Our study demonstrates that Htt is required for cytokinesis and chemotaxis through the regulation of myosin II phosphorylation/dephosphorylation. As cytokinesis proceeds, htt− cells gradually lose myosin II from the cortex of the cleavage furrow and fail to complete cell division, producing unseparated daughter cells connected by a thin cytoplasmic bridge. Similarly, myosin II levels in the cortex are reduced during chemotaxis in htt− cells, leading to decreased migration speed. Recruitment of myosin II to the cortex requires dephosphorylation of myosin II by the phosphatase PP2A. htt− cells displayed a reduction in PP2A activity and contained increased amounts of phosphorylated myosin II. Furthermore, our data also indicate that Htt is required for assembly of myosin II, but not for translocation to the cortex, as a phosphorylation-defective myosin II can be maintained in the cleavage furrow of htt− cells.
PP2A consists of three subunits: catalytic, scaffolding, and regulatory. The scaffolding subunit contains the HEAT domain, which binds to the catalytic subunit. Because Htt contains HEAT domains, it is tempting to speculate that Htt may directly bind to the catalytic subunit through this domain and function as a scaffold protein, modulating the phosphatase activity or its specificity toward myosin II. In addition, Htt may regulate the assembly of PP2A. Although we found no evidence of a physical interaction between Htt and PP2A in coimmunoprecipitation studies (Y. Wang and M. Iijima, unpublished observations), it remains possible that Htt weakly and/or transiently interacts with PP2A to regulate its enzymatic activity, specificity, or assembly. Moreover, the Dictyostelium
genome contains at least six PP2A regulatory subunits (Table S1), suggesting that PP2A likely has many functions and many substrates in Dictyostelium
in addition to myosin II. A recent study showed that a PP2A regulatory subunit, phr2aBα, is required for myosin II assembly (Rai and Egelhoff, 2011
). It would be interesting to test whether and how interactions of catalytic subunits with different regulatory subunits modulate the target specificity, phosphatase activity, and localization.
We found that knockdown of Htt also reduced the activity of PP2A in HeLa cells, suggesting that the role of Htt in PP2A activity is evolutionarily conserved. Although the assembly of mammalian myosin II is also regulated by heavy-chain phosphorylation (Vicente-Manzanares et al., 2009
), it remains to be determined whether PP2A regulates myosin II during cytokinesis or chemotaxis in mammalian cells. Nonetheless, since the loss of normal function of Htt could contribute at least partially to the pathogenesis of Huntington's disease (Harjes and Wanker, 2003
; Caviston and Holzbaur, 2009
), it would be interesting to further investigate the phosphorylation status of PP2A substrates in this disease. In addition, a recent study has shown that a PP2A-B55α complex regulates mitotic exit, most likely by dephosphorylating Cdk1 substrates in human cells (Schmitz et al. 2010
). RNAi knockdown of B55α, a regulatory subunit of this PP2A complex, delays spindle disassembly, chromosome decondensation, and Golgi assembly. These findings and our current study suggest that PP2A plays a key regulatory role in the mitotic phase.
cells can divide on surfaces or in suspension culture. However, suspension growth is highly restrictive for cells with defects in the cytokinetic machinery. As a result, many cytokinetic mutants show quantitative defects under both growth conditions, but only fail at cytokinesis, becoming multinucleated only in suspension culture (Robinson et al., 2001
; Uyeda and Nagasaki 2004
). Therefore htt−
cells, like mhcA
null cells, complete cytokinesis successfully on surfaces by drawing on adhesion properties but fail in suspension culture. However, htt−
cells formed GLCs, unlike mhcA
null cells, which become enlarged and multinucleated. In addition, there are several Dictyostelium
mutants that are defective in cytokinesis, none of which have been reported to form GLCs. Therefore Htt likely acts at an unidentified step during cytokinesis. Our study also suggests a novel role for myosin II in the completion of cytokinesis, in addition to its known role in the initiation and ingression of the cleavage furrow. The failure of myosin II maintenance at later stages of cytokinesis likely blocks abscission in htt−
cells, leading to the formation of GLCs.