A Computational Model of YAP/TAZ Mechanosensing

Abstract

In cell proliferation, stem cell differentiation, chemoresistance, and tissue organization, the ubiquitous role of YAP/TAZ continues to impact our fundamental understanding in numerous physiological and disease systems. YAP/TAZ is an important signaling nexus integrating diverse mechanical and biochemical signals, such as ECM stiffness, adhesion ligand density, or cell-cell contacts, and thus strongly influences cell fate. Recent studies show that YAP/TAZ mechanical sensing is dependent on RhoA-regulated stress fibers. However, current understanding of YAP/TAZ remains limited due to the unknown interaction between the canonical Hippo pathway and cell tension. Furthermore, the multiscale relationship connecting adhesion signaling to YAP/TAZ activity through cytoskeleton dynamics remains poorly understood. To identify the roles of key signaling molecules in mechanical signal sensing and transduction, we present a, to our knowledge, novel computational model of the YAP/TAZ signaling pathway. This model converts extracellular-matrix mechanical properties to biochemical signals via adhesion, and integrates intracellular signaling cascades associated with cytoskeleton dynamics. We perform perturbations of molecular levels and sensitivity analyses to predict how various signaling molecules affect YAP/TAZ activity. Adhesion molecules, such as FAK, are predicted to rescue YAP/TAZ activity in soft environments via the RhoA pathway. We also found that changes of molecule concentrations result in different patterns of YAP/TAZ stiffness response. We also investigate the sensitivity of YAP/TAZ activity to ECM stiffness, and compare with that of SRF/MAL, which is another important regulator of differentiation. In addition, the model shows that the unresolved synergistic effect of YAP/TAZ activity between the mechanosensing and the Hippo pathways can be explained by the interaction of LIM-kinase and LATS. Overall, our model provides a, to our knowledge, novel platform for studying YAP/TAZ activity in the context of integrating different signaling pathways. This platform can be used to gain, to our knowledge, new fundamental insights into roles of key molecular and mechanical regulators on development, tissue engineering, or tumor progression.

Figure 1

YAP/TAZ mechano-sensing signaling pathway network. This is the scheme of the computational signaling cascade model. The ECM mechanical properties are transmitted to intracellular signals via adhesion molecules, such as FAK. Adhesion molecules activation induces RhoA binding with GTP. RhoA-GTP drives the formation of actomyosin via mDia and ROCK activation. mDia is a formin that polymerizes F-actin. ROCK can both activate myosin and induce F-actin assembly through LIMK and cofilin. Eventually, the resultant of F-actin and myosin activity, i.e., stress fibers in 2D systems, leads to the YAP/TAZ nuclear translocation. In comparison with the YAP/TAZ mechanical sensing mechanism, SRF/MAL senses mechanical inputs by being inhibited by G-actin. In addition, the Hippo core component LATS is sensitive to cell-cell interaction. In our model, the synergistic effect between the mechanical sensing and the Hippo pathway is predicted due to the interaction of LATS and LIMK. To see this figure in color, go online.

Figure 2

Molecular intervention of YAP/TAZ activity in stiffness sensing. (a) The results in the computational model regarding molecular interventions are consistent with the experimental results from previous mechanical regulation studies on YAP/TAZ (8). Here the molecular intervention process is mimicked by upregulating or downregulating the corresponding kinetic parameters (see Supporting Materials and Methods). The soft environment here is 0.5 kPa hydrogel for 2D MEC cells, while the stiff environment is 20 kPa hydrogel (8). Blebbist, myosin inhibitor; Y-27632, ROCK inhibitor; C3, RhoA inhibitor; Lat. A, F-actin inhibitor; mDia, overexpression of mDia (a formin that induces F-actin assembly). (b) Local sensitivity analysis of the nondimensionalized model. The local perturbations of adhesion-relevant parameters (C, k_sf, and k_df) are the most robust ones in affecting YAP/TAZ activity in the model. This is the local sensitivity analysis of YAP/TAZ activity in a stiff environment by downregulating the kinetic parameters individually 10%. The upregulating effect is much weaker than this, because most of the molecular activities are saturated. The upregulation in a soft environment also shows that the adhesion is the most robust component in affecting YAP/TAZ activity (Fig. S2). (c and d) Cell stiffness response function, which is a YAP/TAZ activity function regulating cellular behavior such as proliferation dependent on external ECM stiffness. (c) Different amounts of FAK determine the threshold stiffness for cell stiffness sensing by shifting the curves horizontally. FAK0∼1 corresponds to the endogenous FAK amount of NMuMG cell in 3D collagen. Collagen with 25 kPa is a soft environment and 45 kPa is a stiff environment for 3D NMuMG cells. If FAK is overexpressed to ∼3-fold, 20 kPa will appear as a stiff environment for these cells. (d) The 2D MEC and MSC stiffness sensing identifies a 1 kPa ECM environment as soft and a 20 kPa environment as stiff, while in 3D, NMuMG cells sense 25 kPa as soft and 45 kPa as stiff. These stiffness response functions are very alike the ones of manipulating only the FAK total amount of cells in (c). To see this figure in color, go online.

Figure 3

YAP/TAZ activity sensitivity analysis and comparison with SRF/MAL. (a) YAP/TAZ nuclear localization under a different stiffness environment, in comparison with MAL nuclear translocation. Here MAL nuclear translocation is not as robust as YAP/TAZ in response to stiffness change, likely due to YAP/TAZ having a myosin-dependent amplification effect other than the common regulator G-actin/F-actin in SRF/MAL activity. (b) Fold-change of YAP/TAZ and MAL activities depending on the kinetic parameters, such as the activation rate of FAK (k_sf/k_sf0) and RhoA (k_fkp/k_fkp0), when switching matrix from a soft to a stiff one. In addition, YAP/TAZ keeps showing higher sensitivity than MAL translocation fold-change in response to different stiffnesses, and the shape of the activated region reveals that downregulating FAK and RhoA has a more robust effect in regulating the YAP/TAZ nuclear localization than upregulating it. To see this figure in color, go online.

Figure 4

The synergistic effect between the mechano-sensing and the Hippo pathway. (a and b) Adding the interaction between LIMK and LATS, which induces more free active LIMK molecules by adding siLats, YAP/TAZ is activated synergistically compared with inducing F-actin assembly and adding siLats separately. Condition 1, stiff environment; condition 2, soft environment;. condition 3, soft environment with siLats; condition 4, soft environment with siCapZ; condition 5, soft environment with both siLats and siCapZ. CapZ is a capping protein that inhibits F-actin polymerization. Here the effect of siCapZ on soft environment can be quantified as its corresponding YAP/TAZ activity (H(siCapZ)), abstracting the activity of YAP/TAZ in a soft environment (control: H(soft)). The synergistic effect is quantified as H(siCapZ) – H(soft) + H(siLats) – H(soft) > H(siCapZ + siLats) – H(soft), i.e., H(siCapZ) + H(siLats) > H(soft) + H(siCapZ + siLats). (b) With a higher dependence on F-actin than on myosin, the YAP/TAZ showed a more apparent synergistic effect. To see this figure in color, go online.