Nuclear architecture in budding yeast

芽殖酵母的核结构

• 批准号: 8939643
• 负责人: Orna Cohen-Fix
• 金额: $ 59.91万
• 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/8939643
• 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; Exhibits; Gene Silencing; 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; Shapes; System; Temperature; Testing; Transport Vesicles; Universities; Vesicle; Work; Yeasts; base; falls; genome wide association study; high throughput screening; mutant; screening; temperature sensitive 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. To understand what regulated nuclear envelope expansion and how the nuclear envelope extension is confined to the nucleolus in mitotically arrested cells, we are in the process of identifying conditional (temperature sensitive) mutants that either fail to form an extension upon arresting in mitosis, or form multiple extensions at the non-permissive temperature. Thus far we have identified several mutants in the first class. These fall into two groups: mutants that expands the nuclear envelope isometrically and those that fail to expand altogether. We have also identified several mutants that exhibit abnormal nuclear shape upon gene inactivation. The identify of these mutants and the mechanisms by which they affect nuclear morphology are under investigation.

核结构和核功能似乎是齐头并进的，因为核组织的缺陷与衰老和癌症等疾病有关。我们一直使用发芽酵母作为研究核结构的模型系统。酵母细胞核与高等真核生物在两个方面不同：(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；(2)酵母核膜(NE)在整个细胞周期中保持完整，不同于高等真核生物的NE，后者在有丝分裂期间分解，并在染色体分离完成后重新组装。然而，酵母细胞核与高等真核生物的细胞核有重要的共同特征：NE在细胞周期中必须扩张，并且细胞核必须获得并保持与细胞体积成正比的球形体积。在所有的系统中，核子核如何扩展以及是什么决定了核的大小和形状都是有待解决的问题。 我们之前的研究集中在一株Spo7蛋白失活的酵母菌株上。Spo7是一种保守的磷脂合成调节剂；如果没有它，磷脂水平会增加，导致内质网(ER)的扩张。在酵母中，这也伴随着细胞核的某些区域的扩张。特别是，我们能够证明，只有与核仁(核的一个亚室)相关的NE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，有一条核绳将核膜与染色质联系在一起，当磷脂水平增加时，它会阻止NE的扩张。我们随后的研究表明，在膜过剩的条件下(例如，在没有Spo7功能的情况下)，囊泡运输参与了决定核形状的过程。发生这种情况的机制目前正在调查中。 为了更好地了解NE的不同结构域是如何确定的，并发现更多影响核形状的蛋白质，我们与布伦达·安德鲁斯博士(多伦多大学)的实验室合作进行了高通量筛选，其中我们通过酵母缺失集合筛选导致核形状异常的突变。在测试的5000个突变体中，近100个基因的突变导致了核形态的改变。我们目前正在对这个子集进行筛选，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个突变在染色体分离之前导致了有丝分裂的细胞周期延迟。在所有这些突变体中，核的延伸与与核仁相关的去甲肾上腺素一致，很像在sp7突变体中。我们发现，导致细胞周期延迟的突变体的核形态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周期细胞相同的速度积累。我们的数据表明，在有丝分裂延迟期间，尽管染色体分离受阻，细胞仍继续向NE增加膜，在这种情况下，增加的膜并不是均匀地分布在整个细胞核周围，而是NE只扩大了与核仁相邻的区域。监管这一过程的机制目前正在调查中。 为了了解是什么调控了核膜的扩张，以及核膜的延伸是如何限制在有丝分裂停止的细胞中的核仁，我们正在寻找条件(温度敏感)突变体，这些突变体要么在有丝分裂停止时不能形成延伸，要么在不允许的温度下形成多个延伸。到目前为止，我们已经鉴定了几个第一类突变体。这些突变分为两类：一类是核包膜等轴向扩展的突变体，另一类是完全不能扩展的突变体。我们还鉴定了几个突变体，它们在基因失活时表现出异常的核形状。这些突变体的鉴定和它们影响核形态的机制正在调查中。