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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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Reduced Akt levels lead to defects in syncytial mitoses. (A–D) Syncytial stage embryos fixed with methanol to reveal DNA (red), centrosomes (A and B, green), or MTs (C and D, green). (A and C) Wild-type embryos. (B and D) akt embryos show gaps in their cortex, which is devoid of nuclei but contains many centrosomes. Although many spindles look normal, regions of akt embryos show poorly formed spindles that are irregularly spaced (D, arrow). (E) A cross-sectional view of an akt embryo in prometaphase. Arrows indicate spindle poles not connected to the embryonic cortex. (F–I) Gross actin morphology is not perturbed in akt embryos. During interphase in wild-type (F) or akt embryos (G), actin is present in cortical caps. During metaphase in wild-type (H) and akt (I) embryos, actin is present in pseudocleavage furrows. Bars, 10 μm.
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fig2: Reduced Akt levels lead to defects in syncytial mitoses. (A–D) Syncytial stage embryos fixed with methanol to reveal DNA (red), centrosomes (A and B, green), or MTs (C and D, green). (A and C) Wild-type embryos. (B and D) akt embryos show gaps in their cortex, which is devoid of nuclei but contains many centrosomes. Although many spindles look normal, regions of akt embryos show poorly formed spindles that are irregularly spaced (D, arrow). (E) A cross-sectional view of an akt embryo in prometaphase. Arrows indicate spindle poles not connected to the embryonic cortex. (F–I) Gross actin morphology is not perturbed in akt embryos. During interphase in wild-type (F) or akt embryos (G), actin is present in cortical caps. During metaphase in wild-type (H) and akt (I) embryos, actin is present in pseudocleavage furrows. Bars, 10 μm.

Mentions: To investigate the role of Akt during syncytial development, we fixed and stained 1–3-h-old embryos laid by akt mutant mothers to visualize DNA, MTs, and centrosomes. To ensure that the nature of the maternal effect lethality reflects a requirement for Akt protein, we generated heteroallelic flies with the genotype akt1q/akt104226. akt1q/akt104226 flies display maternal effect lethality comparable to akt104226/akt104226 individuals, and all analyses described in this study unless otherwise stated were performed on akt1q/akt104226 embryos (henceforth termed akt embryos). Two main differences to wild-type embryos could be discerned. First, although nuclei were arranged with regular spacing in wild-type embryos, akt embryos showed patches of the cortex devoid of nuclei (Fig. 2, A and B). These patches often still contained centrosomes, which implies that nuclear migration had been successful but that nuclei had subsequently moved into the interior of the embryo (Fig. 2 B). Second, spindles in akt embryos were occasionally bent or short (Fig. 2, C–E). Cross-sectional views of these embryos also showed spindles that appeared to be attached to the cortex by only one pole (Fig. 2 E, arrows).


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)

Reduced Akt levels lead to defects in syncytial mitoses. (A–D) Syncytial stage embryos fixed with methanol to reveal DNA (red), centrosomes (A and B, green), or MTs (C and D, green). (A and C) Wild-type embryos. (B and D) akt embryos show gaps in their cortex, which is devoid of nuclei but contains many centrosomes. Although many spindles look normal, regions of akt embryos show poorly formed spindles that are irregularly spaced (D, arrow). (E) A cross-sectional view of an akt embryo in prometaphase. Arrows indicate spindle poles not connected to the embryonic cortex. (F–I) Gross actin morphology is not perturbed in akt embryos. During interphase in wild-type (F) or akt embryos (G), actin is present in cortical caps. During metaphase in wild-type (H) and akt (I) embryos, actin is present in pseudocleavage furrows. Bars, 10 μm.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2234228&req=5

fig2: Reduced Akt levels lead to defects in syncytial mitoses. (A–D) Syncytial stage embryos fixed with methanol to reveal DNA (red), centrosomes (A and B, green), or MTs (C and D, green). (A and C) Wild-type embryos. (B and D) akt embryos show gaps in their cortex, which is devoid of nuclei but contains many centrosomes. Although many spindles look normal, regions of akt embryos show poorly formed spindles that are irregularly spaced (D, arrow). (E) A cross-sectional view of an akt embryo in prometaphase. Arrows indicate spindle poles not connected to the embryonic cortex. (F–I) Gross actin morphology is not perturbed in akt embryos. During interphase in wild-type (F) or akt embryos (G), actin is present in cortical caps. During metaphase in wild-type (H) and akt (I) embryos, actin is present in pseudocleavage furrows. Bars, 10 μm.
Mentions: To investigate the role of Akt during syncytial development, we fixed and stained 1–3-h-old embryos laid by akt mutant mothers to visualize DNA, MTs, and centrosomes. To ensure that the nature of the maternal effect lethality reflects a requirement for Akt protein, we generated heteroallelic flies with the genotype akt1q/akt104226. akt1q/akt104226 flies display maternal effect lethality comparable to akt104226/akt104226 individuals, and all analyses described in this study unless otherwise stated were performed on akt1q/akt104226 embryos (henceforth termed akt embryos). Two main differences to wild-type embryos could be discerned. First, although nuclei were arranged with regular spacing in wild-type embryos, akt embryos showed patches of the cortex devoid of nuclei (Fig. 2, A and B). These patches often still contained centrosomes, which implies that nuclear migration had been successful but that nuclei had subsequently moved into the interior of the embryo (Fig. 2 B). Second, spindles in akt embryos were occasionally bent or short (Fig. 2, C–E). Cross-sectional views of these embryos also showed spindles that appeared to be attached to the cortex by only one pole (Fig. 2 E, arrows).

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