Regulation of the small GTP-binding protein Rho by cell adhesion and the cytoskeleton
Regulation of the small GTP-binding protein Rho by cell adhesion and the cytoskeleton
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DOI：
10.1093/emboj/18.3.578
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发表时间：
1999-02-01
期刊：
影响因子：
11.4
通讯作者：
Schwartz, MA
中科院分区：
文献类型：
--
作者：
Ren, XD;Kiosses, WB;Schwartz, MA
来自血清的可溶性因子如溶血磷脂酸（LPA）被认为基于它们以Rho依赖性方式诱导肌动蛋白应力纤维和粘着斑的能力来激活小的GTP结合蛋白Rho。细胞粘附到细胞外基质（ECM）也被认为是激活Rho，但这一点一直存在争议，因为很难区分Rho活性的变化与ECM对粘着斑形成的结构贡献。为了解决这些问题，我们建立了一种测定GTP结合的细胞Rho的方法，将Swiss 3T3细胞接种在纤连蛋白包被的培养皿上引起Rho的短暂抑制，随后是Rho激活的阶段。血清大大增强了激活阶段。在血清饥饿的贴壁细胞，LPA诱导瞬时Rho激活，而在悬浮细胞Rho激活持续。此外，悬浮细胞表现出更高的Rho活性比贴壁细胞在血清的存在下。这些数据表明存在一个依赖于粘附的负反馈回路。我们还观察到，细胞松弛素D和秋水仙碱都触发Rho激活，尽管它们对应力纤维和局灶性粘附有相反的作用。我们的研究结果表明，ECM，细胞骨架结构和可溶性因子都有助于调节Rho活性。
Soluble factors from serum such as lysophosphatidic acid (LPA) are thought to activate the small GTP-binding protein Rho based on their ability to induce actin stress fibers and focal adhesions in a Rho-dependent manner. Cell adhesion to extracellular matrices (ECM) has also been proposed to activate Rho, but this point has been controversial due to the difficulty of distinguishing changes in Rho activity from the structural contributions of ECM to the formation of focal adhesions. To address these questions, we established an assay for GTP-bound cellular Rho, Plating Swiss 3T3 cells on fibronectin-coated dishes elicited a transient inhibition of Rho, followed by a phase of Rho activation. The activation phase was greatly enhanced by serum. In serum-starved adherent cells, LPA induced transient Rho activation, whereas in suspended cells Rho activation was sustained. Furthermore, suspended cells showed higher Rho activity than adherent cells in the presence of serum. These data indicate the existence of an adhesion-dependent negative-feedback loop, We also observed that both cytochalasin D and colchicine trigger Rho activation despite their opposite effects on stress fibers and focal adhesions. Our results show that ECM, cytoskeletal structures and soluble factors all contribute to regulation of Rho activity.