverse micelles. Examples of the inverse micellar cubic phase that have been reported to date all have the face-centered Fd3m crystal structure. These include the observation of an Fd3m phase in a lipid extract from Pseudomonas fluorescens25 and hydrated DOPC/diolein (DOG);26 further examples are summarized by Seddon et al.24 In this letter, we report the observation and characterization of an inverted hexagonal (HII) and an Fd3m phase in hydrated DOPC/PI extract mixtures under physiological conditions. The phase behavior of the systems, as described in the Supporting Information, was (21) Epand, R. M. Biochim. Biophys. Acta 1998, 1376, 353–68. (22) Siegel, D. P.; Epand, R. M. Biophys. J. 1997, 73, 3089–111. (23) De Kruijff, B.; Cullis, P. R.; Verkleij, A. J.; Hope, M. J.; Van Echteld, C. J. A.; Taraschi, T. F. Lipid polymorphism and membrane function. In The Enzymes of Biolo-gical Membranes, Martonosi, A. N., Ed. Plenum Press: New York, 1985; Vol. 1, pp 131-204. (24) Seddon, J. M.; Robins, J.; Gulik-Krzywicki, T.; Delacroix, H. Phys. Chem. Chem. Phys. 2000, 2, 4485–4493. (25) Mariani, P.; Rivas, E.; Luzzati, V.; Delacroix, H. Biochemistry 1990, 29, 6799–810. (26) Takahashi, H.; Hatta, I.; Quinn, P. J. Biophys. J. 1996, 70, 1407–11.
elucidated using small-angle X-ray scattering (SAXS). Further details of lipid and buffer composition as well as methodology are provided in the Supporting Information section.
Results and Discussion Figure 2a-c shows the small-angle X-ray scattering patterns obtained for a 10/90 mol % bovine liver PI/DOPC sample (30 wt % buffer solution, incubated at 37 °C) at 2, 174, and 246 h, respectively. Figure 3a-c show the corresponding SAXS data sequence acquired for an 11:89 mol % wheat germ PI/DOPC sample (40 wt % buffer solution) at 2, 170, and 330 h, respectively. These are not excess water systems because the PI/DOPC system will form swollen fluid lamellar systems with increasing hydration up to the limits of detector resolution (data not shown). To ensure that no lipid degradation occurred in this time period, thin layer chromatography (TLC), as described in Supporting Information, was run concurrently with the SAXS measurements. These experiments showed no detectable degradation products. The lamellar phase gives Bragg peaks in the ratio of 1:2:3:4, the HII phase in the ratio of1:3:2:7, and the Fd3m phase in the ratio of3:8:11:12:16 with characteristic intensity distributions (Figure 2).27 Both PI systems undergo similar phase transformations as a function of time following sample (27) Huang, Z.; Seddon, J. M.; Templer, R. H. Chemistry and Physics of Lipids 1996, 82, 53–61.
Letters
Langmuir, Vol. 24, No. 16, 2008 8445
Figure 2. Small-angle diffraction patterns for a 10:90 mol % bovine liver PI/DOPC 30 wt % buffer sample (A) 2, (B) 174, and (C) 246 h after sample homogenization. The SAXS patterns are characteristic of fluid lamellar (LR), inverse hexagonal (HII) and inverse micellar cubic phases, respectively (Fd3m). Integration and fitted peaks are shown in Supporting Information, Figure S2.
Figure 3. Small-angle diffraction patterns for a 11:89 mol % wheat germ liver PI/DOPC 40 wt % buffer sample (A) 2, (B) 170, and (C) 330 h after sample homogenization. The SAXS patterns are characteristic of fluid lamellar (LR), inverse hexagonal (HII), and inverse micellar cubic phases, respectively (Fd3m). Integration and fitted peaks are shown in Supporting Information, Figure S3.
homogenizationsfrom a fluid lamellar to an inverse hexagonal and finally to a micellar cubic phase (Fd3m). Figures 2a and 3a show that after 2 h a pattern characteristic of a lamellar phase is observed: two orders of diffraction are visible in a ratio of 1:2. The bovine liver PI/DOPC system has an interlamellar repeat distance, d, of 52.9 ( 0.1 Å, and the wheat germ PI/DOPC system has d ) 59.7 ( 0.4 Å. These values compare with a value of 55.6 Å reported for a EDOPC/PI (1:1 molar ratio) system.28 With time, these lamellar phases transform from the fluid lamellar phase to an inverse hexagonal phase (Figures 2b and 3b) with characteristic low-angle peaks occurring in the ratio of 1:3:2 with lattice parameters of 62.7 ( 0.9 Å for the bovine liver PI/DOPC system and 71.2 ( 0.9 Å for the wheat germ PI/DOPC assembly. After approximately 10 days, a final transition is seen. Initially this becomes apparent from the observation of a marked decrease in the sharpness of the small angle diffraction peaks of the inverse hexagonal phase as they are replaced by a single broad peak centered around 150 to 160 Å (data not shown) for both the wheat germ and bovine liver PI systems. Typically, after a further 24 to 48 h, this broad scatter resolves itself into a set of diffraction peaks (Figures 2c and 3c) occurring in the ratio of 3: 8: 11: 12: 16. Under polarizing microscopy this phase is isotropic and viscous, characteristic of a cubic lyotropic phase (data not shown). The corresponding indexed radial intensity plots for Figures 2 and 3 are shown in Supporting Information, Figures S2 and S3. Supporting Information, Figures S2 and S3 show the indexing of the SAXS pattern for this cubic phase. The slope gives a lattice parameter of 161.0 ( 2.4 Å for the bovine liver PI/DOPC system and 167.1 ( 3.9 Å for the wheat germ PI/DOPC system. (28) Tarahovsky, Y. S.; Koynova, R.; MacDonald, R. C. Biophys. J. 2004, 87, 1054–64.
The lattice parameter is close to that of 150 ( 1 Å at 26 ( 1 wt % water recorded in a DOPE-DOG mixture.29 The kinetic sequence observed in which the interface tends toward increasing interfacial curvature also supports the identification of an inverse micellar cubic phase. At least five Bragg reflections can be seen for the micellar phase (Supporting Information, Figures S2 and S3), and these index as the 111, 220, 311, 222, 400, and 331 reflections of a cubic phase of cubic aspect 15.27 This sequencing of Bragg peaks means that only one of two cubic space groups can be assigned to this phase, that of the Fd3m (Q227) or Fd3 (Q203). In combination with the peak intensity distribution, lattice parameter, and polarizing microscopy and the work of Luzzati et al.29 and Seddon et al.,30 this suggests that the Fd3m assignment is the correct one for this phase. The structure of the proposed Fd3m phase24,31 shown in Figure 1c, consists of the packing of discrete inverse micellar aggregates. These micellar aggregates, which are quasi-spherical, consist of two different sizes, and the packing motif of this phase is such that eight of the larger inverse micelles are packed among six of the smaller. The majority of previously reported examples of Fd3m phases have contained at least two different components, varying in both their degree of amphiphilicity and desire for curvature. For example, adding diacylglycerols (DAGs) and fatty acids (FAs) (which on their own form sparsely hydrated inverse micelles) to bichained PCs will drive the system toward greater interfacial curvature and at high enough composition into the inverse micellar (29) Luzzati, V.; Vargas, R.; Gulik, A.; Mariani, P.; Seddon, J. M.; Rivas, E. Biochemistry 1992, 31, 279–85. (30) Seddon, J. M. Biochemistry 1990, 29, 7997–8002. (31) Seddon, J. M.; Bartle, E. A.; Mingins, J. J. Phys.: Condens. Matter 1990, 2, SA285-SA290.
8446 Langmuir, Vol. 24, No. 16, 2008
Letters
Figure 4. Diagram illustrating how the imbalance of the forces exerted in the headgroup and chain regions leads to a net monolayer torque tension being applied to the monolayer, the direction of which will depend on which set of forces is greatest. (a) In this instance, the monolayers wish to curve toward the water interface, but such a situation is problematic because of the back-to-back arrangement of the monolayers. (b) Neither half of the sheet is able to attain the desired curvature because they are coupled by the hydrophobic effect and the formation of the resultant voids shown proves too energetically unfavorable. This leads to stored curvature elastic stress in the bilayer.
cubic phase. However, DAG and FA drive the transition from a flat membrane interface to a spherical interface not just because they are weakly hydrophilic but also because they are able to hydrogen bond to PCs at both the carbonyl ester and phosphate groups.31 This hydrogen bonding dehydrates the interface and draws the lipid headgroups together, driving the interface to bend ever more strongly toward the water. The headgroup structure of PI may account for its ability to promote curved interfaces: there are five potential hydrogen bonding sites on the inositol ring compared to only one hydrogen bonding site for both FA and DAG. We hypothesize that direct hydrogen bonding to neighboring PC headgroups, which reduces headgroup repulsions, pulling lipids together with a resulting increase in repulsive pressure to the chain region, may drive the formation of the curved interfaces. The rich headgroup functionality and capability of hydrogen bonding between neighboring headgroups in phosphoinositol lipids has already been implicated in the formation of pHdependent domains in phosphatidylinositol polyphosphate/ phosphatidylcholine mixed vesicles.32 There are also various chemically specific recognition processes in which a lipid is specifically bound by a protein, including that of the inositol phospholipid signaling pathway.33–35 However, there is now a good deal of evidence to suggest that the stresses imparted by lipids in the membrane (illustrated in Figure 4) are able to regulate the activity and function of both peripheral and integral membrane proteins.14,20 This includes the function of enzymes/peripheral membrane proteins that catalyze the synthesis or degradation of (32) Redfern, D. A.; Gericke, A. J. Lipid Res. 2005, 46, 504–15. (33) Berridge, M. J. Biochem. J. 1984, 220, 345–360. (34) Berridge, M. J.; Irvine, R. F. Nature 1984, 312, 317–319. (35) Hokin, M. R., Hokin, L. E. In Metabolism and physiological significance of lipids, Dawson, R. M. C.; Rhodes, D. N., Eds. John Wiley: London, 1964; pp 423-434.
structural components of membranes, transport proteins, and trans-membrane mechanosensitive channels. Because adding PI to DOPC at low mole fractions, albeit under nonexcess water conditions, will drive DOPC (a lipid that would otherwise form flat interfaces) into the Fd3m phase, it has the potential to increase levels of stored curvature elastic stress in membranes (illustrated in Figure 4). The fact that our measurements were not made in excess buffer demands comment. It is noteworthy that for all PC lipids a water composition of 30% (or indeed less) will always give rise to a stable lamellar phase. The addition of very small amounts of PI lipid has therefore clearly increased the propensity for interfacial curvature toward water in a rather dramatic way. The time scales for the transition to the inverse micellar cubic phase are consistent with the time scales for stabilization of this structure seen in other systems, but the observation of a lamellar phase at short times is not. Exactly what processes lead to a metastable lamellar phase remain to be investigated, but we do know that its conversion into a micellar cubic phase is not coupled to the degradation of the PI lipid. The water composition on the inner leaflet of the plasma membrane (where PI resides) is probably not equivalent to excess water for PI lipids in the metastable lamellar phase nor to the 30-40 wt % water that we used here. So whereas the data recorded under water stress may be potentially useful, its biological meaning should be approached with caution in the absence of data at higher water composition. Although the PI headgroup may take part in multiple hydrogen bonds under limited hydration conditions, the inositol ring may have a different orientation and interactions when fully hydrated. For instance, the headgroup could form hydrogen bonds with solvent (water) itself rather than with the phosphate groups of neighboring PC molecules.
Letters
Despite the overall low levels of PI in cells, they are disproportionally important in biology. The observations presented here demonstrate that the phase behavior of this class of lipids warrants further detailed study, particularly given that the rich headgroup functionality of this class of lipids means that systematic modifications to the head groups (e.g., formation of phosphoinositide polyphosphates) could provide a route to biochemically altering the local stresses in membranes. Acknowledgment. This work formed part of the Ph.D. research of X.M. within the Chemical Biology Centre’s Doctoral Training
Langmuir, Vol. 24, No. 16, 2008 8447
Centre, funded by the UK Engineering and Physical Research Council (EPSRC). We also acknowledge the EPSRC for the award of Platform grant GR/S77721, which in part funded this research. Supporting Information Available: Integrated and fitted diffraction patterns as well as lipid structure. This material is available free of charge via the Internet at http://pubs.acs.org. LA801114N
