Connecting cellular and molecular mechanics: An investigation into the micromechanical architecture of living cells

Mechanical properties of living cells are critical for shape, motility and growth, among other physiologically important features and processes. Mechanical properties of whole cells and individual molecular components have been examined and the conclusion of these studies has been that the cytoskeleton determines cellular mechanics. However, the cellular micromechanical architecture, which connects cellular and molecular mechanics, has not been extensively studied in living cells. The goal of my dissertation is to bring together cellular and molecular micromechanics by investigating micromechanical architecture of cells. Here, I first exploit atomic force microscopy (AFM) to study both topography and mechanics of live cells, and evaluate different cell types in order to determine a suitable system to work with. My results identify Madine-Darby canine kidney (MDCK) cells and bovine pulmonary artery endothelial cells (BPAEC) as such. I compare different approaches to the analysis of AFM force measurements on living cells; demonstrate that Epp (point-to-point elastic modulus), FIEL (force integration to equal limits) and stiffness maps are in good agreement. These spatially resolved maps illustrate that the cell body is softer than the cell periphery in confluent monolayers. Additionally, cells appear to be relatively stiffer at smaller indentations than at large indentations, suggesting the presence of a stiff cortex. Next, I study BPAEC established as a useful system to investigate the micromechanical architecture in living cells using AFM and confocal fluorescence microscopy (CFM). The micromechanical architecture of BPAEC is revealed with sub-optical resolution in AFM images, the contrast of which derives in large part from local mechanical differences. The cortex of BPAEC is organized at two length scales: a coarse mesh and a fine mesh, both of which appear to be intertwined. Correlated CFM and AFM experiments in addition to pharmacological treatments show that actin and vimentin, but not microtubules are components of the coarse mesh. Finally, I demonstrate that the coarse mesh of the cortex of BPAEC is highly dynamic and displays two modes of remodeling: intact-boundary-mode where mesh element boundaries remain intact but move at ∼0.08 mum/min allowing the mesh element to change shape; and altered-boundary-mode where new mesh boundaries form and existing ones disappear.