Survey of proteins involved in signal transduction pathway controlling cortical microtubules

参与控制皮质微管的信号转导途径的蛋白质的调查

• 批准号: 07804056
• 负责人: SAKAGUCHI Shuichi
• 金额: $ 1.34万
• 依托单位: Nara Women's University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1995
• 资助国家: 日本
• 起止时间: 1995 至 1996
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07804056/
• 关键词: cortical microtubule; microtubule-binding protein; signal transduction; small GTPase; Cdc42; tubulin; EF1alpha; maize; EF1α; 微小管; 情報伝達系; DCC42; 被子植物; GTP結合タンパク質; 細胞極性

This study aimed at 1) isolation of Arabidopsis mutants defective in cortical microtubule function and 2) immunological detection of proteins homologous to animal proteins that are known to work in signal transduction pathway and their localization on microtubules. In the first project, we have constructed a temperature-sensitive mutant plants library and now are screening mutant lines which show abnormal cortical microtubule structure. In the second project, we surveyed Cdc42 homologs in maize. Cdc42 is a ras-related small GTPase involved in the control of cell polarity and actin cytoskeleton. The protein is found in wide range of organisms from yeasts to mammals, but has not been reported from higher plants. By western blot experiment using anti-human Cdc42 antibody, we found two positive bands at ca.50 kD and ca.25 kD in maize cell lysates. Immunofluorescence observation of maize root tip cells stained by the same antibody revealed that the fluorescence localizes in cytosol and on microtubular structures including cortical microtubules, preprophase bands of microtubules, spindles and phragmoplasts. The fluorescence on microtubular structures is fairly punctate, and therefore, differs from the image of microtubules stained by anti-tubulin antibody. Cdc42 shows weak homology to tubulins and EF1alpha, a known microtubule-binding protein. However, two-dimensional electrophoresis/western blots experiment revealed that the spots recognized by anti-human Cdc42 are all different from the spots for alpha-tubulin, beta-tubulin or EF1alpha, suggesting that the protein(s) recognized by the anti-Cdc42 antibody is novel microtubule-binding protein. Now we are cloning the gene coding for the protein crossreacting to anti-human Cdc42 antibody using lambda phage-based maize cDNA library.

本研究的目的是：1）分离皮质微管功能缺陷的拟南芥突变体；2）免疫学检测与已知在信号转导途径中起作用的动物蛋白同源的蛋白及其在微管上的定位。在第一个项目中，我们构建了温度敏感突变植物库，现在正在筛选表现出异常皮质微管结构的突变株系。在第二个项目中，我们调查了玉米中的 Cdc42 同源物。 Cdc42 是一种与 ras 相关的小 GTP 酶，参与细胞极性和肌动蛋白细胞骨架的控制。该蛋白质存在于从酵母到哺乳动物的多种生物体中，但尚未在高等植物中发现过。通过使用抗人Cdc42抗体的蛋白质印迹实验，我们在玉米细胞裂解物中发现了约50 kD和约25 kD的两条阳性条带。用相同抗体染色的玉米根尖细胞的免疫荧光观察表明，荧光定位于细胞质和微管结构，包括皮层微管、微管前期带、纺锤体和成膜体。微管结构上的荧光相当点状，因此与抗微管蛋白抗体染色的微管图像不同。 Cdc42 与微管蛋白和 EF1α（一种已知的微管结合蛋白）表现出弱同源性。然而，二维电泳/蛋白质印迹实验显示，抗人Cdc42识别的斑点与α-微管蛋白、β-微管蛋白或EF1α的斑点均不同，这表明抗人Cdc42识别的蛋白质抗体是新型微管结合蛋白。现在，我们正在使用基于 lambda 噬菌体的玉米 cDNA 文库克隆编码与抗人 Cdc42 抗体交叉反应的蛋白质的基因。