Spatially periodic discrete contact regions in polylysine-induced erythrocyte-yeast adhesion

Cell-cell adhesion occurs when human erythrocytes and yeast cells are suspended together in suprathreshold concentrations of polylysine in saline. The threshold polycation concentration for adhesion depends on cell concentration and decreases with increasing polycation molecular weight. The threshold concentration was similar for erythrocyte-erythrocyte adhesion and for yeast-erythrocyte adhesion. Transmission electron micrographs show that the erythrocytes adhere to yeast as if to engulf the cell. The regions of close contact between the erythrocyte membrane and the yeast cell walls are spatially discrete. The contact separation distance for the asymmetric erythrocyte-yeast adhesion is very similar to that (0.83 micron) observed when polylysine-induced adhesion occurs in the symmetrical erythrocyte-erythrocyte system. The spacing is attributed to the growth of a squeezing wave as an interfacial instability, on the intercellular aqueous layer. Freeze-fracture electron microscopy of cells that were not fixed during preparation for microscopy confirms the discrete nature of contacts between polylysine treated erythrocytes.     

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.     

Spreading of wheat germ agglutinin-induced erythrocyte contact by formation of spatially discrete contacts.

The time dependence of agglutination and cell-cell contact spreading in human erythrocytes exposed to wheat germ agglutinin (WGA) was characterized by light and electron microscopy. Cells (3 x 10(7)/mL) had a threshold lectin concentration in the range of 0.6-2.0 micrograms/mL for initial cell contact. Spreading was essentially completed within 60 and 2 min in undisturbed and gently agitated suspensions, respectively. The cells in large WGA agglutinates retained features of their initial disk form in contrast to the convex outlines of polycation or polyethylene glycol-induced agglutinates. Spreading of contact area was accompanied by development of a pattern of discrete contact regions separated by a distance of the order of 1 micron. Freeze fracture electron microscopy and studies with ferritin-labeled WGA showed no significant aggregation of intramembrane particles or specific lectin receptors under conditions when contact spreading occurred. It is argued that flow stress effects on cells in suspended agglutinates give rise to a situation where opposite membranes, at the leading edge of cell contact, are separated by a thin aqueous layer. When this intercellular water layer exceeds a critical length, it becomes unstable. The layer breaks up by surface wave development to form an array of intracellular water spaces. Formation of the aqueous spaces causes opposite membrane regions to move synchronously toward each other. Lectin molecules crosslink the wave crests to give spatially periodic contact points.     

Depletion flocculation and depletion stabilization of erythrocytes

At dextran (Mw ≈ 500,000) concentrations from 2 to ≈10%, suspensions of normal human erythrocytes flocculate in small convex agglutinates. At dextran concentrations > 10%, the erythrocytes resegregate in a stable monodisperse suspension. At all these dextran concentrations, the erythrocytes are coated with considerable amounts of dextran. It can be argued that at dextran concentrations from 2 to 10%, as well as at dextran concentrations > 10%, there is a thin layer, which is depleted of dextran, between the dextran layer adsorbed onto the erythrocytes and the bulk dextran solution. It can also be shown that there is a repulsive interaction between the two layers of dextran: one adsorbed and one free. When the adsorbed dextran layer is the most concentrated, stability must ensue, and when the dextran in free solution is the most concentrated, flocculation should occur. Below 7% dextran, the concentration of free dextran is higher than the adsorbed concentration; above 10% dextran that situation is reversed. These data correlate well with the depletion flocculation predicted for the lower concentration and the depletion stabilization predicted for the higher dextran concentration.

     

Divalent cation effects on membrane bending in heated erythrocytes

The thermal fragmentation of human erythrocytes involves either surface wave growth and membrane externalization at the cell rim or membrane internalization at the cell dimple. In symmetrical monovalent electrolytes an increase in membrane internalization at the cell dimple correlates with the decrease in zeta potential arising from surface charge (sialic acid residue) depletion. The influence of divalent cations on thermal fragmentation is examined in this work. The erythrocyte zeta potential decreased when divalent cations replaced some Na⁺ in the cell-suspending phase. The incidence of membrane internalization increased in rank order Ca²⁺>Ba²⁺>Mg²⁺Sr²⁺. Calcium continued to influence the thermal fragmentation of cells highly depleted of sialic acid, suggesting that the ion also interacted with membrane sites other than sialic acid. The divalent cation influence on cell fragmentation was shown to be greater than that due to zeta potential decrease alone. This conclusion was supported by the observation that the divalent cation-induced changes in zeta potential showed much less cation specificity than did the changes induced in the thermal fragmentation pattern. The result implies that the specificity of the divalent cation effects was due to interactions within the erythrocyte shear layer. The possibility that the interaction is with membrane lipids is examined.     

Membrane-membrane interactions: parallel membranes or patterned discrete contacts.

Theoretical and experimental studies of thin liquid films show that, under certain conditions, the film thickness can undergo a sudden transition which gives a stable narrower film or ends in film rupture at spatially periodic points. Theoretical analysis have also indicated that similar transitions might arise in the thin aqueous layer separating interacting membranes. Experiments described here show spatially periodic intermembrane contact points and suggest that spontaneous rapid growth of fluctuations can occur on an intermembrane water layer. Normal and pronase pretreated erythrocytes were exposed to 2% Dextran (450,000 Mr) and the resultant aggregates were examined by light and transmission electron microscopy. Cell electrophoresis measurements were used as an index of pronase modification of the glycocalyx. Erythrocytes exposed to dextran revealed a uniform intercellular separation of parallel membranes. This equilibrium between attractive and repulsive intermembrane forces is consistent with the established Derjaguin, Landau, Verwey, Overbeek (DLVO) model for colloidal particle interaction. In contrast to the above uniform separation a spatial pattern of discrete contact regions was observed in cells coming together in dextran following pronase pretreatment. The lateral contact separation distance was 3.0 microns for mild pronase pretreatment and decreased to 0.85 micron for more extensive pronase pretreatments. The system examined here is seen as a useful experimental model in which to study the principles involved in producing either uniform separation or point contacts between interacting membranes.     

Impacts of soil temperature,phenology and plant community composition on invertebrate herbivory in a natural warming experiment

Species and community-level responses to warming are well documented, with plants and invertebrates known to alter their range, phenology or composition as temperature increases. The effects of warming on biotic interactions are less clearly understood, but can have consequences that cascade through ecological networks. Here, we used a natural soil temperature gradient of 5–35°C in the Hengill geothermal valley, Iceland, to investigate the effects of temperature on plant community composition and plant–invertebrate interactions. We quantified the level of invertebrate herbivory on the plant community across the temperature gradient and the interactive effects of temperature, plant phenology (i.e. development stage) and vegetation community composition on the probability of herbivory for three ubiquitous plant species, Cardamine pratensis, Cerastium fontanum and Viola palustris. We found that the percentage cover of graminoids and forbs increased, while the amount of litter decreased, with increasing soil temperature. Invertebrate herbivory also increased with soil temperature at the plant community level, but this was underpinned by different effects of temperature on herbivory for individual plant species, mediated by the seasonal development of plants and the composition of the surrounding vegetation. This illustrates the importance of considering the development stage of organisms in climate change research given the variable effects of temperature on susceptibility to herbivory at different ontogenetic stages.