**6. Heterogeneous force distribution in single cells subjected to shear pulses**

The shear-induced strains and stresses in the cytoskeleton could be the core of the mechanical response of a cell. Since the cytoskeleton is an anisotropic collection of dynamically cross-linked structural proteins, the cytoskeletal stresses are intrinsically nonuniform. This heterogeneity could be responsible for the nonlinear behavior of cells in response to shear stresses.

#### **6.1 Cytoskeletal force measurements**

The recent development of force probes based on fluorescence resonance energy transfer (FRET) technique [110–113] provides a powerful tool to observe cytoskeletal protein forces in live cells. The probe consists of two mutant fluorophores, a donor and an acceptor, linked with an elastic structure. Mechanical forces change the distance and/or the angle between the donor and the acceptor, resulting in a change in the energy transfer efficiency from donor to the acceptor [112, 113]. These probes were genetically encoded in cross-linking proteins, such as α-actinin and actin, and they are being used to report the strains in cytoskeletal linking proteins when cells are subjected to shear pulses. Using FRET probes, the distribution of protein forces induced by fluid shear stress has been mapped in real time [114].

#### **6.2 Heterogeneous force distribution in single cells**

By inserting the FRET-based force probes in actin-binding protein (α-actinin) in astrocytes, the cytoskeletal protein response to shear stress was measured for the first time. Results show that rapid shear stimuli generate nonuniform protein forces in single cells. **Figure 5** demonstrates that a narrow square shear pulse produced an immediate increase in force in α-actinin at the upstream end of the cell and a small negative force at the downstream end, resulting in a force gradient along the flow direction. In contrast, a slow ramp to the same force amplitude caused a minimal and more uniform change in actinin force. Moreover, the cytoskeleton structure and its prestress states also influence the cell response. The shear pulse produced significant internal force gradients in softer cells that have fewer bundled actin filaments [114]. These experiments demonstrate that uniform shear stress can generate heterogeneous cytoskeletal forces in single cells. Importantly, the time dependence of the stimulus plays a critical role.

While fluid velocity in the flow chamber was precisely controlled, the body stresses produced in the cells depend on cell geometry and the distribution of stresses within the cytoskeleton (we now know that stress distribution is heterogeneous). This heterogeneity likely accounts for the observed variability between cells. Thus, an averaged impact force cannot be used as a parameter to specify the activation of cell activities; the stress at local points can be an order of magnitude higher than the average.

If the local cytoskeletal stresses are the direct cause of Ca2+ rise, then they should be correlated in time and space. This correlation cannot be resolved by simple statistical comparisons. The distribution of forces in the structural proteins and the changes in Ca2+ was measured simultaneously using a Quad-View optical imaging system. It was found that the activation of Ca2+ began in regions of higher strain,

#### **Figure 5.**

*Rapid fluid shear pulse generates nonuniform force distribution in astrocytes. Panel (a) shows fluorescent images (YFP, CFP) and inverse FRET ratio representing actinin forces in an astrocyte cell. (b) Changes in actinin force in two ROIs in response to a square shear pulse (23 dyn/cm<sup>2</sup> , 15 ms), showing that the narrow shear pulse produce tension in actinin at the upstream edge and compression at downstream edge. (c) Finite element analysis model of a viscoelastic cells under fluid shear stress.*

**157**

*Early Cell Response to Mechanical Stimuli during TBI DOI: http://dx.doi.org/10.5772/intechopen.93295*

the cytoskeletal structure is most pronounced.

**7. Shear-induced cell membrane tension**

**7.2 Effect of shear stress on cell membrane tension**

membrane tension [116, 117].

**7.1 The cell membrane**

of fluid shear (23 dyn/cm2

that takes ~150 ms to saturate [122].

which normally occurred at the upstream edge of the cells and propagated from the edge to the somata of the cell as a wave [114]. In animal models, the NMDAR subunits are found primarily in the astrocytic processes [115]. This finding supports the earlier speculations that applied force may primarily affect the processes where

breaking of the bonds or irreversible deformations of the cytoskeleton.

We have noticed that shear stresses above the Ca2+ threshold did not disrupt cell adhesions nor produced any visible changes in cytoskeletal structures, indicating that the activation of Ca2+-permeable ion channels require much lower forces than

Since the mechanosensitive ion channels are membrane bound, changes in cell membrane tension inevitably affect the ion channel configurations that mediate Ca2+ influx. Indeed, it is known that mechanosensitive channels can be opened by

The cell membrane consists of a lipid bilayer incorporating the membrane proteins including integral proteins such as transmembrane ion channels and receptors and peripheral proteins that loosely attach to the outer side of the cell membrane. Through the functional proteins, the cell membrane selectively controls the transport of ions, water, and macromolecules between the intracellular and extracellular compartments. Inside the cell, the lipid bilayer intimately adheres to the cortical cytoskeleton that provides the support for membrane topography and integrity [118]. On the outside is a hair-like structure called the glycocalyx. Depending on the cell type and local environments, the cell membrane may have tension at its resting state, called pretension. Several factors contribute to the pretension, including internal forces exerted by the cytoskeleton, osmotic pressure from the cytosol, and the forces resulting from cell-substrate interactions at adhesions that can be passed by cytoskeleton [119].

The bilayer tension can be measured using lipid-soluble molecular rotor probe FCVJ [120] whose mobility is commonly used to extract the lipid bilayer fluidity or viscosity. An increase in bilayer tension increases the fluidity in cell membrane, causing a decrease fluorescent intensity of probes [121]. We measured the bilayer tension in astrocyte membranes using the molecular rotor probe FCVJ with the above-described microfluidic chip. As demonstrated in **Figure 6(a)**, a square pulse

with higher tension at the upstream edge of the cell and a lower tension (compression) at the distal edge. Both tension and compression recover back to the initial state within ~30 ms. In comparison, the same shear pulse generated a much longerlasting tension in actinin at the upstream edge of the cell. **Figure 6(b)** and **(c)** illustrates these different characteristics. Interestingly, the membrane tension at the front edge increased much slower than compression at the downstream edge, suggesting that there exists a pretension in the membrane probably via the cortical actin cytoskeleton. The pretension resists the effect of shear force at the upstream edge. In addition, buckling (rapid compression) can occur at the downstream edge. It has been shown that buckling of the lipid membrane can occur at a similar timescale

, 400 ms) generates a gradient in the membrane tension,

*Recent Advances in Biomechanics*

**6.2 Heterogeneous force distribution in single cells**

Importantly, the time dependence of the stimulus plays a critical role.

By inserting the FRET-based force probes in actin-binding protein (α-actinin) in astrocytes, the cytoskeletal protein response to shear stress was measured for the first time. Results show that rapid shear stimuli generate nonuniform protein forces in single cells. **Figure 5** demonstrates that a narrow square shear pulse produced an immediate increase in force in α-actinin at the upstream end of the cell and a small negative force at the downstream end, resulting in a force gradient along the flow direction. In contrast, a slow ramp to the same force amplitude caused a minimal and more uniform change in actinin force. Moreover, the cytoskeleton structure and its prestress states also influence the cell response. The shear pulse produced significant internal force gradients in softer cells that have fewer bundled actin filaments [114]. These experiments demonstrate that uniform shear stress can generate heterogeneous cytoskeletal forces in single cells.

While fluid velocity in the flow chamber was precisely controlled, the body stresses produced in the cells depend on cell geometry and the distribution of stresses within the cytoskeleton (we now know that stress distribution is heterogeneous). This heterogeneity likely accounts for the observed variability between cells. Thus, an averaged impact force cannot be used as a parameter to specify the activation of cell activities; the stress at local points can be an order of magnitude higher than the average.

If the local cytoskeletal stresses are the direct cause of Ca2+ rise, then they should

be correlated in time and space. This correlation cannot be resolved by simple statistical comparisons. The distribution of forces in the structural proteins and the changes in Ca2+ was measured simultaneously using a Quad-View optical imaging system. It was found that the activation of Ca2+ began in regions of higher strain,

*Rapid fluid shear pulse generates nonuniform force distribution in astrocytes. Panel (a) shows fluorescent images (YFP, CFP) and inverse FRET ratio representing actinin forces in an astrocyte cell. (b) Changes in* 

*pulse produce tension in actinin at the upstream edge and compression at downstream edge. (c) Finite element* 

*, 15 ms), showing that the narrow shear* 

*actinin force in two ROIs in response to a square shear pulse (23 dyn/cm<sup>2</sup>*

*analysis model of a viscoelastic cells under fluid shear stress.*

**156**

**Figure 5.**

which normally occurred at the upstream edge of the cells and propagated from the edge to the somata of the cell as a wave [114]. In animal models, the NMDAR subunits are found primarily in the astrocytic processes [115]. This finding supports the earlier speculations that applied force may primarily affect the processes where the cytoskeletal structure is most pronounced.

We have noticed that shear stresses above the Ca2+ threshold did not disrupt cell adhesions nor produced any visible changes in cytoskeletal structures, indicating that the activation of Ca2+-permeable ion channels require much lower forces than breaking of the bonds or irreversible deformations of the cytoskeleton.
