Biology subjects

Newberg, J. K.

Publications and source records attributed to Newberg, J. K..

3 recordsLinked to original sources

bioRxiv2020

Modeling stem cell nucleus mechanics using confocal microscopy

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 (MSC) were converted into finite element models and siRNA-mediated LaminA/C depletion isolated chromatin and LaminA/C structures. Using AFM-measured experimental stiffness values, a set of conversion factors were determined for both chromatin and LaminA/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.

bioengineering↗

bioRxiv2020

Low Intensity Vibration Restores Nuclear YAP Levels and Acute YAP Nuclear Shuttling in Mesenchymal Stem Cells Subjected to Simulated Microgravity

Reducing the bone deterioration that astronauts experience in microgravity requires countermeasures that can improve the effectiveness of rigorous and time-expensive exercise regimens under microgravity. 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 cyto-mechanical forces. Here, using YAP nuclear shuttling as a measure of MSC mechanoresponse, we tested the effect of 72 hours of simulated microgravity (SMG) and daily LIV application (LIVDT) on the YAP nuclear entry driven by either acute LIV (LIVAT) or Lysophosphohaditic acid (LPA), applied at the end of the 72h period. We hypothesized that SMG-induced impairment of acute YAP nuclear entry will be alleviated by daily application of LIVDT. Results showed that while both acute LIVAT and 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. Daily dosing of 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 the non-LIVDT treated, SMG only, counterparts. These cell level observations suggest that utilizing daily LIV treatments is a feasible countermeasure for increasing the YAP-mediated anabolic responsiveness of MSCs to subsequent mechanical challenge under SMG.

bioengineering↗

bioRxiv2020

Isolated Nuclei Stiffen in Response to Low Intensity Vibration

The nucleus, central to all cellular activity, relies on both direct mechanical input and its molecular transducers to sense and respond to external stimuli. While it has been shown that isolated nuclei can adapt to applied force ex vivo, the mechanisms governing nuclear mechanoadaptation in response to physiologic forces in vivo remain unclear. To investigate nuclear mechanoadaptation in cells, we developed an atomic force microscopy (AFM) based procedure to probe live nuclei isolated from mesenchymal stem cells (MSCs) following the application of low intensity vibration (LIV) to determine whether nuclear stiffness increases as a result of LIV. Results indicated that isolated nuclei were, on average, 30% softer than nuclei tested within intact MSCs prior to LIV. When the nucleus was isolated following LIV (0.7g, 90Hz, 20min) applied four times (4x) separated by 1h intervals, stiffness of isolated nuclei increased 75% compared to non-LIV controls. LIV-induced nuclear stiffening required functional Linker of Nucleoskeleton and Cytoskeleton (LINC) complex, but was not accompanied by increased levels of the nuclear envelope proteins LaminA/C or Sun-2. While depleting LaminA/C or Sun-1&2 resulted in either a 47% or 39% increased heterochromatin to nuclear area ratio in isolated nuclei, the heterochromatin to nuclear area ratio was decreased by 25% in LIV-treated nuclei compared to controls, indicating LIV-induced changes in the chromatin structure. Overall, our findings indicate that increased apparent cell stiffness in response to exogenous mechanical challenge of MSCs in the form of LIV is in part retained by increased nuclear stiffness and changes in chromatin structure.

bioengineering↗