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null (Ed.)Nuclear mechanics is emerging as a key component of stem cell function and differentiation. While changes in nuclear structure can be visually imaged with confocal microscopy, mechanical characterization of the nucleus and its sub-cellular components require specialized testing equipment. A computational model permitting cell-specific mechanical information directly from confocal and atomic force microscopy of cell nuclei would be of great value. Here, we developed a computational framework for generating finite element models of isolated cell nuclei from multiple confocal microscopy scans and simple atomic force microscopy (AFM) tests. Confocal imaging stacks of isolated mesenchymal stem cells were converted into finite element models and siRNA-mediated Lamin A/C depletion isolated chromatin and Lamin A/C structures. Using AFM-measured experimental stiffness values, a set of conversion factors were determined for both chromatin and Lamin A/C to map the voxel intensity of the original images to the element stiffness, allowing the prediction of nuclear stiffness in an additional set of other nuclei. The developed computational framework will identify the contribution of a multitude of sub-nuclear structures and predict global nuclear stiffness of multiple nuclei based on simple nuclear isolation protocols, confocal images and AFM tests.more » « less
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Thompson, Matthew ; Woods, Kali ; Newberg, Joshua ; Oxford, Julia Thom ; Uzer, Gunes ( , npj Microgravity)
Abstract Reducing the musculoskeletal deterioration that astronauts experience in microgravity requires countermeasures that can improve the effectiveness of otherwise rigorous and time-expensive exercise regimens in space. The ability of low-intensity vibrations (LIV) to activate force-responsive signaling pathways in cells suggests LIV as a potential countermeasure to improve cell responsiveness to subsequent mechanical challenge. Mechanoresponse of mesenchymal stem cells (MSC), which maintain bone-making osteoblasts, is in part controlled by the “mechanotransducer” protein YAP (Yes-associated protein), which is shuttled into the nucleus in response to cyto-mechanical forces. Here, using YAP nuclear shuttling as a measurement outcome, we tested the effect of 72 h of clinostat-induced simulated microgravity (SMG) and daily LIV application (LIVDT) on the YAP nuclear entry driven by either acute LIV (LIVAT) or Lysophosphohaditic acid (LPA), applied after the 72 h period. We hypothesized that SMG-induced impairment of acute YAP nuclear entry would be alleviated by the daily application of LIVDT. Results showed that while both acute LIVATand LPA treatments increased nuclear YAP entry by 50 and 87% over the basal levels in SMG-treated MSCs, nuclear YAP levels of all SMG groups were significantly lower than non-SMG controls. LIVDT, applied in parallel to SMG, restored the SMG-driven decrease in basal nuclear YAP to control levels as well as increased the LPA-induced but not LIVAT-induced YAP nuclear entry over SMG only, counterparts. These cell-level observations suggest that daily LIV treatments are a feasible countermeasure for restoring basal nuclear YAP levels and increasing the YAP nuclear shuttling in MSCs under SMG.