(485ay) Shear Stress Induced Rheological Changes in the Nucleus

Endothelial cells are a single monolayer of cells that line blood vessels and directly experience and respond to shear flow by deforming and aligning in the direction of the shear flow. The deformation of these cells, and all cells, is suggested to decrease the shear stress on the nucleus, the largest organelle which directs the activities of the cell by altering genome expression. Defects in cellular response to shear stress are correlated with early signs of atherosclerosis. Atherosclerosis is an inflammatory response of the blood vessels caused, in part, by altered chemical and mechanical function of the endothelial cells and underlying smooth muscle cells. Atherosclerosis is responsible for most heat attacks and strokes and is the most prominent cause of death in the western world. Here, we aim to better understand the resistance of cells to shear stress by examining the response of the nucleus. We will study the effects of a model aging system to examine the role of aging on the progression of atherosclerosis.

Hutchinson-Gilford Progeria Syndrome (HGPS) is a premature aging disorder resulting in patient death on average at the age of 13 due to an atherosclerotic related event. At the time of death the endothelial cell layer in HGPS patients is denuded exposing the underlying smooth muscle cells which are morphologically altered due to blood flow. HGPS is a laminopathy, meaning it affects the filamentous lamin structure that lies at the inner nuclear membrane. The nuclear lamina is responsible for maintaining nuclear stiffness, nuclear integrity, and aids in chromatin organization. The nuclear lamina provides a physical link between the chromatin inside the nucleus and the nuclear membrane and cytoskeleton. In the case of HGPS a mutant form of lamin A, progerin, is manufactured and assembles into the lamina. Progerin expression results in altered lamin localization, dysmorphic nuclei and stiffer nuclei. Interestingly, the production of progerin is not limited to HGPS patients but occurs sporadically over the lifetime of healthy individuals, resulting in altered lamin localization and dysmorphic nuclei as people age. We are testing the hypothesis that the stiffening of the nuclei with progerin compromises a cells ability to respond to shear stress and may be involved in the development of atherosclerosis due to endothelial cell dysfunction in HGPS patients and otherwise healthy patients.

Mechanotransduction is the process by which external stimuli result in changes in cell behavior and includes the alignment and deformation of cells. Protein expression in the nucleus has been linked to chromatin organization, demonstrating that proteins can only be expressed when the chromatin for the protein of interest is located in specific places in the nucleus. The physical links between the nuclear lamina and the cytoskeleton provides a direct path for external stimuli to affect chromatin organization. These connections suggest that externally experienced mechanical stimuli could directly alter chromatin organization and cell fate.

We have studied the response of progerin expressing cells to shear stress to examine possible roles of a stiffened nucleus in atherosclerosis progression and mechanotransduction. Subcellular effects of shear stress are difficult to predict due to the dampening effects of the cytoskeleton. We label nuclei with RFP-progerin and GFP tagged chromatin binding proteins to produce ~50-100 small, fiducial points within the nucleus. We quantify changes in nuclear organization and subnuclear movement in cells under shear. We show that progerin-expressing nuclei have decreased deformation in flow and do not alter chromatin organization when compared to control cells. We track mean square displacement of nuclear markers to determine changes in nuclear rheological properties and correlate these changes to the diseased cells.

In summation, we track changes in nuclear rheology in cells experiencing shear stress to examine the degree of force transduced to the nuclear interior from outside the cell. We are also examining the effects of a stiffened nuclear lamina associated with a model aging syndrome in attenuating that mechanotransduction force. These results will help in understanding the role of force and shear stress in directly regulating gene expression with the goal of linking aging with a tendency to atherosclerosis.