F-actin rearrangement is regulated by mTORC2/Akt/Girdin in mouse fertilized eggs.
In mouse fertilized eggs, correct assembly and distribution of the actin cytoskeleton are intimately related to cleavage in early-stage embryos. However, in mouse fertilized eggs, mechanisms and involved factors responsible for regulating the actin cytoskeleton are poorly defined. In this study, mTORC2, PKB/Akt and Girdin were found to modulate division of mouse fertilized eggs by regulating distribution of the actin cytoskeleton. RNA interference (RNAi)-mediated depletion of mTORC2, Akt1 or Girdin disrupted F-actin rearrangement and strongly inhibited egg development. PKB/Akt has been proven to be a downstream target of the mTORC2 signalling pathway. Girdin, a newly found actin cross-linker, has been proven to be a downstream target of the Akt signalling pathway. Furthermore, phosphorylation of both Akt1 and girdin was affected by knockdown of mTORC2. Akt1 positively regulated development of the mouse fertilized eggs by girdin-mediated F-actin rearrangement. Thus it seems that girdin could be a downstream target of the Akt1-mediated signalling pathway. Collectively, this study aimed to prove participation of mTORC2/Akt in F-actin assembly in early-stage cleavage of mouse fertilized eggs via the function of girdin.


OBJECTIVE
In mouse fertilized eggs, the proper assembly and distribution of actin cytoskeleton is intimately related with the cleavage of early-stage embryo. However, in mammals, especially in mouse fertilized eggs, the mechanisms and involved factors responsible for regulating the actin cytoskeleton are poorly defined. The aim of this study was to determine the role of mTORC2,PKB/Akt and Girdin in early development of fertilized mouse eggs, via regulating the distribution of actin cytoskeleton.


METHODS
Changes of F-actin after treatting with mTORC2 shRNA, Akt siRNA or Girdin siRNA were observed by Immunofluorescence staining and laser-scanning confocal microscopy. Levels of phosphorylated Girdin at Se1417 were detected by Western immunoblotting. Percentages of cells undergoing division were determined by counting, using a dissecting microscope.


RESULTS
RNA interference (RNAi)-mediated depletion of mTORC2, Akt1 or Girdin disrupts F-actin rearrangement, and remarkably inhibited the development of mouse-fertilized eggs. PKB/Akt has been proved to be a downstream target of the mTORC2 signaling pathway. Girdin, the newly found actin-cross linker, has been proved to be a downstream target of the Akt signaling pathway. Furthermore phosphorylation of both Akt1 and Girdin were affected by knockdown of mTORC2. Akt1 positively regulates the development of mouse-fertilized eggs by Girdin mediated F-actin rearrangement. Girdin could be a downstream target of the Akt1-mediated signaling pathway.


CONCLUSIONS
Collectively, this study aimed to prove the participation of mTORC2/Akt in F-actin assembling in early-stage cleavage of mouse fertilized eggs via the function of Girdin.
Department of Biochemical and Molecular Biology, China Medical University, Shenyang, China.
Didi
D
Wu
Department of Obstetrics and Gynecology, Shengjing Hospital Affiliated to China Medical University, Shenyang, China.
Dahai
D
Yu
Department of Obstetrics and Gynecology, Shengjing Hospital Affiliated to China Medical University, Shenyang, China. wangxx@sj-hospital.org.
Xiuxia
X
Wang
Department of Biochemical and Molecular Biology, China Medical University, Shenyang, China. ybzbiochem@yeah.net.
Bingzhi
B
Yu
D000199:Actins / Q000032:analysis / Q000378:metabolism*; D000818:Animals; D002455:Cell Division; D005260:Female; D000076225:Mechanistic Target of Rapamycin Complex 2; D051379:Mice / Q000196:embryology* / Q000378:metabolism; D008840:Microfilament Proteins / Q000032:analysis / Q000378:metabolism*; D046912:Multiprotein Complexes / Q000032:analysis / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt / Q000032:analysis / Q000378:metabolism*; D058570:TOR Serine-Threonine Kinases / Q000032:analysis / Q000378:metabolism*; D033921:Vesicular Transport Proteins / Q000032:analysis / Q000378:metabolism*; D015053:Zygote / Q000166:cytology / Q000378:metabolism* / Q000648:ultrastructure
D000199:Actins; D000076225:Mechanistic Target of Rapamycin Complex 2; D008840:Microfilament Proteins; D046912:Multiprotein Complexes; D051057:Proto-Oncogene Proteins c-akt; D058570:TOR Serine-Threonine Kinases; D033921:Vesicular Transport Proteins; C527550:Ccdc88a protein, mouse; C514862:Akt1 protein, mouse
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