Cell-substrate interactions and locomotion of Dictyostelium wild-type and mutants defective in three cytoskeletal proteins: a study using quantitative reflection interference contrast microscopy.

摘要

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.