Toshiko Kitanishi-Yumura

Multiple mechanisms for accumulation of myosin II filaments at the equator during cytokinesis.

Total internal reflection fluorescence microscopy revealed how individual bipolar myosin II filaments accumulate at the equatorial region in dividing Dictyostelium cells. Direct observation of individual filaments in live cells provided us with much convincing information. Myosin II filaments accumulated at the equatorial region by at least two independent mechanisms: (i) cortical flow, which is driven by myosin II motor activities and (ii) de novo association to the equatorial cortex. These two mechanisms were mutually redundant. At the same time, myosin II filaments underwent rapid turnover, repeating their association and dissociation with the actin cortex. Examination of the lifetime of mutant myosin filaments in the cortex revealed that the turnover mainly depended on heavy chain phosphorylation and that myosin motor activity accelerated the turnover. Double mutant myosin II deficient in both motor and phosphorylation still accumulated at the equatorial region, although they displayed no cortical flow and considerably slow turnover. Under this condition, the filaments stayed for a significantly longer time at the equatorial region than at the polar regions, indicating that there are still other mechanisms for myosin II accumulation such as binding partners or stabilizing activity of filaments in the equatorial cortex.

Cell-scale dynamic recycling and cortical flow of the actin–myosin cytoskeleton for rapid cell migration

Summary Actin and myosin II play major roles in cell migration. Whereas pseudopod extension by actin polymerization has been intensively researched, less attention has been paid to how the rest of the actin cytoskeleton such as the actin cortex contributes to cell migration. In this study, cortical actin and myosin II filaments were simultaneously observed in migrating Dictyostelium cells under total internal reflection fluorescence microscopy. The cortical actin and myosin II filaments remained stationary with respect to the substratum as the cells advanced. However, fluorescence recovery after photobleaching experiments and direct observation of filaments showed that they rapidly turned over. When the cells were detached from the substratum, the actin and myosin filaments displayed a vigorous retrograde flow. Thus, when the cells migrate on the substratum, the cortical cytoskeleton firmly holds the substratum to generate the motive force instead. The present studies also demonstrate how myosin II localizes to the rear region of the migrating cells. The observed dynamic turnover of actin and myosin II filaments contributes to the recycling of their subunits across the whole cell and enables rapid reorganization of the cytoskeleton.

Concerted Movement of Prestalk Cells in Migrating Slugs of Dictyostelium Revealed by the Localization of Myosin

Localization of myosin in slugs of the cellular slime mold Dictyostelium discoideum was investigated by an immunofluorescence technique. Myosin is thought to provide the molecular machinery for cellular movement. We found that myosin could be visualized as c‐shaped fluorescence at the cortex of prestalk cells in a migrating slug, and that the open regions of all c‐shaped fluorescence point in the direction of the slugs migration. We reported previously that the c‐shaped fluorescence of myosin can be seen at the cortex of the tail region of actively locomoting cells at the unicellular stage (39, 41). These results suggest that prestalk cells move actively in the slug, and that their direction of movement, which can be identified from the polarity of c‐shaped fluorescence, correspond with the direction of the slugs migration. The distribution of c‐shaped fluorescence in slugs during migration, phototaxis and avoidance of ammonia strongly suggests that the slugs behavior is controlled by the concerted movement of prestalk cells.

Turnover and flow of the cell membrane for cell migration

The role of cell membrane dynamics in cell migration is unclear. To examine whether total cell surface area changes are required for cell migration, Dictyostelium cells were flattened by agar-overlay. Scanning electron microscopy demonstrated that flattened migrating cells have no membrane reservoirs such as projections and membrane folds. Similarly, optical sectioning fluorescence microscopy showed that the cell surface area does not change during migration. Interestingly, staining of the cell membrane with a fluorescent lipid analogue demonstrated that the turnover rate of cell membrane is closely related to the cell migration velocity. Next, to clarify the mechanism of cell membrane circulation, local photobleaching was separately performed on the dorsal and ventral cell membranes of rapidly moving cells. The bleached zones on both sides moved rearward relative to the cell. Thus, the cell membrane moves in a fountain-like fashion, accompanied by a high membrane turnover rate and actively contributing to cell migration.