The lateral separation of contacts on erythrocytes agglutinated by polylysine

The form of contact seam (whether a continuous parallel seam or membranes in spatially periodic contact) has been characterized for normal and for neuraminidase pretreated human erythrocytes following adhesion in solutions of polylysine in the molecular mass range 10–225 kDa at concentrations from 0.5 to 1.0 mg/mL. The adhesion contact seam was spatially periodic for all normal control cells in polylysine. The lateral separation of contacts decreased from 1.6 to 0.8 μm as the concentration of 225 kDa polylysine was increased threefold from the adhesion threshold value. The separation distance did not change further even at high polymer concentrations that increased the electrophoretic velocity to positive values over twice the modulus of the velocity of control cells. The probability of cell adhesion decreased at these high polymer concentrations. The lateral contact separation increased and cell adhesion decreased for cells pretreated with neuraminidase. Cell adhesion did not occur when neuraminidase reduced the cell electrophoretic velocity modulus by 30%. Following neuraminidase pretreatments that allowed a small amount of adhesion, the cell contact seam was continuous rather than spatially peridic. The results show that a procedure that increases (e.g., polymer concentration increase) or decreases (e.g., enzyme removal of polycation crosslinking site) attraction leads to shorter (to a limiting value) or longer lateral contact separation, respectively.     

Contact patterns in concanavalin A agglutinated erythrocytes.

Agglutination of human erythrocytes by the lectin concanavalin A is enhanced when the erythrocytes are pretreated with neuraminidase, which removes sialic acids, or with pronase, which degrades both the glycophorins and band 3 protein. In the present work transmission electron microscopy of the enzymatically pretreated erythrocytes shows a regular pattern of interruption of contact between interacting plasma membranes. The lengths characteristic of the pattern were 0.66 and 0.50 microns for pronase- and neuraminidase-pretreated cells, respectively. Agglutination of normal erythrocytes and of neuraminidase-pretreated erythrocytes can be fully reversed by exposure to the competitive inhibitor methyl alpha-D-mannopyranoside. Complete reversal of contact does not occur with pronase-pretreated cells. The comparatively greater tenacity of contact between cells that were treated with pronase before exposure to lectin argues for an involvement of nonspecific interactions in the agglutination process. The results are compared with previously published studies of spatially periodic contact patterns induced by a range of other polymers.     

Interfacial instability and the agglutination of erythrocytes by polylysine

Human erythrocytes have been exposed to poylysine of molecular weight range 4 to 220 kDa and concentration range 0.5 to 2,000 /ml at 37°C. Threshold concentrations for cell agglutination by the polycation have been determined for the samples of different molecular weight. Light and electron micrographs show that, in the erythrocyte agglutinates, cell-cell contact is generally made only at discrete, spatially periodic, regions which are distributed over a significant part of the cell surface. The average spacing between contact regions is 0.83 m. The cell membrane has a wavy profile between contact regions. Agglutination occurs only in cell samples whose electrophoretic mobility is significantly altered by polylysine and, in agreement with a previous report, occurs even when the electrophoretic mobility reaches high positive values. The electrophoretic mobility data implies that agglutination requires some protrusion of polylysine from the cell glycocalyx. We discuss how a resulting net attractive intercellular force could act to destabilize the aqueous layer between two cells, allowing surface wave growth which results in spatially periodic contact regions. Examples of situations where cell and membrane contact might be explained by the general concept of interfacial instability are discussed.     

Phase separation in bimolecular mixed lipid membranes induced by polylysine

We demonstrate, for the first time, polylysine-induced phase separation in a bimolecular lipid membrane of a lecithin/phosphatidylglycerol-mixture by analysing the single channel current fluctuations of gramicidin. The bimodal conductance histograms are direct evidence for the incorporation of the transport system into the two coexisting phases of different composition.     

Electron microscopy of human erythrocytes agglutinated by lectin from Codium fragile ssp. tomentosoides and pseudo-haemagglutinin from Ascophyllum nodosum

Human erythrocytes were exposed to extracts of Codium fragile ssp. tomentosoides, Ascophyllum nodosum, Dolichos biflorus seeds and a solution of purified polyphenols from Ascophyllum nodosum. In each case the erythrocytes appeared to agglutinate. Agglutinates were examined by scanning electron microscopy. Photomicrographs of the erythrocytes exposed to lectin-containing extracts of C. fragile and D. biflorus showed the typical appearance of agglutinated cells. Photomicrographs of the erythrocytes exposed to the crude extract and purified polyphenols from A. nodosum were similar to each other, but quite different in appearance to erythrocytes agglutinated by the lectins from C. fragile and D. biflorus. This evidence adds further support to the contention that lectin presence recorded in brown algal extracts is largely due to ‘pseudoagglutination’ produced by polyphenols contained in these extracts.     

Interaction forces between red cells agglutinated by antibody. I. Theoretical.

A general method of calculating forces, torques, and translational and rotational velocities of rigid, neutrally buoyant spheres suspended in viscous liquids undergoing a uniform shear flow has been given by Arp and Mason (1977). The method is based on the matrix formulation of hydrodynamic resistances in creeping flow by Brenner and O'Neill (1972). We describe the solution of the Brenner-O'Neill force-torque vector equation in terms of the particle and external flow field coordinates and derive expressions for the normal force acting along, and the shear force acting perpendicular to, the axis of the doublet of spheres, the latter explicitly given for the first time. The equations consist of a term comprising force and torque coefficients obtained from the matrices of the hydrodynamic resistances (functions of the distance h between sphere surfaces which have been computed), and terms comprising the orientation of the doublet axis relative to the coordinates of the external flow field and the shear stress (which can be experimentally determined). We have applied the theory to a system of doublets of sphered, hardened human red cells of group A or B antigenic type cross-linked by the corresponding antibody at a fixed interparticle distance. Working from studies of the breakup of doublets of red cells in an accelerating Poiseuille flow, given in the succeeding paper, we are able to compute the hydrodynamic force required to separate the two spheres. Previous work has shown that the theory can be applied to doublets in a variable shear, Poiseuille flow, provided the ratio of particle to tube diameter is small. In calculating the force-torque coefficients it was assumed that the cells are crosslinked by antibody with h = 20 nm.     

ADF/cofilin weakens lateral contacts in the actin filament.

Observed in vivo motility rates can only be accounted for if the rate of actin filament treadmilling in cells is considerably greater than has been quantified for purified actin in vitro. ADF/cofilin is uniquely suited to promote actin dynamics in cells, owing to its remarkable ability to change actin filament structure. In earlier work we showed that human cofilin chanRges filament twist by about 5 degrees per subunit and suggested that this contributes to increased filament turnover. Our initial structural modeling provided some insights into how the longitudinal actin-actin contacts might be disrupted following cofilin-induced twisting. Here we present direct evidence that cofilin also disrupts lateral actin-actin contacts in the filament and suggest a model showing how this could contribute to cofilin's novel effects on actin filament dynamics and assembly.     

Interaction forces between red cells agglutinated by antibody. III. Micromanipulation.

In the flow studies described in two previous papers (Tha, S. P., and H. L. Goldsmith, 1986, Biophys. J. 50:1109-1116; Tha, S. P., J. Shuster, and H. L. Goldsmith, 1986, Biophys. J. 50:1117-1126), hydrodynamic forces of the order of 10(-11) N (mu dyn) were applied to measure the force of separation of doublets of hardened, sphered human red blood cells cross-linked by anti-B antibody. The same cell preparation and hyperimmune antiserum has here been used to carry out experiments with micropipet aspiration techniques. One cell of a doublet was aspirated onto a holding pipet, and a second aspiration pipet was brought into proximity of the other cell so that the two pipets and the doublet were colinear. Suction was then raised until the two cells separated. Some doublets were assembled by aspiration of a singlet, bringing a second singlet into apposition with the first, and releasing it from the pipet which was then withdrawn. Cells could be repeatedly assembled and separated. At 3.56% vol/vol antiserum, the mean normal force of separation was 0.45 +/- 0.11 nN in phosphate-buffered saline suspensions containing 2.5 x 10(4) cells/microliter; at 1.22% vol/vol antiserum, the value was 0.22 +/- 0.11 nN. The above values of the force were approximately 2.5 x greater than those from the flow studies. The data could be fitted to a Poisson distribution with 0.05 nN as the force needed to break a single cross-bridge (c.f. 0.024 nN from the previous hydrodynamic data). The forces of separation of randomly assembled doublets were lower than those of preexisting doublets. Repeated assembly and separation of doublets showed that the cell surfaces are nonuniform in adhesion strength both over the local scale less than 0.25 micron2 and the cell population.     

The membrane skeleton of erythrocytes: models of its effect on lateral diffusion

The membrane skeleton, a network of structural proteins attached to the cytoplasmic surface of the plasma membrane, hinders lateral diffusion of integral proteins. 2. In some types of cells, such as epithelial cells and nerve cells, the obstruction of lateral diffusion by the membrane skeleton is one of the mechanisms by which proteins are localized to domains on the cell surface. 3. The effect of the membrane skeleton on lateral diffusion may involve steric hindrance, transient binding or both. Three pictures of the effect are reviewed, the discrete barrier model, the continuous barrier model and the transient binding model. 4. Experiments to distinguish the models are discussed.     

High gradient magnetic separation of erythrocytes

The high gradient magnetic separation technique has been applied to separate paramagnetic erythrocytes from a cell suspension that also contained diamagnetic cells. Paramagnetism was induced in the red blood cells by oxidizing the iron atoms in the cell hemoglobin to the ferric state (methemoglobin). Diamagnetic cells were either untreated erythrocytes, containing oxyferrohemoglobin, or leukocytes in a suspension of mouse spleen cells. Cell suspensions were passed through a column containing 40 micron diameter stainless steel wire in a high magnetic field (33 kG). The paramagnetic cells were retained on the surface of the wire while the diamagnetic cells passed through. Elution of the paramagnetic cells was accomplished by removing the column from the magnet, in effect turning off the field.     

The spectrin network as a barrier to lateral diffusion in erythrocytes. A percolation analysis.

The spectrin network on the cytoplasmic surface of an erythrocyte can be modeled as a triangular lattice of spectrin tetramers (Tsuji, A., and S. Ohnishi, 1986. Biochemistry. 25:6133-6139). The tetramers act as barriers to protein diffusion, while dissociated dimer pairs, single dimers, and missing tetramers do not. Diffusion in the presence of these barriers is shown to be equivalent to bond percolation on the honeycomb lattice. Monte Carlo calculations for this system then yield the relative diffusion constant of a mobile integral protein as a function of the fraction of spectrin tetramers. At high concentrations of spectrin tetramer, long-range diffusion is blocked, but short-range diffusion is still possible. Monte Carlo calculations yield the average distance over which short-range diffusion can occur, as a function of the fraction of spectrin tetramers. Applications to erythrocyte development and hereditary hemolytic anemia are discussed.     

Efficient gene transfer by histidylated polylysine/pDNA complexes.

Plasmid/polylysine complexes, which are used to transfect mammalian cells, increase the uptake of DNA, but plasmid molecules are sequestered into vesicles where they cannot escape to reach the nuclear machinery. However, the transfection efficiency increases when membrane-disrupting reagents such as chloroquine or fusogenic peptides, are used to disrupt endosomal membranes and to favor the delivery of plasmid into the cytosol. We designed a cationic polymer that forms complexes with a plasmid DNA (pDNA) and mediates the transfection of various cell lines in the absence of chloroquine or fusogenic peptides. This polymer is a polylysine (average degree of polymerization of 190) partially substituted with histidyl residues which become cationic upon protonation of the imidazole groups at pH below 6.0. The transfection efficiency was optimal with a polylysine having 38 +/- 5% of the epsilon-amino groups substituted with histidyl residues; it was not significantly impaired in the presence of serum in the culture medium. The transfection was drastically inhibited in the presence of bafilomycin A1, indicating that the protonation of the imidazole groups in the endosome lumen might favor the delivery of pDNA into the cytosol.     

Different modes of internalization of proteins associated with adhaerens junctions and desmosomes: experimental separation of lateral contacts induces endocytosis of desmosomal plaque material.

The distribution and fate of two junctional complexes, zonula adhaerens and desmosomes, after dissociation of cell-cell contacts is described in MDBK cells. Junctions were split between adjacent cells by treatment with EGTA and proteins associated with the plaques of zonulae adhaerentes and desmosomes were localized by immunological methods. Splitting of these junctions is accompanied by the dislocation of desmosomal plaque protein from the cell periphery and its distribution in punctate arrays over the whole cytoplasm. By contrast, vinculin associated with zonulae adhaerentes is still seen at early times (0.5-1 h) in a conspicuous belt-like structure which, however, is displaced from the plasma membrane. Strong vinculin staining is maintained on leading edges of free cell surfaces. Electron microscopy of EGTA-treated cells exposed to colloidal gold particles reveals the disappearance of junctional structures from the cell periphery and the concomitant appearance of a distinct class of gold particle-containing vesicles which are coated by dense plaques. These vesicle plaques react with antibodies to desmosomal plaque proteins and are associated with filaments of the cytokeratin type. In the same cells, extended dense aggregates are seen which are most probably the membrane-detached vinculin-rich material from the zonula adhaerens . The experiments show that, upon release from their junction-mediated connections with adjacent cells, major proteins associated with the cytoplasmic side of the junctions remain, for several hours, clustered within plaques displaced from the cell surface. While plaque material of adhaerens junctions containing vinculin is recovered in large belt-like aggregates, desmosomal plaque protein remains attached to membrane structures and appears on distinct vesicles endocytotically formed from half-desmosomal equivalents.(ABSTRACT TRUNCATED AT 250 WORDS)