A 'dynamic adder model' for cell size homeostasis in Dictyostelium cells.

After a cell divides into two daughter cells, the total cell surface area of the daughter cells should increase to the original size to maintain cell size homeostasis in a single cell cycle. Previously, three models have been proposed to explain the regulation of cell size homeostasis: sizer, timer, and adder models. Here, we precisely measured the total cell surface area of Dictyostelium cells in a whole cell cycle by using the agar-overlay method, which eliminated the influence of surface membrane reservoirs, such as microvilli and membrane wrinkles. The total cell surface area exponentially increased during interphase, slightly decreased at metaphase, and then increased by approximately 20% during cytokinesis. From the analysis of the added surface area, we concluded that the cell size was regulated by the adder or near-adder model in interphase. This adder model is not caused by a simple cell membrane addition, but is more dynamic due to the rapid cell membrane turnover. We propose a 'dynamic adder model' to explain cell size homeostasis in interphase.

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.

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

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.

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.

Myosin II contributes to the posterior contraction and the anterior extension during the retraction phase in migrating Dictyostelium cells.

Cells must exert force against the substrate to migrate. We examined the vectors (both the direction and the magnitude) of the traction force generated by Dictyostelium cells using an improved non-wrinkling silicone substrate. During migration, the cells showed two 'alternate' phases of locomotory behavior, an extension phase and a retraction phase. In accordance with these phases, two alternate patterns were identified in the traction force. During the extension phase, the cell exerted a 'pulling force' toward the cell body in the anterior and the posterior regions and a 'pushing force' in the side of the cell (pattern 1). During the retraction phase, the cell exerted a 'pushing force' in the anterior region, although the force disappeared in the side and the posterior regions of the cell (pattern 2). Myosin II heavy chain null cells showed a single pattern in their traction force comparable to 'pattern 1', although they still had the alternate biphasic locomotory behavior similar to the wild-type cells. Therefore, the generation of 'pushing force' in the anterior and the cancellation of the traction force in the side and the posterior during the retraction phase were deficient in myosin knock-out mutant cells, suggesting that these activities depend on myosin II via the posterior contraction. Considering all these results, we hypothesized that there is a highly coordinated, biphasic mechanism of cell migration in Dictyostelium.

A mechanism for the intracellular localization of myosin II filaments in the Dictyostelium amoeba

When ATP is added to membrane-cytoskeletons prepared from Dictyostelium amoebae by the method described previously (S. Yumura and T. Kitanishi-Yumura, Cell Struct. Funct. 15, 355­364, 1990), myosin II is released from the membrane-cytoskeletons after contraction. Simultaneously, the heavy chains of myosin II are phosphorylated by a putative myosin II heavy-chain kinase, at foci within the actin network, with the resultant disassembly of filaments. In this study, we examined factors that control the release of myosin II from the membrane-cytoskeletons, on the assumption that inhibition of the release of myosin II keeps the myosin II in the cortical region, and is responsible for the localization of myosin II in the cortical region. The release of myosin II is inhibited at pH values below 6.5. This effect is not due to the inhibition of heavy-chain phosphorylation but is due to the suppression of disassembly of the filaments. In the membrane-cytoskeletons of aggregating cells, the release of myosin II is inhibited by Ca2+, and this effect is enhanced by pretreatment with calmodulin. In the membrane-cytoskeletons of vegetative cells, the release of myosin II is inhibited by pretreatment with calmodulin, and this effect is Ca2+-independent. The inhibition of the release of myosin II by Ca2+ and/or calmodulin is due to the inhibition of heavy-chain phosphorylation, and calmodulin is associated with the foci within the actin network. These results represent a possible mechanism for the intracellular localization of myosin II via regulation of the release of myosin from the cortical region by changes in intracellular pH and/or intracellular concentrations of Ca2+.

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 slug's 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 slug's migration. The distribution of c-shaped fluorescence in slugs during migration, phototaxis and avoidance of ammonia strongly suggests that the slug's behavior is controlled by the concerted movement of prestalk cells.