We have employed an interferometric technique for the local measurement of bending modulus, membrane tension, and adhesion energy of motile cells adhering to a substrate. Wild-type and mutant cells of Dictyostelium discoideum were incubated in a flow chamber. The flow-induced deformation of a cell near its adhesion area was determined by quantitative reflection interference contrast microscopy (RICM) and analyzed in terms of the elastic boundary conditions: equilibrium of tensions and bending moments at the contact line. This technique was employed to quantify changes caused by the lack of talin, a protein that couples the actin network to the plasma membrane, or by the lack of cortexillin I or II, two isoforms of the actin-bundling protein cortexillin. Cells lacking either cortexillin I or II exhibited reduced bending moduli of 95 and 160 k(B)T, respectively, as compared to 390 k(B)T, obtained for wild-type cells. No significant difference was found for the adhesion energies of wild-type and cortexillin mutant cells. In cells lacking talin, not only a strongly reduced bending modulus of 70 k(B)T, but also a low adhesion energy one-fourth of that in wild-type cells was measured.

We used micropipettes to aspirate leading and trailing edges of wild-type and mutant cells of Dictyostelium discoideum. Mutants were lacking either myosin II or talin, or both proteins simultaneously. Talin is a plasma membrane-associated protein important for the coupling between membrane and actin cortex, whereas myosin II is a cytoplasmic motor protein essential for the locomotion of Dictyostelium cells. Aspiration into the pipette occurred above a threshold pressure only. For all cells containing talin this threshold was significantly lower at the leading edge of an advancing cell as compared to its rear end, whereas we found no such difference in cells lacking talin. Wild-type and talin-deficient cells were able to retract from the pipette against an applied suction pressure. In these cells, retraction was preceded by an accumulation of myosin II in the tip of the aspirated cell lobe. Mutants lacking myosin II could not retract, even if the suction pressures were removed after aspiration. We interpreted the initial instability and the subsequent plastic deformation of the cell surface during aspiration in terms of a fracture between the cell plasma membrane and the cell body, which may involve destruction of part of the cortex. Models are presented that characterize the coupling strength between membrane and cell body by a surface energy sigma. We find sigma approximately 0.6(1.6) mJ/m(2) at the leading (trailing) edge of wild-type cells.

Abstract. Actin-binding proteins are known to regulate in vitro the assembly of actin into supramolecular structures, but evidence for their activities in living nonmuscle cells is scarce. Amebae of Dictyostelium discoideum are nonmuscle cells in which mutants defective in several actin-binding proteins have been described. Here we characterize a mutant deficient in the 120-kD gelation factor, one of the most abundant F-actin cross-linking proteins of D. discoideum cells. No F-actin cross-linking activity attributable to the 120-kD protein was detected in mutant cell extracts, and antibodies recognizing different epitopes on the polypeptide showed the entire protein was lacking. Under the conditions used, elimination of the gelation factor did not substantially alter growth, shape, motility, or chemotactic orientation of the cells towards a cAMP source. Aggregates of the mutant developed into fruiting bodies consisting of normally differentiated spores and stalk cells. In cytoskeleton preparations a dense network of actin filaments as typical of the cell cortex, and bundles as they extend along the axis of filopods, were recognized. A significant alteration found was an enhanced accumulation of actin in cytoskeletons of the mutant when cells were stimulated with cyclic AMP. Our results indicate that control of cell shape and motility does not require the fine-tuned interactions of all proteins that have been identified as actin-binding proteins by in vitro assays.

A monoclonal antibody, mAb 47‐19‐2, was used to study the subunit topology of the rod‐shaped alpha‐actinin molecules of Dictyostelium discoideum and to screen for mutants defective in the production of alpha‐actinin. Electron microscopy of rotary‐shadowed alpha‐actinin‐antibody complexes showed binding of mAb 47‐19‐2 to both ends of the alpha‐actinin rods and cleavage of the rods into its subunits, indicating that the two subunits of alpha‐actinin extend in an anti‐parallel mode through the whole length of the rod. The antibody binding sites were located in close proximity to the sites responsible for actin cross‐linking, which is consistent with the blocking activity of the antibody. In a mutant, HG1130, no antibody label was detected in colony blots, and by immunoblotting of mutant proteins separated by SDS‐PAGE, only trace amounts of alpha‐actinin were found. The mutant showed normal binding of antibodies directed against the actin‐binding proteins severin and capping protein. The mutation responsible for the alpha‐actinin defect was recessive and located on linkage group I of the genetic map of D. discoideum. HG1130 cells grew on bacteria at a normal rate and also axenically like cells of the parent strain AX2. After starvation the mutant cells expressed the contact site A glycoprotein, a marker of the aggregation‐competent stage, and reacted chemotactically to cyclic AMP. The aggregation patterns and fruiting bodies of the mutant appeared to be normal. Patching and capping on the surface of HG1130 cells was induced by antibodies against the contact site A glycoprotein.(ABSTRACT TRUNCATED AT 250 WORDS)

Reflection interference contrast microscopy combined with digital image processing was applied to study the motion of Dictyostelium discoideum cells in their pre-aggregative state on substrata of different adhesiveness (glass, albumin-covered glass, and freshly cleaved mica). The temporal variations of the size and shape of the cell/substratum contact area and the time course of advancement of pseudopods protruding in contact with the substratum were analyzed. The major goal was to study differences between the locomotion of wild-type cells and strains of triple mutants deficient in two F-actin cross-linking proteins (alpha-actinin and the 120-kDa gelation factor) and one F-actin fragmenting protein (severin). The size of contact area, AC, of both wild-type and mutant cells fluctuates between minimum and maximum values on the order of minutes, pointing toward an intrinsic switching mechanism associated with the mechanochemical control system. The fluctuation amplitudes are much larger on freshly cleaved mica than on glass. Wild-type and mutant cells exhibit remarkable differences on mica but not on glass. These differences comprise the population median of AC and alterations in pseudopod protrusion. AC is smaller by a factor of two or more for all mutants. Pseudopods protrude slower and shorter in the mutants. It is concluded that cell shape and pseudopods are destabilized by defects in the actin-skeleton, which can be overcompensated by strongly adhesive substrata. Several features of amoeboid cell locomotion on substrata can be understood on the basis of the minimum bending energy concept of soft adhering shells and by assuming that adhesion induces local alterations of the composite membrane consisting of the protein/lipid bilayer on the cell surface and the underlying actin-cortex.