Investigating how nuclear pore complexes regulate aging

描述(申请人提供)：核孔复合体(Npc)调节真核细胞的核和细胞质之间的运输。核祖细胞是核膜上的大型集合体，由核孔蛋白(NUP)组成，具有支架、结构和运输功能。以前的研究表明，随着细胞和生物体的老化，神经前体的组成和功能会恶化，这导致了这样的假设，即这些变化可能解释了衰老细胞中存在的一些有害表型。然而，目前尚不清楚鼻咽癌功能障碍是衰老的原因还是结果。初步数据表明，特定的Nup结构域的缺失调节了酵母中的复制寿命(RLS)。RLS被定义为酵母细胞产生的子代数量，由于几条长寿途径的保守，它经常被用作后生动物衰老的模型。这些初步实验为研究鼻咽癌如何调节衰老提供了基础。我们假设鼻咽癌功能的改变扰乱了衰老调节所必需的因子的亚细胞定位。目的1建议通过测量各种鼻咽癌突变体的寿命和甘油敏感性来进一步定义调节RLS的鼻咽癌成分。将对显示RLSS改变的菌株进行测试，以确定突变是否会改变不同核运输途径的动力学。如果突变体的运输功能发生改变，那么将利用显微切割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随着酵母复制老化，npc和核运输是如何变化的。一种生化技术已经被用来丰富复制老化的酵母细胞，并表明至少有一条核输入途径随着细胞的老化而效率降低。其他途径将使用GFP标记的记者在老化的酵母细胞中进行测试。还将检查NUP，以确定它们在复制老化过程中是否受到氧化损伤、定位错误和/或降解。目的3描述了确定寿命改变的鼻咽癌突变体中哪些细胞功能受到影响的策略。初步数据显示，在多种生物中调节寿命的线粒体，在寿命缩短的细胞中功能较差。多拷贝过表达筛选和RNA-SEQ实验将被用来确定调节RLS的因素和也受NPC功能影响的因素。将进一步测试这些因素，以了解它们是如何调节寿命的，以及它们的活动是如何受到NPC的调节的。[上位性实验也将被用来确定NPC突变是否会影响已知的酵母寿命途径。]

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.]