Ret kinase-mediated mechanical induction of colon stem cells by tumor growth pressure stimulates cancer progression in vivo.

How mechanical stress actively impacts the physiology and pathophysiology of cells and tissues is little investigated in vivo. The colon is constantly submitted to multi-frequency spontaneous pulsatile mechanical waves, which highest frequency functions, of 2 s period, remain poorly understood. Here we find in vivo that high frequency pulsatile mechanical stresses maintain the physiological level of mice colon stem cells (SC) through the mechanosensitive Ret kinase. When permanently stimulated by a magnetic mimicking-tumor growth analogue pressure, we find that SC levels pathologically increase and undergo mechanically induced hyperproliferation and tumorigenic transformation. To mimic the high frequency pulsatile mechanical waves, we used a generator of pulsed magnetic force stimulation in colonic tissues pre-magnetized with ultra-magnetic liposomes. We observed the pulsatile stresses using last generation ultra-wave dynamical high-resolution imaging. Finally, we find that the specific pharmacological inhibition of Ret mechanical activation induces the regression of spontaneous formation of SC, of CSC markers, and of spontaneous sporadic tumorigenesis in Apc mutated mice colons. Consistently, in human colon cancer tissues, Ret activation in epithelial cells increases with tumor grade, and partially decreases in leaking invasive carcinoma. High frequency pulsatile physiological mechanical stresses thus constitute a new niche that Ret-dependently fuels mice colon physiological SC level. This process is pathologically over-activated in the presence of permanent pressure due to the growth of tumors initiated by pre-existing genetic alteration, leading to mechanotransductive self-enhanced tumor progression in vivo, and repressed by pharmacological inhibition of Ret.

The major ß-catenin/E-cadherin junctional binding site is a primary molecular mechano-transductor of differentiation in vivo.

In vivo, the primary molecular mechanotransductive events mechanically initiating cell differentiation remain unknown. Here we find the molecular stretching of the highly conserved Y654-ß-catenin-D665-E-cadherin binding site as mechanically induced by tissue strain. It triggers the increase of accessibility of the Y654 site, target of the Src42A kinase phosphorylation leading to irreversible unbinding. Molecular dynamics simulations of the ß-catenin/E-cadherin complex under a force mimicking a 6 pN physiological mechanical strain predict a local 45% stretching between the two α-helices linked by the site and a 15% increase in accessibility of the phosphorylation site. Both are quantitatively observed using FRET lifetime imaging and non-phospho Y654 specific antibody labelling, in response to the mechanical strains developed by endogenous and magnetically mimicked early mesoderm invagination of gastrulating Drosophila embryos. This is followed by the predicted release of 16% of ß-catenin from junctions, observed in FRAP, which initiates the mechanical activation of the ß-catenin pathway process.

Mechanotransductive cascade of Myo-II-dependent mesoderm and endoderm invaginations in embryo gastrulation.

Animal development consists of a cascade of tissue differentiation and shape change. Associated mechanical signals regulate tissue differentiation. Here we demonstrate that endogenous mechanical cues also trigger biochemical pathways, generating the active morphogenetic movements shaping animal development through a mechanotransductive cascade of Myo-II medio-apical stabilization. To mimic physiological tissue deformation with a cell scale resolution, liposomes containing magnetic nanoparticles are injected into embryonic epithelia and submitted to time-variable forces generated by a linear array of micrometric soft magnets. Periodic magnetically induced deformations quantitatively phenocopy the soft mechanical endogenous snail-dependent apex pulsations, rescue the medio-apical accumulation of Rok, Myo-II and subsequent mesoderm invagination lacking in sna mutants, in a Fog-dependent mechanotransductive process. Mesoderm invagination then activates Myo-II apical accumulation, in a similar Fog-dependent mechanotransductive process, which in turn initiates endoderm invagination. This reveals the existence of a highly dynamic self-inductive cascade of mesoderm and endoderm invaginations, regulated by mechano-induced medio-apical stabilization of Myo-II.

Cells as liquid motors: mechanosensitivity emerges from collective dynamics of actomyosin cortex.

Living cells adapt and respond actively to the mechanical properties of their environment. In addition to biochemical mechanotransduction, evidence exists for a myosin-dependent purely mechanical sensitivity to the stiffness of the surroundings at the scale of the whole cell. Using a minimal model of the dynamics of actomyosin cortex, we show that the interplay of myosin power strokes with the rapidly remodeling actin network results in a regulation of force and cell shape that adapts to the stiffness of the environment. Instantaneous changes of the environment stiffness are found to trigger an intrinsic mechanical response of the actomyosin cortex. Cortical retrograde flow resulting from actin polymerization at the edges is shown to be modulated by the stress resulting from myosin contractility, which in turn, regulates the cell length in a force-dependent manner. The model describes the maximum force that cells can exert and the maximum speed at which they can contract, which are measured experimentally. These limiting cases are found to be associated with energy dissipation phenomena, which are of the same nature as those taking place during the contraction of a whole muscle. This similarity explains the fact that single nonmuscle cell and whole-muscle contraction both follow a Hill-like force-velocity relationship.

Evolutionary conservation of early mesoderm specification by mechanotransduction in Bilateria.

The modulation of developmental biochemical pathways by mechanical cues is an emerging feature of animal development, but its evolutionary origins have not been explored. Here we show that a common mechanosensitive pathway involving ß-catenin specifies early mesodermal identity at gastrulation in zebrafish and Drosophila. Mechanical strains developed by zebrafish epiboly and Drosophila mesoderm invagination trigger the phosphorylation of ß-catenin-tyrosine-667. This leads to the release of ß-catenin into the cytoplasm and nucleus, where it triggers and maintains, respectively, the expression of zebrafish brachyury orthologue notail and of Drosophila Twist, both crucial transcription factors for early mesoderm identity. The role of the ß-catenin mechanosensitive pathway in mesoderm identity has been conserved over the large evolutionary distance separating zebrafish and Drosophila. This suggests mesoderm mechanical induction dating back to at least the last bilaterian common ancestor more than 570 million years ago, the period during which mesoderm is thought to have emerged.

Acto-myosin based response to stiffness and rigidity sensing.

Cells sense the rigidity of their environment and respond to it. Most studies have been focused on the role of adhesion complexes in rigidity sensing. In particular, it has been clearly shown that proteins of the adhesion complexes were stretch-sensitive, and could thus trigger mechano-chemical signaling in response to applied forces. In order to understand how this local mechano-sensitivity could be coordinated at the cell scale, we have recently carried out single cell traction force measurements on springs of varying stiffness. We found that contractility at the cell scale (force, speed of contraction, mechanical power) was indeed adapted to external stiffness, and reflected ATPase activity of non-muscle myosin II and acto-myosin response to load. Here we suggest a scenario of rigidity sensing where local adhesions sensitivity to force could be coordinated by adaptation of the acto-myosin dependent cortical tension at the global cell scale. Such a scenario could explain how spreading and migration are oriented by the rigidity of the cell environment.

Real-time single-cell response to stiffness.

Living cells adapt to the stiffness of their environment. However, cell response to stiffness is mainly thought to be initiated by the deformation of adhesion complexes under applied force. In order to determine whether cell response was triggered by stiffness or force, we have developed a unique method allowing us to tune, in real time, the effective stiffness experienced by a single living cell in a uniaxial traction geometry. In these conditions, the rate of traction force buildup dF/dt was adapted to stiffness in less than 0.1 s. This integrated fast response was unambiguously triggered by stiffness, and not by force. It suggests that early cell response could be mechanical in nature. In fact, local force-dependent signaling through adhesion complexes could be triggered and coordinated by the instantaneous cell-scale adaptation of dF/dt to stiffness. Remarkably, the effective stiffness method presented here can be implemented on any mechanical setup. Thus, beyond single-cell mechanosensing, this method should be useful to determine the role of rigidity in many fundamental phenomena such as morphogenesis and development.

Single-cell response to stiffness exhibits muscle-like behavior.

Living cells sense the rigidity of their environment and adapt their activity to it. In particular, cells cultured on elastic substrates align their shape and their traction forces along the direction of highest stiffness and preferably migrate towards stiffer regions. Although numerous studies investigated the role of adhesion complexes in rigidity sensing, less is known about the specific contribution of acto-myosin based contractility. Here we used a custom-made single-cell technique to measure the traction force as well as the speed of shortening of isolated myoblasts deflecting microplates of variable stiffness. The rate of force generation increased with increasing stiffness and followed a Hill force-velocity relationship. Hence, cell response to stiffness was similar to muscle adaptation to load, reflecting the force-dependent kinetics of myosin binding to actin. These results reveal an unexpected mechanism of rigidity sensing, whereby the contractile acto-myosin units themselves can act as sensors. This mechanism may translate anisotropy in substrate rigidity into anisotropy in cytoskeletal tension, and could thus coordinate local activity of adhesion complexes and guide cell migration along rigidity gradients.