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Activation of an AMP-activated protein kinase is involved in post-diapause development of Artemia franciscana encysted embryos.

Zhu XJ, Dai JQ, Tan X, Zhao Y, Yang WJ - BMC Dev. Biol. (2009)

Bottom Line: However, the intrinsic mechanisms that regulate this process are unclear.Using a phospho-AMPKalpha antibody, AMPK was shown to be phosphorylated in the post-diapause developmental process.Using whole-mount immunohistochemistry, phosphorylated AMPK was shown to be predominantly located in the ectoderm of the early developed embryos in a ring shape; however, the location and shape of the activation region changed as development proceeded.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Cell Biology and Genetics, College of Life Sciences, Zijingang Campus, Zhejiang University, Hangzhou, Zhejiang 310058, PR China. xiao_jingzhu@hotmail.com

ABSTRACT

Background: Cysts of Artemia can remain in a dormant state for long periods with a very low metabolic rate, and only resume their development with the approach of favorable conditions. The post-diapause development is a very complicated process involving a variety of metabolic and biochemical events. However, the intrinsic mechanisms that regulate this process are unclear.

Results: Herein we report the specific activation of an AMP-activated protein kinase (AMPK) in the post-diapause developmental process of Artemia. Using a phospho-AMPKalpha antibody, AMPK was shown to be phosphorylated in the post-diapause developmental process. Results of kinase assay analysis showed that this phosphorylation is essential for AMPK activation. Using whole-mount immunohistochemistry, phosphorylated AMPK was shown to be predominantly located in the ectoderm of the early developed embryos in a ring shape; however, the location and shape of the activation region changed as development proceeded. Additionally, Western blotting analysis on different portions of the cyst extracts showed that phosphorylated AMPKalpha localized to the nuclei and this location was not affected by intracellular pH. Confocal microscopy analysis of immunofluorescent stained cyst nuclei further showed that AMPKalpha localized to the nuclei when activated. Moreover, cellular AMP, ADP, and ATP levels in developing cysts were determined by HPLC, and the results showed that the activation of Artemia AMPK may not be associated with cellular AMP:ATP ratios, suggesting other pathways for regulation of Artemia AMPK activity.

Conclusion: Together, we report evidence demonstrating the activation of AMPK in Artemia developing cysts and present an argument for its role in the development-related gene expression and energy control in certain cells during post-diapause development of Artemia.

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Location of activated AMPK in developing cysts by whole mount immunohistochemistry. Decapsulated embryos developing for 2, 4, 6, 8, 10, and 12 h were fixed in 4% paraformaldehyde, permeated by 0.1% Triton X-100, incubated with phospho-AMPK (Thr172) antibody (a – f), and subsequently detected with the AP-conjugated secondary antibody by NBT/BCIP staining. e' and f' represent control embryos that were not incubated with the primary antibody. Embryos are shown at the same magnification.
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Figure 2: Location of activated AMPK in developing cysts by whole mount immunohistochemistry. Decapsulated embryos developing for 2, 4, 6, 8, 10, and 12 h were fixed in 4% paraformaldehyde, permeated by 0.1% Triton X-100, incubated with phospho-AMPK (Thr172) antibody (a – f), and subsequently detected with the AP-conjugated secondary antibody by NBT/BCIP staining. e' and f' represent control embryos that were not incubated with the primary antibody. Embryos are shown at the same magnification.

Mentions: Using whole-mount immunohistochemistry, we have examined the distribution of activated AMPK in developing embryos (Figure 2). The results also illustrate that AMPK is activated in 4-h embryos and embryos at later stages. Activated AMPK was predominantly located in the ectoderm region of embryos at early stages in a ring shape (Figures 2b–d). As development proceeded, the activated loop enlarged (Figure 2e), and in late embryos that have a pear shape, AMPK was activated in the head region (Figure 2f).


Activation of an AMP-activated protein kinase is involved in post-diapause development of Artemia franciscana encysted embryos.

Zhu XJ, Dai JQ, Tan X, Zhao Y, Yang WJ - BMC Dev. Biol. (2009)

Location of activated AMPK in developing cysts by whole mount immunohistochemistry. Decapsulated embryos developing for 2, 4, 6, 8, 10, and 12 h were fixed in 4% paraformaldehyde, permeated by 0.1% Triton X-100, incubated with phospho-AMPK (Thr172) antibody (a – f), and subsequently detected with the AP-conjugated secondary antibody by NBT/BCIP staining. e' and f' represent control embryos that were not incubated with the primary antibody. Embryos are shown at the same magnification.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2667496&req=5

Figure 2: Location of activated AMPK in developing cysts by whole mount immunohistochemistry. Decapsulated embryos developing for 2, 4, 6, 8, 10, and 12 h were fixed in 4% paraformaldehyde, permeated by 0.1% Triton X-100, incubated with phospho-AMPK (Thr172) antibody (a – f), and subsequently detected with the AP-conjugated secondary antibody by NBT/BCIP staining. e' and f' represent control embryos that were not incubated with the primary antibody. Embryos are shown at the same magnification.
Mentions: Using whole-mount immunohistochemistry, we have examined the distribution of activated AMPK in developing embryos (Figure 2). The results also illustrate that AMPK is activated in 4-h embryos and embryos at later stages. Activated AMPK was predominantly located in the ectoderm region of embryos at early stages in a ring shape (Figures 2b–d). As development proceeded, the activated loop enlarged (Figure 2e), and in late embryos that have a pear shape, AMPK was activated in the head region (Figure 2f).

Bottom Line: However, the intrinsic mechanisms that regulate this process are unclear.Using a phospho-AMPKalpha antibody, AMPK was shown to be phosphorylated in the post-diapause developmental process.Using whole-mount immunohistochemistry, phosphorylated AMPK was shown to be predominantly located in the ectoderm of the early developed embryos in a ring shape; however, the location and shape of the activation region changed as development proceeded.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Cell Biology and Genetics, College of Life Sciences, Zijingang Campus, Zhejiang University, Hangzhou, Zhejiang 310058, PR China. xiao_jingzhu@hotmail.com

ABSTRACT

Background: Cysts of Artemia can remain in a dormant state for long periods with a very low metabolic rate, and only resume their development with the approach of favorable conditions. The post-diapause development is a very complicated process involving a variety of metabolic and biochemical events. However, the intrinsic mechanisms that regulate this process are unclear.

Results: Herein we report the specific activation of an AMP-activated protein kinase (AMPK) in the post-diapause developmental process of Artemia. Using a phospho-AMPKalpha antibody, AMPK was shown to be phosphorylated in the post-diapause developmental process. Results of kinase assay analysis showed that this phosphorylation is essential for AMPK activation. Using whole-mount immunohistochemistry, phosphorylated AMPK was shown to be predominantly located in the ectoderm of the early developed embryos in a ring shape; however, the location and shape of the activation region changed as development proceeded. Additionally, Western blotting analysis on different portions of the cyst extracts showed that phosphorylated AMPKalpha localized to the nuclei and this location was not affected by intracellular pH. Confocal microscopy analysis of immunofluorescent stained cyst nuclei further showed that AMPKalpha localized to the nuclei when activated. Moreover, cellular AMP, ADP, and ATP levels in developing cysts were determined by HPLC, and the results showed that the activation of Artemia AMPK may not be associated with cellular AMP:ATP ratios, suggesting other pathways for regulation of Artemia AMPK activity.

Conclusion: Together, we report evidence demonstrating the activation of AMPK in Artemia developing cysts and present an argument for its role in the development-related gene expression and energy control in certain cells during post-diapause development of Artemia.

Show MeSH
Related in: MedlinePlus