Levenberg S, Katz B Z, Yamada K M, Geiger B
Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot 76100, Israel.
J Cell Sci. 1998 Feb;111 ( Pt 3):347-57. doi: 10.1242/jcs.111.3.347.
In this study we demonstrate that local stimulation of cell surface cadherins or integrins induces a selective enhancement of adherens junction or focal contact assembly, respectively, throughout the cell. N-cadherin transfected CHO cells (CHO-Ncad) were incubated with different ligands including N-cadherin extracellular domain (NEC), anti-N-cadherin antibodies, fibronectin and concanavalin A (ConA), conjugated to synthetic beads. Electron microscopic examination indicated that both cadherin- and integrin-reactive beads bound tightly to the cell surface and were apparently endocytosed after several hours of incubation. The ConA-beads remained largely at the cell surface. Immunofluorescence labeling of the cells with antibodies to different adhesion-associated molecules indicated that both NEC- and anti-N-cadherin-conjugated beads induced a major increase in the level of junction-associated cadherin and beta-catenin labeling and a modest increase in junctional vinculin labeling, compared to untreated cells or cells bound to ConA-beads. FN-conjugated beads, on the other hand, significantly enhanced vinculin labeling at focal contacts and suppressed cadherin and beta-catenin staining in cell-cell junctions. The cadherin-reactive beads specifically stimulated tyrosine phosphorylation at cell-cell junctions, while the FN-beads increased the levels of focal contact-associated phosphotyrosine, as shown by immunofluorescence labeling of the cells for phosphotyrosine. Inhibition of this phosphorylation by genistein resulted in a complete suppression of the effects of both types of beads. These findings indicate that specific cadherin- and integrin-mediated surface interactions can trigger positively cooperative long-range signaling events which lead to the selective assembly of cell-cell or cell-matrix adhesions, and that these signals involve tyrosine phosphorylation.
在本研究中，我们证明局部刺激细胞表面钙黏着蛋白或整合素分别会在整个细胞中选择性增强黏着连接或黏着斑的组装。将转染了N-钙黏着蛋白的CHO细胞（CHO-Ncad）与不同的配体一起孵育，这些配体包括与合成珠结合的N-钙黏着蛋白胞外结构域（NEC）、抗N-钙黏着蛋白抗体、纤连蛋白和伴刀豆球蛋白A（ConA）。电子显微镜检查表明，与钙黏着蛋白和整合素反应的珠子都紧密结合在细胞表面，并且在孵育数小时后明显被内吞。ConA珠主要留在细胞表面。用针对不同黏附相关分子的抗体对细胞进行免疫荧光标记表明，与未处理的细胞或与ConA珠结合的细胞相比，NEC和抗N-钙黏着蛋白结合的珠子都导致连接相关钙黏着蛋白和β-连环蛋白标记水平大幅增加，连接斑蛋白标记略有增加。另一方面，纤连蛋白结合的珠子显著增强了黏着斑处的斑蛋白标记，并抑制了细胞间连接中钙黏着蛋白和β-连环蛋白的染色。如对细胞进行磷酸酪氨酸免疫荧光标记所示，与钙黏着蛋白反应的珠子特异性刺激细胞间连接处的酪氨酸磷酸化，而纤连蛋白珠增加了黏着斑相关磷酸酪氨酸的水平。金雀异黄素对这种磷酸化的抑制导致两种类型珠子的作用完全被抑制。这些发现表明，特定的钙黏着蛋白和整合素介导的表面相互作用可以触发正向协同的远程信号事件，从而导致细胞间或细胞-基质黏附的选择性组装，并且这些信号涉及酪氨酸磷酸化。
