STRUCTURE OF KINETOCHORE-MICROTUBULE INTERACTIONS

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 我们正在使用冷冻固定，冷冻替代细胞，研究有丝分裂细胞中的动粒及其相关微管（MT）的形态。我们已经开发了用于制备良好冷冻的有丝分裂细胞的成功方法（Morphew和McIntosh，J.Micros212：21-25）。三维重建动粒从几个物种已经获得了使用双轴断层扫描。 已被定向和提取使用IMOD中的“切片器”工具的结构的kinetochorse相关的MT结束。 大多数动粒MT的壁在其正端向外张开，这是体外分解MT的形态学特征（Mandelkow等人，JCB 114：977，1991）。扩口的程度已被量化，并发现类似地分布在所有有丝分裂阶段。 这些研究结果表明，在体内的耀斑是不是简单的解体的指示，但可能反映了不同的动态状态，可以由其他细胞过程控制。 动粒MT周围的区域含有细长的纤维，似乎连接在着丝粒异染色质中的MT壁。 这些发现提示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. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. 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.