**1.1. Mechanical properties of cells and their biological significances.**

As a viscoelastic body, the cell exhibits both elastic and viscous characteristics (Kasza, 07). Although these mechanical properties have not been attributed wholly to a single element, such as the cytoskeletal network, the cytoplasm, the cell membrane, or the extracellular network (Janmey et al., 2007), it is agreed that they are determined predominantly by the cytoskeleton, a network of biopolymers in the form of actin filaments, microtubules, and intermediate filaments. The dynamic assembly and disassembly of these biopolymers give the cell the ability to move and to modulate its shape, elasticity, and mechanical strength in responses to mechanical and chemical stimuli from the external environment (Fletcher & Mullins, 2010). Among these cytoskeletal polymers, actin filaments are known to be primarily responsible for the rigidity of the cell. An increase in the concentration of actin filaments typically results in an increase in the rigidity of the cell, which can be characterized by Young's modulus (Satcher Jr & Dewey Jr, 1996).

The cytoskeleton is also essential in regulation of cell signaling and trafficking (Janmey, 1998; Papakonstanti & Stournaras, 2008). In particular, the structure of the cytoskeleton plays an essential role in EGFR signaling and trafficking that is initiated by the binding of epidermal growth factor (EGF) to the EGF receptor (EGFR) (Ridley, 1994; Song et al., 2008). EGF is a protein molecule known to play a crucial role in the regulation of cell growth, proliferation, differentiation and motility. EGFR is a transmembrane receptor that consists of an extracellular ligand-binding domain, a transmembrane domain, an intracellular tyrosine kinase domain, and a C-terminal regulatory domain (Scaltriti & Baselga, 2006). Binding of EGF to the extracellular domain of EGFR leads to the dimerization of EGFR, which in turn stimulates tyrosine kinase activity of the receptors and triggers autophosphorylation of

© 2012 Xi et al., licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2012 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

specific tyrosine residues within the cytoplasmic regulatory domain. The activation of tyrosine kinases initiates multiple downstream signaling pathways such as Ras/Raf-1/MAPK (Scaltriti & Baselga, 2006), PI3Kinase/Akt/mTOR (Ono & Kuwano, 2006), Src/NFKb (Lee C.-W. et al., 2007; Silva, 2004), catenin/cytoskeleton (Yasmeen et al., 2006) and PAK-1 /Rac pathways (McManus et al., 2000).

Dynamic Mechanical Response of Epithelial Cells to Epidermal Growth Factor 173

**Figure 1.** Typical force-displacement curves generated for the approach and retraction of the AFM probe. Approach and retraction correspond to mechanical loading and unloading of the AFM probe on

To indent epithelial cells that are being treated with biologically active molecules, the force applied on the cells by the AFM probe is often kept approximately 50 nN or slightly lower to minimize the adverse effects on the cells caused by the probe. With the force at this level, the probe can have the probing depth that is sufficiently deep (100 to 500 nm) to register the cytoskeleton remodeling (Schillers et al., 2010) but is still shallow enough to avoid influence from the nucleus and the solid substrate on which the cells rest (Melzak et al., 2011). In addition to a low magnitude of loading, the velocity of loading should be kept low enough so that the transient friction interactions between the probe tip and the cell surface are

Force-displacement curves acquired with all of these precautions in place then can be used to estimate values for Young's modulus and energy dissipation of the cell. The Young's modulus of a cell can be extracted from a curve of unloading force displacement with the aid of the Herzian elastic contact model for a conically shaped tip indenting an elastic body


1

where *F* is the applied force, *d* is the deflection of the cantilever, and *k* is the spring constant of the cantilever. Also, *α* is the half angle of the cone-shaped tip, *υ* is the Poisson ratio (taken to be 0.5, for an incompressible material), *δ* is the indentation depth, and *E* is Young's modulus. It should be noted that the force-displacement curves are dependent on the frequency of the probe (Hoffman & Crocker, 2009); this means that the estimated values of

In the estimation of the Young's modulus, the cell is assumed to be an elastic body, i.e., to return all of the energy deposited during the loading portion of the indentation process. However, in reality, the cell is not perfectly elastic but exhibits some dissipative behaviour.

2

*<sup>E</sup> F kd*

2 2

 tan

(1)

the top surface of the cell.

avoided (Alcaraz et al., 2003).

(Touhami et al., 2003):

modulus are not unique.

It is known that EGFR signaling induces drastic morphological changes, such as rounding of cells, induction of membrane ruffling and extension of filopodia (Bretscher, 1989; Chinkers et al., 1981). These changes can be attributed to the remodeling of cytoskeletal structures (Rijken et al., 1991), which may also alter mechanical properties of the cells (Kasza et al., 2007; Stamenovic, 2005). Currently, the connection between cell signalling and alterations of the mechanical properties of cells is still not fully understood in general. Information concerning the effects of EGF stimulation on the mechanical properties of cells will certainly provide insights into this connection. In addition, since EGFR is highly expressed in a variety of human tumors (Dei Tos & Ellis, 2005) and mutations in EGFR can produce aberrant cell signaling that often leads to uncontrolled cell growth and a malignant phenotype, such information will also shed light on the link between cell mechanical properties and human diseases (Bao & Suresh, 2003).

Many highly sensitive techniques have been developed over the years to assess mechanical properties of cells (Addae-Mensah & Wikswo, 2008). These include atomic force microscopy (Smith et al., 2005), magnetic twisting cytometry (Wang et al., 1993), micropipette aspiration (Alexopoulos et al., 2003), optical tweezers (Svoboda & Block, 1994), Shear-flow methods (Usami et al., 1993), particle-tracking microrheology (Wirtz, 2009), cantilever beams (Galbraith & Sheetz, 1997), and others. Each technique probes a cell or cells in a different manner and does not necessarily measure the same aspects of a cell as another technique. Thus, the use of more than one technique to study the same object (i.e., cell) may prove useful. This chapter describes the application of two sensitive techniques, the atomic force microscopy and the quartz crystal microbalance with dissipation monitoring, to the study of the mechanical properties of cells in response to exposure to EGF.

## **1.2. Probing mechanical response of cells with atomic force microscopy**

Atomic force microscopy (AFM) is one of the most popular choices for probing the mechanical properties of cells, because individual cells can be probed in high sensitivity and resolution with a minimum of force (Radmacher Manfred, 2007). To measure the mechanical properties of the cell with AFM, the top surface of a live cell is indented with the sharp tip located at the end of a cantilever (a probe). The cantilever is mounted on a piezoelectric tube that moves the cantilever down and up in the vertical direction toward and away from the surface of the cell. The deflection in the cantilever is typically measured by a laser that tracks a spot on the tip of the cantilever. From the position of the cantilever and its deflection, force-displacement curves during the indentation of the cell by the probe are generated as shown in Figure 1 (Radmacher M., 1997).

/Rac pathways (McManus et al., 2000).

properties and human diseases (Bao & Suresh, 2003).

the mechanical properties of cells in response to exposure to EGF.

shown in Figure 1 (Radmacher M., 1997).

**1.2. Probing mechanical response of cells with atomic force microscopy** 

Atomic force microscopy (AFM) is one of the most popular choices for probing the mechanical properties of cells, because individual cells can be probed in high sensitivity and resolution with a minimum of force (Radmacher Manfred, 2007). To measure the mechanical properties of the cell with AFM, the top surface of a live cell is indented with the sharp tip located at the end of a cantilever (a probe). The cantilever is mounted on a piezoelectric tube that moves the cantilever down and up in the vertical direction toward and away from the surface of the cell. The deflection in the cantilever is typically measured by a laser that tracks a spot on the tip of the cantilever. From the position of the cantilever and its deflection, force-displacement curves during the indentation of the cell by the probe are generated as

specific tyrosine residues within the cytoplasmic regulatory domain. The activation of tyrosine kinases initiates multiple downstream signaling pathways such as Ras/Raf-1/MAPK (Scaltriti & Baselga, 2006), PI3Kinase/Akt/mTOR (Ono & Kuwano, 2006), Src/NFKb (Lee C.-W. et al., 2007; Silva, 2004), catenin/cytoskeleton (Yasmeen et al., 2006) and PAK-1

It is known that EGFR signaling induces drastic morphological changes, such as rounding of cells, induction of membrane ruffling and extension of filopodia (Bretscher, 1989; Chinkers et al., 1981). These changes can be attributed to the remodeling of cytoskeletal structures (Rijken et al., 1991), which may also alter mechanical properties of the cells (Kasza et al., 2007; Stamenovic, 2005). Currently, the connection between cell signalling and alterations of the mechanical properties of cells is still not fully understood in general. Information concerning the effects of EGF stimulation on the mechanical properties of cells will certainly provide insights into this connection. In addition, since EGFR is highly expressed in a variety of human tumors (Dei Tos & Ellis, 2005) and mutations in EGFR can produce aberrant cell signaling that often leads to uncontrolled cell growth and a malignant phenotype, such information will also shed light on the link between cell mechanical

Many highly sensitive techniques have been developed over the years to assess mechanical properties of cells (Addae-Mensah & Wikswo, 2008). These include atomic force microscopy (Smith et al., 2005), magnetic twisting cytometry (Wang et al., 1993), micropipette aspiration (Alexopoulos et al., 2003), optical tweezers (Svoboda & Block, 1994), Shear-flow methods (Usami et al., 1993), particle-tracking microrheology (Wirtz, 2009), cantilever beams (Galbraith & Sheetz, 1997), and others. Each technique probes a cell or cells in a different manner and does not necessarily measure the same aspects of a cell as another technique. Thus, the use of more than one technique to study the same object (i.e., cell) may prove useful. This chapter describes the application of two sensitive techniques, the atomic force microscopy and the quartz crystal microbalance with dissipation monitoring, to the study of

**Figure 1.** Typical force-displacement curves generated for the approach and retraction of the AFM probe. Approach and retraction correspond to mechanical loading and unloading of the AFM probe on the top surface of the cell.

To indent epithelial cells that are being treated with biologically active molecules, the force applied on the cells by the AFM probe is often kept approximately 50 nN or slightly lower to minimize the adverse effects on the cells caused by the probe. With the force at this level, the probe can have the probing depth that is sufficiently deep (100 to 500 nm) to register the cytoskeleton remodeling (Schillers et al., 2010) but is still shallow enough to avoid influence from the nucleus and the solid substrate on which the cells rest (Melzak et al., 2011). In addition to a low magnitude of loading, the velocity of loading should be kept low enough so that the transient friction interactions between the probe tip and the cell surface are avoided (Alcaraz et al., 2003).

Force-displacement curves acquired with all of these precautions in place then can be used to estimate values for Young's modulus and energy dissipation of the cell. The Young's modulus of a cell can be extracted from a curve of unloading force displacement with the aid of the Herzian elastic contact model for a conically shaped tip indenting an elastic body (Touhami et al., 2003):

$$F = kd = \frac{2}{\pi} \bullet \frac{E}{1 \cdot \nu} \delta^2 \tan a \tag{1}$$

where *F* is the applied force, *d* is the deflection of the cantilever, and *k* is the spring constant of the cantilever. Also, *α* is the half angle of the cone-shaped tip, *υ* is the Poisson ratio (taken to be 0.5, for an incompressible material), *δ* is the indentation depth, and *E* is Young's modulus. It should be noted that the force-displacement curves are dependent on the frequency of the probe (Hoffman & Crocker, 2009); this means that the estimated values of modulus are not unique.

In the estimation of the Young's modulus, the cell is assumed to be an elastic body, i.e., to return all of the energy deposited during the loading portion of the indentation process. However, in reality, the cell is not perfectly elastic but exhibits some dissipative behaviour.

This dissipative behavior is manifested as a loss (as heat to the surroundings) of some of the energy stored during loading, and can be seen in the indentation process as hysteresis in a cycle of force displacement (Figure 1). In a cell, energy dissipation is believed to be accomplished by internal friction and/or viscous damping mechanisms (Alcaraz et al., 2003; Smith et al., 2005). In AFM, the mechanical energy dissipated per cycle of indentation is given quantitatively by the area of the hysteresis loop enclosed by the approach and retraction curves (Alcaraz et al., 2003), as shown in Figure 1.
