F-actin rearrangement is regulated by mTORC2/Akt/Girdin in mouse fertilized eggs

Abstract

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.

Objectives: 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.

Materials and 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.

Figure 1

mTORC2 affects rearrangement of the F‐actin cytoskeleton in mouse fertilized eggs. (a) The development model of mouse fertilized eggs. G1 phase: 12–21 hours after injecting hCG; S phase: 21–27 hours after injecting hCG; G2 phase: 27–30 hours after injecting hCG; M phase: 30–33 hours after injecting hCG. (b) Staining for F‐actin (red) and DNA (blue) revealed the organization of the F‐actin cytoskeleton in mouse fertilized eggs transfected with RICTOR shRNA (F‐actin: yellow arrow). Scale bar: 20 μm. (c) Western immunoblotting detection of mTORC2 in 200 mouse fertilized eggs treated with shRNA targeted against mTORC2. WB: Western blotting

Figure 2

Akt1 regulates cell division and F‐actin rearrangement in mouse fertilized eggs. (a) Western immunoblotting detection of Akt1 in 200 mouse fertilized eggs treated with siRNA targeted against Akt1. WB: Western blotting. (b) The division rate in cultured mouse embryos after 0.03 ng of mRNA‐encoding Akt1‐WT, myr‐Akt1, Akt1‐KD or Akt1 siRNA injection. The percentage of cells undergoing cell division and cell survival was calculated after manual counting under a dissecting microscope 35 hours after injection of human chorionic gonadotropin in female mice. The numbers of eggs undergoing cell division (hatched bars) or survival are given above each bar graph. (c) Cortical remodelling of F‐actin is induced by Akt1 activation. Mouse fertilized eggs were stained with rhodamine‐phalloidin (red fluorescence) to visualize F‐actin cytoskeleton. Various scenarios were studied, which included cells injected with mRNA‐encoding Akt1‐WT, myr‐Akt1, Akt1‐KD or siRNA targeted against Akt1. Fertilized eggs were fixed and labelled with rhodamine‐phalloidin (10 or 20 μmol/L; F‐actin labelling, indicated by white arrows) and with Hoechst 33258 (1 mg/ml; DNA labelling) and imaged by laser‐scanning confocal microscope. Scale bar: 20 μm

Figure 3

The mTORC2/Akt1 pathway rearranges the F‐actin cytoskeleton of one‐cell stage fertilized eggs. Microinjection of Rictor shRNA then with myr‐Akt1 mRNA into mouse one‐cell stage embryos. Staining for F‐actin (red) revealed the organization of the F‐actin cytoskeleton in mouse fertilized eggs (F‐actin is shown by the yellow arrow). Scale bar: 20 μm

Figure 4

Girdin is essential for rearrangement of the F‐actin cytoskeleton and the development of mouse fertilized eggs. (a) Depletion of Girdin in mouse one‐cell staged fertilized eggs by siRNA. Total cell extracts from control siRNA‐ and Girdin siRNA‐injected one‐cell staged fertilized eggs were subjected to Western blot analyses and immunodetection with anti‐Girdin, anti‐p‐Girdin, anti‐Akt1 and anti‐actin antibodies. (b) The cleavage rate in cultured mouse embryos after Girdin siRNA injections shows that the total number of eggs undergoing cell division is given above each bar graph from three independent experiments. (c) F‐actin cytoskeleton of embryos derived from fertilized eggs treated with Girdin siRNA. Mouse fertilized eggs were treated with control or Girdin siRNAs and fixed 48 hours later, followed by staining with rhodamine‐phalloidin and anti‐Girdin antibody. In situ validation of the interaction between Girdin and polymerized F‐actin by confocal microscopy is shown. One‐cell stage mouse fertilized eggs were treated with 10 or 20 μmol/L Girdin siRNA. Immunolocalization of Girdin is revealed by green staining (antibody), and immunolocalization of actin is revealed by red staining. Control fertilized eggs (21 hours after hCG): one‐cell arrested embryos derived from fertilized eggs that were injected with Girdin siRNA 21 hours after hCG and cultured for 24 hours; control two‐cell embryo: in control fertilized eggs, F‐actin forms a regular ring in the cell cortex. In embryos derived from fertilized eggs treated with girdin siRNA, polymerized F‐actin was observed in the cytoplasm and formed irregular patches that were scattered randomly in the cortex. Scale bar: 20 μm

Figure 5

The Akt1/ Girdin pathway regulates rearrangement of the F‐actin cytoskeleton of one‐cell stage fertilized eggs. (a) One‐cell stage mouse embryos were first microinjected with Akt1‐WT mRNA, myr‐Akt1 mRNA or siRNA, then stained with rhodamine‐phalloidin (20 μmol/L; actin labelling as shown in red) and anti‐P‐Girdin (1:50; Girdin labelling; as shown in green). Merged colour detection between P‐Girdin and polymerize F‐actin appears as yellow stained images. The areas of co‐localization are highlighted with yellow arrowheads. Scale bar: 20 μm. (b) 200 fertilized eggs were treated with mRNA coding for Akt1‐WT, myr‐Akt1, Akt1‐KD or siRNA against Akt1. Western immunoblot analyses assessed following immunoreaction with anti‐P‐Girdin (upper panel) and anti‐Girdin (lower panel) antibodies are shown. (c) One‐cell stage mouse embryos were first microinjected with 0.03 ng of myr‐Akt1 mRNA as described in the Materials and Methods, and then 1–2 hours later with Girdin siRNA, following which specimens were stained with rhodamine‐phalloidin (20 μmol/L; actin labelling shown in red). Scale bar: 20 μm

Figure 6

The mTORC2/Akt1/Girdin pathway rearranges the F‐actin cytoskeleton in one‐cell stage fertilized eggs. (a) The effect of gene knockdown on the expression of Rictor and the phosphorylation of Girdin‐1417. Extracts of mouse fertilized eggs were resolved by 6% SDS‐PAGE, transferred to nitrocellulose and probed with phosphor‐Girdin‐Ser1417 antibody and Girdin antibody. (b) We examined Girdin phosphorylation in the fertilized eggs treated with Rictor shRNA, WT‐Akt1 mRNA, Rictor shRNA and WT‐Akt1 coinjection. In WT‐Akt1 mRNA treated cells, we noted that Girdin was highly phosphorylated at 1417. But cells treated with Rictor siRNA and WT‐Akt1 exhibited decreased phosphorylation of Girdin 1417

Figure 7

Schematic illustration of the working model for the regulation of F‐actin in mouse fertilized eggs by mTORC2/Akt1/Girdin. Akt1 and Girdin were affected by knockdown of mTORC2. Akt1 positively regulated the development of mouse fertilized eggs by Girdin‐mediated F‐actin remodelling. Girdin protein could be a downstream target of the Akt1 signalling pathway. mTORC2 may regulate Girdin in the development of mouse fertilized eggs. Our current study suggests a critical role of Akt1 as a substrate for mTORC2 and supports an important role for mTORC2 in regulating the development of mouse fertilized eggs through Akt1 and Girdin. Our study indicates that the mTORC2/Akt1/Girdin signalling pathway is crucial for remodelling F‐actin in mouse fertilized eggs