Myosin-II localization is the result of a dynamic steady state in which recruitment is offset by thick filament disassembly. In this model, a signal that possibly emanates from the mitotic spindle instructs the cortex to recruit myosin-II to the furrow cortex and to begin contraction. This leads to the elongation of the cell to the cylindrical form followed by constriction of the furrow. Myosin-II is recruited until enough protein has accumulated and the cell can constrict in the desired manner. More myosin-II is recruited to the furrow cortex when D. discoideum cells are flattened by agar overlay when compared to cells grown under standard conditions on the surface of a culture plate . Flattening a cell by agar overlay may increase the apparent stiffness of the cell by introducing tension into the cortex from increased hydrostatic pressure. Intriguingly, myosin-II null D. discoideum cells divide relatively normally if allowed to adhere to a surface , but myosin-II becomes essential if the adherent cells are grown under a sheet of agarose .
The pathway for myosin-II disassembly is most likely always active and the direction of the steady state depends on whether the signal to recruit myosin-II is active. Consistent with this concept, Yumura  measured the turnover of cortically distributed myosin-II by fluorescence recovery after photobleaching (FRAP) and showed that wild type myosin-II is localized to the cortex with a half-life of about 7 seconds. This cortical half-life is the same regardless of whether the cell is in interphase or undergoing cytokinesis. On the other hand, the 3 × Ala mutant myosin-II has a half-life greater than the several minutes used to track the protein. The quantitation of the distribution of the 3 × Ala GFP-myosin-II verifies that phosphorylation-controlled disassembly is a normal part of myosin-II turnover. The amount of the 3 × Ala myosin-II enrichment in the furrow cortex might represent normal accumulation of myosin-II that is ordinarily offset by the disassembly of the myosin-II thick filaments possibly as a part of a feedback mechanism that senses the mechanical needs of the cell.
The data presented here does not distinguish between recruitment of myosin-II to the cleavage furrow cortex by cortical sliding or recruitment from the cytoplasm. Between the period of cytokinesis involving transition from a spherical cell shape to a cylindrical shape, there is little or no net flux of myosin-II from the cytoplasm to the cortex. A simple interpretation is that the myosin-II simply slides along the cortex from the poles to the cleavage furrow. Indeed, FRAP studies support a model where dynamic myosin-II filaments progressively slide along the cortex while exchanging with the cytoplasmic pool of myosin-II monomers . However, between the cylindrical and dumbbell stages of cell shape, there is a net flux of myosin-II from the cytoplasm to the cortex. This flux from the cytoplasm to the cortex may simply reflect the increase in the surface area (and consequently the cortex volume) to total cell volume ratio and may not be specific to cytokinesis per se. Then, cortical flow could continue to concentrate the myosin-II into the furrow cortex. Since more 3 × Ala myosin-II than wild type myosin-II is recruited to the cleavage furrow cortex, the recruitment mechanism is unlikely to be limiting for the wild type myosin-II. This suggests that an unlimiting regulator molecule may control the localization of myosin-II. A candidate for a catalytic regulator molecule is the PAKa kinase, which is required for myosin-II localization to the cleavage furrow cortex . This kinase could act on enzymes that regulate the myosin-II assembly-state or it could act on a cortical receptor for myosin-II thick filaments.
During cytokinesis, global and spatial mechanical properties are likely used to deform a cell, allowing the constriction of the cell's equator (reviewed in ). These spatial properties include local modulation of stiffness and the generation of contractile force. A localized stiffness increase may define the region that is deformed during a morphological process. The cleavage furrow cortex increases in stiffness above the global stiffness in mammalian cells as determined by atomic force microscopy . This regional increase in stiffness is likely to occur in most or all metazoan and ameboid cells. For example, in D. discoideum, the cortexillin actin-filament crosslinking proteins are recruited to the cleavage furrow where they are thought to contribute to the stiffness of the cleavage furrow cortex [24, 25]. Myosin-II is probably a major contractile force generator. However, direct measurement of this parameter is far more difficult in small cells. In this paper, we have measured the amount of myosin-II that is recruited to the cleavage furrow cortex. This amount is in close agreement with the estimate of the amount of myosin-II that would be required to generate the forces that may be required to cleave a cell.
The mechanical model presented provides a useful framework for conceptualizing the quantitation of myosin-II. However, there are many issues that must be considered before a comprehensive model for contractile force production can be developed. First, it should be noted that myosin-II null cells can divide if given a surface to which to adhere. The mechanism by which these cells divide under these conditions is not known. However, by considering the mechanics of the process one realizes that the cell has the highest need for force generation during elongation and initial furrow ingression. Traction forces may serve well to elongate the cell, allowing the formation of the cylindrical shape. Indeed, myosin-II null cells in the absence of surface attachment fail to elongate, verifying that myosin-II is required for this step when surface traction is not available . Other gelation and solvation factors and possibly an unconventional myosin might complete the process once the cylindrical shape has been acquired (for further discussion see ). Interestingly, during interphase the myosin-II null cells are 30% less stiff in bending modulus and 70% less stiff in surface tension than the wild type parental cells [10, 26]. Thus, by considering the mechanics of division, one realizes that myosin-II null cells probably require significantly less force to divide, making it easier to cleave. Since other proteins are likely to contribute to contractile force production, the quantitation of the amount of myosin-II recruited to the cleavage furrow cortex allows us to estimate how much force can be provided by myosin-II. In fact, there are adequate amounts of myosin-II to generate at least the majority of the forces that are estimated to be required. It is reasonable that the contractile ring may be able to generate greater force than is minimally required. Having the ability to generate more force than is minimally required may have great utility in nature. Environmental factors such as cell-cell contact and hypotonicity might place additional demands on the equatorial contractile apparatus that go beyond the intrinsic stiffness of the cell cortex.
Another issue is that the level of myosin-II activity may be able to vary. In muscle, there is load-dependent recruitment of myosin-II cross-bridges; in other words, the duty ratio of muscle myosin-II can vary (reviewed by ). For our calculations, we considered the duty ratio to be 0.006, which is the value measured for unloaded D. discoideum myosin-II. Load-dependent recruitment of crossbridges for D. discoideum myosin-II has not been examined so it is not known whether this is important for a simple eukaryotic nonmuscle myosin-II. However, this ratio may be able to change in a load-dependent manner such that the amount of myosin-II that is recruited to the cleavage furrow may be able to generate much greater forces than appear to be minimally required. Myosin-II activity might also be predicted to vary as a result of localized activation by myosin light chain phosphorylation. However, in D. discoideum, myosin light chain phosphorylation is not required for cytokinesis so this is unlikely to be a major consideration [28, 29]. Finally, the ratio imaging experiments do not allow us to make conclusions regarding the radial uniformity of the myosin-II in the cleavage furrow cortex. Thus, it is possible that the force generation is not generated in a perfectly symmetrical fashion. However, the contractile force may not need to be generated in an entirely uniform manner to generate the needed forces to stabilize the ingression of the cleavage furrow cortex. The quantitation of amounts of myosin-II that are recruited to the contractile ring is an essential starting point for developing a more detailed but experimentally tractable model for cytokinesis. Given the complex bookkeeping involved in accounting for the contributions of each individual force generator to the precise mechanical needs of every individual cell, it is remarkable that the trends of myosin-II distribution are so closely predicted by the simple theoretical analysis.