Mechanisms of mechanotransduction by specialized low-threshold mechanoreceptors in the guinea pig rectum

The guinea pig rectum, but not the colon, is innervated by a specialized class of distension-sensitive mechanoreceptors that have transduction sites corresponding to rectal intraganglionic laminar endings (rIGLEs). Rectal mechanoreceptors recorded in vitro had low threshold to circumferential stretch, adapted slowly, and could respond within 2 ms to mechanical stimulation by a piezo-electric probe. Antagonists to ionotropic N-methyl-D-aspartate (NMDA; CGS 19755, memantine) and non-NMDA (6,7-dinitroquinoxaline-2,3-dione) glutamate receptors did not affect mechanotransduction. In normal Krebs solution, the P2X purinoreceptor agonist alpha,beta-methylene ATP reduced mechanoreceptor firing evoked by distension but simultaneously relaxed circular smooth muscle and inhibited stretch-induced contractions. Neither ATP nor alpha,beta-methylene ATP affected mechanotransduction when transduction sites were directly compressed with von Frey hairs. The P2 purinoreceptor antagonist pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid did not affect stretch-induced firing but reduced the inhibitory effect of alpha,beta-methylene ATP on stretch-induced firing. Under isometric conditions, blocking synaptic transmission in Ca2+-free solution reduced stretch-evoked firing but not when basal tension was restored to control levels. Under isotonic condition, Ca2+-free solution did not significantly affect load-evoked firing. The blockers of mechanogated and/or transient receptor potential channels, benzamil, Gd3+, SKF 96365, and ruthenium red inhibited stretch-induced firing but, in parallel, significantly reduced stretch-induced contractions. Benzamil and SKF 96365 were able to inhibit mechanotransduction when transduction sites were compressed with von Frey hairs. The results show that mechanotransduction is rapid but does not depend on fast exocytotic release of mediators. It is likely that stretch-activated ion channels on rIGLEs are involved in direct, physical mechanotransduction by rectal low-threshold mechanoreceptors.     

Integrins in mechanotransduction

Mechanical forces are crucial to the regulation of cell and tissue morphology and function. At the cellular level, forces influence cytoskeletal organization, gene expression, proliferation, and survival. Integrin-mediated adhesions are intrinsically mechanosensitive and a large body of data implicates integrins in sensing mechanical forces. We review the relationship between integrins and mechanical forces, the role of integrins in cellular responses to stretch and fluid flow, and propose that some of these events are mechanistically related.     

Interactions of mechanotransduction pathways

Integrins may serve as mechanosensors in endothelial cells (ECs): shear stress causes integrin-Shc association, assembly of the signaling complex and then leads to JNK activation. Flow also mediates selective and cell-specific alterations in vascular cell G-protein expression that correlate with changes in cell-signalling, G-protein functionality and modulate Ca2+ concentration. In this study, we explored the cross-talks between EC membrane mechanosensors, such as integrins, ion channels, and G-proteins in shear stress-induced signal transduction by their specific inhibition. Confluent monolayer of bovine aortic endothelial cells (BAECs) were incubated with or without specific inhibitors prior to shearing experiments. Our results showed an attenuation of integrin-Shc association under shear stress with RGD, and with PTX, but not with BAPTA/AM. The inhibitions of shear-activated JNK are similar for RGD and PTX. However, unlike for integrin association, the chelation of calcium reduced JNK activation. These results provide several lines of evidence of the interactions between different mechanosensors in ECs. First, integrin-Shc association required cell attachment and G-protein activity, but not intracellular calcium. Second, shear-induced JNK activation is regulated by multiple mechano-sensing mechanisms such as integrin, G-protein and calcium concentration.     

Computational modeling of extracellular mechanotransduction

Mechanotransduction may occur through numerous mechanisms, including potentially through autocrine signaling in a dynamically changing extracellular space. We developed a computational model to analyze how alterations in the geometry of an epithelial lateral intercellular space (LIS) affect the concentrations of constitutively shed ligands inside and below the LIS. The model employs the finite element method to solve for the concentration of ligands based on the governing ligand diffusion-convection equations inside and outside of the LIS, and assumes idealized parallel plate geometry and an impermeable tight junction at the apical surface. Using the model, we examined the temporal relationship between geometric changes and ligand concentration, and the dependence of this relationship on system characteristics such as ligand diffusivity, shedding rate, and rate of deformation. Our results reveal how the kinetics of mechanical deformation can be translated into varying rates of ligand accumulation, a potentially important mechanism for cellular discrimination of varying rate-mechanical processes. Furthermore, our results demonstrate that rapid changes in LIS geometry can transiently increase ligand concentrations in underlying media or tissues, suggesting a mechanism for communication of mechanical state between epithelial and subepithelial cells. These results underscore both the plausibility and complexity of the proposed extracellular mechanotransduction mechanism.     

Putting the squeeze on mechanotransduction

Both mechanical and chemical stimuli guide tissue function. In a recent paper, Tschumperlin et al. proposed that pressure acting on airway epithelium elicits mechanotransduction not by directly altering biochemical signaling but by regulating extracellular fluid volume to modulate ligand-receptor interactions.     

Hair-cell mechanotransduction and cochlear amplification

In the inner ear, sensory hair cells not only detect but also amplify the softest sounds, allowing us to hear over an extraordinarily wide intensity range. This amplification is frequency specific, giving rise to exquisite frequency discrimination. Hair cells detect sounds with their mechanotransduction apparatus, which is only now being dissected molecularly. Signal detection is not the only role of this molecular network; amplification of low-amplitude signals by hair bundles seems to be universal in hair cells. "Fast adaptation," the rapid closure of transduction channels following a mechanical stimulus, appears to be intimately involved in bundle-based amplification.     

Cell adhesion receptors in mechanotransduction

Integrins and cadherins are tri-functional: they bind ligands on other cells or in the extracellular matrix, connect to the cytoskeleton inside the cell, and regulate intracellular signaling pathways. These adhesion receptors therefore transmit mechanical stresses and are well positioned to mediate mechanotransduction. Studies of cultured cells have shown that both integrin- and cadherin-mediated adhesion are intrinsically mechanosensitive. Strengthening of adhesions in response to mechanical stimulation may be a central mechanism for mechanotransduction. Studies of developing organisms suggest that these mechanisms contribute to tissue level responses to tension and compression, thereby linking morphogenetic movements to cell fate decisions.     

Integrins and extracellular matrix in mechanotransduction

Integrins bind extracellular matrix fibrils and associate with intracellular actin filaments through a variety of cytoskeletal linker proteins to mechanically connect intracellular and extracellular structures. Each component of the linkage from the cytoskeleton through the integrin-mediated adhesions to the extracellular matrix therefore transmits forces that may derive from both intracellular, myosin-generated contractile forces and forces from outside the cell. These forces activate a wide range of signaling pathways and genetic programs to control cell survival, fate, and behavior. Additionally, cells sense the physical properties of their surrounding environment through forces exerted on integrin-mediated adhesions. This article first summarizes current knowledge about regulation of cell function by mechanical forces acting through integrin-mediated adhesions and then discusses models for mechanotransduction and sensing of environmental forces.     

Neighborly relations: cadherins and mechanotransduction

Cell–cell adhesions are sites where cells experience and resist tugging forces. It has long been postulated, but not directly tested, that cadherin adhesion molecules may serve in mechanotransduction at cell–cell contacts. In this issue, Le Duc et al. (2010. J. Cell Biol. doi: 10.1083/jcb.201001149) provide direct evidence that E-cadherin participates in a mechanosensing pathway that regulates the actomyosin cytoskeleton to modulate cell stiffness in response to pulling force.All of the cells in our body experience force: from the shear stress of blood flow experienced by the vascular endothelium to the tugging of other cells in skeletal muscle. Accordingly, cellular mechanisms exist to preserve tissue integrity by resisting this play of forces. Characteristically, these mechanisms involve adhesion receptors that are mechanically coupled to the cytoskeleton. However, these apparatuses do not simply support passive resistance. Instead, there has been great recent interest in the concept that adhesion receptors contribute to cell signaling pathways, which sense the magnitude of force exerted on cells and trigger cellular responses to those forces (Vogel and Sheetz, 2006). This notion is best established for integrin cell–matrix adhesion molecules in which well-characterized signaling pathways are clearly involved in mechanotransduction, which modifies focal adhesion size in response to force (Balaban et al., 2001) and may ultimately affect processes that range from stem cell differentiation (Engler et al., 2006) to tumor cell progression (Levental et al., 2009).At cell–cell contacts, classical cadherin adhesion molecules play major roles in morphogenesis and in the maintenance of tissue integrity. A role for cadherins in mechanotransduction has often been suspected (Schwartz and DeSimone, 2008) but not directly tested. One challenge in dissecting this problem is to distinguish responses principally elicited by the cadherin from juxtacrine events that occur when adhesion systems bring native cell surfaces into contact with one another. In this issue, Le Duc et al. circumvent this problem by using recombinant cadherin ligands, which contain the entire adhesive ectodomain, to test the capacity for a classical cadherin to participate in mechanosensing. The authors allowed magnetic beads coated with recombinant E-cadherin ectodomains to adhere to the dorsal surfaces of cultured cells. Classical cadherins engage in homophilic interactions via their ectodomains, and ligation of cellular cadherins by these immobilized ligands is a commonly used approach to generate adhesive contacts through E-cadherin alone. They used an oscillating magnetic field to twist the beads, thereby applying shear forces onto the sites of adhesion. By measuring the displacement of the beads in response to twisting stimuli, they could calculate changes in the local stiffness of the adhesive contact of each bead.Strikingly, they found that these adhesive contacts between the cadherin-coated beads and the cells stiffened in response to repetitive twisting force. The magnitude of stiffening increased with the magnitude of the applied force, which is evidence for the existence of a mechanism that could apparently measure the applied force and calibrate a proportionate cellular response. The use of E-cadherin as the ligand for homophilic engagement implied that the cellular cadherin was key to the force-sensing apparatus. This was further substantiated by the demonstration that the stiffening response did not occur when cadherin function was disrupted by removing extracellular calcium or adding a function-blocking antibody. Moreover, stiffening could not be elicited by beads coated with cadherin antibodies, suggesting that a native ligand was required rather than simple binding to the cellular cadherin ectodomain. Moreover, cell stiffening required an intact actomyosin cytoskeleton, implying that it reflected a cellular mechanical response to applied force. Overall, these findings indicate that E-cadherin engaged in homophilic interactions can serve to sense force and trigger a cellular response that involves the actin cytoskeleton, classical hallmarks of a mechanotransduction pathway (Fig. 1).Open in a separate windowFigure 1.E-cadherin mechanotransduction. Forces acting on surface E-cadherin molecules activate mechanosensing processes that lead to proportionate mechanical responses from cells. (1) In this model, E-cadherin engaged in homophilic adhesive interactions acts as a surface receptor for forces that tug on cells. (2) This induces an intracellular signaling cascade, which includes events such as alterations in protein conformation (notably α-catenin) and recruitment of proteins such as vinculin. (3 and 4) The subsequent mechanical response involves the actomyosin cytoskeleton (3), which can alter adhesion stiffness (4) by diverse processes such as changes in cortical organization and contractility. One potential outcome is that this cellular response will be felt as a pulling force by the neighboring cell that initiated the cascade, leading to cooperative interactions between the cells.What do we know of the molecular players in this E-cadherin–activated mechanotransduction pathway? A comprehensive answer to this question must ultimately encompass the signal transduction pathways that are activated by mechanical stimulation of E-cadherin and the elicited downstream cytoskeletal responses. Many different kinds of signaling events are implicated in other forms of mechanosensing, including the Src tyrosine kinase and ion channels (Vogel and Sheetz, 2006), which can be found at cell–cell contacts (Wang et al., 2006). Another molecular paradigm involves alterations in protein conformation in response to applied force, which thereby reveals novel sites for posttranslational modification or protein binding (del Rio et al., 2009). In this regard, a recent study identifies an apparently cryptic site in α-catenin that is sensitive to the cellular force generator myosin II (Yonemura et al., 2010). The study found that junctional staining with a monoclonal antibody directed to the central region of α-catenin was abolished in cells treated with the myosin II inhibitor blebbistatin, although α-catenin protein remained at cell–cell contacts. Notably, the epitope for this monoclonal antibody resides close to the region of α-catenin that can directly bind the actin regulator vinculin. Both Le Duc et al. (2010) and Yonemura et al. (2010) show that the recruitment of vinculin to cell–cell junctions is blebbistatin sensitive. Moreover, Le Duc et al. (2010) demonstrate that cellular stiffening in response to twisting force is reduced in vinculin-deficient cells. This suggests the attractive hypothesis that transmission of force to α-catenin that is incorporated into the E-cadherin complex may alter its conformation and capacity to interact with binding partners such as vinculin. This notion warrants more detailed analysis; however, if experience with integrin mechanotransduction is any guide (Vogel and Sheetz, 2006; Schwartz and DeSimone, 2008), force-induced conformational change in proteins such as α-catenin are likely to be but one part of a more complex network of signal transduction mechanisms.The force-dependent recruitment of vinculin also provides a potential mechanism to coordinate a cytoskeletal response to E-cadherin mechanosensing. Although long known to concentrate at the zonula adherens (as well for its better-known localization in focal adhesions), the precise role that vinculin plays in cell–cell interactions remains enigmatic. Nonetheless, depletion of vinculin reduces cell–cell adhesion and disrupts the integrity of epithelial cell–cell junctions (Peng et al., 2010). Vinculin depletion also perturbs the junctional actin cytoskeleton (Maddugoda et al., 2007), and vinculin has the capacity to bind actin filaments and diverse actin regulators, thereby influencing both filament bundling and dynamics (Le Clainche et al., 2010). However, vinculin is unlikely to be the sole mediator of the cytoskeletal response. Many cytoskeletal regulators act at E-cadherin cell–cell junctions to control actin filament dynamics and organization. A particularly interesting case is nonmuscle myosin II, which was implicated as the dominant cellular force generator in recent studies (Le Duc et al., 2010; Yonemura et al., 2010). However, the contribution of myosin II is likely to be complex, as myosin II is also necessary for the cytoskeletal response to force (Le Duc et al., 2010). Moreover, there is emerging evidence for both contractile and noncontractile functions for myosin II (Choi et al., 2008). Also, the myosin II A and B isoforms can have distinct contributions to E-cadherin clustering and apical actin regulation (Smutny et al., 2010). Thus, myosin II may have several contributions to cadherin mechanotransduction.Finally, what functions might be served by cadherin-based mechanosensing? One possibility is that local stiffening, and perhaps the cytoskeletal response more broadly, might provide a mechanism to strengthen adhesions against potentially disruptive forces. This notion is supported by a recent study by Liu et al. (2010), who analyzed forces at the contacts between pairs of cells grown on micropatterned substrata. Force vectors oriented approximately perpendicular to the cell–cell contacts could be extracted from their data, and strikingly, the authors identified a linear relationship between the magnitude of the forces and the size of the contacts. This appeared to reflect the coordinated action of Rho- and Rac-based signaling pathways. They proposed that force-dependent growth of adhesions may be a mechanism to reduce stress at the contacts and thus preserve their integrity. In addition, it is interesting to consider the possibility that mechanosensing through E-cadherin could provide a mechanism for cells to assess the mechanical properties of their neighboring cells. Cells appear to use integrin-based mechanosensing to assess the stiffness of their surrounding matrix (Schwartz and DeSimone, 2008; Levental et al., 2009). Their ability to use myosin II–based contractility to pull on adhesion sites is likely critical for cells to assess stiffness of their surroundings. However, an important difference between cell–matrix and cell–cell mechanosensing is that although in the former case the environment is passive, in the latter case, it is active (i.e., neighboring cells can pull back). Is the mechanical response of a neighboring cell an important parameter in cadherin-based cell–cell recognition? Clearly, then, the new work of Le Duc et al. (2010) opens many new avenues for understanding the role of mechanosensing in cadherin biology and tissue organization.     

Cadherins and mechanotransduction by hair cells

Mechanotransduction, the conversion of a mechanical stimulus into an electrical signal is crucial for our ability to hear and to maintain balance. Recent findings indicate that two members of the cadherin superfamily are components of the mechanotransduction machinery in sensory hair cells of the vertebrate inner ear. These studies show that cadherin 23 (CDH23) and protocadherin 15 (PCDH15) form several of the extracellular filaments that connect the stereocilia and kinocilium of a hair cell into a bundle. One of these filaments is the tip link that has been proposed to gate the mechanotransduction channel in hair cells. The extracellular domains of CDH23 and PCDH15 differ in their structure from classical cadherins and their cytoplasmic domains bind to distinct effectors, suggesting that evolutionary pressures have shaped the two cadherins for their function in mechanotransduction.     

Development of the hair bundle and mechanotransduction

This review focuses on the cellular and molecular mechanisms underlying the development of the sensory hair bundle, an apical specialisation of the hair cell that is essential for mechanotransduction. The structure, function and development of the hair bundle is described, with an emphasis on the properties and possible roles played by the different link types that interconnect the individual elements of the hair bundle - the multiple stereocilia and the single kinocilium. Studies of mouse and zebrafish mutants have revealed that several classes of molecule are required for the genesis and maintenance of hair-bundle structure. These include cell surface molecules that are associated with the different hair-bundle links, along with myosin motors, scaffolding proteins and an actin cross-linker. Finally we consider how differences in the form and shape of hair bundles within and between different sensory organs are generated.     

The significance of bone microstructure in mechanotransduction

Recent developments in modeling the relationship between bone microstructure and mechanotransduction are reviewed. The focus is on the relationship between the bone microstructure and the mechanosensation mechanism by which osteocytes sense the bone fluid motion propelled by the mechanical loading of the whole bone.     

Balancing forces: architectural control of mechanotransduction

All cells exist within the context of a three-dimensional microenvironment in which they are exposed to mechanical and physical cues. These cues can be disrupted through perturbations to mechanotransduction, from the nanoscale-level to the tissue-level, which compromises tensional homeostasis to promote pathologies such as cardiovascular disease and cancer. The mechanisms of such perturbations suggest that a complex interplay exists between the extracellular microenvironment and cellular function. Furthermore, sustained disruptions in tensional homeostasis can be caused by alterations in the extracellular matrix, allowing it to serve as a mechanically based memory-storage device that can perpetuate a disease or restore normal tissue behaviour.     

Cell mechanotransduction and interactions with biological tissues

The biomechanical mechanisms involved in the processes of tissue remodeling and adaptation are reviewed with emphasis on mechanotransduction at the cellular level. New theoretical models associated with experimental rheological techniques are briefly commented.     

Participation of caveolae in beta1 integrin-mediated mechanotransduction

We previously reported that caveolin-1 is a key component in a beta1 integrin-dependent mechanotransduction pathway suggesting that caveolae organelles and integrins are functionally linked in their mechanotransduction properties. Here, we exposed BAEC monolayers to shear stress then isolated caveolae vesicles form the plasma membrane. While little beta1 integrin was detected in caveolae derived from cells kept in static culture, shear stress induced beta1 integrin transposition to the caveolae. To evaluate the significance of shear-induced beta1 integrin localization to caveolae, cells were pretreated with cholesterol sequestering compounds or caveolin-1 siRNA to disrupt caveolae structural domains. Cholesterol depletion attenuated integrin-dependent caveolin-1 phosphorylation, Src activation and Csk association with beta1 integrin. Reduction of both caveolin-1 protein and membrane cholesterol inhibited downstream shear-induced, integrin-dependent phosphorylation of myosin light chain. Taken together with our previous findings, the data supports the concept that beta1 integrin-mediated mechanotransduction is mediated by caveolae domains.