Junction Contraction Research Articles

Inhibition of negative feedback for persistent epithelial cell–cell junction contraction by p21-activated kinase 3

Actin-mediated mechanical forces are central drivers of cellular dynamics. They generate protrusive and contractile dynamics, the latter of which are induced in concert with myosin II bundled at the site of contraction. These dynamics emerge concomitantly in tissues and even each cell; thus, the tight regulation of such bidirectional forces is important for proper cellular deformation. Here, we show that contractile dynamics can eventually disturb cell–cell junction contraction in the absence of p21-activated kinase 3 (Pak3). Upon Pak3 depletion, contractility induces the formation of abnormal actin protrusions at the shortening junctions, which causes decrease in E-cadherin levels at the adherens junctions and mislocalization of myosin II at the junctions before they enough shorten, compromising completion of junction shortening. Overexpressing E-cadherin restores myosin II distribution closely placed at the junctions and junction contraction. Our results suggest that contractility both induces and perturbs junction contraction and that the attenuation of such perturbations by Pak3 facilitates persistent junction shortening.

Force-dependent intercellular adhesion strengthening underlies asymmetric adherens junction contraction

Tissue morphogenesis arises from the culmination of changes in cell-cell junction length. Mechanochemical signaling in the form of RhoA underlies these ratcheted contractions, which occur asymmetrically. The underlying mechanisms of asymmetry remain unknown. We use optogenetically controlled RhoA in model epithelia together with biophysical modeling to uncover the mechanism lending to asymmetric vertex motion. Using optogenetic and pharmacological approaches, we find that both local and global RhoA activation can drive asymmetric junction contraction in the absence of tissue-scale patterning. We find that standard vertex models with homogeneous junction properties are insufficient to recapitulate the observed junction dynamics. Furthermore, these experiments reveal a local coupling of RhoA activation with E-cadherin accumulation. This motivates a coupling of RhoA-mediated increases in tension and E-cadherin-mediated adhesion strengthening. We then demonstrate that incorporating this force-sensitive adhesion strengthening into a continuum model is successful in capturing the observed junction dynamics. Thus, we find that a force-dependent intercellular "clutch" at tricellular vertices stabilizes vertex motion under increasing tension and is sufficient to generate asymmetries in junction contraction.

Convergent extension requires adhesion-dependent biomechanical integration of cell crawling and junction contraction.

SUMMARYConvergent extension (CE) is an evolutionarily conserved collective cell movement that elongates several organ systems during development. Studies have revealed two distinct cellular mechanisms, one based on cell crawling and the other on junction contraction. Whether these two behaviors collaborate is unclear. Here, using live-cell imaging, we show that crawling and contraction act both independently and jointly but that CE is more effective when they are integrated via mechano-reciprocity. We thus developed a computational model considering both crawling and contraction. This model recapitulates the biomechanical efficacy of integrating the two modes and further clarifies how the two modes and their integration are influenced by cell adhesion. Finally, we use these insights to understand the function of an understudied catenin, Arvcf, during CE. These data are significant for providing interesting biomechanical and cell biological insights into a fundamental morphogenetic process that is implicated in human neural tube defects and skeletal dysplasias.

Mechanosensitive calcium flashes promote sustained RhoA activation during tight junction remodeling.

Epithelial cell-cell junctions remodel in response to mechanical stimuli to maintain barrier function. Previously, we found that local leaks in tight junctions (TJs) are rapidly repaired by local, transient RhoA activation, termed "Rho flares," but how Rho flares are regulated is unknown. Here, we discovered that intracellular calcium flashes and junction elongation are early events in the Rho flare pathway. Both laser-induced and naturally occurring TJ breaks lead to local calcium flashes at the site of leaks. Additionally, junction elongation induced by optogenetics increases Rho flare frequency, suggesting that Rho flares are mechanically triggered. Depletion of intracellular calcium or inhibition of mechanosensitive calcium channels (MSCs) reduces the amplitude of calcium flashes and diminishes the sustained activation of Rho flares. MSC-dependent calcium influx is necessary to maintain global barrier function by regulating reinforcement of local TJ proteins via junction contraction. In all, we uncovered a novel role for MSC-dependent calcium flashes in TJ remodeling, allowing epithelial cells to repair local leaks induced by mechanical stimuli.

Convergent Extension Requires Adhesion-Dependent Biomechanical Integration of Cell Crawling and Junction Contraction

Convergent extension is an evolutionarily conserved collective cell movement that elongates the body axis of all animals and is required for the morphogenesis of several organ systems. Decades of study have revealed two distinct mechanisms of cell movement during CE, one based on cell crawling and the other on junction contraction. How these two behaviors collaborate during CE is not understood. Here, using quantitative live cell imaging we show that these two modes act both independently and in concert during CE, but that cell movement is more effective when the two modes are integrated via mechano-reciprocity. Based on these findings, we developed a novel computational model that for the first time treats crawling and contraction independently. This model not only confirmed the biomechanical efficacy of integrating the two modes, but also revealed for the first time how the two modes -and their integration- are influenced by cell adhesion. Finally, we use these new insights to further understand the complex CE phenotype resulting from loss of the C-cadherin interacting catenin Arvcf. These data are significant for providing new biomechanical and cell biological insights into a fundamental morphogenetic process that is implicated in human neural tube defects and skeletal dysplasias.

RhoA Mediates Epithelial Cell Shape Changes via Mechanosensitive Endocytosis.

Epithelial remodeling involves ratcheting behavior whereby periodic contractility produces transient changes in cell-cell contact lengths, which stabilize to produce lasting morphogenetic changes. Pulsatile RhoA activity is thought to underlie morphogenetic ratchets, but how RhoA governs transient changes in junction length, and how these changes are rectified to produce irreversible deformation, remains poorly understood. Here, we use optogenetics to characterize responses to pulsatile RhoA in model epithelium. Short RhoA pulses drive reversible junction contractions, while longer pulses produce irreversible junction length changes that saturate with prolonged pulse durations. Using an enhanced vertex model, we show this is explained by two effects: thresholded tension remodeling and continuous strain relaxation. Our model predicts that structuring RhoA into multiple pulses overcomes the saturation of contractility and confirms this experimentally. Junction remodeling also requires formin-mediated E-cadherin clustering and dynamin-dependent endocytosis. Thus, irreversible junction deformations are regulated by RhoA-mediated contractility, membrane trafficking, and adhesion receptor remodeling.

Pharmacological restoration of gut barrier function in stressed neonates partially reverses long-term alterations associated with maternal separation.

Intestinal permeability plays an important role in gut-brain axis communication. Recent studies indicate that intestinal permeability increases in neonate pups during maternal separation (MS). The present study aims to determine whether pharmacological inhibition of myosin light chain kinase (MLCK), which regulates tight junction contraction and controls intestinal permeability, in stressed neonates, protects against the long-term effects of MS. Male Wistar rats were exposed to MS (3h per day from post-natal day (PND)2 to PND14) or left undisturbed and received daily intraperitoneal injection of a MLCK inhibitor (ML-7, 5mg/kg) or vehicle during the same period. At adulthood, emotional behaviors, corticosterone response to stress, and gut microbiota composition were analyzed. ML-7 restored gut barrier function in MS rats specifically during the neonatal period. Remarkably, ML-7 prevented MS-induced sexual reward-seeking impairment and reversed the alteration of corticosterone response to stress at adulthood. The effects of ML-7 were accompanied by the normalization of the abundance of members of Lachnospiraceae, Clostridiales, Desulfovibrio, Bacteroidales, Enterorhabdus, and Bifidobacterium in the feces of MS rats at adulthood. Altogether, our work suggests that improvement of intestinal barrier defects during development may alleviate some of the long-term effects of early-life stress and provides new insight on brain-gut axis communication in a context of stress.

Junction contraction for a T-shaped micro-channel to enhance mixing

A design concept of a junction contraction is proposed for a T-shaped microchannel to enhance mixing. The proposed design entails contracting the flow passage at the junction of two fluids in a T-shaped microchannel. It is validated numerically by application to a well known T-shaped microchannel. The proposed junction contraction results in a noticeable improvement in mixing. The improvement of mixing is found to be caused by a vortex flow around the junction contraction. A downward flow is shown to form just before the contraction and is followed by an upward flow. The dependence on the position and the size of contraction is also studied. The best degree of mixing is obtained when the contraction is placed in the range of 50–125μm downstream the junction of the two fluids being mixed. The mixing enhancement is found to increase quadratically with the contraction ratio. However, more contracted junction requires a larger pressure drop.

Mixing in the shear superposition micromixer: three-dimensional analysis.

In this paper, we analyse mixing in an active chaotic advection micromixer. The micromixer consists of a main rectangular channel and three cross-stream secondary channels that provide ability for time-dependent actuation of the flow stream in the direction orthogonal to the main stream. Three-dimensional motion in the mixer is studied. Numerical simulations and modelling of the flow are pursued in order to understand the experiments. It is shown that for some values of parameters a simple model can be derived that clearly represents the flow nature. Particle image velocimetry measurements of the flow are compared with numerical simulations and the analytical model. A measure for mixing, the mixing variance coefficient (MVC), is analysed. It is shown that mixing is substantially improved with multiple side channels with oscillatory flows, whose frequencies are increasing downstream. The optimization of MVC results for single side-channel mixing is presented. It is shown that dependence of MVC on frequency is not monotone, and a local minimum is found. Residence time distributions derived from the analytical model are analysed. It is shown that, while the average Lagrangian velocity profile is flattened over the steady flow, Taylor-dispersion effects are still present for the current micromixer configuration.

Tachykinins mediate noncholinergic excitatory neural responses in the circular muscle of rat proximal colon.

Using the sucrose-gap technique, we attempted to assess a role for tachykinins (TKs) in mediating noncholinergic excitatory junction potential (EJP) and contraction, in the circular muscle of rat proximal colon. Excitatory responses were evoked by submaximal electrical field stimulation (EFS) in the presence of atropine (1 microM), guanethidine (1 microM), indomethacin (10 microM), and N(omega)-nitro-L-arginine methyl ester (L-NAME) (100 microM). The NK1 receptor antagonist, SR 140,333 (up to 3 microM) or the NK2 receptor antagonists, SR 48,968 and MEN 10,627 (up to 5 microM) produced a partial inhibition of the excitatory responses to EFS. The co-administration of the selective NK1 and NK2 receptor antagonists produced additive effects on the responses to EFS. Selective NK1 receptor agonist, [Sar9, Met (O2)11]-substance P, induced depolarization and contraction, antagonized by SR 140,333, but not by NK2 receptor antagonists. NK2 receptor agonist, [betaAla8]-neurokinin A (4-10), also produced electrical and mechanical excitatory effects that were antagonized by SR 48,968 or MEN 10,627, but not by the NK1 receptor antagonist. Our results provide evidence that, in circular muscle of rat colon, endogenous tachykinins are the main excitatory transmitters for nonadrenergic, noncholinergic (NANC) excitation and their action is mediated by both NK1 and NK2 receptors.

Ca2+ role in myogenic and neurogenic activities of canine ileum circular muscle.

The role of Ca2+ in myogenic and neural activation in canine ileum circular muscle (CM) was studied during simultaneous recordings of contractile and electrical activity in cross-sectioned slabs of muscularis externa or of isolated CM with deep muscular plexus (DMP) intact. Ca(2+)-free Krebs solution abolished inhibitory junction potentials (IJP) and contractions before changes in CM membrane potentials and while slow waves (SW) persisted at lower amplitude and frequency. This medium abolished SW more rapidly in isolated CM than in intact muscle strips and affected triggered SW (TSW) by 100-ms pulses recorded near myenteric plexus or near DMP differentially in the full-thickness preparation; TSW did not occur in isolated CM. Ni2+, a nonselective Ca2+ channel antagonist, left IJP unchanged and reduced contractions, frequencies, and amplitudes of spontaneous SW and TSW, but increased their durations. Nifedipine abolished contractions but SW, TSW, and IJP were unaffected. Cyclopiazonic acid (CPA) increased SW frequency, produced spikes on SW plateaus, and increased CM tone, but did not affect IJP or resting membrane potentials. In nifedipine-pretreated strips, CPA decreased SW frequencies and amplitudes, evoked less tone, depolarized membrane potentials, and left IJP unaltered. The neuronal N-type Ca2+ channel blocker omega-conotoxin GVIA abolished IJP, without affecting SW or TSW. We conclude that Ca2+ influx, not through L- or N-type Ca2+ channels, helps initiate ileal SW; L-type Ca2+ channels provide Ca2+ for contraction and N-type Ca2+ channels provide Ca2+ for IJP mediator release. Frequencies of SW may be modulated by uptake of Ca2+ into pacemaker stores.

Mediation of contraction and relaxation by alpha- and beta-adrenoceptors in the ureterovesical junction of the sheep

The effects of alpha- and beta-adrenoceptor agonists and antagonists were studied in the sheep ureterovesical junction. Non-specific adrenergic agonists such as adrenaline and noradrenaline induced contraction in the sheep ureterovesical junction, suggesting a predominance of alpha- over beta-adrenoceptors in this functional unity. An inhibition of the noradrenaline-induced contraction was observed after prior blocking with prazosin (10 −7M and yohimbine (10 −7M), the effect of prazosin being more potent than that of yohimbine. The effect of phenylephrine on alpha 1-adrenoceptors was more potent than that of B-HT 920 on alpha 2-adrenoceptors. Isoproterenol caused a concentration-dependent relaxation that was inhibited by propranolol (10 −6M), pafenolol (10 −5M) and butoxamine (10 −5M). These results suggest that ureterovesical junction contraction is mediated by both alpha, and alpha 2-adrenoceptors, alpha 1 predominating over alpha 2. Relaxation is mediated by beta-adrenoceptors of the beta 1 and beta 2 subtypes.

Neural control of swimming in Aplysia brasiliana. III. Serotonergic modulatory neurons.

1. We describe a group of serotonergic neurons in the pedal ganglia of Aplysia brasiliana and characterize their modulatory effects on the motoneuron input to swimming muscles of the parapodia. Each pedal ganglion contains one cluster of large neurons near its dorsomedial surface that fires in phase with opening (downstroke) of the parapodia; these have been designated parapodial opener-phase (POP) cells. 2. POP cells are large, number 15-20 per ganglion, have peripheral axons in parapodial nerves, have distinctively shaped action potentials, and fire in bursts phasically with motoneurons during the opening, or downstroke portion, of parapodial movement during fictive swimming. Firing individual POP cells with intracellular current indicates that they have no direct detectable effect on muscle, causing neither junction potentials nor contractions. 3. 5,7-Dihydroxytryptamine (5,7-DHT) staining, immunocytochemistry using serotonin (5-HT) antibodies, and direct biochemical measurements revealed that POP cells are serotonergic. Serotonergic nerve endings were also seen in parapodial muscle. 4. Simultaneous intracellular recordings and use of altered divalent concentrations revealed that no detectable direct synaptic interactions exist between POP cells and motor neurons. 5. When POP cells and motoneurons were simultaneously recorded while measuring muscle contractions, it was found that POP cell activity enhances motoneuron-induced tension by 120-900%, averaging around 300%. Variability in the efficacy of individual POP cells suggests that they may influence specific regions or groups of muscle fibers. 6. POP cell activity also significantly increased the rate of relaxation of parapodial muscle contractions, averaging about a 40% reduction in the time required to relax to one-half peak tension. Increased relaxation rate implies a postsynaptic change in muscle behavior. 7. The effectiveness of POP cells to increase contraction tension and relaxation rate was positively correlated with POP cell spike frequency. These effects were slow (seconds) in onset and could persist for a minute or more after cessation of POP firing. Concurrent motoneuron activity is not required for modulation by POP cells. 8. Simultaneous intracellular recording from a POP cell, motoneuron, and muscle fiber revealed that POP cell activity enhanced the amplitude of motoneuron-induced excitatory junction potentials (EJPs). Activity of POP cells did not alter muscle fiber membrane potential, but the experiments left open the possibility that EJP enhancement is presynaptic, postsynaptic, or a combination.(ABSTRACT TRUNCATED AT 400 WORDS)