Investigating how nuclear pore complexes regulate aging

研究核孔复合物如何调节衰老

• 批准号: 8783111
• 负责人: Christopher L Lord
• 金额: $ 5.33万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-12-01 至 2015-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8783111
• 关键词: Accounting; Active Biological Transport; Address; Affect; Age; Aging; Aging-Related Process; Biochemical; Biological; Biological Assay; Biotinylation; Caliber; Cardiovascular Diseases; Cell Aging; Cell Nucleus; Cell Wall; Cell physiology; Cells; Characteristics; Cytoplasm; Data; Daughter; Defect; Development; Diffuse; Eukaryota; Eukaryotic Cell; Event; Exhibits; Face; Fibroblasts; Gene Expression Profile; Genetic; Genetic Epistasis; Glycerol; Humanities; Lamin Type A; Learning; Link; Longevity; Longevity Pathway; Malignant Neoplasms; Measures; Mediating; Microdissection; Mitochondria; Modeling; Molecular; Mothers; Mutation; Nuclear; Nuclear Envelope; Nuclear Import; Nuclear Pore Complex; Nuclear Pore Complex Proteins; Organelles; Organism; Pathway interactions; Patients; Permeability; Phenotype; Physiological; Process; Progeria; Proteins; Rattus; Regulation; Reporter; Risk Factors; Role; Saccharomyces cerevisiae; Saccharomycetales; Severities; Signal Pathway; Streptavidin; Suggestion; Syndrome; Techniques; Testing; Yeasts; age effect; age related; aged; base; cell age; cell type; daughter cell; in vivo; insight; mutant; nervous system disorder; nucleocytoplasmic transport; overexpression; public health relevance; research study; scaffold; screening; small molecule; trafficking; transcriptome sequencing

DESCRIPTION (provided by applicant): Nuclear pore complexes (NPCs) mediate transport between the nucleus and cytoplasm of eukaryotic cells. NPCs are large assemblies in the nuclear membrane that are composed of nucleoporins (Nups), which perform scaffolding, structural, and transport functions. Previous studies have shown the composition and functions of NPCs deteriorate as cells and organisms age, leading to the hypothesis that these changes may account for some of the detrimental phenotypes present in aged cells. It is currently unclear, however, if disrupted NPC function is a cause or effect of aging. Preliminary data demonstrates deletion of specific Nup domains regulates replicative life span (RLS) in the yeast S. cerevisiae. RLS, which is defined as the number of daughters a yeast cell produces, is often utilized as a model for metazoan aging due to the conservation of several longevity pathways. These preliminary experiments provide a basis to study how NPCs regulate aging. We hypothesize that changes in NPC function disrupt the subcellular localization of factors essential for aging regulation. Aim 1 proposes to further define NPC components that regulate RLS by measuring the life spans and glycerol sensitivities of a variety of NPC mutants. Strains that display altered RLSs will be tested to determine if mutations modify the dynamics of different nuclear transport pathways. If transport functions are altered in mutants, then microdissections will be utilized to ascertain whether these pathways also regulate RLS. Strategies described in Aim 2 will show how NPCs and nuclear transport change as yeast replicatively age. A biochemical technique has been utilized to enrich for replicatively aged yeast cells, and indicates at least one nuclear import pathway is less efficient as cells age. Other pathways will be tested using GFP-tagged reporters in aged yeast cells. Nups will also be examined to determine if they are oxidatively damaged, mislocalized, and/or degraded during replicative aging. Aim 3 describes strategies to determine which cellular functions are affected in NPC mutants with altered life spans. Preliminary data shows mitochondria, which regulate longevity in a variety of organisms, are less functional in cells with decreased life spans. A multicopy overexpression screen and RNA-seq experiments will be used to identify factors that regulate RLS and are also affected by NPC function. These factors will be further tested to understand how they regulate longevity and how their activity is regulated by NPCs. [Epistasis experiments will also be used to determine if NPC mutants affect known yeast longevity pathways.]

描述（由申请人提供）：核孔复合物（NPC）介导真核细胞的细胞核和细胞质之间的转运。NPC是核膜中由核孔蛋白（Nups）组成的大型组件，其执行支架，结构和运输功能。以前的研究表明，随着细胞和生物体的衰老，NPC的组成和功能会恶化，从而导致这些变化可能导致衰老细胞中存在的一些有害表型的假设。然而，目前尚不清楚NPC功能的破坏是否是衰老的原因或影响。初步数据表明，特定Nup结构域的缺失调节酵母S.啤酒。RLS被定义为酵母细胞产生的子体的数量，由于几种长寿途径的保守性，通常被用作后生动物衰老的模型。这些初步实验为研究NPC如何调节衰老提供了基础。我们推测，NPC功能的变化破坏了衰老调节所必需的因子的亚细胞定位。目的1提出通过测量各种NPC突变体的寿命和甘油敏感性来进一步确定调节RLS的NPC组分。将对显示RLS改变的菌株进行检测，以确定突变是否改变了不同核转运途径的动力学。如果突变体的运输功能发生改变，则将利用显微解剖来确定这些途径是否也调节RLS。目标2中描述的策略将显示NPC和核转运如何随着酵母复制老化而变化。生物化学技术已被用于富集复制老化的酵母细胞，并表明至少一种核输入途径随着细胞老化而效率降低。其他途径将在老化的酵母细胞中使用GFP标记的报告基因进行测试。还将检查核以确定它们在复制老化期间是否被氧化损伤、错误定位和/或降解。目的3描述了确定哪些细胞功能在具有改变的寿命的NPC突变体中受到影响的策略。初步数据显示，在各种生物体中调节寿命的线粒体在寿命缩短的细胞中功能较弱。多拷贝过表达筛选和RNA-seq实验将用于鉴定调节RLS的因子，并且也受NPC功能的影响。这些因素将被进一步测试，以了解它们如何调节寿命以及它们的活性如何受到NPC的调节。[上位性实验也将用于确定NPC突变体是否影响已知的酵母寿命途径。