Cell motility : the organization of the microfilament system in cultured cells with special emphasis on the cortical weave of actin filaments

Sammanfattning: The microfilament system formed by actin, myosin and associated proteins constitutes the molecular basis for many of the motile phenomena which eukaryotic cells express. The organization of this system varies considerably from cell type to cell type, and is characterized on one hand by strictly ordered, more or less flexible arrangements of actin filaments and on the other by actin present in primarily a nonfilamentous form ready to polymerize in response to certain stimuli. How the microfilament system functions in muscle cells to bring about contraction is fairly well understood but the mechanisms behind non-muscle motility are less well characterized. The relationships between the microfilament system and the other two fiber structures present in eukaryotic cells, microtubules and intermediate filaments, are unknown and the molecular basis for the regulation of the extensive reorganizations of microfilament arrangements, that occur in certain cells in response to internal or external signals, are only beginning to be unravelled. Profilin, a low molecular-weight protein, is one component which appears to be involved in this control system.In this study the cortical arrangements of the microfilament system in glial cells and spreading platelets were visualized by transmission electron microscopy of negatively-stained whole cell mounts. The preservation of the cell ultrastructure was optimized by the development of a detergent-fixative mixture which was combined with the use of sodium silicotungstate as negative-stain. This led to a highly detailed picture of the microfilament arrangement at the leading edge of spreading and translocating cells. In addition, sodium silicotungstate was found to penetrate the plasma membrane of glutaraldehyde-fixed cells, thereby enabling the visualization of actin filaments aligned in parallel to form sheets which appeared to extend in the submembraneous space around the whole cell.Human placental profilin was isolated and partially characterized in order to elicit antibodies for studies of the cellular localization of profilin. For unknown reasons, however, the antibodies obtained were not reactive in immunofluorescence microscopy and the distribution of profilin could thus not be determined. Myosin was localized by immuno-electron microscopy in glial cells which had been exposed to N-ethyl-maleimide. A derangement of actin - possibly a depolymerization - as well as a change in the distribution of myosin was observed as a result of the NEM-treatment.