Though most organisms have two or more 14-3-3 isoforms (mammals have seven), the Dictyostelium genome has just one. It is not surprising then that 14-3-3 is an essential gene in this system.4
However, partial silencing of 14-3-3 to 30% of normal levels using a hairpin plasmid (14-3-3 hp) led to cell cultures that produced multinucleated cells, a hallmark of cytokinesis failure. The dynamics of cleavage furrow contractility and the morphology of the 14-3-3 hp cells as their shaped evolved was highly reminiscent of the very stereotypical myosin II
null cytokinesis. Subsequent proteomics analysis revealed that myosin II heavy chain is a major binding partner for 14-3-3. Follow-up analysis confirmed that 14-3-3 does indeed bind wild type myosin II though not the phosphomimic 3x Asp mutant myosin II, which is myosin II bipolar thick filament (BTF) assembly incompetent.
Three well characterized consensus binding motifs are commonly found in 14-3-3-binding partners.9
These include two phosphorylation-dependent binding motifs (motif 1 and motif 2) and a nonphosphorylation-dependent motif (motif 3). However, other types of interaction sites have also been observed. Interestingly, Dictyostelium myosin II has no predicted consensus motif 1 binding sites, only motifs 2 and 3, most of which are found in the coiled coil tail.4
All of the predicted sites near the 3x Asp mutant sites are of the motif 3 class, suggesting that the 14-3-3-myosin binding interactions may not be directly dependent on phosphorylation. The inability of 14-3-3 to associate with 3x Asp myosin II may be because 3x Asp is severely impaired in its ability to assemble into bipolar thick filaments15
or its cellular distribution is shifted from the cortex to the cytoplasm.16
Alternatively, subtle structural changes in the myosin II tail upon phosphorylation might reduce the affinity of the 14-3-3-myosin II interaction, thereby reducing the association of 14-3-3 with the phosphomimic 3x Asp myosin II.17
Nevertheless, assessment of the myosin II distribution in cells with altered 14-3-3 levels demonstrated that 14-3-3 promotes myosin II BTF remodeling (): depletion of 14-3-3 led to decreased myosin II mobile fraction and clumping, whereas 14-3-3 overexpression led to an increased mobile fraction (). Mobility here refers to the mobile and immobile fractions of myosin II as assessed by fluorescence recovery after photobleaching. Consistent with the RacE dependency for 14-3-3 function, myosin II also shows aggregation in racE null cells. In in vitro myosin II BTF assembly assays, the addition of 14-3-3 reduced the amount of assembled myosin II. The combination of in vivo and in vitro data suggests that 14-3-3 might reduce the affinity or increase accessibility of the myosin II monomers (the functional monomer is the hexamer of two heavy chains, two essential light chains and two regulatory light chains) for the BTF, which in vivo leads to alterations in myosin II mobility that correlates with 14-3-3 levels.
The direct biochemical interactions between myosin II and 14-3-3 suggest an intriguing new twist in myosin II regulation. Much of the known regulation of myosin II BTF assembly occurs through heavy chain phosphorylation and dephosphorylation.15
Clearly, this level of regulation is critical for maintaining a free pool of myosin monomers so that myosin II BTFs may be remodeled during dynamic processes such as cytokinesis and cell motility. Undoubtedly, this represents just part of the story. It remains unknown how myosin II BTF nucleation is controlled and what the cortical receptor/anchor(s) are for myosin II BTFs in most contexts (here, we define the cortex as the composite of cortical actin network and the overlying plasma membrane). Where there is evidence for a receptor, such as in the case of anillin (in metazoans), the structure-function studies raise questions about whether anillin is really an anchor or alternatively one of several anchors.17–19
In a recent study, α4β1 integrins may also provide one possible cortical anchor for myosin IIA in mammalian cells,20
demonstrating that multiple myosin II cortical receptors likely exist. As 14-3-3 is cortically enriched, one possibility is that it forms part of one such cortical receptor for myosin II. By having many receptors, the myosin II BTFs may be nucleated at several sites, yielding smaller BTFs (), which in turn may also lead to greater mobility. The recovery kinetics (τrec
) largely reflect the release of myosin II monomers from the BTFs, which remain unchanged between the different levels of 14-3-3 expression (τrec
= 5 s for WT, 14-3-3 hp and 14-3-3-overexpressing cells) (). By helping set the steady state mobility of the myosin II monomers and by maintaining a uniform distribution of myosin II BTFs, 14-3-3 contributes to cortical mechanics and cell shape changes such as in cytokinesis (). Depletion of 14-3-3 leads to a significant reduction in cortical tension while its overexpression could rescue the mechanical defects associated with racE
deletion or with nocodazole treatment. However, 14-3-3 overexpression failed to alter the cortical mechanics of myosin II
null cells further illustrating that 14-3-3 works through myosin II.