Remodeling of Ordered Membrane Domains by GPI-Anchored

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Remodeling of Ordered Membrane Domains by GPI-Anchored Intestinal Alkaline Phosphatase Marie-Ce´cile Giocondi,† Franc¸ oise Besson,‡ Patrice Dosset,† Pierre-Emmanuel Milhiet,† and Christian Le Grimellec*,† Institut National de la Sante´ et de la Recherche Me´ dicale, Unite´ 554, Montpellier, France, and UniVersite´ de Montpellier, Centre National de la Recherche Scientifique, UMR 5048, Centre de Biochimie Structurale, Montpellier, France, and ICBMS (Institut de Chimie et Biochimie Mole´ culaires et Supramole´ culaires), 43 bouleVard du 11 noVembre 1918, Villeurbanne, 69622, France, CNRS, UMR 5246, Villeurbanne, 69622, France, UniVersite´ de Lyon, Lyon, 69003, France, and UniVersite´ Lyon 1, Lyon, 69003, France ReceiVed March 27, 2007. In Final Form: May 29, 2007 Glycosylphosphatidyl-inositol (GPI)-anchored proteins preferentially localize in the most ordered regions of the cell plasma membrane. Acyl and alkyl chain composition of GPI anchors influence the association with the ordered domains. This suggests that, conversely, changes in the fluid and in the ordered domains lipid composition affect the interaction of GPI-anchored proteins with membrane microdomains. Validity of this hypothesis was examined by investigating the spontaneous insertion of the GPI-anchored intestinal alkaline phophatase (BIAP) into the solid (gel) phase domains of preformed supported membranes made of dioleoylphosphatidylcholine/dipalmitoylphosphatidylcholine (DOPC/DPPC), DOPC/sphingomyelin (DOPC/SM), and palmitoyloleoylphosphatidylcholine/SM (POPC/SM). Atomic force microscopy (AFM) showed that BIAP inserted in the gel phases of the three mixtures. However, changes in the lipid composition of membranes had a marked effect on the protein containing bilayer topography. Moreover, BIAP insertion was associated with a net transfer of phospholipids from the fluid to the gel (DOPC/DPPC) or from the gel to the fluid (POPC/SM) phases. For DOPC/SM bilayers, transfer of lipids was dependent on the homogeneity of the gel SM phase. The data strongly suggest that BIAP interacts with the most ordered lipid species present in the gel phases of phase-separated membranes. They also suggest that GPI-anchored proteins might contribute to the selection of their own microdomain environment.

Introduction Alkaline phosphatases (AP) are a class of exoplasmic membrane proteins catalyzing nonspecific hydrolysis of phosphate monoesters at alkaline pH. They belong to the family of eukaryotic glycosylphosphatidyl-inositol (GPI)-anchored proteins that play very diverse biological functions including hydrolytic enzyme activity, transmembrane signaling, and cell adhesion interaction.1,2 Interestingly, many classical membrane markers such as the AP, 5′-nucleotidase, dipeptidase, aminopeptidase P, are GPI-anchored proteins rather than transmembrane proteins. In kidney brush borders membranes, which constitute the major part of the apical pole of proximal tubule renal epithelial cells, kidney AP (KAP) and other GPI-anchored enzymes are resistant to Triton X100 solubilization whereas transmembrane enzymes are solubilized.3 This GPI-anchored protein resistance to solubilization is a function of the nonionic detergent used. In stably transfected MDCK strain II kidney cell line expressing the GPI-anchored human placental alkaline phosphatase (PLAP), the phosphatase is also recovered from the detergent-resistant membrane fractions (DRMs) which contained other GPI-anchored proteins.4 MDCK cells DRMs are enriched in sphingolipids and cholesterol (Chl), * To whom correspondence should be addressed: Nanostructures and Membrane Complexes, C.B.S., 29 rue de Navacelles, 34090 Montpellier Cedex, France. Tel: (33) 467 41 79 07. Fax: (33) 467 41 79 13. E-mail: [email protected]. † Institut National de la Sante ´ et de la Recherche Me´dicale and Universite´ de Montpellier. ‡ ICBMS and Universite ´ de Lyon. (1) Low, M. G. Biochim. Biophys. Acta 1989, 988, 427-454. (2) Ferguson, M. A. J. Cell Sci. 1999, 112, 2799-2809. (3) Hooper, N. M.; Turner, A. J. Biochem. J. 1988, 250, 865-869. (4) Brown, D. A.; Rose, J. K. Cell 1992, 68, 533-544.

with a molar ratio of glycerophospholipid:sphingolipid:Chl of 1:1:1.4 This molar ratio compares with those determined in apical plasma membrane domains of intestinal5 and renal cells6 and results in a liquid ordered state for the corresponding membrane fractions.7,8 It is not clear to what extent isolation of DRMs supports the hypothesis of a lipid-dependent in-plane heterogeneity of membrane organization. What the relationships are between DRMs and native membrane domains like membrane rafts, a subset category of liquid ordered membrane lipid domains enriched in sphingolipids and Chl working as functional platforms involved in signal transduction and protein sorting,9,10 remains a matter of debate.11-14 In fact, lipid-dependent and cytoskeletondependent,15,16 but also protein-dependent,17 membrane microdomains likely contribute to the lateral heterogeneity of biological membranes. Most of the available model systems, including membrane monolayers, large unilamellar vesicles, giant liposomes, and supported membranes, have been used to better characterize the formation of in-plane lipid-dependent microdomains and AP/ (5) Simons, K.; van Meer, G. Biochemistry 1988, 27, 6197-6202. (6) Carmel, G.; Rodrigue, F.; Carriere, S.; Le Grimellec, C. Biochim. Biophys. Acta 1985, 818, 149-157. (7) Schroeder, R.; London, E.; Brown, D. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 12130-12134. (8) Ahmed, S. N.; Brown, D. A.; London, E. Biochemistry 1997, 36, 1094410953. (9) Simons, K.; Ikonen, E. Nature 1997, 387, 569-572. (10) Pike, L. J. J. Lipid Res. 2006, 47, 1597-1598. (11) London, E.; Brown, D. A. Biochim. Biophys. Acta 2000, 1508, 182-195. (12) Mayor, S.; Riezman, H. Nat. ReV. Mol. Cell Biol. 2004, 5, 110-120. (13) Lagerholm, B. C.; Weinreb, G. E.; Jacobson, K.; Thompson, N. L. Annu. ReV. Phys. Chem. 2005, 56, 309-336. (14) Gallegos, A. M.; Storey, S. M.; Kier, A. B.; Schroeder, F.; Ball, J. M. Biochemistry 2006, 45, 12100-12116.

10.1021/la700892z CCC: $37.00 © 2007 American Chemical Society Published on Web 07/28/2007

Alkaline Phosphatase Modifies Gel Phase Domains

lipid interactions in membranes.18-23 GPI-anchored proteins have the ability to spontaneously insert into the lipid bilayer of cells and liposomes.24,25 Insertion of PLAP into liposomes strongly suggested that, in membranes where fluid (LR) and ordered lipid phases coexist, the PLAP essentially associates with the ordered acyl chains, either in the gel phase (Lβ’) in the absence of Chl or in the liquid ordered phase (Lo) in its presence,26 a situation that might differ in giant liposomes.22 A combination of atomic force microscopy (AFM) with direct addition of GPI-anchored intestinal alkaline phosphatase (BIAP) to the medium bathing dioleoylphosphatidylcholine (DOPC)/sphingomyelin (SM) and DOPC/SM/Chl supported bilayers (SLB) demonstrated the ability of BIAP to spontaneously insert into the Lβ’ and Lo phases, respectively, of supported membranes in the absence of any detergent treatment.23,27 Comparable results were reported for PLAP inserted in liposomes before SLB formation.28 It has been proposed that acyl and alkyl chain length of GPI anchors could determine raft association.29 Thus, the length and order of aliphatic chains in both the fluid and ordered phases are expected to affect the GPI-anchored protein-membrane domains interactions. DOPC is commonly used as the fluid phase phospholipid species in “rafts models” studies. Quantitatively, it is however a very minor species of biological membranes.30 Rather, plasma membrane phosphatidylcholines (PC) contain one saturated and one unsaturated, at the sn-2 position, acyl chains.30 Replacement of DOPC by palmitoyloleoylphosphatidylcholine (POPC), a major natural PC species, markedly affects the formation processes of lipid model rafts.31 In this study, we have compared the behavior of BIAP when added to fluid-ordered phase-separated DOPC/DPPC, DOPC/ SM, and POPC/SM supported bilayers. For SM, bovine brain SM was selected because of its thermotropic behavior close to that of renal epithelial cells SM.6,32 AFM was used to probe the interaction at the mesoscopic scale between BIAP and lipid ordered domains under physiological buffer. Natural SMs exhibit a strong polymorphism.33,34 SLB made of binary SM/PC mixtures (15) Marguet, D.; Lenne, P. F.; Rigneault, H.; He, H. T. EMBO J. 2006, 25, 3446-3457. (16) Lenne, P. F.; Wawrezinieck, L.; Conchonaud, F.; Wurtz, O.; Boned, A.; Guo, X. J.; Rigneault, H.; He, H. T.; Marguet, D. EMBO J. 2006, 25, 3245-3256. (17) Fanning, A. S.; Anderson, J. M. Curr. Opin. Cell Biol. 1999, 11, 432439. (18) Camolezi, F. L.; Daghastanli, K. R.; Magalhaes, P. P.; Pizauro, J. M.; Ciancaglini, P. Int. J. Biochem. Cell Biol. 2002, 34, 1091-1101. (19) Ronzon, F.; Desbat, B.; Buffeteau, T.; Mingotaud, C.; Chauvet, J.-P.; Roux, B. J. Phys. Chem. B 2002, 106, 3307-3315. (20) Cross, B.; Ronzon, F.; Roux, B.; Rieu, J. P. Langmuir 2005, 21, 51495153. (21) Kouzayha, A.; Besson, F. Biochem. Biophys. Res. Commun. 2005, 333, 1315-1321. (22) Kahya, N.; Brown, D. A.; Schwille, P. Biochemistry 2005, 44, 74797489. (23) Milhiet, P.-E.; Giocondi, M.-C.; Baghdadi, O.; Ronzon, F.; Le Grimellec, C.; Roux, B. Single Mol. 2002, 3, 135-140. (24) Moran, P.; Beasley, H.; Gorrell, A.; Martin, E.; Gribling, P.; Fuchs, H.; Gillett, N.; Burton, L. E.; Caras, I. W. J. Immunol. 1992, 149, 1736-1743. (25) Schreier, H.; Moran, P.; Caras, I. W. J. Biol. Chem. 1994, 269, 90909098. (26) Schroeder, R. J.; Ahmed, S. N.; Zhu, Y.; London, E.; Brown, D. A. J. Biol. Chem. 1998, 273, 1150-1157. (27) Milhiet, P. E.; Giocondi, M. C.; Baghdadi, O.; Ronzon, F.; Roux, B.; Le Grimellec. C. EMBO Rep. 2002, 3, 485-490. (28) Saslowsky, D. E.; Lawrence, J.; Ren, X.; Brown, D. A.; Henderson, R. M.; Edwardson, J. M. J. Biol. Chem. 2002, 277, 26966-26970. (29) Benting, J.; Rietveld, A.; Ansorge, I.; Simons, K. FEBS Lett. 1999, 462, 47-50. (30) White, D. The phospholipid composition of mammalian tissues, 2nd ed.; Elsevier: Amsterdam, 1973; Chapter 16, p 441. (31) Giocondi, M. C.; Milhiet, P. E.; Dosset, P.; Le Grimellec, C. Biophys. J. 2004, 86, 861-869. (32) Barenholz, Y. In Physiology of membrane fluidity; Shinitzky, M., Ed.; CRC Press: Boca Raton, 1984; Vol. 1, p 131. (33) Meyer, H. W.; Bunjes, H.; Ulrich, A. S. Chem. Phys. Lipids 1999, 99, 111-123.

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also adopt various structures with frequent immiscibility of the ordered phase characterized by solid-solid (gel-gel) phase separation of SM-enriched domains.33,34 How BIAP interacts with such samples was also studied. Materials and Methods Alkaline Phosphatase Purification. BIAP of fresh bovine intestine mucosa was purified as described by Ronzon et al.19 The enzyme was solubilized in 10 mM Tris-HCl, 150 mM NaCl, 1 mM Mg2+, and 2 mM n-octyl β-D-glucopyranoside, pH 8.5. The prepared BIAP has kept its GPI anchor and presents an apparent molecular mass of about 130000 Da. The specific activity of the purified fraction, determined according to Cyboron and Wuthier,35 was higher than 1000 U/mg. The protein concentration was determined by the method of Bradford36 using bovine serum albumin as standard. Preparation of Supported Bilayers. Supported bilayers (SLB) were made by vesicle fusion, starting from multilamellar vesicles (MLVs), as previously described.37 MLVs were prepared under argon, at 65 °C for DOPC/DPPC (1:1 mol:mol) and DOPC/SM (1:1 mol: mol) and 70 °C for POPC/SM (1:1 mol:mol), in phosphate buffered saline (PBS), pH 7.4, from chloroform/methanol 2/1 (v/v) stock solutions (10 mM) of bovine brain SM, DOPC, and POPC (Avanti Polar Lipids, Alabaster, AL, and Sigma-Aldrich, Saint QuentinFallavier, France) dried under nitrogen gas. Purity of the phospholipids was checked by TLC and the phospholipid concentration was determined according to Mrsny et al.38 Small unilamellar vesicles (SUVs) were prepared, at corresponding temperatures, by extrusion of MLV through polycarbonate membranes (Avanti mini-extruder, AL). To form a supported bilayer, SUVs were deposited on a freshly cleaved mica disk (1/2 in. diameter, JBG-Metafix, Montdidier, France) inserted in a 13 mm holder for swinney syringe (Millipore, Bedford, MA) and incubated at either 65 °C (DOPC) or 70 °C (POPC) for 2 h in a water bath. At the end of the incubation, the samples, always maintained in an aqueous environment, were returned to room temperature, kept overnight, and then carefully rinsed with PBS to remove the SUVs in excess and the loosely adsorbed bilayers. BIAP Insertion into Preformed Supported Bilayers. BIAP was added, at room temperature, to preformed supported bilayers under PBS as previously described.27 Briefly, BIAP, ∼1 mg/mL in solution in solubilization buffer, was sonicated 5 min in a water bath. Then 2-20 µg of protein was added under the microscope to the PBS medium bathing the bilayers. Alternatively, BIAP was first diluted in PBS and the BIAP-containing PBS was used to replace the bilayer original buffer. The two procedures gave similar results. The final concentration of n-octyl β-D-glucopyranoside in the bilayer solution was below 0.2 mM, i.e., less than 1/100 of the cmc. Control experiments showed that n-octyl β-D-glucopyranoside has no significant effect on bilayer topography at such concentrations. Atomic Force Microscopy. The mica supports were glued onto glass coverslips with Super Glue 3 (Loctite) before mounting on a homemade holder screwed on the stage of an inverted microscope (Zeiss) coupled to a Bioscope (Digital Instruments, Santa Barbara, CA). The bilayers were imaged in PBS, at room temperature. Silicon nitride cantilevers, with 0.01 and 0.03 N/m nominal spring constant (Park Scientific Instruments, Sunnyvale, CA), were used in the experiments which were run in contact mode. Unless mentioned, the scanning force was adjusted to below 0.3 nN and readjusted for drift during images acquisition. The scan rate was adjusted between 1 and 3.5 Hz, according to the scan size.

Results BIAP Insertion into DOPC/DPPC Bilayers. AFM imaging of DOPC/DPPC (1:1, mol:mol) SLB, under PBS, confirmed37 (34) Giocondi, M. C.; Boichot, S.; Plenat, T.; Le Grimellec, C. C. Ultramicroscopy 2004, 100, 135-143. (35) Cyboron, G. W.; Wuthier, R. E. J. Biol. Chem. 1981, 256, 7262-7268. (36) Bradford, M. M. Anal. Biochem. 1976, 72, 248-254. (37) Giocondi, M. C.; Vie´, V.; Lesniewska, E.; Milhiet, P. E.; Zinke-Allmang, M.; Le Grimellec, C. Langmuir 2001, 17, 1653-1659. (38) Mrsny, R. J.; Volwerk, J. J.; Griffith, O. H. Chem. Phys. Lipids 1986, 39, 185-191.

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Figure 2. Insertion of BIAP into the homogeneous gel phase of DOPC/SM supported bilayers. AFM height images of DOPC/SM (1:1, mol:mol) before (A) and after 2 h incubation with BIAP (B). Bar: 1 µm. Black arrows point at contaminants. Bright dots corresponding to BIAP were essentially observed at the boundary of gel-fluid domains (white arrows) and randomly distributed in the ordered gel phase.

Figure 1. Insertion of BIAP into gel phase domains of DOPC/ DPPC supported bilayers. AFM height images of DOPC/DPPC (1: 1, mol:mol) before (A) and after 2 h incubation with BIAP (B). Bar: 1 µm. White arrows point to regions with important concentration of BIAP. (C) is a higher magnification scan of the zone in insert in (B). (D) is a merge of A and B, with the orange/pink color corresponding to the increase in ordered domain area.

the existence at room temperature of lighter DPPC ordered gel phase (Lβ’) domains protruding by 0.9 ( 0.2 nm from the fluid (LR), darker, DOPC-enriched phase matrix (Figure 1A). This value is in accordance with the previous estimate of the thickness difference between (LR) DOPC and (Lβ’) DPPC obtained by AFM as well as by NMR and X-ray diffraction studies.23,37,39-41 Comparison of Figure 1A with Figure 1B, obtained after 2 h of incubation of BIAP with the PBS medium bathing the preformed DOPC/DPPC SLB, revealed the presence of numerous small bright dots onto the ordered gel phase domains. These bright dots, emerging from the bilayer surface by ∼2-5 nm, were often forming short alignments, in a direction that differed from the scanning direction, mostly localized at the periphery of the gel phase. The minimal diameter of single bright dots estimated from higher magnification scans (Figure 1C) was 14 ( 2 nm. Taking into account the tip convolution effect, this strongly suggested that the smallest bright dots, which accounted for the majority of the signal, corresponded to single BIAP dimer species. Significantly, AFM detected the presence of BIAP almost exclusively in the gel phase with few particles in the fluid phase. A color overlay where the enriched BIAP zones appear in translucent pink over the control bilayer shows that BIAP insertion was associated with a significant increase in the area occupied by the ordered domains (BIAP/control ) 1.08 ( 0.02; Figure 1D, arrows). This suggested a BIAP-induced displacement of the gel-fluid phase equilibrium with DPPC molecules dissolved in the DOPC matrix moving toward the ordered DPPC-enriched phase. BIAP Insertion into DOPC/SM Bilayers. Replacing DPPC by SM, we observed in ∼75% of our samples a “simple” gelfluid phase separation in the DOPC/SM (1:1, mol:mol) binary (39) Tristram-Nagle, S.; Petrache, H. I.; Nagle, J. F. Biophys. J. 1998, 75, 917-925. (40) Seelig, J.; Seelig, A. Q. ReV. Biophys. 1980, 13, 19-61. (41) El Kirat, K.; Lins, L.; Brasseur, R.; Dufrene, Y. F. Langmuir 2005, 21, 3116-3121.

mixture, with the homogeneous SM-enriched gel phase domains protruding by 0.9 ( 0.2 nm from the DOPC-enriched fluid phase matrix (Figure 2A). Black arrows point to a few small contaminants at the bilayer surface which stay in place during all of the experiment. As previously reported,27 when added to the bathing medium, BIAP spontaneously inserted into preformed DOPC/SM supported bilayers (Figure 2B). Bright dots corresponding to the BIAP hydrophilic part, 14-50 nm in apparent diameter, were essentially observed both at the boundary of gelfluid domains (white arrows) and the others randomly distributed in the ordered gel phase. Some shape remodeling of ordered domains, with the presence of more straight contours, occurred upon BIAP insertion. However, this was not accompanied by a significant change in their area (BIAP/control ) 1.01 ( 0.05). BIAP Insertion into POPC/SM Bilayers. POPC/SM (1:1, mol:mol) supported bilayers also gave gel phase SM ordered domains, with a size in the micrometer range, surrounded by a fluid phase POPC enriched matrix (Figure 3A). They protruded from the POPC bilayer by 0.8 ( 0.1 nm, a value that compared with that measured in DOPC/SM samples. Keeping a large scan (20 µm) range, the BIAP addition resulted, after 1-3 h of incubation, in the formation of structures heterogeneous in size, shape, and distribution among SM domains (Figure 3B). Isolated, small size ( DPPC/DOPC > singlephase SM/DOPC > SM/POPC.66 For the first two mixtures a net transfer into the ordered phases was observed, for the third no net transfer was recorded, and a net phospholipids transfer, from the ordered phase, characterized the fourth mixture. This strongly suggests that BIAP-induced net transfer of phospholipids molecules depends both on the matching between the hydrophobic thickness of the BIAP acyl chains and phospholipids constituting the two phases and on the gradient of lipid order at the interface of the fluid and gel phases. With at least two C16:0 and two C18:0 acyl chains for the dimer, BIAP could constitute a nucleation site for saturated species in the fluid phase region close to the interface and consequently increase the size of SM/ DOPC and DPPC/DOPC ordered domains. On the other hand, in single gel phase SM/ POPC samples, the enrichment in the most ordered species around BIAP would result in their depletion in other parts of the ordered domains and favor the mixing of SM lower melting species with the fluid phase. Experiments using binary mixtures made of SM or PC with different chain lengths and melting temperatures and different GPI proteins will be required to precisely determine the relative importance of lipid order gradient and hydrophobic matching in this mechanism. As discussed above for DOPC/DPPC bilayers, preferential insertion of BIAP at the boundary of domains with decrease in the line tension should be common to all the fluid-ordered phaseseparated mixtures. The concave edges of gel domains further indicate that the decrease in line tension must be more marked for POPC/SM mixtures.67 The images also show that the distribution of BIAP within ordered domains differs according to the bilayer composition. GPI-anchored protein preferentially localized along the periphery of gel domains in DOPC/DPPC and POPC/SM bilayers as opposed to a more random distribution in DOPC/SM samples without gel-gel phase separation. For POPC/SM samples, the alignment of particles in ordered domains suggests the existence of structural defects like those found in (64) Vie´, V.; Van Mau, N.; Lesniewska, E.; Goudonnet, J. P.; Heitz, F.; Le Grimellec, C. Langmuir 1998, 14, 4574-4583. (65) Seelig, J.; Browning, J. L. FEBS Lett. 1978, 92, 41-44. (66) Koynova, R.; Caffrey, M. Biochim. Biophys. Acta 1998, 1376, 91-145. (67) Baumgart, T.; Hess, S. T.; Webb, W. W. Nature 2003, 425, 821-824.

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DOPC/DPPC mixtures along which BIAP migrates at a rate equivalent, or most likely higher, to that of lipid diffusion in a gel phase.49 Replacement of the saturated palmitic by a second unsaturated oleic chain would increase the structural and packing defects within the SM-enriched domains, resulting in enhanced lateral diffusion leading to the apparent BIAP random distribution. Coupling of AFM with fluorescence techniques will be needed to assess the relative contribution of line tension, lipids, and protein diffusion properties.68 Finally, as shown by the force-dependent experiments, imaging of BIAP in domains was highly sensitive to the scanning conditions. In particular, the use of moderate scanning forces, i.e., forces slightly above 300 pN, was sufficient to extract a large number of BIAP from the ordered domains, the most resistant to scanning GPI-anchored molecules being localized at

the gel-fluid interface. This scanning force effect, the way the GPI-anchored protein was introduced into the bilayer, and differences in the hydrophobic part of the anchor could have contributed to the relatively low amount of PLAP visualized in liquid ordered domains by AFM.68 This would be in-line with the observation that acyl and alkyl chain length of GPI anchors play a critical role in raft association.29 In conclusion, the present data show that BIAP spontaneously inserts into the ordered domains of bilayers under fluid-gel phase separation. This insertion can induce a net transfer of phospholipids from one phase to the other whose direction depends on the lipid constituents present in each phase. This strongly suggests that the GPI anchor might contribute to the formation of its most appropriate membrane environment in terms of hydrophobic length and lipid order parameter.

(68) Chiantia, S.; Ries, J.; Kahya, N.; Schwille, P. Chemphyschem 2006, 7, 2409-2418.

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