PARTHENOGENESIS IN XENOPUS EGGS REQUIRES CENTROSOMAL INTEGRITY
PARTHENOGENESIS IN XENOPUS EGGS REQUIRES CENTROSOMAL INTEGRITY
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DOI：
10.1083/jcb.110.2.405
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发表时间：
1990-02-01
影响因子：
7.8
通讯作者：
KARSENTI, E
中科院分区：
文献类型：
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作者：
KLOTZ, C;DABAUVALLE, MC;KARSENTI, E
非洲爪蟾卵产于减数分裂第二中期，缺乏功能性中心体。受精后，精子提供了卵裂发生所需的活性中心体。注射纯化的中心体模拟受精并导致蝌蚪形成（孤雌生殖）。在这项工作中，我们表明，中心体的孤雌生殖活性是由尿素浓度高于2 M灭活。活性的丧失与中心柱的进行性破坏和蛋白质的提取相关。这表明中心体对尿素相对敏感，因为完全的蛋白质解折叠和蛋白质的溶解通常发生在高达8-10 M的尿素浓度下。当存在时，孤雌生殖活性总是与显示其不能被尿素溶解的可粒化部分相关。单性生殖活性逐渐失活的盐浓度高于2 M（NaCl或KCl）。然而，只有少数蛋白质提取这些处理和中心体超微结构不受影响。这表明孤雌生殖活性和中心体结构都能抵抗相对较高的离子强度。事实上，大多数由静电力保持的蛋白质结构被2 M盐解离。在较高的盐浓度下产生的孤雌生殖活性的损失，而中心体的结构不受影响，是一个明显的矛盾。我们解释这一结果意味着中心体的天然状态是通过高离子强度有利于功能变性的力保持在一起的。尿素和盐对中心体结构和活性的影响表明，中心体主要是通过氢键和疏水键结合在一起。在不影响孤雌生殖活性的盐或尿素浓度下，中心体的体外微管成核活性可以失活。由于卵裂需要形成的微管星，我们得出结论，提取或变性的微管成核活性的中心体可以补充存在于卵细胞质中的成分。孤雌生殖和微管成核活性被蛋白酶处理废除，但抵抗核酸酶的作用。由于我们在经RNase处理的中心体中没有发现RNA，因此它们可能不含受保护的RNA。总之，这些结果是一致的想法，即整个或部分的中心体结构作为种子启动中心体复制周期在非洲爪蟾卵。
Xenopus eggs are laid arrested at second metaphase of meiosis lacking a functional centrosome. Upon fertilization, the sperm provides the active centrosome that is required for cleavage to occur. The injection of purified centrosomes mimics fertilization and leads to tadpole formation (parthenogenesis). In this work we show that the parthenogenetic activity of centrosomes is inactivated by urea concentrations higher than 2 M. The loss of activity is correlated with a progressive destruction of the centriolar cylinder and extraction of proteins. This shows that centrosomes are relatively sensitive to urea since complete protein unfolding and solubilization of proteins normally occurs at urea concentrations as high as 8-10 M. When present, the parthenogenetic activity is always associated with a pelletable fraction showing that it cannot be solubilized by urea. The parthenogenetic activity is progressively inactivated by salt concentrations higher than 2 M (NaCl or KCl). However, only a few proteins are extracted by these treatments and the centrosome ultrastructure is not affected. This shows that both parthenogenetic activity and centrosomal structure are resistant to relatively high ionic strength. Indeed, most protein structures held by electrostatic forces are dissociated by 2 M salt. The loss of parthenogenetic activity produced at higher salt concentrations, while the structure of the centrosome is unaffected, is an apparent paradox. We interpret this result as meaning that the native state of centrosomes is held together by forces that favor functional denaturation by high ionic strength. The respective effects of urea and salts on centrosomal structure and activity suggest that the centrosome is mainly held together by hydrogen and hydrophobic bonds. The in vitro microtubule nucleating activity of centrosomes can be inactivated at salt or urea concentrations that do not affect the parthenogenetic activity. Since egg cleavage requires the formation of microtubule asters, we conclude that the extracted or denatured microtubule nucleating activity of centrosomes can be complemented by components present in the egg cytoplasm. Both parthenogenetic and microtubule nucleating activities are abolished by protease treatments but resist nuclease action. Since we find no RNA in centrosomes treated by RNase, they probably do not contain a protected RNA. Taken together, these results are consistent with the idea that the whole or part of the centrosome structure acts as a seed to start the centrosome duplication cycle in Xenopus eggs.