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).