DHA-containing phospholipids control membrane fusion and transcellular tunnel dynamics.

Metabolic studies and animal knockout models point to the critical role of polyunsaturated docosahexaenoic acid (22:6, DHA)-containing phospholipids (DHA-PLs) in physiology. Here, we investigated the impact of DHA-PLs on the dynamics of transendothelial cell macroapertures (TEMs) triggered by RhoA inhibition-associated cell spreading. Lipidomic analyses showed that human umbilical vein endothelial cells (HUVECs) subjected to a DHA diet undergo a 6-fold enrichment in DHA-PLs at the plasma membrane (PM) at the expense of monounsaturated oleic acid-containing PLs (OA-PLs). Consequently, DHA-PL enrichment at the PM induces a reduction in cell thickness and shifts cellular membranes towards a permissive mode of membrane fusion for transcellular tunnel initiation. We provide evidence that a global homeostatic control of membrane tension and cell cortex rigidity minimizes overall changes of TEM area through a decrease of TEM size and lifetime. Conversely, low DHA-PL levels at the PM lead to the opening of unstable and wider TEMs. Together, this provides evidence that variations of DHA-PL levels in membranes affect cell biomechanical properties.

System-Wide Modulation of HECT E3 Ligases with Selective Ubiquitin Variant Probes.

HECT-family E3 ligases ubiquitinate protein substrates to control virtually every eukaryotic process and are misregulated in numerous diseases. Nonetheless, understanding of HECT E3s is limited by a paucity of selective and potent modulators. To overcome this challenge, we systematically developed ubiquitin variants (UbVs) that inhibit or activate HECT E3s. Structural analysis of 6 HECT-UbV complexes revealed UbV inhibitors hijacking the E2-binding site and activators occupying a ubiquitin-binding exosite. Furthermore, UbVs unearthed distinct regulation mechanisms among NEDD4 subfamily HECTs and proved useful for modulating therapeutically relevant targets of HECT E3s in cells and intestinal organoids, and in a genetic screen that identified a role for NEDD4L in regulating cell migration. Our work demonstrates versatility of UbVs for modulating activity across an E3 family, defines mechanisms and provides a toolkit for probing functions of HECT E3s, and establishes a general strategy for systematic development of modulators targeting families of signaling proteins.

Characterization of a highly neutralizing single monoclonal antibody to botulinum neurotoxin type A.

Compared to conventional antisera strategies, monoclonal antibodies (mAbs) represent an alternative and safer way to treat botulism, a fatal flaccid paralysis due to botulinum neurotoxins (BoNTs). In addition, mAbs offer the advantage to be produced in a reproducible manner. We previously identified a unique and potent mouse mAb (TA12) targeting BoNT/A1 with high affinity and neutralizing activity. In this study, we characterized the molecular basis of TA12 neutralization by combining Hydrogen/Deuterium eXchange Mass Spectrometry (HDX-MS) with site-directed mutagenesis and functional studies. We found that TA12 recognizes a conformational epitope located at the interface between the HCN and HCC subdomains of the BoNT/A1 receptor-binding domain (HC ). The TA12-binding interface shares common structural features with the ciA-C2 VHH epitope and lies on the face opposite recognized by ciA-C2- and the CR1/CR2-neutralizing mAbs. The single substitution of N1006 was sufficient to affect TA12 binding to HC confirming the position of the epitope. We further uncovered that the TA12 epitope overlaps with the BoNT/A1-binding site for both the neuronal cell surface receptor synaptic vesicle glycoprotein 2 isoform C (SV2C) and the GT1b ganglioside. Hence, TA12 potently blocks the entry of BoNT/A1 into neurons by interfering simultaneously with the binding of SV2C and to a lower extent GT1b. Our study reveals the unique neutralization mechanism of TA12 and emphasizes on the potential of using single mAbs for the treatment of botulism type A.

Synergistic interaction between pH and NaCl in the limits of germination and outgrowth of Clostridium sporogenes and Group I Clostridium botulinum vegetative cells and spores after heat treatment.

Group I Clostridium botulinum and Clostridium sporogenes are physiologically and genetically closely related. Both are widely distributed in the environment and can cause foodborne botulism. In this work, a physiological study was conducted with 37 isolates from spoiled canned food and five referenced strains of C. sporogenes (three isolates) and Group I C. botulinum (two isolates). Growth limits of vegetative cells were established as a function of pH and NaCl concentration in PYG modified medium (PYGm) at 30 °C for 48 days. The heat resistance of the spores was studied for 2 min and 10 min at 102 °C and 110 °C. This physiological study (pH, NaCl growth limits and heat resistance) allowed the selection of 14 isolates of C. sporogenes (twelve isolates) and Group I C. botulinum (two isolates) representative of the diversity found. This panel of 14 selected isolates (11 isolated from spoiled canned food and three reference strains), were whole genome sequenced, but no association of physiological and genetic characteristics could be detected. Finally, we studied the ability of spores to germinate and grow from 5 isolates (four C. sporogenes and one Group I C. botulinum), under stress conditions generated by pH and NaCl following a low intensity heat treatment. The accumulation of these 3 stresses creates synergies that will strongly reduce the probability of spore growth in pH and salt conditions where they usually proliferate. The effect is progressive as the conditions become drastic: the number of decimal reduction observed increases translating a probability of growth which decreases. This study provides a better understanding of the behaviour of C. sporogenes and Group I C. botulinum isolates and shows how the combination of pH, NaCl and heat treatment can help prevent or minimise foodborne botulism outbreaks.

Cellular microbiology: Bacterial toxin interference drives understanding of eukaryotic cell function.

Intimate interactions between the armament of pathogens and their host dictate tissue and host susceptibility to infection also forging specific pathophysiological outcomes. Studying these interactions at the molecular level has provided an invaluable source of knowledge on cellular processes, as ambitioned by the Cellular Microbiology discipline when it emerged in early 90s. Bacterial toxins act on key cell regulators or membranes to produce major diseases and therefore constitute a remarkable toolbox for dissecting basic biological processes. Here, we review selected examples of recent studies on bacterial toxins illustrating how fruitful the discipline of cellular microbiology is in shaping our understanding of eukaryote processes. This ever-renewing discipline unveils new virulence factor biochemical activities shared by eukaryotic enzymes and hidden rules of cell proteome homeostasis, a particularly promising field to interrogate the impact of proteostasis breaching in late onset human diseases. It is integrating new concepts from the physics of soft matter to capture biomechanical determinants forging cells and tissues architecture. The success of this discipline is also grounded by the development of therapeutic tools and new strategies to treat both infectious and noncommunicable human diseases.

UBTD1 is a mechano-regulator controlling cancer aggressiveness.

Ubiquitin domain-containing protein 1 (UBTD1) is highly evolutionary conserved and has been described to interact with E2 enzymes of the ubiquitin-proteasome system. However, its biological role and the functional significance of this interaction remain largely unknown. Here, we demonstrate that depletion of UBTD1 drastically affects the mechanical properties of epithelial cancer cells via RhoA activation and strongly promotes their aggressiveness. On a stiff matrix, UBTD1 expression is regulated by cell-cell contacts, and the protein is associated with ß-catenin at cell junctions. Yes-associated protein (YAP) is a major cell mechano-transducer, and we show that UBTD1 is associated with components of the YAP degradation complex. Interestingly, UBTD1 promotes the interaction of YAP with its E3 ubiquitin ligase ß-TrCP Consequently, in cancer cells, UBTD1 depletion decreases YAP ubiquitylation and triggers robust ROCK2-dependent YAP activation and downstream signaling. Data from lung and prostate cancer patients further corroborate the in cellulo results, confirming that low levels of UBTD1 are associated with poor patient survival, suggesting that biological functions of UBTD1 could be beneficial in limiting cancer progression.

Conformational Insights into the Control of CNF1 Toxin Activity by Peptidyl-Prolyl Isomerization: A Molecular Dynamics Perspective.

The cytotoxic necrotizing factor 1 (CNF1) toxin from uropathogenic Escherichia coli constitutively activates Rho GTPases by catalyzing the deamidation of a critical glutamine residue located in the switch II (SWII). In crystallographic structures of the CNF1 catalytic domain (CNF1CD), surface-exposed P768 and P968 peptidyl-prolyl imide bonds (X-Pro) adopt an unusual cis conformation. Here, we show that mutation of each proline residue into glycine abrogates CNF1CD in vitro deamidase activity, while mutant forms of CNF1 remain functional on RhoA in cells. Using molecular dynamics simulations coupled to protein-peptide docking, we highlight the long-distance impact of peptidyl-prolyl cis-trans isomerization on the network of interactions between the loops bordering the entrance of the catalytic cleft. The energetically favorable isomerization of P768 compared with P968, induces an enlargement of loop L1 that fosters the invasion of CNF1CD catalytic cleft by a peptide encompassing SWII of RhoA. The connection of the P968 cis isomer to the catalytic cysteine C866 via a ladder of stacking interactions is alleviated along the cis-trans isomerization. Finally, the cis-trans conversion of P768 favors a switch of the thiol side chain of C866 from a resting to an active orientation. The long-distance impact of peptidyl-prolyl cis-trans isomerizations is expected to have implications for target modification.

Dewetting: From Physics to the Biology of Intoxicated Cells.

Pathogenic bacteria colonize or disseminate into cells and tissues by inducing large-scale remodeling of host membranes. The physical phenomena underpinning these massive membrane extension and deformation are poorly understood. Invasive strategies of pathogens have been recently enriched by the description of a spectacular mode of opening of large transendothelial cell macroaperture (TEM) tunnels correlated to the dissemination of EDIN-producing strains of Staphylococcus aureus via a hematogenous route or to the induction of gelatinous edema triggered by the edema toxin from Bacillus anthracis. Remarkably, these highly dynamic tunnels close rapidly after they reach a maximal size. Opening and closure of TEMs in cells lasts for hours without inducing endothelial cell death. Multidisciplinary studies have started to provide a broader perspective of both the molecular determinants controlling cytoskeleton organization at newly curved membranes generated by the opening of TEMs and the physical processes controlling the dynamics of these tunnels. Here we discuss the analogy between the opening of TEM tunnels and the physical principles of dewetting, stemming from a parallel between membrane tension and surface tension. This analogy provides a broad framework to investigate biophysical constraints in cell membrane dynamics and their diversion by certain invasive microbial agents.

New Aspects on Bacterial Effectors Targeting Rho GTPases.

The virulence of highly pathogenic bacteria such as Salmonella, Yersinia, Staphylococci, Clostridia, and pathogenic strains of Escherichia coli involves intimate cross-talks with the host actin cytoskeleton and its upstream regulators. A large number of virulence factors expressed by these pathogens modulate Rho GTPase activities either by mimicking cellular regulators or by catalyzing posttranslational modifications of these small proteins. This impressive convergence of virulence toward Rho GTPases and actin indeed offers pathogens the capacity to breach host defenses and invade their host, while it promotes inflammatory reactions. In return, the study of this targeting of Rho GTPases in infection has been an invaluable source of information in cell signaling, cell biology, and biomechanics, as well as in immunology. Through selected examples, I highlight the importance of recent studies on this crosstalk, which have unveiled new mechanisms of regulation of Rho GTPases; the relationship between cell shape and actin cytoskeleton organization; and the relationship between Rho GTPases and innate immune signaling.

Escherichia coli α-hemolysin counteracts the anti-virulence innate immune response triggered by the Rho GTPase activating toxin CNF1 during bacteremia.

The detection of the activities of pathogen-encoded virulence factors by the innate immune system has emerged as a new paradigm of pathogen recognition. Much remains to be determined with regard to the molecular and cellular components contributing to this defense mechanism in mammals and importance during infection. Here, we reveal the central role of the IL-1ß signaling axis and Gr1+ cells in controlling the Escherichia coli burden in the blood in response to the sensing of the Rho GTPase-activating toxin CNF1. Consistently, this innate immune response is abrogated in caspase-1/11-impaired mice or following the treatment of infected mice with an IL-1ß antagonist. In vitro experiments further revealed the synergistic effects of CNF1 and LPS in promoting the maturation/secretion of IL-1ß and establishing the roles of Rac, ASC and caspase-1 in this pathway. Furthermore, we found that the α-hemolysin toxin inhibits IL-1ß secretion without affecting the recruitment of Gr1+ cells. Here, we report the first example of anti-virulence-triggered immunity counteracted by a pore-forming toxin during bacteremia.

Autophagy is required for CSF-1-induced macrophagic differentiation and acquisition of phagocytic functions.

Autophagy is the process by which superfluous or damaged macromolecules or organelles are degraded by the lysosome. Pharmacologic and genetic evidence indicates that autophagy plays pleiotropic functions in cellular homeostasis, development, survival, and differentiation. The differentiation of human blood monocytes into macrophages is a caspase-dependent process when triggered ex vivo by colony stimulating factor-1. We show here, using pharmacologic inhibitors, siRNA approaches, and Atg7-/- mice, that autophagy initiated by ULK1 is required for proper colony stimulating factor-1-driven differentiation of human and murine monocytes. We also unravel a role for autophagy in macrophage acquisition of phagocytic functions. Collectively, these findings highlight an unexpected and essential role of autophagy during monocyte differentiation and acquisition of macrophage functions.

Transcellular tunnel dynamics: Control of cellular dewetting by actomyosin contractility and I-BAR proteins.

Dewetting is the spontaneous withdrawal of a liquid film from a non-wettable surface by nucleation and growth of dry patches. Two recent reports now propose that the principles of dewetting explain the physical phenomena underpinning the opening of transendothelial cell macroaperture (TEM) tunnels, referred to as cellular dewetting. This was discovered by studying a group of bacterial toxins endowed with the property of corrupting actomyosin cytoskeleton contractility. For both liquid and cellular dewetting, the growth of holes is governed by a competition between surface forces and line tension. We also discuss how the dynamics of TEM opening and closure represent remarkable systems to investigate actin cytoskeleton regulation by sensors of plasma membrane curvature and investigate the impact on membrane tension and the role of TEM in vascular dysfunctions.

Hijacking of Rho GTPases during bacterial infection.

Highly pathogenic bacteria, including Yersinia, Salmonella, E. coli and Clostridia, produce an amazing array of virulence factors that target Rho proteins. These pathogens exploit and/or impair many aspects of Rho protein activities by activating or inhibiting these key molecular switches. Here, we describe examples illustrating how modulation of Rho protein activity is the underlying molecular mechanism used by pathogens to disrupt host epithelial/endothelial barriers, paralyze immune cell migration and phagocytic functions, invade epithelial cells, replicate, and form reservoirs or disseminate in epithelia. Remarkably, emerging evidence points to the capacity of target cells to not only perceive the imbalance of Rho activity induced by virulence factors but also to respond by stimulating the production of anti-microbial responses that alert the host to the pathogenic threat. Furthermore, toxins that activate Rho proteins have been extremely useful in revealing the exquisite cellular regulations of these GTPases, notably by the ubiquitin and proteasome system. Finally, a number of studies indicate that toxins targeting Rho proteins have great potential in the development of new therapeutic tools.

Caveolin-1 protects endothelial cells from extensive expansion of transcellular tunnel by stiffening the plasma membrane.

Large transcellular pores elicited by bacterial mono-ADP-ribosyltransferase (mART) exotoxins inhibiting the small RhoA GTPase compromise the endothelial barrier. Recent advances in biophysical modeling point toward membrane tension and bending rigidity as the minimal set of mechanical parameters determining the nucleation and maximal size of transendothelial cell macroaperture (TEM) tunnels induced by bacterial RhoA-targeting mART exotoxins. We report that cellular depletion of caveolin-1, the membrane-embedded building block of caveolae, and depletion of cavin-1, the master regulator of caveolae invaginations, increase the number of TEMs per cell. The enhanced occurrence of TEM nucleation events correlates with a reduction in cell height due to the increase in cell spreading and decrease in cell volume, which, together with the disruption of RhoA-driven F-actin meshwork, favor membrane apposition for TEM nucleation. Strikingly, caveolin-1 specifically controls the opening speed of TEMs, leading to their dramatic 5.4-fold larger widening. Consistent with the increase in TEM density and width in siCAV1 cells, we record a higher lethality in CAV1 KO mice subjected to a catalytically active mART exotoxin targeting RhoA during staphylococcal bloodstream infection. Combined theoretical modeling with independent biophysical measurements of plasma membrane bending rigidity points toward a specific contribution of caveolin-1 to membrane stiffening in addition to the role of cavin-1/caveolin-1-dependent caveolae in the control of membrane tension homeostasis.

The colibactin-producing Escherichia coli alters the tumor microenvironment to immunosuppressive lipid overload facilitating colorectal cancer progression and chemoresistance.

Intratumoral bacteria flexibly contribute to cellular and molecular tumor heterogeneity for supporting cancer recurrence through poorly understood mechanisms. Using spatial metabolomic profiling technologies and 16SrRNA sequencing, we herein report that right-sided colorectal tumors are predominantly populated with Colibactin-producing Escherichia coli (CoPEC) that are locally establishing a high-glycerophospholipid microenvironment with lowered immunogenicity. It coincided with a reduced infiltration of CD8+ T lymphocytes that produce the cytotoxic cytokines IFN-γ where invading bacteria have been geolocated. Mechanistically, the accumulation of lipid droplets in infected cancer cells relied on the production of colibactin as a measure to limit genotoxic stress to some extent. Such heightened phosphatidylcholine remodeling by the enzyme of the Land's cycle supplied CoPEC-infected cancer cells with sufficient energy for sustaining cell survival in response to chemotherapies. This accords with the lowered overall survival of colorectal patients at stage III-IV who were colonized by CoPEC when compared to patients at stage I-II. Accordingly, the sensitivity of CoPEC-infected cancer cells to chemotherapies was restored upon treatment with an acyl-CoA synthetase inhibitor. By contrast, such metabolic dysregulation leading to chemoresistance was not observed in human colon cancer cells that were infected with the mutant strain that did not produce colibactin (11G5∆ClbQ). This work revealed that CoPEC locally supports an energy trade-off lipid overload within tumors for lowering tumor immunogenicity. This may pave the way for improving chemoresistance and subsequently outcome of CRC patients who are colonized by CoPEC.

Escherichia coli producing CNF1 toxin hijacks Tollip to trigger Rac1-dependent cell invasion.

Rho GTPases, which are master regulators of both the actin cytoskeleton and membrane trafficking, are often hijacked by pathogens to enable their invasion of host cells. Here we report that the cytotoxic necrotizing factor-1 (CNF1) toxin of uropathogenic Escherichia coli (UPEC) promotes Rac1-dependent entry of bacteria into host cells. Our screen for proteins involved in Rac1-dependent UPEC entry identifies the Toll-interacting protein (Tollip) as a new interacting protein of Rac1 and its ubiquitinated forms. We show that knockdown of Tollip reduces CNF1-induced Rac1-dependent UPEC entry. Tollip depletion also reduces the Rac1-dependent entry of Listeria monocytogenes expressing InlB invasion protein. Moreover, knockdown of Tollip, Tom1 and clathrin, decreases CNF1 and Rac1-dependent internalization of UPEC. Finally, we show that Tollip, Tom1 and clathrin associate with Rac1 and localize at the site of bacterial entry. Collectively, these findings reveal a new link between Rac1 and Tollip, Tom1 and clathrin membrane trafficking components hijacked by pathogenic bacteria to allow their efficient invasion of host cells.

Laminin-binding integrins induce Dll4 expression and Notch signaling in endothelial cells.

RATIONALE: Integrins play a crucial role in controlling endothelial cell proliferation and migration during angiogenesis. The Delta-like 4 (Dll4)/Notch pathway establishes an adequate ratio between stalk and tip cell populations by restricting tip cell formation through "lateral inhibition" in response to a vascular endothelial growth factor gradient. Because angiogenesis requires a tight coordination of these cellular processes, we hypothesized that adhesion, vascular endothelial growth factor, and Notch signaling pathways are interconnected. OBJECTIVE: This study was aimed at characterizing the cross-talk between integrin and Notch signaling in endothelial cells. METHODS AND RESULTS: Adhesion of primary human endothelial cells to laminin-111 triggers Dll4 expression, leading to subsequent Notch pathway activation. SiRNA-mediated knockdown of α2ß1 and α6ß1 integrins abolishes Dll4 induction, which discloses a selective integrin signaling acting upstream of Notch pathway. The increase in Foxc2 transcription, triggered by α2ß1 binding to laminin, is required but not sufficient per se for Dll4 expression. Furthermore, vascular endothelial growth factor stimulates laminin γ1 deposition, which leads to integrin signaling and Dll4 induction. Interestingly, loss of integrins α2 or α6 mimics the effects of Dll4 silencing and induces excessive network branching in an in vitro sprouting angiogenesis assay on three-dimensional matrigel. CONCLUSIONS: We show that, in endothelial cells, ligation of α2ß1 and α6ß1 integrins induces the Notch pathway, and we disclose a novel role of basement membrane proteins in the processes controlling tip vs stalk cell selection.

Cytotoxic necrotizing factor 1 hinders colon tumorigenesis induced by colibactin-producing Escherichia coli in ApcMin/+ mice.

Colorectal cancer (CRC) patients are frequently colonized by colibactin-producing Escherichia coli (CoPEC) (>40%), which enhances tumorigenesis in mouse models of CRC. We observed that 50% of CoPEC also contains the cnf1 gene, which encodes cytotoxic necrotizing factor-1 (CNF1), an enhancer of the eukaryotic cell cycle. The impact of its co-occurrence with colibactin (Clb) has not yet been investigated. We evaluated the impact of CNF1 on colorectal tumorigenesis using human colonic epithelial HT-29 cells and CRC-susceptible ApcMin/+ mice inoculated with the CoPEC 21F8 clinical strain (Clb+Cnf+) or 21F8 isogenic mutants (Clb+Cnf-, Clb-Cnf+ and Clb-Cnf-). Infection with the Clb+Cnf- strain induced higher levels of inflammatory cytokines and senescence markers both in vitro and in vivo compared to those induced by infection with the Clb+Cnf+ strain. In contrast, the Clb+Cnf- and Clb+Cnf+ strains generated similar levels of DNA damage in HT-29 cells and in colonic murine tissues. Furthermore, the ApcMin/+ mice inoculated with the Clb+Cnf- strain developed significantly more tumors than the mice inoculated with the Clb+Cnf+ strain or the isogenic mutants, and the composition of their microbiota was changed. Finally, rectal administration of the CNF1 protein in ApcMin/+ mice inoculated with the Clb+Cnf- strain significantly decreased tumorigenesis and inflammation. Overall, this study provides evidence that CNF1 decreases the carcinogenic effects of CoPEC in ApcMin/+ mice by decreasing CoPEC-induced cellular senescence and inflammation.

Parvimonas micra, an oral pathobiont associated with colorectal cancer, epigenetically reprograms human colonocytes.

Recently, an intestinal dysbiotic microbiota with enrichment in oral cavity bacteria has been described in colorectal cancer (CRC) patients. Here, we characterize and investigate one of these oral pathobionts, the Gram-positive anaerobic coccus Parvimonas micra. We identified two phylotypes (A and B) exhibiting different phenotypes and adhesion capabilities. We observed a strong association of phylotype A with CRC, with its higher abundance in feces and in tumoral tissue compared with the normal homologous colonic mucosa, which was associated with a distinct methylation status of patients. By developing an in vitro hypoxic co-culture system of human primary colonic cells with anaerobic bacteria, we show that P. micra phylotype A alters the DNA methylation profile promoters of key tumor-suppressor genes, oncogenes, and genes involved in epithelial-mesenchymal transition. In colonic mucosa of CRC patients carrying P. micra phylotype A, we found similar DNA methylation alterations, together with significant enrichment of differentially expressed genes in pathways involved in inflammation, cell adhesion, and regulation of actin cytoskeleton, providing evidence of P. micra's possible role in the carcinogenic process.

alpha2beta1 integrin controls association of Rac with the membrane and triggers quiescence of endothelial cells.

Integrin receptors and their extracellular matrix ligands provide cues to cell proliferation, survival, differentiation and migration. Here, we show that alpha2beta1 integrin, when ligated to the basement membrane component laminin-1, triggers a proliferation arrest in primary endothelial cells. Indeed, in the presence of strong growth signals supplied by growth factors and fibronectin, alpha2beta1 engagement alters assembly of mature focal adhesions by alpha5beta1 and leads to impairment of downstream signaling and cell-cycle arrest in the G1 phase. Although the capacity of alpha5beta1 to signal for GTP loading of Rac is preserved, the joint engagement of alpha2beta1 interferes with membrane anchorage of Rac. Adapting the 'split-ubiquitin' sensor to screen for membrane-proximal alpha2 integrin partners, we identified the CD9 tetraspanin and further establish its requirement for destabilization of focal adhesions, control of Rac subcellular localization and growth arrest induced by alpha2beta1 integrin. Altogether, our data establish that alpha2beta1 integrin controls endothelial cell commitment towards quiescence by triggering a CD9-dependent dominant signaling.