Apical Contraction Research Articles

Integration of actomyosin contractility with cell-cell adhesion during dorsal closure.

In this work, we combine genetic perturbation, time-lapse imaging and quantitative image analysis to investigate how pulsatile actomyosin contractility drives cell oscillations, apical cell contraction and tissue closure during morphogenesis of the amnioserosa, the main force-generating tissue during the dorsal closure in Drosophila We show that Myosin activity determines the oscillatory and contractile behaviour of amnioserosa cells. Reducing Myosin activity prevents cell shape oscillations and reduces cell contractility. By contrast, increasing Myosin activity increases the amplitude of cell shape oscillations and the time cells spend in the contracted phase relative to the expanded phase during an oscillatory cycle, promoting cell contractility and tissue closure. Furthermore, we show that in AS cells, Rok controls Myosin foci formation and Mbs regulates not only Myosin phosphorylation but also adhesion dynamics through control of Moesin phosphorylation, showing that Mbs coordinates actomyosin contractility with cell-cell adhesion during amnioserosa morphogenesis.

α-Catenin stabilises Cadherin-Catenin complexes and modulates actomyosin dynamics to allow pulsatile apical contraction.

We have investigated how cell contractility and adhesion are functionally integrated during epithelial morphogenesis. To this end, we have analysed the role of α-Catenin, a key molecule linking E-Cadherin-based adhesion and the actomyosin cytoskeleton, during Drosophila embryonic dorsal closure, by studying a newly developed allelic series. We find that α-Catenin regulates pulsatile apical contraction in the amnioserosa, the main force-generating tissue driving closure of the embryonic epidermis. α-Catenin controls actomyosin dynamics by stabilising and promoting the formation of actomyosin foci, and also stabilises DE-Cadherin (Drosophila E-Cadherin, also known as Shotgun) at the cell membrane, suggesting that medioapical actomyosin contractility regulates junction stability. Furthermore, we uncover a genetic interaction between α-Catenin and Vinculin, and a tension-dependent recruitment of Vinculin to amniosersoa apical cell membranes, suggesting the existence of a mechano-sensitive module operating in this tissue.

Emergent material properties of developing epithelial tissues.

BackgroundForce generation and the material properties of cells and tissues are central to morphogenesis but remain difficult to measure in vivo. Insight is often limited to the ratios of mechanical properties obtained through disruptive manipulation, and the appropriate models relating stress and strain are unknown. The Drosophila amnioserosa epithelium progressively contracts over 3 hours of dorsal closure, during which cell apices exhibit area fluctuations driven by medial myosin pulses with periods of 1.5–6 min. Linking these two timescales and understanding how pulsatile contractions drive morphogenetic movements is an urgent challenge.ResultsWe present a novel framework to measure in a continuous manner the mechanical properties of epithelial cells in the natural context of a tissue undergoing morphogenesis. We show that the relationship between apicomedial myosin fluorescence intensity and strain during fluctuations is consistent with a linear behaviour, although with a lag. We thus used myosin fluorescence intensity as a proxy for active force generation and treated cells as natural experiments of mechanical response under cyclic loading, revealing unambiguous mechanical properties from the hysteresis loop relating stress to strain. Amnioserosa cells can be described as a contractile viscoelastic fluid. We show that their emergent mechanical behaviour can be described by a linear viscoelastic rheology at timescales relevant for tissue morphogenesis. For the first time, we establish relative changes in separate effective mechanical properties in vivo. Over the course of dorsal closure, the tissue solidifies and effective stiffness doubles as net contraction of the tissue commences. Combining our findings with those from previous laser ablation experiments, we show that both apicomedial and junctional stress also increase over time, with the relative increase in apicomedial stress approximately twice that of other obtained measures.ConclusionsOur results show that in an epithelial tissue undergoing net contraction, stiffness and stress are coupled. Dorsal closure cell apical contraction is driven by the medial region where the relative increase in stress is greater than that of stiffness. At junctions, by contrast, the relative increase in the mechanical properties is the same, so the junctional contribution to tissue deformation is constant over time. An increase in myosin activity is likely to underlie, at least in part, the change in medioapical properties and we suggest that its greater effect on stress relative to stiffness is fundamental to actomyosin systems and confers on tissues the ability to regulate contraction rates in response to changes in external mechanics.Electronic supplementary materialThe online version of this article (doi:10.1186/s12915-015-0200-y) contains supplementary material, which is available to authorized users.

Synaptopodin Couples Epithelial Apical Contractions to Adherens Junction Maturation

Cell‐cell adhesive contact is a central player in mechanotransduction, providing both mechanical support and a hub for signaling. Yet, the molecular mechanism is poorly defined, partly due to a lack of cell culture model to manipulate tension at cell‐cell contacts. Here, using live cell imaging of α‐actinin, we showed that MDCK cells exhibit many cellular behaviors of epithelial cells in vivo, including (1) oscillating contractility of the junction, (2) pulsatile centripetal cellular contractions, and (3) ratchet constrictions of apical domain. We hypothesized that pulsatile contractions participate in the maturation of junctional complexes. To test our hypothesis, we applied cyclic pulsatile tension to cell‐cell contacts in confluent cell monolayers. Pulsatile intercellular tension induces recruitment of α‐actinin‐4 in a time and tension‐dependent manner without changing the localization of canonical adherens junction proteins, suggesting that a missing factor is involved. We have identified this missing factor as synaptopodin. Synaptopodin recruitment to the junction is also tension‐sensitive, which is necessary for the generation of contractility at cell‐cell adhesions. Recruitment of a‐actinin‐4 strengthens cell‐cell adhesion and promote epithelial permeability barrier. Our studies reveal a molecular system with both mechanical and biochemical inputs and underscore the complexity of junction assembly in epithelial monolayers in vivo and in cultured cell systems.

Myosin-II-mediated cell shape changes and cell intercalation contribute to primitive streak formation.

Primitive streak formation in the chick embryo involves large scale highly coordinated flows of over 100.000 cells in the epiblast. These large scale tissue flows and deformations can be correlated with specific anisotropic cell behaviours in the forming mesendoderm through a combined light-sheet microscopy and computational analysis. Relevant behaviours include apical contraction, elongation along the apical-basal axis followed by ingression as well as asynchronous directional cell intercalation of small groups of mesendoderm cells. Cell intercalation is associated with sequential, directional contraction of apical junctions, the onset, localisation and direction of which correlate strongly with the appearance of active Myosin II cables in aligned apical junctions in neighbouring cells. Use of a class specific Myosin inhibitors and gene specific knockdowns show that apical contraction and intercalation are Myosin II dependent and also reveal critical roles for Myosin I and Myosin V family members in the assembly of junctional Myosin II cables.

Coupling of Apical Contractions and Adherens Junction Maturation by Synaptopodin-Depedent Recruitment of A-Actinin-4

Cell-cell adhesive contact is a central player in mechanotransduction, providing both mechanical support and a hub for signaling. Yet, the molecular mechanism is poorly defined, partly due to a lack of cell culture model to mechanically manipulate tension at cell-cell contacts. Here, using live cell imaging of α-actinin, we showed that MDCK cells exhibit many cellular behaviors of epithelial cells in vivo, including (1) oscillating contractility of the adherens junction, (2) pulsatile centripetal cellular contractions, and (3) ratchet constrictions of apical junctional domain. We hypothesized that pulsatile contractions participate in the maturation of junctional complexes. To test our hypothesis, we applied cyclic pulsatile tension to cell-cell contacts in confluent cell monolayers. We found that pulsatile intercellular tension induces recruitment of α-actinin-4 to the adherens junction in a time and tension-dependent manner without changing the localization of canonical adherens junction proteins, suggesting that a missing factor is involved. We have identified this missing factor as synaptopodin. Synaptopodin recruitment to the adherens junction is also tension-sensitive, which is necessary for generation of contractility at cell-cell adhesions. Recruitment of a-actinin-4 strengthens cell-cell adhesion and promote epithelial permeability barrier. Thus, by controlling the mechanical force generated through apical contractility, epithelial cells can adjust the strength of cell-cell adhesion and the maturation process of adherens junction maturation. Conversely, synaptopodin dictates the efficiency of mechanical-biochemical coupling between contractility and junction assembly through the recruitment of a-actinin-4. Our studies reveal a tunable molecular system with both mechanical and biochemical inputs and underscore the complexity of junction assembly in epithelial monolayers in vivo and in cultured cell systems.

Functional, morphological and electrocardiographical abnormalities in patients with apical hypertrophic cardiomyopathy and apical aneurysm: correlation with cardiac MR

ObjectiveThe prognosis of apical hypertrophic cardiomyopathy (APH) has been benign, but apical myocardial injury has prognostic importance. We studied functional, morphological and electrocardiographical abnormalities in patients with APH and with...

Contractile and mechanical properties of epithelia with perturbed actomyosin dynamics.

Mechanics has an important role during morphogenesis, both in the generation of forces driving cell shape changes and in determining the effective material properties of cells and tissues. Drosophila dorsal closure has emerged as a reference model system for investigating the interplay between tissue mechanics and cellular activity. During dorsal closure, the amnioserosa generates one of the major forces that drive closure through the apical contraction of its constituent cells. We combined quantitation of live data, genetic and mechanical perturbation and cell biology, to investigate how mechanical properties and contraction rate emerge from cytoskeletal activity. We found that a decrease in Myosin phosphorylation induces a fluidization of amnioserosa cells which become more compliant. Conversely, an increase in Myosin phosphorylation and an increase in actin linear polymerization induce a solidification of cells. Contrary to expectation, these two perturbations have an opposite effect on the strain rate of cells during DC. While an increase in actin polymerization increases the contraction rate of amnioserosa cells, an increase in Myosin phosphorylation gives rise to cells that contract very slowly. The quantification of how the perturbation induced by laser ablation decays throughout the tissue revealed that the tissue in these two mutant backgrounds reacts very differently. We suggest that the differences in the strain rate of cells in situations where Myosin activity or actin polymerization is increased arise from changes in how the contractile forces are transmitted and coordinated across the tissue through ECadherin-mediated adhesion. Altogether, our results show that there is an optimal level of Myosin activity to generate efficient contraction and suggest that the architecture of the actin cytoskeleton and the dynamics of adhesion complexes are important parameters for the emergence of coordinated activity throughout the tissue.

S0210101 多細胞力学シミュレーションを用いた上皮組織変形に対するアピカル収縮と細胞増殖の寄与の検討([S021]多細胞・組織への展開を目指した細胞工学,バイオエンジニアリング部門)

S0210101 多細胞力学シミュレーションを用いた上皮組織変形に対するアピカル収縮と細胞増殖の寄与の検討([S021]多細胞・組織への展開を目指した細胞工学,バイオエンジニアリング部門)

Apical hypertrophic cardiomyopathy: chest pain and myocardial perfusion defects result from regional diastolic persistence of hyperdynamic cardiac contractility

Background: Apical hypertrophic cardiomyopathy (HCM) is commonly associated with drug-refractory chest pain. We sought to determine whether, in apical HCM, coronary perfusion time is abbreviated by the diastolic persistence of apical contraction, resulting in impaired myocardial perfusion and chest pain. Methods: 62 apical HCM patients had cardiac magnetic resonance (CMR) scans assessed for stress perfusion (myocardial perfusion reserve index (MPRi)), late gadolinium enhancement (LGE; % of myocardial volume) left ventricular (LV) volumes and LV contractile persistence (% total cardiac cycle) at the LV apex and base. Radial and circumferential strain were assessed. Patients were divided into three groups on the basis of severity of contractile persistence. The interval between earliest and latest systolic peaks was measured from strain data from each of the apical segments. Results: Compared to subjects with the least contractile persistence (C1), those with the most (C3) were more likely to have chest pain (94% vs 63%, p 130ms as a threshold, circumferential dyssynchrony was present in 25% of C1 and 81% of C3 patients (p<0.001) and radial dyssynchrony in 65% of C1 and 95% of C3 patients (p<0.05). In patients with radial dyssynchrony, the earliest peak was most often in the inferior or anterior segments (60%) and the latest in the lateral segment (33%). In patients with circumferential dyssynchrony, the earliest peak was most often in the inferior or anterior segments (59%) and the latest in the lateral segment (41%). Conclusion: In apical HCM, regional persistence of contractility into diastole causes myocardial ischaemia and chest pain. This is the first description of contractile persistence and dyssynchrony as a mechanism for myocardial perfusion abnormalities and presents novel therapeutic opportunities for drug-refractory chest pain in apical HCM.

Mechano-Chemical Coupling Drives Cell Area Oscillations during Morphogenesis

Marie Curie Career Integration Grant (No. PCIG09-GA-2011-293479); Spanish Ministry of Science (No. BFU2011-25828)

Regional differences in actomyosin contraction shape the primary vesicles in the embryonic chicken brain

In the early embryo, the brain initially forms as a relatively straight, cylindrical epithelial tube composed of neural stem cells. The brain tube then divides into three primary vesicles (forebrain, midbrain, hindbrain), as well as a series of bulges (rhombomeres) in the hindbrain. The boundaries between these subdivisions have been well studied as regions of differential gene expression, but the morphogenetic mechanisms that generate these constrictions are not well understood. Here, we show that regional variations in actomyosin-based contractility play a major role in vesicle formation in the embryonic chicken brain. In particular, boundaries did not form in brains exposed to the nonmuscle myosin II inhibitor blebbistatin, whereas increasing contractile force using calyculin or ATP deepened boundaries considerably. Tissue staining showed that contraction likely occurs at the inner part of the wall, as F-actin and phosphorylated myosin are concentrated at the apical side. However, relatively little actin and myosin was found in rhombomere boundaries. To determine the specific physical mechanisms that drive vesicle formation, we developed a finite-element model for the brain tube. Regional apical contraction was simulated in the model, with contractile anisotropy and strength estimated from contractile protein distributions and measurements of cell shapes. The model shows that a combination of circumferential contraction in the boundary regions and relatively isotropic contraction between boundaries can generate realistic morphologies for the primary vesicles. In contrast, rhombomere formation likely involves longitudinal contraction between boundaries. Further simulations suggest that these different mechanisms are dictated by regional differences in initial morphology and the need to withstand cerebrospinal fluid pressure. This study provides a new understanding of early brain morphogenesis.

Transient stress cardiomyopathies in the elderly: Clinical &amp; Pathophysiologic considerations

Transient stress-induced cardiomyopathies have been increasingly recognized and while rare, they tend to affect elderly women more than other demographic groups. One type, often called tako-tsubo cardiomyopathy (TTC), is typically triggered by significant emotional or physical stress and is associated with chest pain, electrocardiogram (ECG) changes and abnormal cardiac enzymes. Significant left ventricular regional wall motion abnormalities usually include an akinetic “ballooning” apex with normal or hyperdynamic function of the base. A second type, often called neurogenic stunned myocardium, typically associated with subarachnoid hemorrhage, also usually presents with ECG changes and positive enzymes, but the typical wall motion abnormalities seen include normal basal and apical left ventricular contraction with akinesis of the mid-cavity in a circumferential fashion. The pathophysiology, clinical care and typical courses, are reviewed.

Active reinforcement of externally imposed folding in amphibians embryonic tissues

Although the folding of epithelial layers is one of the most common morphogenetic events, the underlying mechanisms of this process are still poorly understood. We aimed to determine whether an artificial bending of an embryonic cell sheet, which normally remains flat, is reinforced and stabilized by intrinsic cell transformations. We observed both reinforcement and stabilization in double explants of blastocoel roof tissue from Xenopus early gastrula embryos. The reinforcement of artificial bending occurred over the course of a few hours and was driven by the gradual apical constriction and radial elongation of previously compressed cells situated at the bending arch of the concave layer of explant. Apical constriction was associated with actomyosin contraction and endocytosis-mediated engulfing of the apical cell membranes. Cooperative apical constrictions of the concave layer of cells produced a tensile force that extended over the entire surface of the explant and correlated with apical contraction of the concave side cells. In the explants taken from the anterior regions of the embryo, this reinforcement was more stable and the bending better expressed than in those taken from suprablastoporal areas. The morphogenetic role of cell responses to the bending force is discussed.

McConnell’s sign unveiled

Summary McConnell’s sign is well described in echocardiography as a harbinger of severe RV failure and more extensive arterial thrombus burden but its mechanical basis has never been satifactorily defined despite multiple current theories. CMR RF tissue-tagging now unveils the mechanism. Background Evidence of echocardiographic RV ‘strain’ in the setting of a large, clinically important, often devasting pulmonary embolism (PE) is defined by the presence of paradoxical, often hyperdynamic RV apical contraction in the setting of severely dysfunctional RV function; so denoted ‘McConnell’ ss ign’. Despite its original observation by its namesake in 1996, it has neither been observed via CMR nor more importantly, while many theories exist, mechanistically explained. Hypothesis We hypothesize that McConnell’s sign (MS) is observable utilizing CMR and can be explained via CMR deformational analysis. Methods Modestly stable patients who had CT confirmed large (>one pulmonary branch obstruction and/or saddle PE) were evaluated via echocardiography and CMR. CMR radio-frequency tissue tagging was performed to determine midwall intramyocardial deformation patterns with superimposition of quiver plot technique to define direction and amplitude of myocardial displacements assuming homogenous tissue strain (%S). Results

Changes in the shape of epithelial embryonic cells of the spur-toed frog upon deformation of the cell layer

A quantitative study was made of changes in the shape of cells in double explants of the blastocoel roof of the clawed frog gastrula within the first four hours after artificial bending of explants. It was found that, on the concave (contracted) side of explants, epithelial cells stretched out, and in many of them the apical surface contracted, whereas on the convex (stretched) side the cells remained isodiametric. The maximal difference in the apical index between epithelial cells located on the concave and convex sides was observed after 2 h of explant cultivation; by 2 h the artificially produced curvature of the explant further increased. Endocytosis on the concave side was more active than on the convex side. Experiments with inhibitors modulating the behavior of the actomyosin complex showed that unimpeded functioning of myosin II is more important for the apical contraction and elongation of cells than proper structural organization of the actin backbone.

Usefulness of right ventricular myocardial strain in assessment of response to thrombolytic therapy in acute pulmonary embolism

Acute pulmonary embolism (PE) is a life-threatening illness that is an important cause of morbidity and mortality. Patients with extensive emboli usually have associated right ventricular (RV) dysfunction. With anticoagulation therapy, RV function usually returns to normal. In patients with massive and submassive PE, echocardiography may directly visualize the embolus or more commonly provide evidence of its haemodynamic sequelae. The McConnell's sign involving sparing of RV apical contraction coupled with akinesia of the RV free wall has previously been reported in patients with acute PE. We present a case of a patient who had massive acute PE with severe RV systolic dysfunction that was demonstrated with both conventional Doppler and strain imaging. Strain imaging was also able to demonstrate the rapid improvement of RV function in response to thrombolytic therapy.

Sequential Activation of Apical and Basolateral Contractility Drives Ascidian Endoderm Invagination

Epithelial invagination is a fundamental morphogenetic behavior that transforms a flat cell sheet into a pit or groove. Previous studies of invagination have focused on the role of actomyosin-dependent apical contraction; other mechanisms remain largely unexplored. We combined experimental and computational approaches to identify a two-step mechanism for endoderm invagination during ascidian gastrulation. During Step 1, which immediately precedes invagination, endoderm cells constrict their apices because of Rho/Rho-kinase-dependent apical enrichment of 1P-myosin. Our data suggest that endoderm invagination itself occurs during Step 2, without further apical shrinkage, via a novel mechanism we call collared rounding: Rho/Rho-kinase-independent basolateral enrichment of 1P-myosin drives apico-basal shortening, whereas Rho/Rho-kinase-dependent enrichment of 1P and 2P myosin in circumapical collars is required to prevent apical expansion and for deep invagination. Simulations show that boundary-specific tension values consistent with these distributions of active myosin can explain the cell shape changes observed during invagination both in normal embryos and in embryos treated with pharmacological inhibitors of either Rho-kinase or Myosin II ATPase. Indeed, we find that the balance of strong circumapical and basolateral tension is the only mechanism based on differential cortical tension that can explain ascidian endoderm invagination. Finally, simulations suggest that mesectoderm cells resist endoderm shape changes during both steps, and we confirm this prediction experimentally. Our findings suggest that early ascidian gastrulation is driven by the coordinated apposition of circumapical and lateral endoderm contraction, working against a resisting mesectoderm. We propose that similar mechanisms may operate during other invaginations.

Cell‐ and Tissue‐mechanics of Apical Contraction in Embryonic Epithelia

Apical contraction is one of the principle cellular mechanisms that sculpt epithelial sheets into structures such as tubes, pits, and furrows. In order to investigate the mechanics of apical contraction and epithelial morphogenesis in vertebrates we have developed three methods (chemical‐, electrical‐, and laser‐activation) to induce contraction in embryonic epithelia of the frog Xenopus laevis. We use these methods together with a biomechanical analytical framework to investigate the molecular signaling pathways regulating contraction and the mechanical coupling of these pathways to tissue movements. Thus far, we find pathways that control exogenously induced contractions are similar to the programs that regulate native epithelial morphogenesis. For instance, laser‐activation induces a Ca++ wave, activates RhoGTPase, and drives F‐actin remodeling. Epithelial stiffness and force‐generation measured during electrically induced contraction suggests these two properties are mechanically independent. In contrast to single‐cell studies we have found no evidence that mechanical‐stimulation can induce apical contraction. In addition to our results on the molecular and mechanical regulation of apical contraction our studies suggest morphogenetic processes can be co‐opted by tissue engineers to build replacement tissues and construct artificial organs.

Right Ventricular Involvement and Pulmonary Hypertension in an Elderly Woman With Tako-Tsubo Cardiomyopathy

We describe a case of tako-tsubo cardiomyopathy in an elderly woman with a permanent pacemaker admitted to the ED with chest pain and dyspnea. Coronary angiography revealed normal coronary arteries. Typical left ventricular apical ballooning was demonstrated on contrast ventriculography. Left and right ventricular ballooning with pulmonary artery systolic hypertension was detected by transthoracic echocardiography. Velocity vector imaging and strain analysis showed a typical pattern of regional myocardial apical right ventricular contraction characterized by paradoxical positive longitudinal systolic strain. Biventricular involvement was associated with hemodynamic instability, signs of pulmonary vascular congestion, and bilateral basal pleural effusion. The patient's clinical condition gradually improved, and she was discharged after prolonged hospitalization. Predischarge echocardiography showed substantial recovery of left and right systolic function along with normalization of pulmonary artery systolic pressure. Right ventricular function should be carefully evaluated in patients with tako-tsubo cardiomyopathy, especially in the acute phase.