Analysis on mechanism for microtubule formation in a plant cortical array

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Despite the absence of a conspicuous microtubule organizing center, microtubules in interphase plant cells are present in the cell cortex as a well oriented array. By the support of the Grant-in-Aid for Scientific Research, we show that nucleation requires extant cortical microtubules, onto which cytosolic γ-tubulin is recruited. Microtubule-independent nucleation is rarely observed in living tobacco BY-2 cells and nucleation is minimal in the absence of original microtubules in a cell-free system. In both living cells and the cell-free system, microtubules are nucleated as branches on the extant cortical microtubules. The branch points contain γ-tubulin, which is abundant in the cytoplasm, and microtubule nucleation in the cell free system is prevented by inhibiting γ-tubulin function with specific antibodies. When isolated plasma membrane with microtubules is exposed to purified neuro-tubulin, no microtubules are nucleated, but when the membrane is exposed to a cytosolic extract, γ-tubulin binds microtubules on the membrane, and after a subsequent incubation in neuro-tubulin, microtubules are nucleated on the pre-existing microtubules. We propose that a cytoplasmic γ-tubulin complex shuttles between cytoplasm and the side of a cortical microtubule and has nucleation activity only when bound to the microtubule.