Nuclear Related Responses to Osmotic Challenge in Chondrocytes.

PhDThe application of prolonged mechanical loading to cartilage alters the osmolality of the extracellular environment, with osmotic challenge known to alter the gene expression and the metabolic activity of chondrocytes. However, the mechanisms by which osmolality controls chondrocyte activity remain unclear. Previous study on various cell types, including chondrocytes, showed that hyper-osmotic challenge induces the condensation of chromatin, with highly condensed chromatin often associated with gene poor regions of DNA and gene silencing. The present study investigated the effect of osmotic challenge on chromatin organisation, genome wide gene-expression and the cellular and nuclear deformability of chondrocytes. In order to observe a broad effect of osmotic challenge on the nuclei, the chondrocytes were subjected to a range of hypo- and hyper-osmotic challenge and imaged by confocal microscopy. Chromatin condensation was quantified by the Sobel edge algorithm in MATLAB. Hyper-osmotic challenge on chondrocytes induced an increase in chromatin condensation. Interestingly, the most marked condensation occurred within the osmolality range of articular cartilage in vivo. The effect of osmotic challenge varied between the monolayer cultured and agarose seeded chondrocytes, which may be due to the differences in cytoskeleton organisation between the two culture conditions. Additionally, chromatin condensation induced by hyper-osmotic challenge was shown to be reversible. Marked differences were observed in the deformability of the cell and nucleus in chondrocytes post osmotic challenge, compared to the 300 mOsm/kg conditions typically used for in vitro isolated chondrocyte studies. From the microarray study, the application of 500 mOsm/kg for both 1 and 5 hours altered the gene expression, including the expression of histone related genes, with a higher number of genes affected by the 5 hours hyper-osmotic challenge. The findings of this study suggest that osmotically-induced alterations in nuclei morphology and chromatin structure may provide a direct biophysical mechanism that controls chondrocytes activity

Rescue of DNA damage after constricted migration reveals a mechano-regulated threshold for cell cycle.

Migration through 3D constrictions can cause nuclear rupture and mislocalization of nuclear proteins, but damage to DNA remains uncertain, as does any effect on cell cycle. Here, myosin II inhibition rescues rupture and partially rescues the DNA damage marker γH2AX, but an apparent block in cell cycle appears unaffected. Co-overexpression of multiple DNA repair factors or antioxidant inhibition of break formation also exert partial effects, independently of rupture. Combined treatments completely rescue cell cycle suppression by DNA damage, revealing a sigmoidal dependence of cell cycle on excess DNA damage. Migration through custom-etched pores yields the same damage threshold, with ∼4-µm pores causing intermediate levels of both damage and cell cycle suppression. High curvature imposed rapidly by pores or probes or else by small micronuclei consistently associates nuclear rupture with dilution of stiff lamin-B filaments, loss of repair factors, and entry from cytoplasm of chromatin-binding cGAS (cyclic GMP-AMP synthase). The cell cycle block caused by constricted migration is nonetheless reversible, with a potential for DNA misrepair and genome variation

Nuclear rupture at sites of high curvature compromises retention of DNA repair factors.

The nucleus is physically linked to the cytoskeleton, adhesions, and extracellular matrix-all of which sustain forces, but their relationships to DNA damage are obscure. We show that nuclear rupture with cytoplasmic mislocalization of multiple DNA repair factors correlates with high nuclear curvature imposed by an external probe or by cell attachment to either aligned collagen fibers or stiff matrix. Mislocalization is greatly enhanced by lamin A depletion, requires hours for nuclear reentry, and correlates with an increase in pan-nucleoplasmic foci of the DNA damage marker γH2AX. Excess DNA damage is rescued in ruptured nuclei by cooverexpression of multiple DNA repair factors as well as by soft matrix or inhibition of actomyosin tension. Increased contractility has the opposite effect, and stiff tumors with low lamin A indeed exhibit increased nuclear curvature, more frequent nuclear rupture, and excess DNA damage. Additional stresses likely play a role, but the data suggest high curvature promotes nuclear rupture, which compromises retention of DNA repair factors and favors sustained damage

Constricted migration modulates stem cell differentiation.

Tissue regeneration at an injured site depends on proliferation, migration, and differentiation of resident stem or progenitor cells, but solid tissues are often sufficiently dense and constricting that nuclei are highly stressed by migration. In this study, constricted migration of myoblastic cell types and mesenchymal stem cells (MSCs) increases nuclear rupture, increases DNA damage, and modulates differentiation. Fewer myoblasts fuse into regenerating muscle in vivo after constricted migration in vitro, and myodifferentiation in vitro is likewise suppressed. Myosin II inhibition rescues rupture and DNA damage, implicating nuclear forces, while mitosis and the cell cycle are suppressed by constricted migration, consistent with a checkpoint. Although perturbed proliferation fails to explain defective differentiation, nuclear rupture mislocalizes differentiation-relevant MyoD and KU80 (a DNA repair factor), with nuclear entry of the DNA-binding factor cGAS. Human MSCs exhibit similar damage, but osteogenesis increases-which is relevant to bone and to calcified fibrotic tissues, including diseased muscle. Tissue repair can thus be modulated up or down by the curvature of pores through which stem cells squeeze

Nuclear Lamins in Cancer

Dysmorphic nuclei are commonly seen in cancers and provide strong motivation for studying the main structural proteins of nuclei, the lamins, in cancer. Past studies have also demonstrated the significance of microenvironment mechanics to cancer progression, which is extremely interesting because the lamina was recently shown to be mechanosensitive. Here, we review current knowledge relating cancer progression to lamina biophysics. Lamin levels can constrain cancer cell migration in 3D and thereby impede tumor growth, and lamins can also protect a cancer cell's genome. In addition, lamins can influence transcriptional regulators (RAR, SRF, YAP/TAZ) and chromosome conformation in lamina associated domains. Further investigation of the roles for lamins in cancer and even DNA damage may lead to new therapies or at least to a clearer understanding of lamins as bio-markers in cancer progression