Investigating how nuclear pore complexes regulate aging

Vanderbilt University, Nashville TN

Investigators

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Abstract

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.]