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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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D. melanogaster Akt regulates Zw3 during early embryonic development. (A) Representation of the akt locus showing differential splicing of the three proposed isoforms (A, B, and C) and the site of insertion of the PZ P element (arrow). Coding regions are shown in red and untranslated regions are shown in blue (adapted from Flybase; http://www.flybase.indiana.org). (B) RT-PCR of a 400-bp region of the akt coding region from cDNA extracted from wild-type and akt mutant flies. (C) Western blot of 0–3-h embryo extracts probed with antibodies specific for Akt. CP190 is shown as a loading control. (D) Western blot of embryo extracts probed with an antibody specific for Zw3 phosphorylated on the consensus Akt phosphorylation site (Ser12). (E) Histogram demonstrating the rescue of akt104426 female sterility through the introduction of one copy of sgg1. Error bars indicate the SEM.
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fig1: D. melanogaster Akt regulates Zw3 during early embryonic development. (A) Representation of the akt locus showing differential splicing of the three proposed isoforms (A, B, and C) and the site of insertion of the PZ P element (arrow). Coding regions are shown in red and untranslated regions are shown in blue (adapted from Flybase; http://www.flybase.indiana.org). (B) RT-PCR of a 400-bp region of the akt coding region from cDNA extracted from wild-type and akt mutant flies. (C) Western blot of 0–3-h embryo extracts probed with antibodies specific for Akt. CP190 is shown as a loading control. (D) Western blot of embryo extracts probed with an antibody specific for Zw3 phosphorylated on the consensus Akt phosphorylation site (Ser12). (E) Histogram demonstrating the rescue of akt104426 female sterility through the introduction of one copy of sgg1. Error bars indicate the SEM.

Mentions: Unlike mammals, which possess three Akt genes, D. melanogaster has only one, akt1. A number of mutant alleles have been described: akt1q is an embryonic lethal allele that leads to a nonfunctional protein possessing a point mutation in the ATP-binding domain of the kinase (Staveley et al., 1998), whereas akt104226 is a semilethal female sterility allele originally generated by the insertion of the P(PZ) transposon into the 5′ untranslated region of akt1 (Fig. 1 A; Spradling et al., 1999). We confirmed the P element insertion site using genomic DNA extracted from homozygous akt104226 adult flies (unpublished data) before analyzing the effect this P element had on the expression of Akt. We found these flies had dramatically reduced levels of akt mRNA (Fig. 1 B) and showed a concomitant reduction in Akt protein (Fig. 1 C). Although both egg chambers and oocytes possessed wild-type morphology (unpublished data), embryos laid by akt104226 homozygote mothers failed to hatch and arrested at varied stages of development. A proportion of these embryos could be clearly categorized as pre- or postcellularized (Table I). However, the majority consisted of two main classes: those that appeared to have a variable number of nuclei in the interior of the embryo and those that appeared to have cellularized but possessed wavy MT bundles and many hypercondensed nuclei (Fig. S1 A, available at http://www.jcb.org/cgi/content/full/jcb.200705085/DC1).


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)

D. melanogaster Akt regulates Zw3 during early embryonic development. (A) Representation of the akt locus showing differential splicing of the three proposed isoforms (A, B, and C) and the site of insertion of the PZ P element (arrow). Coding regions are shown in red and untranslated regions are shown in blue (adapted from Flybase; http://www.flybase.indiana.org). (B) RT-PCR of a 400-bp region of the akt coding region from cDNA extracted from wild-type and akt mutant flies. (C) Western blot of 0–3-h embryo extracts probed with antibodies specific for Akt. CP190 is shown as a loading control. (D) Western blot of embryo extracts probed with an antibody specific for Zw3 phosphorylated on the consensus Akt phosphorylation site (Ser12). (E) Histogram demonstrating the rescue of akt104426 female sterility through the introduction of one copy of sgg1. Error bars indicate the SEM.
© Copyright Policy
Related In: Results  -  Collection

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

fig1: D. melanogaster Akt regulates Zw3 during early embryonic development. (A) Representation of the akt locus showing differential splicing of the three proposed isoforms (A, B, and C) and the site of insertion of the PZ P element (arrow). Coding regions are shown in red and untranslated regions are shown in blue (adapted from Flybase; http://www.flybase.indiana.org). (B) RT-PCR of a 400-bp region of the akt coding region from cDNA extracted from wild-type and akt mutant flies. (C) Western blot of 0–3-h embryo extracts probed with antibodies specific for Akt. CP190 is shown as a loading control. (D) Western blot of embryo extracts probed with an antibody specific for Zw3 phosphorylated on the consensus Akt phosphorylation site (Ser12). (E) Histogram demonstrating the rescue of akt104426 female sterility through the introduction of one copy of sgg1. Error bars indicate the SEM.
Mentions: Unlike mammals, which possess three Akt genes, D. melanogaster has only one, akt1. A number of mutant alleles have been described: akt1q is an embryonic lethal allele that leads to a nonfunctional protein possessing a point mutation in the ATP-binding domain of the kinase (Staveley et al., 1998), whereas akt104226 is a semilethal female sterility allele originally generated by the insertion of the P(PZ) transposon into the 5′ untranslated region of akt1 (Fig. 1 A; Spradling et al., 1999). We confirmed the P element insertion site using genomic DNA extracted from homozygous akt104226 adult flies (unpublished data) before analyzing the effect this P element had on the expression of Akt. We found these flies had dramatically reduced levels of akt mRNA (Fig. 1 B) and showed a concomitant reduction in Akt protein (Fig. 1 C). Although both egg chambers and oocytes possessed wild-type morphology (unpublished data), embryos laid by akt104226 homozygote mothers failed to hatch and arrested at varied stages of development. A proportion of these embryos could be clearly categorized as pre- or postcellularized (Table I). However, the majority consisted of two main classes: those that appeared to have a variable number of nuclei in the interior of the embryo and those that appeared to have cellularized but possessed wavy MT bundles and many hypercondensed nuclei (Fig. S1 A, available at http://www.jcb.org/cgi/content/full/jcb.200705085/DC1).

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