2.5D Traction Force Microscopy: Imaging three-dimensional cell forces at interfaces and biological applications.

The forces that cells, tissues, and organisms exert on the surface of a soft substrate can be measured using Traction Force Microscopy (TFM), an important and well-established technique in Mechanobiology. The usual TFM technique (two-dimensional, 2D TFM) treats only the in-plane component of the traction forces and omits the out-of-plane forces at the substrate interfaces (2.5D) that turn out to be important in many biological processes such as tissue migration and tumour invasion. Here, we review the imaging, material, and analytical tools to perform "2.5D TFM" and explain how they are different from 2D TFM. Challenges in 2.5D TFM arise primarily from the need to work with a lower imaging resolution in the z-direction, track fiducial markers in three-dimensions, and reliably and efficiently reconstruct mechanical stress from substrate deformation fields. We also discuss how 2.5D TFM can be used to image, map, and understand the complete force vectors in various important biological events of various length-scales happening at two-dimensional interfaces, including focal adhesions forces, cell diapedesis across tissue monolayers, the formation of three-dimensional tissue structures, and the locomotion of large multicellular organisms. We close with future perspectives including the use of new materials, imaging and machine learning techniques to continuously improve the 2.5D TFM in terms of imaging resolution, speed, and faithfulness of the force reconstruction procedure.

The effect of testosterone treatment on bone mineral density in Klinefelter syndrome: A retrospective cohort study.

BACKGROUND: Although Klinefelter syndrome (KS) is the most frequent sex-hormone disorder, there is ongoing uncertainty about the often associated sex-hormone deficiency, its impact on common comorbidities, and therefore about prevention and treatment. In this study, we focus on bone loss, reported to occur in over 40% of KS patients, and the impact of testosterone replacement therapy (TRT) on this comorbidity. OBJECTIVES: This single-center retrospective cohort study in a tertiary hospital compared the effect of treatment with TRT to no TRT on evolution of bone mineral density (BMD) in KS patients. METHODS: After a medical chart review, a total of 52 KS subjects were included in this study. BMD was measured by dual-energy X-ray absorptiometry (DXA) and expressed as T-scores. RESULTS: The subjects were divided into three groups, according to TRT. In the subgroup that only started TRT after baseline measurements (mean age 31 years), we observed significant gain in BMD T-score at the lumbar spine (0.58 ± 0.60, p = 0.003; mean gain of 0.62% areal BMD per year) and total femur T-score (0.24 ± 0.39, p = 0.041; mean gain of 0.25% areal BMD per year) after a mean follow-up period of 7.5 years. Compared to untreated subjects, a significant difference in evolution was demonstrated at the lumbar level (+0.58 ± 0.60 vs. -0.14 ± 0.42, p = 0.007). In untreated subjects with normal testosterone levels, a loss of BMD (-0.27 ± 0.37, p = 0.029; mean loss of 0.49% areal BMD per year) at the femoral neck was measured. This decline was equal to the predicted loss seen in the general male population. CONCLUSION: TRT results in BMD gain in patients with KS with testosterone deficiency, mainly at the lumbar spine. However, this effect is limited (0.62% per year). Patients who were not treated with TRT because of sufficient endogenous testosterone levels, showed only the predicted age-related bone loss during follow-up. The need for TRT in maintaining bone health in KS should be evaluated on an individual basis according to the degree of sex steroid deficiency.

Mechanical stress driven by rigidity sensing governs epithelial stability.

Epithelia act as a barrier against environmental stress and abrasion and in vivo they are continuously exposed to environments of various mechanical properties. The impact of this environment on epithelial integrity remains elusive. By culturing epithelial cells on 2D hydrogels, we observe a loss of epithelial monolayer integrity through spontaneous hole formation when grown on soft substrates. Substrate stiffness triggers an unanticipated mechanical switch of epithelial monolayers from tensile on soft to compressive on stiff substrates. Through active nematic modelling, we find that spontaneous half-integer defect formation underpinning large isotropic stress fluctuations initiate hole opening events. Our data show that monolayer rupture due to high tensile stress is promoted by the weakening of cell-cell junctions that could be induced by cell division events or local cellular stretching. Our results show that substrate stiffness provides feedback on monolayer mechanical state and that topological defects can trigger stochastic mechanical failure, with potential application towards a mechanistic understanding of compromised epithelial integrity during immune response and morphogenesis.

Mapping cell cortex rheology to tissue rheology and vice versa.

The mechanics of biological tissues mainly proceeds from the cell cortex rheology. A direct, explicit link between cortex rheology and tissue rheology remains lacking, yet would be instrumental in understanding how modulations of cortical mechanics may impact tissue mechanical behavior. Using an ordered geometry built on 3D hexagonal, incompressible cells, we build a mapping relating the cortical rheology to the monolayer tissue rheology. Our approach shows that the tissue low-frequency elastic modulus is proportional to the rest tension of the cortex, as expected from the physics of liquid foams as well as of tensegrity structures. A fractional visco-contractile cortex rheology is predicted to yield a high-frequency fractional visco-elastic monolayer rheology, where such a fractional behavior has been recently observed experimentally at each scale separately. In particular cases, the mapping may be inverted, allowing to derive from a given tissue rheology the underlying cortex rheology. Interestingly, applying the same approach to a 2D hexagonal tiling fails, which suggests that the 2D character of planar cell cortex-based models may be unsuitable to account for realistic monolayer rheologies. We provide quantitative predictions, amenable to experimental tests through standard perturbation assays of cortex constituents, and hope to foster new, challenging mechanical experiments on cell monolayers.

All-in-one rheometry and nonlinear rheology of multicellular aggregates.

Tissues are generally subjected to external stresses, a potential stimulus for their differentiation or remodeling. While single-cell rheology has been extensively studied leading to controversial results about nonlinear response, mechanical tissue behavior under external stress is still poorly understood, in particular, the way individual cell properties translate at the tissue level. Herein, using magnetic cells we were able to form perfectly monitored cellular aggregates (magnetic molding) and to deform them under controlled applied stresses over a wide range of timescales and amplitudes (magnetic rheometer). We explore the rheology of these minimal tissue models using both standard assays (creep and oscillatory response) as well as an innovative broad spectrum solicitation coupled with inference analysis thus being able to determine in a single experiment the best rheological model. We find that multicellular aggregates exhibit a power-law response with nonlinearities leading to tissue stiffening at high stress. Moreover, we reveal the contribution of intracellular (actin network) and intercellular components (cell-cell adhesions) in this aggregate rheology.

The impact of androgen deprivation therapy on bone mineral density in men treated for paraphilic disorder: A retrospective cohort study.

BACKGROUND: Guidelines suggest treating men with paraphilic disorder with androgen-deprivation therapy (ADT). However, little evidence is available about the long-term impact on bone loss and how to manage this adverse event. OBJECTIVES: The aim of this study is to assess the impact of ADT on bone mineral density (BMD) in men treated for paraphilic disorder with the androgen receptor blocker cyproterone acetate (CPA) and/or GnRH agonist triptoreline (GnRHa) and to evaluate the effect of treatment with bisphosphonates. METHODS: Baseline and follow-up dual-energy X-ray absorptiometry scan (DXA-scan) data (lumbar and femoral T-scores) were retrospectively extracted from electronic medical files of paraphilic men who received CPA and/or GnRHa. RESULTS: A total of 53 patients with a mean age of 39.1 years (range 17.5-74.6) were included. Lumbar (-0.39 ± 0.17, Mean ± SEM, p = 0.046), femoral neck (-0.34 ± 0.09, p = 0.002) and total femur (-0.33 ± 0.12, p = 0.014) T-scores decreased significantly in the CPA-only group (n = 13) during a mean follow-up of 6.0 ± 5.3 years. In the GnRHa group (n = 29), T-scores at all sites decreased significantly over 6.6 ± 4.4 years (lumbar: -0.55 ± 0.12, p < 0.001, femoral neck: -0.53 ± 0.09, total femur: -0.44 ± 0.09, p < 0.001). In the group, who received bisphosphonates (n = 11), no significant T-score change was observed (lumbar: -0.25 ± 0.14, p = 0.106, femoral neck -0.15 ± 0.17, p = 0.402, total femur -0.25 ± 0.14, p = 0.106) during 5.0 ± 2.8 years of follow-up. DISCUSSION AND CONCLUSION: Following a mean duration of 6 years of ADT, we observed a significant decline in BMD of approximately half a standard deviation in T-score at lumbar and femoral site. Although the number of patients who received bisphosphonates was limited, this treatment seems to have a positive stabilizing effect on bone density.

Enhanced RhoA signalling stabilizes E-cadherin in migrating epithelial monolayers.

Epithelia migrate as physically coherent populations of cells. Previous studies have revealed that mechanical stress accumulates in these cellular layers as they move. These stresses are characteristically tensile in nature and have often been inferred to arise when moving cells pull upon the cell-cell adhesions that hold them together. We now report that epithelial tension at adherens junctions between migrating cells also increases due to an increase in RhoA-mediated junctional contractility. We found that active RhoA levels were stimulated by p114 RhoGEF (also known as ARHGEF18) at the junctions between migrating MCF-7 monolayers, and this was accompanied by increased levels of actomyosin and mechanical tension. Applying a strategy to restore active RhoA specifically at adherens junctions by manipulating its scaffold, anillin, we found that this junctional RhoA signal was necessary to stabilize junctional E-cadherin (CDH1) during epithelial migration and promoted orderly collective movement. We suggest that stabilization of E-cadherin by RhoA serves to increase cell-cell adhesion to protect against the mechanical stresses of migration. This article has an associated First Person interview with the first author of the paper.

The role of single cell mechanical behavior and polarity in driving collective cell migration.

The directed migration of cell collectives is essential in various physiological processes, such as epiboly, intestinal epithelial turnover, and convergent extension during morphogenesis as well as during pathological events like wound healing and cancer metastasis. Collective cell migration leads to the emergence of coordinated movements over multiple cells. Our current understanding emphasizes that these movements are mainly driven by large-scale transmission of signals through adherens junctions. In this study, we show that collective movements of epithelial cells can be triggered by polarity signals at the single cell level through the establishment of coordinated lamellipodial protrusions. We designed a minimalistic model system to generate one-dimensional epithelial trains confined in ring shaped patterns that recapitulate rotational movements observed in vitro in cellular monolayers and in vivo in genitalia or follicular cell rotation. Using our system, we demonstrated that cells follow coordinated rotational movements after the establishment of directed Rac1-dependent polarity over the entire monolayer. Our experimental and numerical approaches show that the maintenance of coordinated migration requires the acquisition of a front-back polarity within each single cell but does not require the maintenance of cell-cell junctions. Taken together, these unexpected findings demonstrate that collective cell dynamics in closed environments as observed in multiple in vitro and in vivo situations can arise from single cell behavior through a sustained memory of cell polarity.

Author Correction: An optochemical tool for light-induced dissociation of adherens junctions to control mechanical coupling between cells.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

An optochemical tool for light-induced dissociation of adherens junctions to control mechanical coupling between cells.

The cadherin-catenin complex at adherens junctions (AJs) is essential for the formation of cell-cell adhesion and epithelium integrity; however, studying the dynamic regulation of AJs at high spatio-temporal resolution remains challenging. Here we present an optochemical tool which allows reconstitution of AJs by chemical dimerization of the force bearing structures and their precise light-induced dissociation. For the dimerization, we reconstitute acto-myosin connection of a tailless E-cadherin by two ways: direct recruitment of α-catenin, and linking its cytosolic tail to the transmembrane domain. Our approach enables a specific ON-OFF switch for mechanical coupling between cells that can be controlled spatially on subcellular or tissue scale via photocleavage. The combination with cell migration analysis and traction force microscopy shows a wide-range of applicability and confirms the mechanical contribution of the reconstituted AJs. Remarkably, in vivo our tool is able to control structural and functional integrity of the epidermal layer in developing Xenopus embryos.

Testicular Vein Sampling Can Reveal Gonadotropin-Independent Unilateral Steroidogenesis Supporting Spermatogenesis.

Suppressed gonadotropins combined with high-normal serum testosterone concentrations in oligozoospermic men suggest either use of exogenous testosterone or presence of a testosterone-producing tumor. We describe the case of a 31-year-old man referred for primary infertility. Gonadotropins were undetectably low, but testosterone and estradiol were in the high-normal range. Semen analysis showed oligoasthenospermia. He denied using exogenous testosterone. Scrotal ultrasound showed microlithiasis and millimetric hypolucent lesions in the left testis but no intratesticular mass. Human chorionic gonadotropin was low. To investigate unilateral hormone secretion, selective testicular venous sampling was performed. Testosterone and estradiol were clearly higher on the left side than on the right (130 vs 26 nmol/L and 1388 vs 62 pmol/L, respectively), with a left spermatic vein-to-periphery gradient of 4.3 for testosterone and 13 for estradiol; there were no similar gradients on the right side. This finding confirms that all sex steroid secretion came from the left testis. The patient was therefore referred for left orchidectomy. Histopathology revealed multifocal seminoma, germ cell neoplasia in situ, and Leydig cell hyperplasia but no choriocarcinoma. However, gonadotrophin levels increased after orchidectomy, indicating that the source of gonadotropin-independent sex steroid secretion was removed. Testosterone and estradiol decreased to the mid-normal range. Sperm concentration improved. This report thus shows that endogenous testosterone secretion in one testicle supports spermatogenesis without measurable levels of gonadotropins. Selective testicular venous sampling is useful to identify the site of unilateral secretion when the clinical picture is inconclusive. However, histopathology could not reveal the factor that stimulated Leydig cell steroidogenesis.

Myosin II isoforms play distinct roles in adherens junction biogenesis.

Adherens junction (AJ) assembly under force is essential for many biological processes like epithelial monolayer bending, collective cell migration, cell extrusion and wound healing. The acto-myosin cytoskeleton acts as a major force-generator during the de novo formation and remodeling of AJ. Here, we investigated the role of non-muscle myosin II isoforms (NMIIA and NMIIB) in epithelial junction assembly. NMIIA and NMIIB differentially regulate biogenesis of AJ through association with distinct actin networks. Analysis of junction dynamics, actin organization, and mechanical forces of control and knockdown cells for myosins revealed that NMIIA provides the mechanical tugging force necessary for cell-cell junction reinforcement and maintenance. NMIIB is involved in E-cadherin clustering, maintenance of a branched actin layer connecting E-cadherin complexes and perijunctional actin fibres leading to the building-up of anisotropic stress. These data reveal unanticipated complementary functions of NMIIA and NMIIB in the biogenesis and integrity of AJ.

Sustained Oscillations of Epithelial Cell Sheets.

Morphological changes during development, tissue repair, and disease largely rely on coordinated cell movements and are controlled by the tissue environment. Epithelial cell sheets are often subjected to large-scale deformation during tissue formation. The active mechanical environment in which epithelial cells operate have the ability to promote collective oscillations, but how these cellular movements are generated and relate to collective migration remains unclear. Here, combining in vitro experiments and computational modeling, we describe a form of collective oscillations in confined epithelial tissues in which the oscillatory motion is the dominant contribution to the cellular movements. We show that epithelial cells exhibit large-scale coherent oscillations when constrained within micropatterns of varying shapes and sizes and that their period and amplitude are set by the smallest confinement dimension. Using molecular perturbations, we then demonstrate that force transmission at cell-cell junctions and its coupling to cell polarity are pivotal for the generation of these collective movements. We find that the resulting tissue deformations are sufficient to trigger osillatory mechanotransduction of YAP within cells, potentially affecting a wide range of cellular processes.

Collective stresses drive competition between monolayers of normal and Ras-transformed cells.

We study the competition for space between two cell lines that differ only in the expression of the Ras oncogene. The two cell populations are initially separated and set to migrate antagonistically towards an in-between stripe of free substrate. After contact, their interface moves towards the population of normal cells. We interpret the velocity and traction force data taken before and after contact thanks to a hydrodynamic description of collectively migrating cohesive cell sheets. The kinematics of cells, before and after contact, allows us to estimate the relative material parameters for both cell lines. As predicted by the model, the transformed cell population with larger collective stresses pushes the wild type cell population.

A Mechanosensitive RhoA Pathway that Protects Epithelia against Acute Tensile Stress.

Adherens junctions are tensile structures that couple epithelial cells together. Junctional tension can arise from cell-intrinsic application of contractility or from the cell-extrinsic forces of tissue movement. Here, we report a mechanosensitive signaling pathway that activates RhoA at adherens junctions to preserve epithelial integrity in response to acute tensile stress. We identify Myosin VI as the force sensor, whose association with E-cadherin is enhanced when junctional tension is increased by mechanical monolayer stress. Myosin VI promotes recruitment of the heterotrimeric Gα12 protein to E-cadherin, where it signals for p114 RhoGEF to activate RhoA. Despite its potential to stimulate junctional actomyosin and further increase contractility, tension-activated RhoA signaling is necessary to preserve epithelial integrity. This is explained by an increase in tensile strength, especially at the multicellular vertices of junctions, that is due to mDia1-mediated actin assembly.

Kalman Inversion Stress Microscopy.

Although mechanical cues are crucial to tissue morphogenesis and development, the tissue mechanical stress field remains poorly characterized. Given traction force time-lapse movies, as obtained by traction force microscopy of in vitro cellular sheets, we show that the tissue stress field can be estimated by Kalman filtering. After validation using numerical data, we apply Kalman inversion stress microscopy to experimental data. We combine the inferred stress field with velocity and cell-shape measurements to quantify the rheology of epithelial cell monolayers in physiological conditions, found to be close to that of an elastic and active material.

Cell growth, division, and death in cohesive tissues: A thermodynamic approach.

Cell growth, division, and death are defining features of biological tissues that contribute to morphogenesis. In hydrodynamic descriptions of cohesive tissues, their occurrence implies a nonzero rate of variation of cell density. We show how linear nonequilibrium thermodynamics allows us to express this rate as a combination of relevant thermodynamic forces: chemical potential, velocity divergence, and activity. We illustrate the resulting effects of the nonconservation of cell density on simple examples inspired by recent experiments on cell monolayers, considering first the velocity of a spreading front, and second an instability leading to mechanical waves.

From cells to tissue: A continuum model of epithelial mechanics.

A two-dimensional continuum model of epithelial tissue mechanics was formulated using cellular-level mechanical ingredients and cell morphogenetic processes, including cellular shape changes and cellular rearrangements. This model incorporates stress and deformation tensors, which can be compared with experimental data. Focusing on the interplay between cell shape changes and cell rearrangements, we elucidated dynamical behavior underlying passive relaxation, active contraction-elongation, and tissue shear flow, including a mechanism for contraction-elongation, whereby tissue flows perpendicularly to the axis of cell elongation. This study provides an integrated scheme for the understanding of the orchestration of morphogenetic processes in individual cells to achieve epithelial tissue morphogenesis.

Emergence of epithelial cell density waves.

Epithelial cell monolayers exhibit traveling mechanical waves. We rationalize this observation thanks to a hydrodynamic description of the monolayer as a compressible, active and polar material. We show that propagating waves of the cell density, polarity, velocity and stress fields may be due to a Hopf bifurcation occurring above threshold values of active coupling coefficients.

Topological defects in epithelia govern cell death and extrusion.

Epithelial tissues (epithelia) remove excess cells through extrusion, preventing the accumulation of unnecessary or pathological cells. The extrusion process can be triggered by apoptotic signalling, oncogenic transformation and overcrowding of cells. Despite the important linkage of cell extrusion to developmental, homeostatic and pathological processes such as cancer metastasis, its underlying mechanism and connections to the intrinsic mechanics of the epithelium are largely unexplored. We approach this problem by modelling the epithelium as an active nematic liquid crystal (that has a long range directional order), and comparing numerical simulations to strain rate and stress measurements within monolayers of MDCK (Madin Darby canine kidney) cells. Here we show that apoptotic cell extrusion is provoked by singularities in cell alignments in the form of comet-shaped topological defects. We find a universal correlation between extrusion sites and positions of nematic defects in the cell orientation field in different epithelium types. The results confirm the active nematic nature of epithelia, and demonstrate that defect-induced isotropic stresses are the primary precursors of mechanotransductive responses in cells, including YAP (Yes-associated protein) transcription factor activity, caspase-3-mediated cell death, and extrusions. Importantly, the defect-driven extrusion mechanism depends on intercellular junctions, because the weakening of cell-cell interactions in an α-catenin knockdown monolayer reduces the defect size and increases both the number of defects and extrusion rates, as is also predicted by our model. We further demonstrate the ability to control extrusion hotspots by geometrically inducing defects through microcontact printing of patterned monolayers. On the basis of these results, we propose a mechanism for apoptotic cell extrusion: spontaneously formed topological defects in epithelia govern cell fate. This will be important in predicting extrusion hotspots and dynamics in vivo, with potential applications to tissue regeneration and the suppression of metastasis. Moreover, we anticipate that the analogy between the epithelium and active nematic liquid crystals will trigger further investigations of the link between cellular processes and the material properties of epithelia.