Topography of cell-glass apposition revealed by total internal reflection fluorescence of volume markers.

We have developed a new method based on total internal reflection fluorescence to map the shape of the region between glass and the lower surface of a living cell spread upon it. Fluorescently labeled nonadsorbing volume marker molecules that cannot penetrate into the cell are locally stimulated so that they fluoresce only very near the glass/medium interface. The total fluorescence intensity at any point beneath the cell depends on the cell-to-glass separation. Focal contacts appear as dark areas owing to dye exclusion, whereas when the gap exceeds approximately 150 nm, fluorescence asymptotes to the bright background level. Our technique provides greater contrast than does interference reflection microscopy and is free from errors due to cytoplasmic thickness and refractive index inhomogeneities arising from cytoplasmic inclusions. We have shown that sufficiently large molecules suffer steric exclusion from regions accessible to small molecules, which gives new information about lateral penetrability in the apposition region.

Apparent modification of forces between lecithin bilayers.

Small sugar solutes effect variation in the equilibrium separation of lecithin bilayers in aqueous solution. Since sugars have negligible influence on bilayer structure, they probably act by modifying interbilayer forces. The observed widening and narrowing of the bilayer separation is correlated with the predicted weakening and strengthening of the attractive van der Waals forces between lipid bilayers that occurs with increasing sugar concentrations.

Nitric oxide donors inhibit platelet spreading on surfaces coated with fibrinogen but not with fibronectin.

We have studied the effect of nitric oxide (NO) on the interaction of washed human platelets with fibrinogen or fibronectin adsorbed on glass. Platelet contacts were visualized by interference reflection microscopy at 37 degrees C in Tyrode's solution. The areas of spread platelets were measured, using digital video image processing techniques, at times up to 30 minutes after initial surface platelet contact. On fibrinogen spreading was inhibited by NO donors in the order of potency: S-nitroso-acetylpenicillamine > sodium nitroprusside > S-nitroso-glutathione. The inhibitory action of NO donors was prevented by Oxy-haemoglobin, confirming that this inhibition was due to the release of NO. In contrast, NO donors had virtually no effect on platelet spreading on fibronectin. Platelet adhesion to fibrinogen is mediated by GP IIb IIIa and the vitronectin receptor (VNR), while that to fibronectin is via GP IIb IIIa, VNR, Ic IIa and VLA-6. Our results thus indicate that NO inhibits adhesion mediated by GP IIb IIIa and/or VNR but not by the two other receptors. The mechanism of this receptor-specific inhibition of platelet adhesion remains to be elucidated.

Contact signalling and cell motility.

There is now clear evidence that signals can be generated when cells make adhesions. This happens when contacts are made with the extracellular matrix, with other cells or with experimental substrata. It is shown that intracellular signals are triggered in a variety of situations by the aggregation of transmembrane glycoproteins. Such aggregation occurs at adhesion sites, and it is suggested that adhesion molecules which are capable of lateral diffusion become trapped at developing adhesion sites because their headgroups bind to arrays of external ligands. Signals generated by molecular clustering can influence cell spreading and motility via familiar transduction pathways. Although it is not yet possible to reconstruct with confidence the entire sequence of events between initial glycoprotein clustering and motility, there is a lot of information which points to the identities of the molecular ingredients. I shall focus the discussion on the thin peripheral lamellar extensions produced by cell types ranging from amoebae to vertebrate nerve growth cones, and discuss the experimental evidence relating to their cytoskeletal architecture and dynamics. Finally I shall try to construct models of motility consistent with the experimental data, and suggest how lamellar motility may be modulated by signals generated by clustered adhesion molecules.

The interpretation of interference-reflection images of spread cells: significant contributions from thin peripheral cytoplasm.

In interference-reflection microscopy, used for investigating cell-substratum separation, it is commonly believed that cytoplasmic thickness can be ignored, provided a high illuminating numerical aperture (INA) is used. It is shown here that even when a maximal INA is used, cytoplasmic lamellae of I micrometer or less can be major determinants of the image. The leading lamella of spreading tissue cells and large peripheral areas of Dictyostelium discoideum amoebae on adhesive substrata are less than I micrometer thick and it is argued that hitherto unexplained features of the interference images of these cells may be interpreted in terms of the theory used here.

Inhibition of platelet spreading from plasma onto glass by an adsorbed layer of a novel fluorescent-labeled poly(ethylene oxide)/poly(butylene oxide) block copolymer: characteristics of the exclusion zone probed by means of polystyrene beads and macromolecules.

We have investigated the anti-adhesive properties of a newly synthesized fluorescent triblock copolymer containing poly(ethylene oxide). This adsorbs from aqueous solution onto glass that has been rendered hydrophobic. When the polymer-treated surface was exposed to human platelet-rich plasma (PRP) or whole blood at 37 degrees C, platelet adhesion and spreading were prevented. Avid adhesion and rapid platelet spreading occurred along tracks scraped in the adsorbed polymer coating, as seen by video-enhanced interference reflection microscopy. Leukocytes from whole blood are eventually able to adhere to the polymer-treated surface and were seen to remove labeled polymer from their vicinity and accumulate it at the cell body. Interferometry using polystyrene spheres showed that they do not adhere to polymer-coated glass and are unable to approach closer than 70-95 nm. On scraped tracks, beads make molecular contacts with the glass. Because the fully extended solvated (EO)400 arms may extend up to 100 nm from the glass, this suggests that the polymer forms a monolayer with the hydrophilic arms projecting into the water, whereas the hydrophobic (BO)55 segment binds the molecule to the hydrophobic surface. Another tri-bloc copolymer with shorter hydrophilic arms allows particles to approach more closely.

Contacts of chick fibroblasts on glass: results and limitations of quantitative interferometry.

We have examined the contacts made by explanted chick heart and limb bud fibroblasts after 24-48 h on glass, using quantitative interference reflection microscopy (IRM). Contacts beneath very thin cytoplasmic lamellae were avoided because the images of such contacts depend on the thickness of the lamellae. Plaque-like focal contacts, distinguished on the basis of shape and low irradiance (darkness), are intimate adhesions to the substratum. These images can be interpreted if it is assumed that microfilaments associated with the lower membrane increase the local cytoplasmic refractive index. The range of irradiances measured for focal contacts was found to be rather wide, and our modelling shows that the most likely explanation for this is that the images receive variable contributions from the adjacent cytoskeleton. For this reason it is particularly difficult to assign a characteristic thickness for these contacts from IRM data. Close contacts, seen principally as 'grey' regions under migrating cells at the edges of the explants, also show a wide range of irradiances. Unlike focal contacts, it is not necessary to postulate any involvement of the cytoskeleton in their images and they can be modelled as regions where an aqueous glycocalyx zone about 20-30 nm thick separates the membrane bilayer from the glass. Paler grey regions that also look like close contacts are apparently formed where the cell surface has lifted several tens of nanometres from the glass.

Adhesion of red blood cells to charged interfaces between immiscible liquids. A new method.

We have devised a method of making a flat oil/water interface which remains flat on inversion. Cell adhesion to the interface can be observed microscopically. Glutaraldehyde-fixed human red blood cells adhere to the interface between physiological saline and hexadecane containing surface-active behenic acid at pH values below about 7-5. At high pH values, cells are prevented from adhering due to dissociation of the carboxyl groups of behenic acid oriented in the interface. The negative red cells are driven away electrostatically. Adherent and non-adherent cells remain on the aqueous side of the interface and do not appreciably deform it when adherent. Cells are electrostatically attracted to a similar interface containing positively charged octadecyltrimethylammonium ions. Cells also adhere to an interface containing octadecanol, which carries no charge. Underlying both electrostatic repulsion and attraction between red cells and oil/water interfaces is an attractive force which may be of electrodynamic (van der Waals) origin.

Red blood cell adhesion. II. Interferometric examination of the interaction with hydrocarbon oil and glass.

Using both living and glutaraldehyde-fixed red cells, we have examined adhesion to both oil/saline and glass/saline interfaces by interference reflection microscopy. At low ionic strength, 0.4 mM NaCl, fixed cells adherent to the oil/saline interface show first order whitish yellow zones of closest approach which indicate a separation of similar to or approximately 100 nm. Quantitative interferometry in monochromatic light supports this conclusion. As the salt concentration is increased the separation decreases and the final image shows zero-order black which probably indicates molecular contact with the interface. Similar but less reproducible results were obtained with fixed and unfixed cells on glass. Thes observations show that physical interactions alone can be responsible for adhesion in dilute and concentrated salt solutions. It is not, however, believed that the results necessarily imply the existence of adhesion with a gap in physiological concentrations of salt.

Interference reflection microscopy. A quantitative theory for image interpretation and its application to cell-substratum separation measurement.

We propose a quantitative theory of microscope interferometry where the specimen is illuminated by a cone of monochromatic light of solid angle 0 - 100 degree, corresponding to an illuminating numerical aperture of 0 to approximately 1.2. Computed results compare favorably with photometric measurements of fringe irradiance for a water wedge 0 - 2,000-nm thick. The interpretation of cell-substratum interference images is discussed in relation to the theory. We conclude that in assessing cell-glass separation, the cytoplasmic thickness does in general contribute significantly to the final image, but this contribution is minimized at high illuminating apertures. In these circumstances, however, normal incidence theory is inapplicable and the theory for finite illuminating aperture is essential. Neglect of this fact can lead to errors of up to 100% in estimated cell-glass separation.

Elimination of the effects of stray light in measurements by total internal reflection aqueous fluorescence (TIRAF).

Total internal reflection aqueous fluorescence has been shown to be capable of achieving spatial resolution in surface contours of about 1 nm. When used with highly structured objects, errors in measurements can arise from light scattered either by the object or within the body of the microscope. We describe how these errors can be eliminated when studying surface contours of human platelets.

Conformational response of the glycocalyx to ionic strength and interaction with modified glass surfaces: study of live red cells by interferometry.

We have measured separation distances between live human red blood cells and simple or modified glass surfaces, using the finite aperture technique of microscope interferometry. In general, separation increases as the ionic strength falls, in isotonic solutions. Restriction on movement parallel to the glass in all except the most dilute salt solutions, coupled with the absence of Brownian motion, indicates direct molecular contact with the substratum. Thus increased separation must be due to swelling of the glycocalyx under electrostatic forces. However, at approximately less than to 2mM adherent cells show a separation greater than 100 nm, execute Brownian motion and the restriction on lateral motion is less evident. This suggests that secondary minimum adhesion by long-range forces with little or no direct molecular connection occurs at extreme dilution only. Treatment of cells with trypsin reduces separation by up to 40 nm, but the extent to which this reflects reduced double-layer repulsion due to loss of surface charge, as opposed to the reduced opportunity for swelling in a trimmed-down glycocalyx, is unclear. Adhesion at a separation approximately 100 nm in 1 mM buffer after trypsinization supports the view that adhesion can occur without very long glycoprotein connections, but does not prove it. Adhesion to unwettable methylated glass and completely wettable unmethylated glass, with an identical ionic strength dependence of the separation, shows that hydrophilicity is not an absolute requirement. Red cells interact closely at all ionic strengths with glass made polycationic with poly-L-lysine, owing to electrostatic attraction. The interference technique also shows that adherent cells can be spaced from the glass by an intervening layer of previously absorbed serum albumin.

A sharp cell surface conformational transition at low ionic strength changes the nature of the adhesion of enzyme-treated red blood cells to a hydrocarbon interface.

Aldehyde-fixed human red cells have previously been used to assess the roles of electrostatic and electrodynamic forces in adhesion. We have attempted to test the prediction that enzymic removal of cell surface negative charges should increase adhesion in dilute salt solutions by reducing electrostatic repulsion. While this is indeed the case for neuraminidase-treated cells and also for Pronase- and trypsin-treated cells over much of the low ionic strength range, the latter two treatments cause very strong adhesion over a remarkably narrow range of ionic strength centred on 1 mM-NaCl. At 0.5 and 1.5 mM adhesion is negligible. After Pronase treatment a further adhesive peak occurs at 2.5 mM. Electrophoresis of protease-treated cells shows small but clear reductions in mobility at precisely these peak adhesion values. These electrophoretic potential changes are almost certainly not large enough to cause increased adhesion directly, and it is thought that they are second-order changes, symptomatic of a structural rearrangement of the cell surface. How this causes such vastly augmented adhesion is an intriguing problem.

Red blood cell adhesion. I. Determination of the ionic conditions for adhesion to an oil-water interface.

We have examined the adhesion of glutaraldehyde-treated human red blood cells to a clean liquid hydrocarbon interface as a function of sodium chloride concentration. Cells adhere reversibly to the interface over a wide range of concentrations but fail to do so below about 0.1 mM. Adherent cells do not alter the tension of the oil/water interface. These results show that cells can adhere by physical forces in special situations without biochemical interactions. The data provide a basis for calculating the size of the physical forces.

Interaction of red blood cells with a polarized electrode: evidence of long-range intermolecular forces.

We have investigated the electrostatic interaction of glutaraldehyde-fixed human red cells with a polarizable electrode carrying a defined surface charge density which can be varied continuously through a wide range. Cells in a dilute salt solution are unable to adhere to the electrode at high negative charge, but at lower negative charge densities they are reversibly adherent and can be forced off by increasing the negative polarization. Near zero electrode charge they become irreversibly stuck to the electrode and cannot be evicted even at maximum electrode polarization. Calculation of the electrostatic repulsive force using measured charge densities indicates the existence of an attractive force which may be acting over several hundred angstroms.

Red blood cell adhesion. III. Analysis of forces.

The results of experiments on the adhesion of glutaraldehyde-fixed red blood cells to both polarizable metal/saline and liquid hexadecane/saline interfaces have been analysed in terms of physical forces. The results show that the electrostatic repulsions sufficient to prevent adhesion to these test surfaces are remarkably similar, and that a force-balance condition is predicted at cell-substratum separations similar to or approximately 100 nm in 0.4 mM NaCl as found by interferometry. From the repulsive force the size of the attractive force can be found. If this is viewed as an electrodynamic attraction, the force coefficient is found to lie between 5 and 8 x 10(-14) erg (5 and 8 x 10(-21)J), a range in reasonable correspondence with measurements in physical systems.

Interaction between intracellular vacuoles and the cell surface analysed by finite aperture theory interference reflection microscopy.

Using finite aperture theory we have shown that localized very dark areas in the interference reflection images of Dictyostelium discoideum amoebae are due to the close intracellular approach of vesicles and tubular elements of the contractile vacuole system to the plasma membrane adjacent to the substratum. Vesicles interacting in this way become locally deformed to the planar contour of the substratum and are separated from the cell surface membrane by a constant approximately less than 0.1 micron of cytoplasm. Lamellar processes formed by these cells on very adhesive surfaces have identical dimensions. This minimal thickness may be a mechanical consequence of a contractile mechanism which pulls membranes together.

Red blood cells experience electrostatic repulsion but make molecular adhesions with glass.

We have studied the detachment of unfixed red cells from glass coverslips under unit gravity and by centrifugation in buffered isotonic solutions over a range of ionic strengths. Cell-glass contact areas and separation distances were measured by quantitative interference reflection microscopy. Detachment under unit gravity is highly dependent on ionic strength: dilution increases electrostatic repulsion and greatly reduces the proportion of adherent cells. However, even at 1.5 mM some cells stick. Over the range 3-110 mM such adherent cells are progressively removed by increasing centrifugal forces, but in a manner virtually independent of ionic strength. This fact, together with the irreversibility of pre-adherent cells as ionic strength is progressively reduced, as well as the resistance of cells to lateral shearing forces, provide evidence sufficient to reject the notion of secondary minimum adhesion for unfixed cells at any ionic strength down to 1.5 mM. We conclude that all unfixed cells that stick at ionic strengths from 157 to 1.5 mM make molecular contacts with glass. Comparison with long range force calculations suggests that to penetrate the electrostatic repulsion barrier the contact regions are unlikely to have average surface properties. A new method that compares frequency distributions of contact areas with responses to detachment forces shows that detachment forces are not linearly related to contact areas. This lack of relationship is less clearly evident for rigid glutaraldehyde-fixed cells and may therefore depend on the degree of cellular deformability.