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; 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.

这项研究的目的是1）分离拟南芥突变体在皮质微管功能中有缺陷的和2）与动物蛋白同源的蛋白质的免疫学检测，这些蛋白质在信号转导途径中起作用及其在微管上的定位。在第一个项目中，我们已经构建了一个对温度敏感的突变植物文库，现在正在筛选突变线，这些突变型线显示出异常的皮质微管结构。在第二个项目中，我们调查了玉米中的CDC42同源物。 CDC42是与RAS相关的小GTPase，涉及细胞极性和肌动蛋白细胞骨架的控制。该蛋白质在从酵母到哺乳动物的各种生物中都发现，但尚未从较高植物中报道。通过使用抗人CDC42抗体的Western印迹实验，我们在玉米细胞裂解液中发现了两个阳性带，在Ca.50 kd和Ca.25 kd的阳性带中。通过相同抗体染色的玉米根尖端细胞的免疫荧光观察表明，荧光位于细胞质和微管结构中，包括皮质微管，微管，纺锤体，纺锤体和phragmoplasts的前循环。微管结构上的荧光相当点状，因此与抗微管抗体染色的微管的图像不同。 CDC42与微管蛋白和EF1Alpha（一种已知的微管结合蛋白）显示出弱的同源性。然而，二维电泳/蛋白质印迹实验表明，抗人类CDC42识别的斑点与α-微管蛋白，β-微管蛋白或EF1Alpha的斑点都不同，这表明抗CDC42抗体是新颖的蛋白质是新颖的蛋白质。现在，我们使用基于lambda噬菌体的玉米cDNA文库将蛋白质交叉反应的基因克隆到抗人CDC42抗体。