Mesenchymal stem cells (MSCs) maintain the musculoskeletal system by differentiating into multiple lineages, including osteoblasts and adipocytes. Mechanical signals, including strain and low-intensity vibration (LIV), are important regulators of MSC differentiation via control exerted through the cell structure. Lamin A/C is a protein vital to the nuclear architecture that supports chromatin organization and differentiation and contributes to the mechanical integrity of the nucleus. We investigated whether lamin A/C and mechanoresponsiveness are functionally coupled during adipogenesis in MSCs. siRNA depletion of lamin A/C increased the nuclear area, height, and volume and decreased the circularity and stiffness. Lamin A/C depletion significantly decreased markers of adipogenesis (adiponectin, cellular lipid content) as did LIV treatment despite depletion of lamin A/C. Phosphorylation of focal adhesions in response to mechanical challenge was also preserved during loss of lamin A/C. RNA-seq showed no major adipogenic transcriptome changes resulting from LIV treatment, suggesting that LIV regulation of adipogenesis may not occur at the transcriptional level. We observed that during both lamin A/C depletion and LIV, interferon signaling was downregulated, suggesting potentially shared regulatory mechanism elements that could regulate protein translation. We conclude that the mechanoregulation of adipogenesis and the mechanical activation of focal adhesions function independently from those of lamin A/C.

The nucleus, central to cellular activity, relies on both direct mechanical input as well as its molecular transducers to sense external stimuli and respond by regulating intra-nuclear chromatin organization that determines cell function and fate. In mesenchymal stem cells of musculoskeletal tissues, changes in nuclear structures are emerging as a key modulator of their differentiation and proliferation programs. In this review we will first introduce the structural elements of the nucleoskeleton and discuss the current literature on how nuclear structure and signaling are altered in relation to environmental and tissue level mechanical cues. We will focus on state-ofthe-art techniques to apply mechanical force and methods to measure nuclear mechanics in conjunction with DNA, RNA, and protein visualization in living cells. Ultimately, combining realtime nuclear deformations and chromatin dynamics can be a powerful tool to study mechanisms of how forces affect the dynamics of genome function.

Mechanical signals regulate adipogenic differentiation of mesenchymal stem cells (MSCs). Critical to the mechano-regulation of MSCs, Linker of the Nucleoskeleton and Cytoskeleton (LINC) complexes are integral to both nucleo-cytoskeletal signal transduction and structural integrity of the nucleus. The LINC complex is made of Nesprin proteins that associate with the cytoskeleton on the outer nuclear membrane (ONM) and Sun proteins that bound to nuclear lamina and chromatin at the inner nuclear membrane (INM). In addition to their role in the LINC complex function, depletion of Sun1/2 effects chromosomal tethering to the nuclear envelope, nuclear morphology, and chromatin organization. Suggesting that Sun1/2 proteins may regulate chromatin organization and adipogenic differentiation independent of the LINC complex mediated nucleo-cytoskeletal connectivity. To test this hypothesis Sun1/2 depletion was compared to expression of a dominant-negative KASH (dnKASH) domain to decouple nucleus from cytoskeleton by inhibiting Nesprin-SUN association. Sun1/2 depletion inhibited fat droplet formation and production of adipogenic proteins such as Adipoq, which were supported by RNA-seq showing decreased adipogensis. In contrast dnKASH responded oppositely, increasing fat droplet formation, Adipoq and adipogenic gene expression. At the chromatin level, Sun1/2 depletion increased H3K9me3 levels, increased H3K9me3 foci count, and enrichment on Adipoq. No increase of H3K9me3 levels, foci count, or increased H3K9me3 enrichment on Adipoq was found during dnKASH expression. We conclude that physically decoupling of the LINC complex via dnKASH accelerates adipogenesis and that depletion of Sun1/2 increases heterochromatin accrual and inhibits adipogenesis independent of the LINC complex function.

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Quantitative and volumetric assessment of filamentous actin fibers (F-actin) remains challenging due to their interconnected nature, leading researchers to utilize threshold based or qualitative measurement methods with poor reproducibility. Here we introduce a novel machine learning based methodology for accurate quantification and reconstruction of nuclei-associated F-actin. Utilizing a Convolutional Neural Network (CNN), we segment actin filaments and nuclei from 3D confocal microscopy images and then reconstruct each fiber by connecting intersecting contours on cross-sectional slices. This allowed measurement of the total number of actin filaments and individual actin filament length and volume in a reproducible fashion. Focusing on the role of F-actin in supporting nucleocytoskeletal connectivity, we quantified apical F-actin, basal F-actin, and nuclear architecture in mesenchymal stem cells (MSCs) following the disruption of the Linker of Nucleoskeleton and Cytoskeleton (LINC) Complexes. Disabling LINC in mesenchymal stem cells (MSCs) generated F-actin disorganization at the nuclear envelope characterized by shorter length and volume of actin fibers contributing a less elongated nuclear shape. Our findings not only present a new tool for mechanobiology but introduce a novel pipeline for developing realistic computational models based on quantitative measures of F-actin.

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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.