Yu C F, Basson M D
Departments of Surgery, Yale University School of Medicine and CT VA Health Care System, New Haven, Connecticut 06511, USA.
Microsc Res Tech. 2000 Oct 15;51(2):191-203. doi: 10.1002/1097-0029(20001015)51:2<191::AID-JEMT10>3.0.CO;2-1.
We have previously reported that Caco-2 cell motility redistributes FAK, paxillin, and activates p38. However, the subcellular organization of these intracellular signals during cell migration is unclear. We, therefore, investigated the organization of actin, FAK, paxillin, and activated ERK and activated p38 during Caco-2 motility across collagen I, fibronectin, laminin, and tissue culture treated glass. Differential density seeding generated homogeneous static and migrating populations. Expression of actin, FAK, paxillin, phospho-ERK, and phospho-p38 were examined by immunofluorescent staining in static and motile cells. Actin was concentrated toward the peri-nuclear central area of cells migrating on matrix proteins studied. Actin immunoreactivity was decreased in the leading edge of lamellipodia. FAK immunoreactivity was weaker in migrating cells than in static cells on the same matrix. FAK was expressed along cell-cell contacts of both cell populations, but absent in migrating lamellipodia of matrix-cultured cells. Paxillin staining was diffuse in static cells but organized toward migrating lamellipodia in a radial manner. Like FAK, phosphorylated ERK was expressed in the central region of migrating cells but was dramatically decreased at areas of cell-cell contact and free lamellipodia. Fibronectin exerted the greatest effect on ERK activation in all matrix proteins studied. In contrast, phosphorylated p38 staining was stronger in migrating cells on matrix than in static cells on the same matrix. Phosphorylated p38 was expressed in the nuclear of migrating cells and disappeared in the cell-cell contact side and free lamellipodia. Interestingly, the reorganization of these proteins was distinctly different on tissue culture treated glass without a physiologic matrix substrate. For instance, FAK staining increased rather than decreased in motile cells on plastic, and lamellipodial FAK staining could be discerned. Matrix may influence Caco-2 biology during migration not only by triggering intracellular phosphorylation events but also by reorganizing the cytoskeleton and the subcellular localization of these intracellular signals.
我们之前报道过，Caco-2细胞的迁移会使黏着斑激酶（FAK）、桩蛋白重新分布，并激活p38。然而，在细胞迁移过程中这些细胞内信号的亚细胞组织尚不清楚。因此，我们研究了Caco-2细胞在跨I型胶原、纤连蛋白、层粘连蛋白以及经组织培养处理的玻璃迁移过程中，肌动蛋白、FAK、桩蛋白、活化的细胞外信号调节激酶（ERK）和活化的p38的组织情况。差异密度接种产生了均匀的静态和迁移细胞群体。通过免疫荧光染色检测静态和迁移细胞中肌动蛋白、FAK、桩蛋白、磷酸化ERK和磷酸化p38的表达。在研究的基质蛋白上迁移的细胞中，肌动蛋白集中在细胞核周围的中央区域。片状伪足前沿的肌动蛋白免疫反应性降低。在相同基质上，迁移细胞中的FAK免疫反应性比静态细胞中的弱。FAK在两种细胞群体的细胞间接触部位均有表达，但在基质培养细胞的迁移片状伪足中不存在。桩蛋白染色在静态细胞中呈弥漫性，但以放射状方式向迁移的片状伪足聚集。与FAK一样，磷酸化的ERK在迁移细胞的中央区域表达，但在细胞间接触部位和游离片状伪足处显著减少。在所有研究的基质蛋白中，纤连蛋白对ERK激活的作用最大。相比之下，磷酸化p38染色在基质上的迁移细胞中比在相同基质上的静态细胞中更强。磷酸化p38在迁移细胞的细胞核中表达，在细胞间接触部位和游离片状伪足处消失。有趣的是，在没有生理基质底物的经组织培养处理的玻璃上，这些蛋白质的重新组织明显不同。例如，在塑料上的迁移细胞中FAK染色增加而非减少，并且可以辨别出片状伪足处的FAK染色。基质可能不仅通过触发细胞内磷酸化事件，还通过重组细胞骨架和这些细胞内信号的亚细胞定位来影响Caco-2细胞在迁移过程中的生物学行为。
