CHARACTERIZATION OF THE MEIOTIC SPINDLE POLE IN BUDDING YEAST

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， 因此可以在其他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。 在芽殖酵母中，纺锤体极体（SPB）是细胞中唯一的微管组织中心。然而，在减数分裂期间，SPB的细胞质面从微管成核的位点转化为膜形成的位点。这种转化是通过募集至少四种减数分裂特异性蛋白质来完成的，这些蛋白质取代γ-微管蛋白复合物并形成一种新的结构，称为减数分裂外斑（MOP），其作为从头膜形成的位点。MOP是一种高度有序的结构，已经鉴定了定位于MOP的几种蛋白质，但它们如何排列以及它们如何促进膜形成尚不清楚。 我们有免疫标记的S.具有与GFP融合的不同纺锤体极体蛋白的酿酒酵母。 Ady 4p和Spo 74 p都定位于MOP，并已显示出促进原孢子膜生长。该项目的目标是开发这种膜组织中心的高分辨率结构模型。复合物的小尺寸（直径为300- 400 nm）使得免疫EM和EM断层扫描对于这种分析至关重要。通过将这些技术与缺少MOP的单个蛋白组分的突变体以及基于荧光的分析结果相结合，将产生MOP组织的稳健模型。这种模型将是理解MOP如何促进细胞内膜形成的长期目标的重要一步。我们已经开始表征野生型细胞中前孢子膜（PSM）形成的第一阶段以及Spo 14缺失突变体中PSM形成的缺乏。

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.