Limits...
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.

Show MeSH

Related in: MedlinePlus

Centrosome separation is inhibited after inactivation of Akt or APC2. (A) Live confocal analysis of embryos expressing α-tubulin–GFP. Examples of the angle of centrosome separation 20 s before NEB in wild-type (WT) embryos, akt1q/akt104226 embryos (akt), embryos injected with anti-Akt antibodies (α-Akt), and embryos expressing an allele of APC2 unable to bind to Arm (apc2Δs). Bar, 10 μm. (B) Histogram representing the mean angle of centrosome separation before NEB in embryos. Data for each class was obtained from 100 individual nuclei. Errors bars indicate the SEM. See Videos 4–8 (available at http://www.jcb.org/cgi/content/full/jcb.200705085/DC1).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2234228&req=5

fig6: Centrosome separation is inhibited after inactivation of Akt or APC2. (A) Live confocal analysis of embryos expressing α-tubulin–GFP. Examples of the angle of centrosome separation 20 s before NEB in wild-type (WT) embryos, akt1q/akt104226 embryos (akt), embryos injected with anti-Akt antibodies (α-Akt), and embryos expressing an allele of APC2 unable to bind to Arm (apc2Δs). Bar, 10 μm. (B) Histogram representing the mean angle of centrosome separation before NEB in embryos. Data for each class was obtained from 100 individual nuclei. Errors bars indicate the SEM. See Videos 4–8 (available at http://www.jcb.org/cgi/content/full/jcb.200705085/DC1).

Mentions: In wild-type embryos expressing α-tubulin–GFP, nuclei can also be followed as dark circles in which soluble tubulin is excluded. (Fig. 6 A and Video 4, available at http://www.jcb.org/cgi/content/full/jcb.200705085/DC1). During telophase and interphase, centrosomes migrate around nuclei until they lie approximately opposite one another (Robinson et al., 1999). After NEB, MTs invade the nucleus, capture chromosomes, and form a stable bipolar mitotic spindle (Video 4). To compare the MT dynamics in wild-type and akt embryos, we developed software able to identify and track the nuclei in which tubulin is excluded and the foci of fluorescent tubulin corresponding to the centrosomes. This package is based on similar principles to those previously described for our automated chromosome tracking software and allows specific biologically relevant parameters to be determined without observer error or bias (see Materials and methods; Yau and Wakefield, 2007).


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)

Centrosome separation is inhibited after inactivation of Akt or APC2. (A) Live confocal analysis of embryos expressing α-tubulin–GFP. Examples of the angle of centrosome separation 20 s before NEB in wild-type (WT) embryos, akt1q/akt104226 embryos (akt), embryos injected with anti-Akt antibodies (α-Akt), and embryos expressing an allele of APC2 unable to bind to Arm (apc2Δs). Bar, 10 μm. (B) Histogram representing the mean angle of centrosome separation before NEB in embryos. Data for each class was obtained from 100 individual nuclei. Errors bars indicate the SEM. See Videos 4–8 (available at http://www.jcb.org/cgi/content/full/jcb.200705085/DC1).
© Copyright Policy
Related In: Results  -  Collection

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

fig6: Centrosome separation is inhibited after inactivation of Akt or APC2. (A) Live confocal analysis of embryos expressing α-tubulin–GFP. Examples of the angle of centrosome separation 20 s before NEB in wild-type (WT) embryos, akt1q/akt104226 embryos (akt), embryos injected with anti-Akt antibodies (α-Akt), and embryos expressing an allele of APC2 unable to bind to Arm (apc2Δs). Bar, 10 μm. (B) Histogram representing the mean angle of centrosome separation before NEB in embryos. Data for each class was obtained from 100 individual nuclei. Errors bars indicate the SEM. See Videos 4–8 (available at http://www.jcb.org/cgi/content/full/jcb.200705085/DC1).
Mentions: In wild-type embryos expressing α-tubulin–GFP, nuclei can also be followed as dark circles in which soluble tubulin is excluded. (Fig. 6 A and Video 4, available at http://www.jcb.org/cgi/content/full/jcb.200705085/DC1). During telophase and interphase, centrosomes migrate around nuclei until they lie approximately opposite one another (Robinson et al., 1999). After NEB, MTs invade the nucleus, capture chromosomes, and form a stable bipolar mitotic spindle (Video 4). To compare the MT dynamics in wild-type and akt embryos, we developed software able to identify and track the nuclei in which tubulin is excluded and the foci of fluorescent tubulin corresponding to the centrosomes. This package is based on similar principles to those previously described for our automated chromosome tracking software and allows specific biologically relevant parameters to be determined without observer error or bias (see Materials and methods; Yau and Wakefield, 2007).

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