STRUCTURE OF KINETOCHORE-MICROTUBULE INTERACTIONS

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。我们使用冷冻固定、冷冻替代的细胞来研究有丝分裂细胞中着丝点及其相关微管（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. Micros 212:21-25）。利用双轴断层扫描获得了几种物种的着丝点的三维重建。使用IMOD中的“切片器”工具对着丝酶相关的MT端结构进行了定向和提取。大多数着丝点mt的细胞壁在正端向外伸展，这是体外解体mt的形态学特征（Mandelkow et al., JCB 114:977, 1991）。燃烧的程度已被量化，并发现在所有有丝分裂阶段分布相似。这些发现表明，体内的燃烧并不仅仅表明分解，而可能反映了一种不同的动态状态，这种动态状态可以由其他细胞过程控制。着丝点MT周围的区域含有细长的原纤维，在着丝点异染色质中似乎连接MT壁。这些发现提示了染色体与有丝分裂纺锤体结合的新机制。

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.