A novel FRET-force biosensor for nucleoporin gp210 reveals that the nuclear pore complex experiences mechanical tension

Abstract The nuclear pore complex (NPC) is a large multi-protein structure that enables movement of macromolecules, such as mRNA and proteins, between the nucleoplasm and cytoplasm. There has been great interest in how the physical state of the NPC can influence nuclear-cytoplasmic transport. The hypothesis that the NPC may be mechanosensitive is supported by prior reports showing that the diameter of the NPC increases with nuclear envelope stretch as well as increased ECM stiffness. We therefore sought to develop a biosensor-based approach to determine if the NPC experiences mechanical tension. Using a previously developed FRET-force biosensor, known as TSmod, we developed a gp210 tension sensor. gp210 is a transmembrane nucleoporin, which may serve to anchor the NPC into the nuclear envelope. Using a CRISPR knock-in strategy, we developed a HeLa cell line which expresses the gp210 tension sensor at endogenous levels. Using this sensor, we observed that the biosensor exhibited FRET changes that were consistent with increased force in response to osmotically induced nuclear swelling. Cell attachment, the nuclear LINC complex, ECM stiffness, chromatin condensation, and actomyosin contractility were all observed to influence gp210 forces. Surprisingly, gp210 forces were increased with chromatin relaxation and myosin light chain kinase inhibition, indicating that NPC forces may be differentially regulated from forces on the LINC complex. Our data support a hypothesis where nuclear strain, rather than cytoskeletal forces, is the predominant source for NPC forces. Our studies provide the first direct measurements of protein-level mechanical forces on the NPC. We anticipate that the gp210 force sensor will be of use for future studies of NPC mechanobiology. Competing Interest Statement The authors have declared no competing interest. Footnotes New data is provided with updated gp210 control sensors. Additionally, we have revised our experiments to disrupt of the LINC complex to use siRNA knockdown of SUN1 and SUN2 instead of DN KASH. Finally we have updated the manuscript text to correct minor typographical errors.