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Protein–protein and protein–lipid interactions in domain-assembly: Lessons from giant unilamellar vesicles
Biochimica et Biophysica Acta 1798 (2010) 1392–1398
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Biochimica et Biophysica Acta j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / b b a m e m
Review
Protein–protein and protein–lipid interactions in domain-assembly: Lessons from giant unilamellar vesicles Nicoletta Kahya ⁎ Department of Cell Biology, University Medical Center Groningen, Antonius Deusinglaan 1, 9713 AV Groningen, The Netherlands
a r t i c l e
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Article history: Received 8 December 2009 Received in revised form 2 February 2010 Accepted 21 February 2010 Available online 6 March 2010 Keywords: Giant unilamellar vesicle Lipid raft Membrane protein reconstitution Optical microscopy
a b s t r a c t Giant Unilamellar Vesicles (GUVs) provide a key model membrane system to study lipid–lipid and lipid– protein interactions, which are relevant to vital cellular processes, by (single-molecule) optical microscopy. Here, we review the work on reconstitution techniques for membrane proteins and other preparation methods for developing GUVs towards most suitable close-to-native membrane systems. Next, we present a few applications of protein-containing GUVs to study domain assembly and protein partitioning into raft-like domains. © 2010 Elsevier B.V. All rights reserved.
Contents 1. 2. 3.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . Giant unilamellar vesicles for optical microscopy . . . . . . . . . . . Membrane proteins in giant unilamellar vesicles . . . . . . . . . . . 3.1. Techniques from purified components. . . . . . . . . . . . . 3.1.1. Peptide-induced membrane fusion . . . . . . . . . . 3.1.2. Dehydration and electroformation . . . . . . . . . . 3.2. Techniques from native cellular membrane fractions . . . . . . 4. Lateral distribution of membrane proteins in domain-forming bilayers . 5. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Introduction As an active interface between different biological compartments, membranes guarantee an efficient exchange of matter, energy and/or signals. For this purpose, such an interface has to be designed as a very dynamic system, yet with a non-random distribution of its components. The enormous structural variety of lipids and proteins brings about a Abbreviations: SPT, single particle tracking; FCS, fluorescence correlation spectroscopy; FRET, Föster resonance energy transfer; GUVs, giant unilamellar vesicles; BLMs, black lipid membranes; BR, bacteriorhodopsin; DOPC, dioleoyl-phosphatidylcholine; SM, sphingomyelin (C18:0); APP, amyloid precursor protein; BACE, beta-site-amyloid cleaving enzyme; LUVs, large unilamellar vesicles; DRMs, detergent resistant membranes; PLAP, placental alkaline phosphatase ⁎ Tel.: + 31 50 3636168; fax: + 31 50 3632728. E-mail address: [email protected]. 0005-2736/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.bbamem.2010.02.028
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large spatial and temporal heterogeneity in membrane organization, which may be related to the way the cell accurately regulates biological processes at and around membranes in space and time. The highly intricate architecture of cellular membranes represents a drawback in determining the biological functions of specific lipids and proteins in the top-down approach, by looking at organisms and/or living cells, mainly because of the large number of interfering events simultaneously occurring at the spot of interest. Therefore, a complementary approach with a bottom-up strategy is highly desirable. Within this context, model membranes are key systems to isolate the biological machinery and identify its function. From a methodological point of view, tracking lipids and proteins in space and time represents a big challenge. The small size of lipids makes it technically difficult to follow their trajectories in a reliable and non-invasive way (i.e. the size of the label is often comparable to
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the size of the lipid itself). On the other hand, membrane proteins may be easier to track but the biological implications of their lateral organization and association/dissociation behaviors are, in many cases, tightly linked to the lipid environment, in ways that are up to date not well understood [1]. In recent years, optical microscopy techniques have greatly contributed to broaden our knowledge of membranes, in particular with the development of techniques, which work at the singlemolecule regime (Single Particle Tracking [2] — SPT and Fluorescence (Cross)-Correlation Spectroscopy [3,4] — FCS) and Föster Resonance Energy Transfer (FRET) [5]. In this paper, we focus on Giant Unilamellar Vesicles (GUVs) as model membranes of choice for protein reconstitution and lipid– protein interaction studies by optical microscopy (Section 2). We review the reconstitution techniques developed so far for embedding membrane proteins into GUVs (Section 3). Next, we present a few applications of protein-containing GUVs to study domain assembly and protein partitioning into raft-like domains (Section 4). Finally, we draw some conclusions. 2. Giant unilamellar vesicles for optical microscopy In order to study events occurring at and around membranes by means of optical microscopy, the bilayer has to span the illumination spot in the focal plane, whose lateral dimension goes with the half of the wavelength of light (∼250 nm in the visible range). Compared to other model membranes, which have been developed throughout the years and applied to optical microscopy, GUVs are the most biomimetic (Fig. 1). Monolayers [6] provide a regular and stable structure and their composition can be accurately controlled. However, their ability to mimic biomembranes can be questioned, as they lack the second leaflet to form a bilayer. The first successful attempt to make membranes with a bilayer structure was reported by Müller et al. [7] with the Black Lipid Membranes (BLMs). Two compartments are
Fig. 1. 3D projection of GUVs composed of DOPC, labeled with 0.1% of DiI-C18 (A), SM/DOPC/cholesterol 1:1:1 (molar ratio), labeled with 0.1% of DiI-C18 (B), SM, labeled with 0.1% of DiI-C18. Note that in the bleached dark spot fluorescence does not recover over time (C), SM/DOPC/cholesterol 22:33:33 (molar ratio) and 10 mol% of ceramide (C18:0), labeled with 0.1% of DiD-C18 (D). Scale bars: 10 μm (A), 10 μm (B), 5 μm (C), 10 μm (D).
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separated by a thin partition (septum) and communicate through an aperture (100–200 μm in diameter) in this partition. An organic solution of lipids is brushed over the hole and then the compartments are filled with an aqueous medium. Dispersion of the solvent in the aqueous medium eventually leaves a bilayer on the hole. In practice, however, there are many technical limitations, which prevent the formation of an ideal BLM and lead to an irregular bilayer structure [8]. A very interesting alternative to BLMs is represented by solidsupported bilayers. The early concept was based on the fusion of small unilamellar vesicles (diameter b100 nm), which can be deposited onto the hydrophilic surface of a clean polar substrate, such as glass or mica. This results in a supported membrane [9] floating on an ultra-thin (∼1–2 nm) water layer. In general, the absence of a “bulk aqueous medium” between the bilayer and the solid substrate may give rise to secondary lipid–substrate interactions and, thereby, to artifacts. In particular, many membrane proteins protrude from the bilayer surface into the water phase, much further than 1–2 nm, implying their soluble residues interact with the solid substrate. Some of these drawbacks have been partially solved by designing a tethered bilayer [10], which is composed of a solid substrate, a tethering layer (e.g. a soft polymer cushion) and a lipid bilayer. The soft and hydrophilic polymer should keep the lipid bilayer far apart from the rigid support, and thereby, preserve membrane fluidity. However, membrane proteins embedded in such a lipid matrix interact with the polymer and appear to be only partially mobile [10]. The artifacts potentially found in solid-supported bilayers are absent in GUVs, which are spherical closed single bilayers, freely floating in aqueous solution [11,12]. They are suitable for (single-molecule) optical microscopy and exhibit a cell-like size, as their size ranges from 10 to 100 μm in diameter. The most common method for the production of GUVs of consistent and reproducible unilamellarity is the electroformation method, in which a dry lipid film is rehydrated in the presence of an alternating electric field for typically 1–3 h [11,12]. GUVs obtained with this method can be studied in situ (as immobilized on the electrodes, either Pt wires or ITO-coated glass coverslips) or can be detached from the electrodes and transferred into other aqueous environments. When transferred to other environments, GUVs may be observed as freely moving (e.g. to study membrane fluctuations and measure mechanical properties such as bending elasticity [13]), or they can be immobilized onto glass surfaces, which are coated with several chemicals, e.g. poly-Llysine [14], casein [15], albumin [16], protein anchors such as avidin/ streptavidin [17], or with lipid patterns [18], to study the effect of specific molecular interactions onto membrane adhesion properties, as relevant to cell–cell and cell–matrix interactions. From all of these studies, it is evident that immobilizing vesicles onto a surface might change both the collective membrane properties (e.g. mechanical) and the molecular self-organization within the bilayer. Researchers engaging with these methodologies need to be aware of these effects (and/or potential artifacts) on membrane structure and dynamics. GUVs can also be trapped by a micropipette for investigating membrane stretching elasticity [19] and for pulling tubes to study membrane budding [20,21]. Although very reproducible and reliable, the GUV electroformation method poses a few limitations, which have, in some cases, limited the applicability of GUVs, such as: 1) low vesicle yield in buffers of high ionic strength and/or physiological ion concentrations, 2) absence of transbilayer asymmetry, 3) inability to control and determine the exact composition of individual vesicles, and 4) difficulty to include membrane proteins into the electroformation procedure. Recently, as to point 1) and 2), efforts have been taken to attain high GUV electroformation yields in physiological buffers [22] and to show that transbilayer asymmetry was preserved by starting from red blood cell ghosts [23]. As to point 3), the variability of composition between vesicles exists and varies depending on type of lipids, temperature and composition of aqueous environment. It is, therefore, good practice and, at times, essential to the data interpretation, to discuss and/or provide an estimate of the variability between individual vesicles [see, for
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instance, 24,25]. As to point 4), the use of organic solvents and the dehydration step in the electroformation easily lead to denaturation of membrane proteins. As discussed in the following section, various reconstitution procedures have been developed over the years to circumvent this problem. 3. Membrane proteins in giant unilamellar vesicles Several methods have been developed to incorporate membrane proteins into GUVs. This section will briefly describe the most common methodologies employed to reconstitute membrane proteins into GUVs and will be followed by examples of how these methods have led to a deeper understanding for how proteins function in membranes. As mentioned in the previous section, the most common method for the production of GUVs of consistent and reproducible unilamellarity is the electroformation method, in which a dry lipid film is rehydrated in the presence of an alternating current electric field [11,12]. This methodology was extended to include drying membrane proteins with the lipid such that it incorporated proteins into the GUVs upon electroswelling. This was first achieved by Manneville et al. [26] for bacteriorhodopsin. However, the method is not applicable in general to membrane proteins, as dissolving such proteins in organic solvents and subsequently drying them lead, in general, to misfolding and denaturation. 3.1. Techniques from purified components 3.1.1. Peptide-induced membrane fusion Another methodology for incorporating membrane proteins into electroformed GUVs is to fuse the GUVs with small preformed proteoliposomes. This method was developed by Kahya et al. [27] and shown for the reconstitution of bacteriorhodopsin (BR) from purple membranes of Halobacterium salinarium. BR was first reconstituted into Large Unilamellar Vesicles (LUVs), which contained a fusion peptide (WAE) and were, thereby, able to fuse with preformed giant unilamellar vesicles. After full membrane fusion of BR-containing LUVs with GUVs, as shown by lipid mixing and water content mixing, BR was then fully reconstituted into GUVs and shown to retain its proton-pumping activity (Fig. 2). Compared to other reconstitution methods, this technique is less straightforward, as it requires multiple steps and it employs peptide-induced membrane fusion, which imposes some constraints on the lipid composition of the vesicles and might potentially interfere with the function of the protein of interest. An elegant example of how this method can be applied to monitoring of lipid–protein and protein–protein interactions in lipid bilayers at physiological concentrations was demonstrated by Kriegsman et al. [28]. They reconstituted the complex of the photoreceptor NpSRII, which consists of seven-transmembrane α-helices, and its cognate transducer NpHtrII, which contains two transmembrane α-helices and a large cytoplasmic domain. They could measure the lateral diffusion of the distinct membrane proteins and quantify the degree of oligomerization. In fact, heterodimeric complexes were characterized quantitatively with dual-color fluorescence correlation spectroscopy
and the intermolecular binding between NpSRII and NpHtrII in the functional unit (with stoichiometry 2:2) was found to be extremely strong, as complexes formed in bilayers with low molar protein:lipid ratios (∼1:2,000,000). 3.1.2. Dehydration and electroformation One of the simplest techniques to produce giant proteoliposomes consists of dehydration of preformed proteoliposomes followed by spontaneous swelling upon rehydration with a buffer [29–31]. The major advantage of such a technique is that vesicles can be prepared with a wide range of buffers. However, the vesicles produced from the rehydration are very heterogeneous and mainly of multilamellar nature. Nonetheless, this method has proved useful in pulling lipid tubes from vesicles, which have been rehydrated from a multilamellar lipid base [32,33]. By using micropipettes, these tubes can be extended to inflate new GUVs and even to create vast lipid tubule networks. A further development of this reconstitution technique involves the partial dehydration of preformed proteoliposomes followed by controlled rehydration via electroformation. Compared to peptideinduced vesicle fusion, partial dehydration of proteoliposomes followed by electroformation is more straightforward and does not require fusogenic molecules. It also results in consistent and homogeneous preparation of GUVs. The method was first applied by Girard et al. [34,35] to incorporate the Ca2+-ATPase as well as bacteriorhodopsin into GUVs. A similar technique, which makes use of partial dehydration of proteoliposomes in the presence of a cryoprotectant that prevents protein misfolding and/or denaturation during drying, has been developed to reconstitute human placental alkaline phosphatase (PLAP) into GUVs (Fig. 3) and examine the protein lateral partitioning in distinct lipid phases [36]. The same reconstitution technique was later applied to investigate the proteolytic activity of the β-secretase, beta-site-amyloid cleaving enzyme (BACE) [37], and its lateral partitioning in liquid-ordered and liquid-disordered lipid phases. Over the years, many variants of this reconstitution technique have been successfully applied to obtain giant proteolipomes (for examples see [38,39]). We and others have reconstituted a number of membrane proteins with very diverse topology, from GPI-anchored proteins (for example PLAP [36], human prion), to transmembrane model peptides (WALP, see [40]), singlespanning membrane proteins (for example BACE [37], syntaxin [38], synaptobrevin [38]) and multi-spanning membrane proteins in various oligomeric states (for example glutamate transporter, mechanosensitive channel, lactose transporter and lactose permease; see [40]). The wide applicability of this reconstitution technique also includes a large variety of lipid compositions, buffers and, in general, physiological conditions. Recently, an elegant application of this reconstitution method was provided by Streicher et al. [41], who developed an in vitro system to mimic the first steps of integrinmediated cell spreading, and, thereby, studying fundamental aspects of cell adhesion, as implicated into cell–cell contacts in tissue and/or in interaction of cells with the extracellular matrix. Integrin from human platelets was functionally reconstituted into GUVs, thereby
Fig. 2. Protein reconstitution into GUVs by peptide-induced membrane fusion: (A) confocal image of GUV after fusion with BR-containing LUVs. The fluorescence signal is given by AF-488labeled BR; (B) activity assay for BR reconstituted into GUVs. The fluorescence signal is given by the pH-sensitive dye pyrene. The fluorescence level from the lumen of GUVs is lower than outside of the GUV, which implies a more acidic pH in the GUVs lumen and, therefore, proving a proton-pumping activity of BR [27].
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Fig. 3. Protein reconstitution into GUVs by partial dehydration followed by electroformation: confocal image of GUVs composed of DOPC, in which AF-488 labeled BR was reconstituted [36]. Scale bar: 10 μm.
allowing for a detailed investigation of the mechanical static as well as dynamic aspects of adhesion of such vesicles onto fibrinogen-coated surfaces.
3.2. Techniques from native cellular membrane fractions One of the most exciting developments in GUV research lies on the reconstitution of membrane proteins in their native membrane environment. This was achieved by extracting native membranes from cellular fractions, without the removal of the proteins, and by applying the electroformation procedure directly to the membrane fractions to prepare GUVs [42]. Recently, we have explored such options with respect the transmembrane receptors, ion channels and ATPases found in all eukaryotic cells. Such proteins include the Na/K-ATPase, the voltage-gated sodium channel, inositol 1,4,5-triphosphate receptor, CD44, L1CAM and the Na/Ca exchanger, just to name a few. Specifically, we focused on the Na/K-ATPase, which plays a crucial role in generating energy, maintaining osmotic balance within a cell, participating in cellular signaling, and anchoring of cytoskeletal components to the plasma membrane [43]. We were interested in investigating the coupling of actin filaments to the membrane bilayer, as in the native membrane. To this end, membrane proteins reconstituted into GUVs were essential for binding of the cytoskeleton, as the anchoring of spectrin/ankyrin and actin filaments to the membrane has been shown to require membrane proteins, among others the Na/K-ATPase [43]. Previous studies had focused on actin polymerization inside (or outside) GUVs made from simple lipid compositions [44–48]. In our system, we reconstituted the whole complex lipid–protein system necessary to achieve spectrin/ankyrin-mediated binding of actin filaments to the membrane. We set out to prepare GUVs from porcine brain membrane fractions, in physiological conditions [49], by using the partial dehydration and rehydration via electroformation. Such a preparation yielded consistent and reproducible GUVs with diameters of 10–100 μm (Fig. 4). The presence of Na/K-ATPase was demonstrated by binding of a mouse monoclonal antibody directed against Na/K-ATPase and with a secondary goat-anti-mouse antibody which was conjugated to the Cy5 fluorescent dye. GUVs incubated in the absence of primary antibody, or with a non-specific primary mouse monoclonal antibody did not stain the GUVs [49]. The presence of Na/K-ATPase in these fractions was also confirmed via immunoblot analysis. Subsequently, actin filaments were reconstituted into such GUVs and proved to specifically bind to the membrane bilayer (Fig. 4, [49]). Current investigations employ such a system to study the mechanical aspects of the membrane–cytoskeleton coupling in a close-to-native system. In addition, consistent and reproducible GUVs can also be prepared by partial native membrane dehydration followed by electroformation starting from internal organelle fractions, as we were able to
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Fig. 4. Confocal images of GUVs prepared from porcine brain membrane extracts with a highly enriched fraction of spectrin/ankyrin, and labeled with 0.1% of DiD-C18 (red) in the presence of actin (visualized with phalloidin-Alexa 488 — green) [49]. Scale bar: 10 μm.
obtain GUVs with excellent yield from endosomal fractions of HeLa cells (N. Kahya, unpublished results). Other groups have meanwhile provided evidence that electroformation is the method of choice for yielding GUVs from native membrane fractions. Montes et al. have recently shown that GUVs can be prepared from red blood cell ghosts in physiological buffers [23,35]. Interestingly, GUVs prepared with this method maintained the original transbilayer asymmetry of the red blood cell membranes, as shown by immunofluorescent staining of GUVs against the cytoplasmic domain of band III. These novel applications pave the way to elucidating specific molecular interactions in the membrane architecture and its dynamic organization needed to carry out essential cellular functions. Finally, it is worth to mention an alternative method to obtain giant liposomes from plasma membranes, which is cell “blebbing” (see for example Baumgart et al. [50]), essentially a chemicallyinduced vesiculation of cells. Although promising, more studies are needed to understand and interpret the results, as to what is the nature of the overall mechanism of vesicle formation and the effect of the chemicals employed on the cell wall and surroundings.
4. Lateral distribution of membrane proteins in domain-forming bilayers Lipid–protein and protein–protein interactions have been shown to play a key role in regulating the function of biological machinery's at the membrane and in its surroundings (see for instance [51–54]. One of the most exciting ways for nature to tightly regulate in time and space intraand intermolecular interactions at the membrane is through spatiotemporal segregation of certain components to so-called “rafts” [55–57] and the exclusion of unwanted components from them. Rafts were defined as “small (10–200 nm), heterogeneous, highly dynamic, steroland sphingolipid-enriched domains that compartmentalize cellular processes. Small rafts can sometimes be stabilized to form larger platforms through protein–protein interactions” from the (Keystone Symposium of Lipid Rafts and Cell Function, held in 2006). Many studies have added to the understanding of the mechanisms of raft formation, which is most likely dependent on a very fine balance of specific lipid– protein interactions and protein–protein associations [58,59]. Cellular membranes are very crowded and, on average, proteins populate the bilayer up to one half of the total membrane mass [60]. By adopting a bottom-up approach and, thereby, starting from a minimum of membrane components to study specific physico-chemical properties of rafts, it is, simply not enough to examine pure lipid systems. We set out to develop biophysical tools to test some ideas concerning rules and structural requirements, which are responsible for targeting membrane proteins to lipid environments of specific chemistry. We could then test whether and how the lipid matrix influences the mechanisms of function of membrane proteins.
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Putative raft-associated and non-raft proteins were reconstituted into domain-exhibiting GUVs [36–38] (Fig. 5). Their spatial organization was observed by optical imaging and FCS. The human placental alkaline phosphatase (PLAP) was abundantly found in detergent resistant membranes (DRMs) after treatment with Triton X-100 at 4 °C [61,62]. However, it mainly associated with liquid-disordered DOPC-enriched phases in GUVs composed of DOPC/SM/cholesterol (1:1:1) (see Fig. 5A), as shown by FCS and by counterstaining the raftlike liquid-ordered and SM-enriched phase with GM1-bound fluorescent cholera toxin [36]. Similar GPI-anchored proteins, such as the bovine intestine alkaline phosphatase, exhibited a comparable behavior (N. Kahya and S. Morandat, unpublished results). FCS measurements of local protein density in distinct phases revealed that at most 25–30% of PLAP partitioned into the liquid-ordered phase. Furthermore, antibody-mediated cross-linking caused the protein to associate more (up to 50%) with the liquid-ordered phase [36]. Taken together, this data showed that PLAP exhibits a higher affinity for the raft-like liquid-ordered phase compared to other membrane proteins, such as syntaxin [38], synaptobrevin [38] and
Fig. 5. (A) Confocal image of GUVs composed of SM/DOPC/cholesterol 1:1:1 (molar) and GPI-anchored rhodamine-labeled PLAP (Rho-PLAP) [36]. Scale bar: 10 μm. (B) Confocal image of GUVs composed of SM/DOPC/cholesterol 1:1:1 and Cy5-labeled syntaxin [38]. Scale bar: 10 μm. (C) Confocal image of GUVs composed of SM/DOPC/ cholesterol 1:1:1 and 0.1% of GM1 containing Cy5-labeled BACE (Cy5-BACE) — red channel [37]. Scale bar: 10 μm. (D) Confocal image as in (C): fluorescence signal from AF488-labeled cholera toxin (AF488-CTXB) — green channel [36]. Scale bar: 10 μm. (E, F) Confocal images of GUVs composed of SM/DOPC/cholesterol 1:1:1, AF488labeled GPI-anchored human prion (green) and 0.1% of DiI-C18 (red) (N. Kahya and P. Schwille, unpublished results). Scale bar: 10 μm.
bacteriorhodopsin [36], which were fully associated with the liquiddisordered phase (Fig. 5B). The different partitioning behavior is however not simply related to the nature of binding to the membrane (GPI-anchor versus transmembrane domain). The fact that subtler rules play a crucial role in determining the protein affinity for specific lipid phases is inferred by the following two observations. First, among GPIanchored proteins, PLAP exhibited similar partitioning behavior to its corresponding bovine form, as mentioned above. However, the GPI-anchored human prion protein, involved in Creutzfeld–Jakob disease, which was reconstituted into GUVs composed of DOPC/SM/ cholesterol (1:1:1 molar ratios), was fully associated with the raft-like liquid-ordered and SM-enriched phase (Fig. 5E–F, green), as opposed to the DiI-C18 (Fig. 5E–F, red), which stained the liquid-disordered phase (N. Kahya and P. Schwille, unpublished results). Second, among single-spanning membrane proteins, we found significant differences between partition coefficients for liquidordered phases. In this respect, as shown in GUV studies with the same lipid composition, syntaxin and synaptobrevin were fully excluded from liquid-ordered phases, whereas BACE, a membrane protease responsible for the cleavage of the amyloid precursor protein (APP) at its β-site, associated with liquid-ordered phases for 15–20% [37]. To make the picture more complex, when GM1 was included in the lipid composition and was cross-linked with cholera toxin, more BACE associated with the liquid-ordered phase. On the confocal microscope, BACE was shown to be evenly distributed on the vesicle surface, although distinct lipid phases still coexisted, as demonstrated by the segregation of CTXB into one lipid phase (Fig. 5C–D; see [37]). This suggests either a specific GM1–BACE interaction, which drags BACE towards the liquid-ordered phase as a result of the GM1 crosslinking or a rearrangement of the lipid phases, which increases BACE affinity for the ordered phase. Although we still have too little statistics to set specific rules for lipid and protein clustering, it is clear that the structural determinants of protein segregation into and/or exclusion from specific membrane environments are more complex than simple structural factors such as GPI-anchor versus single-span or multi-span. Likely, subtle differences in water–bilayer interface, protein thickness, nature and structure of soluble domains will also play a role in the protein organization in diverse lipid environments. More systematic studies are needed to nail down the subtle structural features that drive proteins into raft-like domains or keep them away from them. Thanks to recent advances in GUV research and electroformation methods, we have now plenty of tools to carry out functionally relevant studies in membranes of increasing complexity. In this respect, interesting developments in membrane studies propose the idea of isolating the native membrane and looking at lipid and protein segregation into GUVs, which would then contain the full natural lipid and protein composition. The first study following this strategy deals with the pulmonary surfactant, the lipid–protein material, which stabilizes the respiratory surface of the lungs and which mainly contains equimolar amounts of unsaturated and saturated phospholipids and cholesterol. De la Serna et al. prepared GUVs from native pulmonary surfactant to study the lateral organization and found the coexistence of two distinct micrometer-sized fluid phases [42]. The lateral domain pattern was shown to be dependent on the presence of cholesterol. Interestingly, the spreading properties of the native pulmonary surfactant were also greatly affected by cholesterol extraction. It was therefore possible to establish a link between the observed domain assembly and the physiological function of the pulmonary surfactant. 5. Conclusions Giant unilamellar vesicles have been widely applied to understand the physic-chemical properties of membranes and electroformation
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has been demonstrated over the years as the method of choice to obtain GUVs from both purified membrane components and natural membrane fractions. Systematic studies under controlled experimental conditions can be now carried out in physiologically relevant environments. In addition, it has been shown that under controlled conditions GUVs from natural membranes maintain the original transbilayer asymmetry. This rich toolbox will allow us to better understand the physicchemical properties of the complex membrane architecture and the intricate interactions of the membrane with the extracellular matrix and/or the cytoskeleton. Acknowledgements We thank Lucie Kalvodova and Salvo Chiantia for useful discussions. References [1] B. de Kruijff, Lipid polymorphism and membrane function, in: A.N. Martonosi (Ed.), The Enzymes of Biological Membranes, 2nd ed., Membrane Structure and Dynamics, vol. 1, Plenum Press, New York, 1985, pp. 131–204. [2] G.J. Schütz, H. Schindler, T. 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Contents lists available at ScienceDirect
Biochimica et Biophysica Acta j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / b b a m e m
Review
Protein–protein and protein–lipid interactions in domain-assembly: Lessons from giant unilamellar vesicles Nicoletta Kahya ⁎ Department of Cell Biology, University Medical Center Groningen, Antonius Deusinglaan 1, 9713 AV Groningen, The Netherlands
a r t i c l e
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Article history: Received 8 December 2009 Received in revised form 2 February 2010 Accepted 21 February 2010 Available online 6 March 2010 Keywords: Giant unilamellar vesicle Lipid raft Membrane protein reconstitution Optical microscopy
a b s t r a c t Giant Unilamellar Vesicles (GUVs) provide a key model membrane system to study lipid–lipid and lipid– protein interactions, which are relevant to vital cellular processes, by (single-molecule) optical microscopy. Here, we review the work on reconstitution techniques for membrane proteins and other preparation methods for developing GUVs towards most suitable close-to-native membrane systems. Next, we present a few applications of protein-containing GUVs to study domain assembly and protein partitioning into raft-like domains. © 2010 Elsevier B.V. All rights reserved.
Contents 1. 2. 3.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . Giant unilamellar vesicles for optical microscopy . . . . . . . . . . . Membrane proteins in giant unilamellar vesicles . . . . . . . . . . . 3.1. Techniques from purified components. . . . . . . . . . . . . 3.1.1. Peptide-induced membrane fusion . . . . . . . . . . 3.1.2. Dehydration and electroformation . . . . . . . . . . 3.2. Techniques from native cellular membrane fractions . . . . . . 4. Lateral distribution of membrane proteins in domain-forming bilayers . 5. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Introduction As an active interface between different biological compartments, membranes guarantee an efficient exchange of matter, energy and/or signals. For this purpose, such an interface has to be designed as a very dynamic system, yet with a non-random distribution of its components. The enormous structural variety of lipids and proteins brings about a Abbreviations: SPT, single particle tracking; FCS, fluorescence correlation spectroscopy; FRET, Föster resonance energy transfer; GUVs, giant unilamellar vesicles; BLMs, black lipid membranes; BR, bacteriorhodopsin; DOPC, dioleoyl-phosphatidylcholine; SM, sphingomyelin (C18:0); APP, amyloid precursor protein; BACE, beta-site-amyloid cleaving enzyme; LUVs, large unilamellar vesicles; DRMs, detergent resistant membranes; PLAP, placental alkaline phosphatase ⁎ Tel.: + 31 50 3636168; fax: + 31 50 3632728. E-mail address: [email protected]. 0005-2736/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.bbamem.2010.02.028
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large spatial and temporal heterogeneity in membrane organization, which may be related to the way the cell accurately regulates biological processes at and around membranes in space and time. The highly intricate architecture of cellular membranes represents a drawback in determining the biological functions of specific lipids and proteins in the top-down approach, by looking at organisms and/or living cells, mainly because of the large number of interfering events simultaneously occurring at the spot of interest. Therefore, a complementary approach with a bottom-up strategy is highly desirable. Within this context, model membranes are key systems to isolate the biological machinery and identify its function. From a methodological point of view, tracking lipids and proteins in space and time represents a big challenge. The small size of lipids makes it technically difficult to follow their trajectories in a reliable and non-invasive way (i.e. the size of the label is often comparable to
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the size of the lipid itself). On the other hand, membrane proteins may be easier to track but the biological implications of their lateral organization and association/dissociation behaviors are, in many cases, tightly linked to the lipid environment, in ways that are up to date not well understood [1]. In recent years, optical microscopy techniques have greatly contributed to broaden our knowledge of membranes, in particular with the development of techniques, which work at the singlemolecule regime (Single Particle Tracking [2] — SPT and Fluorescence (Cross)-Correlation Spectroscopy [3,4] — FCS) and Föster Resonance Energy Transfer (FRET) [5]. In this paper, we focus on Giant Unilamellar Vesicles (GUVs) as model membranes of choice for protein reconstitution and lipid– protein interaction studies by optical microscopy (Section 2). We review the reconstitution techniques developed so far for embedding membrane proteins into GUVs (Section 3). Next, we present a few applications of protein-containing GUVs to study domain assembly and protein partitioning into raft-like domains (Section 4). Finally, we draw some conclusions. 2. Giant unilamellar vesicles for optical microscopy In order to study events occurring at and around membranes by means of optical microscopy, the bilayer has to span the illumination spot in the focal plane, whose lateral dimension goes with the half of the wavelength of light (∼250 nm in the visible range). Compared to other model membranes, which have been developed throughout the years and applied to optical microscopy, GUVs are the most biomimetic (Fig. 1). Monolayers [6] provide a regular and stable structure and their composition can be accurately controlled. However, their ability to mimic biomembranes can be questioned, as they lack the second leaflet to form a bilayer. The first successful attempt to make membranes with a bilayer structure was reported by Müller et al. [7] with the Black Lipid Membranes (BLMs). Two compartments are
Fig. 1. 3D projection of GUVs composed of DOPC, labeled with 0.1% of DiI-C18 (A), SM/DOPC/cholesterol 1:1:1 (molar ratio), labeled with 0.1% of DiI-C18 (B), SM, labeled with 0.1% of DiI-C18. Note that in the bleached dark spot fluorescence does not recover over time (C), SM/DOPC/cholesterol 22:33:33 (molar ratio) and 10 mol% of ceramide (C18:0), labeled with 0.1% of DiD-C18 (D). Scale bars: 10 μm (A), 10 μm (B), 5 μm (C), 10 μm (D).
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separated by a thin partition (septum) and communicate through an aperture (100–200 μm in diameter) in this partition. An organic solution of lipids is brushed over the hole and then the compartments are filled with an aqueous medium. Dispersion of the solvent in the aqueous medium eventually leaves a bilayer on the hole. In practice, however, there are many technical limitations, which prevent the formation of an ideal BLM and lead to an irregular bilayer structure [8]. A very interesting alternative to BLMs is represented by solidsupported bilayers. The early concept was based on the fusion of small unilamellar vesicles (diameter b100 nm), which can be deposited onto the hydrophilic surface of a clean polar substrate, such as glass or mica. This results in a supported membrane [9] floating on an ultra-thin (∼1–2 nm) water layer. In general, the absence of a “bulk aqueous medium” between the bilayer and the solid substrate may give rise to secondary lipid–substrate interactions and, thereby, to artifacts. In particular, many membrane proteins protrude from the bilayer surface into the water phase, much further than 1–2 nm, implying their soluble residues interact with the solid substrate. Some of these drawbacks have been partially solved by designing a tethered bilayer [10], which is composed of a solid substrate, a tethering layer (e.g. a soft polymer cushion) and a lipid bilayer. The soft and hydrophilic polymer should keep the lipid bilayer far apart from the rigid support, and thereby, preserve membrane fluidity. However, membrane proteins embedded in such a lipid matrix interact with the polymer and appear to be only partially mobile [10]. The artifacts potentially found in solid-supported bilayers are absent in GUVs, which are spherical closed single bilayers, freely floating in aqueous solution [11,12]. They are suitable for (single-molecule) optical microscopy and exhibit a cell-like size, as their size ranges from 10 to 100 μm in diameter. The most common method for the production of GUVs of consistent and reproducible unilamellarity is the electroformation method, in which a dry lipid film is rehydrated in the presence of an alternating electric field for typically 1–3 h [11,12]. GUVs obtained with this method can be studied in situ (as immobilized on the electrodes, either Pt wires or ITO-coated glass coverslips) or can be detached from the electrodes and transferred into other aqueous environments. When transferred to other environments, GUVs may be observed as freely moving (e.g. to study membrane fluctuations and measure mechanical properties such as bending elasticity [13]), or they can be immobilized onto glass surfaces, which are coated with several chemicals, e.g. poly-Llysine [14], casein [15], albumin [16], protein anchors such as avidin/ streptavidin [17], or with lipid patterns [18], to study the effect of specific molecular interactions onto membrane adhesion properties, as relevant to cell–cell and cell–matrix interactions. From all of these studies, it is evident that immobilizing vesicles onto a surface might change both the collective membrane properties (e.g. mechanical) and the molecular self-organization within the bilayer. Researchers engaging with these methodologies need to be aware of these effects (and/or potential artifacts) on membrane structure and dynamics. GUVs can also be trapped by a micropipette for investigating membrane stretching elasticity [19] and for pulling tubes to study membrane budding [20,21]. Although very reproducible and reliable, the GUV electroformation method poses a few limitations, which have, in some cases, limited the applicability of GUVs, such as: 1) low vesicle yield in buffers of high ionic strength and/or physiological ion concentrations, 2) absence of transbilayer asymmetry, 3) inability to control and determine the exact composition of individual vesicles, and 4) difficulty to include membrane proteins into the electroformation procedure. Recently, as to point 1) and 2), efforts have been taken to attain high GUV electroformation yields in physiological buffers [22] and to show that transbilayer asymmetry was preserved by starting from red blood cell ghosts [23]. As to point 3), the variability of composition between vesicles exists and varies depending on type of lipids, temperature and composition of aqueous environment. It is, therefore, good practice and, at times, essential to the data interpretation, to discuss and/or provide an estimate of the variability between individual vesicles [see, for
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instance, 24,25]. As to point 4), the use of organic solvents and the dehydration step in the electroformation easily lead to denaturation of membrane proteins. As discussed in the following section, various reconstitution procedures have been developed over the years to circumvent this problem. 3. Membrane proteins in giant unilamellar vesicles Several methods have been developed to incorporate membrane proteins into GUVs. This section will briefly describe the most common methodologies employed to reconstitute membrane proteins into GUVs and will be followed by examples of how these methods have led to a deeper understanding for how proteins function in membranes. As mentioned in the previous section, the most common method for the production of GUVs of consistent and reproducible unilamellarity is the electroformation method, in which a dry lipid film is rehydrated in the presence of an alternating current electric field [11,12]. This methodology was extended to include drying membrane proteins with the lipid such that it incorporated proteins into the GUVs upon electroswelling. This was first achieved by Manneville et al. [26] for bacteriorhodopsin. However, the method is not applicable in general to membrane proteins, as dissolving such proteins in organic solvents and subsequently drying them lead, in general, to misfolding and denaturation. 3.1. Techniques from purified components 3.1.1. Peptide-induced membrane fusion Another methodology for incorporating membrane proteins into electroformed GUVs is to fuse the GUVs with small preformed proteoliposomes. This method was developed by Kahya et al. [27] and shown for the reconstitution of bacteriorhodopsin (BR) from purple membranes of Halobacterium salinarium. BR was first reconstituted into Large Unilamellar Vesicles (LUVs), which contained a fusion peptide (WAE) and were, thereby, able to fuse with preformed giant unilamellar vesicles. After full membrane fusion of BR-containing LUVs with GUVs, as shown by lipid mixing and water content mixing, BR was then fully reconstituted into GUVs and shown to retain its proton-pumping activity (Fig. 2). Compared to other reconstitution methods, this technique is less straightforward, as it requires multiple steps and it employs peptide-induced membrane fusion, which imposes some constraints on the lipid composition of the vesicles and might potentially interfere with the function of the protein of interest. An elegant example of how this method can be applied to monitoring of lipid–protein and protein–protein interactions in lipid bilayers at physiological concentrations was demonstrated by Kriegsman et al. [28]. They reconstituted the complex of the photoreceptor NpSRII, which consists of seven-transmembrane α-helices, and its cognate transducer NpHtrII, which contains two transmembrane α-helices and a large cytoplasmic domain. They could measure the lateral diffusion of the distinct membrane proteins and quantify the degree of oligomerization. In fact, heterodimeric complexes were characterized quantitatively with dual-color fluorescence correlation spectroscopy
and the intermolecular binding between NpSRII and NpHtrII in the functional unit (with stoichiometry 2:2) was found to be extremely strong, as complexes formed in bilayers with low molar protein:lipid ratios (∼1:2,000,000). 3.1.2. Dehydration and electroformation One of the simplest techniques to produce giant proteoliposomes consists of dehydration of preformed proteoliposomes followed by spontaneous swelling upon rehydration with a buffer [29–31]. The major advantage of such a technique is that vesicles can be prepared with a wide range of buffers. However, the vesicles produced from the rehydration are very heterogeneous and mainly of multilamellar nature. Nonetheless, this method has proved useful in pulling lipid tubes from vesicles, which have been rehydrated from a multilamellar lipid base [32,33]. By using micropipettes, these tubes can be extended to inflate new GUVs and even to create vast lipid tubule networks. A further development of this reconstitution technique involves the partial dehydration of preformed proteoliposomes followed by controlled rehydration via electroformation. Compared to peptideinduced vesicle fusion, partial dehydration of proteoliposomes followed by electroformation is more straightforward and does not require fusogenic molecules. It also results in consistent and homogeneous preparation of GUVs. The method was first applied by Girard et al. [34,35] to incorporate the Ca2+-ATPase as well as bacteriorhodopsin into GUVs. A similar technique, which makes use of partial dehydration of proteoliposomes in the presence of a cryoprotectant that prevents protein misfolding and/or denaturation during drying, has been developed to reconstitute human placental alkaline phosphatase (PLAP) into GUVs (Fig. 3) and examine the protein lateral partitioning in distinct lipid phases [36]. The same reconstitution technique was later applied to investigate the proteolytic activity of the β-secretase, beta-site-amyloid cleaving enzyme (BACE) [37], and its lateral partitioning in liquid-ordered and liquid-disordered lipid phases. Over the years, many variants of this reconstitution technique have been successfully applied to obtain giant proteolipomes (for examples see [38,39]). We and others have reconstituted a number of membrane proteins with very diverse topology, from GPI-anchored proteins (for example PLAP [36], human prion), to transmembrane model peptides (WALP, see [40]), singlespanning membrane proteins (for example BACE [37], syntaxin [38], synaptobrevin [38]) and multi-spanning membrane proteins in various oligomeric states (for example glutamate transporter, mechanosensitive channel, lactose transporter and lactose permease; see [40]). The wide applicability of this reconstitution technique also includes a large variety of lipid compositions, buffers and, in general, physiological conditions. Recently, an elegant application of this reconstitution method was provided by Streicher et al. [41], who developed an in vitro system to mimic the first steps of integrinmediated cell spreading, and, thereby, studying fundamental aspects of cell adhesion, as implicated into cell–cell contacts in tissue and/or in interaction of cells with the extracellular matrix. Integrin from human platelets was functionally reconstituted into GUVs, thereby
Fig. 2. Protein reconstitution into GUVs by peptide-induced membrane fusion: (A) confocal image of GUV after fusion with BR-containing LUVs. The fluorescence signal is given by AF-488labeled BR; (B) activity assay for BR reconstituted into GUVs. The fluorescence signal is given by the pH-sensitive dye pyrene. The fluorescence level from the lumen of GUVs is lower than outside of the GUV, which implies a more acidic pH in the GUVs lumen and, therefore, proving a proton-pumping activity of BR [27].
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Fig. 3. Protein reconstitution into GUVs by partial dehydration followed by electroformation: confocal image of GUVs composed of DOPC, in which AF-488 labeled BR was reconstituted [36]. Scale bar: 10 μm.
allowing for a detailed investigation of the mechanical static as well as dynamic aspects of adhesion of such vesicles onto fibrinogen-coated surfaces.
3.2. Techniques from native cellular membrane fractions One of the most exciting developments in GUV research lies on the reconstitution of membrane proteins in their native membrane environment. This was achieved by extracting native membranes from cellular fractions, without the removal of the proteins, and by applying the electroformation procedure directly to the membrane fractions to prepare GUVs [42]. Recently, we have explored such options with respect the transmembrane receptors, ion channels and ATPases found in all eukaryotic cells. Such proteins include the Na/K-ATPase, the voltage-gated sodium channel, inositol 1,4,5-triphosphate receptor, CD44, L1CAM and the Na/Ca exchanger, just to name a few. Specifically, we focused on the Na/K-ATPase, which plays a crucial role in generating energy, maintaining osmotic balance within a cell, participating in cellular signaling, and anchoring of cytoskeletal components to the plasma membrane [43]. We were interested in investigating the coupling of actin filaments to the membrane bilayer, as in the native membrane. To this end, membrane proteins reconstituted into GUVs were essential for binding of the cytoskeleton, as the anchoring of spectrin/ankyrin and actin filaments to the membrane has been shown to require membrane proteins, among others the Na/K-ATPase [43]. Previous studies had focused on actin polymerization inside (or outside) GUVs made from simple lipid compositions [44–48]. In our system, we reconstituted the whole complex lipid–protein system necessary to achieve spectrin/ankyrin-mediated binding of actin filaments to the membrane. We set out to prepare GUVs from porcine brain membrane fractions, in physiological conditions [49], by using the partial dehydration and rehydration via electroformation. Such a preparation yielded consistent and reproducible GUVs with diameters of 10–100 μm (Fig. 4). The presence of Na/K-ATPase was demonstrated by binding of a mouse monoclonal antibody directed against Na/K-ATPase and with a secondary goat-anti-mouse antibody which was conjugated to the Cy5 fluorescent dye. GUVs incubated in the absence of primary antibody, or with a non-specific primary mouse monoclonal antibody did not stain the GUVs [49]. The presence of Na/K-ATPase in these fractions was also confirmed via immunoblot analysis. Subsequently, actin filaments were reconstituted into such GUVs and proved to specifically bind to the membrane bilayer (Fig. 4, [49]). Current investigations employ such a system to study the mechanical aspects of the membrane–cytoskeleton coupling in a close-to-native system. In addition, consistent and reproducible GUVs can also be prepared by partial native membrane dehydration followed by electroformation starting from internal organelle fractions, as we were able to
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Fig. 4. Confocal images of GUVs prepared from porcine brain membrane extracts with a highly enriched fraction of spectrin/ankyrin, and labeled with 0.1% of DiD-C18 (red) in the presence of actin (visualized with phalloidin-Alexa 488 — green) [49]. Scale bar: 10 μm.
obtain GUVs with excellent yield from endosomal fractions of HeLa cells (N. Kahya, unpublished results). Other groups have meanwhile provided evidence that electroformation is the method of choice for yielding GUVs from native membrane fractions. Montes et al. have recently shown that GUVs can be prepared from red blood cell ghosts in physiological buffers [23,35]. Interestingly, GUVs prepared with this method maintained the original transbilayer asymmetry of the red blood cell membranes, as shown by immunofluorescent staining of GUVs against the cytoplasmic domain of band III. These novel applications pave the way to elucidating specific molecular interactions in the membrane architecture and its dynamic organization needed to carry out essential cellular functions. Finally, it is worth to mention an alternative method to obtain giant liposomes from plasma membranes, which is cell “blebbing” (see for example Baumgart et al. [50]), essentially a chemicallyinduced vesiculation of cells. Although promising, more studies are needed to understand and interpret the results, as to what is the nature of the overall mechanism of vesicle formation and the effect of the chemicals employed on the cell wall and surroundings.
4. Lateral distribution of membrane proteins in domain-forming bilayers Lipid–protein and protein–protein interactions have been shown to play a key role in regulating the function of biological machinery's at the membrane and in its surroundings (see for instance [51–54]. One of the most exciting ways for nature to tightly regulate in time and space intraand intermolecular interactions at the membrane is through spatiotemporal segregation of certain components to so-called “rafts” [55–57] and the exclusion of unwanted components from them. Rafts were defined as “small (10–200 nm), heterogeneous, highly dynamic, steroland sphingolipid-enriched domains that compartmentalize cellular processes. Small rafts can sometimes be stabilized to form larger platforms through protein–protein interactions” from the (Keystone Symposium of Lipid Rafts and Cell Function, held in 2006). Many studies have added to the understanding of the mechanisms of raft formation, which is most likely dependent on a very fine balance of specific lipid– protein interactions and protein–protein associations [58,59]. Cellular membranes are very crowded and, on average, proteins populate the bilayer up to one half of the total membrane mass [60]. By adopting a bottom-up approach and, thereby, starting from a minimum of membrane components to study specific physico-chemical properties of rafts, it is, simply not enough to examine pure lipid systems. We set out to develop biophysical tools to test some ideas concerning rules and structural requirements, which are responsible for targeting membrane proteins to lipid environments of specific chemistry. We could then test whether and how the lipid matrix influences the mechanisms of function of membrane proteins.
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Putative raft-associated and non-raft proteins were reconstituted into domain-exhibiting GUVs [36–38] (Fig. 5). Their spatial organization was observed by optical imaging and FCS. The human placental alkaline phosphatase (PLAP) was abundantly found in detergent resistant membranes (DRMs) after treatment with Triton X-100 at 4 °C [61,62]. However, it mainly associated with liquid-disordered DOPC-enriched phases in GUVs composed of DOPC/SM/cholesterol (1:1:1) (see Fig. 5A), as shown by FCS and by counterstaining the raftlike liquid-ordered and SM-enriched phase with GM1-bound fluorescent cholera toxin [36]. Similar GPI-anchored proteins, such as the bovine intestine alkaline phosphatase, exhibited a comparable behavior (N. Kahya and S. Morandat, unpublished results). FCS measurements of local protein density in distinct phases revealed that at most 25–30% of PLAP partitioned into the liquid-ordered phase. Furthermore, antibody-mediated cross-linking caused the protein to associate more (up to 50%) with the liquid-ordered phase [36]. Taken together, this data showed that PLAP exhibits a higher affinity for the raft-like liquid-ordered phase compared to other membrane proteins, such as syntaxin [38], synaptobrevin [38] and
Fig. 5. (A) Confocal image of GUVs composed of SM/DOPC/cholesterol 1:1:1 (molar) and GPI-anchored rhodamine-labeled PLAP (Rho-PLAP) [36]. Scale bar: 10 μm. (B) Confocal image of GUVs composed of SM/DOPC/cholesterol 1:1:1 and Cy5-labeled syntaxin [38]. Scale bar: 10 μm. (C) Confocal image of GUVs composed of SM/DOPC/ cholesterol 1:1:1 and 0.1% of GM1 containing Cy5-labeled BACE (Cy5-BACE) — red channel [37]. Scale bar: 10 μm. (D) Confocal image as in (C): fluorescence signal from AF488-labeled cholera toxin (AF488-CTXB) — green channel [36]. Scale bar: 10 μm. (E, F) Confocal images of GUVs composed of SM/DOPC/cholesterol 1:1:1, AF488labeled GPI-anchored human prion (green) and 0.1% of DiI-C18 (red) (N. Kahya and P. Schwille, unpublished results). Scale bar: 10 μm.
bacteriorhodopsin [36], which were fully associated with the liquiddisordered phase (Fig. 5B). The different partitioning behavior is however not simply related to the nature of binding to the membrane (GPI-anchor versus transmembrane domain). The fact that subtler rules play a crucial role in determining the protein affinity for specific lipid phases is inferred by the following two observations. First, among GPIanchored proteins, PLAP exhibited similar partitioning behavior to its corresponding bovine form, as mentioned above. However, the GPI-anchored human prion protein, involved in Creutzfeld–Jakob disease, which was reconstituted into GUVs composed of DOPC/SM/ cholesterol (1:1:1 molar ratios), was fully associated with the raft-like liquid-ordered and SM-enriched phase (Fig. 5E–F, green), as opposed to the DiI-C18 (Fig. 5E–F, red), which stained the liquid-disordered phase (N. Kahya and P. Schwille, unpublished results). Second, among single-spanning membrane proteins, we found significant differences between partition coefficients for liquidordered phases. In this respect, as shown in GUV studies with the same lipid composition, syntaxin and synaptobrevin were fully excluded from liquid-ordered phases, whereas BACE, a membrane protease responsible for the cleavage of the amyloid precursor protein (APP) at its β-site, associated with liquid-ordered phases for 15–20% [37]. To make the picture more complex, when GM1 was included in the lipid composition and was cross-linked with cholera toxin, more BACE associated with the liquid-ordered phase. On the confocal microscope, BACE was shown to be evenly distributed on the vesicle surface, although distinct lipid phases still coexisted, as demonstrated by the segregation of CTXB into one lipid phase (Fig. 5C–D; see [37]). This suggests either a specific GM1–BACE interaction, which drags BACE towards the liquid-ordered phase as a result of the GM1 crosslinking or a rearrangement of the lipid phases, which increases BACE affinity for the ordered phase. Although we still have too little statistics to set specific rules for lipid and protein clustering, it is clear that the structural determinants of protein segregation into and/or exclusion from specific membrane environments are more complex than simple structural factors such as GPI-anchor versus single-span or multi-span. Likely, subtle differences in water–bilayer interface, protein thickness, nature and structure of soluble domains will also play a role in the protein organization in diverse lipid environments. More systematic studies are needed to nail down the subtle structural features that drive proteins into raft-like domains or keep them away from them. Thanks to recent advances in GUV research and electroformation methods, we have now plenty of tools to carry out functionally relevant studies in membranes of increasing complexity. In this respect, interesting developments in membrane studies propose the idea of isolating the native membrane and looking at lipid and protein segregation into GUVs, which would then contain the full natural lipid and protein composition. The first study following this strategy deals with the pulmonary surfactant, the lipid–protein material, which stabilizes the respiratory surface of the lungs and which mainly contains equimolar amounts of unsaturated and saturated phospholipids and cholesterol. De la Serna et al. prepared GUVs from native pulmonary surfactant to study the lateral organization and found the coexistence of two distinct micrometer-sized fluid phases [42]. The lateral domain pattern was shown to be dependent on the presence of cholesterol. Interestingly, the spreading properties of the native pulmonary surfactant were also greatly affected by cholesterol extraction. It was therefore possible to establish a link between the observed domain assembly and the physiological function of the pulmonary surfactant. 5. Conclusions Giant unilamellar vesicles have been widely applied to understand the physic-chemical properties of membranes and electroformation
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has been demonstrated over the years as the method of choice to obtain GUVs from both purified membrane components and natural membrane fractions. Systematic studies under controlled experimental conditions can be now carried out in physiologically relevant environments. In addition, it has been shown that under controlled conditions GUVs from natural membranes maintain the original transbilayer asymmetry. This rich toolbox will allow us to better understand the physicchemical properties of the complex membrane architecture and the intricate interactions of the membrane with the extracellular matrix and/or the cytoskeleton. Acknowledgements We thank Lucie Kalvodova and Salvo Chiantia for useful discussions. References [1] B. de Kruijff, Lipid polymorphism and membrane function, in: A.N. Martonosi (Ed.), The Enzymes of Biological Membranes, 2nd ed., Membrane Structure and Dynamics, vol. 1, Plenum Press, New York, 1985, pp. 131–204. [2] G.J. Schütz, H. Schindler, T. 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