Department of Medical Biophysics, University of Toronto, Canada.
Exp Cell Res. 2011 Feb 15;317(4):539-51. doi: 10.1016/j.yexcr.2010.12.005. Epub 2010 Dec 11.
Numb is an endocytic adaptor protein that regulates internalization and post-endocytic trafficking of cell surface proteins. In polarized epithelial cells Numb is localized to the basolateral membrane, and recent work has implicated Numb in regulation of cell adhesion and migration, suggesting a role for Numb in epithelial-mesenchymal transition (EMT). We depleted MDCK cells of Numb and examined the effects downstream of EMT-promoting stimuli. While knockdown of Numb did not affect apicobasal polarity, we show that depletion of Numb destabilizes E-cadherin-based cell-cell adhesion and promotes loss of epithelial cell morphology. In addition, Numb knockdown in MDCK cells potentiates HGF-induced lamellipodia formation and cell dispersal. Examination of Rac1-GTP levels in Numb knockdown cells revealed hyperactivation of Rac1 following extracellular calcium depletion and HGF stimulation, which corresponds with enhanced loss of cell adhesions and lamellipodia formation. Furthermore, inhibition of Rac1 in Numb depleted cells stabilized cell-cell contacts following depletion of extracellular calcium. Together, these data indicate that Numb acts to suppress Rac1-GTP accumulation, and its loss leads to increased sensitivity toward extracellular signals that disrupt cell-cell adhesion to induce epithelial-mesenchymal transition (EMT) and cell dispersal.
Numb 是一种内吞衔接蛋白，可调节细胞表面蛋白的内化和内化后的转运。在极化的上皮细胞中，Numb 定位于基底外侧膜，最近的研究表明 Numb 参与了细胞黏附和迁移的调节，提示它在上皮间质转化（EMT）中发挥作用。我们敲低 MDCK 细胞中的 Numb 并检测 EMT 促进刺激的下游效应。虽然 Numb 的敲低不影响顶底极性，但我们发现 Numb 的耗竭会破坏基于 E-钙黏蛋白的细胞间黏附，并促进上皮细胞形态的丧失。此外，Numb 在 MDCK 细胞中的敲低会增强 HGF 诱导的片状伪足形成和细胞分散。检测 Numb 敲低细胞中的 Rac1-GTP 水平表明，细胞外钙耗竭和 HGF 刺激后 Rac1 的过度激活，与细胞黏附的增强丧失和片状伪足的形成相对应。此外，在细胞外钙耗竭后，抑制 Rac1 可稳定 Numb 耗尽细胞中的细胞间接触。总之，这些数据表明 Numb 抑制 Rac1-GTP 的积累，其缺失会导致对破坏细胞间黏附以诱导上皮间质转化（EMT）和细胞分散的细胞外信号的敏感性增加。
