(339e) Investigating Changes in Cellular Based Forces in Monolayers By Tracking Sub-Nuclear Sensors

Authors: 
Armiger, T., Carnegie Mellon University
Dahl, K. N., Carnegie Mellon University
Reinhart-King, C., Vanderbilt University
In mechanobiological processes, cells generate forces to modulate stiffness and are responsive to mechanical perturbations. Measuring mechanical forces within monolayers of cells is particularly challenging where there are unknown balances of forces among cells and between the cells and the substrate. This is further complicated when considering heterogeneities within a monolayer with additional spatial variations. However, these mechanical heterogeneous variations in the monolayer are of interest since they lead to cellular transformations in development, wound healing, and disease. Additionally, the extent that forces propagate through a given cell to its nucleus can alter the expression of genes through the process of mechanotransduction. Current techniques for cellular force measurement often require an extracellular probe, which the cells perturb. Forces are then calculated from these perturbations. The use of external probes, or specific extracellular environments, for force measurement limits current techniques to particular experimental set ups, often limiting their biological relevance. Here we present a novel technique, termed Sensors from IntraNuclear Kinetics (SINK), which uses the cell nucleus as a relative force sensor. SINK utilizes fluorescently tagged genome bound proteins, whose motion is observed in live cells and tracked over time. SINK takes advantage of the interconnectedness of the cytoskeleton to the nucleus where we show the extent to which molecular motors as well as nuclear to cytoskeletal connections alter chromatin movement. We further show the utility of SINK by investigating how structural changes affect force propagation through cells as they transition from isolated cells to confluent monolayers. Additionally, we investigate the extent that substrate stiffness affects force propagation in monolayers, demonstrating that cells sense their underlying substrate, which ultimately influences motion within the nucleus. Finally, we begin to show how point defects in a relatively homogeneous monolayer affect surrounding cells, with future applications as a disease model for cancer or cardiovascular diseases, as these often stem from point defects in tissues. Because SINK relies on the cell nucleus, it could be extended to in situ and in vivo systems, or may be used in conjunction with current force measurement techniques to get a more complete understanding of how cells propagate force from focal adhesions, through the cytoskeleton, and ultimately into the nucleus where the chromatin fibers are affected. Thus, the future utility of SINK lies in the ability to investigate the extent to which physiologically based forces affect genome expression through physical perturbation of the chromatin fiber.