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Akt regulates centrosome migration and spindle orientation in the early Drosophila melanogaster embryo.

Buttrick GJ, Beaumont LM, Leitch J, Yau C, Hughes JR, Wakefield JG - J. Cell Biol. (2008)

Bottom Line: Here we find that, in the Drosophila melanogaster early embryo, reduced levels of the protein kinase Akt result in incomplete centrosome migration around cortical nuclei, bent mitotic spindles, and loss of nuclei into the interior of the embryo.We also show that reduced levels of Akt result in mislocalization of APC2 in postcellularized embryonic mitoses and misorientation of epithelial mitotic spindles.Together, our results suggest that Akt regulates a complex containing Zw3, Armadillo, APC2, and EB1 and that this complex has a role in stabilizing MT-cortex interactions, facilitating both centrosome separation and mitotic spindle orientation.

View Article: PubMed Central - PubMed

Affiliation: Department of Zoology, University of Oxford, Oxford OX1 3PS, England, UK.

ABSTRACT
Correct positioning and morphology of the mitotic spindle is achieved through regulating the interaction between microtubules (MTs) and cortical actin. Here we find that, in the Drosophila melanogaster early embryo, reduced levels of the protein kinase Akt result in incomplete centrosome migration around cortical nuclei, bent mitotic spindles, and loss of nuclei into the interior of the embryo. We show that Akt is enriched at the embryonic cortex and is required for phosphorylation of the glycogen synthase kinase-3beta homologue Zeste-white 3 kinase (Zw3) and for the cortical localizations of the adenomatosis polyposis coli (APC)-related protein APC2/E-APC and the MT + Tip protein EB1. We also show that reduced levels of Akt result in mislocalization of APC2 in postcellularized embryonic mitoses and misorientation of epithelial mitotic spindles. Together, our results suggest that Akt regulates a complex containing Zw3, Armadillo, APC2, and EB1 and that this complex has a role in stabilizing MT-cortex interactions, facilitating both centrosome separation and mitotic spindle orientation.

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The localization of EB1 is altered in akt embryos. Wild-type (A and B) and akt104226/akt104226 (C and D) embryos fixed and stained with antibodies to α-tubulin and EB1. The ability of EB1 to bind centrosomes and spindles during metaphase is similar in wild-type and akt embryos. However, although wild-type embryos accumulate EB1 at the embryonic cortex and at centrosomes during interphase, akt embryos only accumulate EB1 on centrosomes. Bar, 10 μm.
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fig8: The localization of EB1 is altered in akt embryos. Wild-type (A and B) and akt104226/akt104226 (C and D) embryos fixed and stained with antibodies to α-tubulin and EB1. The ability of EB1 to bind centrosomes and spindles during metaphase is similar in wild-type and akt embryos. However, although wild-type embryos accumulate EB1 at the embryonic cortex and at centrosomes during interphase, akt embryos only accumulate EB1 on centrosomes. Bar, 10 μm.

Mentions: It has previously been suggested that the cortical APC2–Arm complex might interact with MTs through the conserved MT + Tip protein EB1 (Allan and Nathke, 2001). D. melanogaster EB1 has multiple roles in centrosome migration, spindle formation, and kinetochore function (Rogers et al., 2002). Mammalian EB1 and APC have been shown to directly bind one another and act together during chromosome alignment (Su et al., 1995; Green et al., 2005). EB1 has also been shown to be capable of interacting with the MT motor protein dynein, which, during syncytial development, is present at the cortex and provides the force required for centrosome separation (Berrueta et al., 1999; Sharp et al., 2000; Cytrynbaum et al., 2003). To investigate whether Akt acts on centrosome separation through EB1, we fixed wild-type and akt embryos with formaldehyde and stained them with antibodies specific to D. melanogaster EB1. In wild-type embryos, EB1 localized to spindles during metaphase, showing enrichment at the plus ends of MTs as described previously (Fig. 8 A; Rogers et al., 2002). During interphase, however, EB1 was present both at centrosomes and the embryonic cortex, where it was enriched at the actin caps (Fig. 8 B). As a second specific antibody to EB1 gave identical results, this cortical localization is unlikely to be a fixation artifact (unpublished data). In akt embryos, although the localization of EB1 to MTs and centrosomes during metaphase and interphase was unperturbed, the cortical enrichment at the actin caps during interphase was completely abolished (Fig. 8, C and D). Akt therefore appears to be required for the cortical localization of EB1.


Akt regulates centrosome migration and spindle orientation in the early Drosophila melanogaster embryo.

Buttrick GJ, Beaumont LM, Leitch J, Yau C, Hughes JR, Wakefield JG - J. Cell Biol. (2008)

The localization of EB1 is altered in akt embryos. Wild-type (A and B) and akt104226/akt104226 (C and D) embryos fixed and stained with antibodies to α-tubulin and EB1. The ability of EB1 to bind centrosomes and spindles during metaphase is similar in wild-type and akt embryos. However, although wild-type embryos accumulate EB1 at the embryonic cortex and at centrosomes during interphase, akt embryos only accumulate EB1 on centrosomes. Bar, 10 μm.
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Related In: Results  -  Collection

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fig8: The localization of EB1 is altered in akt embryos. Wild-type (A and B) and akt104226/akt104226 (C and D) embryos fixed and stained with antibodies to α-tubulin and EB1. The ability of EB1 to bind centrosomes and spindles during metaphase is similar in wild-type and akt embryos. However, although wild-type embryos accumulate EB1 at the embryonic cortex and at centrosomes during interphase, akt embryos only accumulate EB1 on centrosomes. Bar, 10 μm.
Mentions: It has previously been suggested that the cortical APC2–Arm complex might interact with MTs through the conserved MT + Tip protein EB1 (Allan and Nathke, 2001). D. melanogaster EB1 has multiple roles in centrosome migration, spindle formation, and kinetochore function (Rogers et al., 2002). Mammalian EB1 and APC have been shown to directly bind one another and act together during chromosome alignment (Su et al., 1995; Green et al., 2005). EB1 has also been shown to be capable of interacting with the MT motor protein dynein, which, during syncytial development, is present at the cortex and provides the force required for centrosome separation (Berrueta et al., 1999; Sharp et al., 2000; Cytrynbaum et al., 2003). To investigate whether Akt acts on centrosome separation through EB1, we fixed wild-type and akt embryos with formaldehyde and stained them with antibodies specific to D. melanogaster EB1. In wild-type embryos, EB1 localized to spindles during metaphase, showing enrichment at the plus ends of MTs as described previously (Fig. 8 A; Rogers et al., 2002). During interphase, however, EB1 was present both at centrosomes and the embryonic cortex, where it was enriched at the actin caps (Fig. 8 B). As a second specific antibody to EB1 gave identical results, this cortical localization is unlikely to be a fixation artifact (unpublished data). In akt embryos, although the localization of EB1 to MTs and centrosomes during metaphase and interphase was unperturbed, the cortical enrichment at the actin caps during interphase was completely abolished (Fig. 8, C and D). Akt therefore appears to be required for the cortical localization of EB1.

Bottom Line: Here we find that, in the Drosophila melanogaster early embryo, reduced levels of the protein kinase Akt result in incomplete centrosome migration around cortical nuclei, bent mitotic spindles, and loss of nuclei into the interior of the embryo.We also show that reduced levels of Akt result in mislocalization of APC2 in postcellularized embryonic mitoses and misorientation of epithelial mitotic spindles.Together, our results suggest that Akt regulates a complex containing Zw3, Armadillo, APC2, and EB1 and that this complex has a role in stabilizing MT-cortex interactions, facilitating both centrosome separation and mitotic spindle orientation.

View Article: PubMed Central - PubMed

Affiliation: Department of Zoology, University of Oxford, Oxford OX1 3PS, England, UK.

ABSTRACT
Correct positioning and morphology of the mitotic spindle is achieved through regulating the interaction between microtubules (MTs) and cortical actin. Here we find that, in the Drosophila melanogaster early embryo, reduced levels of the protein kinase Akt result in incomplete centrosome migration around cortical nuclei, bent mitotic spindles, and loss of nuclei into the interior of the embryo. We show that Akt is enriched at the embryonic cortex and is required for phosphorylation of the glycogen synthase kinase-3beta homologue Zeste-white 3 kinase (Zw3) and for the cortical localizations of the adenomatosis polyposis coli (APC)-related protein APC2/E-APC and the MT + Tip protein EB1. We also show that reduced levels of Akt result in mislocalization of APC2 in postcellularized embryonic mitoses and misorientation of epithelial mitotic spindles. Together, our results suggest that Akt regulates a complex containing Zw3, Armadillo, APC2, and EB1 and that this complex has a role in stabilizing MT-cortex interactions, facilitating both centrosome separation and mitotic spindle orientation.

Show MeSH
Related in: MedlinePlus