Open Access Thesis
Date of Award
Karen A. Beningo
The mechanical environment of a cell and its tissue can impact multiple biological processes including development, wound healing, and metastasis. Specific cellular behaviors influenced by the mechanical microenvironment include differentiation, morphology, apoptosis, migration, and proliferation. In this thesis I have focused specifically on the effect of environmental stiffness and applied mechanical forces on cellular migration and proliferation, respectively. Using two different applications, both tailored to evaluate the mechanical forces alone on cellular behavior, I attempted to simulate the mechanical composition of the in vivo tissue microenvironments in vitro using polyacrylamide hydrogels. To test whether cells maintain a mechanical memory for a specific stiffness in vitro, we utilized a substrate that differentially polymerizes with variant levels of UV exposure and analyzed the directional migration patterns upon different rigidities. These substrates did not show any particular directional preference for migration, however cells did seem to be able to sense variation in stiffness based on the results of a morphology assay. It is unknown whether the cells were unable to sense differences in neighboring stiffnesses due to the extracellular matrix or to the hydrogel itself. To examine the proliferation rates of cells given an applied mechanical stimulus, we created hydrogels embedded with magnetic microbeads that provided a tugging and pulling motion mimicking the effects of adherent cells on their neighboring environment. The observed increase in proliferation upon mechanical stimulation was dependent on the presence of fibronectin coated to the hydrogel surface, indicating that this protein is essential for the mechanosensing response of cells. I hypothesized that compacted conformations of fibronectin are released during mechanical stimulation, opening cryptic binding sites for cells to adhere to. I tested the presence of these cryptic binding sites by chemically crosslinking the ECM prior to stimulation, as well as adding the competitive peptide arginine-glycine-aspartic acid (RGD). Both of these resulted in the decrease in proliferation rate during stimulation but had no effect in control cells. The surface receptor protein responsible for activating these cascades is still unknown. After testing the activity level of â1 integrin, a known mechanosensor and binding partner to fibronectin, there was no difference in the activity of this particular integrin subunit, strongly suggesting this is not the integrin activated by our mechanical stimulus. Protein activity studies found that the phosphorylation state of both Focal Adhesion Kinase (FAK), as well as, Extracellular Signal-Regulated Kinase (ERK) are increased upon stimulation, indicating that these two signaling cascades lead to the increase in the cell cycle activity. Further studies are required to determine the link between the fibronectin cryptic sites and the downstream signaling cascades activated during stimulation. Both of these cell behavioral studies will help to better understand the extent of impact the mechanical environment has on living tissue systems.
Alger, Tara Shane, "Influence Of Mechanical Cues And The Extracellular Matrix On Cell Migration Patterns And The Proliferation Rates Of Cells" (2013). Wayne State University Theses. 225.