The MTORC2/Akt/Girdin Signaling Pathway Is Crucial For Actin Remodeling In Mouse Fertilized Eggs

Objective: Actin filaments(microfilaments)regulate many dynamic events during oocyte meiotic maturation and fertilization.Processes such as sperm incorporation,cortical granule exocytosis,spindle rotation(in the mouse),second polar body emission,and contractile ring formation during cleavage.However,in mammals,and especially in mouse fertilized eggs,the mechanism regulating the F-actin cytoskeleton was poorly defined.mTORC2 is a multimeric kinase composed of the mammalian target of rapamycin kinase(mTOR),m Sin1,m LST8,and rictor.It has shown functions in controlling cell growth and actin cytoskeletal assembly and is insensitive to rapamycin.In NIH3T3 fibroblasts siRNA-mediated knockdown of mTOR,Rictor,or m LST8 prevents actin polymerization and cell spreading.In human neutrophils,inhibition of mTORC2 function by Rictor knockdown also leads to cell polarity defects and uniform cortical F-actin accumulation.Together,these studies highlight the possibility that mTORC2 may have effects on the actin cytoskeleton.However,whether mTORC2 impacts microfilament aggregation of mouse fertilized eggs and alters this aggregation of microfilament assembly requires empirical determination.Although the direct targets of mTORC2 that mediate signaling to the actin cytoskeleton are unknown,mTORC2 signaling to the actin cytoskeleton may involve PKC? and the small GTPases Rho and Rac.In He La cells,the morphology of the actin cytoskeleton in PKC-?knockdown cells is similar to that of Rictor knockdown cells.Recent findings show mTORC2 directly phosphorylated Akt/PKB on Ser473.mTORC2 is responsible for insulin-induced Akt Ser-473 phosphorylation in 3T3-L1 adipocytes.Akt,which was also known as protein kinase B(PKB),was also a serine/threonine kinase.Studies indicated that Akt1 promoted cell motility in mammalian fibroblasts and tumor cells predominantly by reorganizing actin filaments.Our early studies reported Akt1 was expressed in mouse fertilized eggs and revealed that Akt1 over-expression in mouse fertilized eggs promoted cell division.Further studies performed in our laboratory revealed that mTORC2 was critical for the phosphorylation of Akt1 /PKB at Ser473 during embryogenesis,and Akt1 was positively regulated by mTORC2.Based on this,we hypothesized that mTORC2 might alter the aggregation of mouse fertilized egg actin cytoskeleton by phosphorylation of Ser473 PKB/Akt site.A search for Akt1-binding proteins led to the identification of Girdin(girders of actin filament),also known as Akt1 phosphorylation enhancer(APE)or G-interacting vesicle associated protein(GIV).Girdin was a large 220-k Da protein with unique amino and carboxylterminal domains flanking a long coiled-coil region.Girdin formed oligomers through its amino-terminal domain and coiled-coil region.The carboxyl-terminal domain contained the actin-binding site and the phosphatidylinositol phosphate-binding motif located near the Akt1 phosphorylation site(serine 1416).Therefore,Girdin was postulated to crosslink actin filaments and to anchor them to the plasma membrane in quiescent cells.Jiang et al.demonstrated that the knockdown of Girdin in breast cancer cells led to an increase in the number of cells,which demonstrate rugged cortical actin filaments,and disruption of stress fibers.siRNA-mediated knockdown of Girdin leads to the disruption of stress fibers in Vero fibroblasts.In addition,the cells lost their shape and formed rugged boundaries.Enomoto et al reported that the Girdin CT1 domain is anchored at the plasma membrane via binding to phosphoinositides,where it subsequently crosslinks actin filaments and anchors cortical actin at the plasma membrane.Since the phosphoinositide binding site is located near the Akt phosphorylation site of the CT1 domain of Girdin,it is indicated that Akt1 might control the localization of Girdin by regulation of its phosphoinositide binding property.Ni w et al reported that Girdin regulates the migration and invasion of glioma cells via the PI3K-Akt signaling pathway.The results provide a theoretical foundation for the development of anticancer drugs.Based on the above results,We speculate that Girdin may play a key role in regulating actin filaments and thus the modulation cell division in mouse fertilized eggs in an Akt1-dependent manner.In this report,we showed that RNA interference mediated dampening of the expression of mTORC2,Akt1 or Girdin disrupts actin filaments,and remarkably inhibits the development of mouse fertilized eggs.Furthermore,we found that Akt1 positively regulates the development of mouse fertilized eggs by Girdin-mediated actin remodeling.Thus,we speculate that Girdin protein may be a downstream target of the Akt1 signaling pathway.Furthermore,we also found that Akt1 and Girdin-mediated phosphorylation were affected by knockdown of mTORC2 and that mTORC2 may regulate Girdin in the development of mouse fertilized eggs.Our observations collectively indicate that the mTORC2/Akt1/Girdin signaling pathway is crucial for actin remodeling in mouse fertilized eggs.Methods: 1.Collection and culture of one-cell stage mouse embryos 2.In vitro transcription 3.mRNA/sh RNA microinjection and observation of the mouse embryos 4.RNA interference 5.Western immunoblotting 6.Immunofluorescence staining and laser-scanning confocal microscopy 7.Statistical analysisResults: 1.mTORC2 affects rearrangement of the F-actin cytoskeleton in mouse fertilized eggs To explore the function of mTORC2 in mouse fertilized eggs,w We used sh RNA to silence the gene expression of RICTOR.Mouse one-cell stage embryos at the G1 phase were cultured in M16 medium after microinjection of Rictor sh RNA.After 20 h,the embryos were collected to quantity gene and protein expression of RICTOR.To clarify the roles of mTORC2 in F-actin rearrangement,we measured the intracellular distribution and rearrangement of F-actin by immunofluorescence confocal microscopy.In control mouse fertilized eggs,F-actin localized to the cell cortex explicitily around contractile ring.By contrast,RICTOR knockdown fertilized eggs showed cytoplasmic actin aggregates and less prominent presence of contractile ring.Using siRNA to down-regulate gene expression,we almost completely inhibited RICTOR protein expression in mouse fertilized eggs.2.Akt1 regulates cell division and F-actin rearrangement in mouse fertilized eggs We investigated whether Akt1 affects cell division and F-actin rearrangement.This was done by culturing one-cell stage mouse fertilized eggs in M16 medium after microinjection of the eggs with 0.03 ng mRNA of Akt1-WT,myr-Akt1(constitutively consistent active form of Akt1),KD-Akt1 or 20?M Akt1 siRNA.In the myr-Akt1 construct,the Akt1 coding region is fused to a myristoylation sequence from pp60 csk.The dominant inhibitory mutant of Akt1 that we used in this study is a kinase-deficient Akt1 that results from the substitution of alamine for lysine at position 179 in the canonical ATP-binding domain.This mutant is not only catalytically inactive but has been shown to inhibit the activity and actions of endogenous Akt1.The control for these procedures was microinjection of fertilized eggs with scrambled sequence siRNA or TE buffer.The effect of Akt1 siRNA on Akt1 protein expression was evaluated by Western immunoblot analysis.The cleavage of fertilized mouse eggs in each group was calculated after manual counting under a dissecting microscope 35 h after h CG injection.In the control groups,61.89% of embryos reached the two-cell stage 35 h after the injection of h CG.Only 29.73% and 26.81% of embryos microinjected with KD-Akt1 mRNA or 20?M Akt1 siRNA reached the two-cell stage 35 h after the h CG injection,respectively.In contrast,the cleavage rate was up to 92.30% 35 h after the h CG injection with mRNA of myr-Akt1.These results demonstrated that Akt1 knockdown or KD-Akt1 clearly interfered with the division of one-cell stage fertilized eggs.Further,microinjection of myr-Akt1 prompted the division of mouse fertilized eggs.Next,we measured the intracellular distribution and organization of F-actin in each experimental group by immunofluorescence confocal microscopy.In the Akt1-WT and myr-Akt1 group,there were a distinct increase in the stain of F-actin around contractile ring after injection,Akt1 mRNAs prompted microfilament aggregation,thus facilitating advanced cell division.When the fertilized eggs over-expressed kinase-deficient Akt1 or cells were microinjected with Akt1 siRNA,the stain weakened around contractile ring and cells appeared to display an abnormal pattern of cell division.Akt1 expression reduced aggregation and affected the state of the microfilaments and thus affected normal cell division.These results suggest that Akt1 can indeed affect the early development of fertilized eggs by affecting the rearrangement of the F-actin cytoskeleton.3.The mTORC2/ Akt1 pathway rearranges the F-actin cytoskeleton of one-cell stage fertilized eggs To explore the function of mTORC2 and Akt1 in mouse fertilized eggs,we also monitored Akt1 phosphorylation.Using siRNA to down-regulate gene expression,we almost completely inhibited RICTOR protein expression in mouse fertilized eggs.We found that Akt1 Ser 473 phosphorylation was abolished by Rictor's knockdown.RNA interference– mediated suppression of rictor inhibited Akt1 Ser473.To further explore the relation of mTORC2 and Akt1,We first microinjected one-cell stage fertilized eggs with Rictor sh RNA,and then with the mRNA encoding wild-type Akt1.Next,we observed the rearrangement of the actin cytoskeleton by immunofluorescence confocal microscopy.We found the phenotype seems to be rescued.Our findings indicated that RICTORC2 regulates the organization of the actin cytoskeleton and that Akt1 is a mediator of this function.4.Girdin is essential for rearrangement of the F-actin cytoskeleton and the development of Mouse Fertilized Eggs A search for binding proteins of Akt1 led to the identification of Girdin,an actin-binding protein.Thus,we examined the functions of Girdin on the rearrangement of the F-actin cytoskeleton and the development of mouse fertilized eggs.We microinjected several Girdin small(21 nucleotide)interfering RNAs(Girdin siRNA)and 2-nucleotide,irrelevant or scrambled sequence RNAs(control siRNA)into mouse fertilized eggs at the G1 phase of cell cycle.According the development model of mouse fertilized eggs we microinjected Girdin siRNA into one-cell stage fertilized eggs at G1 stage of the cell cycle about 18-20 h after h CG.Fertilized eggs were collected the next day(38-42)hours after the h CG injection.Western immunoblot analyses showed that microinjected with the Girdin siRNAs(20?M)effectively reduced the expression levels of Girdin.The percentage of cell division and death in each group was calculated after manual counting under a dissecting microscope 35 h after of h CG.In the control groups,about 83% of the embryos reached the two-cell stage 35 h after the injection of h CG,and there was no significant difference between the two control groups(P>0.05). Microinjection of embryos with 10?M Girdin siRNA resulted in 28.59% of the embryos reaching the two-cell stage at 35 h following h CG injection.However,only 23.91% reached the two-cell stage when microinjected with 20?M Girdin siRNA.Depletion of Girdin inhibited cleavage of mouse fertilized eggs.To test whether Girdin promotes cross-linking of F-actin filaments,we examined the effects of Girdin knockdown on the rearrangement of the F-actin cytoskeleton.Immunofluorescent staining with anti-Girdin Ab showed that Girdin expression was very low in the Girdin siRNA-microinjected fertilized eggs.Staining of F-actin-rich structures with phalloidin revealed that The correct orientation of the filament is affected and can not be gathered at the contractile ring F-actin was disrupted iin the Girdin siRNA-microinjected mouse fertilized eggs.Moreover,F-actin rich structures lost their shape.The depletion of Girdin disrupted the rearrangement of the actin cytoskeleton in mouse eggs,resulting in abnormal cleavage and asymmetrical cytokinesis.These observations suggested that Girdin is essential for rearrangement of F-actin filaments.5.The Akt1/ Girdin pathway regulates rearrangement of the F-actin cytoskeleton of one-cell stage fertilized eggs We used Scansite software(http://scansite.mit.edu)and the Clustal W(1.83)multiple sequence alignment program(http://www.ebi.ac.uk/clustalw/)to predict relevant target sites of Akt1 on mouse Girdin.Using this approach,we found that mouse Girdin had a serine residue at position 1417(Ser 1417),which corresponded to Ser 1416 of human Girdin,which was also a residue that was phosphorylated by human Akt1.To gain insight into the role of phosphorylation of Girdin by Akt1,we assessed the location of phosphorylated Girdin in fertilized eggs by staining with anti-P-Girdin 1417 Ab.One-cell stage mouse embryos were first microinjected with Akt1-WT mRNA,myr-Akt1 mRNA or Akt1 siRNA,then stained with rhodamine-phalloidin and anti-PGirdin 1417 Ab.We found that the actin filaments and P-Girdin were clustered in the cell membrane and the cleavage furrow.We next asked whether anti-P-Girdin Ab could detect endogenously phosphorylated Girdin in response to activation by Akt1.We found that treatment with myr-Akt1 caused significant increases in phosphorylation of Girdin 1417,whereas only minor phosphorylation was seen with WT-Akt1.In addition,treatment with KD-Akt1 mRNA or siRNA-mediated Akt1 knockdown blocked phosphorylation of Girdin.These results strongly suggested that Akt1 could directly phosphorylate Girdin on Ser1417 and promoted its function in mouse fertilized eggs.In additional studies,we first microinjected one-cell stage eggs with myr-Akt1 mRNA,as mentioned above,followed by microinjected with Girdin siRNA.Embryos in each experiment were assessed by immunofluorescence confocal microscopy for their F-actin organization 20 h after microinjection.As anticipated,we detected the disrupted microfilament aggregation after microinjected of Akt1 mRNA and Girdin siRNA.Similar effects were seen in Girdin siRNA-microinjected groups.These findings indicate that Girdin and Akt1 mediated phosphorylation play major roles in the development of mouse fertilized eggs.Taken together,our observations indicate that the Akt1 / Girdin pathway regulates division of one-cell stage fertilized eggs.6.The mTORC2/ Akt1 / Girdin pathway rearranges the F-actin cytoskeleton in one-cell stage fertilized eggs. To further explore a role for m TOR complexes in the regulation of the Girdin,We examined Girdin phosphorylation in the Rictor knockdown fertilized eggs.In control fertilized eggs we noted that Girdin was highly phosphorylated at 1417.Mouse fertilized eggs treated with Rictor shRNA exhibited decreased phosphorylation of Girdin 1417.Thus,knockdown of mTORC2 prevented Girdin phosphorylation.Furthermore,we examined Girdin phosphorylation in the Rictor shRNA and WT-Akt1 m RNA injection fertilized eggs.In WT-Akt1 mRNA treated fertilized eggs,we noted that Girdin was highly phosphorylated at 1417.But fertilized eggs treated with Rictor si RNA and WT-Akt1 exhibited decreased phosphorylation of Girdin 1417.Our dates indicated that Akt1 and Girdin-mediated phosphorylation were affected by knockdown of mTORC2 and that mTORC2 may regulate Akt1 and Girdin in the development of mouse fertilized eggs.