Adducin promotes micrometer-scale organization of β2-spectrin in lateral membranes of bronchial epithelial cells
Adducin promotes micrometer-scale organization of β2-spectrin in lateral membranes of bronchial epithelial cells
复制标题
DOI：
10.1091/mbc.e07-08-0818
复制
发表时间：
2008-02-01
影响因子：
3.3
通讯作者：
Bennett, Vann
中科院分区：
文献类型：
--
作者：
Abdi, Khadar M.;Bennett, Vann
内收蛋白促进血影蛋白-肌动蛋白复合物的组装，并且是钙调蛋白、蛋白激酶C和rho激酶调节的靶标。我们在这里证明，内收蛋白是需要通过与β 2-血影蛋白的相互作用，以稳定人支气管上皮细胞（HBE）的预形成的侧膜。我们使用Tet-on调节的诱导型小干扰RNA（siRNA）系统从汇合的HBE细胞中耗尽α-内收蛋白。消耗α-内收蛋白导致在有丝分裂期间正常膜生物发生后血影蛋白的去污剂溶解度增加。相反，β 2-血影蛋白的耗竭导致侧膜内收蛋白的丢失。抗siRNA的α-内收蛋白阻止了侧膜的丧失，但前提是α-内收蛋白保留了介导血影蛋白-肌动蛋白相互作用的MARCKS结构域。在MARCKS结构域中的蛋白激酶C磷酸化位点处具有S/D取代的内收蛋白的拟磷酸化版本在拯救中不活跃。我们发现，内收蛋白调制远程组织的侧膜的基础上几个标准。首先，内收蛋白耗尽细胞的侧膜高度降低，曲率增加，并扩展到基底表面。此外，E-钙粘蛋白-GFP，这通常是有限的横向流动性，迅速扩散的距离高达10 μ m。我们的结论是，内收蛋白通过血影蛋白发挥作用，提供了一种新的机制来调节支气管上皮细胞侧膜的全球性。
Adducin promotes assembly of spectrin-actin complexes, and is a target for regulation by calmodulin, protein kinase C, and rho kinase. We demonstrate here that adducin is required to stabilize preformed lateral membranes of human bronchial epithelial (HBE) cells through interaction with beta 2-spectrin. We use a Tet-on regulated inducible small interfering RNA (siRNA) system to deplete alpha-adducin from confluent HBE cells. Depletion of alpha-adducin resulted in increased detergent solubility of spectrin after normal membrane biogenesis during mitosis. Conversely, depletion of beta 2-spectrin resulted in loss of adducin from the lateral membrane. siRNA-resistant alpha-adducin prevented loss of lateral membrane, but only if alpha-adducin retained the MARCKS domain that mediates spectrin-actin interactions. Phosphomimetic versions of adducin with S/D substitutions at protein kinase C phosphorylation sites in the MARCKS domain were not active in rescue. We find that adducin modulates long-range organization of the lateral membrane based on several criteria. First, the lateral membrane of adducin-depleted cells exhibited reduced height, increased curvature, and expansion into the basal surface. Moreover, E-cadherin-GFP, which normally is restricted in lateral mobility, rapidly diffuses over distances up to 10 mu m. We conclude that adducin acting through spectrin provides a novel mechanism to regulate global properties of the lateral membrane of bronchial epithelial cells.