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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 dynamics of centrosome migration in akt embryos. (A) Purpose designed automated tracking software was used to identify and follow centrosome movement in wild-type or akt embryos. Data were obtained from 26 wild-type and 16 akt1q/akt104226 centrosomal pairs. Error bars show the SEM based on one standard deviation. (B) Stills from time-lapse videos of cycle 11 wild-type (wt) and akt1q/akt104226 (akt) embryos. In the akt embryo, a centrosome that appears to have detached from the nuclear envelope does not initially contribute to spindle formation (arrows). The bent, short spindle formed recaptures the centrosome and is capable of chromosome segregation. Nonetheless, the resulting nuclei move into the interior of the embryo during the following interphase. See Videos 9 and 10 (available at http://www.jcb.org/cgi/content/full/jcb.200705085/DC1). Bar, 10 μm.
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fig7: The dynamics of centrosome migration in akt embryos. (A) Purpose designed automated tracking software was used to identify and follow centrosome movement in wild-type or akt embryos. Data were obtained from 26 wild-type and 16 akt1q/akt104226 centrosomal pairs. Error bars show the SEM based on one standard deviation. (B) Stills from time-lapse videos of cycle 11 wild-type (wt) and akt1q/akt104226 (akt) embryos. In the akt embryo, a centrosome that appears to have detached from the nuclear envelope does not initially contribute to spindle formation (arrows). The bent, short spindle formed recaptures the centrosome and is capable of chromosome segregation. Nonetheless, the resulting nuclei move into the interior of the embryo during the following interphase. See Videos 9 and 10 (available at http://www.jcb.org/cgi/content/full/jcb.200705085/DC1). Bar, 10 μm.

Mentions: To analyze the exact cause of the impaired centrosome migration seen in akt embryos, we used our software to track the centrosomes over time. In agreement with previously published data, separation of centrosomes during interphase in wild-type cycle 12 embryos appeared to be composed of two components (Fig. 7 A; Sharp et al., 2000; Cytrynbaum et al., 2003). An initial fast hyperbolic separation led to an intercentrosomal distance of 4–5 μm. This was followed by a near linear rate of 0.01 μm/s, resulting in full separation of centrosomes by 6–7 μm just before NEB (Fig. 7 A and Video 4). We found that the profile of centrosome separation in akt embryos differed from the wild type in two respects. First, the mean initial rate of separation appeared to be slower. Second, the secondary, linear rate of separation appeared to be absent. Consequently, centrosomes completed their movements ∼240 s before NEB, after they had reached a separation of ∼4–5 μm, and remained at this approximate distance away from each other until after NEB (Fig. 7 A and Video 5). Of these two differences, however, only the second was statistically significant. Thus, although centrosome separation may be compromised in akt embryos in general, it is the terminal stages of this process that are clearly altered in comparison to wild-type embryos.


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 dynamics of centrosome migration in akt embryos. (A) Purpose designed automated tracking software was used to identify and follow centrosome movement in wild-type or akt embryos. Data were obtained from 26 wild-type and 16 akt1q/akt104226 centrosomal pairs. Error bars show the SEM based on one standard deviation. (B) Stills from time-lapse videos of cycle 11 wild-type (wt) and akt1q/akt104226 (akt) embryos. In the akt embryo, a centrosome that appears to have detached from the nuclear envelope does not initially contribute to spindle formation (arrows). The bent, short spindle formed recaptures the centrosome and is capable of chromosome segregation. Nonetheless, the resulting nuclei move into the interior of the embryo during the following interphase. See Videos 9 and 10 (available at http://www.jcb.org/cgi/content/full/jcb.200705085/DC1). Bar, 10 μm.
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Related In: Results  -  Collection

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

fig7: The dynamics of centrosome migration in akt embryos. (A) Purpose designed automated tracking software was used to identify and follow centrosome movement in wild-type or akt embryos. Data were obtained from 26 wild-type and 16 akt1q/akt104226 centrosomal pairs. Error bars show the SEM based on one standard deviation. (B) Stills from time-lapse videos of cycle 11 wild-type (wt) and akt1q/akt104226 (akt) embryos. In the akt embryo, a centrosome that appears to have detached from the nuclear envelope does not initially contribute to spindle formation (arrows). The bent, short spindle formed recaptures the centrosome and is capable of chromosome segregation. Nonetheless, the resulting nuclei move into the interior of the embryo during the following interphase. See Videos 9 and 10 (available at http://www.jcb.org/cgi/content/full/jcb.200705085/DC1). Bar, 10 μm.
Mentions: To analyze the exact cause of the impaired centrosome migration seen in akt embryos, we used our software to track the centrosomes over time. In agreement with previously published data, separation of centrosomes during interphase in wild-type cycle 12 embryos appeared to be composed of two components (Fig. 7 A; Sharp et al., 2000; Cytrynbaum et al., 2003). An initial fast hyperbolic separation led to an intercentrosomal distance of 4–5 μm. This was followed by a near linear rate of 0.01 μm/s, resulting in full separation of centrosomes by 6–7 μm just before NEB (Fig. 7 A and Video 4). We found that the profile of centrosome separation in akt embryos differed from the wild type in two respects. First, the mean initial rate of separation appeared to be slower. Second, the secondary, linear rate of separation appeared to be absent. Consequently, centrosomes completed their movements ∼240 s before NEB, after they had reached a separation of ∼4–5 μm, and remained at this approximate distance away from each other until after NEB (Fig. 7 A and Video 5). Of these two differences, however, only the second was statistically significant. Thus, although centrosome separation may be compromised in akt embryos in general, it is the terminal stages of this process that are clearly altered in comparison to wild-type embryos.

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