Microtubule-Nucleation Sites on Nuclei in Higher Plant Cells

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The nucleation and the elongation of microtubules from isolated nuclei of higher plant cells were investigated. Isolated intact nuclei failed to nucleate microtubules from their surface when they were incubated with purified tubulin from plant or animal sources. However, frozen-thawed nuclei or nuclear particles obtained by gentle homogenization of nuclei nucleated microtubules and nucleated microtubules elongated radially from the surface of nuclei or from the nuclear materials. Microtubules radiating from the nuclear particles were extremely shorter than those radiating from frozen-thawed nuclei. The washing of the nuclear particles diminished the ability of the particles to nucleate microtubules. The ability of the washed nuclear particles to nucleate microtubules was restored by the addition of the soluble fraction of a nuclear homogenate. The staining with a monoclonal antibody specific for plant tubulin of the complexes of radiating microtubules-prepared by successive polymerization of animal tubulin and plant tubulin revealed that microtubules in the complex incorporated tubulin at their proximal ends. This result indicates that the mode of incorporation of tubulin in frozen-thawed nuclei or the nuclear particles is different from that in pericentriolar bodies in animal cells. The partially purified microtubulenucleation particles contained a peptide having an apparent molecular weight of 50 kd as a main constitution, which cross-reacted specifically with monoclonal antibody against alpha-tubulin. 2-D PAGE analysis showed the 50 kd peptide to be separated into two peptides, one of which was assumed to be phosphorylated. Therefore, the essential factor in the soluble fraction is possible to be estimated to be protein kinase or phosphoprotein phosphatase. The action of such essential factor may enable the 50 kd peptide to change from inactive form to active form.