Using Microinterferometry to Study the Function of the Cytoskeleton in Cell Motility
Monday, January 4, 1999 - 9:30am - 10:30am
Graham Dunn (King's College)
A major problem in characterising the locomotion of freely-moving, isolated cells in culture is the large variability between individual cells and the large variation in motile characteristics with time. DRIMAPS (Digitally Recorded Interference Microscopy with Automatic Phase Shifting) is a technique developed at the Randall Institute that overcomes this problem by enabling data from large numbers of cells to be gathered automatically and analysed by computer. The images that it produces are calibrated maps of the distribution of cellular material (dry mass) within all cells in the field of view. This is the only system that can automatically and reliably detect the exact location of the margins of living, unstained cells, even in thinly spread vertebrate cells. Time lapse sequences of images recorded over many hours and stored digitally provide data for many different forms of motion analysis and also enable the growth (increase in dry mass) of individual cells to be monitored. Three projects will be described to illustrate the use of the technique to study cytoskeletal function. In the first, we have used specific drugs to alter the dynamics of microtubule assembly. Microinterferometry has revealed rapid, microtubule-dependent fluctuations of the cell margin that give rise to much larger, low frequency changes in cell spreading and motility. The second project, in collaboration with Michelle Peckham of Leeds University, is the analysis of motility in mouse myoblasts transfected to express foreign or defective components of the cytoskeleton. Transfection with a foreign beta-cardiac myosin II suppresses cell motility whereas overexpression of human beta-actin promotes protrusion and locomotion. Finally, in collaboration with David Critchley of Leicester University, we have studied the subtle effects of knockout mutants of talin and vinculin in undifferentiated cells derived from mouse embryos.