concanavalin-a and 3-3--dithiobis(sulfosuccinimidyl-propionate)

Cooperativity of molecular adhesion has been proposed as a mechanism for enhanced binding strength of adhesion molecules on the cell surface. Direct evidence for its mechanism, however, has been lacking until now. Atomic force microscopy (AFM) was used to measure the adhesive strength between concanavalin A (Con A) coupled to an AFM tip and Con A receptors on the surface of NIH3T3 fibroblast cells. Cross-linking of receptors with either glutaraldehyde or 3, 3'-dithio-bis(sulfosuccinimidylproprionate) (DTSSP) led to an increase in adhesion that could be attributed to enhanced cooperativity among adhesion complexes. An increase in loading rate due to greater stiffness of fixed cells also contributed to the twofold increase in binding strength. These results show that receptor cross-linking can greatly contribute to a total increase in cell adhesion by creating a shift toward cooperative binding of receptors.

Topics: 3T3 Cells; Animals; Cell Adhesion; Concanavalin A; Cross-Linking Reagents; Glutaral; Mice; Microscopy, Atomic Force; Models, Biological; Receptors, Cell Surface; Receptors, Concanavalin A; Succinimides

To shed light on the architecture of the cytoskeleton, we used the atomic force microscope (AFM) to measure the elasticity, viscoelasticity, and plasticity of L929 cells. The initial elastic response (Young's modulus approximately 4,000 Pa) of the cells to an applied force was followed by a slow compression of the cytoskeleton (tau 1/2 approximately equal to 10 s). When force application was terminated, the cytoskeleton underwent a sudden partial decompression and a subsequent slow, incomplete recovery. The role of the cytoskeletal elements in cell mechanics was accessed in AFM measurements carried out on cells treated with cytochalasin D, nocodazole, or colcemid. Cytochalasin D treatment reduced both elasticity (approximately 45%) and cytoplasmic viscosity (approximately 65%), whereas cells treated with nocodazole or colcemid exhibited a marked increase in elasticity (approximately 100%) and a slight increase in viscosity (approximately 15%). The AFM force measurements also provided evidence that the cell membrane and the cytoskeleton are mechanically coupled. Tightly adherent cells were stiffer than cells that were loosely attached. Moreover, cells crosslinked with either glutaraldehyde, 3, 3'-dithiobis [sulfosuccinimidylpropionate] (DTSSP), or Concanavalin A were more rigid than untreated cells. It is of interest that cells crosslinked with Concanavalin A, but not DTSSP, displayed plastic behaviors that may reflect the induction of cytoskeletal reorganization by Concanavalin A.

Topics: Actins; Animals; Antineoplastic Agents; Cell Line; Cell Membrane; Concanavalin A; Cross-Linking Reagents; Cytochalasin D; Cytoskeletal Proteins; Cytoskeleton; Demecolcine; Elasticity; Fibroblasts; Glutaral; Membrane Proteins; Mice; Microscopy, Atomic Force; Microtubules; Nocodazole; Nucleic Acid Synthesis Inhibitors; Succinimides; Viscosity