Spatiotemporal analysis of the behavior of the small GTPase, Rho

小 GTP 酶 Rho 行为的时空分析

• 批准号: 13670115
• 负责人: YONEMURA Shigenobu
• 金额: $ 2.62万
• 依托单位: RIKEN
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2001
• 资助国家: 日本
• 起止时间: 2001 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13670115/
• 关键词: Rho; Microvilli; TCA; LPA; EGF; cleavage furrow; 可視化; 低分子量Gタンパク質; 抗体; 局在; TCA固定

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.

我们筛选了抗Rho抗体和固定方案的组合，用于Rho在细胞内的精确定位。发现几种商业抗体和我们自己生产的抗体在适当的固定方案如TCA固定下是可靠的。对于该筛选，我们用抗myc抗体和抗Rho抗体双重染色表达Myc-RhoA的培养细胞。可靠的抗Rbo抗体应该像抗Myc抗体一样染色细胞。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积聚在侧膜上。在成纤维细胞中，Rho几乎均匀地分布在整个细胞质中。在胞质分裂过程中，Rho通常集中在卵裂沟。在组织中，虽然Rho在微绒毛上的浓度是明显的，但Rho在细胞内的定位取决于细胞类型而不同，这可能反映了组织中细胞的功能差异。生化数据表明，Rho从细胞质易位到质膜上的激活。我们第一次使用LPA或EGF刺激的培养细胞观察到这种易位。在这些因子的刺激下，Rho在30秒内从细胞质迅速募集到质膜。

项目成果

S.Kikuchi, M.Hata, K.Fukumotoその他: "Radixin deficiency causes conjugated hyperbilirubinemia with loss of Mrp2 from bile canalicular membranes"Nature Genet.. 24. 320-325 (2002)

S.Kikuchi、M.Hata、K.Fukumoto 等人：“Radixin 缺乏导致结合性高胆红素血症，并导致胆小管膜 Mrp2 缺失”Nature Genet.. 24. 320-325 (2002)

S. Kikuchi: "Radixin deficiency causes conjugated hyperbilirubinemia with loss of Mrp2 from bile canalicular membranes"Nature Genet.. 24. 320-325 (2002)

S. Kikuchi：“Radixin 缺乏导致结合性高胆红素血症，并导致胆小管膜中 Mrp2 的丢失”Nature Genet.. 24. 320-325 (2002)

S.Kikuchi: "Radixin deficiency causes conjugated hyperbilirubinemia with loss of Mrp2 from bile canalicular membranes"Nature Genet.. 24. 320-325 (2002)

S.Kikuchi：“Radixin 缺乏导致结合性高胆红素血症，并导致胆小管膜中 Mrp2 的丢失”Nature Genet.. 24. 320-325 (2002)

Eda, M: "Rho-dependent transfer of Citron-kinase to the cleavage furrow of dividing cells"J.Cell Sci.. 10. 3273-3284 (2001)

Eda, M：“香橼激酶向分裂细胞分裂沟的 Rho 依赖性转移”J.Cell Sci.. 10. 3273-3284 (2001)

J.Fujikura: "Differentiation of embryonic stem cells is induced by GATA factors"Genes & Dev.. 16. 748-789 (2002)

J.Fujikura：“胚胎干细胞的分化是由GATA因子诱导的”基因