Professor University of Nebraska-Lincoln Lincoln, Nebraska, United States
Introduction: The linker of nucleoskeleton and cytoskeleton (LINC) complex composed of Nesprin and SUN proteins physically links cytoskeleton to nucleoskeleton at the nuclear envelope, thereby transmitting external mechanical forces to the nucleus. In addition to its role in force transmission, recent pioneering studies have proposed that the LINC complex contributes to the regulation of nucleocytoplasmic trafficking of transcriptional regulators, such as yes-associated protein (YAP). YAP is a mechanosensitive transcriptional co-activator acting primarily through the translocation from cytoplasm to nucleus for its activation. Nuclear translocation of YAP and its binding with TEAD triggers a variety of transcriptional activities to regulate cell proliferation and growth, differentiation, and resultant tissue homeostasis or pathology. Here, we investigated how the LINC complex contributes to the regulation of nucleocytoplasmic trafficking of YAP in mesenchymal stem cells (MSCs) under physiologically relevant cyclic mechanical stretch loading.
Materials and
Methods: Murine MSCs (C3H10T1/2) were exposed to cyclic stretching (10% strain, 1 Hz frequency) using a FX-5000 Tension device (Flexcell). Type-I collagen-coated stretchable membrane (BioFlex, Flexcell) was used for cell culture and stretching. To test the role of Nesprin and SUN, cell stretch tests were conducted with Nesprin and SUN-specific siRNAs and control siRNA. YAP nuclear import was assessed with immunofluorescent imaging as well as immunoblotting of nuclear extract via using a Nuclear and Cytoplasmic Extraction Reagents Kit (Thermo Fisher, PI78835). In immunoblotting, YAP content in the nucleus was normalized by that of lamin B1. In addition, nuclear pore complex (NPC) was imaged by immunofluorescence to test the effect of mechanical stretch loading on NPC activation.
Results, Conclusions, and Discussions: We observed that cyclic stretch loading at a physiological level induced YAP nuclear import in MSCs, in both immunofluorescent imaging and immunoblotting of nuclear extract. Furthermore, the mechanical stretch loading-induced YAP nuclear transport was significantly negated by Nesprin1 siRNA (Fig. 1A,B), suggesting the role of Nesprin-mediated force transmission in the nucleocytoplasmic trafficking of YAP. Also, the reinforcement of actin stress fibers in response to stretch stimulation was abrogated by Nesprin1 knockdown (not shown). Our results provide the role of Nesprin, the cytoskeleton and nucleoskeleton connector at the outer nuclear membrane, in stretch-induced YAP nuclear translocation in association with stretch generated F-actin. Next, we assessed the effect of mechanical stretching on the activation of NPC and evaluated the role of SUN in this process. We observed that NPC expression was increased at the nuclear envelope by stretch loading, which effect was suppressed by SUN depletion with siRNA (Fig. 1C). This suggests that force transmission through SUN, the cytoskeleton and nucleoskeleton connector at the perinuclear space and inner nuclear membrane, may be required for the activation of NPC to facilitate YAP nuclear import. In conclusion, we have identified the vital contribution of the LINC complex, Nesprin-SUN, in the cyclic stretch loading-induced nuclear translocalization of YAP in MSCs. Our data suggest that YAP activation by stretch is achieved at least via Nesprin-mediated F-actin formation under stretch and its regulation of YAP nuclear import. Also, SUN mediation of the stretch-induced NPC activation appeared to play a role in the stretch induction of YAP nuclear import. Considering the critical importance of YAP nuclear import-triggered transcriptional MSC fate decision, our data propose a significant implication in MSC mechanobiology by deciphering the underlying mechanosensing mechanism of YAP nucleocytoplasmic trafficking.
Acknowledgements (Optional): We thank the funding support given to J.Y.L. from NIH/NIGMS COBRE Nebraska Center for Integrated Biomolecular Communication (NCIBC), University of Nebraska Collaboration Initiative, NE DHHS Stem Cell Research Project (Stem Cell 2023-04), University of Nebraska Biomedical Research Seed Grants.
