THE DEVELOPMENT OF FRET TO STUDY THE STRUCTURE OF THE SPINDLE POLE BODY

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 中心体是细胞的微管组织中心。在细胞分裂期间，位于有丝分裂纺锤体相反两端的两个中心体，通过锚定附着在动粒上的微管来定位DNA。因此，中心体确定了染色体分离的极性，这是细胞基因组正常遗传所需的关键步骤。在酵母中，中心体称为纺锤体极体(SPB)。SPB含有10种主要结构蛋白。原子分辨率下SPB的详细结构尚未解决。然而，通过结合电子显微镜、双杂交分析和遗传学，已经获得了该结构的轮廓。 我们建议通过进一步发展荧光共振能量转移(FRET)在蛋白质复合体研究中的应用来探索SPB的结构。要在两个荧光分子之间发生FRET，它们必须在80埃以内。这一距离要求使FRET成为衡量两种蛋白质接近程度的极佳方法。这个项目建立在我们开发的检测CFP和YFP标记蛋白之间的FRET的微观方法的基础上。已知定位于SPB的蛋白质将被标记为YFP或CFP。将改进图像采集和分析。我们将扩大我们的研究，以检查破坏SPB组织的SPB突变体。

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. The centrosome is the microtubule organizing center of the cell. During cell division two centrosomes, at opposite ends of the mitotic spindle, position the DNA by anchoring the microtubules attached to the kinetochores. The centrosomes thereby establish the polarity of chromosome segregation, a critical step required for the proper inheritance of the cellular genome. In yeast the centrosome is referred to as the spindle pole body (SPB). The SPB contains 10 major structural proteins. A detailed structure of the SPB at atomic resolution has not been solved. However an outline of the structure has been achieved through a combination of electron microscopy, two-hybrid analysis, and genetics. We propose to probe the structure of the SPB by further developing the application of fluorescence resonance energy transfer (FRET) to the study of protein complexes. For FRET to occur between two fluorescent molecules, they must be within 80 angstroms. This distance requirement makes FRET an excellent measure of the proximity of two proteins. This project builds on our development of microscopic methods to detect FRET between CFP- and YFP-tagged proteins. Proteins known to localize to the SPB will be tagged with either YFP or CFP. Improvements in image acquisition and analysis will be developed. We will extend our study to examine SPB mutants that disrupt the organization of the SPB.