Motility and substratum adhesion of Dictyostelium wild-type and cytoskeletal mutant cells: a study by RICM/bright-field double-view image analysis

Abstract

To investigate the dynamics of cell-substratum adhesion during locomotion, a double-view optical technique and computer-assisted image analysis has been developed which combines reflection interference contrast microscopy (RICM) with bright-field imaging. The simultaneous recording of cell-substratum contact and cell body contour has been applied to aggregation-competent cells of Dictyostelium discoideum. These cells are distinguished from cells at earlier stages of development by small areas of contact to a substratum. Three questions have been addressed in analysing the locomotion of aggregation-competent cells. (1) What is the relationship between changes in the shape of cells and their contact to a substratum during a chemotactic response? (2) What is the relationship between protrusion and retraction of the cell body, and between local attachment and detachment? (3) Are there differences between wild-type and mutant cells that lack certain cytoskeletal proteins? During a chemotactic response the front region of the amoeba can bend towards the gradient of attractant without being supported by its contact with a surface, which excludes the necessity for gradients of adhesion for the response. The finding that in locomoting cells protrusion of the leading edge often precedes retraction establishes a pioneer role for the front region. The finding that gain of contact area precedes loss provides evidence for the coordination of interactions between the cell surface and a substratum. For comparison with wild-type, aggregation-competent triple mutant cells have been used that lack two F-actin crosslinking proteins, alpha-actinin and 120 kDa gelation factor, and an actin filament fragmenting protein, severin. Disturbances in the spatial and temporal control of cytoskeletal activities have been unravelled in the mutant by RICM and quantified by cross-correlation analysis of attachment and detachment vectors. In order to detect these disturbances, it was essential to analyse cell locomotion on the weakly adhesive surface of freshly cleaved mica.