Assembly and Regulation of Yeast Spindle Poles

项目总结 准确地传递遗传信息是生长、增殖和发育所必需的 组织和有机体。忠实的染色体分离涉及许多事件，包括复制 在后生动物和纺锤体中称为中心体的微管组织中心(MTOC) (SPBS)在真菌中，每个细胞周期一次且仅一次。为了使细胞质微管装置 从MTOCs发出以访问核中的染色体，核膜(NE)也必须 在细胞分裂过程中被重塑。在某些细胞类型中，NE完全或部分分解，而在另一些类型中 仍然完好无损。中心体和SPB对这两种机制的适应不同，如NE所示 SPBS在酿酒酵母和裂殖酵母中的插入。尽管着丝粒 和SPB在结构上是不同的，这两种类型的MTOC都复制、生长并与NE相互作用。中的缺陷 这些事件中的任何一种都可能导致纺锤体错误，这通常会导致染色体的获得或丢失 (非整倍体)。酵母中的非整倍体通常是可以容忍的，但在人类中，它经常与癌症有关 由于未发现的隐性突变的变化或蛋白质复合体的变化。这项建议旨在 阐明细胞用来将中心体/SPB复制限制在每个细胞周期一次的保守原理 确保MTOC达到的大小足以使成核能力足以进行染色体分离 将中心体/SPBS插入或系在NE上。我们创新的双色结构照明显微镜(SIM) 单粒子平均(SPA)方法使我们的工作脱颖而出，因为我们能够解决SPB 这些事件所需的特征和复制中间体，而不是用电子观察到的 显微镜、生化、遗传学或其他超分辨方法。在这项工作中，我们建立在 我们已经进行的观察，并通过配对荧光共振能量进一步扩展了成像技术 使用SIM卡进行转接(FRET)。这一进展使我们能够研究SPB过程中蛋白质之间的相互作用 复制并与成熟SPB中的蛋白质-蛋白质相互作用进行比较，以了解中心体是如何 队形被控制了。我们的初步数据表明，物理相互作用是动态和变化的 在整个SPB复制周期中，我们将通过检查磷酸化和 其他依赖细胞周期的修饰。使用SIM，我们可以可视化SPB孔-环状结构 将可溶性的SPB锚定在NE中。这种结构和相关的孔隙形成的机制 核孔复合体的形成还知之甚少。因为我们可以观察到酵母中的SPB孔，所以我们可以 剖析细胞用来在NE上形成这个洞的分子事件。我们的总体目标是确定 协调中心体复制和DNA复制的机制(Aim1)，阐明了 允许SPB插入NE的事件(目标2)，并研究SPB的大小和微管成核 对照(目标3)结合遗传学和分子方法进行成像。

PROJECT SUMMARY Accurate transmission of genetic information is required for growth, proliferation and development of tissues and organisms. Faithful segregation of chromosomes involves many events, including the duplication of microtubule organizing centers (MTOCs), known as centrosomes in metazoans and spindle pole bodies (SPBs) in fungi, once and only once per cell cycle. In order for the cytoplasmic microtubule apparatus emanating from the MTOCs to access the chromosomes in the nucleus, the nuclear envelope (NE) must also be remodeled during cell division. In some cell types the NE entirely or partially disassembles while in others it remains intact. Centrosomes and SPBs adapt differently to these two mechanisms, as illustrated by the NE insertion of SPBs in both Saccharomyces cerevisiae and Schizosacchromyces pombe. Although centrosomes and SPBs are structurally distinct, both types of MTOCs duplicate, grow and interact with the NE. Defects in any of these events could lead to spindle errors, which often result in the gain or loss of a chromosome (aneuploidy). Aneuploidy in yeast often can be tolerated, but in humans it is frequently associated with cancer due to changes in uncovered recessive mutations or alterations in protein complexes. This proposal seeks to elucidate conserved principles used by the cell to restrict centrosome/SPB duplication to once per cell cycle, to ensure the MTOC reaches a size where nucleation capacity is sufficient for chromosome segregation and to insert or tether centrosomes/SPBs to the NE. Our innovative two-color structured illumination microscopy (SIM) with single-particle averaging (SPA) approach sets our work apart because we are able to resolve SPB features and duplication intermediates required for these events that were not observed using electron microscopy, biochemical, genetic or other super-resolution methods. In this work, we build upon the observations we have made and further extend imaging technology by pairing fluorescence resonance energy transfer (FRET) with SIM. This advancement allows us to study protein-protein interactions during SPB duplication and compare them to protein-protein interactions in a mature SPB to understand how centrosome formation is controlled. Our preliminary data suggests that physical interactions are dynamic and change throughout the SPB duplication cycle, an idea that we will further investigate by examining phosphorylation and other cell cycle-dependent modifications. Using SIM, we can visualize the SPB pore—the ring-like structure that anchors the soluble SPB in the NE. The mechanisms by which this structure and the related pore at nuclear pore complexes form is poorly understood. Because we can observe the SPB pore in yeast, we can dissect the molecular events used by cells to create this hole in the NE. Our overall objective is to determine mechanisms that coordinate centrosome duplication with DNA replication (Aim1), elucidate the molecular events that allow the SPB to insert into the NE (Aim 2) and study how SPB size and microtubule nucleation is controlled (Aim 3) using imaging in combination with genetic and molecular methods.