gbpC和gbpD基因在盘基网柄菌细胞趋化性和趋电性运动中的差异研究

目的 趋化性和趋电性是细胞定向迁移的主要方式,并在生物有机体的生理和病理过程中发挥重要作用,但二者存在差异。本文对盘基网柄菌gbpC和gbpD基因在细胞趋电性和趋化性中的作用进行对比研究,以寻找两种迁移方式之间的新差异。方法 将gbpC基因突变株gefT-和gbpD基因突变株gefU-分别置于场强为12 V/cm的直流电场中,分析细胞在电场中的运动方向及运动速度,探讨细胞的趋电性变化;利用电穿孔技术将标记F-actin的Lifeact-GFP质粒转化进入细胞,用荧光显微镜观察活细胞运动时F-actin的分布;用蛋白质印迹技术定量分析细胞的肌球蛋白调节轻链（RLC）在受直流电场刺激后的磷酸化变化情况。结果 gefT-突变株细胞极化消失,但保持与野生型类似的趋电性;gefU-突变株细胞发生超极化,但趋电性显著降低。在直流电场中,突变株细胞和野生型细胞的F-actin主要分布在伪足部位。在电场作用下,细胞株的肌球蛋白RLC磷酸化变化情况存在差异,即野生型细胞以时间依赖的方式发生磷酸化,gefT-突变株细胞先急剧下降,然后再上升,gefU-突变株细胞却以时间依赖方式脱磷酸化。结论 本研究表明gbpC和gbpD基因在盘基网柄菌趋化性和趋电性中的作用不同,暗示了电信号与化学信号确实通过不同的机理指导细胞的定向迁移。

gbpC and gbpD Have Differential Effects on Chemotaxis and Electrotaxis in Dictyostelium

Objective Chemotaxis and electrotaxis are the primary mechanisms underlying directed cell migration, and they play important roles in the physiology and pathology of organisms. However, there are differences between the two. This paper presents a comparative study of the roles of Dictyostelium discoideum genes gbpC and gbpD in cell electrotaxis and chemotaxis in order to gain further insight into the differences between these two mechanisms of migration.Methods The gbpC/gefT- mutant strain and gbpD/gefU- mutant strain were placed in a 12 V/cm direct current (DC) electric field to investigate the direction and velocity of cell movement and the changes in cell electrotaxis; Lifeact-GFP (F-actin) was electroporated into cells and the distribution of F-actin during cell movement was observed under a fluorescence microscope; Western blot was used to quantify the phosphorylation of myosin regulatory light chain (RLC) in cells stimulated by DC electric fields.Results The gefT- mutant cells lost polarization but retained electrotaxis at a level similar to the wild-type cells; the gefU- mutant cells showed hyperpolarization but significantly reduced electrotaxis; in a DC field, F-actin was predominantly distributed in the pseudopods in both mutant and wild-type cells; there were differences in myosin RLC phosphorylation between the cell lines in electric fields. Phosphorylation was time-dependent in wild-type cells, whereas phosphorylation first decreased rapidly and then increased in gefT- mutant cells. Time-dependent dephosphorylation occurred in gefU- mutant cells.Conclusion Our findings indicate that gbpC and gbpD play differntal roles between the chemotaxis and electrotaxis of Dictyostelium discoideum and provide further evidence that electrical and chemical signals drive the directed migration of cells through different mechanisms.