The Role Of PI3K/Akt/ERK Signaling Pathway In The Electrotaxis In Dictyostelium Discoideum

Cell migration plays an important role in physiological and pathological processes such as embryonic development,tissue formation,wound healing,and cancer.The phenomenon that cells migrate toward the positive or negative electrode of the electric field under direct current stimulation is called cell electrotaxis.However,the mechanism of cell response to electric field is not clear.Using the model organism Dictyostelium discoideum as the research object,it has been found that the electrotaxis of cells is regulated by genes and proteins.In this project,mitogen-activated protein kinase(MAPK)and phosphalyl inositol-3-kinase(PI3K)in Dictyostelium discoideum were used as the research objects.CRISPR/Cas9 gene editing technology was used to knock out their coding genes,and the relationship between genes and cell electrotaxis was explored.The wild-type cells AX2 and erk A~-mutants were placed in a DC electric field with an electric field intensity of 12 V/cm,respectively.The cell trajectory within 30min was recorded by a real-time video system,and the cell movement was analyzed by analysis software.The results showed that the Directedness of AX2 in wild-type cells was 0.85±0.02,the Euclidian Persistence was 0.36±0.01,the Speed was 3.34±0.08μm/min,and the Velocity was 1.26±0.05μm/min.The Directedness of erk A~-mutant was 0.89±0.01,the Euclidian Persistence index was 0.43±0.01(P<0.05,n=200),the Speed was 4.36±0.09μm/min,and the Velocity was 1.9±0.06μm/min(P<0.001,n=200).The above results showed that erk A did not affect the direction of cell movement in the electric field.The wild-type cells and the mutant cells were placed in a DC electric field with an electric field intensity of 12 V/cm,respectively.After recording the movement trajectory of the cells within 30 min with a real-time video recording system,the cell movement was statistically analyzed using analysis software.The results showed that the mutant also migrated towards the negative electrode of the electric field in the DC electric field.Among them,the Directedness of the pik A~-mutant was 0.95±0.01(P<0.05,n=200),the Euclidian Persistence was 0.60±0.01(P<0.001,n=200),the Speed was 4.85±0.20μm/min(P<0.001,n=200),and the Velocity was 3.04±0.17μm/min(P<0.001,n=200);the Directedness of pik B~-mutant was 0.94±0.01(P<0.05,n=200),the Euclidian Persistence was 0.62±0.01(P<0.001,n=200),the Speed was 5.48±0.15μm/min(P<0.001,n=200),and the Velocity was 3.46±0.13μm/min(P<0.001,n=200).The Directedness of pik C~-mutant was 0.53±0.04(P<0.001,n=200),the Euclidian Persistence was 0.32±0.01,the Speed was 4.57±0.10μm/min(P<0.001,n=200),and the Velocity was 1.54±0.05μm/min(P<0.01,n=200).The Directedness of pik F~-mutant was 0.62±0.04(P<0.01,n=200),the Euclidian Persistence was 0.44±0.01(P<0.01,n=200),the Speed was 4.38±0.12μm/min(P<0.001,n=200),and the Velocity was 1.97±0.07μm/min(P<0.001,n=200).The Directedness of pik G~-cells was 0.74±0.02,the Euclidian Persistence was 0.43±0.01(P<0.01,n=200),the Speed was 2.91±0.08μm/min(P<0.05,n=200),and the Velocity was 1.35±0.06μm/min.The Directedness of pik H~-cells was 0.89±0.01(P<0.001,n=200),the Euclidian Persistence was 0.47±0.01μm/min(P<0.001,n=200),and Speed was 5.11±0.13μm/min(P<0.001,n=200),the Velocity was 2.49±0.09μm/min(P<0.001,n=200).The above results indicate that the six PI3K coding genes play different roles in the electrotaxis of Dictyostelium discoideum.In order to explore the role of PI3K/Akt/ERK signaling pathways in cell electrotaxis,the expression of ERK and Akt in mutant strains was investigated by Western Blot.The results showed that the expression of p-Akt in erk A~-mutant cells was increased compared with that in wild-type cells.The expression of p-Akt in pik A~-and pik B~-mutants was also significantly higher than that in wild-type cells,and the expression of p-ERKB in pik A~-and pik B~-mutants was significantly increased.The above results indicate that the high expression of p-Akt in the mutant promotes cell movement,and the high expression of p-ERKB in the mutant promotes cell response to the electric field.Conclusion:The two MAPK encoding genes in Dictyostelium discoideum have different effects on cell electrotaxis;the six genes encoding PI3K play different roles in cell electrotaxis,among which pik A and pik B inhibit cell response to electric field and cell movement speed.