Nuclear architecture in budding yeast

芽殖酵母的核结构

• 批准号: 7734250
• 负责人: Orna Cohen-Fix
• 金额: $ 23.51万
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7734250
• 关键词: Affect; Aging; Animal Model; Animals; Architecture; Biological Models; Caenorhabditis elegans; Cell Cycle; Cell Death; Cell Nucleolus; Cell Nucleus; Cell Survival; Cells; Chromatin; Code; Condition; Defect; Disease; Elements; Endoplasmic Reticulum; Eukaryota; Eukaryotic Cell; Genes; Genetic; Growth; Hand; Human; Induced Mutation; Lamins; Lead; Malignant Neoplasms; Membrane; Mitosis; Morphology; Mutation; Nuclear; Nuclear Envelope; Nuclear Lamin; Nuclear Structure; Nucleolar Proteins; Phospholipids; Play; Process; Proteins; Rest; Role; Role playing therapy; Saccharomycetales; Shapes; Translating; Visual; Yeasts; base; mutant

Nuclear architecture and nuclear function appear to go hand in hand: defects in nuclear organization are associated with aging and diseases such as cancer. We have been using budding yeast as a model system to study nuclear architecture. The yeast nucleus differs from that of higher eukaryotes in two aspects: (1) yeast lack lamins, proteins that play a major structural role in shaping the nucleus in cells of metazoans. There is also accumulating evidence to suggest that lamins contribute to various nuclear processes. (2) The yeast nuclear membrane remains intact throughout the cell cycle, unlike nuclear membrane of higher eukaryotes, which breaks down during mitosis. Nonetheless, in an earlier study from our lab (Campbell et al, 2006), we showed that the shape of the yeast nucleus is determined by three factors: the composition of the nuclear membrane, the shape of the chromatin, and the presence of an unidentified nuclear structure that tethers the nuclear membrane to the chromatin, akin to nuclear lamins of higher eukaryotes. Our previous study focused on a yeast strain in which the Spo7 protein was inactivated. Spo7 is a regulator of phospholipids synthesis; in its absence phospholipids levels increase, leading to the expansion of the endoplasmic reticulum (ER) and certain regions of the nucleus. In particular, we were able to show that only the membrane associated with the nucleolus (a sub-compartment of the nucleus) expands, whereas the rest of the nuclear membrane remains juxtaposed to the bulk of the chromatin. This led to the hypothesis that in yeast there is a nuclear tether that associates the nuclear membrane to the chromatin and resists membrane expansion when phospholipids levels increase. Based on this hypothesis we assume that inactivating this tether will further alter nuclear shape, to the point where nuclear functions will be severely compromised, and this will be reflected in reduced cell viability. Thus, we sought mutations whose combination with a spo7 mutation leads to cell death or severe growth defects.We conducted a screen for randomly induced mutations that cause cell death in a yeast background lacking Spo7 function (known as a yeast synthetic lethal screen). This was followed by a secondary screen for mutants that have an altered nuclear shape. So far we have identified over a dozen mutations that lead to an altered nuclear shape, two of which are in genes coding for nucleolar protein. We are in the process of identifying the remaining genes. Once identified, we will study how these proteins affect nuclear shape and what role they play in coordinating nuclear architecture with nuclear function.

核结构和核功能似乎是齐头并进的：核组织的缺陷与衰老和癌症等疾病有关。我们一直使用发芽酵母作为研究核结构的模型系统。酵母核与高等真核生物的核在两个方面不同：(1)酵母缺乏层蛋白，层蛋白在后生动物细胞中对细胞核的形成起主要结构作用。也有越来越多的证据表明，板层对各种核过程都有贡献。(2)酵母核膜在整个细胞周期中保持完整，与高等真核生物的核膜不同，核膜在有丝分裂期间破裂。尽管如此，在我们实验室的早期研究中(Campbell等人，2006年)，我们表明酵母核的形状由三个因素决定：核膜的组成，染色质的形状，以及是否存在一种将核膜与染色质捆绑在一起的未知核结构，类似于高等真核生物的核层。我们之前的研究集中在一株Spo7蛋白失活的酵母菌株上。Spo7是磷脂合成的调节剂；如果没有Spo7，磷脂水平会增加，导致内质网(ER)和细胞核的某些区域扩张。特别是，我们能够证明，只有与核仁相关的膜(核的一个亚室)扩张，而核膜的其余部分仍然与染色质的大部分并列。这导致了一种假设，即在酵母中，存在一种核系带，它将核膜与染色质联系在一起，并在磷脂水平增加时抵制核膜的扩张。基于这一假设，我们假设，使这种系链失活会进一步改变核的形状，以至于核功能将严重受损，这将反映在细胞存活率的降低上。因此，我们寻找与sp7突变结合导致细胞死亡或严重生长缺陷的突变。我们在缺乏Spo7功能的酵母背景下进行了随机诱导突变的筛选(称为酵母合成致死筛选)。随后对核形状发生变化的突变体进行了二次筛查。到目前为止，我们已经确定了十几种导致核形状改变的突变，其中两种是在编码核仁蛋白的基因中。我们正在鉴定剩下的基因。一旦确定，我们将研究这些蛋白质如何影响核形状，以及它们在协调核结构和核功能方面发挥什么作用。