Multiphase coalescence mediates Hippo pathway activation.

The function of biomolecular condensates is often restricted by condensate dissolution. Whether condensates can be suppressed without condensate dissolution is unclear. Here, we show that upstream regulators of the Hippo signaling pathway form functionally antagonizing condensates, and their coalescence into a common phase provides a mode of counteracting the function of biomolecular condensates without condensate dissolution. Specifically, the negative regulator SLMAP forms Hippo-inactivating condensates to facilitate pathway inhibition by the STRIPAK complex. In response to cell-cell contact or osmotic stress, the positive regulators AMOT and KIBRA form Hippo-activating condensates to facilitate pathway activation. The functionally antagonizing SLMAP and AMOT/KIBRA condensates further coalesce into a common phase to inhibit STRIPAK function. These findings provide a paradigm for restricting the activity of biomolecular condensates without condensate dissolution, shed light on the molecular principles of multiphase organization, and offer a conceptual framework for understanding upstream regulation of the Hippo signaling pathway.

Toll Receptor-Mediated Hippo Signaling Controls Innate Immunity in Drosophila.

The Hippo signaling pathway functions through Yorkie to control tissue growth and homeostasis. How this pathway regulates non-developmental processes remains largely unexplored. Here, we report an essential role for Hippo signaling in innate immunity whereby Yorkie directly regulates the transcription of the Drosophila IκB homolog, Cactus, in Toll receptor-mediated antimicrobial response. Loss of Hippo pathway tumor suppressors or activation of Yorkie in fat bodies, the Drosophila immune organ, leads to elevated cactus mRNA levels, decreased expression of antimicrobial peptides, and vulnerability to infection by Gram-positive bacteria. Furthermore, Gram-positive bacteria acutely activate Hippo-Yorkie signaling in fat bodies via the Toll-Myd88-Pelle cascade through Pelle-mediated phosphorylation and degradation of the Cka subunit of the Hippo-inhibitory STRIPAK PP2A complex. Our studies elucidate a Toll-mediated Hippo signaling pathway in antimicrobial response, highlight the importance of regulating IκB/Cactus transcription in innate immunity, and identify Gram-positive bacteria as extracellular stimuli of Hippo signaling under physiological settings.

YAP-VGLL4 antagonism defines the major physiological function of the Hippo signaling effector YAP.

The Hippo-YAP signaling pathway plays a critical role in development, homeostasis, regeneration, and tumorigenesis by converging on YAP, a coactivator for the TEAD family DNA-binding transcription factors, to regulate downstream transcription programs. Given its pivotal role as the nuclear effector of the Hippo pathway, YAP is indispensable in multiple developmental and tissue contexts. Here we report that the essentiality of YAP in liver and lung development can be genetically bypassed by simultaneous inactivation of the TEAD corepressor VGLL4. This striking antagonistic epistasis suggests that the major physiological function of YAP is to antagonize VGLL4. We further show that the YAP-VGLL4 antagonism plays a widespread role in regulating Hippo pathway output beyond normal development, as inactivation of Vgll4 dramatically enhanced intrahepatic cholangiocarcinoma formation in Nf2-deficient livers and ameliorated CCl4-induced damage in normal livers. Interestingly, Vgll4 expression is temporally regulated in development and regeneration and, in certain contexts, provides a better indication of overall Hippo pathway output than YAP phosphorylation. Together, these findings highlight the central importance of VGLL4-mediated transcriptional repression in Hippo pathway regulation and inform potential strategies to modulate Hippo signaling in cancer and regenerative medicine.

WWTR1(TAZ)-CAMTA1 reprograms endothelial cells to drive epithelioid hemangioendothelioma.

Epithelioid hemangioendothelioma (EHE) is a poorly understood and devastating vascular cancer. Sequencing of EHE has revealed a unique gene fusion between the Hippo pathway nuclear effector TAZ (WWTR1) and the brain-enriched transcription factor CAMTA1 in ∼90% of cases. However, it remains unclear whether the TAZ-CAMTA1 gene fusion is a driver of EHE, and potential targeted therapies are unknown. Here, we show that TAZ-CAMTA1 expression in endothelial cells is sufficient to drive the formation of vascular tumors with the distinctive features of EHE, and inhibition of TAZ-CAMTA1 results in the regression of these vascular tumors. We further show that activated TAZ resembles TAZ-CAMTA1 in driving the formation of EHE-like vascular tumors, suggesting that constitutive activation of TAZ underlies the pathological features of EHE. We show that TAZ-CAMTA1 initiates an angiogenic and regenerative-like transcriptional program in endothelial cells, and disruption of the TAZ-CAMTA1-TEAD interaction or ectopic expression of a dominant negative TEAD in vivo inhibits TAZ-CAMTA1-mediated transformation. Our study provides the first genetic model of a TAZ fusion oncoprotein driving its associated human cancer, pinpointing TAZ-CAMTA1 as the key driver and a valid therapeutic target of EHE.

Assembling a Hippo: the evolutionary emergence of an animal developmental signaling pathway.

Decades of work in developmental genetics has given us a deep mechanistic understanding of the fundamental signaling pathways underlying animal development. However, little is known about how these pathways emerged and changed over evolutionary time. Here, we review our current understanding of the evolutionary emergence of the Hippo pathway, a conserved signaling pathway that regulates tissue size in animals. This pathway has deep evolutionary roots, emerging piece by piece in the unicellular ancestors of animals, with a complete core pathway predating the origin of animals. Recent functional studies in close unicellular relatives of animals and early-branching animals suggest an ancestral function Hippo pathway of cytoskeletal regulation, which was subsequently co-opted to regulate proliferation and animal tissue size.

Spatial organization of Hippo signaling at the plasma membrane mediated by the tumor suppressor Merlin/NF2.

Although Merlin/NF2 was discovered two decades ago as a tumor suppressor underlying Neurofibromatosis type II, its precise molecular mechanism remains poorly understood. Recent studies in Drosophila revealed a potential link between Merlin and the Hippo pathway by placing Merlin genetically upstream of the kinase Hpo/Mst. In contrast to the commonly depicted linear model of Merlin functioning through Hpo/Mst, here we show that in both Drosophila and mammals, Merlin promotes downstream Hippo signaling without activating the intrinsic kinase activity of Hpo/Mst. Instead, Merlin directly binds and recruits the effector kinase Wts/Lats to the plasma membrane. Membrane recruitment, in turn, promotes Wts phosphorylation by the Hpo-Sav kinase complex. We further show that disruption of the actin cytoskeleton promotes Merlin-Wts interactions, which implicates Merlin in actin-mediated regulation of Hippo signaling. Our findings elucidate an important molecular function of Merlin and highlight the plasma membrane as a critical subcellular compartment for Hippo signal transduction.

Structural basis for Mob1-dependent activation of the core Mst-Lats kinase cascade in Hippo signaling.

The Mst-Lats kinase cascade is central to the Hippo tumor-suppressive pathway that controls organ size and tissue homeostasis. The adaptor protein Mob1 promotes Lats activation by Mst, but the mechanism remains unknown. Here, we show that human Mob1 binds to autophosphorylated docking motifs in active Mst2. This binding enables Mob1 phosphorylation by Mst2. Phosphorylated Mob1 undergoes conformational activation and binds to Lats1. We determine the crystal structures of phospho-Mst2-Mob1 and phospho-Mob1-Lats1 complexes, revealing the structural basis of both phosphorylation-dependent binding events. Further biochemical and functional analyses demonstrate that Mob1 mediates Lats1 activation through dynamic scaffolding and allosteric mechanisms. Thus, Mob1 acts as a phosphorylation-regulated coupler of kinase activation by virtue of its ability to engage multiple ligands. We propose that stepwise, phosphorylation-triggered docking interactions of nonkinase elements enhance the specificity and robustness of kinase signaling cascades.

Machine learning for reparameterization of four-site water models: TIP4P-BG and TIP4P-BGT.

Parameterizing an effective water model is a challenging issue because of the difficulty in maintaining a comprehensive balance among the diverse physical properties of water with a limited number of parameters. The advancement in machine learning provides a promising path to search for a reliable set of parameters. Based on the TIP4P water model, hence, about 6000 molecular dynamics (MD) simulations for pure water at 1 atm and in the range of 273-373 K are conducted here as the training data. The back-propagation (BP) neural network is then utilized to construct an efficient mapping between the model parameters and four crucial physical properties of water, including the density, vaporization enthalpy, self-diffusion coefficient and viscosity. Without additional time-consuming MD simulations, this mapping operation could result in sufficient and accurate data for high-population genetic algorithm (GA) to optimize the model parameters as much as possible. Based on the proposed parameterizing strategy, TIP4P-BG (a conventional four-site water model) and TIP4P-BGT (an advanced model with temperature-dependent parameters) are established. Both the water models exhibit excellent performance with a reasonable balance among the four crucial physical properties. The relevant mean absolute percentage errors are 3.53% and 3.08%, respectively. Further calculations on the temperature of maximum density, isothermal compressibility, thermal expansion coefficient, radial distribution function and surface tension are also performed and the resulting values are in good agreement with the experimental values. Through this water modeling example, the potential of the proposed data-driven machine learning procedure has been demonstrated for parameterizing a MD-based material model.

Regulating the mechanical properties of nanocrystalline nickel via molybdenum segregation: an atomistic study.

The effects of segregation of impurity molybdenum (Mo) atoms on the tensile mechanical properties of nanocrystalline nickel (Ni) are investigated with molecular dynamics simulation. The results show that the segregation of Mo atoms induces an obvious increase in the elastic modulus and strength of nanocrystalline Ni, and the strengthening effect is more significant with smaller grain size. When the grain size decreases below a critical value, at which the softening occurs in non-segregated Ni-Mo alloy, no evident softening phenomenon is observed in Mo-segregated systems. Furthermore, based on a bicrystal configuration, it is found that Mo atoms segregating to the grain boundary reduce the energy and mobility of the grain boundary, increasing the grain boundary stability and thus accommodating the strengthening. The present findings will shed light on the fabrication of high strength nanocrystalline materials by controlling the segregation of atoms.

Aggregation of nanoparticles regulated by mechanical properties of nanoparticle-membrane system.

The aggregation of nanoparticles (NPs) on the cell membrane is crucial for the cellular uptake process and has important biological implications in protein-membrane interactions. In this paper, we systematically investigate how the aggregation is regulated by the mechanical properties of the NP-membrane system, including the membrane tension, and the size and shape of the NPs. Results show that when NPs aggregate parallel to the cell membrane, increasing the membrane tension will modulate the membrane-mediated interaction between the NPs from attractive to attractive-repulsive and finally to purely repulsive. In contrast, the membrane-mediated interaction is attractive and independent of the membrane tension when the NPs aggregate to a tubular configuration. For the aggregation of NPs of different sizes, the large-size NP is wrapped to a greater extent than the small-size NP. For the aggregation of nonspherical NPs, low aspect ratio and weak NP-membrane adhesion strength lead to the side-to-side configuration, whereas a system with a high aspect ratio and strong NP-membrane adhesion strength prefers the tip-to-tip configuration. Importantly, NPs of different sizes and anisotropic shapes are found to facilitate the aggregation process by reducing the energy barrier that should be overcome during the aggregation. The results reveal the mechanism of the aggregation of NPs on the cell membrane and provide guidelines to the design of NP-based drug delivery systems.

Divergent effect of electric fields on the mechanical property of water-filled carbon nanotubes with an application as a nanoscale trigger.

Polar water molecules exhibit extraordinary phenomena under nanoscale confinement. Through the application of an electric field, a water-filled carbon nanotube (CNT) that has been successfully fabricated in the laboratory is expected to have distinct responses to the external electricity. Here, we examine the effect of electric field direction on the mechanical property of water-filled CNTs. It is observed that a longitudinal electric field enhances, but the transverse electric field reduces the elastic modulus and critical buckling stress of water-filled CNTs. The divergent effect of the electric field is attributed to the competition between the axial and circumferential pressures induced by polar water molecules. Furthermore, it is notable that the transverse electric field could result in an internal pressure with elliptical distribution, which is an effective and convenient approach to apply nonuniform pressure on nanochannels. Based on pre-strained water-filled CNTs, we designed a nanoscale trigger with an evident and rapid height change initiated by switching the direction of the electric field. The reported finding provides a foundation for an electricity-controlled property of nanochannels filled with polar molecules and provides an insight into the design of nanoscale functional devices.

The Hippo signaling pathway restricts the oncogenic potential of an intestinal regeneration program.

Although a developmental role for Hippo signaling in organ size control is well appreciated, how this pathway functions in tissue regeneration is largely unknown. Here we address this issue using a dextran sodium sulfate (DSS)-induced colonic regeneration model. We find that regenerating crypts express elevated Yes-associated protein (YAP) levels. Inactivation of YAP causes no obvious intestinal defects under normal homeostasis, but severely impairs DSS-induced intestinal regeneration. Conversely, hyperactivation of YAP results in widespread early-onset polyp formation following DSS treatment. Thus, the YAP oncoprotein must be exquisitely controlled in tissue regeneration to allow compensatory proliferation and prevent the intrinsic oncogenic potential of a tissue regeneration program.

Hetero interface and twin boundary mediated strengthening in nano-twinned Cu//Ag multilayered materials.

Based on molecular dynamics simulations, tensile mechanical properties and plastic deformation mechanisms of nano-twinned Cu//Ag multilayered materials are investigated in this work. Simulation results show that, due to the stronger strengthening effect of the twin boundary than both the cube-on-cube and hetero-twin interfaces between Cu and Ag layers, the strength increases with the increase of layer thickness for nano-twinned Cu//Ag multilayered materials with a constant twin spacing, while it decreases with the increase of layer thickness for twin-free ones. The strength of hetero-twin multilayered materials is higher than that of the cube-on-cube samples due to the different hetero interfacial configurations. The confined layer slip of dislocation is found to be the dominant plastic deformation mechanism for twin-free hetero-twin multilayered materials and the strength versus twin spacing in nano-twinned samples follows the conventional Hall-Petch relationship. These findings will shed light on the understanding of the plastic deformation mechanisms and the fabrication of high strength nano-twinned multilayered metallic materials.

Controllable deformation of salt water-filled carbon nanotubes using an electric field with application to molecular sieving.

Precisely controlling the deformation of carbon nanotubes (CNTs) has practical application in the development of nanoscale functional devices, although it is a challenging task. Here, we propose a novel method to guide the deformation of CNTs through filling them with salt water and applying an electric field. With the electric field along the axial direction, the height of CNTs is enlarged by the axial electric force due to the internal ions and polar water molecules. Under an electric field with two mutually orthogonal components, the transverse electric force could further induce the bending deformation of CNTs. Based on the classical rod and beam theories, two mechanical models are constructed to verify and quantitatively describe the relationships between the tension and bending deformations of CNTs and the electric field intensity. Moreover, by means of the electric field-driven tension behavior of CNTs, we design a stretchable molecular sieve to control the flow rate of mixed gas and collect a single high-purity gas. The present work opens up new avenues in the design and fabrication of nanoscale controlling units.

Free-end adaptive nudged elastic band method for locating transition states in minimum energy path calculation.

A free-end adaptive nudged elastic band (FEA-NEB) method is presented for finding transition states on minimum energy paths, where the energy barrier is very narrow compared to the whole paths. The previously proposed free-end nudged elastic band method may suffer from convergence problems because of the kinks arising on the elastic band if the initial elastic band is far from the minimum energy path and weak springs are adopted. We analyze the origin of the formation of kinks and present an improved free-end algorithm to avoid the convergence problem. Moreover, by coupling the improved free-end algorithm and an adaptive strategy, we develop a FEA-NEB method to accurately locate the transition state with the elastic band cut off repeatedly and the density of images near the transition state increased. Several representative numerical examples, including the dislocation nucleation in a penta-twinned nanowire, the twin boundary migration under a shear stress, and the cross-slip of screw dislocation in face-centered cubic metals, are investigated by using the FEA-NEB method. Numerical results demonstrate both the stability and efficiency of the proposed method.

Wrapping of nanoparticles by the cell membrane: the role of interactions between the nanoparticles.

A fundamental understanding of the interactions between nanoparticles (NPs) and the cell membrane is essential to improve the performance of the NP-based biomedical applications and assess the potential toxicity of NPs. Despite the great progress in understanding the interaction between individual NP and the membrane, little is known about the interaction between multiple NPs and the membrane. In this work, we investigate the wrapping of two parallel elongated NPs by the membrane, taking the NP-NP electrostatic interaction and van der Waals (vdW) interaction into consideration. Three types of NPs, namely the rigid NPs with circular and elliptic cross-sections and the deformable NPs, are systematically investigated. The results show that the electrostatic interaction would enhance the tendency of the independent wrapping and inhibit the rotation of the elongated and equally charged NPs with elliptic cross-sections. Under the vdW interaction, the competition of the NP-NP adhesion and the membrane elastic energies with the NP-membrane adhesion energy leads the NPs to be wrapped cooperatively or independently. For the system with elongated NPs with elliptic cross-sections, the NPs are more likely to be wrapped independently as the shapes become more anisotropic and the NPs would rotate to contact each other with the flat sides in the cooperative wrapping configuration. Moreover, the soft NPs are more likely to be wrapped cooperatively compared with the stiff NPs. These results may provide guidelines to control the internalization pathway of NPs and improve the efficiency of NP-based drug delivery systems.

Innate immune and proinflammatory signals activate the Hippo pathway via a Tak1-STRIPAK-Tao axis.

The Hippo pathway controls developmental, homeostatic and regenerative tissue growth, and is frequently dysregulated in various diseases. Although this pathway can be activated by innate immune/inflammatory stimuli, the underlying mechanism is not fully understood. Here, we identify a conserved signaling cascade that leads to Hippo pathway activation by innate immune/inflammatory signals. We show that Tak1, a key kinase in innate immune/inflammatory signaling, activates the Hippo pathway by inducing the lysosomal degradation of Cka, an essential subunit of the STRIPAK PP2A complex that suppresses Hippo signaling. Suppression of STRIPAK results in the activation of Hippo pathway through Tao-Hpo signaling. We further show that Tak1-mediated Hippo signaling is involved in processes ranging from cell death to phagocytosis and innate immune memory. Our findings thus reveal a molecular connection between innate immune/inflammatory signaling and the evolutionally conserved Hippo pathway, thus contributing to our understanding of infectious, inflammatory and malignant diseases.

The apical transmembrane protein Crumbs functions as a tumor suppressor that regulates Hippo signaling by binding to Expanded.

The Hippo signaling pathway regulates organ size and tissue homeostasis from Drosophila to mammals. At the core of the Hippo pathway is a kinase cascade extending from the Hippo (Hpo) tumor suppressor to the Yorkie (Yki) oncoprotein. The Hippo kinase cascade, in turn, is regulated by apical membrane-associated proteins such as the FERM domain proteins Merlin and Expanded (Ex), and the WW- and C2-domain protein Kibra. How these apical proteins are themselves regulated remains poorly understood. Here, we identify the transmembrane protein Crumbs (Crb), a determinant of epithelial apical-basal polarity in Drosophila embryos, as an upstream component of the Hippo pathway in imaginal disk growth control. Loss of Crb leads to tissue overgrowth and target gene expression characteristic of defective Hippo signaling. Crb directly binds to Ex through its juxtamembrane FERM-binding motif (FBM). Loss of Crb or mutation of its FBM leads to mislocalization of Ex to basolateral domain of imaginal disk epithelial cells. These results shed light on the mechanism of Ex regulation and provide a molecular link between apical-basal polarity and tissue growth. Furthermore, our studies implicate Crb as a putative cell surface receptor for Hippo signaling by uncovering a transmembrane protein that directly binds to an apical component of the Hippo pathway.

The PIWI-specific insertion module helps load longer piRNAs for translational activation essential for male fertility.

PIWI-clade proteins harness piRNAs of 24-33 nt in length. Of great puzzles are how PIWI-clade proteins incorporate piRNAs of different sizes and whether the size matters to PIWI/piRNA function. Here we report that a PIWI-Ins module unique in PIWI-clade proteins helps define the length of piRNAs. Deletion of PIWI-Ins in Miwi shifts MIWI to load with shorter piRNAs and causes spermiogenic failure in mice, demonstrating the functional importance of this regulatory module. Mechanistically, we show that longer piRNAs provide additional complementarity to target mRNAs, thereby enhancing the assembly of the MIWI/eIF3f/HuR super-complex for translational activation. Importantly, we identify a c.1108C>T (p.R370W) mutation of HIWI (human PIWIL1) in infertile men and demonstrate in Miwi knock-in mice that this genetic mutation impairs male fertility by altering the property of PIWI-Ins in selecting longer piRNAs. These findings reveal a critical role of PIWI-Ins-ensured longer piRNAs in fine-tuning MIWI/piRNA targeting capacity, proven essential for spermatid development and male fertility.

Water diffusion inside carbon nanotubes: mutual effects of surface and confinement.

The mutual effects of two crucial features of carbon nanotubes (CNTs) (surface and confinement) on the temperature-dependent water diffusion are studied through molecular dynamics simulations. A two-stage diffusion mechanism is detected in the CNTs of diameter smaller than 12.2 Å, which becomes obscure as the temperature increases. This peculiar phenomenon can be ascribed to the cooperation of the small confinement and the periodic surface. The diffusion coefficient of the confined water exhibits a nonmonotonic dependence on the confinement size and an unexpected increase inside the large CNTs (compared to that of bulk water). These anomalous behaviors can be attributed to the competition of the smooth surface and the small confinement. Considering the mutual effects, an empirical formula is proposed on the basis of two groups of numerical examples, whose results indicate that the confinement effect will dominate over the surface effect until the CNT diameter increases up to ~16 Å, whereas thereafter the surface effect becomes dominant and finally both of them vanish gradually.