Response of two temperate scleractinian corals to projected ocean warming and marine heatwaves.

The Mediterranean Sea is a hotspot of global change, particularly exposed to ocean warming and the increasing occurrence of marine heatwaves (MHWs). However, experiments based on long-term temperature data from the field are scarce. Here, we investigate the response of the zooxanthellate coral Cladocora caespitosa and the azooxanthellate coral Astroides calycularis to future warming and MHWs based on 8 years of in situ data. Corals were maintained in the laboratory for five months under four temperature conditions: Warming (3.2°C above the in situ mean from 2012 to 2020), Heatwave (temperatures of 2018 with two heatwaves), Ambient (in situ mean) and Cool (deeper water temperatures). Under the Warming treatment, some C. caespitosa colonies severely bleached and A. calycularis colonies presented necrosis. Cladocora caespitosa symbiosis was impaired by temperature with a decrease in the density of endosymbiotic algae and an increase in per cent whiteness in all the treatments except for the coolest. Recovery for both species was observed through different mechanisms such as regrowth of polyps of A. calycularis and recovery of pigmentation for C. caespitosa. These results suggest that A. calycularis and C. caespitosa may be resilient to heat stress and can recover from physiological stresses caused by heatwaves in the laboratory.

Reduction of cortical pulling at mitotic entry facilitates aster centration.

Equal cell division relies upon astral microtubule-based centering mechanisms, yet how the interplay between mitotic entry, cortical force generation and long astral microtubules leads to symmetric cell division is not resolved. We report that a cortically located sperm aster displaying long astral microtubules that penetrate the whole zygote does not undergo centration until mitotic entry. At mitotic entry, we find that microtubule-based cortical pulling is lost. Quantitative measurements of cortical pulling and cytoplasmic pulling together with physical simulations suggested that a wavelike loss of cortical pulling at mitotic entry leads to aster centration based on cytoplasmic pulling. Cortical actin is lost from the cortex at mitotic entry coincident with a fall in cortical tension from ∼300pN/µm to ∼100pN/µm. Following the loss of cortical force generators at mitotic entry, long microtubule-based cytoplasmic pulling is sufficient to displace the aster towards the cell center. These data reveal how mitotic aster centration is coordinated with mitotic entry in chordate zygotes.

The -KTS splice variant of WT1 is essential for ovarian determination in mice.

Sex determination in mammals depends on the differentiation of the supporting lineage of the gonads into Sertoli or pregranulosa cells that govern testis and ovary development, respectively. Although the Y-linked testis-determining gene Sry has been identified, the ovarian-determining factor remains unknown. In this study, we identified -KTS, a major, alternatively spliced isoform of the Wilms tumor suppressor WT1, as a key determinant of female sex determination. Loss of -KTS variants blocked gonadal differentiation in mice, whereas increased expression, as found in Frasier syndrome, induced precocious differentiation of ovaries independently of their genetic sex. In XY embryos, this antagonized Sry expression, resulting in male-to-female sex reversal. Our results identify -KTS as an ovarian-determining factor and demonstrate that its time of activation is critical in gonadal sex differentiation.

What Is the Best Candidate to Replace the Tarsus? A Biomechanical, Histological, and Optical Study Comparing Five Grafts.

Purpose: Reconstruction of the posterior lamella after eyelid tumor removal is challenging and not consensual. Tarsus is the most suitable graft, but is only available in small amounts. Herein, we aim to determine the most appropriate way to replace the tarsus by comparing the biomechanical, histological, and optical properties of five commonly used grafts. Methods: This study was conducted at the University hospital of Nice between June 2019 and June 2020. Five posterior lamella grafts (tarsus, conchal cartilage, sclera, hard palate, and dermis) were harvested in five fresh frozen cadavers. Biomechanical properties were assessed by tractometry. Collagen and elastin fibers were analyzed by using histological analysis and optical characterization with the second harmonic generation imaging. Results: The mean Young's modulus was 8.92 MPa (range, 2.90-22.90 MPa), 1.05 MPa (range, 0.39-1.76 MPa), 8.72 MPa (range, 2.0-23.50 MPa), 2.57 MPa (range, 0.41-4.35 MPa), and 1.44 MPa (range, 0.71-2.30 MPa) for the tarsus, the conchal cartilage, the sclera, the hard palate mucosa, and the dermis, respectively. The mean tensile strength was 3 MPa (range, 1.70-6.88 MPa), 0.54 MPa (range, 0.13-0.79 MPa), 2.87 MPa (range, 1.23-5.40 MPa), 1.4 MPa (range, 0.21-2.40 MPa) and 1.0 MPa (range, 0.46-1.43 MPa) for the tarsus, the conchal cartilage, the sclera, the hard palate mucosa, and the dermis, respectively. Hard palate mucosa was the closest to the tarsus regarding the ratio of elastin and collagen fibers. The average second harmonic generation intensity was 221 arbitrary units (a.u.) (range, 165-362 a.u.), 182 a.u. (range, 35-259 a.u.), 369 a.u. (range, 206-533 a.u.), 108 a.u. (range, 34-208 a.u.), and 244 a.u. (range, 195-388 a.u.) for the tarsus, the conchal cartilage, the sclera, the hard palate mucosa, and the dermis, respectively. The hard palate mucosa and the dermis were the closest to the tarsus regarding the collagen fiber size and orientation, respectively. Conclusions: By attributing 2 points for each characteristic (biomechanical, histological, and optical), the hard palate mucosa and the sclera seem to be the most suitable grafts to replace the tarsus. Translational Relevance: The aim of this article was to assess the biomechanical, histological and optical characteristics of five of the most commonly used tarsal grafts; this may be helpful in decisions for clinical practice.

Regulation of the collagen IV network by the basement membrane protein perlecan is crucial for squamous epithelial cell morphogenesis and organ architecture.

All epithelia have their basal side in contact with a specialized extracellular matrix, the basement membrane (BM). During development, the BM contributes to the shaping of epithelial organs via its mechanical properties. These properties rely on two core components of the BM, collagen type IV and perlecan/HSPG2, which both interact with another core component, laminin, the initiator of BM assembly. While collagen type IV supplies the BM with rigidity to constrain the tissue, perlecan antagonizes this effect. Nevertheless, the number of organs that has been studied is still scarce, and given that epithelial tissues exhibit a wide array of shapes, their forms are bound to be regulated by distinct mechanisms. This is underscored by mounting evidence that BM composition and assembly/biogenesis is tissue-specific. Moreover, previous reports have essentially focused on the mechanical role of the BM in morphogenesis at the tissue scale, but not the cell scale. Here, we took advantage of the robust conservation of core BM proteins and the limited genetic redundancy of the Drosophila model system to address how this matrix shapes the wing imaginal disc, a complex organ comprising a squamous, a cuboidal and a columnar epithelium. With the use of a hypomorphic allele, we show that the depletion of Trol (Drosophila perlecan) affects the morphogenesis of the three epithelia, but particularly that of the squamous one. The planar surface of the squamous epithelium (SE) becomes extremely narrow, due to a function for Trol in the control of the squamous shape of its cells. Furthermore, we find that the lack of Trol impairs the biogenesis of the BM of the SE by modifying the structure of the collagen type IV lattice. Through atomic force microscopy and laser surgery, we demonstrate that Trol provides elasticity to the SE's BM, thereby regulating the mechanical properties of the SE. Moreover, we show that Trol acts via collagen type IV, since the global reduction in the trol mutant context of collagen type IV or the enzyme that cross-links its 7S -but not the enzyme that cross-links its NC1- domain substantially restores the morphogenesis of the SE. In addition, a stronger decrease in collagen type IV achieved by the overexpression of the matrix metalloprotease 2 exclusively in the BM of the SE, significantly rescues the organization of the two other epithelia. Our data thus sustain a model in which Trol counters the rigidity conveyed by collagen type IV to the BM of the SE, via the regulation of the NC1-dependant assembly of its scaffold, allowing the spreading of the squamous cells, spreading which is compulsory for the architecture of the whole organ.

COL0RME: Super-resolution microscopy based on sparse blinking/fluctuating fluorophore localization and intensity estimation.

To overcome the physical barriers caused by light diffraction, super-resolution techniques are often applied in fluorescence microscopy. State-of-the-art approaches require specific and often demanding acquisition conditions to achieve adequate levels of both spatial and temporal resolution. Analyzing the stochastic fluctuations of the fluorescent molecules provides a solution to the aforementioned limitations, as sufficiently high spatio-temporal resolution for live-cell imaging can be achieved using common microscopes and conventional fluorescent dyes. Based on this idea, we present COL0RME, a method for covariance-based super-resolution microscopy with intensity estimation, which achieves good spatio-temporal resolution by solving a sparse optimization problem in the covariance domain and discuss automatic parameter selection strategies. The method is composed of two steps: the former where both the emitters' independence and the sparse distribution of the fluorescent molecules are exploited to provide an accurate localization; the latter where real intensity values are estimated given the computed support. The paper is furnished with several numerical results both on synthetic and real fluorescence microscopy images and several comparisons with state-of-the art approaches are provided. Our results show that COL0RME outperforms competing methods exploiting analogously temporal fluctuations; in particular, it achieves better localization, reduces background artifacts, and avoids fine parameter tuning.

Proteomic analysis identified LBP and CD14 as key proteins in blood/biphasic calcium phosphate microparticle interactions.

Immediately upon implantation, scaffolds for bone repair are exposed to the patient's blood. Blood proteins adhere to the biomaterial surface and the protein layer affects both blood cell functions and biomaterial bioactivity. Previously, we reported that 80-200 µm biphasic calcium phosphate (BCP) microparticles embedded in a blood clot, induce ectopic woven bone formation in mice, when 200-500 µm BCP particles induce mainly fibrous tissue. Here, in a LC-MS/MS proteomic study we compared the differentially expressed blood proteins (plasma and blood cell proteins) and the deregulated signaling pathways of these osteogenic and fibrogenic blood composites. We showed that blood/BCP-induced osteogenesis is associated with a higher expression of fibrinogen (FGN) and an upregulation of the Myd88- and NF-κB-dependent TLR4 signaling cascade. We also highlighted the key role of the LBP/CD14 proteins in the TLR4 activation of blood cells by BCP particles. As FGN is an endogenous ligand of TLR4, able to modulate blood composite stiffness, we propose that different FGN concentrations modify the blood clot mechanical properties, which in turn modulate BCP/blood composite osteoactivity through TLR4 signaling. The present findings provide an insight at the protein level, into the mechanisms leading to an efficient bone reconstruction by blood/BCP composites. STATEMENT OF SIGNIFICANCE: Upon implantation, scaffolds for bone repair are exposed to the patient's blood. Blood proteins adhere to bone substitute surface and this protein layer affects both biomaterial bioactivity and bone healing. Therefore, for the best outcome for patients, it is crucial to understand the molecular interactions between blood and bone scaffolds. Biphasic calcium phosphate (BCP) ceramics are considered as the gold standard in bone reconstruction surgery. Here, using proteomic analyses we showed that the osteogenic properties of 80-200 µm BCP particles embedded in a blood clot is associated with a higher expression of fibrinogen. Fibrinogen upregulates the Myd88- and NF-κB-dependent TLR4 pathway in blood cells and, BCP-induced TLR4 activation is mediated by the LBP and CD14 proteins.

Fibronectin Extra Domains tune cellular responses and confer topographically distinct features to fibril networks.

Cellular fibronectin (FN; also known as FN1) variants harboring one or two alternatively spliced so-called extra domains (EDB and EDA) play a central bioregulatory role during development, repair processes and fibrosis. Yet, how the extra domains impact fibrillar assembly and function of the molecule remains unclear. Leveraging a unique biological toolset and image analysis pipeline for direct comparison of the variants, we demonstrate that the presence of one or both extra domains impacts FN assembly, function and physical properties of the matrix. When presented to FN-null fibroblasts, extra domain-containing variants differentially regulate pH homeostasis, survival and TGF-ß signaling by tuning the magnitude of cellular responses, rather than triggering independent molecular switches. Numerical analyses of fiber topologies highlight significant differences in variant-specific structural features and provide a first step for the development of a generative model of FN networks to unravel assembly mechanisms and investigate the physical and functional versatility of extracellular matrix landscapes.This article has an associated First Person interview with the first author of the paper.

Mechanical force-induced morphology changes in a human fungal pathogen.

BACKGROUND: The initial step of a number of human or plant fungal infections requires active penetration of host tissue. For example, active penetration of intestinal epithelia by Candida albicans is critical for dissemination from the gut into the bloodstream. However, little is known about how this fungal pathogen copes with resistive forces upon host cell invasion. RESULTS: In the present study, we have used PDMS micro-fabrication to probe the ability of filamentous C. albicans cells to penetrate and grow invasively in substrates of different stiffness. We show that there is a threshold for penetration that corresponds to a stiffness of ~ 200 kPa and that invasive growth within a stiff substrate is characterized by dramatic filament buckling, along with a stiffness-dependent decrease in extension rate. We observed a striking alteration in cell morphology, i.e., reduced cell compartment length and increased diameter during invasive growth, that is not due to depolarization of active Cdc42, but rather occurs at a substantial distance from the site of growth as a result of mechanical compression. CONCLUSIONS: Our data reveal that in response to this compression, active Cdc42 levels are increased at the apex, whereas active Rho1 becomes depolarized, similar to that observed in membrane protrusions. Our results show that cell growth and morphology are altered during invasive growth, suggesting stiffness dictates the host cells that C. albicans can penetrate.

A Feed-Forward Mechanosignaling Loop Confers Resistance to Therapies Targeting the MAPK Pathway in BRAF-Mutant Melanoma.

Aberrant extracellular matrix (ECM) deposition and stiffening is a physical hallmark of several solid cancers and is associated with therapy failure. BRAF-mutant melanomas treated with BRAF and MEK inhibitors almost invariably develop resistance that is frequently associated with transcriptional reprogramming and a de-differentiated cell state. Melanoma cells secrete their own ECM proteins, an event that is promoted by oncogenic BRAF inhibition. Yet, the contribution of cancer cell-derived ECM and tumor mechanics to drug adaptation and therapy resistance remains poorly understood. Here, we show that melanoma cells can adapt to targeted therapies through a mechanosignaling loop involving the autocrine remodeling of a drug-protective ECM. Analyses revealed that therapy-resistant cells associated with a mesenchymal dedifferentiated state displayed elevated responsiveness to collagen stiffening and force-mediated ECM remodeling through activation of actin-dependent mechanosensors Yes-associated protein (YAP) and myocardin-related transcription factor (MRTF). Short-term inhibition of MAPK pathway also induced mechanosignaling associated with deposition and remodeling of an aligned fibrillar matrix. This provided a favored ECM reorganization that promoted tolerance to BRAF inhibition in a YAP- and MRTF-dependent manner. Matrix remodeling and tumor stiffening were also observed in vivo upon exposure of BRAF-mutant melanoma cell lines or patient-derived xenograft models to MAPK pathway inhibition. Importantly, pharmacologic targeting of YAP reversed treatment-induced excessive collagen deposition, leading to enhancement of BRAF inhibitor efficacy. We conclude that MAPK pathway targeting therapies mechanically reprogram melanoma cells to confer a drug-protective matrix environment. Preventing melanoma cell mechanical reprogramming might be a promising therapeutic strategy for patients on targeted therapies. SIGNIFICANCE: These findings reveal a biomechanical adaptation of melanoma cells to oncogenic BRAF pathway inhibition, which fuels a YAP/MRTF-dependent feed-forward loop associated with tumor stiffening, mechanosensing, and therapy resistance. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/10/1927/F1.large.jpg.

In vitro characterization of a novel magnetic fibrin-agarose hydrogel for cartilage tissue engineering.

The encapsulation of cells into biopolymer matrices enables the preparation of engineered substitute tissues. Here we report the generation of novel 3D magnetic biomaterials by encapsulation of magnetic nanoparticles and human hyaline chondrocytes within fibrin-agarose hydrogels, with potential use as articular hyaline cartilage-like tissues. By rheological measurements we observed that, (i) the incorporation of magnetic nanoparticles resulted in increased values of the storage and loss moduli for the different times of cell culture; and (ii) the incorporation of human hyaline chondrocytes into nonmagnetic and magnetic fibrin-agarose biomaterials produced a control of their swelling capacity in comparison with acellular nonmagnetic and magnetic fibrin-agarose biomaterials. Interestingly, the in vitro viability and proliferation results showed that the inclusion of magnetic nanoparticles did not affect the cytocompatibility of the biomaterials. What is more, immunohistochemistry showed that the inclusion of magnetic nanoparticles did not negatively affect the expression of type II collagen of the human hyaline chondrocytes. Summarizing, our results suggest that the generation of engineered hyaline cartilage-like tissues by using magnetic fibrin-agarose hydrogels is feasible. The resulting artificial tissues combine a stronger and stable mechanical response, with promising in vitro cytocompatibility. Further research would be required to elucidate if for longer culture times additional features typical of the extracellular matrix of cartilage could be expressed by human hyaline chondrocytes within magnetic fibrin-agarose hydrogels.

NK Cell and Fibroblast-Mediated Regulation of Skin Squamous Cell Carcinoma Invasion by CLEC2A Is Compromised in Xeroderma Pigmentosum.

The ability of cancer cells to invade and disseminate can be affected by components of the surrounding microenvironment. To identify dermal components that regulate the growth of epidermal carcinomas, we studied the genetic disease called xeroderma pigmentosum that bears mutations in genes involved in the nucleotide excision repair of DNA. Patients with xeroderma pigmentosum are more prone to develop cutaneous tumors than the general population and their dermal fibroblasts display the features of dermal cancer-associated fibroblasts, which promote the invasion of keratinocytes. Here, we report that 3-dimensional dermal cultures of fibroblasts from healthy donors but not from patients with xeroderma pigmentosum complementation group C express CLEC2A, which is the ligand of the activating NK cell receptor NKp65. A similar loss of CLEC2A was observed in sporadic dermal cancer-associated fibroblasts and upon the culture of fibroblasts with cutaneous squamous cell carcinoma-conditioned medium. Using an innovative 3-dimensional organotypic skin culture model that contain NK cells in addition to fibroblasts and squamous cell carcinoma cells, we unveiled a key role of CLEC2A that orchestrates a crosstalk between fibroblasts and NK cells, thereby leading to the control of squamous cell carcinoma invasion. These findings indicate that CLEC2A-expressing dermal fibroblasts play a major role in immune surveillance of the skin.

Quantifying Tubulin Concentration and Microtubule Number Throughout the Fission Yeast Cell Cycle.

The fission yeast Schizosaccharomycespombe serves as a good genetic model organism for the molecular dissection of the microtubule (MT) cytoskeleton. However, analysis of the number and distribution of individual MTs throughout the cell cycle, particularly during mitosis, in living cells is still lacking, making quantitative modelling imprecise. We use quantitative fluorescent imaging and analysis to measure the changes in tubulin concentration and MT number and distribution throughout the cell cycle at a single MT resolution in living cells. In the wild-type cell, both mother and daughter spindle pole body (SPB) nucleate a maximum of 23 ± 6 MTs at the onset of mitosis, which decreases to a minimum of 4 ± 1 MTs at spindle break down. Interphase MT bundles, astral MT bundles, and the post anaphase array (PAA) microtubules are composed primarily of 1 ± 1 individual MT along their lengths. We measure the cellular concentration of αß-tubulin subunits to be ~5 µM throughout the cell cycle, of which one-third is in polymer form during interphase and one-quarter is in polymer form during mitosis. This analysis provides a definitive characterization of αß-tubulin concentration and MT number and distribution in fission yeast and establishes a foundation for future quantitative comparison of mutants defective in MTs.

Nanometric axial resolution of fibronectin assembly units achieved with an efficient reconstruction approach for multi-angle-TIRF microscopy.

High resolution imaging of molecules at the cell-substrate interface is required for understanding key biological processes. Here we propose a complete pipeline for multi-angle total internal reflection fluorescence microscopy (MA-TIRF) going from instrument design and calibration procedures to numerical reconstruction. Our custom setup is endowed with a homogeneous field illumination and precise excitation beam angle. Given a set of MA-TIRF acquisitions, we deploy an efficient joint deconvolution/reconstruction algorithm based on a variational formulation of the inverse problem. This algorithm offers the possibility of using various regularizations and can run on graphics processing unit (GPU) for rapid reconstruction. Moreover, it can be easily used with other MA-TIRF devices and we provide it as an open-source software. This ensemble has enabled us to visualize and measure with unprecedented nanometric resolution, the depth of molecular components of the fibronectin assembly machinery at the basal surface of endothelial cells.

Migraine-Associated TRESK Mutations Increase Neuronal Excitability through Alternative Translation Initiation and Inhibition of TREK.

It is often unclear why some genetic mutations to a given gene contribute to neurological disorders and others do not. For instance, two mutations have previously been found to produce a dominant negative for TRESK, a two-pore-domain K+ channel implicated in migraine: TRESK-MT, a 2-bp frameshift mutation, and TRESK-C110R. Both mutants inhibit TRESK, but only TRESK-MT increases sensory neuron excitability and is linked to migraine. Here, we identify a new mechanism, termed frameshift mutation-induced alternative translation initiation (fsATI), that may explain why only TRESK-MT is associated with migraine. fsATI leads to the production of a second protein fragment, TRESK-MT2, which co-assembles with and inhibits TREK1 and TREK2, two other two-pore-domain K+ channels, to increase trigeminal sensory neuron excitability, leading to a migraine-like phenotype in rodents. These findings identify TREK1 and TREK2 as potential molecular targets in migraine and suggest that fsATI should be considered as a distinct class of mutations.

Gallium enhances reconstructive properties of a calcium phosphate bone biomaterial.

Calcium phosphate (CaP)-based biomaterials are commonly used in bone reconstructive surgery to replace the damaged tissue, and can also serve as vectors for local drug delivery. Due to its inhibitory action on osteoclasts, the semi-metallic element gallium (Ga) is used for the systemic treatment of disorders associated with accelerated bone resorption. As it was demonstrated that Ga could be incorporated in the structure of CaP biomaterials, we investigated the biological properties of Ga-loaded CaP biomaterials. Culturing bone cells on Ga-CaP, we observed a decrease in osteoclast number and a downregulation of late osteoclastic markers expression, while Ga-CaP upregulated the expression of osteoblastic marker genes involved in the maturation of bone matrix. We next investigated in vivo bone reconstructive properties of different Ga-loaded biomaterials using a murine bone defect healing model. All implanted biomaterials showed a good osseointegration into the surrounding host tissue, accompanied by a successful bone ingrowth and bone marrow reconstruction, as evidenced by histological analysis. Moreover, quantitative micro-computed tomography analysis of implants revealed that Ga enhanced total defect filling. Lastly, we took advantage for the first time of a particular mode of non-linear microscopy (second harmonic generation) to quantify in vivo bone tissue reconstruction within a CaP bone substitute. By doing so, we showed that Ga exerted a positive impact on mature organized collagen synthesis. As a whole, our data support the hypothesis that Ga represents an attractive additive to CaP biomaterials for bone reconstructive surgery. Copyright © 2017 John Wiley & Sons, Ltd.

Counterbalancing anti-adhesive effects of Tenascin-C through fibronectin expression in endothelial cells.

Cellular fibronectin (FN) and tenascin-C (TNC) are prominent development- and disease-associated matrix components with pro- and anti-adhesive activity, respectively. Whereas both are present in the tumour vasculature, their functional interplay on vascular endothelial cells remains unclear. We have previously shown that basally-oriented deposition of a FN matrix restricts motility and promotes junctional stability in cultured endothelial cells and that this effect is tightly coupled to expression of FN. Here we report that TNC induces FN expression in endothelial cells. This effect counteracts the potent anti-adhesive activity of TNC and leads to the assembly of a dense highly-branched subendothelial matrix that enhances tubulogenic activity. These findings suggest that pro-angiogenic remodelling of the perivascular matrix may involve TNC-induced upregulation of FN in endothelial cells.

Signalling crosstalk at the leading edge controls tissue closure dynamics in the Drosophila embryo.

Tissue morphogenesis relies on proper differentiation of morphogenetic domains, adopting specific cell behaviours. Yet, how signalling pathways interact to determine and coordinate these domains remains poorly understood. Dorsal closure (DC) of the Drosophila embryo represents a powerful model to study epithelial cell sheet sealing. In this process, JNK (JUN N-terminal Kinase) signalling controls leading edge (LE) differentiation generating local forces and cell shape changes essential for DC. The LE represents a key morphogenetic domain in which, in addition to JNK, a number of signalling pathways converges and interacts (anterior/posterior -AP- determination; segmentation genes, such as Wnt/Wingless; TGFß/Decapentaplegic). To better characterize properties of the LE morphogenetic domain, we sought out new JNK target genes through a genomic approach: 25 were identified of which 8 are specifically expressed in the LE, similarly to decapentaplegic or puckered. Quantitative in situ gene profiling of this new set of LE genes reveals complex patterning of the LE along the AP axis, involving a three-way interplay between the JNK pathway, segmentation and HOX genes. Patterning of the LE into discrete domains appears essential for coordination of tissue sealing dynamics. Loss of anterior or posterior HOX gene function leads to strongly delayed and asymmetric DC, due to incorrect zipping in their respective functional domain. Therefore, in addition to significantly increasing the number of JNK target genes identified so far, our results reveal that the LE is a highly heterogeneous morphogenetic organizer, sculpted through crosstalk between JNK, segmental and AP signalling. This fine-tuning regulatory mechanism is essential to coordinate morphogenesis and dynamics of tissue sealing.

Fibronectin-guided migration of carcinoma collectives.

Functional interplay between tumour cells and their neoplastic extracellular matrix plays a decisive role in malignant progression of carcinomas. Here we provide a comprehensive data set of the human HNSCC-associated fibroblast matrisome. Although much attention has been paid to the deposit of collagen, we identify oncofetal fibronectin (FN) as a major and obligate component of the matrix assembled by stromal fibroblasts from head and neck squamous cell carcinomas (HNSCC). FN overexpression in tumours from 435 patients corresponds to an independent unfavourable prognostic indicator. We show that migration of carcinoma collectives on fibrillar FN-rich matrices is achieved through αvß6 and α9ß1 engagement, rather than α5ß1. Moreover, αvß6-driven migration occurs independently of latent TGF-ß activation and Smad-dependent signalling in tumour epithelial cells. These results provide insights into the adhesion-dependent events at the tumour-stroma interface that govern the collective mode of migration adopted by carcinoma cells to invade surrounding stroma in HNSCC.

Heterodimerization within the TREK channel subfamily produces a diverse family of highly regulated potassium channels.

Twik-related K(+) channel 1 (TREK1), TREK2, and Twik-related arachidonic-acid stimulated K(+) channel (TRAAK) form the TREK subfamily of two-pore-domain K(+) (K2P) channels. Despite sharing up to 78% sequence homology and overlapping expression profiles in the nervous system, these channels show major differences in their regulation by physiological stimuli. For instance, TREK1 is inhibited by external acidification, whereas TREK2 is activated. Here, we investigated the ability of the members of the TREK subfamily to assemble to form functional heteromeric channels with novel properties. Using single-molecule pull-down (SiMPull) from HEK cell lysate and subunit counting in the plasma membrane of living cells, we show that TREK1, TREK2, and TRAAK readily coassemble. TREK1 and TREK2 can each heterodimerize with TRAAK, but do so less efficiently than with each other. We functionally characterized the heterodimers and found that all combinations form outwardly rectifying potassium-selective channels but with variable voltage sensitivity and pH regulation. TREK1-TREK2 heterodimers show low levels of activity at physiological external pH but, unlike their corresponding homodimers, are activated by both acidic and alkaline conditions. Modeling based on recent crystal structures, along with mutational analysis, suggests that each subunit within a TREK1-TREK2 channel is regulated independently via titratable His. Finally, TREK1/TRAAK heterodimers differ in function from TRAAK homodimers in two critical ways: they are activated by both intracellular acidification and alkalinization and are regulated by the enzyme phospholipase D2. Thus, heterodimerization provides a means for diversifying functionality through an expansion of the channel types within the K2P channels.