Here, we found that the inhibition of the Rho-signaling abolished mDia2 localization at the cleavage furrow and induced its accumulation at the central spindle, and further that the Rho-binding activity of mDia2 is essential for its function in execution of cytokinesis (). We also found that the ARR/DID and CC domain, the N-terminal region of mDia2 without GBD alone, can localize at the cleavage furrow (). We performed a pulldown assay using the fragment containing this domain and identified anillin as a novel binding partner of mDia2 (). The anillin directly binds to mDia2 and its binding was competitive with DAD toward the autoinhibitory interaction of mDia2 (). Finally, we found that the interaction between anillin and mDia2 was not only required for the mDia2 localization at the cleavage furrow (), but also important for cytokinesis (). Our findings have thus clarified the localization mechanisms of mDia2 in cytokinesis and further revealed a heretofore unnoticed interaction of an actin nucleater, mDia2, and an essential scaffold in cytokinesis, anillin. We discussed these issues in detail below.
First, although the formin family of proteins have been characterized as a major component of cytokinesis in animal cells (Castrillon and Wasserman, 1994
; Swan et al., 1998
; Watanabe et al., 2008
), its localization and regulation mechanisms during this process have not yet been revealed. Here we have used siRNA for RhoGEF Ect2 and found that the inhibition of the Rho-signaling diminished the mDia2 localization at the cleavage furrow and induced its accumulation at the central spindle (A). We also found that the cytokinesis failure induced by mDia2 RNAi was not effectively rescued by the expression of the Rho-binding-defective mutant of mDia2 (C). These results suggest that targeting and function of mDia2 in cytokinesis are indeed regulated by Rho, which is consistent with the previous report showing the regulation of other Diaphanous-related formin (DRF) proteins by RhoGTPases (Seth et al., 2006
). The mDia2 localization at the central spindle and midbody indicates associations of mDia2 with microtubules. Previous reports suggested that mDia2 directly or indirectly binds to tips of microtubules in interphase via FH domains (Wen et al., 2004
; Bartolini et al., 2008
). However, we found that the C-terminal region of mDia2 and not those suggested previously is required for the localization at the central spindle and midbody (). As for the central spindle localization, previous studies also reported that one of other Rho effectors involved in cytokinesis, citron kinase, was transferred to the cleavage furrow in an Rho-dependent manner and localized at the central spindle on Rho inactivation (Eda et al., 2001
), and that the citron kinase at the central spindle made a complex with KIF14 and PRC1 at the central spindle (Gruneberg et al., 2006
). It is thus tempting to speculate that mDia2 responds to Rho-signaling and is transferred from the central spindle to the cleavage furrow, and this mechanism may be common among several Rho effectors.
Second, we found that Rho and the Rho-binding do not solely determine the mDia2 localization in cytokinesis. Notably, we found that GFP-mDia2 lacking the GBD still localized at the cleavage furrow (, A–C). These constructs have ARR/DID and DD domains that are also called Formin homology 3 (FH3) domain and are important for the localization of several formin family proteins (Carnahan and Gould, 2003
; Brandt et al., 2007
; Rincon et al., 2009
), suggesting that the FH3 domains of each formin family protein contributes to its localization and that such localization is driven by binding of this domain to context-dependent interaction partners. We performed pulldown assays using the mDia2 fragment containing the above region, and identified anillin as a binding partner (D). Anillin is essential in cytokinesis in most cell types, which functions as a cross-linker of other cytoskeltal components in the contractile ring (D'Avino, 2009
). Both anillin and formin family proteins have been studied as important components of the contractile ring in cytokinesis (Eggert et al., 2006
), but their physical interaction has not been revealed. Here, we have revealed the physical interaction between anillin and one of the formin family proteins, mDia2 ( and ). The N-terminal region of anillin, which directly binds to mDia2 ( and A), is well conserved in anillin of various vertebrates, but has not been characterized before.
Third, we found that this N-terminal fragment of anillin can compete in binding to the DID domain of mDia2 with DAD of mDia2 (B). This indicates a possibility that mDia2 can be activated by anillin through the disruption of DID–DAD interaction. However, we suggest that mDia2 binds to anillin after active Rho binds to mDia2 to release the autoinhibitory interaction and stabilizes the open conformation, since the Rho-binding-defective mDia2 did not localize at the cleavage furrow and the addition of a mutation into DAD region, which structurally opens mDia, induced the accumulation of mDia2 at the cleavage furrow (, A and C, bottom panels). Our results are thus similar to the targeting mechanism of mDia1 suggested by Brandt et al. (2007)
, who showed that IQGAP1 binds to mDia1 after the RhoA-mediated release of autoinhibition of mDia1 and regulates phagocytotic processes. The specific interaction between mDia and mDia binding partners and their involvement in the activation of the mDia proteins are thus key issues in controlling mDia function. Structural analyses of the interaction between mDia proteins and each interaction partner will be useful to fully understand how mDia activity is controlled by each binding partner.
Finally, we provide the evidence that the interaction between anillin and mDia2 is important in cytokinesis ( and ). We found in the anillin-RNAi rescue experiments using the anillin lacking the mDia2-binding region that mDia2 did not localize at the cleavage furrow even if RhoA localize as in control-RNAi cells (, B–D), and that the anillin lacking the mDia2-binding region did not rescue the cytokinesis failure as full-length anillin (). These results suggest that localization of RhoA at the cleavage furrow is not enough for the localization of mDia2 at the cleavage furrow, and that, in addition to it, the binding between mDia2 and anillin is required for the localization of mDia2 and execution of cytokinesis. Because anillin binds several cytoskeletal proteins and functions as an essential scaffold in cytokinesis (D'Avino, 2009
), our findings suggest that mDia2 is also anchored in the anillin scaffold so that it can function effectively within this scaffold. In fact, the cytokinesis phenotypes of mDia2-RNAi cells and that of anillin-RNAi cells were similar in that the both cells showed the unstabilized contractile ring and abnormal contraction during cytokinesis (Straight et al., 2005
; Watanabe et al., 2008
). It is thus interesting to speculate that anillin may regulate actomyosin-ring dynamics locally by linking the active mDia2 and the contractile ring components such as actin and myosin. Based on these findings, we propose a model. When Rho is activated at the equatorial cortex of the dividing cells, mDia2 is first targeted there by activated Rho. Anillin then binds to the DID domain of this open form of mDia2 and stabilizes its localization and activation. The stabilized mDia2 can provide F-actin scaffold for the contraction of the contractile ring in cytokinesis.