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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 axis of spindle formation is perturbed in cellularized akt embryos. (A and B) Single plane confocal analysis of 2–4-h wild-type and akt104226 embryos fixed and stained with anti-tubulin antibodies. (A) Mitotic spindles in wild-type embryos appear parallel to the embryonic cortex. (B) In akt embryos, mitotic spindles are positioned in many different orientations. (C) Quantitative analysis of spindle orientation in wild-type and akt embryos. Data were obtained from 100 spindles from four wild-type and akt embryos. Error bars indicate the SEM. Blue, wild-type embryos; red, akt embryos. (D) Diagram of the results shown in C. (E and F) 2–4-h wild-type (E) and akt104226 (F) embryos fixed and stained with antibodies to α-tubulin and APC2. Arrows indicate mitotic spindles oriented perpendicular to the most embryonic cortex. Bar, 10 μm.
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fig9: The axis of spindle formation is perturbed in cellularized akt embryos. (A and B) Single plane confocal analysis of 2–4-h wild-type and akt104226 embryos fixed and stained with anti-tubulin antibodies. (A) Mitotic spindles in wild-type embryos appear parallel to the embryonic cortex. (B) In akt embryos, mitotic spindles are positioned in many different orientations. (C) Quantitative analysis of spindle orientation in wild-type and akt embryos. Data were obtained from 100 spindles from four wild-type and akt embryos. Error bars indicate the SEM. Blue, wild-type embryos; red, akt embryos. (D) Diagram of the results shown in C. (E and F) 2–4-h wild-type (E) and akt104226 (F) embryos fixed and stained with antibodies to α-tubulin and APC2. Arrows indicate mitotic spindles oriented perpendicular to the most embryonic cortex. Bar, 10 μm.

Mentions: As some akt embryos complete cellularization and undergo normal gastrulation (Table I), we also investigated whether mitotic cells in embryos exhibit any defects in spindle formation. 2–4-h-old wild-type and akt embryos were fixed and stained for α-tubulin to show MTs (Fig. 9, A and B). After cellularization, mitosis occurs asynchronously in specific mitotic domains (Foe, 1989). Importantly, in wild-type embryos, these cell divisions are usually oriented parallel to the surface of the embryo (Fig. 9 A). However, when we investigated the morphology of spindles from akt embryos, we found they were not uniformly oriented in relation to the cortex (Fig. 9 B). Images taken along the z axis of embryos at 1-μm intervals were used to assess the relative vertical displacement of the two poles. In wild-type embryos, the mean vertical displacement between the two spindle poles was ∼1 μm (Fig. 9 C). By assuming a mean spindle length of 10 μm pole-to-pole, this corresponds to a typical orientation of 6° from the horizontal (Fig. 9 D). In akt embryos, the mean vertical displacement between spindle poles was ∼5 μm (Fig. 9 C). This displacement varied widely, with some spindles having both poles in the same plane and some apparently perpendicular to the plane of the epithelium. The mean vertical displacement in akt embryos corresponds to a spindle at an angle of 29° to the horizontal (Fig. 9 D).


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 axis of spindle formation is perturbed in cellularized akt embryos. (A and B) Single plane confocal analysis of 2–4-h wild-type and akt104226 embryos fixed and stained with anti-tubulin antibodies. (A) Mitotic spindles in wild-type embryos appear parallel to the embryonic cortex. (B) In akt embryos, mitotic spindles are positioned in many different orientations. (C) Quantitative analysis of spindle orientation in wild-type and akt embryos. Data were obtained from 100 spindles from four wild-type and akt embryos. Error bars indicate the SEM. Blue, wild-type embryos; red, akt embryos. (D) Diagram of the results shown in C. (E and F) 2–4-h wild-type (E) and akt104226 (F) embryos fixed and stained with antibodies to α-tubulin and APC2. Arrows indicate mitotic spindles oriented perpendicular to the most embryonic cortex. Bar, 10 μm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2234228&req=5

fig9: The axis of spindle formation is perturbed in cellularized akt embryos. (A and B) Single plane confocal analysis of 2–4-h wild-type and akt104226 embryos fixed and stained with anti-tubulin antibodies. (A) Mitotic spindles in wild-type embryos appear parallel to the embryonic cortex. (B) In akt embryos, mitotic spindles are positioned in many different orientations. (C) Quantitative analysis of spindle orientation in wild-type and akt embryos. Data were obtained from 100 spindles from four wild-type and akt embryos. Error bars indicate the SEM. Blue, wild-type embryos; red, akt embryos. (D) Diagram of the results shown in C. (E and F) 2–4-h wild-type (E) and akt104226 (F) embryos fixed and stained with antibodies to α-tubulin and APC2. Arrows indicate mitotic spindles oriented perpendicular to the most embryonic cortex. Bar, 10 μm.
Mentions: As some akt embryos complete cellularization and undergo normal gastrulation (Table I), we also investigated whether mitotic cells in embryos exhibit any defects in spindle formation. 2–4-h-old wild-type and akt embryos were fixed and stained for α-tubulin to show MTs (Fig. 9, A and B). After cellularization, mitosis occurs asynchronously in specific mitotic domains (Foe, 1989). Importantly, in wild-type embryos, these cell divisions are usually oriented parallel to the surface of the embryo (Fig. 9 A). However, when we investigated the morphology of spindles from akt embryos, we found they were not uniformly oriented in relation to the cortex (Fig. 9 B). Images taken along the z axis of embryos at 1-μm intervals were used to assess the relative vertical displacement of the two poles. In wild-type embryos, the mean vertical displacement between the two spindle poles was ∼1 μm (Fig. 9 C). By assuming a mean spindle length of 10 μm pole-to-pole, this corresponds to a typical orientation of 6° from the horizontal (Fig. 9 D). In akt embryos, the mean vertical displacement between spindle poles was ∼5 μm (Fig. 9 C). This displacement varied widely, with some spindles having both poles in the same plane and some apparently perpendicular to the plane of the epithelium. The mean vertical displacement in akt embryos corresponds to a spindle at an angle of 29° to the horizontal (Fig. 9 D).

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