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Intracellular bodies such as nucleoli, Cajal bodies and various signalling assemblies represent membraneless organelles, or condensates, that form via liquid–liquid phase separation (LLPS) 1 , 2 . Biomolecular interactions—particularly homotypic interactions mediated by self-associating intrinsically disordered protein regions—are thought to underlie the thermodynamic driving forces for LLPS, forming condensates that can facilitate the assembly and processing of biochemically active complexes, such as ribosomal subunits within the nucleolus. Simplified model systems 3 – 6 have led to the concept that a single fixed saturation concentration is a defining feature of endogenous LLPS 7 – 9 , and has been suggested as a mechanism for intracellular concentration buffering 2 , 7 , 8 , 10 . However, the assumption of a fixed saturation concentration remains largely untested within living cells, in which the richly multicomponent nature of condensates could complicate this simple picture. Here we show that heterotypic multicomponent interactions dominate endogenous LLPS, and give rise to nucleoli and other condensates that do not exhibit a fixed saturation concentration. As the concentration of individual components is varied, their partition coefficients change in a manner that can be used to determine the thermodynamic free energies that underlie LLPS. We find that heterotypic interactions among protein and RNA components stabilize various archetypal intracellular condensates—including the nucleolus, Cajal bodies, stress granules and P-bodies—implying that the composition of condensates is finely tuned by the thermodynamics of the underlying biomolecular interaction network. In the context of RNA-processing condensates such as the nucleolus, this manifests in the selective exclusion of fully assembled ribonucleoprotein complexes, providing a thermodynamic basis for vectorial ribosomal RNA flux out of the nucleolus. This methodology is conceptually straightforward and readily implemented, and can be broadly used to extract thermodynamic parameters from microscopy images. These approaches pave the way for a deeper understanding of the thermodynamics of multicomponent intracellular phase behaviour and its interplay with the nonequilibrium activity that is characteristic of endogenous condensates. Heterotypic multicomponent interactions are shown to dominate the liquid–liquid phase separation that enables the formation of intracellular condensates.

The pancreas originates from two epithelial evaginations of the foregut, which consist of multipotent epithelial progenitors that organize into a complex tubular epithelial network. The trunk domain of each epithelial branch consists of bipotent pancreatic progenitors (bi-PPs) that give rise to both duct and endocrine lineages, whereas the tips give rise to acinar cells 1 . Here we identify the extrinsic and intrinsic signalling mechanisms that coordinate the fate-determining transcriptional events underlying these lineage decisions 1 , 2 . Single-cell analysis of pancreatic bipotent pancreatic progenitors derived from human embryonic stem cells reveal that cell confinement is a prerequisite for endocrine specification, whereas spreading drives the progenitors towards a ductal fate. Mechanistic studies identify the interaction of extracellular matrix (ECM) with integrin α5 as the extracellular cue that cell-autonomously, via the F-actin–YAP1–Notch mechanosignalling axis, controls the fate of bipotent pancreatic progenitors. Whereas ECM–integrin α5 signalling promotes differentiation towards the duct lineage, endocrinogenesis is stimulated when this signalling cascade is disrupted. This cascade can be disrupted pharmacologically or genetically to convert bipotent pancreatic progenitors derived from human embryonic stem cells to hormone-producing islet cells. Our findings identify the cell-extrinsic and intrinsic mechanotransduction pathway that acts as gatekeeper in the fate decisions of bipotent pancreatic progenitors in the developing pancreas. Single-cell analysis reveals that interactions with the extracellular matrix via integrin α5 and mechanotransducer YAP1 determine whether pancreatic progenitors develop along the duct or endocrine lineages.

The E3 ligase Cbl-b acts on TAM tyrosine kinase receptors and has a critical role in the regulation of natural killer (NK) cell rejection of metastatic tumours; a small molecule TAM kinase inhibitor is shown to enhance the anti-metastatic NK cell activity. Controlling NK cell anti-metastatic activity This study describes a previously unknown role for the E3 ubiquitin ligase Cbl-b as part of a regulatory pathway in innate natural killer (NK) cells that licenses them to spontaneously reject cancer metastases. Genetic loss of Cbl-b or inactivation of its E3 ligase activity in mice allows NK cells to suppress growth of both multiple primary tumours and distant tumour metastases. The effect is mediated via members of the TAM family tyrosine kinase receptors, and treatment of wild-type NK cells with a small-molecule TAM inhibitor conferred therapeutic NK cell activity against metastatic melanomas. This suggests a possible approach for NK-cell-based anti-metastatic therapy in humans and at the same time explains the anti-metastatic properties of the widely used anticoagulant warfarin. Tumour metastasis is the primary cause of mortality in cancer patients and remains the key challenge for cancer therapy 1 . New therapeutic approaches to block inhibitory pathways of the immune system have renewed hopes for the utility of such therapies 2 . Here we show that genetic deletion of the E3 ubiquitin ligase Cbl-b (casitas B-lineage lymphoma-b) or targeted inactivation of its E3 ligase activity licenses natural killer (NK) cells to spontaneously reject metastatic tumours. The TAM tyrosine kinase receptors Tyro3, Axl and Mer (also known as Mertk) were identified as ubiquitylation substrates for Cbl-b. Treatment of wild-type NK cells with a newly developed small molecule TAM kinase inhibitor conferred therapeutic potential, efficiently enhancing anti-metastatic NK cell activity in vivo . Oral or intraperitoneal administration using this TAM inhibitor markedly reduced murine mammary cancer and melanoma metastases dependent on NK cells. We further report that the anticoagulant warfarin exerts anti-metastatic activity in mice via Cbl-b/TAM receptors in NK cells, providing a molecular explanation for a 50-year-old puzzle in cancer biology 3 . This novel TAM/Cbl-b inhibitory pathway shows that it might be possible to develop a ‘pill’ that awakens the innate immune system to kill cancer metastases.

YAP has previously been identified as an oncogene that promotes cell growth, but now it is shown to restrict stem cell expansion during regeneration in the mouse intestine, suggesting that it may function as a tumour suppressor in colon cancer. Conflicting growth effects of YAP protein YAP is the main transcriptional effector of the growth-suppressive Hippo signalling pathway. It has been identified as an oncogene that promotes cell growth, and has emerged as a potential antitumour target. Surprisingly, Fernando Camargo and colleagues now find that in the mouse intestine, YAP actually suppresses growth, acting to restrict stem cell expansion during regeneration by limiting Wnt signalling. Consequently, loss of YAP leads to hyperplasia and microadenomas during regeneration. YAP is found to be downregulated in some human colon cancer, and its expression can reduce the growth of human colorectal cancer xenografts, suggesting that it may function as a tumour suppressor in colon cancer. A remarkable feature of regenerative processes is their ability to halt proliferation once an organ’s structure has been restored. The Wnt signalling pathway is the major driving force for homeostatic self-renewal and regeneration in the mammalian intestine. However, the mechanisms that counterbalance Wnt-driven proliferation are poorly understood. Here we demonstrate in mice and humans that yes-associated protein 1 (YAP; also known as YAP1)—a protein known for its powerful growth-inducing and oncogenic properties 1 , 2 —has an unexpected growth-suppressive function, restricting Wnt signals during intestinal regeneration. Transgenic expression of YAP reduces Wnt target gene expression and results in the rapid loss of intestinal crypts. In addition, loss of YAP results in Wnt hypersensitivity during regeneration, leading to hyperplasia, expansion of intestinal stem cells and niche cells, and formation of ectopic crypts and microadenomas. We find that cytoplasmic YAP restricts elevated Wnt signalling independently of the AXIN–APC–GSK-3β complex partly by limiting the activity of dishevelled (DVL). DVL signals in the nucleus of intestinal stem cells, and its forced expression leads to enhanced Wnt signalling in crypts. YAP dampens Wnt signals by restricting DVL nuclear translocation during regenerative growth. Finally, we provide evidence that YAP is silenced in a subset of highly aggressive and undifferentiated human colorectal carcinomas, and that its expression can restrict the growth of colorectal carcinoma xenografts. Collectively, our work describes a novel mechanistic paradigm for how proliferative signals are counterbalanced in regenerating tissues. Additionally, our findings have important implications for the targeting of YAP in human malignancies.

Sudipto Roy, Carol Wicking, Carsten Bergmann and colleagues report that mutations in DZIP1L cause autosomal recessive polycystic kidney disease (ARPKD). Through studies of mouse and zebrafish models of DZIP1L loss of function, the authors demonstrate that DZIP1L is required for proper function of the periciliary diffusion barrier. Autosomal recessive polycystic kidney disease (ARPKD), usually considered to be a genetically homogeneous disease caused by mutations in PKHD1 , has been associated with ciliary dysfunction. Here, we describe mutations in DZIP1L , which encodes DAZ interacting protein 1-like, in patients with ARPKD. We further validated these findings through loss-of-function studies in mice and zebrafish. DZIP1L localizes to centrioles and to the distal ends of basal bodies, and interacts with septin2, a protein implicated in maintenance of the periciliary diffusion barrier at the ciliary transition zone. In agreement with a defect in the diffusion barrier, we found that the ciliary-membrane translocation of the PKD proteins polycystin-1 and polycystin-2 is compromised in DZIP1L -mutant cells. Together, these data provide what is, to our knowledge, the first conclusive evidence that ARPKD is not a homogeneous disorder and further establish DZIP1L as a second gene involved in ARPKD pathogenesis.

This study finds that the Hippo pathway is essential for gut epithelial regeneration and tumour initiation; the Hippo component Yap holds off differentiation of intestinal stem cells to Paneth cells to promote a survival and self-renewal regenerative program through activation of the Egfr pathway. Role of Yap in tissue regeneration The Hippo pathway, an influence on cell fate determination and tissue growth during development, has emerged as an important regulator of tissue regeneration in the adult. Routine gut epithelium self-renewal is controlled by Wnt signalling pathways in intestinal stem cells, but how the gut regenerates after injury has been unclear. Jeffrey Wrana and colleagues show that a second signalling pathway — Hippo — is required for intestinal epithelium to recover after exposure to ionizing radiation. The Hippo component Yap holds off the differentiation of intestinal stem cells to Paneth cells to promote a survival and self-renewal regenerative program through activation of a third pathway, Egfr. This Yap-driven regeneration pathway is also shown to participate in tumorigenesis. The gut epithelium has remarkable self-renewal capacity that under homeostatic conditions is driven by Wnt signalling in Lgr5 + intestinal stem cells (ISCs) 1 . However, the mechanisms underlying ISC regeneration after injury remain poorly understood. The Hippo signalling pathway mediates tissue growth and is important for regeneration 2 , 3 . Here we demonstrate in mice that Yap, a downstream transcriptional effector of Hippo, is critical for recovery of intestinal epithelium after exposure to ionizing radiation. Yap transiently reprograms Lgr5 + ISCs by suppressing Wnt signalling and excessive Paneth cell differentiation, while promoting cell survival and inducing a regenerative program that includes Egf pathway activation. Accordingly, growth of Yap-deficient organoids is rescued by the Egfr ligand epiregulin, and we find that non-cell-autonomous production of stromal epiregulin may compensate for Yap loss in vivo . Consistent with key roles for regenerative signalling in tumorigenesis, we further demonstrate that Yap inactivation abolishes adenomas in the Apc Min mouse model of colon cancer, and that Yap-driven expansion of Apc −/− organoids requires the Egfr module of the Yap regenerative program. Finally, we show that in vivo Yap is required for progression of early Apc mutant tumour-initiating cells, suppresses their differentiation into Paneth cells, and induces a regenerative program and Egfr signalling. Our studies reveal that upon tissue injury, Yap reprograms Lgr5 + ISCs by inhibiting the Wnt homeostatic program, while inducing a regenerative program that includes activation of Egfr signalling. Moreover, our findings reveal a key role for the Yap regenerative pathway in driving cancer initiation.

Mitochondrial role in innate immunity The stimulation of a subset of surface Toll-like receptors (TLRs), transmembrane proteins of the innate immune system that recognize microbe-derived molecules, is shown to induce production of reactive oxygen for bacterial killing by the mitochondrial pathway. When the 'bacterial' TLRs (TLR1, 2 and 4) bind to a ligand they promote the recruitment of mitochondria to macrophage phagosomes and induce upregulation of mitochondrial reactive oxygen species (mROS). This work implicates mROS as important components of antibacterial responses and further establishes mitochondria as hubs for innate immune signalling. Reactive oxygen species (ROS) are essential components of the innate immune response against intracellular bacteria and it is thought that professional phagocytes generate ROS primarily via the phagosomal NADPH oxidase machinery 1 . However, recent studies have suggested that mitochondrial ROS (mROS) also contribute to mouse macrophage bactericidal activity, although the mechanisms linking innate immune signalling to mitochondria for mROS generation remain unclear 2 , 3 , 4 . Here we demonstrate that engagement of a subset of Toll-like receptors (TLR1, TLR2 and TLR4) results in the recruitment of mitochondria to macrophage phagosomes and augments mROS production. This response involves translocation of a TLR signalling adaptor, tumour necrosis factor receptor-associated factor 6 (TRAF6), to mitochondria, where it engages the protein ECSIT (evolutionarily conserved signalling intermediate in Toll pathways), which is implicated in mitochondrial respiratory chain assembly 5 . Interaction with TRAF6 leads to ECSIT ubiquitination and enrichment at the mitochondrial periphery, resulting in increased mitochondrial and cellular ROS generation. ECSIT- and TRAF6-depleted macrophages have decreased levels of TLR-induced ROS and are significantly impaired in their ability to kill intracellular bacteria. Additionally, reducing macrophage mROS levels by expressing catalase in mitochondria results in defective bacterial killing, confirming the role of mROS in bactericidal activity. These results reveal a novel pathway linking innate immune signalling to mitochondria, implicate mROS as an important component of antibacterial responses and further establish mitochondria as hubs for innate immune signalling.

The RNA-editing enzyme adenosine deaminase acting on RNA 1 (ADAR1) limits the accumulation of endogenous immunostimulatory double-stranded RNA (dsRNA) 1 . In humans, reduced ADAR1 activity causes the severe inflammatory disease Aicardi–Goutières syndrome (AGS) 2 . In mice, complete loss of ADAR1 activity is embryonically lethal 3 – 6 , and mutations similar to those found in patients with AGS cause autoinflammation 7 – 12 . Mechanistically, adenosine-to-inosine (A-to-I) base modification of endogenous dsRNA by ADAR1 prevents chronic overactivation of the dsRNA sensors MDA5 and PKR 3 , 7 – 10 , 13 , 14 . Here we show that ADAR1 also inhibits the spontaneous activation of the left-handed Z-nucleic acid sensor ZBP1. Activation of ZBP1 elicits caspase-8-dependent apoptosis and MLKL-mediated necroptosis of ADAR1-deficient cells. ZBP1 contributes to the embryonic lethality of Adar -knockout mice, and it drives early mortality and intestinal cell death in mice deficient in the expression of both ADAR and MAVS. The Z-nucleic-acid-binding Zα domain of ADAR1 is necessary to prevent ZBP1-mediated intestinal cell death and skin inflammation. The Zα domain of ADAR1 promotes A-to-I editing of endogenous Alu elements to prevent dsRNA formation through the pairing of inverted Alu repeats, which can otherwise induce ZBP1 activation. This shows that recognition of Alu duplex RNA by ZBP1 may contribute to the pathological features of AGS that result from the loss of ADAR1 function. In addition to its role in suppressing MDA5 and PKR activation, ADAR1 is a negative regulator of ZPB1-mediated apoptosis and necroptosis, providing insights into the pathology of Aicardi–Goutières syndrome.

The expansion of the white adipose tissue (WAT) in obesity goes along with increased mechanical, metabolic and inflammatory stress. How adipocytes resist this stress is still poorly understood. Both in human and mouse adipocytes, the transcriptional co-activators YAP/TAZ and YAP/TAZ target genes become activated during obesity. When fed a high-fat diet (HFD), mice lacking YAP/TAZ in white adipocytes develop severe lipodystrophy with adipocyte cell death. The pro-apoptotic factor BIM, which is downregulated in adipocytes of obese mice and humans, is strongly upregulated in YAP/TAZ-deficient adipocytes under HFD, and suppression of BIM expression reduces adipocyte apoptosis. In differentiated adipocytes, TNFα and IL-1β promote YAP/TAZ nuclear translocation via activation of RhoA-mediated actomyosin contractility and increase YAP/TAZ-mediated transcriptional regulation by activation of c-Jun N-terminal kinase (JNK) and AP-1. Our data indicate that the YAP/TAZ signaling pathway may be a target to control adipocyte cell death and compensatory adipogenesis during obesity. The expansion of the white adipose tissue during obesity is accompanied by increased cellular stress, but factors that protect adipocytes from cell death are not well known. Here the authors report that the transcriptional co-activators YAP and TAZ are activated in adipocytes during obesity, which increases adipocyte survival through the proapoptotic factor BIM.

Type 2 diabetes (T2D) is characterized by a deficiency in β cell mass, increased β cell apoptosis, and extracellular accumulation of islet amyloid derived from islet amyloid polypeptide (IAPP), which β cells coexpress with insulin. IAPP expression is increased in the context of insulin resistance, the major risk factor for developing T2D. Human IAPP is potentially toxic, especially as membrane-permeant oligomers, which have been observed to accumulate within β cells of patients with T2D and rodents expressing human IAPP. Here, we determined that β cell IAPP content is regulated by autophagy through p62-dependent lysosomal degradation. Induction of high levels of human IAPP in mouse β cells resulted in accumulation of this amyloidogenic protein as relatively inert fibrils within cytosolic p62-positive inclusions, which temporarily averts formation of toxic oligomers. Mice hemizygous for transgenic expression of human IAPP did not develop diabetes; however, loss of β cell-specific autophagy in these animals induced diabetes, which was attributable to accumulation of toxic human IAPP oligomers and loss of β cell mass. In human IAPP-expressing mice that lack β cell autophagy, increased oxidative damage and loss of an antioxidant-protective pathway appeared to contribute to increased β cell apoptosis. These findings indicate that autophagy/lysosomal degradation defends β cells against proteotoxicity induced by oligomerization-prone human IAPP.