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Abstract
Every day physical activity results in mechanical forces acting on the body. These forces are transferred from the tissue level to the cellular level through the extracellular matrix. The propagation continues further within the cell as the forces are transferred from the extracellular matrix to the cytoskeleton, and finally to the nucleus. The mechanical cues are perceived both physically and biochemically directing gene activation and cell function. However, high impact forces seen in trauma and perturbed mechanical stimulation in disease states, not only compromise the tissue and cellular microenvironment, but also affect the nuclear structural integrity as a growing body of evidence has proven the nucleus to be a mechanosensitive organelle.
This thesis aims to better define alterations in the biophysical features and the biochemical cues disrupted with mechanical force leading to altered nuclear structural integrity. Initially, interested in understanding the nucleus as a mechanosensor and the ensembled response to mechanical loading through the characterization of biophysical features, we examined nuclear perturbations in primary murine neurons subjected to high impact loads in an in vitro model of traumatic axonal injury through a custom-built device and new methods of defining nuclear motion. Live cell imaging of the neuronal nucleus after high impact loading revealed decreased chromatin dynamics, demonstrating a mechanosensitivity of the soma immediately after load. Further interested in distilling the effects of mechanical stimulation in altering a cell’s phenotype, we studied the effects of perturbed loading. In a simplified in vitro model of cardiac disease using primary murine cardiac fibroblast, we showed that aberrant mechanical stimulation is an extrinsic factor in a mechanically-induced premature senescent-like phenotype. Furthermore, alterations in the nuclear envelope and related proteins (i.e., Emerin) could act as key elements in the initiation of this premature senescent-like state. To study how mechanical forces alter chromatin dynamics, we created an imaging tool called CrisprView, based on the CRISPR-Cas9 editing system. Using an in-house designed sgRNA library, we show fluorescent tagging of the telomeres, and Gapdh and Srf genomic loci. As a proof-of-concept study, we tracked telomere movement in real-time and found significant differences of motion in relation to location within the nucleus, and are thus poised to perform future studies using this tool to study chromatin dynamics in response to mechanical loading. Through in vitro studies of high impact loading and perturbed mechanical stimulation, we were able to characterize the nuclear perturbations for an improved understanding of chromatin dynamics and the initiation of stress-induced senescent pathways in altering nuclear integrity.
