Survey of proteins involved in signal transduction pathway controlling cortical microtubules

本研究旨在1)分离拟南芥皮层微管功能缺陷突变体，2)免疫检测已知参与信号转导通路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，我们建立了一个温度敏感突变植物文库，目前正在筛选皮质微管结构异常的突变系。在第二个项目中，我们调查了Cdc42在玉米中的同源物。Cdc42是一种与ras相关的小GTPase，参与控制细胞极性和肌动蛋白细胞骨架。这种蛋白质存在于从酵母到哺乳动物的各种生物中，但尚未在高等植物中发现。用抗人Cdc42抗体进行western blot实验，发现玉米细胞裂解液在ca.50 kD和ca.25 kD处有两条阳性带。用同一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，包括皮质微管、微管前体带、纺锤体和膜质体。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图像不同。Cdc42与微管蛋白和ef1 α（一种已知的微管结合蛋白）具有弱同源性。然而，双向电泳/western blots实验显示，抗人Cdc42抗体识别的斑点均不同于α -微管蛋白、β -微管蛋白或ef1 α的斑点，提示抗Cdc42抗体识别的蛋白是新型微管结合蛋白。目前，我们正在利用基于λ噬菌体的玉米cDNA文库克隆抗人Cdc42抗体交叉反应蛋白的编码基因。

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.