Closure of wounds and gaps in tissues is fundamental for the correct development and physiology of multicellular organisms and, when misregulated, may lead to inflammation and tumorigenesis. To re-establish tissue integrity, epithelial cells exhibit coordinated motion into the void by active crawling on the substrate and by constricting a supracellular actomyosin cable. Coexistence of these two mechanisms strongly depends on the environment. However, the nature of their coupling remains elusive because of the complexity of the overall process. Here we demonstrate that epithelial gap geometry in both in vitro and in vivo regulates these collective mechanisms. In addition, the mechanical coupling between actomyosin cable contraction and cell crawling acts as a large-scale regulator to control the dynamics of gap closure. Finally, our computational modelling clarifies the respective roles of the two mechanisms during this process, providing a robust and universal mechanism to explain how epithelial tissues restore their integrity.

Multicellularity in animals requires dynamic maintenance of cell-cell contacts. Intercellularly ligated cadherins recruit numerous proteins to form supramolecular complexes that connect with the actin cytoskeleton and support force transmission. However, the molecular organization within such structures remains unknown. Here we mapped protein organization in cadherin-based adhesions by superresolution microscopy, revealing a multi-compartment nanoscale architecture, with the plasma membrane-proximal cadherin-catenin compartment segregated from the actin cytoskeletal compartment, bridged by an interface zone containing vinculin. Vinculin position is determined by α-catenin, and upon activation, vinculin can extend ˜30 nm to bridge the cadherincatenin and actin compartments, while modulating the nanoscale positions of the actin regulators, zyxin and VASP. Vinculin conformational activation requires tension and tyrosine

Using a new equibiaxial strain device, we investigated strain-induced Ca2+ signals and their relation to lamellar body (LB) exocytosis in single rat alveolar type II (AT II) cells. The strain device allows observation of single cells while inducing strain to the entire substratum. AT II cells tolerated high strain amplitudes up to 45% increase in cell surface area (Delta CSA) without release of lactate dehydrogenase or ATP. Strain exceeding a threshold of approximately 8% Delta CSA resulted in a transient rise of the cytoplasmic Ca2+ concentration in some cells. Higher strain levels increased the fraction of Ca2+-responding cells. The occurrence of strain-induced Ca2+ signals depended on cell-cell contacts, because lone cells (i.e., cells without cell-cell contacts) did not exhibit Ca2+ signals. Above threshold, the amplitude of the Ca2+ signal as well as the number of stimulated LB fusions correlated well with the amplitude of strain. Furthermore, stimulated LB fusions occurred only in cells exhibiting a Ca2+ signal; 50 microM Gd3+ in the bath affected neither Ca2+ signals nor fusions. Intracellular Ca2+ release was triggered at higher strain amplitudes and inhibited by thapsigargin. Removal of bath Ca2+ completely inhibited Ca2+ signals and fusions. We conclude that strain of AT II cells stimulates a Ca2+ entry pathway that is highly sensitive to strain and a prerequisite for subsequent Ca2+ release. Both mechanisms result in a graded response of fusions to strain. Our data also allow us to introduce the term "effective strain" as the physiologically relevant portion of the strain amplitude.

Collective migration of cells is of fundamental importance for a number of biological functions such as tissue development and regeneration, wound healing and cancer metastasis. The movement of cell groups consisting of multiple cells connected by cell-cell junctions depends on both extracellular and intercellular contacts. Epithelial cell assemblies are thus regulated by a cross-talk between cell-substrate and cell-cell interactions. Here, we investigated the onset of collective migration in groups of cells as they expand from a few cells into large colonies as a function of extracellular matrix (ECM) protein coating. By varying the amount of ECM presented to the cells, we observe that the mode of colony expansion, as well as their overall geometry, is strongly dependent on substrate adhesiveness. On high ECM protein coated surfaces, cells at the edges of the colonies are well spread exhibiting large outward-pointing protrusive activity, whereas cellular colonies display more circular and convex shapes on less adhesive surfaces. Actin structures at the edge of the colonies also show different organizations with the formation of lamellipodial structures on highly adhesive surfaces and a pluricellular actin cable on less adhesive ones. The analysis of traction forces and cell velocities within the cellular assemblies confirm these results. By increasing ECM protein density, cells exert higher traction forces together with a higher outward motility at the edges. Furthermore, tuning cell-cell adhesion of epithelial cells modified the mode of expansion of the colonies. Finally, we used a recently developed computational model to recapitulate the emergent experimental behaviors of expanding cell colonies and extract that the main effect of the different cell-substrate interactions is on the ability of edge cells to form outward lamellipodia-driven motility. Overall, our data suggest that switching behaviors of epithelial cell assemblies result in a tug-of-war between friction forces at the cell-substrate interface and cell-cell interactions.

Adherens junctions connect the actin cytoskeleton of neighboring cells through transmembrane cadherin receptors and a network of adaptor proteins. The interactions between these adaptors and cadherin as well as the activity of actin regulators localized to adherens junctions are tightly controlled to facilitate cell junction assembly or disassembly in response to changes in external or internal forces and/or signaling. Phosphorylation of tyrosine, serine, or threonine residues acts as a switch on the majority of adherens junction proteins, turning “on” or “off” their interactions with other proteins and/or their enzymatic activity. Here, we provide an overview of the kinases and phosphatases regulating phosphorylation of adherens junction proteins and bring examples of phosphorylation events leading to the assembly or disassembly of adherens junctions, highlighting the important role of phosphorylation switches in regulating their dynamics.

Alveolar type II (AT II) cells are in close contact with an air-liquid interface (IAL). This contact may be of considerable physiological relevance; however, no data exist to provide a satisfying description of this specific microenvironment. This is mainly due to the experimental difficulty to manipulate and analyze cell-air contacts in a specific way. Therefore, we designed assays to quantify cell viability, Ca 2ϩ changes, and exocytosis in the course of interface contact and miniaturized IAL devices for direct, subcellular, and real-time analyses of cell-interface interactions by fluorescence microscopy or interferometry. The studies demonstrated that the sole presence of an IAL is not sensed by the cells. However, when AT II cells are forced into closer contact with it, they respond promptly with sustained Ca 2ϩ signals and surfactant exocytosis before the occurrence of irreversible cell damage. This points to a paradoxical situation: a potential threat and potent stimulus for the cells. Furthermore, we found that the signalling mechanism underlying sensation of an I AL can be sufficiently explained by mechanical forces. These results demonstrate that the IAL itself can play a major, although so-far neglected, role in lung physiology, particularly in the regulatory mechanisms related with surfactant homeostasis. Moreover, they also support a general new concept of mechanosensation in the lung. mechanical stress; pneumocytes; strain; stretch; surfactant THE ALVEOLAR EPITHELIUM is covered by a thin and continuous layer of water (5). This aqueous layer, referred to as hypophase or alveolar lining fluid (ALF), introduces a considerable physical instability to the millions of alveolar invaginations, tending on overall to force the air out of the lungs. As a consequence, the great alveolar corner cells (or alveolar type II cells; AT II) synthesize and release surfactant into the ALF, from where it transits to the air-liquid interface (I AL ) and creates a highly surface-active coat (40). The importance of this protective coat is best demonstrated in the premature infant lung, where deficiency causes alveolar collapse with life-threatening consequences unless medicated by surfactant administration strategies (17).An important and almost unique aspect of the AT II cellspecific microenvironment is the presence of air. Not astonishingly, therefore, studies introducing such an I AL in AT II cell culture systems reported dramatic effects on cell function, morphology, protein expression, and ion channel activities (13,29). In general, the AT II cells have a higher phenotypic stability and seem to preserve characteristic cell functions for longer periods than compared with standard culture conditions. In particular, biosynthesis and secretion of surfactant is stable over several weeks. Furthermore, reculturing with exposure to air reverses the loss of differentiated AT II cell phenotype observed in submerged cultures (13). This effect of an I AL is indeed remarkable, though not readily intelligible. In particular, the phy...

IntroductionLevosimendan is an extensively investigated inodilator showing also cardioprotective and antiinflammatory effects. The aim of our study was to explore the influence of levosimendan on polymorphonuclear leucocytes (PMN), a main source of reactive oxygen species, in vitro and in patients with acute heart failure or septic myocardial depression.MethodsPMN isolated from healthy volunteers were incubated with levosimendan in vitro. After stimulation with N-formyl-Met-Leu-Phe (fMLP) or phorbol 12-myristate 13-acetate (PMA) respiratory burst was quantified using a fluorescent dye. Apoptosis and expression of cell adhesion molecules of PMN were measured by flow cytometry. For determination of in vivo effects patients with acute heart failure (n = 16) or septic cardiac failure (n = 9) receiving levosimendan treatment were enrolled consecutively. PMN were isolated to measure respiratory burst activity before treatment as well as one and two hours after initiation of levosimendan administration. Furthermore inflammatory, hemodynamic and renal function parameters were obtained.ResultsIn vitro, levosimendan suppressed respiratory burst activity in fMLP or PMA stimulated PMN in a dose dependent manner by 30 ± 11% (P < 0.001) at 100 ng/mL and by 27 ± 17% (P < 0.001) at 1000 ng/mL respectively. Markers of apoptosis and PMN cell adhesion molecule expression remained unaffected by levosimendan treatment.In vivo, levosimendan treatment for two hours resulted in a significant reduction of PMA stimulated oxidative burst by 45% (P < 0.01) and fMLP stimulated oxidative burst by 49% (P < 0.05) in patients with acute heart failure. In patients suffering from septic shock levosimendan treatment decreased oxidative burst activity in unstimulated, fMLP and PMA stimulated PMN by 48% (P < 0.05), 46% (P < 0.01) and 43% (P < 0.01) respectively.ConclusionsLevosimendan appears to exert distinct immunomodulatory effects by decreasing oxidative burst activity of PMN. This property might contribute to the previously described cardioprotective effects of the drug.

Pulmonary surfactant is essential for lung function. It is assembled, stored and secreted as particulate entities (lamellar body-like particles; LBPs). LBPs disintegrate when they contact an air-liquid interface, leading to an instantaneous spreading of material and a decline in surface tension. Here, we demonstrate that the film formed by the adsorbed material spontaneously segregate into distinct ordered and disordered lipid phase regions under unprecedented near-physiological conditions and, unlike natural surfactant purified from bronchoalveolar lavages, dynamically reorganized into highly viscous multilayer domains with complex three-dimensional topographies. Multilayer domains, in coexistence with liquid phases, showed a progressive stiffening and finally solidification, probably driven by a self-driven disassembly of LBPs from a sub-surface compartment. We conclude that surface film formation from LBPs is a highly dynamic and complex process, leading to a more elaborated scenario than that observed and predicted by models using reconstituted, lavaged, or fractionated preparations.