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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 timing of syncytial mitoses is not affected in akt embryos. Chromosome dynamics in wild-type and akt1q/akt104226 embryos expressing GFP-histone. (A) Histogram representing the percentage of nuclei that move into the interior of the embryo (nuclear fallout) during cycles 11, 12, and 13. Data were obtained from 20 wild-type and 20 akt1q/akt104226 embryos. (B) Graphical representation of an idealized graph applied to chromosomes throughout mitosis (Yau and Wakefield, 2007). Parameters of interest are automatically obtained using this software, such as (i) rate of chromosome condensation, (ii) length of metaphase, and (iii) rate of chromosome segregation. (C) Chromosome dynamics during cycle 12 in wild-type (black) and akt1q/akt104226 (red) embryos. Each line is the mean of 10 nuclei from a single embryo. Data were obtained from seven different wild-type or akt1q/akt104226 embryos. Similar results were found for embryos in cycles 11 and 13 (unpublished data). See Videos 2 and 3 (available at http://www.jcb.org/cgi/content/full/jcb.200705085/DC1).
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fig5: The timing of syncytial mitoses is not affected in akt embryos. Chromosome dynamics in wild-type and akt1q/akt104226 embryos expressing GFP-histone. (A) Histogram representing the percentage of nuclei that move into the interior of the embryo (nuclear fallout) during cycles 11, 12, and 13. Data were obtained from 20 wild-type and 20 akt1q/akt104226 embryos. (B) Graphical representation of an idealized graph applied to chromosomes throughout mitosis (Yau and Wakefield, 2007). Parameters of interest are automatically obtained using this software, such as (i) rate of chromosome condensation, (ii) length of metaphase, and (iii) rate of chromosome segregation. (C) Chromosome dynamics during cycle 12 in wild-type (black) and akt1q/akt104226 (red) embryos. Each line is the mean of 10 nuclei from a single embryo. Data were obtained from seven different wild-type or akt1q/akt104226 embryos. Similar results were found for embryos in cycles 11 and 13 (unpublished data). See Videos 2 and 3 (available at http://www.jcb.org/cgi/content/full/jcb.200705085/DC1).

Mentions: To investigate the defects in mitosis observed in fixed akt embryos in more detail, we analyzed nuclei, MT, and centrosome dynamics using time-lapse confocal microscopy. In wild-type embryos expressing histone H2B-GFP, the synchronous mitoses occur with high fidelity, although nuclei occasionally (0.8%, n = 1,357) move into the interior of the embryo (Fig. 5 A and Video 2 available at http://www.jcb.org/cgi/content/full/jcb.200705085/DC1). In agreement with our observations in fixed cells, akt embryos showed an approximately fivefold increase in nuclear fallout (3.7%, n = 1.953; Fig. 5 A and Video 3). However, a quantitative analysis of chromosome dynamics showed that key mitotic parameters such as the rate of chromosome condensation, the length of time spent in metaphase, and the rate of chromosome segregation were similar in both wild-type and akt embryos (Fig. 5, B and C).


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 timing of syncytial mitoses is not affected in akt embryos. Chromosome dynamics in wild-type and akt1q/akt104226 embryos expressing GFP-histone. (A) Histogram representing the percentage of nuclei that move into the interior of the embryo (nuclear fallout) during cycles 11, 12, and 13. Data were obtained from 20 wild-type and 20 akt1q/akt104226 embryos. (B) Graphical representation of an idealized graph applied to chromosomes throughout mitosis (Yau and Wakefield, 2007). Parameters of interest are automatically obtained using this software, such as (i) rate of chromosome condensation, (ii) length of metaphase, and (iii) rate of chromosome segregation. (C) Chromosome dynamics during cycle 12 in wild-type (black) and akt1q/akt104226 (red) embryos. Each line is the mean of 10 nuclei from a single embryo. Data were obtained from seven different wild-type or akt1q/akt104226 embryos. Similar results were found for embryos in cycles 11 and 13 (unpublished data). See Videos 2 and 3 (available at http://www.jcb.org/cgi/content/full/jcb.200705085/DC1).
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Related In: Results  -  Collection

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

fig5: The timing of syncytial mitoses is not affected in akt embryos. Chromosome dynamics in wild-type and akt1q/akt104226 embryos expressing GFP-histone. (A) Histogram representing the percentage of nuclei that move into the interior of the embryo (nuclear fallout) during cycles 11, 12, and 13. Data were obtained from 20 wild-type and 20 akt1q/akt104226 embryos. (B) Graphical representation of an idealized graph applied to chromosomes throughout mitosis (Yau and Wakefield, 2007). Parameters of interest are automatically obtained using this software, such as (i) rate of chromosome condensation, (ii) length of metaphase, and (iii) rate of chromosome segregation. (C) Chromosome dynamics during cycle 12 in wild-type (black) and akt1q/akt104226 (red) embryos. Each line is the mean of 10 nuclei from a single embryo. Data were obtained from seven different wild-type or akt1q/akt104226 embryos. Similar results were found for embryos in cycles 11 and 13 (unpublished data). See Videos 2 and 3 (available at http://www.jcb.org/cgi/content/full/jcb.200705085/DC1).
Mentions: To investigate the defects in mitosis observed in fixed akt embryos in more detail, we analyzed nuclei, MT, and centrosome dynamics using time-lapse confocal microscopy. In wild-type embryos expressing histone H2B-GFP, the synchronous mitoses occur with high fidelity, although nuclei occasionally (0.8%, n = 1,357) move into the interior of the embryo (Fig. 5 A and Video 2 available at http://www.jcb.org/cgi/content/full/jcb.200705085/DC1). In agreement with our observations in fixed cells, akt embryos showed an approximately fivefold increase in nuclear fallout (3.7%, n = 1.953; Fig. 5 A and Video 3). However, a quantitative analysis of chromosome dynamics showed that key mitotic parameters such as the rate of chromosome condensation, the length of time spent in metaphase, and the rate of chromosome segregation were similar in both wild-type and akt embryos (Fig. 5, B and C).

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