Power-Law Rheology of Isolated Nuclei with Deformation Mapping of Nuclear Substructures

Force-induced changes in genome expression as well as remodeling of nuclear architecture in development and disease motivate a deeper understanding of nuclear mechanics. Chromatin and green fluorescent protein-lamin B dynamics were visualized in a micropipette aspiration of isolated nuclei, and both were shown to contribute to viscoelastic properties of the somatic cell nucleus. Reversible swelling by almost 200% in volume, with changes in salt, demonstrates the resilience and large dilational capacity of the nuclear envelope, nucleoli, and chromatin. Swelling also proves an effective way to separate the mechanical contributions of nuclear elements. In unswollen nuclei, chromatin is a primary force-bearing element, whereas swollen nuclei are an order of magnitude softer, with the lamina sustaining much of the load. In both cases, nuclear deformability increases with time, scaling as a power law—thus lacking any characteristic timescale—when nuclei are either aspirated or indented by atomic force microscopy. The nucleus is stiff and resists distortion at short times, but it softens and deforms more readily at longer times. Such results indicate an essentially infinite spectrum of timescales for structural reorganization, with implications for regulating genome expression kinetics

Multi-scale cellular engineering: From molecules to organ-on-a-chip.

Recent technological advances in cellular and molecular engineering have provided new insights into biology and enabled the design, manufacturing, and manipulation of complex living systems. Here, we summarize the state of advances at the molecular, cellular, and multi-cellular levels using experimental and computational tools. The areas of focus include intrinsically disordered proteins, synthetic proteins, spatiotemporally dynamic extracellular matrices, organ-on-a-chip approaches, and computational modeling, which all have tremendous potential for advancing fundamental and translational science. Perspectives on the current limitations and future directions are also described, with the goal of stimulating interest to overcome these hurdles using multi-disciplinary approaches

Surface probe measurements of the elasticity of sectioned tissue, thin gels and polyelectrolyte multilayer films : correlations between substrate stiffness and cell adhesion

Surface probe measurements of the elasticity of thin-film matrices as well as biological samples prove generally important to understanding cell attachment across such systems. To illustrate this, sectioned arteries were probed by Atomic Force Microscopy (AFM) within the smooth muscle cell (SMC)-rich medial layer, yielding an apparent Young’s modulus Emedia ~ 5-8 kPa. Polyacrylamide gels with Egel spanning several-fold above and below this range were then cast 5-70 μm thick and coated with collagen: SMC spreading shows a hyperbolic dependence in projected cell area versus Egel. The modulus that gives half-max spreading is E1/2-spread ~ 8-10 kPa, proving remarkably close to Emedia. More complex, layer-by-layer micro-films of poly(L-lysine)/hyaluronic acid were also tested and show equivalent trends of increased SMC spreading with increased stiffness. Adhesive spreading of cells thus seems to correlate broadly with the effective stiffness of synthetic materials and tissues

Myotubes differentiate optimally on substrates with tissue-like stiffness : pathological implications for soft or stiff microenvironments

Contractile myocytes provide a test of the hypothesis that cells sense their mechanical as well as molecular microenvironment, altering expression, organization, and/or morphology accordingly. Here, myoblasts were cultured on collagen strips attached to glass or polymer gels of varied elasticity. Subsequent fusion into myotubes occurs independent of substrate flexibility. However, myosin/actin striations emerge later only on gels with stiffness typical of normal muscle (passive Young\u27s modulus, E ~12 kPa). On glass and much softer or stiffer gels, including gels emulating stiff dystrophic muscle, cells do not striate. In addition, myotubes grown on top of a compliant bottom layer of glass-attached myotubes (but not softer fibroblasts) will striate, whereas the bottom cells will only assemble stress fibers and vinculin-rich adhesions. Unlike sarcomere formation, adhesion strength increases monotonically versus substrate stiffness with strongest adhesion on glass. These findings have major implications for in vivo introduction of stem cells into diseased or damaged striated muscle of altered mechanical composition

High shear stress enhances endothelial permeability in the presence of the risk haplotype at 9p21.3

Single nucleotide polymorphisms (SNPs) are exceedingly common in non-coding loci, and while they are significantly associated with a myriad of diseases, their specific impact on cellular dysfunction remains unclear. Here, we show that when exposed to external stressors, the presence of risk SNPs in the 9p21.3 coronary artery disease (CAD) risk locus increases endothelial monolayer and microvessel dysfunction. Endothelial cells (ECs) derived from induced pluripotent stem cells of patients carrying the risk haplotype (R/R WT) differentiated similarly to their non-risk and isogenic knockout (R/R KO) counterparts. Monolayers exhibited greater permeability and reactive oxygen species signaling when the risk haplotype was present. Addition of the inflammatory cytokine TNF alpha further enhanced EC monolayer permeability but independent of risk haplotype; TNF alpha also did not substantially alter haplotype transcriptomes. Conversely, when wall shear stress was applied to ECs in a microfluidic vessel, R/R WT vessels were more permeable at lower shear stresses than R/R KO vessels. Transcriptomes of sheared cells clustered more by risk haplotype than by patient or clone, resulting in significant differential regulation of EC adhesion and extracellular matrix genes vs static conditions. A subset of previously identified CAD risk genes invert expression patterns in the presence of high shear concomitant with altered cell adhesion genes, vessel permeability, and endothelial erosion in the presence of the risk haplotype, suggesting that shear stress could be a regulator of non-coding loci with a key impact on CAD

Rapidly Self-Renewing Human Multipotent Marrow Stromal Cells (hMSC) Express Sialyl Lewis X and Actively Adhere to Arterial Endothelium in a Chick Embryo Model System

There have been conflicting observations regarding the receptors utilized by human multipotent mesenchymal bone marrow stromal cells (hMSC) to adhere to endothelial cells (EC). To address the discrepancies, we performed experiments with cells prepared with a standardized, low-density protocol preserving a sub-population of small cells that are rapidly self-renewing.Sialyl Lewis X (SLeX) and α4 integrin expression were determined by flow cytometry. Fucosyltransferase expression was determined by quantitative realtime RT-PCR. Cell adhesion assays were carried out with a panel of endothelial cells from arteries, veins and the microvasculature in vitro. In vivo experiments were performed to determine single cell interactions in the chick embryo chorioallantoic membrane (CAM). The CAM is a well-characterized respiratory organ allowing for time-lapse image acquisition of large numbers of cells treated with blocking antibodies against adhesion molecules expressed on hMSC.hMSC expressed α4 integrin, SLeX and fucosyltransferase 4 and adhered to human EC from arteries, veins and the microvasculature under static conditions in vitro. In vivo, hMSC rolled on and adhered to arterioles in the chick embryo CAM, whereas control melanoma cells embolized. Inhibition of α4 integrin and/or SLeX with blocking antibodies reduced rolling and adhesion in arterioles and increased embolism of hMSC.The results demonstrated that rapidly self-renewing hMSC were retained in the CAM because they rolled on and adhered to respiratory arteriolar EC in an α4 integrin- and SLeX-dependent manner. It is therefore important to select cells based on their cell adhesion receptor profile as well as size depending on the intended target of the cell and the injection route