Effects of gibberellins on proteins that cross-link microtubules to the plasma membrane

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1. Isolation of a microtubule-plasma membrane cross-linking protein from tobacco cultured cells - Tubulin in the extract prepared from miniprotoplasts that had been prepared from protoplasts of tobacco BY-2 cells polymerized into microtubules in the absence of any microtubule-stabilizing agents. The polymerized microtubules formed bundles and between adjacent microtubules in the bundle cross-bridges were observed. A protein of 65Kd was copurified with tubulin by assembly and disassembly method. The protein was found to have the ability to cross-link microtubules. Immunofluorescence microscopy using antibodies against the 65Kd protein revealed that this protein is codistributed with microtubules in tobacco BY-2 cells.2. Cross-bridge structures between microtubules and plasma membrane in tobacco cultured cells - Membrane ghosts prepared from tobacco BY-2 cells have the ability to bind microtubules that were polymerized in the extract prepared from tobacco BY-2 miniprotoplasts. Treatment with 0.6 M KCl or trypsin eliminated the ability of the ghosts to bind microtubules, indicating a factor that is necessary for the binding is protein and is extracted by high concentrations of KCl. In the presence of taxol, a microtubule-stabilizing agent, microtubules polymerized in the extract were bound to the ghosts. The factor eliminated by KCl seems to have a microtubule-stabilizing activity. Microtubules polymerized in the extract attached to the ghost in the presence of taxol, but microtubules polymerized from purified tubulin didn't, indicating the presence in the extract of factor(s) that is necessary for the attachment of microtubules to the membrane.3. Microtubule-stabilizing effect of gibberellin A_3 (GA_3) in maize mesocotyl cells - GA_3 stabilized microtubules in mesocotyl cells of maize seedlings. Microtubules in protoplasts prepared from mesocotyls of GA_3-treated seedlings were more stable than those in protoplasts prepared from GA_3-untreated seedlings.