Noncanonical WNT5A controls the activation of latent TGF-ß to drive fibroblast activation and tissue fibrosis.

Transforming growth factor ß (TGF-ß) signaling is a core pathway of fibrosis, but the molecular regulation of the activation of latent TGF-ß remains incompletely understood. Here, we demonstrate a crucial role of WNT5A/JNK/ROCK signaling that rapidly coordinates the activation of latent TGF-ß in fibrotic diseases. WNT5A was identified as a predominant noncanonical WNT ligand in fibrotic diseases such as systemic sclerosis, sclerodermatous chronic graft-versus-host disease, and idiopathic pulmonary fibrosis, stimulating fibroblast-to-myofibroblast transition and tissue fibrosis by activation of latent TGF-ß. The activation of latent TGF-ß requires rapid JNK- and ROCK-dependent cytoskeletal rearrangements and integrin αV (ITGAV). Conditional ablation of WNT5A or its downstream targets prevented activation of latent TGF-ß, rebalanced TGF-ß signaling, and ameliorated experimental fibrosis. We thus uncovered what we believe to be a novel mechanism for the aberrant activation of latent TGF-ß in fibrotic diseases and provided evidence for targeting WNT5A/JNK/ROCK signaling in fibrotic diseases as a new therapeutic approach.

Fiber alignment in 3D collagen networks as a biophysical marker for cell contractility.

Cells cultured in 3D fibrous biopolymer matrices exert traction forces on their environment that induce deformations and remodeling of the fiber network. By measuring these deformations, the traction forces can be reconstructed if the mechanical properties of the matrix and the force-free matrix configuration are known. These requirements limit the applicability of traction force reconstruction in practice. In this study, we test whether force-induced matrix remodeling can instead be used as a proxy for cellular traction forces. We measure the traction forces of hepatic stellate cells and different glioblastoma cell lines and quantify matrix remodeling by measuring the fiber orientation and fiber density around these cells. In agreement with simulated fiber networks, we demonstrate that changes in local fiber orientation and density are directly related to cell forces. By resolving Rho-kinase (ROCK) inhibitor-induced changes of traction forces, fiber alignment, and fiber density in hepatic stellate cells, we show that the method is suitable for drug screening assays. We conclude that differences in local fiber orientation and density, which are easily measurable, can be used as a qualitative proxy for changes in traction forces. The method is available as an open-source Python package with a graphical user interface.

Collective forces of tumor spheroids in three-dimensional biopolymer networks.

We describe a method for quantifying the contractile forces that tumor spheroids collectively exert on highly nonlinear three-dimensional collagen networks. While three-dimensional traction force microscopy for single cells in a nonlinear matrix is computationally complex due to the variable cell shape, here we exploit the spherical symmetry of tumor spheroids to derive a scale-invariant relationship between spheroid contractility and the surrounding matrix deformations. This relationship allows us to directly translate the magnitude of matrix deformations to the total contractility of arbitrarily sized spheroids. We show that our method is accurate up to strains of 50% and remains valid even for irregularly shaped tissue samples when considering only the deformations in the far field. Finally, we demonstrate that collective forces of tumor spheroids reflect the contractility of individual cells for up to 1 hr after seeding, while collective forces on longer timescales are guided by mechanical feedback from the extracellular matrix.

Durotropic Growth of Pollen Tubes.

To reach the female gametophyte, growing pollen tubes must penetrate different tissues within the pistil, the female reproductive organ of a flower. Past research has identified various chemotropic cues that guide pollen tubes through the transmitting tract of the pistil, which represents the longest segment of its growth path. In addition, physical mechanisms also play a role in pollen tube guidance; however, these processes remain poorly understood. Here we show that pollen tubes from plants with solid transmitting tracts actively respond to the stiffness of the environment. We found that pollen tubes from Nicotiana tabacum and other plant species with a solid or semisolid transmitting tract increase their growth rate in response to an increasing matrix stiffness. By contrast, pollen tubes from Lilium longiflorum and other plant species with a hollow transmitting tract decrease their growth rate with increasing matrix stiffness, even though the forces needed to maintain a constant growth rate remain far below the maximum penetration force these pollen tubes are able to generate. Moreover, when confronted with a transition from a softer to a stiffer matrix, pollen tubes from N. tabacum display a greater ability to penetrate into a stiffer matrix compared with pollen tubes from L. longiflorum, even though the maximum force generated by pollen tubes from N. tabacum (11 µN) is smaller than the maximum force generated by pollen tubes from L. longiflorum (36 µN). These findings demonstrate a mechano-sensitive growth behavior, termed here durotropic growth, that is only expressed in pollen tubes from plants with a solid or semisolid transmitting tract and thus may contribute to an effective pollen tube guidance within the pistil.

Treatment of keratinocytes with 4-phenylbutyrate in epidermolysis bullosa: Lessons for therapies in keratin disorders.

BACKGROUND: Missense mutations in keratin 5 and 14 genes cause the severe skin fragility disorder epidermolysis bullosa simplex (EBS) by collapsing of the keratin cytoskeleton into cytoplasmic protein aggregates. Despite intense efforts, no molecular therapies are available, mostly due to the complex phenotype of EBS, comprising cell fragility, diminished adhesion, skin inflammation and itch. METHODS: We extensively characterized KRT5 and KRT14 mutant keratinocytes from patients with severe generalized EBS following exposure to the chemical chaperone 4-phenylbutyrate (4-PBA). FINDINGS: 4-PBA diminished keratin aggregates within EBS cells and ameliorated their inflammatory phenotype. Chemoproteomics of 4-PBA-treated and untreated EBS cells revealed reduced IL1ß expression- but also showed activation of Wnt/ß-catenin and NF-kB pathways. The abundance of extracellular matrix and cytoskeletal proteins was significantly altered, coinciding with diminished keratinocyte adhesion and migration in a 4-PBA dose-dependent manner. INTERPRETATION: Together, our study reveals a complex interplay of benefits and disadvantages that challenge the use of 4-PBA in skin fragility disorders.

Breast Cancer Cells Adapt Contractile Forces to Overcome Steric Hindrance.

Cell migration through the extracellular matrix is governed by the interplay between cell-generated propulsion forces, adhesion forces, and resisting forces arising from the steric hindrance of the matrix. Steric hindrance in turn depends on matrix porosity, matrix deformability, cell size, and cell deformability. In this study, we investigate how cells respond to changes in steric hindrance that arise from altered cell mechanical properties. Specifically, we measure traction forces, cell morphology, and invasiveness of MDA-MB 231 breast cancer cells in three-dimensional collagen gels. To modulate cell mechanical properties, we either decrease nuclear deformability by twofold overexpression of the nuclear protein lamin A or we introduce into the cells stiff polystyrene beads with a diameter larger than the average matrix pore size. Despite this increase of steric hindrance, we find that cell invasion is only marginally inhibited, as measured by the fraction of motile cells and the mean invasion depth. To compensate for increased steric hindrance, cells employ two alternative strategies. Cells with higher nuclear stiffness increase their force polarity, whereas cells with large beads increase their net contractility. Under both conditions, the collagen matrix surrounding the cells stiffens dramatically and carries increased strain energy, suggesting that increased force polarity and increased net contractility are functionally equivalent strategies for overcoming an increased steric hindrance.

The role of focal adhesion anchoring domains of CAS in mechanotransduction.

CAS is a docking protein, which was shown to act as a mechanosensor in focal adhesions. The unique assembly of structural domains in CAS is important for its function as a mechanosensor. The tension within focal adhesions is transmitted to a stretchable substrate domain of CAS by focal adhesion-targeting of SH3 and CCH domain of CAS, which anchor the CAS protein in focal adhesions. Mechanistic models of the stretching biosensor propose equal roles for both anchoring domains. Using deletion mutants and domain replacements, we have analyzed the relative importance of the focal adhesion anchoring domains on CAS localization and dynamics in focal adhesions as well as on CAS-mediated mechanotransduction. We confirmed the predicted prerequisite of the focal adhesion targeting for CAS-dependent mechanosensing and unraveled the critical importance of CAS SH3 domain in mechanosensing. We further show that CAS localizes to the force transduction layer of focal adhesions and that mechanical stress stabilizes CAS in focal adhesions.

Cell Adhesion on Surface-Functionalized Magnesium.

The biocompatibility of commercially pure magnesium-based (cp Mg) biodegradable implants is compromised of strong hydrogen evolution and surface alkalization due to high initial corrosion rates of cp Mg in the physiological environment. To mitigate this problem, the addition of corrosion-retarding alloying elements or coating of implant surfaces has been suggested. In the following work, we explored the effect of organic coatings on long-term cell growth. cp Mg was coated with aminopropyltriehtoxysilane + vitamin C (AV), carbonyldiimidazole (CDI), or stearic acid (SA). All three coatings have been previously suggested to reduce initial corrosion and to enhance protein adsorption and hence cell adhesion on magnesium surfaces. Endothelial cells (DH1+/+) and osteosarcoma cells (MG63) were cultured on coated samples for up to 20 days. To quantify Mg corrosion, electrochemical impedance spectroscopy (EIS) was measured after 1, 3, and 5 days of cell culture. We also investigated the speed of initial cell spreading after seeding using fluorescently labeled fibroblasts (NIH/3T3). Hydrogen evolution after contact with cell culture medium was markedly decreased on AV- and SA-coated Mg compared to uncoated Mg. These coatings also showed improved cell adhesion and spreading after 24 h of culture comparable to tissue-treated plastic surfaces. On AV-coated cp Mg, a confluent layer of endothelial cells formed after 5 days and remained intact for up to 20 days. Together, these data demonstrate that surface coating with AV is a viable strategy for improving long-term biocompatibility of cp Mg-based implants. EIS measurements confirmed that the presence of a confluent cell layer increased the corrosion resistance.