Osteoprotegerin is sensitive to actomyosin tension in human periodontal ligament fibroblasts.

Periodontal ligament fibroblasts (PdLFs) are an elongated cell type in the periodontium with matrix and bone regulatory functions which become abnormal in periodontal disease (PD). Here we found that the normally elongated and oriented PdLF nucleus becomes rounded and loses orientation in a mouse model of PD. Using in vitro micropatterning of cultured primary PdLF cell shape, we show that PdLF elongation correlates with nuclear elongation and the presence of thicker, contractile F-actin fibers. The rounded nuclei in mouse PD models in vivo are, therefore, indicative of reduced actomyosin tension. Inhibiting actomyosin contractility by inhibiting myosin light chain kinase, Rho kinase or myosin ATPase activity, in cultured PdLFs each consistently reduced messenger RNA levels of bone regulatory protein osteoprotegerin (OPG). Infection of cultured PdLFs with two different types of periodontal bacteria (Porphyromonas gingivalis and Fusobacterium nucleatum) failed to recapitulate the observed nuclear rounding in vivo, upregulated nonmuscle myosin II phosphorylation and downregulated OPG. Collectively, our results add support to the hypothesis that PdLF contractility becomes decreased and contributes to disease progression in PD.

Apical cell protrusions cause vertical deformation of the soft cancer nucleus.

Breast cancer nuclei have highly irregular shapes, which are diagnostic and prognostic markers of breast cancer progression. The mechanisms by which irregular cancer nuclear shapes develop are not well understood. Here we report the existence of vertical, apical cell protrusions in cultured MDA-MB-231 breast cancer cells. Once formed, these protrusions persist over time scales of hours and are associated with vertically upward nuclear deformations. They are absent in normal mammary epithelial cells (MCF-10A cells). Microtubule disruption enriched these protrusions preferentially in MDA-MB-231 cells compared with MCF-10A cells, whereas inhibition of nonmuscle myosin II (NMMII) abolished this enrichment. Dynamic confocal imaging of the vertical cell and nuclear shape revealed that the apical cell protrusions form first, and in response, the nucleus deforms and/or subsequently gets vertically extruded into the apical protrusion. Overexpression of lamin A/C in MDA-MB-231 cells reduced nuclear deformation in apical protrusions. These data highlight the role of mechanical stresses generated by moving boundaries, as well as abnormal nuclear mechanics in the development of abnormal nuclear shapes in breast cancer cells.

Nuclear size changes caused by local motion of cell boundaries unfold the nuclear lamina and dilate chromatin and intranuclear bodies.

The mechanisms by which mammalian nuclear shape and size are established in cells, and become abnormal in disease states are not understood. Here, we tracked motile cells that underwent systematic changes in cell morphology as they moved from 1-D to 2-D micro-patterned adhesive domains. Motion of the cell boundaries during cell motility caused a dynamic and systematic change in nuclear volume. Short time scales (∼1 h) distinguished the dilation of the nucleus from the familiar increase that occurs during the cell cycle. Nuclear volume was systematically different between cells cultured in 3-D, 2-D and 1-D environments. Dilation of the nuclear volume was accompanied by dilation of chromatin, a decrease in the number of folds in the nuclear lamina, and an increase in nucleolar volume. Treatment of 2-D cells with non-muscle myosin-II inhibitors decreased cell volume, and proportionately caused a decrease in nuclear volume. These data suggest that nuclear size changes during cell migration may potentially impact gene expression through the modulation of intranuclear structure.

Dynamic Adaptation in Extant Porins Facilitates Antibiotic Tolerance in Energetic Escherichia coli.

Bacteria can tolerate antibiotics despite lacking the genetic components for resistance. The prevailing notion is that tolerance results from depleted cellular energy or cell dormancy. In contrast to this view, many cells in the tolerant population of Escherichia coli can exhibit motility - a phenomenon that requires cellular energy, specifically, the proton-motive force (PMF). As these motile-tolerant cells are challenging to isolate from the heterogeneous tolerant population, their survival mechanism is unknown. Here, we discovered that motile bacteria segregate themselves from the tolerant population under micro-confinement, owing to their unique ability to penetrate micron-sized channels. Single-cell measurements on the motile-tolerant population showed that the cells retained a high PMF, but they did not survive through active efflux alone. By utilizing growth assays, single-cell fluorescence studies, and chemotaxis assays, we showed that the cells survived by dynamically inhibiting the function of existing porins in the outer membrane. A drug transport model for porin-mediated intake and efflux pump-mediated expulsion suggested that energetic tolerant cells withstand antibiotics by constricting their porins. The novel porin adaptation we have uncovered is independent of gene expression changes and may involve electrostatic modifications within individual porins to prevent extracellular ligand entry.

Viscous shaping of the compliant cell nucleus.

The cell nucleus is commonly considered to be a stiff organelle that mechanically resists changes in shape, and this resistance is thought to limit the ability of cells to migrate through pores or spread on surfaces. Generation of stresses on the cell nucleus during migration and nuclear response to these stresses is fundamental to cell migration and mechano-transduction. In this Perspective, we discuss our previous experimental and computational evidence that supports a dynamic model, in which the soft nucleus is irreversibly shaped by viscous stresses generated by the motion of cell boundaries and transmitted through the intervening cytoskeletal network. While the nucleus is commonly modeled as a stiff elastic body, we review how nuclear shape changes on the timescale of migration can be explained by simple geometric constraints of constant nuclear volume and constant surface area of the nuclear lamina. Because the lamina surface area is in excess of that of a sphere of the same volume, these constraints permit dynamic transitions between a wide range of shapes during spreading and migration. The excess surface area allows the nuclear shape changes to mirror those of the cell with little mechanical resistance. Thus, the nucleus can be easily shaped by the moving cell boundaries over a wide range of shape changes and only becomes stiff to more extreme deformations that would require the lamina to stretch or the volume to compress. This model explains how nuclei can easily flatten on surfaces during cell spreading or elongate as cells move through pores until the lamina smooths out and becomes tense.

The Nucleus Bypasses Obstacles by Deforming Like a Drop with Surface Tension Mediated by Lamin A/C.

Migrating cells must deform their stiff cell nucleus to move through pores and fibers in tissue. Lamin A/C is known to hinder cell migration by limiting nuclear deformation and passage through confining channels, but its role in nuclear deformation and passage through fibrous environments is less clear. Cell and nuclear migration through discrete, closely spaced, slender obstacles which mimic the mechanical properties of collagen fibers are studied. Nuclei bypass slender obstacles while preserving their overall morphology by deforming around them with deep local invaginations of little resisting force. The obstacles do not impede the nuclear trajectory and do not cause rupture of the nuclear envelope. Nuclei likewise deform around single collagen fibers in cells migrating in 3D collagen gels. In contrast to its limiting role in nuclear passage through confining channels, lamin A/C facilitates nuclear deformation and passage through fibrous environments; nuclei in lamin-null (Lmna-/- ) cells lose their overall morphology and become entangled on the obstacles. Analogous to surface tension-mediated deformation of a liquid drop, lamin A/C imparts a surface tension on the nucleus that allows nuclear invaginations with little mechanical resistance, preventing nuclear entanglement and allowing nuclear passage through fibrous environments.

Local, transient tensile stress on the nuclear membrane causes membrane rupture.

Cancer cell migration through narrow constrictions generates compressive stresses on the nucleus that deform it and cause rupture of nuclear membranes. Nuclear membrane rupture allows uncontrolled exchange between nuclear and cytoplasmic contents. Local tensile stresses can also cause nuclear deformations, but whether such deformations are accompanied by nuclear membrane rupture is unknown. Here we used a direct force probe to locally deform the nucleus by applying a transient tensile stress to the nuclear membrane. We found that a transient (∼0.2 s) deformation (∼1% projected area strain) in normal mammary epithelial cells (MCF-10A cells) was sufficient to cause rupture of the nuclear membrane. Nuclear membrane rupture scaled with the magnitude of nuclear deformation and the magnitude of applied tensile stress. Comparison of diffusive fluxes of nuclear probes between wild-type and lamin-depleted MCF-10A cells revealed that lamin A/C, but not lamin B2, protects the nuclear membranes against rupture from tensile stress. Our results suggest that transient nuclear deformations typically caused by local tensile stresses are sufficient to cause nuclear membrane rupture.

Repair of nuclear ruptures requires barrier-to-autointegration factor.

Cell nuclei rupture following exposure to mechanical force and/or upon weakening of nuclear integrity, but nuclear ruptures are repairable. Barrier-to-autointegration factor (BAF), a small DNA-binding protein, rapidly localizes to nuclear ruptures; however, its role at these rupture sites is unknown. Here, we show that it is predominantly a nonphosphorylated cytoplasmic population of BAF that binds nuclear DNA to rapidly and transiently localize to the sites of nuclear rupture, resulting in BAF accumulation in the nucleus. BAF subsequently recruits transmembrane LEM-domain proteins, causing their accumulation at rupture sites. Loss of BAF impairs recruitment of LEM-domain proteins and nuclear envelope membranes to nuclear rupture sites and prevents nuclear envelope barrier function restoration. Simultaneous depletion of multiple LEM-domain proteins similarly inhibits rupture repair. LEMD2 is required for recruitment of the ESCRT-III membrane repair machinery to ruptures; however, neither LEMD2 nor ESCRT-III is required to repair ruptures. These results reveal a new role for BAF in the response to and repair of nuclear ruptures.