CHARACTERIZATION OF THE MEIOTIC SPINDLE POLE IN BUDDING YEAST

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。 在芽殖酵母中，纺锤体是细胞中唯一的微管组织中心。然而，在减数分裂过程中，SPB的细胞质表面从微管成核的部位转变为膜形成的部位。这种转化是通过招募至少四个减数分裂特异蛋白来完成的，这些蛋白取代了伽马-微管蛋白复合体，形成了一种新的结构，称为减数分裂外斑(MOP)，它是从头开始膜形成的场所。拖把是一个高度有序的结构，已经鉴定了几种定位于拖把的蛋白质，但它们是如何排列的，以及它们如何促进膜的形成尚不清楚。我们有免疫标记的酿酒酵母菌株，它们有不同的纺锤体极体蛋白与GFP融合。Ady4P和Spo74P都定位在拖把上，并被证明能促进孢子膜的生长。本项目的目标是开发这个膜组织中心的高分辨率结构模型。小尺寸的复合体(直径300-400 nm)使得免疫电子显微镜和电子显微镜断层扫描对于这一分析是必不可少的。通过将这些技术与缺乏MOP个别蛋白质成分的突变体以及基于荧光的分析结果相结合，将产生一个稳健的MOP组织模型。这样的模型将是理解MOP如何促进细胞内膜形成的长期目标的重要一步。我们已经开始对野生型细胞形成前孢子膜的第一阶段以及Spo14缺失突变体中缺乏前孢子膜的形成进行了表征。

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. In budding yeast, the spindle pole body (SPB) is the sole microtubule organizing center of the cell. During meiosis, however, the cytoplasmic face of the SPB is converted from a site of microtubule nucleation to a site of membrane formation. This transformation is accomplished by the recruitment of at least four meiosis-specific proteins that displace the gamma-tubulin complex and form a novel structure, termed the meiotic outer plaque (MOP), which serves as a site of de novo membrane formation. The MOP is a highly ordered structure and several proteins that localize to the MOP have been identified, but how they are arranged and how they promote membrane formation is not known. We have immuno labeled strains of S. cerevisiae that have different spindle pole body proteins fused with GFP. Both Ady4p and Spo74p localize to the MOP and have been shown to promote prospore membrane growth. The goal of this project is to develop a high-resolution structural model of this membrane organizing center. The small size of the complex (300-400nm in diameter) make Immuno-EM and EM tomography essential for this analysis. By combining these techniques with mutants lacking individual protein components of the MOP, as well as results from fluorescence-based analysis, a robust model of the organization of the MOP will be generated. Such a model will be an important step in the long-term goal of understanding how the MOP promotes the formation of intracellular membranes. We have begun to characterize the first stages of pro-spore membrane (PSM) formation in wild type cells as well as the lack of PSM formation in Spo14 deletion mutants.