Physiology and Developmental Biology Brigham Young University, 574 WIDB Provo, UT 84602, USA.
Physiology and Developmental Biology Brigham Young University, 574 WIDB Provo, UT 84602, USA.
Exp Cell Res. 2014 Aug 15;326(2):187-200. doi: 10.1016/j.yexcr.2014.04.011. Epub 2014 Apr 26.
Epithelial scattering occurs when cells disassemble cell-cell junctions, allowing individual epithelial cells to act in a solitary manner. Epithelial scattering occurs frequently in development, where it accompanies epithelial-mesenchymal transitions and is required for individual cells to migrate and invade. While migration and invasion have received extensive research focus, how cell-cell junctions are detached remains poorly understood. An open debate has been whether disruption of cell-cell interactions occurs by remodeling of cell-cell adhesions, increased traction forces through cell substrate adhesions, or some combination of both processes. Here we seek to examine how changes in adhesion and contractility are coupled to drive detachment of individual epithelial cells during hepatocyte growth factor (HGF)/scatter factor-induced EMT. We find that HGF signaling does not alter the strength of cell-cell adhesion between cells in suspension, suggesting that changes in cell-cell adhesion strength might not accompany epithelial scattering. Instead, cell-substrate adhesion seems to play a bigger role, as cell-substrate adhesions are stronger in cells treated with HGF and since rapid scattering in cells treated with HGF and TGFβ is associated with a dramatic increase in focal adhesions. Increases in the pliability of the substratum, reducing cells ability to generate traction on the substrate, alter cells׳ ability to scatter. Further consistent with changes in substrate adhesion being required for cell-cell detachment during EMT, scattering is impaired in cells expressing both active and inactive RhoA mutants, though in different ways. In addition to its roles in driving assembly of both stress fibers and focal adhesions, RhoA also generates myosin-based contractility in cells. We therefore sought to examine how RhoA-dependent contractility contributes to cell-cell detachment. Inhibition of Rho kinase or myosin II induces the same effect on cells, namely an inhibition of cell scattering following HGF treatment. Interestingly, restoration of myosin-based contractility in blebbistatin-treated cells results in cell scattering, including global actin rearrangements. Scattering is reminiscent of HGF-induced epithelial scattering without a concomitant increase in cell migration or decrease in adhesion strength. This scattering is dependent on RhoA, as blebbistatin-induced scattering is reduced in cells expressing dominant-negative RhoA mutants. This suggests that induction of myosin-based cellular contractility may be sufficient for cell-cell detachment during epithelial scattering.
上皮细胞的散射发生在细胞分离细胞-细胞连接时，允许单个上皮细胞以单独的方式起作用。上皮细胞的散射在发育过程中经常发生，它伴随着上皮-间质转化，并且是单个细胞迁移和入侵所必需的。虽然迁移和入侵受到了广泛的研究关注，但细胞-细胞连接的分离仍然知之甚少。一个悬而未决的争论是，细胞-细胞相互作用的破坏是通过细胞-细胞黏附的重塑、通过细胞基质黏附的牵引力增加还是这两个过程的某种组合来发生的。在这里，我们试图研究在肝细胞生长因子 (HGF)/散射因子诱导的 EMT 过程中，细胞黏附力和收缩力的变化如何偶联以驱动单个上皮细胞的分离。我们发现，HGF 信号不改变悬浮细胞之间细胞-细胞黏附的强度，这表明细胞-细胞黏附强度的变化可能不会伴随上皮细胞的散射。相反，细胞-基质黏附似乎起更大的作用，因为用 HGF 处理的细胞中的细胞-基质黏附更强，并且由于用 HGF 和 TGFβ 处理的细胞中快速散射与粘着斑的急剧增加相关联。基底的柔韧性增加，降低了细胞在基质上产生牵引力的能力，改变了细胞的散射能力。进一步与 EMT 过程中细胞-细胞分离所需的细胞基质黏附的变化一致，在表达活性和非活性 RhoA 突变体的细胞中，散射受到损害，尽管方式不同。除了在驱动应力纤维和粘着斑的组装中的作用外，RhoA 还在细胞中产生肌球蛋白依赖性收缩力。因此，我们试图研究 RhoA 依赖性收缩力如何促进细胞-细胞分离。Rho 激酶或肌球蛋白 II 的抑制对细胞产生相同的效果，即在 HGF 处理后抑制细胞散射。有趣的是，在 blebbistatin 处理的细胞中恢复肌球蛋白依赖性收缩力会导致细胞散射，包括全局肌动蛋白重排。散射类似于 HGF 诱导的上皮细胞散射，而没有伴随的细胞迁移增加或黏附力降低。这种散射依赖于 RhoA，因为 blebbistatin 诱导的散射在表达显性负性 RhoA 突变体的细胞中减少。这表明诱导肌球蛋白依赖性细胞收缩力可能足以在上皮细胞散射过程中分离细胞。
