Membrane bending energy and shape determination of phospholipid vesicles and red blood cells

A procedure is developed to calculate red blood cell and phospholipid vesicle shapes within the bilayer couple model of the membrane. The membrane is assumed to consist of two laterally incompressible leaflets which are in close contact but unconnected. Shapes are determined by minimizing the membrane bending energy at a given volume of a cell (V), given average membrane area (A) and given difference of the areas of two leaflets (A). Different classes of shapes exist in parts of the v/a phase diagram, where v and a are the volume and the leaflet area difference relative to the sphere with area A. The limiting shapes are composed of sections of spheres with only two values allowed for their radii. Two low energy axisymmetrical classes, which include discocyte and stomatocyte shapes are studied and their phase diagrams are analyzed. For v=0.6, the discocyte is the lowest energy shape, which transforms by decreasing a continuously into a stomatocyte. The spontaneous membrane curvature (C ₀) and compressibility of membrane leaflest can be incorporated into the model.A model, where A is free and C ₀ determines the shapes at given V and A, is also studied. In this case, by decreasing C ₀, a discocyte transforms discontinuously into an almost closed stomatocyte.     

Influence of thermally driven surface undulations on tethers formed from bilayer membranes

Tether formation is a powerful method to study the mechanical properties of soft lipid bilayer membranes. The force required to maintain a tether at a given length depends upon both membrane elastic properties and tension. In this report, we develop a theoretical analysis that considers the contribution of thermally driven surface undulations and the corresponding entropically driven tensions on the conformation of tethers formed from unaspirated lipid vesicles. In this model, thermal undulations of the vesicle surface provide the excess area required for tether formation. Energy minimization demonstrates the dependence of equilibrium tether conformation on membrane tension and provides an analytical relationship between tether force and radius. If the contributions of nonlocal bending are not considered, an analytical relationship between tether force and length can also be obtained. The predictions of the model are compared to recently reported experimental data, and a value for the initial vesicle tension is obtained. Since most analyses of tether formation from cells and unaspirated vesicles neglect the contributions of nonlocal bending, the appropriateness of this assumption is analyzed. The effect of surface microvesiculations on the tether force-length relation is also considered.     

Membrane bending modulus and adhesion energy of wild-type and mutant cells of Dictyostelium lacking talin or cortexillins.

We have employed an interferometric technique for the local measurement of bending modulus, membrane tension, and adhesion energy of motile cells adhering to a substrate. Wild-type and mutant cells of Dictyostelium discoideum were incubated in a flow chamber. The flow-induced deformation of a cell near its adhesion area was determined by quantitative reflection interference contrast microscopy (RICM) and analyzed in terms of the elastic boundary conditions: equilibrium of tensions and bending moments at the contact line. This technique was employed to quantify changes caused by the lack of talin, a protein that couples the actin network to the plasma membrane, or by the lack of cortexillin I or II, two isoforms of the actin-bundling protein cortexillin. Cells lacking either cortexillin I or II exhibited reduced bending moduli of 95 and 160 k(B)T, respectively, as compared to 390 k(B)T, obtained for wild-type cells. No significant difference was found for the adhesion energies of wild-type and cortexillin mutant cells. In cells lacking talin, not only a strongly reduced bending modulus of 70 k(B)T, but also a low adhesion energy one-fourth of that in wild-type cells was measured.     

The viscoelasticity of membrane tethers and its importance for cell adhesion

Cell adhesion mechanically couples cells to surfaces. The durability of individual bonds between the adhesive receptors and their ligands in the presence of forces determines the cellular adhesion strength. For adhesive receptors such as integrins, it is a common paradigm that the cell regulates its adhesion strength by altering the affinity state of the receptors. However, the probability distribution of rupture forces is dependent not only on the affinity of individual receptor-ligand bonds but also on the mechanical compliance of the cellular anchorage of the receptor. Hence, by altering the anchorage, the cell can regulate its adhesion strength without changing the affinity of the receptor. Here, we analyze the anchorage of the integrin VLA-4 with its ligand VCAM-1. For this purpose, we develop a model based on the Kelvin body, which allows one to quantify the mechanical properties of the adhesive receptor's anchorage using atomic force microscopy on living cells. As we demonstrate, the measured force curves give valuable insight into the mechanics of the cellular anchorage of the receptor, which is described by the tether stiffness, the membrane rigidity, and the membrane viscosity. The measurements relate to a tether stiffness of k_t = 1.6 μN/m, an initial membrane rigidity of k_i = 260 μN/m, and a viscosity of μ = 5.9 μN·s/m. Integrins exist in different activation states. When activating the integrin with Mg²⁺, we observe altered viscoelastic parameters of k_t = 0.9 μN/m, k_i = 190 μN/m, and μ = 6.0 μ N·s/m. Based on our model, we postulate that anchorage-related effects are common regulating mechanisms for cellular adhesion beyond affinity regulation.     

Isolation of progenitor cells from cord blood using adhesion matrices

The aim of this study was to develop optimal conditions for selective adhesion and isolation of mesenchymal progenitor cells (PCs) from cord blood and to determine their potential for osteogenic differentiation. Mononuclear cells (MNCs) were isolated by Ficoll-Paque gradient and plated onto 48-well culture plates precoated with: human or bovine collagen type I, human collagen type IV, fibronectin or matrigel. Cultures were incubated in αMEM containing fetal calf serum. Viability of the adherent cells was determined by alamarBlue® assay after 2, 3, and 4 weeks. After 4 weeks in culture, cells were typsinized and replated. Primary cultures were analyzed by histochemistry and third passage cells by FACS. Isolated fibroblast-like cells were cultured in the presence of osteogenic factors and differentiation determined by Alizarin Red S staining, RT-PCR and electron dispersive spectroscopy (EDS). MNCs adhered to all types of matrices with the greatest adhesion rates on fibronectin. These cells were CD45⁺, CD105⁺, CD14⁺, CD49a⁺, CD49f⁺, CD44⁺ and CD34⁻. The highest incidence of PCs was observed on fibronectin and polystyrene. Passages were CD45⁻, CD14⁻, CD34⁻ and weakly CD105⁺. Primary cultures expressed endothelial/macrophage RNA markers whether cultured on fibronectin or polystyrene and these markers decreased upon passage. The best osteogenic differentiation was observed in MPCs cultured in osteogenic medium containing Vit D₃ and FGF9. These cells expressed the bone-related mRNA, collagen type I, core binding factor I (Cbfa I), osteocalcin and osteopontin. EDS of deposits produced by these cells demonstrated a calcium/phosphate ratio parallel to hydroxyapatite. It was concluded that fibronectin increased adhesion rates and isolation potential of cord blood mesenchymal progenitor cells.     

Isolation of progenitor cells from cord blood using adhesion matrices

The aim of this study was to develop optimal conditions for selective adhesion and isolation of mesenchymal progenitor cells (MPCs) from cord blood and to determine their potential for osteogenic differentiation. Mononuclear cells (MNCs) were isolated by Ficoll-Paque gradient and plated onto 48-well culture plates precoated with: human or bovine collagen type I, human collagen type IV, fibronectin or matrigel. Cultures were incubated in αMEM containing fetal calf serum. Viability of the adherent cells was determined by alamarBlue® assay after 2, 3, and 4 weeks. After 4 weeks in culture, cells were typsinized and replated. Primary cultures were analyzed by histochemistry and third passage cells by FACS. Isolated fibroblast-like cells were cultured in the presence of osteogenic factors and differentiation determined by Alizarin Red S staining, RT-PCR and electron dispersive spectroscopy (EDS). MNCs adhered to all types of matrices with the greatest adhesion rates on fibronectin. These cells were CD45⁺, CD105⁺, CD14⁺, CD49a⁺, CD49f⁺, CD44⁺ and CD34⁻. The highest incidence of progenitor cells (PC) was observed on fibronectin and polystyrene. Passages were CD45⁻, CD14⁻, CD34⁻ and weakly CD105⁺. Primary cultures expressed endothelial/macrophage RNA markers whether cultured on fibronectin or polystyrene and these markers decreased upon passage. The best osteogenic differentiation was observed in MPCs cultured in osteogenic medium containing vitamin D₃ and FGF9. These cells expressed the bone-related mRNA, collagen type I, core binding factor I (Cbfa I), osteocalcin and osteopontin. EDS of deposits produced by these cells demonstrated a calcium/phosphate ratio parallel to hydroxyapatite. It was concluded that fibronectin increased adhesion rates and isolation potential of cord blood mesenchymal progenitor cells.     

Structure of membrane tethers and their role in fusion

Vesicular transport between different membrane compartments is a key process in cell biology required for the exchange of material and information. The complex machinery that executes the formation and delivery of transport vesicles has been intensively studied and yielded a comprehensive view of the molecular principles that underlie the budding and fusion process. Tethering also represents an essential step in each trafficking pathway. It is mediated by Rab GTPases in concert with so‐called tethering factors, which constitute a structurally diverse family of proteins that share a similar role in promoting vesicular transport. By simultaneously binding to proteins and/or lipids on incoming vesicles and the target compartment, tethers are thought to bridge donor and acceptor membrane. They thus provide specificity while also promoting fusion. However, how tethering works at a mechanistic level is still elusive. We here discuss the recent advances in the structural and biochemical characterization of tethering complexes that provide novel insight on how these factors might contribute the efficiency of fusion.     

Isolation and purification of a cell adhesion factor from crayfish blood cells

Isolated granular haemocytes (blood cells) from the crayfish Pacifastacus leniusculus attached and spread in vitro on coverslips coated with a lysate of crayfish haemocytes. No cell adhesion activity was detected in crayfish plasma. The cell adhesion activity was only present in haemocyte lysates in which the prophenoloxidase (proPO) activating system (Soderhall and Smith, 1986a, b) had been activated; either by lipopolysaccharide (LPS), the beta-1,3-glucan laminarin, or by preparing the lysate in 5 mM Ca2+. Both lysates of granular or of semigranular haemocytes could mediate adhesion. After A23187-induced exocytosis of the granular cells, cell adhesion activity could be generated in the secreted material if it was incubated with laminarin. The factor responsible for cell adhesion was isolated from an active haemocyte lysate and purified by ammonium sulfate precipitation, cation exchange chromatography and Con A-Sepharose; it had a molecular mass of approximately 76 kD on an SDS-polyacrylamide gel. An antibody to this 76-kD band inhibited cell adhesion. Ca2+ was necessary in the medium for the cells to adhere to the adhesion factor. With cyanide or azide, the cells attached but failed to spread. It is suggested that in vivo the cell adhesion factor is stored in the secretory granules of the semigranular and the granular cells in a putative inactive pro-form, which can be released during exocytosis and, in the presence of beta- 1,3-glucans or LPS, be activated outside the cells to mediate cell attachment and spreading, processes of essential importance in arthropod host defense.     

Membrane transformation during malaria parasite release from human red blood cells

Three opposing pathways are proposed for the release of malaria parasites from infected erythrocytes: coordinated rupture of the two membranes surrounding mature parasites; fusion of erythrocyte and parasitophorus vacuolar membranes (PVM); and liberation of parasites enclosed within the vacuole from the erythrocyte followed by PVM disintegration. Rupture by cell swelling should yield erythrocyte ghosts; membrane fusion is inhibited by inner-leaflet amphiphiles of positive intrinsic curvature, which contrariwise promote membrane rupture; and without protease inhibitors, parasites would leave erythrocytes packed within the vacuole. Therefore, we visualized erythrocytes releasing P. falciparum using fluorescent microscopy of differentially labeled membranes. Release did not yield erythrocyte ghosts, positive-curvature amphiphiles did not inhibit release but promoted it, and release of packed merozoites was shown to be an artifact. Instead, two sequential morphological stages preceded a convulsive rupture of membranes and rapid radial discharge of separated merozoites, leaving segregated internal membrane fragments and plasma membrane vesicles or blebs at the sites of parasite egress. These results, together with the modulation of release by osmotic stress, suggest a pathway of parasite release that features a biochemically altered erythrocyte membrane that folds after pressure-driven rupture of membranes.     

Membrane molecules involved in adhesion properties of cultured sertoli cells

Membrane components involved in adhesion properties of cultured Sertoli cells have been studied by a combination of immunological and biochemical methods. An antiserum prepared against Sertoli cells induced reversible rounding and detachment of the cells from the culture dishes. The cell surface morphology during detachment was studied by scanning electron microscopy and indirect immunofluorescence. A Triton soluble fraction of crude membrane preparations inhibited the antibody-induced detachment. The antibodies recognized a restricted number of membrane glycoproteins [detectable as prominent bands on Sodium dodecylsulphate polyacrilamide gel electrophoresis (SDS-PAGE), M_r 170, 140, 80, and 48K] both in the Triton soluble fraction of crude membrane preparation and on intact Sertoli cells. The data suggest that the molecules involved in adhesion properties of cultured Sertoli cells are integral membrane glycoproteins exposing antigenic determinants at the cell surface.     

Minimum energy analysis of membrane deformation applied to pipet aspiration and surface adhesion of red blood cells.

An experimental procedure is demonstrated which can be used to determine the interfacial free energy density for red cell membrane adhesion and membrane elastic properties. The experiment involves micropipet aspiration of a flaccid red blood cell and manipulation of the cell proximal to a surface where adhesion occurs. A minimum free energy method is developed to model the equilibrium contour of unsupported membrane regions and to evaluate the partial derivatives of the total free energy, which correspond to the micropipet suction force and the interfacial free energy density of adhesion. It is shown that the bending elasticity of the red cell membrane does not contribute significantly to the pressure required to aspirate a flaccid red cell. Based on experimental evidence, the upper bound for the bending or curvature elastic modulus of the red cell membranes is 10-12 ergs (dyn-cm). Analysis of the adhesion experiment shows that interfacial free energy densities for red cell adhesion can be measured from a lower limit of 10-4 ergs/cm2 to an upper limit established by the membrane tension for lysis of 5-10 ergs/cm2.     

Membrane permeability equations and their solutions for red cells

Summary The mathematical equations for the transport of nonelectrolytes across cell membranes are critically examined and cast in forms suitable for solution which involve fewer approximations than has heretofore been commonly done. For the case of red cells, the equations are developed to include the effect of the variation in apparent nonosmotic water owing to the variation in hemoglobin concentration as the cell swells or shrinks. Two methods of solution of the equations are developed and studied and sample calculations are provided. It is shown that the solutions to the linearized equations commonly found in the literature are insufficiently accurate for some purposes and this inaccuracy is avoided by the methods given here. The importance of retaining the effects of variations in apparent nonosmotic water and in solute volume in the cell is demonstrated.     

Methods for measuring amounts of energy available from banksia inflorescences

Several methods have been used to remove nectar from banksia inflorescences to measure amounts of energy available, but these methods have been poorly described in the literature. Each method has advantages and disadvantages with respect to the proportion of nectar removed and the time taken to remove nectar. Power-driven aspirators probably remove the most nectar, followed by syringes and capillary tubes. However, it often takes more than 20 min to sample an inflorescence using these methods. Centrifuging inflorescences by placing them in plastic bags and swinging them by hand on a rope takes only about 5 min anil allows many more inflorescences to be sampled per time. Centrifuging. however, removes only about 70% of the nectar so that the benefits gained by faster sampling are countered by reduction in accuracy. Nevertheless, amounts of nectar extracted by centrifuging provide a reliable index of the amounts present, and so it is possible to make Qualitative comparisons of inflorescences sampled at different times, from different locations, or from different experimental treatments.     

Membrane changes associated with lysis of red blood cells by hypochlorous acid

This study was carried out to investigate HOCl-induced lysis of human erythrocytes. Using reagent HOCl with isolated red cells, we showed that the rate of lysis was dependent on the dose of HOCl per red cell rather than on the concentration of oxidant. The process was inhibited by scavengers such as methionine and taurine, but only if they were present at the time of addition of HOCl. Lysis was preceded by a decrease in cell density, a change in the deformability of the membrane as evidence by ektacytometry, and an increase in K⁺-leak. Electron microscopy showed extensive disruption of the membrane. Increasing doses of HOCl caused progressive loss of membrane thiols, bu complete thiol oxidation by N-ethylmaleimide did not result in an equivalent rate of lysis. Restoration of oxidised thiols by incubation with glucose did not significantly alter the pattern of lysis. Taken together, these results suggest that thiol oxidation was not responsible for HOCl-mediated lysis. There was evidence of increasing crosslinking of membrane proteins on electrophoresis, only some of which was due to the formation of disulfides. TLC of the membrane lipids indicated that there may be formation of chlorohydrins by reaction of HOCl with the fatty acid double bonds. This reaction results in the formation of a more polar species which, if formed, would be extremely disrupting to the lipid bilayer. The results indicate that HOCl-mediated damage to the membrane proteins or to the lipid bilayer comprises an initial damaging event that sets the cells on a path toward eventual lysis.     

Membrane glycoprotein and surface free energy changes in hypoxic fibroblast cells

Hypoxia affects the biochemistry of mammalian cells and thus alters their sensitivity to subsequent chemo- and radiotherapy. When V79 Chinese hamster lung fibroblasts were grown under conditions of extreme hypoxia (less than 10 ppm O2) there was a significant shift in the membrane glycoprotein composition. Scanning electron microscopy revealed altered cell surface morphology including loss of pseudopodial projections. Experiments to determine changes in interfacial free energy of these cells using equilibrium two phase systems of poly(ethylene glycol) (PEG) and dextran were carried out. Test fluid droplets of the denser dextran-rich phase were formed on layers of cells in the PEG-rich phase as the bathing medium, and the contact angles the droplets made with the cell layers were measured from photomicrographs. The contact angles on cells in the plateau phase increased significantly with time of exposure to hypoxia, from 25 degrees (zero time) to 35 degrees (6 h) to 60 degrees (9 h). Contact angles on cells in the exponential phase increased from 80 degrees (zero time) to 150 degrees after 20 h of hypoxia. It appears that the altered contact angles reflect changes in cell surface hydrophobicity that may, in part, reflect alterations in the membrane glycoprotein composition.     

Membrane potentials of vallisneria leaf cells and their relation to photosynthesis

A study has been made of the effects of the inhibitors carbonylcyanide m-chlorophenylhydrazone (CCCP), 3-(3,4-dichlorophenyl)-1,1-dimethyl urea (DCMU), and of anoxia on the light-sensitive membrane potential of Vallisneria leaf cells. The present results are compared with the known effects of these inhibitors on ion transport and photosynthesis (Prins 1974 Ph.D thesis). The membrane potential is composed of a diffusion potential plus an electrogenic component. The electrogenic potential is about −13 millivolts in the dark and −80 millivolts in the light. The inhibitory effect of DCMU and CCCP on the electrogenic mechanisms strongly depends on the light intensity used, the inhibition being less at a higher light intensity. This is of significance in view of the often conflicting results obtained with these inhibitors. With ion transport in Vallisneria the electrogenic pump derives its energy from phosphorylation; however, the process which causes the initial light-induced hyperpolarization and the process that keeps the membrane potential at a steady hyperpolarized state in the light have different energy requirements. The action of photosystem I alone is sufficient to induce the initial hyperpolarization. For continuous operation in the light the activity of photosystem II also is needed.     

Membrane and cytoplasmic resistivity properties of normal and sickle red blood cells

The cytoplasmic resistivities and membrane breakdown potentials of normal (AA), sickle-cell-trait (AS), and sickle (SS) red blood cells have been measured by the biophysical methodology of resistive pulse spectroscopy over a range of osmolalities. At isotonicity, the average membrane breakdown potentials are virtually identical for the three types of cells occurring at about 1150 V/cm. Average isotonic cytoplasmic resistivities are somewhat higher for the SS cells (166.7±7.49 ohm-cm) compared to the AA (147.6±1.98 ohm-cm) or AS cells (148.7±1.79 ohm-cm). As medium osmolality is varied, the differences in resistive properties become enlarged, especially at very low and very high osmolalities. At high osmolalities, both types of sickle cells show a large increase in internal resistivity compared to the normals; at low osmolality, the SS samples exhibit a distinctly different membrane breakdown characteristic, decreasing in this parameter, whereas the other two groups increase. Of the 15 SS samples tested, three displayed much higher cytoplasmic resistivities at isotonicity: 218.2±5.25 ohm-cm, compared to an average of 153.5±3.46 ohm-cm for the other 12. The relationship between these high resistivities and the subfraction of irreversibly sickled cells in the sample is discussed.     

Membrane and cytoplasmic resistivity properties of normal and sickle red blood cells

The cytoplasmic resistivities and membrane breakdown potentials of normal (AA), sickle-cell-trait (AS), as sickle (SS) red blood cells have been measured by the biophysical methodology of resistive pulse spectroscopy over a range of osmolalities. At isotonicity, the average membrane breakdown potentials are virtually identical for the three types of cells occurring at about 1150 V/cm. Average isotonic cytoplasmic resistivities are somewhat higher for the SS cells (166.7 +/- 7.49 ohm-cm) compared to the AA (147.6 +/- 1.98 ohm-cm) or AS cells (148.7 +/- 1.79 ohm-cm). As medium osmolality is varied, the differences in resistive properties become enlarged, especially at very low and very high osmolalities. At high osmolalities, both types of sickle cells show a large increase in internal resistivity compared to the normals; at low osmolality, the SS samples exhibit a distinctly different membrane breakdown characteristic, decreasing in this parameter, whereas the other two groups increase. Of the 15 SS samples tested, three displayed much higher cytoplasmic resistivities at isotonicity: 218.2 +/- 5.25 ohm-cm, compared to an average of 153.5 +/- 3.46 ohm-cm for the other 12. The relationship between these high resistivities and the subfraction of irreversibly sickled cells in the sample is discussed.