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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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Akt localizes to the embryonic cortex in a cell cycle–dependent manner. (A) Western blot of 0–3-h wild-type embryos and embryos containing UAS-Akt-turbo-GFP driven by Nanos Gal4. Akt-tGFP is expressed at similar levels to endogenous Akt. Molecular mass (kD) is indicated on the left. (B) Live confocal analysis of embryos expressing Akt-tGFP under the UAS-Gal4 system. Akt-tGFP appears to cycle between cortical caps (0 and 1,770 s) and pseudocleavage furrows (710 and 930 s). Time is indicated in seconds. See Video 1 (available at http://www.jcb.org/cgi/content/full/jcb.200705085/DC1). Bar, 10 μm.
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fig4: Akt localizes to the embryonic cortex in a cell cycle–dependent manner. (A) Western blot of 0–3-h wild-type embryos and embryos containing UAS-Akt-turbo-GFP driven by Nanos Gal4. Akt-tGFP is expressed at similar levels to endogenous Akt. Molecular mass (kD) is indicated on the left. (B) Live confocal analysis of embryos expressing Akt-tGFP under the UAS-Gal4 system. Akt-tGFP appears to cycle between cortical caps (0 and 1,770 s) and pseudocleavage furrows (710 and 930 s). Time is indicated in seconds. See Video 1 (available at http://www.jcb.org/cgi/content/full/jcb.200705085/DC1). Bar, 10 μm.

Mentions: Activated Akt is known to associate to the plasma membrane using its conserved pleckstrin homology domain. As commercial antibodies that recognize D. melanogaster Akt in Western blots failed to show any specific localization in early embryos (unpublished data), we investigated the localization of Akt in flies expressing an Akt-turbo-GFP fusion protein using the maternal UAS/Gal4 system (see Materials and methods). 0–3-h embryos collected from nanos-Gal4:UAS-Akt-tGFP flies (hereafter termed Akt-tGFP embryos) express Akt-tGFP at levels similar to that of endogenously expressed Akt (Fig. 4 A). Live analysis of cycle 12 Akt-tGFP embryos showed that Akt-tGFP localized to cortical patches, which is reminiscent of actin caps during interphase (Figs. 2 F and 4 B; and Video 1, available at http://www.jcb.org/cgi/content/full/jcb.200705085/DC1; Sullivan and Theurkauf, 1995). As the cycle progressed, these caps spread and migrated away from the cortex until they become enriched in hexagonal arrangements that appeared similar to metaphase pseudocleavage furrows. Eventually these hexagons extended before dissolving completely. Akt-tGFP localization then returned to cortical caps (Fig. 4 B and Video 1). Thus, Akt is dynamically recruited to the embryonic cortex with similar dynamics to actin, placing it in the correct subcellular location to regulate Zw3, Arm, and 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)

Akt localizes to the embryonic cortex in a cell cycle–dependent manner. (A) Western blot of 0–3-h wild-type embryos and embryos containing UAS-Akt-turbo-GFP driven by Nanos Gal4. Akt-tGFP is expressed at similar levels to endogenous Akt. Molecular mass (kD) is indicated on the left. (B) Live confocal analysis of embryos expressing Akt-tGFP under the UAS-Gal4 system. Akt-tGFP appears to cycle between cortical caps (0 and 1,770 s) and pseudocleavage furrows (710 and 930 s). Time is indicated in seconds. See Video 1 (available at http://www.jcb.org/cgi/content/full/jcb.200705085/DC1). Bar, 10 μm.
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Related In: Results  -  Collection

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fig4: Akt localizes to the embryonic cortex in a cell cycle–dependent manner. (A) Western blot of 0–3-h wild-type embryos and embryos containing UAS-Akt-turbo-GFP driven by Nanos Gal4. Akt-tGFP is expressed at similar levels to endogenous Akt. Molecular mass (kD) is indicated on the left. (B) Live confocal analysis of embryos expressing Akt-tGFP under the UAS-Gal4 system. Akt-tGFP appears to cycle between cortical caps (0 and 1,770 s) and pseudocleavage furrows (710 and 930 s). Time is indicated in seconds. See Video 1 (available at http://www.jcb.org/cgi/content/full/jcb.200705085/DC1). Bar, 10 μm.
Mentions: Activated Akt is known to associate to the plasma membrane using its conserved pleckstrin homology domain. As commercial antibodies that recognize D. melanogaster Akt in Western blots failed to show any specific localization in early embryos (unpublished data), we investigated the localization of Akt in flies expressing an Akt-turbo-GFP fusion protein using the maternal UAS/Gal4 system (see Materials and methods). 0–3-h embryos collected from nanos-Gal4:UAS-Akt-tGFP flies (hereafter termed Akt-tGFP embryos) express Akt-tGFP at levels similar to that of endogenously expressed Akt (Fig. 4 A). Live analysis of cycle 12 Akt-tGFP embryos showed that Akt-tGFP localized to cortical patches, which is reminiscent of actin caps during interphase (Figs. 2 F and 4 B; and Video 1, available at http://www.jcb.org/cgi/content/full/jcb.200705085/DC1; Sullivan and Theurkauf, 1995). As the cycle progressed, these caps spread and migrated away from the cortex until they become enriched in hexagonal arrangements that appeared similar to metaphase pseudocleavage furrows. Eventually these hexagons extended before dissolving completely. Akt-tGFP localization then returned to cortical caps (Fig. 4 B and Video 1). Thus, Akt is dynamically recruited to the embryonic cortex with similar dynamics to actin, placing it in the correct subcellular location to regulate Zw3, Arm, and 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