(683e) Mechanical Enhancement of VEGF Intracellular Signaling in Endothelial Cells

A majority of intracellular signaling that triggers changes in gene expression is propagated by chemical cascades that occur as distinct temporal and spatial events within the cell. However, there are other aspects of DNA structure which regulate genome expression including epigenetic modifications, higher order chromatin compaction as well as gene positioning and movement. Our work is aimed at investigating global genome scale reorganization during physiological processes and its role in genome regulation. Specifically, we focus on intracellular mechanical signaling processes that influence gene expression by altering nuclear organization. We examine changes in cytoskeletal force generation, propagation of force through the nuclear envelope and changes to the chromatin in the nuclear interior. To do this, we visualize stimulated actin reorganization in live cells and the associated active nuclear deformations through image analysis of nuclear envelope fluctuations. Further, the propagation of these forces to the nuclear interior is examined using particle tracking of fluorescently-tagged sub-nuclear markers, where we utilize the mechanical-functional coupling of chromatin to monitor mechanical changes as a means of inferring functional changes. Additionally, we investigate the associated changes in chromatin condensation state using fluorescence lifetime imaging microscopy and DNA texture analysis.

Our results indicate that we can decouple the effects of chromatin condensation and intranuclear active stresses arising from intracellular force generation using the particle tracking experiments through calculation of the mean squared displacement (MSD) fit to a power-law rheological model MSD(τ) = D_effτ^α, as is consistent with cell rheological modeling. These results demonstrate that intracellular force generation through the cytoskeleton and its propagation to the nuclear interior is a dominant means of chromatin agitation for nuclear reorganization. Additionally, our work indicates these effects are largely dominated by motor activity, with the actin network serving as a primary driver of intranuclear agitation via myosin-II. These findings are extended to VEGF-stimulated angiogenesis, which is known to induce a dramatic transition in gene expression. The results provide mechanistic details underlying the nuclear reorganization and subsequent turnover in gene expression, where early intracellular force generation induces chromatin agitation leading to chromatin decondensation and reorganization at the long time scales coinciding with changes in expression. Thus, our work highlights the effects of intracellular force generation on chromatin rearrangement and agitation as a means of altering higher order genome organization during physiological changes. These mechanical events occur as part of the underlying intracellular chemical signaling processes and act to enhance the efficacy of the response that brings about changes in gene expression.