The Cellular Mechanoresponse: Single-Cell Studies by Atomic Force Microscopy

Cells in their native environment are bombarded by mechanical signals ranging from strains within a developing embryo to stiffening of diseased tissue. How these extracellular mechanical signals are converted to biological activity on the cellular scale is a complex and unresolved problem in biology with implications in development and disease. This dissertation focuses on development and implementation of Atomic Force Microscopy (AFM)-based techniques to probe the interactions of cells with the mechanical microenvironment and use of these techniques towards characterizing and explaining the cellular mechanoresponse.We began by integrating a DNA-based adhesion technology with AFM that enables the manipulation of cells by a cantilever without influencing cell viability or signaling. This technique surpasses existing approaches both in the tunability and magnitude of adhesion strength to allow single-cell de-adhesion experiments that measure cell-ligand bonds without cell-surface rupture. The first step in the cellular mechanoresponse is the translation of an extracellular mechanical signal to an intracellular mechanical signal. We used high-resolution three-dimensional multi-particle tracking to measure how local stress applied by an AFM cantilever is propagated through an adherent cell. We observe a distance-dependent propagation on the timescale of seconds and that required an intact cytoskeletal network. This slow stress propagation is consistent with a poroelastic description of the cell that controls the timescales and lengthscales over which external stresses can be transmitted through cells.Recent studies have demonstrated that cells exhibit stiffness-dependent behaviors over long timescales, but the mechanism of how cells sense stiffness over short timescales remains particularly elusive. To study early events in stiffness sensing, we developed a feedback algorithm that enables dynamic and reversible control of the stiffness exposed to a single cell. We employ this stiffness clamp technique to study the contractile response of cells to sudden changes in extracellular stiffness, as defined by the cantilever. We find that the cell contraction rate adapts by accelerating upon a step decrease in stiffness or decelerating upon a step increase, both on a timescale of seconds. This seconds-timescale adaptation is independent of focal adhesion signaling, but it depends strongly on cell contractility suggesting that extracellular stiffness signals are filtered by the viscoelastic cytoskeleton.Together, the techniques described here provide novel and tunable control of the mechanical signals presented to and measured from a single cell with AFM precision. The results obtained using these techniques describe important timescales and considerations towards understanding the cellular mechanoresponse