Nuclear architecture in budding yeast

芽殖酵母的核结构

• 批准号: 7967652
• 负责人: Orna Cohen-Fix
• 金额: $ 27.35万
• 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/7967652
• 关键词: Affect; Aging; Animal Model; Animals; Architecture; Biological Models; Caenorhabditis elegans; Candidate Disease Gene; Cell Cycle; Cell Death; Cell Nucleolus; Cell Nucleus; Cell Survival; Cells; Cellular biology; Centrosome; Chromatin; Chromosome Segregation; Code; Collaborations; Collection; DNA repair protein; Defect; Disease; Electron Microscopy; Elements; Endoplasmic Reticulum; Eukaryota; Exhibits; Genes; Genetic; Genetic Screening; Goals; Growth; Hand; Human; Induced Mutation; Lamins; Lead; Malignant Neoplasms; Membrane; Methodology; Microtubules; Mitosis; Morphology; Movement; Mutation; Nature; Nuclear; Nuclear Envelope; Nuclear Lamin; Nuclear Pore Complex; Nuclear Structure; Pathway interactions; Phospholipids; Play; Process; Protein Biosynthesis; Proteins; Rest; Role; Saccharomyces cerevisiae; Saccharomycetales; Screening procedure; Shapes; Structure; Testing; Translating; Yeasts; base; functional group; mutant; spindle pole body

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 phospholipid 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. We are in the process of studying how these proteins affect nuclear shape and what role they play in coordinating nuclear architecture with nuclear function. We also combined mutations in candidate genes with the spo7 mutation, looking for reduced growth in the double mutant. The candidate genes were selected based on the function and localization of the protein for which they code; most were involved in processes that take place near or at the nuclear membrane. Through this screen we found a mutant that was known to affect spindle pole body function. The yeast spindle pole body is equivalent to the metazoan centrosome and it acts in nucleating spindle microtubules during chromosome segregation and in nucleating cytoplasmic microtubules that are needed for nuclear movement. The mechanism by which the spindle pole body is inserted and maintained in the nuclear membrane is not known, but this is a question of immense importance because the mechanism of nuclear membrane insertion could apply to other nuclear membrane structures, such as nuclear pore complexes. We found that additional mutations known to abrogate spindle pole body assembly also relay on the Spo7 pathway for viability. These findings suggest that the composition of the nuclear membrane affects the function of nuclear membrane associated structures. Our studies are now focused on understanding the nature of this defect, using cell biology and genetic methodologies. We are also collaborating with Dr. Sue Jaspersen in order to look at spindle pole bodies by electron microscopy and to determine the relationship between nuclear membrane composition and the assembly of integral membrane structures. Finally, in collaboration with Dr. Brenda Anderws' lab, we screened through the yeast deletion collection for mutations that cause abnormal nuclear shape. Of the 5000 mutants tested, mutations in over two hundred genes resulted in altered nuclear morphology. We are currently screening through this subset to determine functional relationships between these genes. This subset contains a wide range of functional groups but is enriched in genes involved in certain processes, including DNA repair and protein synthesis. Our goal is to determine which of these genes contribute directly to nuclear architecture and which do so in an indirect manner.

核结构和核功能似乎密切相关：核组织的缺陷与衰老和癌症等疾病有关。我们一直使用芽殖酵母作为模型系统来研究核结构。酵母细胞核与高等真核生物的细胞核在两个方面不同：（1）酵母缺乏核纤层蛋白，核纤层蛋白是在后生动物细胞核形成中起主要结构作用的蛋白质。也有越来越多的证据表明核纤层蛋白有助于各种核过程。(2)酵母的核膜在整个细胞周期中保持完整，不像高等真核生物的核膜在有丝分裂过程中破裂。尽管如此，在我们实验室的早期研究中（坎贝尔等人，2006年），我们表明酵母细胞核的形状由三个因素决定：核膜的组成，染色质的形状，以及存在一种将核膜与染色质连接在一起的未识别的核结构，类似于高等真核生物的核纤层蛋白。我们先前的研究集中在Spo 7蛋白失活的酵母菌株上。Spo 7是磷脂合成的调节剂;在其缺失的情况下，磷脂水平增加，导致内质网（ER）和细胞核某些区域的扩张。特别是，我们能够证明，只有与核仁（细胞核的一个亚区室）相关的膜扩张，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链，该核系链将核膜与染色质联系在一起，并在磷脂水平增加时抵抗膜扩张。基于这一假设，我们假设使该系链失活将进一步改变核形状，达到核功能将严重受损的程度，这将反映在细胞活力降低上。因此，我们寻找与spo 7突变组合导致细胞死亡或严重生长缺陷的突变。我们进行了一个随机诱导的突变，导致细胞死亡的酵母背景缺乏Spo 7功能的屏幕（称为酵母合成致死屏幕）。随后对具有改变的核形状的突变体进行二次筛选。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。我们正在研究这些蛋白质如何影响核形状，以及它们在协调核结构与核功能方面发挥什么作用。 我们还将候选基因中的突变与spo 7突变相结合，寻找双突变体中生长减少的情况。候选基因是根据它们编码的蛋白质的功能和定位来选择的;大多数基因参与了发生在核膜附近或核膜上的过程。通过这个筛选，我们发现了一个已知影响纺锤体极体功能的突变体。酵母纺锤体极体相当于后生动物的中心体，它在染色体分离过程中起着使纺锤体微管成核的作用，并在细胞核运动所需的细胞质微管成核中起作用。纺锤体极体插入并保持在核膜中的机制尚不清楚，但这是一个非常重要的问题，因为核膜插入的机制可以应用于其他核膜结构，如核孔复合物。我们发现，已知废除纺锤体极体组装的其他突变也依赖于Spo 7通路的活力。这些发现表明，核膜的组成影响核膜相关结构的功能。我们的研究现在集中在利用细胞生物学和遗传学方法来了解这种缺陷的性质。我们还与Sue Jaspersen博士合作，通过电子显微镜观察纺锤体极体，并确定核膜组成与整体膜结构组装之间的关系。 最后，与Brenda Anderws博士的实验室合作，我们通过酵母删除集合筛选导致异常核形状的突变。在测试的5000个突变体中，超过200个基因的突变导致了核形态的改变。我们目前正在通过这个子集进行筛选，以确定这些基因之间的功能关系。这个子集包含广泛的功能基团，但富含参与某些过程的基因，包括DNA修复和蛋白质合成。我们的目标是确定这些基因中哪些直接影响核结构，哪些以间接方式影响核结构。