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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 regulation of APC2 and Arm is altered in akt embryos. (A) Immunoprecipiation of Arm from 0–3-h wild-type and akt104226 embryo extracts. Pros-35 is shown as a control. T, total extract; S, supernatant; P, pellet. (B) The mobility of APC2 in akt104226 embryos can be altered by treatment with λ phosphatase. (C) Wild-type and akt104226 embryos fixed and stained to visualize DNA and APC2. In akt embryos, APC2 fails to localize. Bar, 10 μm.
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fig3: The regulation of APC2 and Arm is altered in akt embryos. (A) Immunoprecipiation of Arm from 0–3-h wild-type and akt104226 embryo extracts. Pros-35 is shown as a control. T, total extract; S, supernatant; P, pellet. (B) The mobility of APC2 in akt104226 embryos can be altered by treatment with λ phosphatase. (C) Wild-type and akt104226 embryos fixed and stained to visualize DNA and APC2. In akt embryos, APC2 fails to localize. Bar, 10 μm.

Mentions: Previously published work has shown that during syncytial mitoses, Zw3 acts to regulate the localization of a complex containing Arm (the D. melanogaster homologue of β-catenin) and the APC-related protein APC2/E-APC (McCartney et al., 2001; Cliffe et al., 2004). In addition, embryos laid by females homozygous for a mutation in the apc2 gene resulting in the deletion of the Arm-binding repeats (apc2ΔS) also displayed patches of embryonic cortex devoid of nuclei (McCartney et al., 2001). Therefore, we decided to investigate whether Akt has a role in regulating the Arm–APC2 complex through Zw3 during embryogenesis. By immunoprecipitating Arm from wild-type and akt104226 embryo extracts, we were able to coprecipitate APC2 (Fig. 3 A). However, although Arm and APC2 were complexed both in wild-type and akt104226 extracts, total levels of Arm were substantially reduced in akt104226 extracts (Fig. 3 A). Additionally, Arm-associated APC2, which ran as a single band on Western blots in wild-type embryos, was present as a smear in akt104226 embryos (Fig. 3, A and B). This extended mobility could be reversed upon incubation with λ phosphatase, possibly suggesting that APC2 is highly phosphorylated in embryos deficient in Akt (Fig. 3 B). To further address what effect decreased Akt activity might have on this complex, we compared the localization of APC2 in wild-type and akt embryos. During interphase in wild-type embryos, APC2 was present at the embryonic cortex, as has been found previously (Fig. 3 C; McCartney et al., 1999; Townsley and Bienz, 2000; McCartney et al., 2001). However, in akt embryos, this cortical localization was disrupted and APC2 was found entirely in the cytoplasm (Fig. 3 C). Thus, in the early embryo, Akt appears to regulate the proteins levels of Arm and the localization of APC2.


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 regulation of APC2 and Arm is altered in akt embryos. (A) Immunoprecipiation of Arm from 0–3-h wild-type and akt104226 embryo extracts. Pros-35 is shown as a control. T, total extract; S, supernatant; P, pellet. (B) The mobility of APC2 in akt104226 embryos can be altered by treatment with λ phosphatase. (C) Wild-type and akt104226 embryos fixed and stained to visualize DNA and APC2. In akt embryos, APC2 fails to localize. Bar, 10 μm.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2234228&req=5

fig3: The regulation of APC2 and Arm is altered in akt embryos. (A) Immunoprecipiation of Arm from 0–3-h wild-type and akt104226 embryo extracts. Pros-35 is shown as a control. T, total extract; S, supernatant; P, pellet. (B) The mobility of APC2 in akt104226 embryos can be altered by treatment with λ phosphatase. (C) Wild-type and akt104226 embryos fixed and stained to visualize DNA and APC2. In akt embryos, APC2 fails to localize. Bar, 10 μm.
Mentions: Previously published work has shown that during syncytial mitoses, Zw3 acts to regulate the localization of a complex containing Arm (the D. melanogaster homologue of β-catenin) and the APC-related protein APC2/E-APC (McCartney et al., 2001; Cliffe et al., 2004). In addition, embryos laid by females homozygous for a mutation in the apc2 gene resulting in the deletion of the Arm-binding repeats (apc2ΔS) also displayed patches of embryonic cortex devoid of nuclei (McCartney et al., 2001). Therefore, we decided to investigate whether Akt has a role in regulating the Arm–APC2 complex through Zw3 during embryogenesis. By immunoprecipitating Arm from wild-type and akt104226 embryo extracts, we were able to coprecipitate APC2 (Fig. 3 A). However, although Arm and APC2 were complexed both in wild-type and akt104226 extracts, total levels of Arm were substantially reduced in akt104226 extracts (Fig. 3 A). Additionally, Arm-associated APC2, which ran as a single band on Western blots in wild-type embryos, was present as a smear in akt104226 embryos (Fig. 3, A and B). This extended mobility could be reversed upon incubation with λ phosphatase, possibly suggesting that APC2 is highly phosphorylated in embryos deficient in Akt (Fig. 3 B). To further address what effect decreased Akt activity might have on this complex, we compared the localization of APC2 in wild-type and akt embryos. During interphase in wild-type embryos, APC2 was present at the embryonic cortex, as has been found previously (Fig. 3 C; McCartney et al., 1999; Townsley and Bienz, 2000; McCartney et al., 2001). However, in akt embryos, this cortical localization was disrupted and APC2 was found entirely in the cytoplasm (Fig. 3 C). Thus, in the early embryo, Akt appears to regulate the proteins levels of Arm and the localization of APC2.

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