STRUCTURE OF KINETOCHORE-MICROTUBULE INTERACTIONS

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。我们正在使用冷冻固定、冷冻替代细胞来研究有丝分裂细胞中动粒及其相关微管(MT)的形态。我们已经开发了成功的方法来制备冷冻良好的有丝分裂细胞(Morphew和McIntosh，J.Micros212：21-25)。利用双轴层析成像获得了几个物种的动点的三维重建。动粒相关MT末端的结构已经用IMOD中的“切片器”工具进行了定位和提取。大多数动粒MT的外壁在其正端向外张开，这是体外分解MT的一种形态特征(Mandelkow等人，JCB 114：977,1991)。闪光的程度已经被量化，并发现在所有有丝分裂阶段都是相似的分布。这些发现表明，体内的发光并不是简单的拆卸，而是可能反映了一种可以由其他细胞过程控制的不同的动态状态。动粒MT周围的区域含有细长的纤维，这些纤维似乎连接着丝粒异染色质中的MT壁。这些发现表明了染色体与有丝分裂纺锤体结合的一种新机制。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. We are using cryofixed, freeze-substituted cells to study the morphology of kinetochores and their associated microtubule (MT) in mitotic cells. We have developed successful methods for preparing well-frozen mitotic cells (Morphew and McIntosh, J. Micros 212:21-25). 3-D reconstructions of kinetochores from several species have been obtained using dual-axis tomography. The structures of kinetochore-associated MT ends have been oriented and extracted using the "slicer" tool in IMOD. The walls of most kinetochore MTs flare outward at their plus ends, a morphology characteristic of disassembling MTs in vitro (Mandelkow et al., JCB 114:977, 1991). The degree of flaring has been quantified and found to be similarly distributed at all mitotic stages. These findings suggest that flaring in vivo is not simply indicative of disassembly, but may reflect a different dynamic state that can be controlled by other cellular processes. The regions around kinetochore MTs contain slender fibrils that appear to connect the MT walls in the centromeric heterochromatin. These findings suggest a novel mechanism for the binding of chromosomes to the mitotic spindle.