(18b) 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)=D_eff*τ^β), 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.