Nuclear architecture in budding yeast

芽殖酵母的核结构

• 批准号: 8553564
• 负责人: Orna Cohen-Fix
• 金额: $ 52.07万
• 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/8553564
• 关键词: Affect; Aging; Animal Model; Architecture; Biological Models; Caenorhabditis elegans; Cell Cycle; Cell Cycle Regulation; Cell Cycle Stage; Cell Nucleolus; Cell Nucleus; Cell Volumes; Cells; Chromatin; Chromosome Segregation; Collaborations; Collection; Cytology; Data; Defect; Disease; Elements; Endoplasmic Reticulum; Eukaryota; Genes; Genetic Screening; Goals; Investigation; Knowledge; Lamins; Lipids; Malignant Neoplasms; Membrane; Mitosis; Mitotic; Morphology; Mutate; Mutation; Nuclear; Nuclear Envelope; Outcome; Pathology; Pathway interactions; Phospholipids; Play; Process; Proteins; Regulation; Rest; Role; Saccharomycetales; Screening procedure; Shapes; System; Testing; Transport Vesicles; Universities; Vesicle; Work; Yeasts; base; genome wide association study; high throughput screening; mutant; trafficking

Nuclear architecture and nuclear function appear to go hand in hand, as 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, and (2) the yeast nuclear envelope (NE) remains intact throughout the cell cycle, unlike the NE of higher eukaryotes, which breaks down during mitosis and reassembles after chromosome segregation is complete. Nonetheless, the yeast nucleus shares important features with nuclei of higher eukaryotes: the NE has to expand during the course of the cells cycle, and the nucleus has to acquire and maintain a spherical shape of a volume proportional to cell volume. How the NE expands and what determine nuclear size and shape are questions that remain to be resolved in all systems. Our previous studies focused on a yeast strain in which the Spo7 protein was inactivated. Spo7 is a conserved regulator of phospholipids synthesis; in its absence phospholipids levels increase, leading to the expansion of the endoplasmic reticulum (ER). In yeast, this is also accompanied by expansion of certain regions of the nucleus. In particular, we were able to show that only the NE 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 NE expansion when phospholipid levels increase. Our subsequent studied revealed that vesicle trafficking is involved in determining nuclear shape under conditions of excess membrane (e.g. in the absence of Spo7 function). The mechanism by which this happens is currently under investigation. To gain a better understanding of how the different domains of the NE are determined and to uncover additional proteins that affect nuclear shape, we conducted a high throughput screen in collaboration with Dr. Brenda Anderws' lab (University of Toronto), in which we screened through the yeast deletion collection for mutations that cause abnormal nuclear shape. Of the 5000 mutants tested, mutations in nearly one hundred genes resulted in altered nuclear morphology. We are currently screening through this subset to determine functional relationships between these genes and how they affect nuclear shape. Among the mutations that led to abnormal nuclear shape were roughly 40 mutants that caused a cell cycle delay in mitosis, prior to chromosome segregation. In all of these mutants the nucleus developed an extension that coincided with the NE associated with the nucleolus, much like in the spo7 mutant. We showed that the altered nuclear morphology in mutants that cause a cell cycle delay was not due to a direct involvement of these genes in nuclear architecture, but rather that a mitotic delay, in and of itself, leads to altered nuclear shape. Delay in other cell cycle stages does not result in changes to nuclear morphology. We also found that during a mitotic delay, phospholipids continue to accumulate at the same rate as in wild type cycling cells. Our data suggest that during a mitotic delay, cell continue to add membrane to the NE despite the block to chromosome segregation, and that under these circumstances the added membrane is not distributed evenly around the entire nucleus, but rather the NE expands only the region adjacent to the nucleolus. The mechanism that regulates this process is currently under investigation.

核结构和核功能似乎齐头并进，因为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有所不同：(1) 酵母缺乏核纤层蛋白，即在后生动物细胞核形成过程中起主要结构作用的蛋白质；(2) 酵母核膜 (NE) 在整个细胞周期中保持完整，这与高等真核生物的 NE 不同，后者在有丝分裂期间分解并在染色体分离后重新组装。 完全的。尽管如此，酵母细胞核与高等真核生物的细胞核具有相同的重要特征：NE必须在细胞周期过程中扩张，并且细胞核必须获得并保持体积与细胞体积成比例的球形。 NE 如何扩展以及什么决定核的大小和形状是所有系统中仍有待解决的问题。 我们之前的研究重点是 Spo7 蛋白失活的酵母菌株。 Spo7 是磷脂合成的保守调节因子；在缺乏磷脂的情况下，磷脂水平增加，导致内质网（ER）扩张。在酵母中，这也伴随着细胞核某些区域的扩张。特别是，我们能够证明，只有与核仁（细胞核的一个子区室）相关的 NE 扩张，而核膜的其余部分仍然与大部分染色质并列。这导致了这样的假设：在酵母中存在一种核系链，它将核膜与染色质联系起来，并在磷脂水平增加时抵抗 NE 扩张。我们随后的研究表明，在膜过多的情况下（例如，在缺乏 Spo7 功能的情况下），囊泡运输参与确定核形状。目前正在研究发生这种情况的机制。 为了更好地了解 NE 的不同结构域是如何确定的，并发现影响核形状的其他蛋白质，我们与 Brenda Anderws 博士的实验室（多伦多大学）合作进行了高通量筛选，通过酵母缺失集合筛选导致异常核形状的突变。在测试的 5000 个突变体中，近一百个基因的突变导致了核形态的改变。我们目前正在筛选这个子集，以确定这些基因之间的功能关系以及它们如何影响核形状。 在导致核形状异常的突变中，大约有 40 个突变体在染色体分离之前导致细胞周期有丝分裂延迟。在所有这些突变体中，细胞核都形成了与核仁相关的 NE 一致的延伸，就像 spo7 突变体一样。我们表明，导致细胞周期延迟的突变体中核形态的改变并不是由于这些基因直接参与核结构，而是有丝分裂延迟本身导致了核形状的改变。其他细胞周期阶段的延迟不会导致核形态的变化。我们还发现，在有丝分裂延迟期间，磷脂继续以与野生型循环细胞相同的速率积累。我们的数据表明，在有丝分裂延迟期间，尽管染色体分离受到阻碍，细胞仍继续向 NE 添加膜，并且在这种情况下，添加的膜不会均匀分布在整个细胞核周围，而是 NE 仅扩展与核仁相邻的区域。目前正在研究调节这一过程的机制。