Google Scholar. Basic Aspects and Applications of Lipids and Protein Dynamics. 110, 4476–4481 (2013). These folded domains consist of coiled areas of the spectrin strings that expand when the spectrin is stretched beyond its natural length as shown in the figure below. However, the low purity and low degree of order in the RBC pellets likely prohibited a more detailed structural analysis at this time. The preparation protocol presented in this paper produces highly oriented, multi-lamellar RBC membranes on silicon wafers, which are highly suited to provide detailed molecular level information. Steck, T. L. The Organization of Proteins in the Human Red Blood Cell Membrane a Review. Molecular structure of the RBC membranes was analyzed by high resolution X-ray diffraction. Department of Physics and Astronomy, McMaster University, Hamilton, ON, Canada, Sebastian Himbert, Richard J. Alsop, Markus Rose, Alexander Dhaliwal & Maikel C. Rheinstädter, Department of Experimental Physics, Saarland University, Saarbrücken, Germany, Sebastian Himbert, Lars Kaestner & Christian Wagner, Research Center for Molecular Imaging and Screening, Saarland University, Homburg/Saar, Germany, Department of Chemistry and Chemical Biology, McMaster University, Hamilton, ON, Canada, Department of Pathology and Molecular Medicine, McMaster University, Hamilton, Canada, McMaster Immunology Research Center, McMaster University, Hamilton, Canada, Michael G. DeGroote Institute for Infectious Disease Research, McMaster University, Hamilton, Canada, You can also search for this author in The preparation protocol is schematically depicted in Fig. Chem. membrane is the only membrane of RBCs and con-sequently it can be isolated without contamination by internal membranes; c) a number of diseases have been associated with defects in the structure of RBC membrane. Circ. 3(a). MEMBRANE CARBOHYDRATES They occur only on the external surface of We present direct experimental evidence that aspirin incorporates into the head group region of erythrocytic membranes and leads to an increase in lipid tail distances and a decrease in membrane width, indicating increased membrane fluidity. modeling along with the folded domains. 263, 488–489 (1994). Biol. The lipid in-plane peaks are the result of a hexagonal packing of the lipid tails in the hydrophobic membrane core (planar group p6)16. Arch. As you can see the membrane of the red blood cell is very important not only to its structural performance but also to its ultimate goal of delivering oxygen. 229, 286–90 (1993). 4(e). For this wafer, 100 μL of concentrated ghosts solution was applied onto a 10 × 10 mm2 hydrophobic silicon wafer mounted on a leveled hot plate at a temperature of 40 °C. 6, e25954 (2011). Tristram-Nagle, S., Liu Y., Legleiter, J. This process was repeated twice. 11, 7748–61 (2015). J. This eight-fold change of thickness is consistent with the view that the membrane consists of plaques which … Glycophorin A,B, C - gives RBC neg charge, location of antigens, anchors cytoskeleton tobilayer. RBCME : The functional red cell membrane is composed of a cholesterol and phospholipid bilayer anchored by integral proteins to an elastic cytoskeletal network. Biophys. During this washing, most of the hemoglobin is removed, resulting in a transparent, colorless solution. You are using a browser version with limited support for CSS. Zhang, Y., Alsop, R. J., Soomro, A., Yang, F. C. & Rheinstädter, M. C. Effect of shampoo, conditioner and permanent waving on the molecular structure of human hair. Hemoglobin shows characteristic absorption lines at 335 ± 0.4 nm, 416.4 ± 0.2 nm 543 ± 0.8 nm and 577 ± 0.4 nm80. In order to prepare a sufficiently diluted RBC solution, 50 μL of the erythrocytes fraction was mixed with 1 mL PBS. RBC from CAPD patients also showed a normal SOD activity, a more than adequate vitamin E status, and a peculiar pattern of membrane lipids, with reduced polyunsaturated fatty acids (p less than 0.001) and increased monounsaturated fatty acids (p less than 0.001). The supernatant was removed and the tube was refilled with buffer solution to the 1 mL mark of the tube. When the membrane form factor F(qz) is measured at several qz values, a continuous function, T(qz), which is proportional to F(qz), can be fitted to the data15,81. After this process, a clear separation between an erythrocyte fraction and a plasma fraction was observed. This correlation peak is observed in the in-plane data in Fig. In contrast to the previous fatty acids, higher RBC membrane arachidonic acid levels are significantly associated with an increased risk of SCC of the skin (OR, 1.11 per 1 mg unit change; 95% CI, 1.04-1.18). To overcome this constraint, we prepared highly oriented stacks of RBC membranes on silicon wafers and studied their molecular properties in-vitro using high resolution X-ray diffraction. X-ray diffraction is not sensitive to monomeric integral or peripheral peptides, but instead to larger integral peptide regions (the packing of peptide helices in the membrane core). Grazer, W. B. ASA has a pronounced effect on the RBC membranes in our study: by reducing the thickness of the lo regions it leads to a more uniform and overall more fluid membrane structure, where the ld and lo domains now have identical membrane thicknesses. The results present evidence for nanometer-sized domains of coiled-coils peptides, as well as liquid ordered (lo) and liquid disordered (ld) lipid patches, and give a detailed picture of the molecular organization in these domains. The in-plane diffraction signals in Fig. Halfway between actin anchors there are what are known as suspension complexes (SC). Rheinstädter, M. C., Ollinger, C., Fragneto, G., Demmel, F. & Salditt, T. Collective dynamics of lipid membranes studied by inelastic neutron scattering. 17, 1795–1809 (2010). CAS  Biochem. Shattil, S. J. Our current concept of the red cell membrane envisions it as a composite structure in which a membrane envelope composed of cholesterol and phospholipids is secured to an elastic network of skeletal proteins via transmembrane proteins. From these results, RBC membranes consist of small, nanometer sized domains of lo and ld lipids and coiled-coil α-helical peptides. 81, 7564–7568 (1984). Lett. The electron density corresponding to the  = 51.6 Å spacing (green curve) agrees well with the electron density reported for fluid lipid bilayers, where the structure of the lipid tails in the hydrophobic membrane core is dominated by gauche-defects, as reported for instance by18,19. Med. & Raphael, R. M. NSAID injury to the gastrointestinal tract: evidence that NSAIDs interact with phospholipids to weaken the hydrophobic surface barrier and induce the formation of unstable pores in membranes. Rheinstädter, M. C. & Mouritsen, O. G. Small-scale structures in fluid cholesterol-lipid bilayers. Mohandas, N. & Gallagher, P. G. Red cell membrane: past, present, and future. This is essential for the shape and reversible deformability of RBC. Level I, the spectrin domains, were modeled as linear springs. These interactions form the shape, deformability, and proper ion balance of the cell. & Brisson, A. J. 207, 1481–1483 (1980). While RBC membranes were previously reported to consist of ~52% proteins and ~40% lipids (including cholesterol)4, we observe a higher fraction of lipids (and cholesterol) and fewer peptides. 12, 3165–3176 (2016). Dodge, J. T., Mitchell, C. & Hanahan, D., J. 3b) agrees qualitatively with the early electron density in ref. The electron rich head group can be identified by the absolute maximum in the electron density profile at |z| ~ 22 Å. ρ monotonically decreases towards the bilayer center at z = 0, where CH3 groups typically reside, with a low electron density. This research was approved by the Hamilton Integrated Research Ethics Board (HIREB) under approval number 1354-T. 256, 7528–7535 (1981). 2(b) by radial integration using Hermans orientation function, as described in the Materials and Methods Section. The combination of oriented membranes and state-of-the-art diffraction equipment and analysis gives unprecedented insight into the structure of these RBC membranes. J. Crick, F. Is α-keratin a coiled coil? This beam geometry provides optimal illumination of the solid supported membrane samples to maximize the scattering signal. sites. Errors for calculated parameters, such as peak area, were then calculated by applying the proper error propagation. 200 μL of the diluted blood solution, the ghosts solution and the RBC solution were filled in the chambers of the plate. The densities for the lo and ld lipid domains agree well with densities reported in the literature. 40% -lipids, 52% - protein, 8% - carbohydrates. To create a hydrophilic state, the wafers were cleaned by immersion in an sulfuric acid mixture (volume fraction of 70% concentrated H2SO4, 30% H2O2 at 40 °C, Piranha solution) for 30 min on a 3D orbital shaker (VWR) set to tilt angle 1 and speed 15). Red cell membrane The red cell membrane consists of: Proteins~50% Lipids ~ 40% Carbohydrates~10% General structure of a Cell Membrane Composition of the red cell membrane. It presents a well-documented and well-illustrated comprehensive picture of clinical manifestations of red blood cell disorders. The imaging of cholera toxin. The setup is schematically shown in (a). Amyloid-β 25-35 peptides aggregate into cross-β sheets in unsaturated anionic lipid membranes at high peptide concentrations. Google Scholar. When present in the body, aspirin (acetylsalicylic acid, ASA) and its metabolites interact with the cyclo-oxygenase (COX) pathway. In the meantime, to ensure continued support, we are displaying the site without styles By using the values in Table 1, areas of lo and ld lipids can be estimated to 38 and 50 Å2, and 95 Å2 (2π ⋅ (11/2)2) for the peptides. 58, 1421–1428 (2006). Figure S2(a) in the Supplementary Material displays the q|| position of the lo and ld lipid in-plane peaks as function of aspirin concentration. L.K. The X-ray diffraction measurements present direct experimental evidence that RBC membranes consist of nanometer sized lo and ld lipid domains, as well as integral α-helical coiled-coil peptide domains. Scientific Reports The white blood cells and platelets form a layer between those two fractions. Beutler, E., West, C. & Blume, K. The removal of leukocytes and platelets from whole blood. Armstrong, C. L. et al. Folded domains in the spectrin safeguard against premature breakage of the spectrin skeleton increasing overall malleability of the membrane. This is, in particular, a consequence of the lack of suitable experimental techniques. Structures of ~5 μm were observed, indicative of the presence of actin. functional lipid domains16,17,56,57,58,59,60. Chem. To create a hydrophobic silicon surface, the wafers were pre-treated by sonication in dichloromethane (DCM) at 40 °C for 25 min. Biophys. 2(d) to 30:45:25 (lo lipids:ld lipids:coiled peptides). Scientific Reports Based on the protocol for the preparation of red blood cell ghosts, small RBC vesicles were produced and applied onto functionalized silicon chips and annealed into multi-lamellar, planar membranes. The average thickness of the peptide domains of 40.6 Å is compatible with the thickness of the membranes and support the assignment to integral peptides. The Molecular Structure of Human Red Blood Cell Membranes from Highly Oriented, Solid Supported Multi-Lamellar Membranes. Plenum Press (1966). A. ADS  2(d). The authors confirm that all methods were performed in accordance with the relevant guidelines and regulations. USA. The total area of the RBC membranes is calculated to (2 ⋅ 10−6) ⋅ (6 ⋅ 1023) ⋅ (58 ⋅ 10−20) m2 ≈ 0.7 m2. Cell Biol. The data are well fit by three series of Bragg peaks corresponding to three different lamellar spacings assigned to lo and ld lipid domains (green and blue) and coiled-coil α-helical peptide domains (red). The main features of this motif is a ~10.8 Å (q|| ~ 0.58 Å−1) equatorial reflection corresponding to the spacing between adjacent coiled-coils32,33,34. Soft Matter. 11, 688–699 (2010). Level II dealt The well developed Bragg peaks along the out-of-plane axis in Fig. Distances and areas in the lo domains are smaller, as lipid tails in their all-trans configuration are straighter and pack tighter than ld tails, dominated by gauche defects. CAS  PBS was added to the precipitate to achieve a volume of 10 mL and centrifuged at 3,000 g for 10 min. Values for the domain size, ξ, are listed in Table 1. Unsaturated lipids were reported to order in a structure with slightly larger nearest neighbor tail distances, leading to an acyl-chain correlation peak at ~1.3 Å−1, as reported for DOPC and POPC25,26. (eds. Once an analytical expression for T(qz) has been determined from fitting the experimental peak intensities, the phases vn can be assessed from T(qz). The red blood cell membrane was fluorescently labeled in part (a) using 1,1′-Dioctadecyl-3,3,3′,3′-Tetramethylindocarbocyanine Perchlorate (DiI). Properties and even existence of rafts are, however, a topic of intense debate in the literature57,59,60. BBA-Biomembranes. S.H., M.C.R. J. Pharm. These consist of ankyrin, band 3, and protein 4.2. The experimental errors were determined as follows: Errors for peak positions, peak width and peak height are determined as the fit standard errors, corresponding to 95% confidence bounds, equivalent to 2 standard deviations, σ. These shapes are likely related to the presence of a cytoskeleton, whose main components are spectrin and actin in RBC75. Kučerka, N., Nieh, M. P. & Katsaras, J. Fluid phase lipid areas and bilayer thicknesses of commonly used phosphatidylcholines as a function of temperature. Describe RBC osmotic balance – esp. In view of this, RBC membrane composition was analyzed in diabetic patients. Clin. As the membranes were oriented with their membrane plane parallel to the silicon substrate, the in-plane and out-of-plane structure was determined separately, but simultaneously. Soft matter. Nova ; 17–42 (2016). Simons, K. & Ikonen, E. Functional rafts in cell membranes. A lipid is a cellular unit with a head and a tail. USA. Figure 2(b) shows typical 2-dimensional X-ray diffraction data. Rev. The results were compared with healthy age and sex matched control groups. The slow drying resulted in a smoother film, however, still incomplete coverage of the substrate, as shown in Fig. The integrated peak intensities, In, are multiplied by qn to receive the form factors, F(qn)15,81. Ultravioletvisible spectroscopy (UV-vis) was obtained using a M1000Pro Plate reader from Tecan. 1848, 805–812 (2015). Three dz-spacings were determined:  = 59.2 Å,  = 51.6 Å and  = 40.6 Å. (c) and (d) show hydrophilic wafers after slow drying and slow drying and annealing, respectively. 7-20 (a). & Leonenko, Z. If the membrane is somehow damaged or its flexibility decreased it follows that the transportation of the cell will be inhibited and its usefulness negated. When the cells are disc-shaped, the membranes is 5.0±1.0 nm thick; when the cells are in the form of a sphere (induced by heating), the membrane is 40±10 nm thick. The number of stacked RBC membranes on one of these chips can be estimated as follows: 100 μL of the 7 mg/mL RBC contain ~2 ⋅ 10−6 mol (when assuming an average molecular weight of the membranes of 400 g/mol). Toppozini, L. et al. The basic structure of the erythrocyte membrane cytoskeleton has been worked out, although some details remain obscure. Spherocytosis is a genetic disease that affects the membrane of a red blood cell. 12 The lipid portion, equal parts of cholesterol and phospholipids, forms a bilayer universal to all animal cells (see Figure 13-9). Lecture Objectives 1. The experiments thus locate aspirin inside the head group region of the RBC membranes, in agreement with results in model phospholipid bilayers15,36,37. Colloid Interface Sci. When erythrocytes are affected by Spherocytosis they often will rupture prematurely and lead to anemia (shortage of RBCs) which can cause lots of problems for the human body. These domains are likely enriched in cholesterol, making them more ordered and thicker16,17. RBC Membrane Evaluation, B Aliases Lists additional common names for a test, as an aid in searching Band 3 EMA Binding Osmotic Fragility Eosin-5-maleimide Eosin RBC membrane Hereditary spherocytosis Hereditary pyropoikilocytosis 2(a). PubMed Central  Blood. ", "Spectrin folding vs. unfolding reactions and RBC membrane stiffness", "A hybrid model for erythrocyte membrane: a single unit of protein network coupled with lipid bilayer. Basic Aspects and Applications of Lipid and Protein Dynamics. How to cite this article: Himbert, S. et al. 2(c) indicate a lamellar organization of the RBC membranes on the substrate. The images show side and top views. No. Upon the addition of aspirin, the electron density increases at z ~ 22.8 Å. Using this technique, we first simulated RBC dynamics in capillary flow and found that the protein density variation and bilayer- skeleton interaction forces are much lower than those in micropipette aspiration, and the maximum interaction force occurs at the trailing edge. Structure and fluctuations of a single floating lipid bilayer. Note that there is no risk of sample damage using this in-house technique because of the large beam size and relatively low intensity of the X-ray beam as compared to synchrotron sources. Each wafer was then thoroughly rinsed with ~50 mL of ultra pure water with a resistivity of 18.2 MΩ ⋅ cm. Aspirin is found to partition the lo lipid domains of RBC membranes and locate in the head group region, at |z|-values of 22.8 Å. 18, 440–447 (2013). By assigning the scattering signals to the different phases, we could determine structural parameters for these domains separately. 86, 740–743 (2001). Alsop, R. J., Schober, R. & Rheinstädter, M. C. Swelling of Phospholipid Membranes by Divalent Metal Ions Depends on the Location of the Ions in the Bilayers. (1) are on a relative scale. 17, 619–627 (2010). Cell Biol. The average size of the different lipid and peptide domains was estimated from the widths of the corresponding in-plane correlation peaks in Fig. Sci. 100 μL of the 7 mg/mL RBC contain ~2 ⋅ 10−6 mol (when assuming an average molecular weight of the membranes of 400 g/mol). These cells are produced by hemolysis and have been named red blood cell (RBC) ghosts based on their appearance under the microscope. Google Scholar. As indicated below, the variation in size and shape of the ghosts, and the presence of an actin network likely prevents the formation of well defined, solid supported multi-lamellar RBC. 62, 123–126 (2004). Lett. Alsop, R. J., et al. Yang, F. C., Peters, R. D., Dies, H. & Rheinstädter, M. C. Hierarchical, self-similar structure in native squid pen. ADS  All membranes were prepared on single-side polished silicon wafers. S2(b) in the Supplementary Material). Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. PLOS ONE. Soft matter. This process of centrifugation and removal of the supernatant was repeated 4 times. The corresponding data is shown in Fig. RBC Membrane Lipids. There is only a small effect of aspirin on the electron density of the ld domains, as shown in Fig. Rep. 6, 22425 (2016). The peaks at q|| = 1.35 Å−1 and q|| = 1.55 Å−1 are in good agreement with structural features reported in model lipid membranes in their well ordered gel and fluid phases, where the lipids tails take an all-trans conformation (gel) or are dominated by gauche defects (fluid). A combined study using phospholipases and freeze-etch electron microscopy. Chen, S. H. et al. The following picture shows the overall structure of the lipid bilayer attached to the protein skeleton. These helical regions are likely part of larger trans-membrane proteins. 9, 6804–6813 (2013). Can make ghosts = resealed plasma membranes. (c) The location of the ASA molecule is determined by comparing the electron density of a pure RBC membrane with a low concentration of 1 mM ASA. Simons, K. & Gerl, M. J. Revitalizing membrane rafts: new tools and insights. 1808, 2761–2771 (2011). The red cell membrane is deformable, mediated in part by the elastic properties (coiling and uncoiling) of the structural proteins that maintain its shape and stability. The skeleton forms an extensive web around the bilayer and is joined to it at what are called junction complexes (JC). Rev. The structure of the multi-lamellar, solid supported RBC membranes is pictured in Fig. This ASA concentration is elevated as compared to plasma concentrations of typically less than 1 mol%, however, comparable to ASA concentrations typically used in the literature79. (b) Comparison of UV-vis absorbance curves at different stages within ghost preparation. Göttinger Nachrichten Math. prepared RBC samples and refined the preparation protocol. Data is well fit by 3 series of Bragg peaks, corresponding to lo, ld and peptide domains, in agreement with pure RBC membranes. Correspondence to To determine the degree of orientation of the membranes in the stack the correlation peak intensities were integrated as function of the meridonal angle φ (the angle relative to the qz axis). Table 3 presents a second approach to analyzing the association of RBC membrane fatty acid profile and risk of skin SCC. This density profile is well described by α-helical coiled-coil peptides, which are embedded in the membranes20, as will be discussed below, and was assigned to domains of integral proteins. & Cooper, R. A. Membrane microviscosity and human platelet function. (d) The in-plane signals show three correlation peaks corresponding to the packing of α-helices in the peptide domains (ap = 10.83 Å), and packing distances of ld (ald = 5.39 Å) and lo lipid tails (alo = 4.69 Å) in the hydrophobic membrane core. The electron density profile of the  = 59.2 Å domain (blue curve) agrees well with a lipid bilayer with lipids in a well ordered, gel-like state with their lipid chains in an all-trans configuration14,15. List some RBC morphologies as they relate to membrane composition, arrangement, and function and correlate to related conditions Aspirin is the most common NSAID and is known to interact with membranes15,68. Electron density profiles of the lo lipid domain for RBC membranes and RBC membranes +1 mM aspirin are shown in Fig. 2 μL, 3 μL, 4 μL, 5 μL, and 6 μL of this solution were added to 100 μL of the final RBC solution resulting in acetylsalicylic acid concentrations of 1 mM, 1.5 mM, 2 mM, 2.5 mM and 3 mM. The ghost solution was analyzed by fluorescence microscopy, as shown in Fig. 6(c): the absorption of light in the visible and adjacent (near-UV and near-infrared) ranges is detected. Mikhalyov, I. While this protocol is well established and widely used in blood cell investigations (see, for instance74, for a recent review), the ghost solution produced by this protocol did not result in well developed multi-lamellar membrane stacks when applied on silicon wafers. Structure of cholesterol in lipid rafts. Each suspension complex acts to pin the spectrin down to the bilayer. The peptide domain shows an almost constant density in the hydrophobic membrane core, indicative of trans-membrane peptides.
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