MEKK1-dependent phosphorylation of calponin-3 tunes cell contractility.

MEKK1 (also known as MAP3K1), which plays a major role in MAPK signaling, has been implicated in mechanical processes in cells, such as migration. Here, we identify the actin-binding protein calponin-3 as a new MEKK1 substrate in the signaling that regulates actomyosin-based cellular contractility. MEKK1 colocalizes with calponin-3 at the actin cytoskeleton and phosphorylates it, leading to an increase in the cell-generated traction stress. MEKK1-mediated calponin-3 phosphorylation is attenuated by the inhibition of myosin II activity, the disruption of actin cytoskeletal integrity and adhesion to soft extracellular substrates, whereas it is enhanced upon cell stretching. Our results reveal the importance of the MEKK1-calponin-3 signaling pathway to cell contractility.

Design and construction of an equibiaxial cell stretching system that is improved for biochemical analysis.

We describe the design and validation of an equibiaxial stretching device in which cells are confined to regions of homogeneous strain. Using this device, we seek to overcome a significant limitation of existing equibiaxial stretching devices, in which strains are not homogeneous over the entire region of cell culture. We cast PDMS in a mold to produce a membrane with a cylindrical wall incorporated in the center, which was used to confine cell monolayers to the central membrane region subjected to homogeneous equibiaxial strain. We demonstrated that the presence of the wall to hold the culture medium did not affect strain homogeneity over the majority of the culture surface and also showed that cells adhered well onto the PDMS membranes. We used our device in cyclic strain experiments and demonstrated strain-dependent changes in extracellular signal-regulated kinase (ERK) and tyrosine phosphorylation upon cell stretching. Furthermore, we examined cell responses to very small magnitudes of strain ranging from 1% to 6% and were able to observe a graduated increase in ERK phosphorylation in response to these strains. Collectively, we were able to study cellular biochemical response with a high degree of accuracy and sensitivity to fine changes in substrate strain. Because we have designed our device along the lines of existing equibiaxial stretching technologies, we believe that our innovations can be incorporated into existing systems. This device would provide a useful addition to the set of tools applied for in vitro studies of cell mechanobiology.