384289 Alterations of Structure and Mechanics in Endothelial Cells Treated with VEGF
Endothelial cells treated with vascular endothelial growth factor (VEGF) show dramatic changes in gene expression and a range of altered phenotypic changes. While the chemical signaling pathways associated with VEGF treatment are well known, we are able to highlight numerous structural and mechanical changes in the cytoskeleton and nucleus, which may accentuate signaling pathways. Generally, stimulated changes in gene expression are accompanied by chromatin remodeling, including the reorganization of genes and regulatory factors. We investigate the physical mechanisms driving chromatin dynamics and reorganization in human endothelial cells in response to VEGF. We visualize stimulated cytoskeletal reorganization in live cells and monitor the associated active nuclear deformations. Within the nucleus, we investigate chromatin movements using particle tracking of chromatin-bound probes and quantify chromatin condensation state using fluorescence lifetime imaging microscopy (FLIM).
We find the magnitude of chromatin fluctuations varies inversely with the extent of chromatin condensation. Our results suggest chromatin decondensation facilitates transcriptional activation through this enhanced mobility in addition to increased transcription factor accessibility for the recruitment to and establishment of transcription sites. Further, we show the temporal dynamics of chromatin fluctuations are enhanced beyond thermal motion by cytoskeletal force generation propagated through the LINC complex and into the nuclear interior. These findings suggest that chromatin condensation and cytoskeletal force generation play distinct functional roles in regulating intranuclear movements.
We extend these findings to VEGF stimulation, where we observe enhanced chromatin dynamics. Early in the VEGF response, we observe actin reorganization and the formation of stress fibers around the nucleus which coincides with increased nuclear deformations. The corresponding enhanced chromatin dynamics early on are derived primarily from an enhanced diffusive exponent reflecting this increased cytoskeleton-driven chromatin agitation. However, at later times the increase in active forces dissipates and returns to control levels, yet enhanced chromatin dynamics persist due to large scale chromatin decondensation as consistent with the induction of the majority of VEGF-responsive genes on this time scale and confirmed via FLIM. Our findings demonstrate a plausible mechanism wherein mechanics, through both force-induced chromatin agitation and chromatin decondensation, may play a role in the kinetic and transport mechanisms associated with stimulated changes in chromatin organization and gene expression as a complement to biochemical signaling cascades. Thus, our work suggests cells may mechanically modulate chromatin dynamics in response to stimuli, which carries vast implications for genome function and regulation including mechanisms for direct mechanical force propagation to the nuclear interior as part of the mechanotranduction response to external forces.