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

开发微品研究主轴杆体结构

• 批准号: 7602186
• 负责人: ERIC MULLER
• 金额: $ 4.88万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-09-01 至 2008-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7602186
• 关键词: Cell division; Cells; Centrosome; Chromosome Segregation; Complex; Computer Retrieval of Information on Scientific Projects Database; Cyclophosphamide/Fluorouracil/Prednisone; DNA; Development; Electron Microscopy; Fluorescence Resonance Energy Transfer; Funding; Genome; Grant; Image; Institution; Kinetochores; Localized; Measures; Methods; Microscopic; Microtubule-Organizing Center; Microtubules; Mitotic spindle; Positioning Attribute; Proteins; Research; Research Personnel; Resolution; Resources; Source; Structural Protein; Structure; United States National Institutes of Health; Yeasts; genetic analysis; mutant; spindle pole body; yeast two hybrid system

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.

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 中心体是细胞的微管组织中心。在细胞分裂过程中，两个中心体，在有丝分裂纺锤体的两端，通过锚定微管连接到动粒来定位DNA。因此，中心体建立了染色体分离的极性，这是细胞基因组正确遗传所需的关键步骤。在酵母中，中心体被称为纺锤体极体（SPB）。SPB包含10个主要结构蛋白。原子分辨率下SPB的详细结构尚未解决。然而，通过电子显微镜，双杂交分析和遗传学的结合，已经实现了结构的轮廓。 我们建议通过进一步发展荧光共振能量转移（FRET）在蛋白质复合物研究中的应用来探测SPB的结构。为了使FRET发生在两个荧光分子之间，它们必须在80埃内。这种距离要求使得FRET成为两种蛋白质接近程度的极好测量。该项目建立在我们开发的显微镜方法来检测CFP-和YFP-标记的蛋白质之间的FRET。已知定位于SPB的蛋白质将用YFP或CFP标记。将改进图像采集和分析。我们将扩展我们的研究，以检查SPB突变体，破坏组织的SPB。