Many types of cells are sensitive to the mechanical properties of their environment. In cultures of fibroblasts and endothelial cells, for example, it has been shown that cell morphology and motility are sensitive to substrate elasticity¹ and are related to the traction forces exerted by cells.^2,3 It was shown that in human airway soft muscle cells, the stiffness of the cell itself increases in proportion to the traction forces the cell exerts on the substrate.⁴ Taken together, these results suggest that, at confluence, cells will exhibit different mechanical behavior than at sparse densities because in a confluent layer the ‘mechanical environment’ is active; it is composed of a substrate and neighboring cells. To explore collective mechanical behavior of cells at confluent densities, we cultured cos-7 epithelial cells on PDMS (Young’s modulus ~10 kPa), and monitored the motion of beads embedded below the elastomer surface (Figure 1).

The cells were monitored for ~10-12 hours and kept at 37^o C and 5% CO₂. Images were acquired every two minutes and processed using standard particle tracking algorithms. To characterize the dynamics of bead motion, the dynamic structure factor was calculated (Figure 2A). The diagonal band of intensity, shown as slices in Figure 2E, indicates that there is a collective excitation that propagates with a speed of approximately 13.9 mm per hour (Figure 2B). The inverse half width of this excitation, corresponding to an excitation lifetime, exhibits peaks at approximately 0.25 mm-1 and 0.45 mm-1, corresponding to long-lived structural correlations of wavelength ~25 mm and 14 mm (Figure 2D). The amplitude of this excitation decays like e^-q/q_d, where q_d ~0.214 mm-1, corresponding to a correlation distance of ~29.3 mm, or about one cell diameter. Tentatively, we interpret this as a reflection of the strong coupling between nearest neighbors, beyond which there is no direct mechanical interaction between cells.

It is difficult to directly visualize such a collective excitation in real space, but by reducing the dimensions of the system, it is possible to watch excitations propagate across a single file line of cells. We use soft lithography to create elastic micropatterns⁵, which forces the cells to adhere on soft strips of PDMS with embedded fluorescent beads (Figure 3). We observed astoundingly coherent bead motion, illustrated by Figure 4A, which is a plot of selected representative tracks with mean bead position labeled on the right.

Such motion can be directly seen in this movie. Within a given strip, beads on opposite sides of the field of view move in phase over the length of the measurement, approximately 10 hours. This striking cooperative behavior demonstrates that mechanical signals can be transmitted between cells over large distances, likely coupled to active changes in intracellular biochemistry and gene expression.

In the set of tracks shown in Figure 4A, there is a quick jump in bead position at around 3.5 hours. This jump shows up as a peak in the speed of the beads, as seen in figure 4B. Plotting the position of the bead at peak speed versus the time of the peak speed, we see the excitation propagating across the cell layer. This excitation propagates at a very rapid speed of 1.3 mm per second, about 1000 times faster than the 4 mm per hour mean bead speed in the row. This fast propagationsuggests that the slow, coherent motion arises from faster mechanical s, allowing cells nearly 1mm apart to cooperate, synchronously generating forces.

For more information, contact:

Thomas E. Angelini

McKay Laboratory 525

9 Oxford St.

Cambridge, MA 02138

Tel: (617) - 495 - 3705

Fax: (617) - 496 - 3088

angelini@deas.harvard.edu

References:

1.             Pelham, R. J., Jr. & Wang, Y. Cell locomotion and focal adhesions are regulated by substrate flexibility. Proc Natl Acad Sci USA 94, 13661-5 (1997).

2.             Harris, A. K., Wild, P. & Stopak, D. Silicone rubber substrata: a new wrinkle in the study of cell locomotion. Science 208, 177-9 (1980).

3.             Tan, J. L. et al. Cells lying on a bed of microneedles: an approach to isolate mechanical force. Proc Natl Acad Sci U S A 100, 1484-9 (2003).

4.             Wang, N. et al. Cell prestress. I. Stiffness and prestress are closely associated in adherent contractile cells. Am J Physiol Cell Physiol 282, C606-16 (2002).

5.             Gray, D. S., Tien, J. & Chen, C. S. Repositioning of cells by mechanotaxis on surfaces with micropatterned Young's modulus. J Biomed Mater Res A 66, 605-14 (2003).