**3. Conclusions**

180 Viscoelasticity – From Theory to Biological Applications

before and after the 60-min EGF treatment, respectively.

**2.4. Comparison of AFM with QCM-D results** 

cells to EGFR signaling are regionally specific.

modulus of the top region of the cells, it is reasonable to assume that the increases in dissipation and hysteresivity were also related to the increase in cortical actin filaments.

The basal area of the cell monolayer, i.e., the area probed by the QCM-D, was also examined by means of fluorescence imaging. In this case, the stress fibers, which are actin filaments that reside in the bottom portion of the cells and are involved in the formation of focal adhesions attaching the cell to the substrate, were studied. As shown in Figures 10A and 10B, the cells displayed a decrease in amount and size of stress fibers after 60 min of exposure to EGF. Thus the decrease in dissipation observed by means of the QCM-D technique can be reasonably assumed to be related to a reduction in number and size of the actin stress fibers in the basal region of the cells. In addition, this reduction in stress fibers suggests a loss of adhesion and/or contact between the cells and the solid substrate, which is consistent with the EGF-induced cell

rounding and retracting that has been reported previously (Chinkers et al., 1979; 1981).

**Figure 10.** Remodeling of actin filaments in a monolayer of A431 cells induced by the treatment of 10 nM of EGF at 37°C. (A) and (B) show fluorescence-stained stress fibers at the basal area of the cell layer

The two sensitive techniques, AFM and QCM-D, described in this chapter probe two different regions of cells in a monolayer on a solid surface. The nature of the AFM limits it to probing the top surface and immediately underlying volume of individual cells. The nature of the QCM-D limits it to probing the basal areas of the cells in a monolayer. The AFM technique revealed an increase in Young's modulus, energy dissipation, and hysteresivity in response to EGF, while the QCM-D technique revealed a decrease in energy dissipation factor. The fluorescence studies of the changes in actin in the top and bottom surfaces upon

Because both hysteresivity (*η*) and the change in energy dissipation factor (∆*D*) represent the ratio between the dissipated energy and the elastic energy, these two quantities are analogous to each other (Collinsworth et al., 2002). The fact that the opposite trends in them were obtained upon exposure of the cells to EGF is perhaps not unexpected (Figure 5 and 7A). For a monolayer of cells on a solid surface, the bottoms of the cells are restricted in shape by contact with a solid surface, while the tops of the cells have no such restrictions. The bottom surfaces of the cells may therefore exhibit unique responses to the presence of the solid surface and the tops of the cells may not. Thus, contrasts in mechanical properties and in the number, size, and form of actin filaments signify that the mechanical responses of

exposure to EGF corroborate the differences observed in mechanical properties.

In this chapter, the examples were provided to illustrate how to employ a combination of AFM and QCM-D to characterize the mechanical behavior of cells in response to exposure to EGF. This unique combination allowed the comparative assessment of the upper volume of the cell bodies as well as the basal areas of the cells. Results from both parts of the A431 cells reveal a regionally specific mechanical behavior of the cells, which can be attributed to the distinct cytoskeleton structures utilized by the cells to alter the local structure in response to EGF stimulation. The signaling pathways that mediate the remodeling of the cytoskeleton in the upper volume of the cell bodies are likely closely coupled to those in the basal areas of the cells. There is a clear correlation between the time-dependent mechanical response and the dynamic process of EGFR signaling.

Overall, the combination of AFM and QCM-D is able to provide a more complete and refined mechanical profile of the cells during the dynamic cell signaling process than either technique alone. The use of combined techniques to track real-time cell signaling based on the measurement of cellular mechanical response in a label-free manner is a powerful approach for investigating the role of EGFR in causing abnormal cell behavior. This combined, real-time approach may have the potential to be applied to the study of other types of receptor-mediated cell signaling and trafficking. This approach should contribute to the fundamental understanding of the correlation between cell function and cell mechanical properties.
