The use of polyacrylamide hydrogels to study the effects of matrix stiffness on nuclear envelope properties

Matrix-derived mechanical cues influence cell proliferation, motility, and differentiation. Recent findings clearly demonstrate that the nuclear envelope (NE) adapts and remodels in response to mechanical signals, including matrix stiffness, yet a plethora of studies have been performed on tissue culture plastic or glass that have a similar stiffness to cortical bone. Using methods that allow modulation of matrix stiffness will provide further insight into the role of the NE in physiological conditions and the impact of changes in stiffness observed during ageing and disease on cellular function. In this chapter, we describe the polyacrylamide hydrogel system, which allows fabrication of hydrogels with variable stiffness to better mimic the environment experienced by cells in most tissues of the body

Mapping the Nonreciprocal Micromechanics of Individual Cells and the Surrounding Matrix Within Living Tissues

The biomechanical properties of the extracellular matrix (ECM) play an important role in cell migration, gene expression, and differentiation. Biomechanics measurements of ECM are usually performed on cryotomed tissue sections. However, studies on cell/matrix interplay are impossible to perform due to disruptions in cell viability and tissue architecture from freeze-thaw cycling. We developed a technique to map the stiffness of living cells and surrounding matrix by atomic force microscopy and use fluorescence microscopy to relate those properties to changes in matrix and cell structure in embryonic and adult tissues in situ. Stiffness mapping revealed significant differences between vibratomed (living) and cryotomed tissues. Isolated cells are softer than those in native matrix, suggesting that cell mechanics are profoundly influenced by their three-dimensional environment and processing state. Viable tissues treated by hyaluronidase and cytochalasin D displayed targeted disruption of matrix and cytoskeletal networks, respectively. While matrix stiffness affected cellular stiffness, changes in cell mechanics did not reciprocally influence matrix stiffness