Department of Physiology and Biophysics, Boston University School of Medicine, Boston, MA 02118-2518.
Mol Biol Cell. 2013 Sep;24(18):2820-33. doi: 10.1091/mbc.E12-12-0884. Epub 2013 Jul 17.
Cooperation between cadherins and the actin cytoskeleton controls the formation and maintenance of cell-cell adhesions in epithelia. We find that the molecular motor protein myosin-1c (Myo1c) regulates the dynamic stability of E-cadherin-based cell-cell contacts. In Myo1c-depleted Madin-Darby canine kidney cells, E-cadherin localization was dis-organized and lateral membranes appeared less vertical with convoluted edges versus control cells. In polarized monolayers, Myo1c-knockdown (KD) cells were more sensitive to reduced calcium concentration. Myo1c separated in the same plasma membrane fractions as E-cadherin, and Myo1c KD caused a significant reduction in the amount of E-cadherin recovered in one peak fraction. Expression of green fluorescent protein (GFP)-Myo1c mutants revealed that the phosphatidylinositol-4,5-bisphosphate-binding site is necessary for its localization to cell-cell adhesions, and fluorescence recovery after photobleaching assays with GFP-Myo1c mutants revealed that motor function was important for Myo1c dynamics at these sites. At 18°C, which inhibits vesicle recycling, Myo1c-KD cells accumulated more E-cadherin-positive vesicles in their cytoplasm, suggesting that Myo1c affects E-cadherin endocytosis. Studies with photoactivatable GFP-E-cadherin showed that Myo1c KD reduced the stability of E-cadherin at cell-cell adhesions. We conclude that Myo1c stabilizes E-cadherin at adherens junctions in polarized epithelial cells and that the motor function and ability of Myo1c to bind membrane are critical.
钙黏蛋白与肌动蛋白细胞骨架的相互作用控制着上皮细胞中细胞-细胞黏附的形成和维持。我们发现，分子马达蛋白肌球蛋白-1c（Myo1c）调节着基于 E-钙黏蛋白的细胞-细胞接触的动态稳定性。在 Myo1c 耗尽的 Madin-Darby 犬肾细胞中，E-钙黏蛋白的定位变得紊乱，与对照细胞相比，侧向膜看起来不那么垂直，边缘呈卷曲状。在极化的单层细胞中，Myo1c 敲低（KD）细胞对钙离子浓度降低更为敏感。Myo1c 与 E-钙黏蛋白分离在相同的质膜部分，Myo1c KD 导致回收的 E-钙黏蛋白量在一个峰部分显著减少。绿色荧光蛋白（GFP）-Myo1c 突变体的表达表明，磷酸肌醇-4,5-二磷酸结合位点对于其在细胞-细胞黏附处的定位是必需的，并且 GFP-Myo1c 突变体的光漂白后荧光恢复测定表明，马达功能对于这些位点的 Myo1c 动力学很重要。在 18°C 下，它抑制囊泡再循环，Myo1c-KD 细胞在其细胞质中积累了更多的 E-钙黏蛋白阳性囊泡，这表明 Myo1c 影响 E-钙黏蛋白的内吞作用。用光活化 GFP-E-钙黏蛋白进行的研究表明，Myo1c KD 降低了 E-钙黏蛋白在黏附连接点的稳定性。我们得出结论，Myo1c 在上皮细胞极化过程中稳定了黏着连接点处的 E-钙黏蛋白，并且 Myo1c 的运动功能和结合膜的能力是至关重要的。
