Spatiotemporal analysis of the behavior of the small GTPase, Rho

我们筛选了抗Rho抗体和固定方案的组合，以精确定位细胞内的Rho。一些商业抗体和我们自己生产的抗体被发现是可靠的固定方案，如TCA固定。为了进行筛选，我们用抗myc抗体和抗rho抗体对表达myc - rhoa的培养细胞进行双重染色。可靠的抗rbo抗体应该像抗myc抗体那样染色细胞。使用有效的抗体，我们将Rho定位在细胞和组织内。在培养的上皮细胞中，Rho积聚在外侧膜上。在成纤维细胞中，Rho几乎均匀地分布在细胞质中。在细胞质分裂过程中，Rho一般集中在卵裂沟处。在组织中，虽然微绒毛处的Rho浓度很明显，但细胞内的Rho定位因细胞类型不同而不同，这可能反映了组织中细胞功能的差异。生化数据表明，Rho在激活时从细胞质转移到质膜。我们首次使用LPA或EGF刺激的培养细胞来观察这种易位。在这些因子的刺激下，Rho在30秒内迅速从细胞质招募到质膜。

We screened combinations of anti-Rho antibodies and fixation protocols for precise localization of Rho within cells. Several commercial antibodies and antibodies produced by ourselves were found to be reliable with appropriate fixation protocols such as TCA fixation. For this screening, we doubly stained Myc-RhoA-expressing cultured cells with an anti-myc antibody and an anti-Rho antibody. Reliable anti-Rbo antibodies should stain the cells as anti-Myc antibody does. Using valid antibodies, we localized Rho within cells and tissues. In cultured epithelial cells, Rho was accumulated on lateral membranes. In fibroblastic cells, Rho was distributed almost evenly throughout the cytoplasm. During cytokinesis, Rho was generally concentrated at the cleavage furrow. In tissues, although Rho concentration at microvilli was obvious, Rho localization within cells differed depending on cell type, probably reflecting the functional difference of the cells in tissues. Biochemical data indicated that Rho translocates from the cytoplasm to the plasma membrane on its activation. We visualized this translocation for the first time using cultured cells stimulated with LPA or EGF. On stimulatioh with these factors, Rho was rapidly recruited from the cytoplasm to the plasma membrane within 30 seconds.