Replacement of Widefield Imaging System

项目摘要 这项研究的主要目的是确定蛋白酶体沿着减数分裂染色体的作用。的 26 S蛋白酶体是所有真核生物蛋白质降解的主要场所。它是一个分隔的， 蛋白酶是一种多组分蛋白酶，存在于细胞质和细胞核中。胞质蛋白酶体 在消除调节蛋白和错误折叠蛋白中的功能已经被认识了很长时间。功能 对细胞核中蛋白酶体的了解要少得多。我们最近发现在减数分裂I期间， 26 S蛋白酶体以进化上保守的方式被募集到染色体上， 同源配对、突触和重组。使用高分辨率显微镜，遗传学 和蛋白质组学方法，我们正在追求以这一发现为基础的三个目标：首先，我们正在使用 严格控制的条件等位基因，以系统地表征不同的减数分裂过程中的作用， 蛋白酶体组分。第二，我们正在鉴定蛋白酶体的底物， 染色体配对和重组，提供了对一类分子的见解，需要 降解以确保正常的减数分裂进程。第三，我们正在确定蛋白酶体的决定因素， 补充到染色体上。在缺乏功能性染色体分离缺陷的情况下， 蛋白酶体强调更好地了解染色体对生殖健康的重要性， 相关蛋白水解我们的分析也与蛋白酶体抑制剂具有 变得越来越重要。我们的许多方法都涉及高分辨率和活细胞成像， 真核芽殖酵母的模型。在这份补充提案中，我们要求提供资金， 高分辨率、实时成像系统，以取代我们老化的现有显微镜。我们当前系统 给了我们宝贵的见解，但已变得不可靠，并接近其寿命结束。一个国家的- 艺术成像系统将使我们能够保持和进一步加强高影响力的研究计划， 染色体传递的机制。

PROJECT SUMMARY The central aim of the parent grant is to identify roles of the proteasome along meiotic chromosomes. The 26S proteasome is the main site of protein degradation in all eukaryotes. It is a compartmentalized, multicomponent protease that resides both in the cytoplasm and the nucleus. Cytoplasmic proteasome functions in eliminating regulatory and misfolded proteins have been recognized for a long time. Functions of the proteasome in the nucleus are much less understood. We recently discovered that during meiosis I, the 26S proteasome is recruited in an evolutionarily conserved manner to chromosomes where it controls homolog pairing, synapsis and recombination. Using a combination of high-resolution microscopy, genetic and proteomic approaches, we are pursuing three aims that build on this discovery: First, we are using tightly controlled conditional alleles to systematically characterize roles during meiosis of different proteasome components. Second, we are identifying substrates of the proteasome relevant to chromosome pairing and recombination, providing insights into a class of molecules that need to be degraded to ensure normal meiotic progression. Third, we are identifying determinants for proteasome recruitment to chromosomes. Meiotic chromosome segregation defects in absence of a functional proteasome emphasize the importance for reproductive health of a better understanding of chromosome- associated proteolysis. Our analysis is also relevant to cancer therapy where proteasome inhibitors have become increasingly important. Many of our approaches involve high resolution and live-cell imaging in the model eukaryote budding yeast. In this supplement proposal, we are requesting funds for an advanced high-resolution, live imaging system as replacement for our aging current microscope. Our current system has given us valuable insights but has become unreliable and is near the end of its lifetime. A state-of-the- art imaging system will enable us to maintain and further enhance a high impact research program into mechanisms of chromosome transmission.