Rho- and rac-dependent assembly of focal adhesion complexes and actin filaments in permeabilized fibroblasts: an essential role for ezrin/radixin/moesin proteins.
Rho- and rac-dependent assembly of focal adhesion complexes and actin filaments in permeabilized fibroblasts: an essential role for ezrin/radixin/moesin proteins.
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DOI：
10.1083/jcb.138.4.927
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发表时间：
1997-08-25
期刊：
影响因子：
--
通讯作者：
Hall A
中科院分区：
文献类型：
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作者：
Mackay DJ;Esch F;Furthmayr H;Hall A
小GTP酶Rho和Rac调节肌动蛋白丝组装和整合素粘附复合物的形成，分别在哺乳动物细胞中产生应力纤维和板状伪足。虽然已经使用酵母双杂交和亲和纯化技术鉴定了许多可能介导这些反应的候选效应物，但它们的细胞作用仍不清楚。我们现在描述一种生物测定法，允许Rho和Rac信号通路的组件被识别。在鸟苷5′-O-（3-硫代三磷酸）（GTPγS）存在下，毛地黄皂苷对血清饥饿的Swiss 3 T3细胞的透化通过激活内源性Rho和Rac诱导肌动蛋白丝和粘着斑复合物组装。透化后6分钟加入GTPγS时，这些反应消失，但可以使用浓缩的胞质提取物重建。我们已经实现了猪脑胞质溶胶中存在的活性的10，000倍纯化，蛋白质序列分析显示其含有膜突蛋白。使用重组蛋白，我们表明，膜突蛋白和它的近亲埃兹蛋白和radixin可以重建应力纤维组装，皮质肌动蛋白聚合和焦点复合物的形成响应于Rho和Rac的激活。
The small GTPases Rho and Rac regulate actin filament assembly and the formation of integrin adhesion complexes to produce stress fibers and lamellipodia, respectively, in mammalian cells. Although numerous candidate effectors that might mediate these responses have been identified using the yeast two-hybrid and affinity purification techniques, their cellular roles remain unclear. We now describe a biological assay that allows components of the Rho and Rac signaling pathways to be identified. Permeabilization of serum-starved Swiss 3T3 cells with digitonin in the presence of guanosine 5′-O-(3-thiotriphosphate) (GTPγS) induces both actin filament and focal adhesion complex assembly through activation of endogenous Rho and Rac. These responses are lost when GTPγS is added 6 min after permeabilization, but can be reconstituted using concentrated cytosolic extracts. We have achieved a 10,000-fold purification of the activity present in pig brain cytosol and protein sequence analysis shows it to contain moesin. Using recombinant proteins, we show that moesin and its close relatives ezrin and radixin can reconstitute stress fiber assembly, cortical actin polymerization and focal complex formation in response to activation of Rho and Rac.