Chagnon-Lasard, with a team from the University of Ottawa, have developed a new microdevice that applies force gradients to cells. With the device, they have shown that the cellular cytoskeleton can not only sense differences in strain forces being applied over an area, it will actually realign itself in the direction of least stress.
The new PDMS* stretching micro device is an intricate little tool: it is able to mimic in-vivo strain events by creating multiple non-uniform strain gradients, meaning a range of strain intensities over a specific area. As well as areas of strain, the device can create gradients of strain on single cell scales. It can even switch the direction of primary strain, which has allowed the researchers to probe the relationship between force and actin dynamics. All the while, maintaining a constant base level of strain previously shown to induce cell reorientation in the direction of strain.
Why it’s important
Understanding how our cells sense and respond to physical cues is important to better understand when these responses go wrong in disease, as well as help researchers to engineer more specific devices for studying these pathologies in the lab. Not only is the humble cell able to respond to biochemical cues and elicit a functional response such as movement or growth, but we also know that the cells will take into account the mechanical forces in the microenvironment.
Mechanotransduction In vivo/at the tissue level
Previous analysis of tissue sections and cell layers, tells us that cells within a tissue structure exposed to cyclic strain such as heart, muscle and lung tissue, are able to organise themselves in the direction of primary strain. In these particular tissues, the strain on the cells will come from stretching of the tissues. In order to avoid significant stress the adherent cells will reorient themselves in the direction of the strain.
Previous studies have attempted to measure the impact of these forces on cellular mechanisms, however reproducing the complex array of strain imposed upon these cells in-vivo has been difficult. This system addresses that difficulty.
The experiment and a clever way to quantitate
The researchers used Human Foreskin Fibroblasts adhered to the PDMS membrane for 15 hours, allowing them to form a strong layer of cells. This layer of cells was then cyclically stretched for 11 hours, inducing maximum strain across the membrane ranging from 2% – 10%mm-1.
Importantly, the researchers developed a programme that was able to measure the strain at any given point on the membrane. Using a series of equations based on the displacement of fluorescent beads embedded in the membrane as a measure for amplitude of strain. This allows them to determine the orientation response of each cell individually in relation to the local environment rather than based on the strain over the entire membrane.
Using this technique the researchers found that cells are able to sense multiple physical cues simultaneously, and will arrange themselves in the most favourable orientation based on the principal strain as well as the strain gradient across the membrane.
How are the cells able to sense these complex strain gradients and respond accordingly?
Chagnon-Lesard and colleagues firstly looked at the effect of the cells own cytoskeleton on reorientation. To investigate this they looked at myosin-II (a key cytoskeletal protein) activity and found that inhibiting contractility prevented the stretch induced alignment in these cells.
Secondly, they thought that the cells focal adhesions, the sites of the cell membrane that are specifically engineered to be in contact with the extra cellular matrix, could be playing a role and this could be working in conjunction with the myosin-II contractility. The protein vinculin was used as a marker for focal adhesions and the vinculin rich structures were seen to align with the direction of the cell. However, when myosin contractility was once again inhibited this correlation of focal adhesions and cell direction was disrupted.
These results together show that cells will change their orientation to avoid large positive strains, as well as strain gradient. Here the researchers have been able to induce and quantify a strain gradient on a cellular level and determine the ability of cells to sense both the maximal strain as well as the strain gradient. This response is reliant on myosin-II contractility. This study ties together bioengineering, functional assays and bio-mechanistic analysis to present an interesting and important finding on cell responses to cyclic stress.
Journal article can be found here
Written by Brooke Lumicisi