Intercellular recognition: quantitation of initial binding events.

Abstract

The hypothesis that intercellular adhesion can be subdivided into two separable phenomena-an initial recognition event and a subsequent stabilization-is supported by the use of a cell binding assay that provides a quantitative measure of intercellular binding strengths. Radioactive single cells are brought into contact with cell monolayers at 40C in sealed compartments. The compartments are inverted and a centrifugal force is then applied to dislodge the probe cells from the monolayers. By varying the speed of centrifugation, the force maintaining associations between embryonic chicken neural retina cells was determined to be on the order of 10-5 dyne. Topographic specificities of single neural retina cells for retinal monolayers from various regions of the retina were detected with this assay and corresponded to those observed in more traditional assays at 37C. Also observed were two time-and temperature-dependent stabilization processes in which the force required for dislodgment increased. One of the stabilization processes was sensitive to dinitrophenol and was inactive at 4TC; the second was still active in metabolically blocked cells. The metabolic-dependent process resulted in interactions at least 13 times as strong as the initial binding. The metabolic-independent process resulted in about a 2-fold increase in binding strength and had a temperature dependence similar to that of membrane diffusional phenomena.Precise cellular interactions play an essential role in the assembly of tissues during embryogenesis and are attributed, in part, to adhesive specificities (1). Studies by Umbreit and Roseman (2) on embryonic chicken tissues in vitro suggested that cell adhesion could be subdivided into an initial reversible phase and a subsequent stabilizing process. Reversible adhesions were characterized by their dissociation by small fluid shear forces. The stabilizing process was strongly dependent on time and temperature and led to more permanent bonds between cells.Data obtained by using a newly developed cell binding assay support these results and suggest that interactions determining recognition specificity take place during the reversible phase ofcell interactions. In addition, the assay provides a quantitative measure of intercellular binding strengths. Radioactive probe cells are brought into contact with monolayers in sealed compartments by a low centrifugal force. The compartments are then inverted in the centrifuge and the force required to dislodge the probe cells from the monolayers is determined. Probe cells were found to bind at 4°C with a strength that resisted about 10' dyne of dislodgment force per cell and exhibited recognition specificity and sensitivity to trypsin and calcium. When the bound cells were incubated for 8 min at 37°C, the resistance to dislodgment was at least 13 times stronger than that of the associations formed at 4°C. This stabilization was dependent on the cells' being metabolically active.With metabolism inhibited, a second type of strengthening ofcell binding wa...

Coaction of intercellular adhesion and cortical tension specifies tissue surface tension

“…However, because the interaction between actin and cadherin-mediated adhesion is a highly dynamic process that is regulated by α-catenin (29,30), it is possible to argue that increasing cadherin expression significantly increases cortical tension and γ∕β remains unknown. Similarly, when we down-regulate surface tension by cytoskeletal drugs, both cortical tension and adhesive energy are decreased because cadherin bonds are stabilized by the cortical network (31)(32)(33). One attempt to dissect the connection of cortical tension and adhesion of individual cells was reported by Krieg et al (19) using AFM measurements.…”

Stimulation of integrin-mediated adhesion of T lymphocytes and monocytes: two mechanisms with divergent biological consequences.

“…This is analogous to ~Ilbfl3, where the "inactive" (low affinity) form is competent for adhesion to immobilized fibrinogen (55, 56) but does not bind soluble fibrinogen. Cytoskdetal organization and subsequent cell spreading dramatically increase the resistance of cells to shear stresses, resulting in stronger adhesion under experimental conditions (57)(58)(59). Phorbol esters are known to affect these events.…”

Wheatgerm agglutinin-mediated toxicity in pancreatic cancer cells

“…In cases of neuraminidase pretreatment, suspended cells were incubated at 2 × 10 6 per ml in serum-free medium with neuraminidase from Vibrio cholerae at 50 mU ml -1 and 37°C over 1 h. The cells were then washed twice with complete medium, checked for viability with trypan blue, counted and plated into 96-well plates as described. For experiments involving immobilized lectin, a technique modified after McClay et al (1981) was used. WGA in PBS solution at 2-80 µg ml -1 was loaded into uncoated 96-well plastic plates at 50 µl per well for 1 h at room temperature.…”