MICROTUBULE ASSEMBLY FACTORS FROM XENOPUS EGGS

拟议的研究调查的机制， 微管组装在卵细胞的过程中受到调节， 非洲蛙Xenopus laevis 该方法涉及 影响微管组装因素的研究 体外，并可能在体内调节组装。 两个这样 因素已经确定。 XMAP，一个促进 微管组装，已经从鸡蛋中纯化了1000倍。 XMAP特异性地促进微管的延长， 通过改变微管蛋白的损失和添加速率来结束。 xMAP 是一个M期磷蛋白，这表明磷酸化 调节XMAP功能。 第二个因素，在非洲爪蟾身上发现的 卵母细胞，特异性抑制微管+末端的延长。 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。 我们会留意 XMAP定位于特定类别的微管。 (2)如何 XMAP是否在加端促进组装而不影响 负末端，以及它的末端特异性如何影响自由 微管？ XMAP对微管蛋白表达的影响 在负端的增加和损失将被直接测量， XMAP对微管成核的影响， 稳定状态下的微管动力学。 (3)磷酸化是否 调节XMAP功能，从而调节微管 组装？ 磷酸化和去磷酸化的XMAP将被 在体外测定微管蛋白组装的促进作用，以确定 磷酸化是否影响XMAP活性。 (4)什么蛋白质 或因子负责抑制微管组装， 卵母细胞 抑制剂将被分离，并且凝胶过滤或 免疫沉淀法将用于确定它是否结合微管蛋白 单体的 (5)在减数分裂过程中抑制剂是如何失活的？ ~（35）S-蛋氨酸的~（32）PO_4代谢标记卵母细胞 和免疫沉淀将用于研究失活的 通过降解或翻译后修饰抑制剂。 （六） XMAP相关蛋白或其他细胞中发现的抑制剂 或者在其他生物体中？ 将使用功能测定来 筛选其他细胞类型的特征性加端效应， 这些因素对微管组装的影响。 此外，具体 抗血清将用于筛选相关蛋白质， 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.