Analysis on mechanism for microtubule formation in a plant cortical array

尽管没有明显的微管组织中心，间期植物细胞中的微管在细胞皮质中以定向排列的形式存在。在科学研究补助金的支持下，我们发现成核需要现存的皮质微管，胞质γ-微管蛋白被招募到这些微管上。在活的烟草BY-2细胞中很少观察到不依赖于微管的成核，在无细胞系统中没有原始微管的情况下，成核很少。在活细胞和无细胞系统中，微管在现存的皮质微管上以分支的形式成核。分支点含有大量存在于细胞质中的γ-微管蛋白，通过用特异性抗体抑制γ-微管蛋白的功能来阻止无细胞系统中的微管成核。当分离的具有微管的质膜与纯化的神经微管蛋白作用时，没有微管形成核，但当细胞质提取物作用于膜时，γ-微管蛋白将微管结合在膜上，随后在神经管蛋白中孵育后，在预先存在的微管上形成微管。我们认为，细胞质γ-微管蛋白复合体穿梭于细胞质和皮质微管的侧面之间，只有在与微管结合时才具有成核活性。

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.