MICROTUBULE ASSEMBLY FACTORS FROM XENOPUS EGGS

这项拟议的研究调查了 微管组装在卵的细胞内受到调节 非洲爪蛙。该方法涉及到 人体微管组装影响因素的研究 在体外，这可能调节体内的组装。两个这样的人 已经确定了影响因素。XMAP，一个促进 微管组装，已经从卵子中纯化了1000倍。 XMAP特别促进微管的伸长- 通过改变微管蛋白丢失和添加的速率来结束。XMAP 是一种M期磷酸化蛋白，提示磷酸化 调节xMAP功能。第二个因素，发现于非洲爪哇 卵母细胞，特别是抑制微管加端的延长。 拟议的实验解决了以下问题 关于这些因素：(1)xMAP是否与微管结合 体外和体内？免疫荧光和电子显微镜 将用于可视化xMAP与微管的结合 无论是在体外还是在完整的卵子中。我们将会关注 将xMAP定位于特定类别的微管。(2)如何 XMAP是否促进了加端的组装而不影响 负端，以及它的末端专一性如何影响自由 微管？XMAP对微管蛋白表达的影响 负端的附加和损失将直接测量，如 XMAP对微管形核和微管形成的影响 稳定状态下的微管动力学。(3)进行磷酸化 调节xMAP功能，从而调节微管 集合？磷酸化和去磷酸化的xMAP将被 体外测定促进微管蛋白组装以确定 磷酸化是否影响xMAP活性。(4)什么蛋白质 OR因子抑制微管组装的作用 卵母细胞？抑制剂将被分离，凝胶过滤或 免疫沉淀将被用来确定它是否与微管蛋白结合 单体。(5)该抑制物在减数分裂过程中是如何失活的？ ~(35)S-蛋氨酸~(32)PO_4对卵母细胞的代谢标记 免疫沉淀将被用来研究失活的 通过降解或翻译后修饰来抑制。(6) 是否与xMAP相关的蛋白质或在其他细胞中发现的抑制物 类型，还是在其他生物体中？功能分析将被用来 筛选其他类型的单元格，以获得 这些因素对微管组装有影响。此外，具体来说， 将使用抗血清通过以下方式筛选相关蛋白质 免疫荧光和免疫印迹。

The proposed research investigates the mechanisms by which microtubule assembly is regulated during the cell in eggs of the African frog Xenopus laevis. The approach involves the investigation of factors which affect microtubule assembly in vitro, and which might regulate assembly in vivo. Two such factors have been identified. XMAP, a factor which promotes microtubule assembly, has been purified 1000-fold from eggs. XMAP specifically promotes elongation of the microtubule plus- ends by altering both the rate of tublin loss and addition. XMAP is an M-phase phosphorprotein, suggesting that phosphorylation regulates XMAP function. The second factor, found in Xenopus oocytes, specifically inhibits elongation of microtubule plus-ends. The proposed experiments address the following questions regarding these factors: (1) Does XMAP bind to microtubules in vitro and in vivo? Immunofluorescence and electron microscopy will be used to visualize the binding of XMAP to microtubules both in vitro and in the intact egg. Attention will be paid to the localizaton of XMAP to specific classes of microtubules. (2) How does XMAP promote assembly at the plus-end without affecting the minus-end, and how does its end specificity affect free microtubules? The effect of XMAP on the rates of tubulin addition and loss at the minus-end will be directly measured, as will the effect of XMAP on microtubule nucleation and microtubule dynamics at steady state. (3) Does phosphorylation regulate XMAP function, and thereby regulate microtubule assembly? Phosphorylated and dephosphorylated XMAP will be assayed for promotion of tubulin assembly in vitro to determine whether phosphorylation affects XMAP activity. (4) What protein or factor is responsible for inhibition of microtubule assembly in the oocyte? The inhibitor will be isolated, and gel-filtration or immunoprecipitation will be used to determine if it binds tubulin monomer. (5) How is the inhibitor inactivated during meiosis? Metabolic labeling of oocytes with 32PO4 of the 35S-methionine and immunoprecipitation will be used to study inactivation of the inhibitor by degradation or post-translational modification. (6) Are proteins related to XMAP or the inhibitor found in other cell types, or in other organisms? A functional assay will be used to screen other cell types for the characteristic plus-end effects of these factors on microtubule assembly. In addition, specific antisera will be used to screen for related proteins by immunofluorescence and immunoblotting.