Cadherins establish mechano-resistant junctions between fibrogenic fibroblasts

During wound healing and in a variety of fibrocontractive diseases, fibroblasts differentiate into contractile myofibroblasts by acquiring stress fibers and by expressing α-smooth muscle actin (α-SMA). Myofibroblasts of wound granulation tissue, in contrast to dermal fibroblasts, join stress fibers at sites of cadherin-type intercellular adherens junctions (AJs). However, the function of myofibroblast AJs, their molecular composition, and the mechanisms of their formation are largely unknown. We demonstrate here that fibroblasts change cadherin expression from N-cadherin in early wounds to OB-cadherin in contractile wounds, populated with α-SMA-positive myofibroblasts. A similar shift occurs during myofibroblast differentiation in culture and seems to be responsible for the homotypic segregation of α-SMA-positive and -negative fibroblasts in suspension. By measuring the adhesion strength between suspended cells with a laser tweezers, we show that fibroblast adhesion is cadherin-mediated since anti-OB-cadherin and anti-N-cadherin peptides reduce the adhesion of both myofibroblasts and fibroblasts respectively. The switch from N- to OB-cadherin expression results in 1.3-fold increased adhesion strength despite reduced cadherin expression levels. Moreover, AJs of plated myofibroblasts are reinforced by α-SMA-mediated contractile activity, resulting in high mechanical resistance as demonstrated by subjecting cell pairs to hydrodynamic forces in a flow chamber. A peptide that inhibits α-SMA-mediated contractile force causes the reorganization of large stripe-like AJs to belt-like contacts as shown for enhanced green fluorescent protein-α-catenin-transfected cells and is associated with a reduced mechanical resistance. Anti-OB-cadherin but not anti-N-cadherin peptides reduce the contraction of myofibroblast-populated collagen gels, suggesting that AJs are instrumental for myofibroblast contractile activity. Using atomic force microscopy we further investigate the difference between N- and OB-cadherin adhesion strength on the single molecule level and show that part of the stronger adhesion of myofibroblasts is accomplished because single OB-cadherin bonds resist 2-fold higher forces compared with single N-cadherin junctions. By assessing the adhesion force between recombinant cadherin dimers and between native cadherins in the membrane of spread fibroblasts, we demonstrate that cadherin bonds are reinforced over time, displaying a hierarchy of three distinct adhesion forces. By modulating the degree of lateral cadherin diffusion and of F-actin organization we can attribute this resulting three force states to the single molecule bond rather than to cadherin cluster formation; this is consistent with a new model of homotypic cadherin ectodomain interaction. Notably, association with actin filaments enhances cadherin adhesion strength on the single molecule level by up to 3-fold; actin depolymerization reduces single bond strength to the level of cadherin constructs missing the cytoplasmic domain. Hence, fibroblasts reinforce intercellular contacts by: 1) switching from N- to OB-cadherin expression, 2) increasing the strength of single molecule bonds in three distinct steps, and 3) actin-promoted intrinsic activation of cadherin extracellular binding. We propose that this plasticity adapts fibroblast adhesions to the changing mechanical microenvironment of tissue under remodeling.

Coordination of Myofibroblast Contraction in Vitro

Lysianne Follonier Castella

The aim of this thesis is to elucidate the common mechanisms that regulate myofibroblast contraction and intercellular communication, with a particular focus on the role of Ca2+ signalling. Synthesis and remodelling of collagenous matrix by myofibroblasts are key elements in the healing process of injured organs. Deregulation of myofibroblast activity and excessive remodelling result in hypertrophic scarring and organ fibrosis. Myofibroblasts are specialised fibroblasts with particularly contractile stress fibres and features that resemble smooth muscle cells. Although myofibroblast contraction is acknowledged being responsible for wound closure and tissue contracture, the underlying regulation mechanisms are unclear. In particular, it remains elusive to what extent changes in the cytosolic Ca2+, a central regulator of smooth muscle contraction, regulate myofibroblast activities. Previous studies have revealed that cultured fibroblasts and myofibroblasts exhibit spontaneous cytosolic Ca2+ oscillations but a link with cell contraction has not been established. In the first part of this thesis, we are testing the hypothesis that cytosolic Ca2+ oscillations regulate myofibroblast contraction. For this, we use analysis of Ca2+ dynamics with fluorescent indicators, tracking of stress-fibre-linked microbeads on the myofibroblast surface and quantification of subcellular pulling events with atomic force microscopy. This combined approach demonstrates that myofibroblasts exhibit periodic (∼100 seconds) Ca2+ oscillations that control small (∼400 nm) and weak (∼100 pN) contractions. Using deformable culture substrate to assess cell isometric tension, we further demonstrate that myofibroblast contraction is regulated at two levels: variations of intracellular Ca2+ mediate contractions of dorsal stress fibres whereas the Ca2+-independent Rho kinase pathway is responsible for maintaining overall isometric cell tension. In the second part of this thesis, we investigate whether the observed subcellular contractions and Ca2+ oscillations may play a role in coordinating the activities between directly contacting myofibroblasts. Rational for this hypothesis is that myofibroblasts in densely populated wound granulation tissue form two types of intercellular junctions: adherens junctions that mechanically couple stress fibres between cells, and gap junctions that provide electrochemical coupling. Both junctions have been shown to play a role in modulating the contraction of wound tissue and of myofibroblast collagen gel cultures but their precise roles are still elusive. To fill this gap in knowledge, we analyse the coordination of Ca2+ oscillations between adjacent fibroblasts and myofibroblasts, respectively. Intercellular communication is modulated with pharmacological treatments acting on either mechanical or electrochemical coupling, as well as on the cell's contractile apparatus and mechanosensitive membrane channels. Our results demonstrate that stress-fibre associated adherens junctions mechanically coordinate myofibroblast activities within a cell population. This mechanism implies that the transmission of contractile events from one cell induces a Ca2+ influx through mechanosensitive ion channels in its neighbour and there triggers a new contraction. Based on these results we suggest that, at the tissue level, myofibroblasts remodel collagen by Ca2+-dependent micro-contractile events that add up to macroscopic tissue contracture, whereas Rho kinase maintains cell isometric tension. Mechanical coupling via adherens junctions may simultaneously improve the remodelling of cell-dense tissue by coordinating the activity of myofibroblasts.

EPFL2010

Interplay between cytoskeletal forces, membrane tension and shape in rapidly migrating cells

Chiara Gabella

Cell migration is a fundamental process in all animal cells. The ability of cell to migrate is crucial for many pathological and physiological conditions such as morphogenesis, the immune response to inflammatory or vascular diseases and cancer metastasis. Actin polymerization pressure and membrane tension are two major forces defining the dynamics of cell leading edge during migration: actin polymerization pushes the membrane forward to drive the motion, while membrane tension is believed to counteract and control protrusion. Recent studies implicated membrane tension in control of cell spreading, limiting lateral extension of the cell, confining protrusion to a single leading edge, and crushing actin network during retraction at the cell rear; however, direct experimental evidence is conflicting. Moreover, actin polymerization and membrane tension are not directly opposing each other, since membrane tension is oriented along the membrane surface, while actin polymerization is directed generally parallel to the substrate. Thus the shape of the leading edge may have important, albeit as yet unknown effect on the force balance. To understand how these forces are orchestrated at the cell’s leading edge, we employed the model system of fish epithelial keratocytes which are spontaneously highly motile and are characterized by a remarkably stable shape and constant protrusion rate, making any changes due to experimental treatments traceable and accessible for quantitative analysis. We set out to measure actin protrusion rate, membrane tension, and three dimensional shape of these cells subjected to manipulation of osmotic pressure, cytoskeletal contractility and drastic shape perturbations (cell wounding). To accomplish this goal, we first developed a simple and reliable technique to measure cell height and volume. This method is based on exclusion of fluorescent dye from the volume of the cell: the decrease of the fluorescent signal with respect to the background is proportional to the height and the volume of the object. We calibrated the technique using a glass microfabricated pattern with steps of defined heights and validated it by comparing our measurements with the ones obtained by atomic force microscopy (AFM). The method is fast and allows precise monitoring of height and volume of rapidly migrating cells, and do not require any modification to the standard epifluorescence setup. We applied our method to measure the real-time volume dynamics of migrating fish epidermal keratocytes subjected to osmotic stress and cytoskeletal perturbations. We then analyzed the relationship between shape of the cell, protrusion rate and membrane tension. We find that protrusion rate does not correlate with membrane tension, but, instead, is strongly correlated with cell roundness. We rationalized the relationship between cell roundness and protrusion velocity using the framework developed for wetting phenomena. The contact angle that the cell forms with the substrate defines the contribution of membrane tension to the force balance at the leading edge: the higher the contact angle, the less load is applied from the membrane to the actin network and therefore the faster is actin protrusion. We validated this concept by manufacturing topographically modified surfaces and showing that the leading edge of the cell exhibits pinning on substrate ridges - a phenomenon characteristic of spreading of liquid drops. These results demonstrate the role of contact angle at the leading edge in control of protrusion, which has important implications for cell shape dynamics and cell migration in 3D. We have also analyzed protrusion velocity in the case of high planar curvature of the advancing edge which was accomplished by making small circular wounds in keratocyte’s lamellipodia. The fact that these wounds presenting negative planar curvature close faster than the protrusion rate at the edge of the cell, is consistent with the important role of membrane curvature in the control of protrusion velocity. Our study underscores the fundamental importance of membrane configuration for cellular force balance and edge dynamics. It provides a novel framework for understanding the relationship between global cell shape and its local dynamics, of the balance between lamellipodia protrusion and blebbing, and for modulation of cell shape and motion by substrate topology.

EPFL2013

The role of recruited bone-marrow derived mesenchymal stem cells in the establishment of the tumor stroma and a growth-supportive environment

Jean-Paul Abbühl

The Mesenchymal Stem Cell (MSC) is a self-renewing multipotent progenitor originally found in the bone marrow. It has drawn strong interest from translational research because of the multipotency of MSCs, their immunosuppression, their intrinsic homing and their promotion of wound healing in human patients. Currently available methods rely on in vitro culture for the isolation and expansion of MSCs and most studies used these cells for their investigation. Fibroblasts constitute the connective tissue and support epithelial or hematopoietic cells. Although they have already been extensively investigated, it is not clear whether stem cells exist in vivo which specifically regenerate fibroblasts. MSCs are a potential candidate, because of their fibroblastic shape in vitro, their differentiation towards several mesodermal lineages and their supportive function. We aim to characterize the in vivo differentiation potential of MSCs toward fibroblasts in normal and tumor tissues. A new transplantation approach was developed in order to transfer labeled MSCs in the bone marrow and assess their recruitment and lineage contribution in vivo. Long-term engraftment was accomplished by isolating and transplanting these cells within one day. To this end, strategies for their purification, preconditioning of the host and transplantation methodologies were established during this thesis. We were able to enrich MSCs over 1000 fold by combining a new extraction protocol, a negative selection by MACS and a positive selection by FACS. Cloning of sorted MSCs was performed to confirm their multipotency at single cell level. GFP-expressing MSCs were able to engraft in host mice tolerant for GFP and their expansion in vivo was stimulated by block of GSK3 activity. Altogether, the chimeric MSC model was used to track the recruitment of bone-marrow derived MSCs into spontaneous breast tumors. The tumor microenvironment encompasses several non-tumorigenic cells interacting with cancer cells. Among them, cancer-associated fibroblasts are known to participate in carcinogenesis. Whilst this heterogeneous stromal population has been extensively investigated, their source(s) remain elusive. I now propose tumoral MSCs as one source of cancer-associated fibroblast, based on their ability to self-renew and reconstitute stromal heterogeneity upon serial transplantation. Finally, I introduced the new concept that the multipotency of MSCs is subjugated by cancer cells to meet their requirements for enhanced tumor progression and prepare for metastasis. I found MSCs present in the tumor microenvironment to differentiate into cancer-associated osteoblasts and produce hydroxyapatite-mineralization in vivo. Microcalcification is an accepted poor prognostic feature in breast cancer patients but its function and origin is not documented. I showed that hydroxyapatite was able to promote proliferation of tumor cells and increased intracellular calcium concentration. Lastly, cancer-associated osteoblast and hydroxyapatite were able to promote tumor growth and metastasis in vivo. To conclude, bone marrow derived MSCs are circulating stem cells that participate in the establishment of the pathogenic stroma in breast cancer. Owing to their multipotency, MSCs give rise to cancer-associated osteoblasts that induce pro-malignant mineralization in situ. Subsequently, cancer cells harbor increased intracellular calcium concentration, enhanced proliferation and metastasis. We foresee the development of new stroma-targeted therapies that prevent the mineralization of tumor tissue and reduce metastasis in breast cancer patients.

EPFL2014