Force Mapping in Epithelial Cell Migration
Olivia du Roure, Alexandre Saez, Axel Buguin, Robert H. Austin, Philippe Chavrier, Pascal Silberzan, and Benoit Ladoux
Supporting Figure 7: http://www.pnas.org/cgi/data/0408482102/DC1/1
Supporting Figure 8: http://www.pnas.org/cgi/data/0408482102/DC1/2
Supporting Movie: http://www.pnas.org/content/vol0/issue2005/images/data/0408482102/DC1/08482Movie1.mpg
Mechanotransduction plays a role in a number of cell processes. As such, it is important to gain a better understanding of the forces and stresses that a cell exerts on its substrate. To quantify the magnitude of these forces, an array of PDMS micro-posts (1-2 μm in diameter) were fabricated and cells were cultured on the array. Each post acts as a linear spring where the deflection of the top of the post is directly related to the force acting on the top of the post. Thus, by monitoring the deflection of each post, the corresponding traction forces and stresses can be calculated.
Prior experiments using an array of microposts to detect cell traction forces limited some cell functions. To combat this issue, the posts were spaced far closer together (2 μm to 4 μm center to center). As a result of this increased post density, cell adhesion, locomotion and proliferation were similar to the behavior of cells on regular 2D substrates. Thus, cell forces generated during these processes could be studied.
For single cells, the greatest traction forces occur around the edges of cells with far less mechanical activity underneath the nucleus and middle of the cell. While the average traction stress transmitted by single cells was approximately .6 nN/μm2, the maximal stress was much larger with a magnitude of 3.8 ± .1 nN/μm2. For a cell monolayer, the largest forces were found at the edge of the monolayer, oriented normally to the edge in the direction opposite of advancement. Furthermore, larger stresses occurred at cell-cell interactions in the monolayer while lower stresses developed underneath cell nuclei.
A number of prior methods for quantifying cell traction forces are limited in their ability to accurately estimate the magnitude and location of these forces. Meanwhile, prior post-arrays limited and affected cell behavior, distorting the information extracted. The importance of this research is twofold: one, the ability of cells to behave normally on an array of posts is demonstrated and two, cell traction forces and stresses were quantified more accurately during cell processes due to the localization of forces on singular posts. Accurately quantifying the magnitude of traction stresses during cell processes is integral to many applications in tissue engineering.
5 comments:
What criteria did the researchers use to determine that the cells cultured on the posts did not differ significantly from a normal 2D culture? Was it their doubling time, their manner of adhesion, movement? It would be interesting to use cells with GFP-labeled cytoskelatal proteins to observe how the structure and dynamics of the actin network changes as the amount of force the cell exerts on its substrate changes.
Ryan,
To determine if the micro-post array affected cell behaviors, cells were cultured on both an array substrate and a normal (control) 2D substrate. The percentage of the surface covered by cells was monitored with time. As there was no difference (statistically) between the two substrates, the authors inferred that the post array did not interfere with cell behaviors (adhesion, locomotion, proliferation, etc.). The data from this is included in supporting figure 7.
The authors actually did monitor some protein activity; however, they did not go into much detail as to their findings. One result mentioned was that actin organization was found to be directly related to cell traction stresses.
I understand that PDMS is an elastomer, and thus rubber-like and easy to bend. As a result my question is how high are these posts and would the height change any conclusions?
Second, has there been any papers that use the Atomic Force Microscope to examine the forces and stresses of the cell?
AJMach,
The height of the posts in this paper are ~10 um tall. Changing the height would affect the stiffness of the posts as stiffness 'k' is proportional to L^-3 (where L is the height). Also, there is always the possibility of the height being changed enough such that the stiffness is no longer linearly related to the displacement. This would be an issue in terms of quantification of cell traction forces. However, for the heights and displacements considered in this paper, it is assumed that the linear relationship is acceptable.
AFMs cannot be used to quantify traction forces; however, they have been used to quantify attachment forces of cells. Specifically, the post-array is used to determine the cell traction forces, which is the forces a cell exerts on its substrate to generate locomotion (or simply to mechanically examine the micro-environment). Meanwhile, attachment force corresponds to the strength with which a cell holds onto is substrate. To quantify this, AFMs have been used to 'push' cells and see how much force is necessary to get cells to detach from the substrate.
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