Sawin K E, Mitchison T J
Department of Biochemistry and Biophysics and Pharmacology, University of California, San Francisco 94143.
J Cell Biol. 1991 Mar;112(5):925-40. doi: 10.1083/jcb.112.5.925.
We have used Xenopus egg extracts to study spindle morphogenesis in a cell-free system and have identified two pathways of spindle assembly in vitro using methods of fluorescent analogue cytochemistry. When demembranated sperm nuclei are added to egg extracts arrested in a mitotic state, individual nuclei direct the assembly of polarized microtubule arrays, which we term half-spindles; half-spindles then fuse pairwise to form bipolar spindles. In contrast, when sperm nuclei are added to extracts that are induced to enter interphase and arrested in the following mitosis, a single sperm nucleus can direct the assembly of a complete spindle. We find that microtubule arrays in vitro are strongly biased towards chromatin, but this does not depend on specific kinetochore-microtubule interactions. Indeed, although we have identified morphological and probably functional kinetochores in spindles assembled in vitro, kinetochores appear not to play an obligate role in the establishment of stable, bipolar microtubule arrays in either assembly pathway. Features of the two pathways suggest that spindle assembly involves a hierarchy of selective microtubule stabilization, involving both chromatin-microtubule interactions and antiparallel microtubule-microtubule interactions, and that fundamental molecular interactions are probably the same in both pathways. This in vitro reconstitution system should be useful for identifying the molecules regulating the generation of asymmetric microtubule arrays and for understanding spindle morphogenesis in general.
我们利用非洲爪蟾卵提取物在无细胞体系中研究纺锤体形态发生，并运用荧光类似物细胞化学方法在体外鉴定出两条纺锤体组装途径。当将去膜精子细胞核添加到处于有丝分裂状态停滞的卵提取物中时，单个细胞核指导极化微管阵列的组装，我们将其称为半纺锤体；然后半纺锤体两两融合形成双极纺锤体。相比之下，当将精子细胞核添加到被诱导进入间期并在随后的有丝分裂中停滞的提取物中时，单个精子细胞核可指导完整纺锤体的组装。我们发现体外的微管阵列强烈偏向染色质，但这并不依赖于特定的动粒 - 微管相互作用。实际上，尽管我们在体外组装的纺锤体中鉴定出了形态学上以及可能功能上的动粒，但在任一组装途径中，动粒似乎在稳定双极微管阵列的建立过程中并非起必不可少的作用。这两条途径的特征表明，纺锤体组装涉及选择性微管稳定的层级结构，包括染色质 - 微管相互作用和平行微管 - 微管相互作用，并且两条途径中基本的分子相互作用可能相同。这种体外重建系统对于鉴定调节不对称微管阵列生成的分子以及总体上理解纺锤体形态发生应该是有用的。
