FORMIN stable kinetochore-microtubule attachments

Abstract

Formins are well-known for promoting actin assembly, but they also play a lesser-studied role in microtubule stabilization. In this issue of Developmental Cell, Cheng et al. (2011) demonstrate that the formin homology protein mDia3 is regulated by Aurora B Kinase, and contributes to the generation of kinetochore-microtubule attachments in mitosis.

Accurate segregation of duplicated chromosomes to daughter cells during mitosis is dependent upon capture of chromosomes by spindle microtubules at a multi-component protein structure built at centromeric DNA, the kinetochore. Stable microtubule attachment at kinetochores is dependent on the highly conserved KNL1/MIS12/NDC80 (KMN) protein network (reviewed by Santaguida and Musacchio, 2009). However in addition to this core complex, a growing cast of accessory proteins appear to make important contributions to microtubule attachment at the kinetochore. An interesting study in 2004 demonstrated that mDia3, a Diaphanous related formin (Drf), localizes to kinetochores and is required for proper mitotic chromosome alignment (Yasuda et al., 2004). Formin homology (FH) proteins are best characterized for their ability to nucleate the assembly of unbranched F-actin filaments. F-actin nucleation by these proteins is dependent on a well-conserved formin homology domain 2 (FH2) (Figure 1A). Mutations to the Drosophila formin homology gene, Diaphanous, result in cytokinetic defects, and mammalian Diaphanous related formins including mDia1, mDia2 and mDia3 have been implicated in both actin- and microtubule-dependent processes (Chesarone et al., 2010). The mechanism by which mDia3 controls chromosome alignment has been elusive. Also unclear is whether this role for mDia3 is dependent on its actin nucleation function or on the more nascently characterized role for Drfs in regulating microtubule stability (Bartolini et al., 2008). A new study by Cheng et al. in this issue of Developmental Cell provides insight to the basis for mDia3's involvement in chromosome alignment (Cheng et al., 2011).

Figure 1. Regulation of kinetochore-microtubule attachment by the formin mDia3.

(A) Domain architecture of mDia3. Predicted Aurora B kinase phosphorylation sites are indicated by yellow polygons. Of the four sites, S196 was confirmed to be phosphorylated in vivo (Cheng et al., 2011).

(B–C) Proposed model for Aurora B kinase regulation of kinetochore-microtubule attachment stability. The blue oval represents a gradient of Aurora B with high levels of the kinase concentrated at the inner centromere (CEN). Kinetochore components that are known to be targets of Aurora B include Hec1 of the NDC80 complex (brown), KNL1 (gray), Dsn1 of the MIS12 complex (green), and mDia3 (purple). (B) In prometaphase, high Aurora B kinase activity phosphorylates kinetochore targets to destabilize kinetochore-microtubule attachments. (C) In metaphase, kinetochore targets escape the Aurora B gradient, stabilizing kinetochore-microtubule attachments.

Using mutants of mDia3 that are defective in nucleating actin (Figure 1), Cheng et al. first demonstrate that the actin nucleation activity of mDia3 is not required for normal chromosome alignment. Thus, while several studies have suggested a role for F-actin in mitotic chromosome segregation, it would appear that mDia3 does not control chromosome alignment through regulation of F-actin assembly. Having ruled out actin assembly, the authors then turned to assessing the effects of mDia3 depletion on microtubule stabilization. Depletion of mDia3 was shown to decrease the stability of kinetochore microtubules without gross disruption of kinetochore organization (Cheng et al., 2011). This raises the question as to how kinetochore-localized mDia3 controls microtubule stability. Previous studies suggest at least two possible mechanisms for microtubule stabilization by mammalian Dia-family proteins. One mode of microtubule stabilization entails recruitment by mDia of the microtubule stabilizing factors EB1, a microtubule plus-end binding protein, and the adenomatous polyposis coli (APC) tumor suppressor protein (Chesarone et al., 2010). Like mDia3, both EB1 and APC localize to kinetochores and are also required for proper chromosome alignment and segregation (Pellman, 2001). This observation is supportive of a model whereby mDia3 recruits EB1 and APC to kinetochores to regulate microtubule stability. mDia2 and as shown by Cheng et al., mDia3, also directly bind and stabilize microtubules independently of EB1 and APC, at least in vitro (Bartolini et al., 2008; Cheng et al., 2011). Intriguingly, Cheng et al. now show that this latter mode of microtubule stabilization by mDia3 is regulated by Aurora B kinase, a protein essential for ensuring the fidelity of chromosome segregation through its role in promoting kinetochore-microtubule turnover and attachment error correction (Santaguida and Musacchio, 2009).

Several kinetochore targets for Aurora B kinase have been identified, and phosphorylation of some of these, such as proteins of the KMN network, has been shown to destabilize kinetochore-microtubule attachments in vivo (Guimaraes et al., 2008; Welburn et al., 2010). Based on these precedents, mDia3 was examined as a potential substrate for Aurora B. Of four (T66, S196, S820, T882) putative Aurora B phosphorylation sites in mDia3, at least one (S196) was shown to be phosphorylated in an Aurora B dependent manner in vivo. Significantly, mDia3 phosphorylated by Aurora B kinase or mDia3 phospho-mimetics failed to bind or stabilize microtubules in vitro. Likewise, mDia3 phosphomimetics failed to rescue chromosome alignment defects and loss of kinetochore microtubule stability when expressed in human cultured cells depleted of endogenous mDia3. Also of note, high levels of S196 phosphorylated mDia3 were observed at kinetochores during early mitosis but levels were reduced on kinetochores of chromosomes aligned at the metaphase plate (Cheng et al., 2011). In total, these data lead the authors to propose a model in which Aurora B acts to phosphorylate mDia3 and destabilize erroneous kinetochore-microtubule attachments during early mitosis; whereas unphosphorylated mDia3, which remains at the kinetochore at metaphase, acts to promote stable microtubule attachments (Figure 1B). The contribution of EB1 and APC function to mDia3-mediated kinetochore microtubule attachment downstream of Aurora B regulation in cells remains ambiguous since phosphorylation of mDia3 does not appear to affect the interaction between mDia3 and EB1/APC in vitro (Cheng et al., 2011).

Interestingly, Cheng et al. observed that mDia3 mutants in which putative Aurora B phosphorylation sites were converted to alanines failed to support normal chromosome alignment when expressed in cells. The root cause of the chromosome alignment abnormalities observed with the mDia3 phosphomutants remains unclear and will be important to address in future studies. One possibility is that dynamic Aurora B phosphorylation of mDia3 is required during mitosis, and mutants of mDia3 which cannot be phosphorylated may produce hyper-stable attachments and consequently chromosome alignment defects. A similar mode of regulation for the core kinetochore component, Hec1 (of the NDC80 complex), by Aurora B has recently been proposed (Deluca et al., 2011).

Yasuda et al. previously suggested that mDia3 regulates chromosome alignment downstream of the Rho-family GTPase, Cdc42 (Yasuda et al., 2004). mDia3, like other mammalian Drfs, is subject to autoinhibition which generally requires relief through Rho-family GTPase association (Figure 1A). How mDia3 is activated at kinetochores and the requirement for Rho-family GTPases in the process remains a subject of debate, as Cheng et al. report that inhibition of Cdc42 activity had no effect on chromosome alignment in their hands. Interestingly, Cdc42 was recently shown to be involved in maintenance of the histone H3 variant CENP-A at centromeres (Lagana et al., 2010). Coimmunoprecipiation experiments have suggested an interaction between mDia3 and CENP-A, and it is not clear whether the role for Cdc42 at centromeres is also mDia3-dependent (Yasuda et al., 2004). It remains possible therefore that mDia3 and Cdc42 may be involved in kinetochore function in as of yet unexplored ways. Further studies will be needed to address these issues.

Finally, one challenge for future studies will be to understand how the functions of mDia3 and the emerging cast of other players involved in microtubule attachment at the kinetochore are coordinated with those of the core kinetochore components such as the KMN network. This new study by Cheng et al. strongly supports the notion that Aurora B kinase plays a key role in integrating these functions.