Nebe B, Bohn W, Sanftleben H, Rychly J
Department of Internal Medicine, University of Rostock, Germany.
Exp Cell Res. 1996 Nov 25;229(1):100-10. doi: 10.1006/excr.1996.0348.
In most cases epithelial cells reveal a polarized distribution of integrin receptors in basolateral domains of the plasma membrane. To evaluate the functional state of integrin receptors in these restricted sites we were interested in the physical association of integrins with the cytoskeleton. Basically, we extracted cells with Triton X-100 to obtain detergent insoluble cytoskeleton fractions and used monoclonal antibodies for the detection of integrins linked to the cytoskeleton. We found that no permanent physical integrin-cytoskeleton associations exist in a confluent culture of the hepatocyte cell line mHepR1. However, incubation with anti-integrin antibodies and cross linking with a secondary antibody induced a physical linkage of beta1 as well as of different alpha subunits to the cytoskeleton. The association of integrins with the cytoskeleton was also inducible in suspended cells, which was detected in flow cytometric analyses and indicates that the formation of a physical integrin-cytoskeleton connection is independent of the localization of integrins, cell shape, and adhesion on a substrate. Using the Ca2+ chelators BAPTA-AM and EGTA, we found that intracellular calcium is a necessary prerequisite to induce a connection of integrins to the cytoskeleton. ATP or tauroursodeoxycholic acid, which provoke an intracellular calcium elevation, partly induced the formation of an integrin-cytoskeleton linkage. These results indicate the obvious role of intracellular calcium in integrin-dependent outside-in as well as inside-out signaling.
在大多数情况下，上皮细胞的整合素受体在质膜的基底外侧区域呈极化分布。为了评估这些受限位点处整合素受体的功能状态，我们对整合素与细胞骨架的物理关联感兴趣。基本上，我们用Triton X-100提取细胞以获得去污剂不溶性细胞骨架组分，并使用单克隆抗体检测与细胞骨架相连的整合素。我们发现，在肝细胞系mHepR1的汇合培养物中不存在永久性的整合素-细胞骨架物理关联。然而，用抗整合素抗体孵育并与二抗交联可诱导β1以及不同α亚基与细胞骨架发生物理连接。整合素与细胞骨架的关联在悬浮细胞中也可诱导，这在流式细胞术分析中得以检测，表明整合素-细胞骨架物理连接的形成与整合素的定位、细胞形状以及在底物上的黏附无关。使用Ca2+螯合剂BAPTA-AM和EGTA，我们发现细胞内钙是诱导整合素与细胞骨架连接的必要前提。ATP或牛磺熊去氧胆酸可引起细胞内钙升高，部分诱导整合素-细胞骨架连接的形成。这些结果表明细胞内钙在整合素依赖性的外向内以及内向内信号传导中具有明显作用。
