Submitted to the Department of Chemical Engineering.
Thesis (Ph.D.)--Stanford University, 2013.
Physical forces are ubiquitous in cellular biology and effect functionality at various different scales. Recent studies have shown that forces influence processes such as growth and development, aneurysm formation, atherosclerosis, wound healing and cancer metastasis. In particular, the exquisitely tuned mechanical crosstalk between the cells and the extracellular matrix (ECM) ensures proper biological form and function. One of the most important process that is modulated by mechanical force is the proteolysis of the ECM. There are several conflicting studies that describe the effect of applied load on ECM proteins proteolysis. To reconcile these discrepancies, and elucidate the effect of force on ECM protein proteolysis, the first part of the thesis uses a high-throughput single-molecule magnetic tweezers assay to study the effect of force on collagen proteolysis by matrix metalloproteinase 1 (MMP-1) and Clostridium collagenase. These studies reveal that the proteolysis of homotrimeric collagen I by MMP-1 increases ~80 fold by the application of 12 pN of force, and the proteolysis of heterotrimeric collagen I by MMP-1 increases ~10 fold by the application of 15 pN of force. However, the application of similar forces does not alter the proteolysis rate of heterotrimeric collagen I by Clostridium collagenase. Detailed analysis of our data suggests that the collagen trimer has to "unwind" prior to proteolysis by MMP-1 while a similar unwinding mechanism is unnecessary for Clostridium collagenase, likely due to its more open active site. To investigate the effect of force on proteolysis on a larger scale, we studied the proteolysis of fibrin gels by plasmin under elongational load. Fibrin gels are highly elastic, undergoing strains > 200% before rupturing, and are thus well suited for studying the effect of strain on ECM degradation. We find that the application of ~180% strain leads to an 8-fold decrease in proteolysis and a marked anisotropy in diffusivity along the axes parallel and perpendicular to strain. We present an analytical model that relates strain-induced hindered diffusion to the observed decrease in bulk proteolysis rates. The second part of the thesis presents novel methods to quantify cell-generated ECM displacements in soft 3-D fibrin matrices. The advent of traction force microscopy in 1995 provided a powerful means to unravel the role of mechanical forces in cell migration, force generation, and matrix stiffness sensing. However, the large majority of these studies are done on stiff 2-D substrates, whereas cells are mostly found in soft 3-D environments in vivo. At present there is very little information about how cells generate forces in soft 3-D environments. We utilized a 3-D cell culture assay with fluorescently labeled fibrin gels to study how cells deform a soft 3-D matrix. We show that the cells use dynamic protrusions to deform the surrounding 3-D matrix, and that myosin localization causes this deformation. Moreover, we find that the cells can additionally deform the surrounding matrix by actively modulating their volume. The findings of this thesis can influence further research that elucidates how cells mechanically and proteolytically deform the surround extracellular matrix in biological processes such as growth, development, wound healing and cancer metastases.