ANLN缺失后Hela细胞的力学特性与骨架蛋白变化分析

Mechanical properties and cytoskeletal protein changes after ANLN deficiency

背景:目前关于ANLN蛋白在细胞分裂间期调节细胞形态、细胞-细胞间接完整性以及胞质分裂中稳定收缩环的作用已经明确,但其对细胞力学性能和骨架蛋白的影响还鲜有报道。目的:探讨ANLN缺失对分裂间期细胞力学特性与骨架蛋白的影响。方法:通过原子力显微镜分别测量正常Hela细胞与siRNA敲降ANLN的Hela细胞的表面弹性模量和破膜力;采用激光共聚焦显微镜观察正常Hela细胞与siRNA敲降ANLN的Hela细胞的肌球蛋白ⅡA以及肌动蛋白纤维分布特征。结果与结论:①敲降ANLN的Hela细胞的表面弹性模量明显高于未经处理的正常Hela细胞,与敲降ANLN的细胞相比,正常细胞的表面弹性模量更倾向于极性分布(两极间逐步降低),但两组细胞长轴边缘区域的破膜力并没有明显差别;②ANLN敲降组细胞在边缘位置有较低的肌球蛋白ⅡA分布;③ANLN敲降组近底层细胞-细胞连接区域的肌动蛋白纤维趋向更散乱,并且细胞内纤维束沿长轴排列不明显,中层有细胞间隙变大的倾向;④结果说明,敲降ANLN对细胞的边缘区域影响最大,ANLN的缺失会导致细胞边缘区域更频繁的重构,细胞需要聚集更多的骨架蛋白及其结合蛋白来稳定细胞状态,导致了更高的表面弹性模量。

BACKGROUND:To date,ANLN has definite roles in altering cell shape,regulating cell-cell junction integrity in interphase and stabilizing actomyosin contractile rings in cytokinesis,but its effects on cell mechanical properties and on cytoskeletal proteins have rarely been reported.OBJECTIVE:To investigate the effect of ANLN deletion on the mechanical properties and cytoskeleton of interphase Hela cells.METHODS:Surface elastic modulus and membrane rupture force of normal untreated Hela cells and ANLN RNA stably knocked down Hela cells were measured by atomic force microscopy.We screened for the cells that stably expressed mCherry-Myosin II A,and observed the distribution characteristics of cytoskeletal proteins by laser scanning confocal microscopy.RESULTS AND CONCLUSION:(1)The elastic modulus of Hela cells with ANLN stably knocked down was significantly higher than that of normal Hela cells,and the elastic modulus of normal cells were more prone to polar distribution(gradually decreasing between the two poles)than that of ANLN knockdown Hela cells.However,there was no significant difference in the membrane rupture force at the long-axis edge region between the two groups.(2)Myosin IIA lowly expressed in the marginal region of ANLN knockdown cells.(3)The actin fibers tended to be scattered in the near-bottom cell-cell junction region of the ANLN knockdown group,and there were no obvious intracellular fibers bundles arranging along the long axis.The cell gap tended to enlarge in the middle layer.To conclude,ANLN knockdown cells have the greatest impact in the marginal region,the deficiency of ANLN leads to a more frequent remodeling in the cell marginal region,and the cells need to accumulate more cytoskeletal proteins and binding proteins to stabilize the cell state,resulting in higher modulus of elastics.