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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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A model of how Akt contributes to centrosome separation in the syncytial blastoderm. (A) In wild-type embryos, cortical Akt phosphorylates Zw3, ensuring it remains inactive. A complex of Arm and APC2 is able to interact with the actin cortex and with the MT + Tip protein EB1. The stable interaction between the cortex and MTs allows cortical dynein to generate the force required for full centrosome separation. (B) In embryos in which akt levels are severely reduced, Zw3 is no longer phosphorylated. The active Zw3 kinase can now phosphorylate both APC2 and Arm. Phosphorylated Arm is targeted for degradation, disrupting the cortical complex. EB1, although able to bind MTs, cannot stably associate with the actin cortex in the absence of cortical Arm–APC2. Cortical dynein is able to transiently interact with MTs and generate force. However, the force required for full centrosome separation is now greater than can be generated in the absence of Arm–APC2. Consequently, centrosome separation stops before completion.
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fig10: A model of how Akt contributes to centrosome separation in the syncytial blastoderm. (A) In wild-type embryos, cortical Akt phosphorylates Zw3, ensuring it remains inactive. A complex of Arm and APC2 is able to interact with the actin cortex and with the MT + Tip protein EB1. The stable interaction between the cortex and MTs allows cortical dynein to generate the force required for full centrosome separation. (B) In embryos in which akt levels are severely reduced, Zw3 is no longer phosphorylated. The active Zw3 kinase can now phosphorylate both APC2 and Arm. Phosphorylated Arm is targeted for degradation, disrupting the cortical complex. EB1, although able to bind MTs, cannot stably associate with the actin cortex in the absence of cortical Arm–APC2. Cortical dynein is able to transiently interact with MTs and generate force. However, the force required for full centrosome separation is now greater than can be generated in the absence of Arm–APC2. Consequently, centrosome separation stops before completion.

Mentions: In our model, we suggest that during the initial stages of centrosome separation, cortical dynein is able to act on MTs that transiently associate with the cell cortex (Fig. 10, A and B). However, as centrosomes continue to separate, the MTs require a more stable interaction with the cortex to contend with the increased force acting upon them. Through phosphorylating and inactivating Zw3 at the embryonic cortex, Akt would maintain sufficient levels of Arm and APC2 at the cortex facilitating a stable interaction with EB1 and MTs. Dynein can therefore continue to exert force on the cortex, centrosomes, and nuclear envelope, and centrosome separation can progress to completion (Fig. 10 A). In akt mutants, Zw3 is derepressed, allowing it to phosphorylate and disrupt the cortical complex. Arm is degraded, cortical APC2 and EB1 are lost, and MTs are only transiently associated with dynein. Consequently, the force generated during the final stages of centrosome separation would be insufficient for full separation and centrosomes would stall (Fig. 10 B).


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)

A model of how Akt contributes to centrosome separation in the syncytial blastoderm. (A) In wild-type embryos, cortical Akt phosphorylates Zw3, ensuring it remains inactive. A complex of Arm and APC2 is able to interact with the actin cortex and with the MT + Tip protein EB1. The stable interaction between the cortex and MTs allows cortical dynein to generate the force required for full centrosome separation. (B) In embryos in which akt levels are severely reduced, Zw3 is no longer phosphorylated. The active Zw3 kinase can now phosphorylate both APC2 and Arm. Phosphorylated Arm is targeted for degradation, disrupting the cortical complex. EB1, although able to bind MTs, cannot stably associate with the actin cortex in the absence of cortical Arm–APC2. Cortical dynein is able to transiently interact with MTs and generate force. However, the force required for full centrosome separation is now greater than can be generated in the absence of Arm–APC2. Consequently, centrosome separation stops before completion.
© Copyright Policy
Related In: Results  -  Collection

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

fig10: A model of how Akt contributes to centrosome separation in the syncytial blastoderm. (A) In wild-type embryos, cortical Akt phosphorylates Zw3, ensuring it remains inactive. A complex of Arm and APC2 is able to interact with the actin cortex and with the MT + Tip protein EB1. The stable interaction between the cortex and MTs allows cortical dynein to generate the force required for full centrosome separation. (B) In embryos in which akt levels are severely reduced, Zw3 is no longer phosphorylated. The active Zw3 kinase can now phosphorylate both APC2 and Arm. Phosphorylated Arm is targeted for degradation, disrupting the cortical complex. EB1, although able to bind MTs, cannot stably associate with the actin cortex in the absence of cortical Arm–APC2. Cortical dynein is able to transiently interact with MTs and generate force. However, the force required for full centrosome separation is now greater than can be generated in the absence of Arm–APC2. Consequently, centrosome separation stops before completion.
Mentions: In our model, we suggest that during the initial stages of centrosome separation, cortical dynein is able to act on MTs that transiently associate with the cell cortex (Fig. 10, A and B). However, as centrosomes continue to separate, the MTs require a more stable interaction with the cortex to contend with the increased force acting upon them. Through phosphorylating and inactivating Zw3 at the embryonic cortex, Akt would maintain sufficient levels of Arm and APC2 at the cortex facilitating a stable interaction with EB1 and MTs. Dynein can therefore continue to exert force on the cortex, centrosomes, and nuclear envelope, and centrosome separation can progress to completion (Fig. 10 A). In akt mutants, Zw3 is derepressed, allowing it to phosphorylate and disrupt the cortical complex. Arm is degraded, cortical APC2 and EB1 are lost, and MTs are only transiently associated with dynein. Consequently, the force generated during the final stages of centrosome separation would be insufficient for full separation and centrosomes would stall (Fig. 10 B).

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