Tozeren A, Wu S, Hoxter B, Xu W, Adamson E D, Byers S W
Biomedical Engineering Program, Catholic University of America, Washington, DC 20064, USA.
Cell Adhes Commun. 1998 Jan;5(1):49-59. doi: 10.3109/15419069809005598.
Vinculin, a 117-kDa protein, is a constituent of adhesion plaques and adherence junctions in non-muscle cells. We investigated the role of vinculin on the physical strength of cell-cell adhesion by conducting disaggregation assays on aggregates of parental wild-type F9 mouse embryonal carcinoma cells (clone BIM), two vinculin-depleted F9 cell lines, gamma 227 and gamma 229, and a reconstituted gamma 229 cell line (R3) that re-express vinculin. Immunoblotting demonstrated that the four cell lines used in the study had similar expressions of the cell-cell adhesion molecule E-cadherin and associated membrane proteins alpha- and beta-catenin. Double immunofluorescence analysis showed that, in contrast to the vinculin-null cell lines. BIM and R3 cells expressed abundant vinculin at the cell margins in adhesion plaques and in cell-cell margins that also contained actin. Laminar flow assays showed that both the vinculin-positive and vinculin-negative cell aggregates that were formed in culture in the course of 24 to 48 hours largely remained intact despite the imposition of shear flow at high shear rates. Since laminar flow imposed on cell aggregates act to separate cells from each other, our data indicate that F9 cells that were adherent to a substrate formed strong cell-cell adhesion bonds independent of vinculin expression. On the other hand, aggregates of vinculin-depleted gamma 229 and gamma 227 cells that were formed in suspension during a two-hour static incubation at 37 degrees C were desegregated more easily with the imposition of shear flow than the BIM and R3 cell aggregates formed under identical conditions. Loss of vinculin was associated with a reduction in cell-cell adhesion strength only among those cells lacking contact to a substrate. Overall, the results indicate that vinculin is not needed for forming strong cell-cell adhesion bonds between neighboring carcinoma cells which are adherent to the basal lamina.
纽蛋白是一种117千道尔顿的蛋白质，是非肌肉细胞中黏着斑和黏附连接的组成成分。我们通过对亲代野生型F9小鼠胚胎癌细胞（克隆BIM）、两种纽蛋白缺失的F9细胞系（γ227和γ229）以及重新表达纽蛋白的重组γ229细胞系（R3）的聚集体进行解离分析，研究了纽蛋白对细胞间黏附物理强度的作用。免疫印迹表明，研究中使用的四种细胞系具有相似的细胞间黏附分子E-钙黏蛋白以及相关膜蛋白α-连环蛋白和β-连环蛋白的表达。双重免疫荧光分析显示，与纽蛋白缺失的细胞系相比，BIM和R3细胞在黏着斑的细胞边缘以及也含有肌动蛋白的细胞间边缘表达丰富的纽蛋白。层流分析表明，在24至48小时的培养过程中形成的纽蛋白阳性和纽蛋白阴性细胞聚集体，尽管在高剪切速率下施加了剪切流，但大部分仍保持完整。由于施加在细胞聚集体上的层流作用是使细胞彼此分离，我们的数据表明，附着于底物的F9细胞形成了独立于纽蛋白表达的强细胞间黏附键。另一方面，在37℃下两小时静态孵育期间在悬浮液中形成的纽蛋白缺失的γ229和γ227细胞聚集体，与在相同条件下形成的BIM和R3细胞聚集体相比，在施加剪切流时更容易解聚。仅在那些与底物缺乏接触的细胞中，纽蛋白的缺失与细胞间黏附强度的降低有关。总体而言，结果表明，对于附着于基膜的相邻癌细胞之间形成强细胞间黏附键，纽蛋白并非必需。
