Buendia B, Draetta G, Karsenti E
EMBL, Cell Biology, Heidelberg, Germany.
J Cell Biol. 1992 Mar;116(6):1431-42. doi: 10.1083/jcb.116.6.1431.
Isolated centrosomes nucleate microtubules when incubated in pure tubulin solutions well below the critical concentration for spontaneous polymer assembly (approximately 15 microM instead of 60 microM). Treatment with urea (2-3 M) does not severely damage the centriole cylinders but inactivates their ability to nucleate microtubules even at high tubulin concentrations. Here we show that centrosomes inactivated by urea are functionally complemented in frog egg extracts. Centrosomes can then be reisolated on sucrose gradients and assayed in different concentrations of pure tubulin to quantify their nucleating activity. We show that the material that complements centrosomes is stored in a soluble form in the egg. Each frog egg contains enough material to complement greater than 6,000 urea-inactivated centrosomes. The material is heat inactivated above 56 degrees C. One can use this in vitro system to study how the microtubule nucleating activity of centrosomes is regulated. Native centrosomes require approximately 15 microM tubulin to begin nucleating microtubules, whereas centrosomes complemented in interphase extracts begin nucleating microtubules around 7-8 microM tubulin. Therefore, the critical tubulin concentrations for polymer assembly off native centrosomes is higher than that observed for the centrosomes first denatured and then complemented in egg extracts. In vivo, the microtubule nucleating activity of centrosomes seems to be regulated by phosphorylation at the onset of mitosis (Centonze, V. E., and G. G. Borisy. 1990. J. Cell Sci. 95:405-411). Since cyclins are major regulators of mitosis, we tested the effect of adding bacterially produced cyclins to interphase egg extracts. Both cyclin A and B activate an H1 kinase in the extracts. Cyclin A-associated kinase causes an increase in the microtubule nucleating activity of centrosomes complemented in the extract but cyclin B does not. The critical tubulin concentration for polymer assembly off centrosomes complemented in cyclin A-treated extracts is similar to that observed for centrosomes complemented in interphase extracts. However, centrosomes complemented in cyclin A treated extracts nucleate much more microtubules at high tubulin concentration. We define this as the "capacity" of centrosomes to nucleate microtubules. It seems that the microtubule nucleating activity of centrosomes can be defined by two distinct parameters: (a) the critical tubulin concentration at which they begin to nucleate microtubules and (b) their capacity to nucleate microtubules at high tubulin concentrations, the latter being modulated by phosphorylation.
当在远低于自发聚合组装临界浓度（约15微摩尔而非60微摩尔）的纯微管蛋白溶液中孵育时，分离出的中心体可形成微管。用尿素（2 - 3摩尔）处理不会严重损害中心粒圆柱体，但会使其形成微管的能力失活，即使在高微管蛋白浓度下也是如此。在此我们表明，被尿素失活的中心体在蛙卵提取物中能得到功能互补。然后可在蔗糖梯度上重新分离中心体，并在不同浓度的纯微管蛋白中进行检测，以量化其成核活性。我们发现，能使中心体互补的物质以可溶形式储存在卵中。每个蛙卵所含的物质足以使超过6000个被尿素失活的中心体得到互补。该物质在56摄氏度以上会被热失活。人们可以利用这个体外系统来研究中心体的微管成核活性是如何被调控的。天然中心体需要约15微摩尔的微管蛋白才能开始形成微管，而在间期提取物中得到互补的中心体在约7 - 8微摩尔微管蛋白时就开始形成微管。因此，从天然中心体上进行聚合物组装的临界微管蛋白浓度高于先变性然后在卵提取物中得到互补的中心体所观察到的浓度。在体内，中心体的微管成核活性在有丝分裂开始时似乎受磷酸化调控（森通泽，V. E.，和G. G. 博里西。1990。《细胞科学杂志》95：405 - 411）。由于细胞周期蛋白是有丝分裂的主要调节因子，我们测试了向间期卵提取物中添加细菌产生的细胞周期蛋白的效果。细胞周期蛋白A和B都能激活提取物中的H1激酶。与细胞周期蛋白A相关的激酶会使提取物中得到互补的中心体的微管成核活性增加，但细胞周期蛋白B则不会。在经细胞周期蛋白A处理的提取物中得到互补的中心体进行聚合物组装的临界微管蛋白浓度与在间期提取物中得到互补的中心体所观察到的浓度相似。然而，在经细胞周期蛋白A处理的提取物中得到互补的中心体在高微管蛋白浓度下能形成更多微管。我们将此定义为中心体形成微管的“能力”。看来中心体的微管成核活性可以由两个不同的参数来定义：（a）它们开始形成微管时的临界微管蛋白浓度，以及（b）它们在高微管蛋白浓度下形成微管的能力，后者受磷酸化调节。
