Modulation of nuclear pore complex structure and function by nuclear envelope tension

点击翻译按钮获取中文摘要

Project Summary/Abstract Mechanical forces play a critical role in regulating numerous biological processes, including development and tissue homeostasis. At the cellular level, changes in mechanical forces impact nuclear function. For example, cells respond to mechanical inputs by altering the localization of transcription factors, chromosome organization, and gene expression. Despite these insights, how forces applied to the nucleus are decoded to affect downstream cellular responses remains poorly defined. Nuclear pore complexes (NPCs) are massive protein channels that control all molecular exchange across the nuclear envelope (NE). Thus, NPCs may serve as the intermediary that translates mechanical signals by functionally responding to changes in NE tension. I hypothesize that tension on the NE alters the conformation of NPCs, which affects the NPC central transport channel in a manner that influences its transport properties. Here, I will directly explore this hypothesis in Aim 1 by testing how the NPC responds to changes in NE tension using two complementary approaches, electron tomography (ET) and fluorescence resonance energy transfer (FRET) between FRET pairs engineered into the NPC scaffold. While ET will be used to examine changes in nuclear pore diameter, FRET will be used to examine dynamic changes in NPC structure in living cells under various tensional states induced using genetic and environmental perturbations. In a complementary but independent Aim 2, I will test how NE tension impacts the function of NPCs by interrogating the localization and dynamics (using fluorescence recovery after photobleaching (FRAP)) of a series of fluorescent protein reporters, which I can place in the context of changes in NPC conformation observed in Aim 1. These in vivo experiments will be performed alongside an in vitro approach using optical tweezers to observe how diffusion across the NPC responds to direct modulation of NE tension on purified nuclei. Completion of these Aims will determine how NE tension affects the structure and function of NPCs and establish a new mechanosensing pathway in cells.