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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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Subcellular location of activated AMPK. A. Supernatant (S) and pellet (P) fractions were prepared using buffer K (pH 6.0 or 8.0) from 4-h incubated embryos and separated by 10% SDS-PAGE. Proteins were also transferred to PVDF membranes and detected using phospho-AMPK (Thr172) antibody. The detected AMPK is indicated by an arrow. B. Confocal microscopy confirms that AMPK localizes in the nuclei when activated. Nuclei were double-stained using DAPI (a) and FITC-conjugated goat anti-rabbit IgG (b) after incubation with phospho-AMPK (Thr172) antibody. c, merged image of a and b. d, d' low magnification image of a field of nuclei double-stained with FITC-conjugated goat anti-rabbit IgG (d) and DAPI (d').
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Figure 3: Subcellular location of activated AMPK. A. Supernatant (S) and pellet (P) fractions were prepared using buffer K (pH 6.0 or 8.0) from 4-h incubated embryos and separated by 10% SDS-PAGE. Proteins were also transferred to PVDF membranes and detected using phospho-AMPK (Thr172) antibody. The detected AMPK is indicated by an arrow. B. Confocal microscopy confirms that AMPK localizes in the nuclei when activated. Nuclei were double-stained using DAPI (a) and FITC-conjugated goat anti-rabbit IgG (b) after incubation with phospho-AMPK (Thr172) antibody. c, merged image of a and b. d, d' low magnification image of a field of nuclei double-stained with FITC-conjugated goat anti-rabbit IgG (d) and DAPI (d').

Mentions: The subcellular location of activated AMPK is closely related to its function. Thus, we used a cell fraction system to separate pellet and supernatant proteins of embryos [22]. The pellet fraction loaded on SDS-PAGE contains nuclei and yolk platelets, while the supernatant contains cellular proteins. As shown in Figure 3A, activated AMPK was detectable on Western blots of pellet extracts, but not in supernatant extracts. Immunofluorescent staining of nuclei and confocal microscopy further confirmed that activated AMPK was localized in the nucleus (Figure 3B; Additional file 1).


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)

Subcellular location of activated AMPK. A. Supernatant (S) and pellet (P) fractions were prepared using buffer K (pH 6.0 or 8.0) from 4-h incubated embryos and separated by 10% SDS-PAGE. Proteins were also transferred to PVDF membranes and detected using phospho-AMPK (Thr172) antibody. The detected AMPK is indicated by an arrow. B. Confocal microscopy confirms that AMPK localizes in the nuclei when activated. Nuclei were double-stained using DAPI (a) and FITC-conjugated goat anti-rabbit IgG (b) after incubation with phospho-AMPK (Thr172) antibody. c, merged image of a and b. d, d' low magnification image of a field of nuclei double-stained with FITC-conjugated goat anti-rabbit IgG (d) and DAPI (d').
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Subcellular location of activated AMPK. A. Supernatant (S) and pellet (P) fractions were prepared using buffer K (pH 6.0 or 8.0) from 4-h incubated embryos and separated by 10% SDS-PAGE. Proteins were also transferred to PVDF membranes and detected using phospho-AMPK (Thr172) antibody. The detected AMPK is indicated by an arrow. B. Confocal microscopy confirms that AMPK localizes in the nuclei when activated. Nuclei were double-stained using DAPI (a) and FITC-conjugated goat anti-rabbit IgG (b) after incubation with phospho-AMPK (Thr172) antibody. c, merged image of a and b. d, d' low magnification image of a field of nuclei double-stained with FITC-conjugated goat anti-rabbit IgG (d) and DAPI (d').
Mentions: The subcellular location of activated AMPK is closely related to its function. Thus, we used a cell fraction system to separate pellet and supernatant proteins of embryos [22]. The pellet fraction loaded on SDS-PAGE contains nuclei and yolk platelets, while the supernatant contains cellular proteins. As shown in Figure 3A, activated AMPK was detectable on Western blots of pellet extracts, but not in supernatant extracts. Immunofluorescent staining of nuclei and confocal microscopy further confirmed that activated AMPK was localized in the nucleus (Figure 3B; Additional file 1).

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