HUMAN CHROMOSOMES AND CENTRIOLES AS NUCLEATING SITES FOR INVITRO ASSEMBLY OF MICROTUBULES FROM BOVINE BRAIN TUBULIN
HUMAN CHROMOSOMES AND CENTRIOLES AS NUCLEATING SITES FOR INVITRO ASSEMBLY OF MICROTUBULES FROM BOVINE BRAIN TUBULIN
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DOI：
10.1083/jcb.67.1.189
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发表时间：
1975-01-01
影响因子：
7.8
通讯作者：
BRINKLEY, BR
中科院分区：
文献类型：
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作者：
MCGILL, M;BRINKLEY, BR
用浓度为0.06-0.10 μ g/ml的秋水仙胺处理HeLa细胞导致有丝分裂的不可逆停滞。秋水仙胺逮捕的细胞含有很少的微管，和许多动粒和中心粒是免费的微管协会。当这些细胞暴露于含有TritonX-100和牛脑微管蛋白的微管重组缓冲液中时，在37 ℃时，大量微管在中期染色体的所有动粒处重新组装，并与中心粒对结合。当牛脑微管蛋白从重组系统中消除时，微管未能在这些位点组装。类似地，当EGTA从重组系统中被消除时，微管也不能重新组装。这些结果是一致的，与其他调查在体外微管组装，并表明，HeLa染色体和中心粒可以作为成核网站的微管组装从脑微管蛋白。在微管蛋白重组过程中，染色体和中心粒都从它们的C-中期构型移位，表明它们的运动是微管形成的直接结果。虽然这两个kinetochore和中心粒相关的微管组装和运动发生，我们没有观察到直接延伸的微管从kinetochores的中心粒。该系统将被证明对哺乳动物细胞中纺锤体微管形成和染色体运动的实验研究是有用的。从分离的微管蛋白亚基（12，17）体外组装微管的方法的发展为微管组织中心（MTOC）在各种真核细胞中的作用提供了重要的新信息。鸡脑微管蛋白在衣原体和海胆精子的鞭毛轴丝和基体上的组装表明，几个物种的微管结构可以作为外源微管蛋白重组的成核位点（1，11，13）。最近，聚合-
Treatment of HeLa cells with Colcemid at concentrations of 0.06-0.10/~ g/ml leads to irreversible arrest in mitosis. Colcemid-arrested cells contained few microtubules, and many kinetochores and centrioles were free of microtubule association. When these cells were exposed to microtubule reassembly buffer containing Triton X-100 and bovine brain tubulin at 37~ numerous microtubules were reassembled at all kinetochores of metaphase chromosomes and in association with centriole pairs. When bovine brain tubulin was eliminated from the reassembly system, microtubules failed to assemble at these sites. Similarly, when EGTA was eliminated from the reassembly system, microtubules failed to polymerize. These results are consistent with other investigations of in vitro microtubule assembly and indicate that HeLa chromosomes and centrioles can serve as nucleating sites for the assembly of microtubules from brain tubulin. Both chromosomes and centrioles became displaced from their C-metaphase configurations during tubulin reassembly, indicating that their movements were a direct result of microtubule formation. Although both kinetochore-and centrioleassociated microtubules were assembled and movement occurred, we did not observe direct extension of microtubules from kinetochores to centrioles. This system should prove useful for experimental studies of spindle microtubule formation and chromosome movement in mammalian cells.The development of procedures for the in vitro assembly of microtubules from isolated tubulin subunits (12, 17) has provided important new information on the role of microtubule organizing centers (MTOC) in a variety of eukaryotic cells. The assembly of chick brain tubulin onto flagellar axonemes and basal bodies from Chlamydomonas and sea urchin sperm demonstrated that microtubular structures from several species can serve as nucleating sites for exogenous tubulin reassembly (1, 11, 13). More recently, polymeriza-