(760a) Microrheology of VEGF-Stimulated Nuclear Reorganization in Endothelial Cells

Introduction: Genome regulation requires delicately
balanced temporal control for proper cellular homeostasis and adaptation. Although
DNA is a linear genetic code, recent studies have illuminated that genome
organization within the nucleus aids in regulation¹, and there is
evidence that gene translocation within the nucleus leads to differential
expression². The correlation of gene expression with gene position
and movement in the mechanically stiff nucleus³ suggests there is
bio-rheological coupling impacting gene expression. We examine the effects of
extracellular biochemical stimulation on nuclear organization. These signals
are typically examined as chemical signaling cascades that activate nuclear
transcription factors leading to gene expression. However, stimulation by
certain factors simultaneously reorganizes cellular structures via motor
proteins, causing mechanical force propagation to the nucleus. We quantify the
relationship between the chemical factors and genome reorganization to compare these
rheological changes with the accompanying changes in gene expression. This
exciting idea that intracellular force generation correlates with gene
expression as manifested by rheological changes allows us to answer fundamental
cell biology questions using microrheology.

Materials and Methods: We examine vascular endothelial growth factor
(VEGF) stimulated angiogenesis of human umbilical endothelial cells (HUVECs).
Cells were transfected with GFP-tagged sub-nuclear markers
and were treated with up to 50 ng/mL
of VEGF. Using the fiducial GFP particles, nuclear
movements were tracked in live cells. Particle trajectories were determined (using
custom software⁵ to remove translocation and rotation) to calculate
the mean square displacement (MSD) versus time, which was fit to a power-law
model (MSD(τ)=D_eff*τ^β), consistent with cell rheological
modeling³.

Results and Discussion: Our results indicate that VEGF stimulation
of HUVECs results in an effective intranuclear
softening corresponding to increased MSD magnitudes at
all time points (p < 0.001 from unstimulated
control). This effective softening corresponds to chromatin decondensation
and reorganization that decreases the resistance to motion. This decondensation is coupled with increased motor protein-driven
cytoskeletal stress on the nucleus. This stress has
been suggested to increase nucleoplasmic agitation to
enhance DNA and protein diffusion and, thus, collision frequency to drive the
favorable binding events necessary for transcriptional activation. In addition,
the VEGF stimulated response is time dependent, with significant attenuation in
the response after 2.5 hours. These results are consistent with a recent DNA
microarray study showing a more than two-fold upregulation
of 139 genes following VEGF stimulation, with 53 genes directly induced by VEGF
within the first 2 hours of exposure⁴.

Conclusions: Understanding genome organization within the
nucleus and the mechanisms underlying stimulated transitions in gene expression
are vital to elucidating genome function. Our results indicate that the large turnover
in gene expression from VEGF stimulation corresponds to an effective nuclear
softening from global chromatin agitation, rather than a purely localized and
gene-specific effect. Thus, our work fortifies the dynamic view of the genome,
suggesting stimulated gene repositioning that likely involves motor protein-driven
cytoskeletal stress by correlating known VEGF-stimulated
changes in gene expression with global nuclear reorganization through microrheology.

¹T. Misteli, Cell. 128, 787 (2007). ²S.M. Gasser, Science.
296, 1412 (2002). ³K.N.
Dahl et al., Biophys.
J. 89, 2855 (2005). ⁴M. Abe et al., Angiogenesis.
4, 289 (2001). ⁵G.
Yang et al., Journal of Cell Biology. 182, 631-639 (2008).