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

点击翻译按钮获取中文摘要

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.