Survey of proteins involved in signal transduction pathway controlling cortical microtubules

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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.