Survey of proteins involved in signal transduction pathway controlling cortical microtubules

本研究的目的是：1）分离拟南芥皮层微管功能缺陷突变体; 2）免疫学检测已知在信号转导途径中起作用的动物蛋白同源蛋白及其在微管上的定位。在第一个项目中，我们已经构建了温度敏感突变体植物库，目前正在筛选皮层微管结构异常的突变株系。在第二个项目中，我们调查了玉米中的Cdc 42同系物。Cdc 42是一种ras相关的小G蛋白，参与细胞极性和肌动蛋白细胞骨架的控制。该蛋白存在于从酵母到哺乳动物的广泛生物中，但尚未在高等植物中报道。用抗人Cdc 42抗体进行蛋白质印迹实验，在玉米细胞裂解液中发现约50 kD和约25 kD的两条阳性条带。用该抗体染色的玉米根尖细胞的免疫荧光观察显示，荧光定位于细胞质和微管结构，包括皮层微管，微管的前期带，纺锤体和成膜体。微管结构上的荧光是相当点状的，因此，与抗微管蛋白抗体染色的微管的图像不同。Cdc 42与微管蛋白和已知的微管结合蛋白EF 1 α具有较弱的同源性。然而，双向电泳/蛋白质印迹实验显示，抗人Cdc 42识别的斑点均不同于α-微管蛋白、β-微管蛋白或EF 1 α的斑点，表明抗人Cdc 42抗体识别的蛋白质是新的微管结合蛋白。目前，我们正在利用λ噬菌体的玉米cDNA文库克隆编码抗人Cdc 42抗体交叉反应蛋白的基因。

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.