Cytokinesis shape change occurs over a fast five-minute time-span, requiring considerable cortical remodeling during this time-frame. Much insight has come from studying the myosin heavy chain kinases, which phosphorylate the critical threonines in the tail of myosin II heavy chain to control bipolar thick filament (BTF) assembly [25
]. Most likely what these enzymes do is set the level of the free pool of myosin II monomers (~80%) in the cell. This free pool then is maintained through flux of the assembled and disassembled BTF states and is required to ensure that the contractile network is turned over during processes like motility and cytokinesis. 14-3-3 also impacts this dynamic, preventing the myosin II from forming larger order assemblies and shifting the assembly equilibrium. Importantly, the cellular concentrations of myosin II in the BTF state and the 14-3-3 dimer are identical (0.7 μM), indicating that hypotheses of direct biochemical modulation of BTF assembly by 14-3-3 are reasonable. Four scenarios may be envisioned for how 14-3-3 might modulate BTF dynamics. First, by binding to unphosphorylated monomers, 14-3-3 may suppress myosin II thick filament nucleation and/or elongation so that fewer well structured BTFs are assembled. Second, 14-3-3 could bind transiently to the mature BTF, lowering individual monomer affinity and helping it to release from the BTF. Third, the in vivo
system may be more complex where 14-3-3 binds to the BTF, putting the myosin monomers in a configuration that makes them more ideal substrates for myosin heavy chain kinases (MHCK). As MHCK was identified as a potential 14-3-3 protein interactor in our mass spectrometric analysis, such a tertiary complex of myosin BTF-MHCK-14-3-3 might be formed. Fourth, 14-3-3 might form part of the myosin II BTF cortical receptor/anchoring complex. In no system is it well established how myosin II BTFs are anchored to the cortex. In dividing 14-3-3hp cells, the myosin II aggregates appear to dislodge from the cortex, suggesting 14-3-3 might contribute to cortical anchoring. Overall, this system is highly dynamic where all of the measured time-scales occur on the low seconds. The data also demonstrate that if myosin II BTFs are not distributed uniformly, they cannot contribute to cortex mechanics and therefore the 14-3-3hp cortical tension and cytokinesis morphology closely resembles that of the myoII
null cells. These observations may prove to be generalized to other systems as mammalian myosin II appeared on the list of potential 14-3-3σ interactors in a proteomics study [16
]. Furthermore, 14-3-3 may contribute to myosin II activation in many ways as 14-3-3 has been implicated in regulating mammalian myosin light chain phosphatase [28
14-3-3 also functions downstream of RacE: its cortical localization depends on RacE, its solubility depends on RacE and GTP, and 14-3-3 overexpression in RacE
nulls partially rescues the growth, cortical tension and cytokinesis defects. Though we were unable at this point to demonstrate direct binding between 14-3-3 and RacE, these data demonstrate that 14-3-3 links at least indirectly to RacE. These data also suggest that the cortical localization of 14-3-3 reflects where active RacE is found. RacE is uniformly distributed around the plasma membrane during cell division [29
] and may regulate one or more of the global/polar-module proteins [1
]. Significantly, 14-3-3 enriches all along the cell cortex, but is reduced in the cleavage furrow region. Since myosin II was still able to accumulate in the cleavage furrow cortex in the 14-3-3hp cells, active RacE may promote 14-3-3 accumulation in the polar cortex where it helps remodel the myosin II BTFs.
14-3-3 may also modify the function of several other cortical actin-associated proteins such as dynacortin, fimbrin, coronin, enlazin and LimE [30
]. Several of these proteins contain predicted consensus 14-3-3 binding motifs and are found in comparable concentrations as 14-3-3. Fimbrin appeared on the list of potential interactors (Fig. S4B
); however, we have been unable so far to recapitulate the fimbrin-14-3-3 interaction using standard co-immunoprecipitation assays. This is possibly due to fimbrin’s short cortical association time (260-ms) [3
]. Interestingly, 14-3-3 proteins often bind to phosphorylated ligands, and a number of phosphorylated proteins reside in the cortex (e.g.
dynacortin is a phosphoprotein [1
]). Similarly, sea urchin embryos accumulate multiple cortical phosphoproteins during cell division, and this accumulation is inhibited by nocodazole-treatment [31
Finally, microtubules contribute to cortical mechanics, probably through a signaling pathway that includes 14-3-3. In return, 14-3-3 contributes to the steady-state microtubule structures and modulates cortical mechanics through RacE and myosin II (). Therefore, this is not a linear pathway, but what appears to be a circular (feedback) system between the microtubules and the cortex. The combination of these observations then begs the question whether the manner in which the spindle regulates the cortex is really through a feedback system where the spindle directs the cortex and the cortex directs the spindle [32
]. In this case, cytokinesis symmetry breaking may occur through bidirectional communication between these two structures. Similar ideas have been suggested elsewhere [33
]. Overall, 14-3-3 coordinates three major cytoskeletal elements – microtubules, actin, and myosin II – to control two critical processes, cytokinesis fidelity and cortical mechanics.