Study of the role of substrate stiffness and force transmission to the nucleus in nucleocytoplasmic transport, nuclear pore conformation, genome organization and gene expression

Abstract

[eng] The application of mechanical force to the nucleus has been recently shown to regulate important functions, including nucleocytoplasmic transport (NCT), chromatin organization, and gene expression. However, how substrate rigidity and the subsequent transmission of force to the nucleus impacts nuclear function and dynamics is still poorly understood. Here we focus on two aspects: the effect of nuclear force in NPCs and in chromatin organization and gene expression. We show how knockdown (KD) of two key NPC structural components, namely NUP155 and NUP153, decrease the accumulation of the transcriptional regulator (TR) YAP/TAZ to the nucleus as well as disrupt NCT in mammalian cells. With the aim to analyze structural changes in nuclear pores we perform super-resolution microscopy (SRM) and establish a method to image cells on substrates of different stiffness. In this set up and using U-2 osteosarcoma (U-2 OS) cells we do not observe rigidity or KD-dependent changes in NPC size. However, we suspect that these results arise from cell type or NUP-specific conditions and propose new candidates to test our new hypothesis. In addition, we use substrate stiffness to characterize via Hi-C and RNA-sequencing the force-dependent changes in chromatin state and transcription. Our results show that biomechanical manipulation using mutants to impair mechanosensitive NCT as well as substrate stiffness affects the functional organization of topologically associated domains (TADs) as well as the expression of genes linked to the cytoskeleton, focal adhesions, and the NPC.