摘要
目的磷脂酰肌醇3激酶(PI3Ks)通过调控肌动蛋白在细胞定向运动中发挥重要作用。然而,PI3Ks的结构和功能很复杂,人们对PI3Ks在细胞趋电性运动中的作用并不完全清楚。因此,本文以模式生物盘基网柄菌细胞为实验材料,探究其中的PI3K1和PI3K2在细胞趋电性运动中的作用。方法首先利用CRISPR/Cas9系统介导分别构建PI3K1编码基因pikA基因敲除突变株和PI3K2编码基因pikB基因敲除突变株;随后将2个突变株置于强度为12 V/cm的直流电场中,记录并分析两个突变株的趋电性。结果数据分析显示,野生型细胞在直流电场中的方向指数为(0.86±0.03),而pikA-和pikB-突变株在直流电场中的运动方向指数分别为(0.95±0.02)和(0.94±0.03);此外,野生型细胞在电场中的平均轨迹速度(3.34±0.08)μm/min,而pikA-和pikB-突变株的平均轨迹速度分别为(4.85±0.20)μm/min和(5.48±0.15)μm/min,t检验表明突变株和野生型的方向性指数和运动速度都存在极显著的差异。蛋白质印迹实验结果显示,pikA-和pikB-突变株中磷酸化Akt和磷酸化ERK都显著增加。结论PI3K1和PI3K2在盘基网柄菌细胞趋电性运动中可能通过增加Akt和ERK的活性发挥抑制作用。
Objective Phosphatidylinositol 3 kinases(PI3Ks)play an important role in cell directional movement by regulating F-actin.However,the structure and function of PI3Ks are complex.The role of PI3Ks in cell electrotaxis is not fully understood.Therefore,in this study,the model organism Dictyostelium discoideum cells were used as experimental materials to explore the role of PI3K1 and PI3K2 in electrotaxis.Methods Firstly,PI3K1 coding gene pikA knockout mutant and PI3K2 coding gene pikB knockout mutant were constructed by CRISPR/Cas9 system.Secondly,two mutants were placed in a DC electric field with a strength of 12 V/cm and the electrotaxis were analyzed.Results Data analysis showed that the direction index of wild-type cells in DC electric field was(0.86±0.03),while the direction index of pikA-and pikB-mutants in DC electric field was(0.95±0.02)and(0.94±0.03),respectively.In addition,the average trajectory speed of wild-type cells in the electric field was(3.34±0.08)μm/min,while the average trajectory speed of pikA-and pikB-mutants were(4.85±0.20)μm/min and(5.48±0.15)μm/min,respectively.The t test showed that there were significant differences in the directedness index and speed between the mutant and wild type.Western blot results showed that both phosphorylated Akt and phosphorylated ERK were significantly increased in pikA-and pikB-mutants.Conclusion PI3K1 and PI3K2 may inhibit the electrotaxis of Dictyostelium discoideum cells by increasing the activity of Akt and ERK.
作者
葛晓雪
蒋锐达
王晓燕
高晶
高润池
GE Xiao-Xue;JIANG Rui-Da;WANG Xiao-Yan;GAO Jing;GAO Run-Chi(School of Life Science,Yunnan Normal University,Kunming 650500,China;School of Agriculture,Yunnan University,Kunming 650500,China;Engineering Research Center of Sustainable Development and Utilization of Biomass Energy,Ministry of Education,Kunming 650500,China)
出处
《生物化学与生物物理进展》
SCIE
CAS
CSCD
北大核心
2024年第3期685-695,共11页
Progress In Biochemistry and Biophysics
基金
国家自然科学基金(32260224,31960144)资助项目。
