282870 Nuclear Mechanics of VEGF-Stimulated Endothelial Cells
The packaging of the genome into higher order structures of chromatin and chromosomes has been well established. However, the complementary knowledge of this hierarchical organization within the nucleus has proven elusive. Recent evidence indicates that genome organization plays a role in gene regulation and, further, that gene position within the nucleus may correlate with expression. In response to a stimulus, chromatin must decondense and reorganize for protein binding and transcription. The signal transduction cascades leading to stimulated gene expression have been a major focus of research, but stimulated cytoskeletal rearrangement frequently occurs in parallel to the biochemical signaling leading to mechanical force on the nucleus that has been implicated in chromatin agitation. We hypothesize that this aids in gene expression through motor protein-induced cytoskeletal stresses on the nucleus that act globally and nonspecifically to move gene domains (strain) and alter their probability of expression. Our work aims to correlate these cytoskeletal stresses with the underlying nuclear strain and known changes in gene expression through particle-tracking microrheology.
As a model system, we focus on vascular endothelial growth factor (VEGF) stimulated angiogenesis in human umbilical vein endothelial cells (HUVECs), which is a well-characterized and direct pathway linked to known changes in gene expression and cytoskeletal rearrangement. We transfect cells with GFP-tagged sub-nuclear markers and track them in live cells during treatment with up to 50 ng/mL VEGF. Particle trajectories are obtained (using previously published custom software), from which we compute the mean square displacement (MSD) versus time. The MSD is fit to a power-law model (MSD(τ)=Deff*τβ), as consistent with previous work. In response to VEGF stimulation, the nuclear interior undergoes an effective softening evinced by an increase in MSD magnitudes over all time points (p < 0.001 compared to unstimulated controls). This effective softening is consistent with global chromatin decondensation necessary for nuclear reorganization of the genome and the transition in gene expression. It also results from an increased driving force from the motor protein-driven cytoskeletal stress necessary to enhance gene and protein motion as well as the frequency of binding events to increase the probability of transcription. Thus, our results suggest global nuclear reorganization is necessary for stimulated gene expression despite the fact that chemical stimulation only leads to the upregulation of specific genes. This lends credibility to the dynamic view of nuclear organization as a means of regulating the genome, with implications for cytoskeletal stress in driving stimulated genome rearrangement.
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