http://informahealthcare.com/mbc ISSN: 0968-7688 (print), 1464-5203 (electronic) Mol Membr Biol, 2014; 31(2–3): 47–57 ! 2014 Informa UK Ltd. DOI: 10.3109/09687688.2014.896485

REVIEW ARTICLE

Membrane rafts of the human red blood cell Annarita Ciana, Cesare Achilli, and Giampaolo Minetti

Abstract

Keywords

The cell type of election for the study of cell membranes, the mammalian non-nucleated erythrocyte, has been scarcely considered in the research of membrane rafts of the plasma membrane. However, detergent-resistant-membranes (DRM) were actually first described in human erythrocytes, as a fraction resisting solubilization by the nonionic detergent Triton X-100. These DRMs were insoluble entities of high density, easily pelleted by centrifugation, as opposed to the now accepted concept of lipid raft-like membrane fractions as material floating in low-density regions of sucrose gradients. The present article reviews the available literature on membrane rafts/DRMs in human erythrocytes from an historical point of view, describing the experiments that provided the solution to the above described discrepancy and suggesting possible avenue of research in the field of membrane rafts that, moving from the most studied model of living cell membrane, the erythrocyte’s, could be relevant also for other cell types.

Cholesterol, DRM, erythrocytes, granulocytes, lipid rafts, nonionic detergents, proteolysis History Received 24 August 2013 Revised 17 February 2014 Accepted 17 February 2014 Published online 10 April 2014

Abbreviations: RBC: red blood cell; DRMs: detergent resistant membranes; PS: phosphatidylserine; GPI: glycosyl phosphatidyl inositol; GSLs: glycosphingolipids; TX100: Triton X-100

Introduction A number of thorough and authoritative reviews have appeared over the years concerning lipid rafts (Brown, 2006; Edidin, 2001, 2003; Fantini et al., 2002; Grzybek et al., 2005; Harder & Engelhardt, 2004; Henderson et al., 2004; Mayor & Rao, 2004; Munro, 2003; Pike, 2004, 2006, 2009; Simons & Ikonen, 1997; Sonnino & Prinetti, 2013). The purpose of the present paper is to give an historical perspective on the connection between lipid rafts research and the investigation of the RBC as a paradigm model of plasma membranes. As we will see, the two lines of investigation, which entail basic and fundamental issues in cell biology, such as membrane composition, topology and topography, ran parallel during most of the pioneering years. The two lines then departed, and each followed a separate trail until they were lately reunited. Despite this, the lipid rafts hypothesis is still far from being corroborated by evidence obtained from studies of the paradigmatic erythrocyte membrane. In the present review, the term ‘‘membrane rafts’’ will be used throughout, in adherence to the consensus reached at the Keystone Symposium in 2006 on the definition of the ‘‘noumenal’’ native rafts as ‘‘Membrane rafts. . .’’, which

Correspondence: Dr Giampaolo Minetti, PhD, University of Pavia, Department of Biology and Biotechnology ‘‘Lazzaro Spallanzani’’, Laboratories of Biochemistry, via Bassi, 21 - 27100 Pavia, Italy. Tel: +39 0382 987891. Fax: +39 0382 987240. E-mail: [email protected]

‘‘. . .are small (10–200 nm), heterogeneous, highly dynamic, sterol- and sphingolipid-enriched domains that compartmentalize cellular processes. Small rafts can sometimes be stabilized to form larger platforms through protein-protein and protein-lipid interactions’’ (Pike, 2006). Consequently, the ‘‘detergent resistant membranes’’ (DRMs) that have been historically likened to the native lipid rafts, and can only be considered a poor ‘‘phenomenal’’ manifestation of the ‘‘noumenal’’ entity, will be defined as ‘‘lipid-raft-like’’ membrane fractions.

Protein and lipid sorting to the cell plasma membrane

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Laboratories of Biochemistry, Department of Biology and Biotechnology ‘‘Lazzaro Spallanzani’’, University of Pavia, Pavia, Italy

Although the concept of lipid domains in biological membranes was already proposed more than thirty years ago (Karnovsky et al., 1982; Morrisett et al., 1975), the now familiar definition of membrane rafts as local, lateral inhomogeneities in the lipid and protein composition of the plasma membrane, was coined relatively late in respect to the time when the studies from which it drew were conducted (Simons & Ikonen, 1997). Most of such studies were, in fact, the foundations of cell biology, dealing with such issues as membrane biogenesis, subcellular organelles, protein sorting, and intracellular vesicular trafficking. Their origin could be placed back in the 1940s, with the pioneering work of researchers such as Albert Claude (Claude, 1974), Christian de Duve (de Duve, 1974), George Palade (Palade, 1974), and, later, Gu¨nter Blobel (Blobel, 1999), to name only a few who received the Nobel Prize for their discoveries. In the specific,

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the notion that not only proteins but also lipids could be sorted not only transversally but also laterally in the cell, came from the observation that, in polarized cells, the apical membrane is enriched in glycosphingolipids (GSLs) with respect to the basolateral membrane (Simons & Fuller, 1985). The mechanism of delivery of GSLs to the apical domain was shown to be based on vesicular transport (van Meer et al., 1987). What was also striking is that the glycosyl-phosphatidyl-inositol (GPI)-anchored proteins are sorted exclusively to the apical membrane in polarized cells (Lisanti et al., 1988). It was therefore originally proposed by Simons and van Meer that the GPI-linked proteins and GSLs followed the same sorting pathway to the apical domain of the plasma membrane. More precisely, they proposed that apicallydestined membrane proteins associate with ‘‘patches’’ of GSLs already in the Golgi apparatus (where the biosynthesis of GSLs takes place), whereas glycerophospholipids and cholesterol are synthesized in the endoplasmic reticulum (Holthuis et al., 2001), and are packaged together in vesicles directed to the cell apex (Simons & van Meer, 1988). That lateral segregation of sphingolipids could occur in membranes, and that cholesterol could play a role, were notions already proposed in the late 1970s (Hong-wei & McConnell, 1975; Karnovsky et al., 1982; Recktenwald & McConnell, 1981). Moreover, the high propensity of GSLs to self-associate and form aggregates when mixed with phospholipids was also well established (Thompson & Tillack, 1985). Therefore, the question was whether the GSLs and the GPIlinked proteins are indeed associated in the apical domain of polarized cells. Here is where the use of nonionic detergents came into play in the study of lipid microdomains. It was already known that GPI-linked proteins are resistant to solubilization with nonionic detergents such as Triton X-100 (TX100). The reason for this insolubility was rather mysterious, since GPI-linked proteins are inserted in the plasma membrane with the lipid anchor of the phosphoinositol, which should easily partition in the soluble micellar phase induced by the detergent. Moreover, as the polypeptide portion of these proteins is entirely located in the extracellular space, they should not be retained in larger supramolecular complexes by interaction with the cytoskeleton. Not only was the insolubility of GPI-linked proteins an established fact, but also GSLs and sphingomyelin were long known to be resistant to solubilization by TX100 (Yu et al., 1973). The logical question that ensued, i.e. whether the GPIlinked proteins and GSLs are indeed associated in the detergent insoluble-material, was addressed experimentally by Brown and Rose in a work that became the reference method for the isolation of lipid rafts as DRMs (Brown & Rose, 1992). Although the term ‘‘lipid rafts’’ was introduced later, these entities were most commonly obtained as detergent-resistant membranes. It is factual that a significant fraction of the 44100 articles that show up in Pubmed (http:// www.ncbi.nlm.nih.gov/sites/entrez?db¼pubmed) at a search of the string ‘‘lipid rafts’’ as of August 2013 are conducted on material isolated as DRMs. However, the notion of DRMs conveyed for some time the concept of an insoluble entity which was easily pelleted by centrifugation, and only later it was considered as a good representation of ‘‘lipid rafts’’ as material floating in low-density regions of sucrose gradients.

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Although other nonionic detergents have been considered for isolation of DRMs (Crepaldi Domingues et al., 2009; Domingues et al., 2010; Schuck et al., 2003), and caution should be used in drawing conclusions from results obtained with only one type (Lingwood & Simons, 2007), TX100 is by far the most used. A protocol was ‘‘coded’’ in the Brown and Rose article (Brown & Rose, 1992), which entailed lysing MDCK cells grown to confluence in a 150 mm dish (approximately 108 cells (Rosen & Misfeldt, 1980, Sargiacomo et al., 1993)) for 20 min on ice with a total of 2 ml of 25 mM Tris-Cl (pH 7.5), 150 mM NaCl, 5 mM EDTA, 1% TX100. Interestingly, no mention to the use of antiproteases was made in the original paper. The sample was then homogenized with eight strokes of a Dounce homogenizer, brought to 40% sucrose using an 80% solution of sucrose in the same buffer but without TX100, and placed in an ultracentrifuge tube. A linear sucrose gradient (5–30% in the same buffer without TX100) was layered on the sample and the tube spun at 200 000 g for 15–22 h at 4  C (note the long centrifugation time). At the end of the centrifugation, 1 ml fractions were collected from the tube for subsequent analysis. The detergent-resistant material was recovered as a visible band (different refractive index) in the low-density region of the gradient (1.081 g/cm3). This is the ‘‘coded’’ procedure which was followed by countless works on DRM/lipid rafts in the years that followed.

Detergent resistance of the RBC: The early years It may seem strange that the cell type of election for the study of plasma membranes, the RBC, was not considered in the research of rafts of the plasma membrane. However DRMs complying with the definition accepted for some time for ‘‘lipid rafts’’ as detergent insoluble-membrane, except for the buoyant density, were actually first described in the RBC. In fact, Steck and co-workers first described a TX100resistant fraction of the RBC membrane after treatment of ghosts [carefully prepared to ‘‘minimize contamination of the ghosts with proteinases’’ (Fairbanks et al., 1971), see below] with cold TX100 and sucrose density gradient fractionation: The detergent resistant fraction was enriched in sphingomyelin and glycosphingolipids and, most importantly, was isolated as a high-density fraction in sucrose gradients, because it was associated with the spectrin membraneskeleton (Yu et al., 1973). The density of this fraction ranged from 1.14 to 1.20 g/cm3, depending on the lipid to protein ratio. These DRMs were observed with the electron microscope. This is important, because it was shown that these structures were not simply aggregates of dispersed lipid and proteins but they derived from the original ghosts. A cautionary note informed about the peculiar fragility of the material, important for the discussion to follow: ‘‘Triton-extracted membranes are fragile, being disrupted by shearing and aggregated by pelleting’’. Their appearance was that of ‘‘aggregated masses of filaments surrounding more densely staining (presumably lipids) vesicles and fragments’’ (Yu et al., 1973). ‘‘The sheetlike and vesicular profiles in the Triton residues [. . .] demonstrate that the retained lipids are not merely bound to protein but form apolar associations with one another which

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DOI: 10.3109/09687688.2014.896485

withstand detergent action’’. It was also clear to the Authors the possible artifactual origin of these detergent-resistant lipid clusters, an issue that has not yet been resolved (Lichtenberg et al., 2005): ‘‘Recent physical studies of mixed phospholipid [. . .] and cholesterol phospholipid [. . .] systems have advanced the concept of clustering of disparate lipid species in membranes. One might speculate on whether the sphingolipid segments observed in the Triton X-100 residues reflect such clusters, and whether these aggregates arise by demixing following detergent action or exist in some form in the original ghost’’ (Yu et al., 1973). A few years later, confirmatory results and further insights were provided by Michael P. Sheetz (Sheetz, 1979). He aimed at characterizing the binding of integral membrane proteins to the cytoskeleton, and therefore his main focus was on the isolation of the TX100-insoluble cytoskeletons from the detergent-soluble components. The data showed that, after treatment of RBCs with TX100 under the conditions that were later adopted for the isolation of lipid raft-like material (1% TX100, 4  C, ultracentrifugation in sucrose density gradient), the insoluble material collected after ultracentrifugation as a light-scattering band had a density of approximately 1.20 g/cm3 (Figure 1) and a protein-to-phospholipid ratio of approximately 2, and was enriched in sphingomyelin. The conclusion is that the detergent-resistant fraction of the RBC membrane remains tightly anchored to the cytoskeleton when RBCs are subjected to the operational protocol that was later adopted for the isolation of lipid raft-like entities as light-density fractions in sucrose gradients. It can be seen that the concentration of TX100 that was originally chosen for the protocol of lipid raft isolation appears to be rather arbitrary when considering that it is located, in the graph of Figure 1, on the steepest region of the ‘‘phospholipid content/detergent concentration’’ curve, where small variations in detergent concentration could determine large changes in phospholipid content, and hence buoyant density of the material. It could be wondered whether this fact could contribute to the variability of the lipid raft-like material isolated from different cell types or even from the same cell type from different laboratories.

Figure 1. Graph extracted and adapted from (Sheetz, 1979) with permission from Elsevier. The graph shows the buoyant density (black, filled circles) and the phosphate content (empty squares) of cytoskeletons extracted from whole RBCs at various TX100 concentrations, at 4  C. The vertical line marked ‘‘50 mg/ml of cells’’ refers to the TX100 concentration most commonly used in extracting lipid rafts from human erythrocytes (either with or without carbonate, see text for details).

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The original identification of lipid raft-like domains in the RBCs has been later acknowledged, although these DRMs were not isolated as a low-density fraction like in other cell types (Brown & London, 1997). In even more recent years, building on the work of Steck and co-workers (Yu et al., 1973), Bu¨tikofer and co-workers showed that, in RBCs, GPIlinked proteins are enriched in the TX100-insoluble fraction. Their detergent-insoluble fraction, like the one described by Steck and co-workers and later by Sheetz, ‘‘. . . contained most of the major skeletal proteins, i.e. spectrin, ankyrin, protein 4.1, and actin; whereas band 3, i.e. the major integral membrane protein, together with protein 4.2 and band 6, were preferentially present in the soluble fraction’’ (Civenni et al., 1998). These authors were also already conscious that their GPI-anchored protein-rich, detergent-resistant material ‘‘was found to band at 31% (wt/vol) sucrose [. . .] as compared with the detergent-insoluble material from other membranes which has been found to band at 15% to 25% (wt/vol) sucrose’’ (Civenni et al., 1998). The reference to ‘‘other membranes’’ is also to the work of Brown and Rose on GPI-linked proteins and GSL sorting to the apical domain of MDCK cells, as mentioned above (Brown & Rose, 1992).

The isolation of low-density DRMs from human RBCs The detergent resistant membranes isolated from cell types other than the RBC were called, in those years, in a number of ways: GEMs (Glycolipid-Enriched Membranes) (Rodgers et al., 1994), DIGs (Detergent-Insoluble Glycolipid-enriched domains) (Parton & Simons, 1995), TIFF (Triton-Insoluble Floating Fraction) (Kurzchalia et al., 1995), TIM (TritonInsoluble Membranes) (Garcı´a-Carden˜a et al., 1996) or LDTI (Low-Density Triton-Insoluble fractions) (Parolini et al., 1996). Common to all these definitions is the reference to material floating in the low-density region of a sucrose gradient. In the light of what was already known from studies of detergent-treated RBC membranes, it was therefore surprising that, from a certain point onward, low-density DRMs could be obtained also from RBCs, without the requirement for additional maneuvers. The turning point in this story is represented by an article from Haldar and co-workers (Lauer et al., 2000), whose results on human RBCs subjected to detergent treatment were clearly at variance with those published in the three previous decades, because DRMs could be obtained from RBCs as TX100-resistant, low-density material. However, the contradiction was not noticed, and many other articles appeared in which lipid raft-like fractions were isolated from RBCs as low-density TX100-insoluble material, according to the original operational definition of Brown and Rose (Fricke et al., 2003; Kamata et al., 2008; Koumanov et al., 2005; Lach et al., 2012; Murphy et al., 2004; Rivas & Gennaro, 2003; Rodi et al., 2006; Salzer & Prohaska, 2001; Salzer et al., 2002; Samuel et al., 2001; Wilkinson et al., 2008). Most of these studies, however, are plagued by an artifactual condition dependent on granulocyte contamination and proteolysis (see below). We observed previously, in fact, that low-density DRMs could be isolated from RBC ghosts only with the addition of carbonate to the detergent-treated membranes (Ciana et al., 2005), while all other studies on lipid rafts in

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RBCs did not include this treatment. The reason for this discrepancy was not clear. Only a couple of other works mentioned the use of carbonate for the isolation of lowdensity, TX100-resistant raft-like material from human RBCs (Nagao et al., 2002; Tokumasu et al., 2009). This situation is illustrated in Figure 2 where a typical sucrose gradient, loaded with TX100-treated whole human RBCs, is shown after 16 h ultracentrifugation. In the absence of carbonate, the translucent band that contains the detergent-insoluble material, remains at the interface between 40% and 30% sucrose, indicating that it is associated with the membrane-skeleton, as previously described (tube 1, arrow) (Civenni et al., 1998; Sheetz, 1979; Yu et al., 1973). Conversely, the detergentinsoluble fraction floats at the expected density for lipid raftlike material only if the sample is treated with carbonate (tube 2, arrow).

The solution to the riddle: A story of proteolysis Since the original description of the problematic isolation of low-density DRMs from human RBCs, where carbonate was introduced as a necessary ingredient (Ciana et al., 2005), several other articles appeared where apparently similar lowdensity DRMs were still isolated from RBCs without the carbonate step (Kamata et al., 2008; Lach et al., 2012; Rodi et al., 2006; Wilkinson et al., 2008). We made several attempts to rationalize this discrepancy, by varying the isolation protocol or the metabolic status of the cells, for instance using stored or fresh RBCs, density-separated young and old RBCs, fully oxygenated or deoxygenated cells, buffers of various composition, pH and ionic strength, but without success. We then realized that the use of RBCs obtained by washing the blood with physiologic buffers, instead of RBCs purified by effective leukodepletion procedures, resulted in the possibility of isolating the lipid raft-like

Figure 2. Isolation of lipid raft-like material from whole human RBCs. Pure RBCs were obtained by filtration of blood through cellulose and incubated (1.25  109 cells) for 30 min at 4  C in HKM buffer (10 mM HEPES, 150 mM KCl, 4.5 mM NaCl, 1 mM MgCl2) containing 1% (w/v) TX100, in a final volume of 625 ml. The sample was then mixed with an equal volume of 80% (w/v) sucrose in HKM (tube 1) or in 0.3 M K2CO3 (tube 2) and transferred to an ultracentrifuge tube. Sucrose solutions in HKM [2.5 ml of 30% (w/v) sucrose followed by 1.25 ml 5% (w/v) sucrose] were layered on the samples and the tubes spun for 16 h at 225 000 gmax. Lower arrow indicates the interface between 40% and 30% sucrose. Upper arrow indicates the interface between 30% and 5% sucrose.

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fraction without the requirement for carbonate, provided that the centrifugation time of the sucrose gradients lasted at least 4 h (Figure 3, tube 1). It was shown that responsible of the phenomenon was the action of hydrolases released by contaminating white cells. In fact, pre-treatment of the cell suspension with diisopropylfluoro-phosphate (DFP) rendered it impossible the subsequent isolation of low-density DRMs without the addition of carbonate (Figure 3, tube 2). At this point we went to some length to examine the relative effectiveness of washing or filtering the blood in purifying RBCs. In fact, the vast majority of RBC studies are conducted on RBCs obtained by a few washes of blood with physiologic solutions: It turned out that this procedure, while removing almost all the mononuclear cells, it leaves a significant amount of granulocytes mixed with the RBCs (Achilli et al., 2011): Its leukodepletion factor is ‘‘log 1’’, i.e. it reduces white cells by only a factor of 10 (but the eliminated cells are mostly lymphocytes, not granulocytes), as opposed to ‘‘log 4’’ leukodepletion factors achieved with efficient filters. More than 95% of circulating granulocytes are of the polymorphonuclear neutrophil type (PMN). The enormous hydrolytic power contained in PMN granules is unleashed by the action of TX100 and, in the absence of effective inhibitors, can damage the RBC structures. Several highly sensitive membrane-skeletal proteins are heavily proteolysed under these conditions, while spectrin apparently undergoes only limited proteolysis (see below), and the membraneskeleton is progressively reduced to smaller fragments. When these fragments no longer act as ballast for the associated sphingolipid- and cholesterol-rich domains, these can migrate to lower density regions in the sucrose gradients upon centrifugation (Ciana et al., 2011).

Figure 3. Isolation of lipid raft-like material from whole human RBCs. RBCs were not purified by filtration through cellulose, but only washed three times with PBS. RBCs (1.25  109) were pre-treated (tube 2) or not (tube 1) with 5 mM DFP for 10 min at room temperature, washed extensively with PBS and incubated for 30 min at 4  C in HN buffer (5 mM HEPES, 150 mM NaCl, 4.5 mM KCl) containing 1% (w/v) TX100, in a final volume of 625 ml. The sample was then mixed with an equal volume of 80% (w/v) sucrose in HN and transferred to an ultracentrifuge tube. Sucrose solutions in HN [2.5 ml of 30% (w/v) sucrose followed by 1.25 ml 5% (w/v) sucrose] were layered on the samples and the tubes spun for 4 h at 225 000 gmax. The arrow indicates the low-density DRM fraction.

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Several hours are commonly needed for the contaminating proteases to act at 4  C, hence the long centrifugation times usually adopted in the protocols. It is wrongly assumed that the high speed and long centrifugation times are necessary for the density separation of lipid raft-like material in the sucrose gradients. However, we have shown that only 30 min are sufficient to effect this separation when carbonate is used (Ciana et al., 2011). In fact, low-density DRMs can be isolated from carefully leukodepleted (‘‘log 3’’), and DFPpretreated, RBC populations only after treatment with carbonate. The resulting material is largely devoid of membrane-skeletal and associated proteins. Previously published characterizations of lipid raft-like material from RBCs are inconsistent, because flawed by the consequences of proteolysis of membrane and membrane-skeletal proteins. It is not possible to exclude that proteolysis could have affected the results of DRM isolation also in other cell types where anti-proteases were not used or used ineffectively, or when cells express high levels of proteases (for instance Parolini et al., 1996; de Gassart et al., 2003). Most of the work in this respect has still to be done, whose results will probably help refining the characterization of lipid raft-like fractions and their proteome. In fact, there is strong evidence that DRMs are associated with cytoskeletal components to a different degree in various cell types. Protocols for DRM isolation from various cell types often include some sort of maneuver in addition to detergent treatment, which is evidently required for dislodging the membrane domains from underlying anchoring structures. The energy input to the system for achieving this result is never quantified: Mechanical manipulation is at best defined as the number of ‘‘strokes’’ with homogenizers, or the number of passages though narrow gauge needles, even ultrasound treatment, or a combination of such maneuvers, when reported. It is clear the impossibility to define reproducible protocols. This may be the reason why the existence of DRMs with a broad range of buoyant densities, related to different degrees of association with the cytoskeleton, or different amounts of associated cytoskeletal portions, has been recognized in some more rigorous studies (Bae et al., 2004; Ishmael et al., 2007; Lillemeier et al., 2006; Nebl et al., 2002).

Effects of carbonate on DRM isolation Although pioneering works contemplated the use of various salts and alkali to perturb ‘‘coulombian’’ interactions among proteins and between proteins and the membrane (Steck & Yu, 1973), it is not easy to find the rationale in the literature for the use of carbonate in this context and, in particular, for the isolation of DRMs. To the best of our knowledge, the only work that mentions carbonate treatment in addition to TX100 as a necessary (and not optional, as reported in Salzer & Prohaska, 2001) step of the experimental protocol for the isolation of DRMs from human erythrocytes is that of Dvorak and co-workers (Nagao et al., 2002). In that work, which is the first description of the modification of RBC DRMs by the malarial plasmodium, carbonate was used for the isolation of lipid raft-like material, to ‘‘separate integral raft-associated proteins from peripheral proteins which are partially associated with DRMs’’. Significantly, carbonate was necessary

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because those Authors worked with RBCs in culture for infection with the plasmodium parasite, and therefore their RBC samples were previously purified from leukocytes (Nagao et al., 2002). Carbonate treatment was previously described as a selective treatment for the isolation, in the absence of detergents, of caveolae. With this technique, these plasma membrane entities were for the first time shown to be distinct from the microdomains rich in sphingolipids and GPI-linked proteins (Sargiacomo et al., 1993; Song et al., 1996). Probably the first occurrence where carbonate was purposely used for effecting the dissociation of proteins from membranes was in the work of Lazarow and co-workers, where a of non-destructive procedure was described for releasing the protein content and the peripheral proteins associated with the membrane of subcellular organelles (mitochondria, peroxisomes, rough and smooth microsomes) (Fujiki et al., 1982a, 1982b). The purified membranes, after treatment with 100 mM carbonate at 0  C could be pelleted and were converted from closed vesicles to open sheets. Ribosomes could be also removed from rough endoplasmic reticulum using this one-step procedure. The key factor in the action of carbonate is the pH, not the ionic strength of the solution. Due to the buffering capacity of carbonate, pH control is easier than with a solution of alkali alone (Fujiki et al., 1982a), and this is the property we exploited in our work on the isolation of DRMs from RBC ghosts (Ciana et al., 2005). Because of the concerns about the possible artifactual origin of lipid rafts due to detergent action, a number of detergent-free methods for raft isolation have been proposed, some of which contemplate the use of carbonate, essentially based on a mechanical disgregation of the membrane, whether induced by sonication or shearing, followed by density fractionation (Macdonald & Pike, 2005). Due to the variability in the results, however, the plethora of different detergentbased or detergent-free methods that have been proposed, and especially the absolute lack of quantification of the (mechanical) energy involved in the manipulation of the cell sample, only add to the complexity of the matter (see also below). Our attempts to isolate DRMs from RBC ghosts using only carbonate have been unsuccessful (Ciana et al., 2005). Although we were able to demonstrate that DRMs can be obtained free of membrane-skeletal and other associated proteins (band 3, glycophorin C), thanks to inhibition of proteolysis and to the use of optimized (high K+- and Mg2+containing) buffers (Ciana et al., 2011), DRMs with associated minute amounts of membrane-skeletal components were again obtained occasionally, even using carefully purified and DFP-pretreated RBCs. The phenomenon is apparently related to a partial fragmentation of the membrane-skeleton, probably resulting from a variable sensitivity of different cell samples to mechanical stress. It is in fact possible to reduce the spectrin in DRMs by limiting the shearing, by pipetting, of the membrane-skeletons during mixing with the detergent and sucrose solutions. But the outcome of these optimized maneuvers cannot always be predicted (unpublished data). The phenomenon remains, therefore, largely stochastic in nature, and its elucidation requires further analysis. It is, nonetheless, independent of proteolysis of membrane proteins.

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The mesomorphic state of the detergent-resistant membrane It is not the purpose of the present chapter to go into details about the biophysics of the lipid phase of plasma membranes and lipid rafts. Authoritative and comprehensive reviews have appeared on this subject (Brown & London, 2000; Munro, 2003; Pike, 2004; Sonnino & Prinetti, 2013). It will be mentioned, however, what is known about the mesomorphic states of the RBC plasma membrane in connection with a model compatible with the existence of membrane rafts. Membrane bilayers composed of a single phospholipid undergo temperature dependent transition between a solid (gel) phase and a liquid-crystalline phase at a characteristic, well defined transition temperature (Tm). In the solidified gel state the acyl chains of the phospholipids are tightly packed in an ordered structure, whereas in the liquid-crystalline phase the acyl chains are more loosely packed, thus conferring to the system the property of a liquid ‘‘disordered’’ state (variably defined as ld or lc). In the plasma membrane of cells, however, this situation is much more complex, due to the presence of different classes of phospholipids, with acyl chains of variable length and degree of saturation, of sphingolipids, and cholesterol. Different phases could exist, beside the more familiar solid-ordered and liquid-disordered. In particular, a different type of ordered phase originates from the interaction between saturated acyl chains of phospholipids and cholesterol. This mesomorphic state is intermediate between the solid-like gel phase and the lc state. It was originally predicted by theoretical calculations based on cholesterol-containing binary bilayers, by Ipsen et al. (1987), and it was termed liquid-ordered (lo). Experimental validations of the model came shortly thereafter (Almeida et al., 1992; Vist & Davis, 1990). Sphingolipids are even more suited to the formation of lo phases when interacting with cholesterol, because saturation is prevalent in their acyl chains. Moreover, sphingolipids are capable of forming hydrogen bonding between their ‘‘head groups’’, because, contrary to the glycerophospholipids, they possess a hydroxyl group that can act as the hydrogen donor, further stabilizing the lo domain (Simons & Fuller, 1985). Immiscible fluid phases have been recognized to coexist at certain levels of cholesterol in mixtures of glycerophospholipids/cholesterol and, especially, of sphingolipids/cholesterol (Sankaram & Thompson, 1990), likely

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reflecting different regions of the bilayer where different liquid phases coexist in the ld or lo states (Figure 4). Knowing the Tm of the lipid species occurring in the RBC membrane, some of them could well be in the gel state even at 37  C: At least for some of them the Tm is so high that a small decrease induced by cholesterol should not have a great impact (Boggs, 1980). The schematic description presented above is functional for understanding the results of direct measurement of the ordered state of DRMs in RBCs. By using EPR spectroscopy with spin labeled lipid molecules (n-doxyl-stearic acid spin labels, SASL, where n ¼ 5 or n ¼ 16), we showed for the first time that the lipid bilayer of DRMs isolated from whole human RBCs by sucrose gradient fractionation is in a higher ordered state with regard to the plasma membrane, compatible with the existence in the RBC membrane of liquid ordered domains that could be isolated with TX100 (Crepaldi Domingues et al., 2009). We also showed that DRMs could be isolated by TX100 treatment of whole RBCs even at physiological temperature (37  C). With regard to the lipid component, these DRMs were quantitatively (cholesterol content) and qualitatively (order parameter measured with EPR and spin probes) similar to those extracted at 4  C, but they were different in terms of protein content (Domingues et al., 2010). Thus, our EPR study indicated that the mesomorphic state of DRMs is compatible with that of a liquid-ordered domain, irrespective of the temperature at which the isolation is carried out. In the DRMs obtained from RBCs, cholesterol is present at approximately 54–57 mol% in respect to total DRM lipids (whereas cholesterol molar ratio in the plasma membrane is approximately 45%). Although our EPR measurements were conducted at a temperature (25  C) at which sphingomyelin should be in a gel state, being 32  C its Tm (Radhakrishnan et al., 2001), the DRM state should be liquidordered even at 25  C, because of the 450 mol% cholesterol content. Interestingly, when DRMs obtained from cholesteroldepleted RBCs were analyzed, the order parameter measured by EPR at 25  C with 5-SASL was significantly higher than the corresponding values for DRMs obtained from noncholesterol-depleted RBCs. This could be related to the existence of a more solid-ordered phase, due to the higher proportion of sphingomyelin in these samples, consequent to the relative cholesterol depletion (Domingues et al., 2010).

Figure 4. Physical states of the lipid bilayer. Here a lipid bilayer made of a single phospholipid is in a solid gel-like, ordered state when the temperature is below the Tm of the phospholipid, and in a liquid-crystalline (lc), or liquid-disordered (ld) state when the temperature is above the Tm. An additional mesomorphic state has been described when cholesterol is present in the system above a certain molar ratio over phospholipids. In this case the cholesterol molecule (black oval) can fluidize the solid-ordered phase and render more ordered the liquid-disordered phase, generating an intermediate state, which was termed liquid-ordered (lo) phase. The bilayer in the lo phase is thicker than that in the lc or in the solid-ordered phase. See text for additional details. Redrawn from (Munro, 2003).

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Of course, these results do not prove the existence of membrane rafts with the properties described for DRMs, in the plasma membrane of RBCs, an issue which is still open to discussion, and which has been addressed also by methods that potentially allow direct visualization of native rafts in the membrane, the topic of the next section. However, the fact that the liquid-ordered state can exist at physiological temperature, exactly because of the presence of cholesterol, makes it possible, in principle, its direct visualization with the appropriate methodology. With regard to the data on the lipid moiety of DRMs obtained from RBCs that are present in the literature, it could be argued that, in those studies at high risk of contamination by hydrolases (where unfiltered blood was used), some hydrolytic events might have affected certain classes of phospho- or sphingolipids, thus leading to artifactual results in terms of composition and physical state of the membrane raft bilayer. The results obtained with our protocol, however, are accurate to the extent that the population of RBCs was highly purified by leukodepletion (Ciana et al., 2013; Minetti et al., 2013).

Direct visualization of lipid rafts The difficulties and controversy still existing around the problem of direct visualization of membrane rafts will not be discussed here, because thorough reviews have appeared (Jacobson & Dietrich, 1999; Jacobson et al., 2007; Sonnino & Prinetti, 2013). Concerning light microscopy, two main opposing views exist on the issue: On the one hand, that conventional fluorescence microscopy is able to detect punctate patterns of membrane rafts, containing specific sets of proteins, and on the other that such patterns are not visible because either their dimensions are well below the resolution limit of light microscopy (0.25 mm) or the fluorescent probe used for visualization (usually a fluorescentlylabeled GPI-linked protein) is not sufficiently concentrated in the raft phase to provide a good signal. Although electron microscopy has the required resolution for detecting local membrane inhomogeneity in the nanoscale, the various maneuvers required for sample preparation, dehydration, fixation, immune-labeling with multivalent probes, render the technique prone to the generation of artifacts. Within this context, we will limit this part to reviewing the published attempts to directly visualize membrane rafts in RBCs. Despite the relative simplicity of the cell model, the erythrocyte does not seem to help solving the problems described above. Early work by Rodgers & Glaser (1991), using fluorescence microscopy and fluorescently-labeled phospholipid analogs reported about the existence of large lipid domains in the membrane of rabbit erythrocytes and erythrocyte ghosts. It was excluded that these micrometric-scale entities could be ‘‘gel-phase’’ domains. Since the domains could not be seen in vesicles made from lipids extracted from ghosts, it was concluded that the proteins were mostly responsible for the organization of lipids in such large scale domains. Some concern comes from the fact that the fluorescent probes were obtained by substituting bulky fluorochromes in the 2-position of the glycerol moiety, thus creating particularly

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non-physiological probes that could have had their own partitioning properties in the plane of the membrane. Of the few next published articles, some fail to see, by conventional fluorescence microscopy, any lateral partition/ aggregation of certain raft markers, such as the ganglioside GM1, unless patching was induced experimentally by means of multivalent ligands (Mro´wczynska & Ha¨gerstrand, 2008), whereas they show a patchy localization of GPI-linked proteins (CD-59), supporting the view that membrane rafts are heterogeneous in nature. It should be mentioned a more recent attempt to directly visualize membrane rafts in the erythrocyte membrane using a fluorescently (BODIPY) labeled GM1 moiety that was detected by fluorescence video microscopy. Again, the probe was found to be uniformly distributed when observed at room temperature, whereas it seemed to cluster at 4  C. When spectrofluorometry was used, the probe exhibited a red shift that was interpreted as the clustering of probe molecules. With this approach, and with the additional observation that extraction of cholesterol with methyl-b-cyclodextrin eliminated the red shift, it was possible to document the existence of lipid rafts also at 37  C, despite conventional fluorescence microscopy only resulted in a uniform distribution of fluorescence in the membrane (Mikhalyov & Samsonov, 2011). Attempts to visualize membrane rafts in RBCs by atomic force microscopy have been made (Cai et al., 2012), but one wonders whether this technique, even though it was applied on whole cells in a physiologic buffer, would be adequate to reveal such highly dynamic and elusive entities. A more promising technique has been developed, called STED (stimulated emission depletion) far-field fluorescence nanoscopy that is able to detect single diffusing molecules in nanosized areas in the plasma membrane of living cells. New insights on the properties of erythrocyte membrane rafts could come from the application of the technique to this cell type (Eggeling et al., 2009).

Figure 5. A spectrin tetramer, with two spectrin heterodimers associated head-to-head. The regions marked with circles contain the a-spectrin 8, 9 and 10 triple-helical repeats and the b-spectrin 12, 13 and 14 triplehelical repeats where high-affinity binding sites for PS map (An et al., 2004). The binding site for ankyrin (‘‘ank’’) is located in the 15th helical repeat of b-spectrin (Bennett & Baines, 2001).

A. Ciana et al.

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Figure 6. A portion of the membrane-skeleton is shown, as obtained after a hypothetical solubilization of the membrane with 1% TX100 under the conditions for obtaining DRMs but with the omission of carbonate, so that DRMs remain associated with the spectrin skeleton. The cartoon depicts the hypothetical direct interaction of DRMs with the spectrin dimer in a region where the highest affinity of spectrin for certain lipid classes was previously described, near the head-to-head association of spectrin dimers into tetramers. The same region in the spectrin dimer also contains the binding site for ankyrin, so that its interaction with ankyrin and the DRM is mutually exclusive. The various proteins are only approximately drawn to scale, but their oligomeric state and interactions are respectful of the current knowledge. See text for additional explanations.

Association of membrane rafts with the membrane-skeleton: Identification of the anchor What keeps the sphingolipid-cholesterol rich, detergentresistant fraction of the RBC membrane tightly associated with the membrane-skeleton in a pH- and ionic strengthsensitive manner is still not known. An interesting hint came

from our study of DRMs obtained, in the absence of carbonate, from neutrophil-contaminated RBC suspensions. Under these conditions, DRMs are released only when proteolysis affects spectrin. Inhibition of proteolysis of spectrin but not of ankyrin or protein 4.1, with eglin-C (a selective inhibitor of neutrophil elastase and cathepsin G) blocks the release of DRMs.

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We propose, on the basis of evidence collected before (Ciana et al., 2011), that the lipid rafts may be anchored to the membrane-skeleton via direct interaction with the spectrin molecule. Our line of reasoning is as follows. We concentrate, in particular, on the region in the spectrin dimer that was reported to display high affinity for phosphatidylserine (PS). This domain, located near the head of the heterodimer (defined as the region containing the N-terminal end of a-spectrin and the C-terminal end of b-spectrin) and containing also the binding site for ankyrin, contributes with repeats 8–10 of a-spectrin and repeats 12–14 of b-spectrin to the binding to PS (An et al., 2004) (see Figure 5). Aminophospholipids have been shown to be present to a significant extent in DRMs from human and ruminant erythrocytes (Koumanov et al., 2005). Thus, it could be that PS act as the anchor for lipid rafts to spectrin. The objection that the hypothetical region for membrane raft binding in the spectrin dimer coincides with the binding site for ankyrin can be overcome by observing that there are roughly 2.2  105 copies of spectrin dimers per cell (Steck, 1974) but only approximately 1.1  105 copies of ankyrin (Bennett & Stenbuck, 1979). Therefore, half the binding sites for ankyrin in the spectrin meshwork would be free at any given time and could be hypothetically engaged in the binding to membrane rafts without the steric hindrance of ankyrin. This scenario is depicted in Figure 6, where a portion of the membraneskeleton is shown as if resulting from the treatment of the cell with TX100, which solubilizes most of the lipid bilayer. The detergent-resistant phase remains attached to the skeleton at the level of the mentioned head region of the spectrin dimer, only to those dimers that are not already engaged with ankyrin (Figure 6). Support to this model comes from experiments where DRMs were obtained from erythrocytes that were not leukodepleted and treated with TX100 but not with carbonate. Under these conditions, DRMs can be isolated after ultracentrifugation for more than 4 h in sucrose gradients, thanks to the action of proteases from contaminating neutrophils that partially break down the membrane-skeleton, thus permitting the floating of DRMs in low-density regions of the gradients. DRMs can be dislodged from the membrane-skeleton only when proteolysis begins to affect spectrin. If only protein 4.1 and ankyrin are proteolysed, something that happens early because of their particular sensitivity, DRMs are not released. What we reproducibly found was that, surprisingly, only approximately 50% of b-spectrin was proteolysed under conditions where DRMs were released from the membrane-skeleton, as if DRMs protected the other 50% of b-spectrin from the attack of proteases during the isolation (Ciana et al., 2011). The reason why a-spectrin appeared not to be affected by proteolysis is unknown, but it could be related to an intrinsically higher resistance to hydrolysis of this polypeptide under the peculiar experimental conditions described. Experiments will be performed to ascertain whether PS exposure consequent to Ca2+ loading of RBCs might induce rearrangements in the membrane-skeleton-lipid bilayer interactions with the dissociation of membrane rafts from the membrane-skeleton.

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Conclusions The purpose of the present review was to highlight the central role that the erythrocyte plasma membrane has represented in the history of cell biology studies and to encourage a reconsideration of its centrality in the light of recent discoveries. Issues such as the existence of lateral inhomogeneities in the lipid phase, the partitioning of different classes of proteins into these membrane rafts, and their interaction with sub-membranous scaffolding structures, are still debated, and important insights may still come from the continuous investigation of the plasma membrane of mammalian erythrocytes as a model of election for the study of biological membranes.

Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper. Funds for the preparation of the present work were from the University of Pavia, the Italian ‘‘Ministero dell’Universita` e della Ricerca’’ (PRIN grant to G.M.), and from the CARIPLO Foundation Project N. 2011-2099 ‘‘Toxicology of engineered nanoparticles: Analysis of their potential thrombotic, inflammatory and haemolytic effects’’.

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