| This thesis explores the role of extracellular matrix stiffness on guiding mesenchymal stem cell migration and the polarized organization of the internal cellular structure. Cells do not have eyes, and therefore must physically and directly pull on its surrounding matrix in order to perceive its stiffness or elasticity. Close attention must be placed to how the matrix stiffness is probed by the cell, specifically the length scales at which this is occurring. From this perspective, micro-elasticities felt by cells will vary for each tissue, and specifically for mesenchymal stem cells that have to egress from the bone marrow and find their way to a final destination where they differentiate or immunosuppress. Undoubtedly, these cells must experience heterogeneity in the stiffness of matrix they experience along their way during migration. Indeed, gradients in matrix elasticity have been measured, notably the heart which has undergone a myocardial infarction. We employ several in vitro systems to test whether these cells durotax (migration from soft to stiff matrix) and find cell accumulation on stiffer matrix that is not due to a difference in proliferation. To date, there is only speculation on how this occurs, and this thesis helps elucidate some molecular insight on this process. The myosin-IIB (MIIB) isoform only polarizes to the cell rear in migrating cells on stiff matrix but not on soft matrix. Not only does soft matrix seem to suppress MIIB polarity, but also the phosphorylation of myosin-IIA's heavy chain tail at serine 1943 was upregulated on soft matrix. Phosphorylation of this site decreased the amount of acto-myosin filament assembly and experimentation with phosphomimetic and non-phosphorylateable mutants reinforced this conclusion by evaluating these mutants' ability for filament integration, as well as their mobility and solubilities inside the cells. Upon further investigation, the phosphorylation at this serine influenced the MIIB's polarization and amounts of phosphorylation and polarization were finally related to durotaxic ability. Additionally, the extent of microtubule and centrosomal organization within these cells was influenced by matrix stiffness. Soft matrix seems to randomize the internal cytoskeletal structure, while stiff matrix promotes organization and polarization that commonly observed in migrating cells. |
