Mechanobiology: how mechanics regulate life

Selected Questions:

1. If indentation induces the unfolding of nuclear envelope wrinkles, do you think the unfolding of wrinkles affect the nuclear pore complexes embedded in the nuclear envelope? How might these changes affect the regulation of nucleocytoplasmic exchanges (such as protein and RNA translocation)?

2. Nuclear mechanotransduction is a key process in the mechanical coupling of the extracellular signaling and nuclear response mainly through gene expression. Do you think that wrinkles are important for this nuclear mechanotransduction? Would the nucleus somehow be able to spatially decode the wrinkles and transduce them into a response through gene expression? 

3. What is anti-Zender model and why this model is selected for fitting? How do you explain the fact that, for positive curvatures, there is a linear relationship between curvature and relative E while for negative curvatures the relationship is exponential (see Figure 4G)? Could this be explained with the anti-Zener viscoelastic model? Along the same lines, in Figure 2B while the bead 'pokes' a bit deeper with each cycle, the irreversible deformation decreases over time. How does the combination of viscous flow and elastic response lead to this pattern?

4. What new information on the connection between nuclear stiffness and nuclear envelope wrinkle unfolding may be gained from the finite element simulations? How did the authors fit the parameters of the model? How can we distinguish between passive structural changes and active processes in the simulation results?

5. Could you please comment on the magnitude of forces typically experienced by the cell nucleus under physiological conditions? Compared to the experimental force (10 nN) in this paper, are they stronger or weaker?

6. Could you explain how the beads were internalized by the cell? How did the authors tether the beads to the nuclear envelope? How would the size or geometry of the bead affect NE wrinkle unfolding (i.e., how would the data change if they used a smaller, larger, or elongated particle)?

7. The Nuclear Envelope (NE) displacement is plotted over 5 consecutive cycles of force indicating a reduction in the deformability of the NE, and therefore a stiffening of the envelope (Figure 2B). Do we expect a maximal stiffening of the NE? In other words, in the process of nuclear stiffening induced by repeated mechanical stimulation, does the irreversible deformation of the nuclear membrane reach an upper limit, causing the nuclear membrane to lose its ability to further harden or deform? If yes, what would be the long-term implications when the external force is no longer present?

8. The quantification of NE wrinkles is achieved by calculating curvature, which is done by visualizing the NE with marker dye under microscopy and utilizing Plugin Kappa. Would these factors, such as the expression level of the NE marker, the resolution of the dye and microscopy (given that Figure 3C seems to show moderate resolution), as well as the robustness of Plugin Kappa, act as confounding variables potentially affecting the curvature calculation? Did the authors validate the robustness of this method using some positive controls (e.g., cases where the NE is expected to exhibit more or fewer wrinkles, with corresponding changes in curvature)? Why did the authors use the absolute value of curvature?