Tranqui L, Usson Y, Marie C, Block M R
Laboratoire d'Etude des Systèmes Adhesifs Cellulaires, A.T.I.P.E. de l'URA 1178 du CNRS, Grenoble, France.
J Cell Sci. 1993 Sep;106 ( Pt 1):377-87. doi: 10.1242/jcs.106.1.377.
We have investigated the dynamics between free fibronectin receptors and clusters of them organized into adhesion plaques on CHO cells using the ability of these free integrins to be endocytosed and recycled to the plasma membrane. Indirect inhibition of the endocytic cycle by monensin resulted in the subsequent internalization of free receptors, which we followed by indirect immunostaining and confocal microscopy. Consequently, all the adhesive structures that were in equilibrium with free integrins became progressively disorganized. The cellular morphological changes were analyzed and correlated with the distribution of cell-substratum contacts viewed by confocal images obtained after immunostaining with antibodies raised against the fibronectin receptor, talin, vinculin and actin. After cell adhesion to fibronectin, blockage of the endocytic cycle induced disruption of the adhesion plaques that were mainly localized at the cell periphery, and disappearance of the stress fibers. However, the cells remained firmly attached to the substratum through focal contacts localized in the central part of the cell. These central focal contacts, but not the peripheral adhesion plaques, could form when the vesicular traffic was blocked prior to adhesion and they allowed the cells to attach and flatten onto the substratum. Whereas both adhesive structures contained the same receptors linked to talin and vinculin, the central adhesive structures were attached to a short stretch of actin but never permitted the organization of stress fibers.
我们利用这些游离整合素被内吞并循环至质膜的能力，研究了游离纤连蛋白受体与在CHO细胞上组织形成黏着斑的受体簇之间的动态变化。莫能菌素对内吞循环的间接抑制导致游离受体随后被内化，我们通过间接免疫染色和共聚焦显微镜对其进行追踪。因此，所有与游离整合素处于平衡状态的黏附结构逐渐变得紊乱。分析了细胞形态变化，并将其与用针对纤连蛋白受体、踝蛋白、纽蛋白和肌动蛋白的抗体进行免疫染色后通过共聚焦图像观察到的细胞-基质接触分布相关联。细胞黏附于纤连蛋白后，内吞循环的阻断导致主要位于细胞周边的黏着斑被破坏，应力纤维消失。然而，细胞通过位于细胞中央部分的黏着斑仍牢固地附着于基质。这些中央黏着斑，而非周边黏附斑，可在黏附前囊泡运输被阻断时形成，并且它们使细胞能够附着并铺展在基质上。虽然两种黏附结构都含有与踝蛋白和纽蛋白相连的相同受体，但中央黏附结构附着于一小段肌动蛋白，且从不允许应力纤维的形成。
