Poster Q10 - Nuclear deformation regulates subcellular YAP transport dynamics in cancer associated fibroblasts

Introduction: Yes Associated Protein (YAP), a transcription cofactor, has been identified as one of the primary mechanosensors for cells. High substrate stiffness is attributed to driving YAP to the nucleus wherein it mediates expression of a wide range of genes. Cells cultured on stiff 2D substrates exert high intracellular force, resulting in mechanical deformation of their nuclei. This nuclear deformation (ND) plays a crucial role in the transport of YAP from the cytoplasm to the nucleus. However, cells in vivo are in soft 3D environment with potentially much lower intracellular forces. Whether and how cells may deform their nuclei in 3D for YAP localization remains unclear. Here, we explored the effects of matrix stiffness, cell force, and ND on nuclear localization of YAP. We also developed a stochastic model that uses cell force history to simulate YAP kinetics [1].

Materials and

Methods: We cultured human colon cancer associated fibroblasts (CAFs) on 2D (Fig. 1A), 2.5D (Fig. 1B, a hybrid system where cells are in contact with 2D substrate at the base and 3D collagen matrix on the apical surface), and 3D (Fig. 1C) substrates with various stiffness. This method allowed us to separately control extracellular mechanics and nuclear deformation. To measure cell forces on 2D and 2.5D substrates, we used traditional traction force microscopy (Fig. 1A-B). For cells in 3D collagen, we microfabricated a sensor with PDMS that integrates a tissue with cell(s) that is connected to a sensitive spring that reads cell force using force equilibrium laws (Fig. 1C-D) [2].

Results, Conclusions, and Discussions: On 2D, our results agree with previous studies that found substrate stiffness, total force, cell spreading area and F-actin stress fibers to correlate with nuclear YAP. In 3D, however, there is no clear correlation. This raises the question whether there is a common mechanism of YAP activation across all dimensionalities. Indeed, we found that nuclear translocation of YAP depends on the degree of ND irrespective of dimensionality, stiffness, and total force (Fig. 2A-D). ND induced by the perinuclear force, not the total force, and nuclear membrane curvature correlates strongly with YAP activation (Fig. 2E-H). Immunostained slices of human tumors further supported the association between ND and nuclear YAP, suggesting ND as a potential biomarker for YAP activation in tumors [1].
Additionally, we conducted quantitative analysis of the force dynamics of CAFs on 2D and 3D substrates to construct a stochastic model of YAP kinetics that predicts the time-dependent nucleo-cytoplasmic YAP transport in response to cellular force. Monte Carlo simulations of YAP dynamics showed that YAP in CAFs can switch between active and inactive states, and the persistence time of active YAP shows a power law dependence with respect to the activation thresholds. This means that the probability (i.e. time fraction) of YAP being localized in nucleus decreases as the activation threshold increases (Fig. 3) [1].
Different sets of genes may require a different level (threshold) of nuclear YAP to induce the intended effects. Therefore, persistence times of YAP above certain thresholds may play important roles in determining the downstream effects of YAP signaling. Short-term YAP activation (lower persistence times) may lead to transient changes in gene expression and downstream signaling, while long-term YAP activation (higher persistence times) may lead to more sustained changes and potentially deleterious effects. To our knowledge, this study provides the first glimpse into probability and persistence times of YAP activation in CAFs [1].
Overall, the study provides biophysical insights into the mechanisms of YAP activation, its persistence and probability. The results can be useful in clinical applications targeting YAP-dependent signaling, as understanding the precise kinetics of YAP activation and downstream signaling may have important therapeutic implications.

Acknowledgements (Optional):

References:
[1] B. Emon, M.S.H. Joy, L. Lalonde, A. Ghrayeb, U. Doha, L. Ladehoff, R. Brockstein, C. Saengow, R.H. Ewoldt, M.T.A. Saif, Nuclear deformation regulates YAP dynamics in cancer associated fibroblasts, Acta Biomater 173 (2024) 93–108. https://doi.org/10.1016/J.ACTBIO.2023.11.015.
[2] B. Emon, Z. Li, M.S.H. Joy, U. Doha, F. Kosari, M.T.A. Saif, A novel method for sensor-based quantification of single/multicellular force dynamics and stiffening in 3D matrices, Sci Adv 7 (2021) eabf2629. https://doi.org/10.1126/sciadv.abf2629.