The fibronectin cell attachment sequence Arg-Gly-Asp-Ser promotes focal contact formation during early fibroblast attachment and spreading.
The fibronectin cell attachment sequence Arg-Gly-Asp-Ser promotes focal contact formation during early fibroblast attachment and spreading.
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DOI：
10.1083/jcb.104.3.573
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发表时间：
1987-03
期刊：
影响因子：
--
通讯作者：
Lark MW
中科院分区：
文献类型：
--
作者：
Singer II;Kawka DW;Scott S;Mumford RA;Lark MW
培养的成纤维细胞在血清或纤连蛋白（FN）包被的基质上附着和铺展期间形成与肌动蛋白微丝束（MFB）相关的焦点接触（FC）。为了确定最小细胞粘附受体识别信号Arg-Gly-Asp-Ser（RGDS）是否足以促进FC和MFB形成，将无血清培养基中的大鼠（NRK）、仓鼠（Nil 8）和小鼠（Balb/c 3 T3）成纤维细胞铺在用含有RGDS的小合成肽衍生的基底上。这些文化进行了研究，用干涉反射显微镜检测FC，Normarski光学识别MFB，和免疫荧光显微镜观察内源性FN纤维的形成。到1小时，72-78%的接种在含RGDS肽上的NRK和Nil 8细胞具有没有伴随FN纤维的病灶接触，而这些成纤维细胞在对照肽上缺乏FC。早期FC形成之后，在4小时出现一致的MFB和共线FN纤维形成纤维连接。在用天然FN或75，000-D FN-细胞结合片段包被的基底上的NRK和Nil 8培养物显示出FC和MFB形成的相似动力学。相比之下，Balb/c 3 T3小鼠成纤维细胞接种在Gly-Arg-Gly-Asp-Ser肽衍生的底物上，或覆盖有75，000-D FN细胞结合片段的盖玻片上，在FC形成方面有缺陷。这些结果表明，底物连接的RGDS序列与细胞表面粘附受体的表观结合足以促进早期局灶性接触形成，随后在一些但不是所有成纤维细胞系中出现纤连蛋白。
Cultured fibroblasts form focal contacts (FCs) associated with actin microfilament bundles (MFBs) during attachment and spreading on serum- or fibronectin (FN)-coated substrates. To determine if the minimum cellular adhesion receptor recognition signal Arg-Gly-Asp-Ser (RGDS) is sufficient to promote FC and MFB formation, rat (NRK), hamster (Nil 8), and mouse (Balb/c 3T3) fibroblasts in serum-free media were plated on substrates derivatized with small synthetic peptides containing RGDS. These cultures were studied with interference reflection microscopy to detect FCs, Normarski optics to identify MFBs, and immunofluorescence microscopy to observe endogenous FN fiber formation. By 1 h, 72-78% of the NRK and Nil 8 cells plated on RGDS-containing peptide had focal contacts without accompanying FN fibers, while these fibroblasts lacked FCs on control peptide. This early FC formation was followed by the appearance of coincident MFBs and colinear FN fibers forming fibronexuses at 4 h. NRK and Nil 8 cultures on substrates coated with native FN or 75,000-D FN-cell binding fragment showed similar kinetics of FC and MFB formation. In contrast, the Balb/c 3T3 mouse fibroblasts plated on Gly-Arg-Gly-Asp-Ser peptide-derivatized substrates, or on coverslips coated with 75,000-D FN cell-binding fragment, were defective in FC formation. These results demonstrate that the apparent binding of substrate-linked RGDS sequences to cell surface adhesion receptors is sufficient to promote early focal contact formation followed by the appearance of fibronexuses in some, but not all, fibroblast lines.