Molecular mechanisms for regulation of microtubule architecture

将哺乳动物培养细胞的中心体加入非洲爪蟾M期卵提取液中，发现非洲爪蟾p48（EF-1 α）在中心体中积累。抗微管药物不能抑制p48的积累，表明微管不参与p48的积累过程。我们利用抗p220单克隆抗体成功克隆了非洲爪蟾微管相关蛋白p220的cDNA。推测的p220蛋白氨基酸序列与哺乳动物的MAP 4蛋白氨基酸序列相似，提示p220蛋白与MAP 4蛋白具有同源性。其中一种抗体命名为W8-C3，在间期细胞中只识别中心体，但在M期细胞中除了中心体外还与纺锤体微管反应。用W8-C3筛选非洲爪蟾cDNA表达文库，筛选到了几个阳性克隆。对其中一个克隆的部分测序显示，它是人类PCM-1的爪蟾同源物。然后，我们分离了编码非洲爪蟾PCM-1的全长cDNA克隆，并测定了其核苷酸序列。我们现在正在研究非洲爪蟾PCM-1在细胞周期中调控微管动力学和组织的功能。

When centrosomes form mammalian cultured cells were add to Xenopus M phase egg extracts, Xenopus p48 (EF-1alpha) was found to accumulate to the centrosomes. This accumulation of p48 was not inhibited by anti-microtubule drugs, indicating that microtubules are not involved in the process of accumulation.We succeeded in cDNA cloning of Xenopus microtubule-associated protein p220 by using anti-p-220 monoclonal antibodies. The deduced amino acid sequence of p220 was similar to that of mammalian MAP4, suggesting that p220 is homologous to MAP4.We produced several monoclonal antibodies against Xenopus centrosomes. One of the antibodies, named W8-C3, recognized centrosomes alone in interphase cells, but reacted with spindle microtubules in addition to centrosomes in M phase cells. We isolated a few positive clones by screening Xenopus cDNA expression library with W8-C3. Sequencing part of one of the clones revealed that it is a Xenopus homolog of human PCM-1. Then, we isolated a full-length cDNA clone encoding Xenopus PCM-1 and determined its nucleotide sequence. We are now examining functions of Xenopus PCM-1 in regulation of microtubule dynamics and organization during cell cycle.