Biology subjects

Williams, C.

Publications and source records attributed to Williams, C..

At least 91 records · Page 5Linked to original sources

bioRxiv2021

Estimating the glutamate transporter surface density in mouse hippocampal astrocytes

Glutamate transporters preserve the spatial specificity of synaptic transmission by limiting glutamate diffusion away from the synaptic cleft, and prevent excitotoxicity by keeping the extracellular concentration of glutamate at low nanomolar levels. Glutamate transporters are abundantly expressed in astrocytes, and previous estimates have been obtained about their surface expression in astrocytes of the rat hippocampus and cerebellum. Analogous estimates for the mouse hippocampus are currently not available. In this work, we derive the surface density of astrocytic glutamate transporters in mice of different ages via quantitative dot blot. We find that the surface density of glial glutamate transporters is similar in 7-8 week old mice and rats. In mice, the levels of glutamate transporters increase until about 6 months of age and then begin to decline slowly. Our data, obtained from a combination of experimental and modeling approaches, show that there are stark differences in the density of expression of glutamate transporters across different sub-cellular compartments, indicating that the extent to which astrocytes limit extrasynaptic glutamate diffusion depends not only on their level of synaptic coverage, but also on the identity of the astrocyte compartment in contact with the synapse. Together, these findings provide information on how heterogeneity in the spatial distribution of glutamate transporters in the plasma membrane of hippocampal astrocytes my alter glutamate receptor activation out of the synaptic cleft. Author summaryIn this work, we use experimental and modeling approaches to estimate the surface density of the glial glutamate transporters GLAST and GLT-1 in mouse hippocampal astrocytes. The results show that the surface density of glutamate transporters in hippocampal astrocytes is similar in 7-8 week old mice and rats. In mice, GLAST and GLT-1 reach their peak expression at ~ 6 months of age. Their expression, however, varies widely across different sub-cellular compartments. These findings indicate that the identity of the sub-cellular compartment of an astrocyte in contact with a glutamatergic synapses, in addition to the extent of its synaptic coverage, is a main factor to limit glutamate spillover and receptor activation at a distance from the release site.

neuroscience↗

bioRxiv2021

Rigidifying a de novo enzyme increases activity and induces a negative activation heat capacity

Conformational sampling profoundly impacts the overall activity and temperature dependence of enzymes. Peroxidases have emerged as versatile platforms for high value biocatalysis owing to their broad palette of potential biotransformations. Here, we explore the role of conformational sampling in mediating a de novo peroxidases activity. We demonstrate that 2,2,2-triflouoroethanol (TFE) affects the equilibrium of enzyme conformational states, tending towards a more globally rigid structure. This is correlated with increases both stability and activity. Notably, these effects are concomitant with the emergence of curvature in the temperature-activity profile, trading off activity gains at ambient temperature with losses at high temperatures. We apply macromolecular rate theory (MMRT) to understand enzyme temperature dependence data. These data point to an increase in protein rigidity associated with a difference in the distribution of protein dynamics between the ground and transition state. We compare the thermodynamics of the de novo enzyme activity to those of a natural peroxidase, horseradish peroxidase. We find that the native enzyme resembles the rigidified de novo enzyme in terms of the thermodynamics of enzyme catalysis and the putative distribution of protein dynamics between the ground and transition state. The addition of TFE apparently causes C45 to behave more like the natural enzyme. Our data suggest robust, generic strategies for improving biocatalytic activity by manipulating protein rigidity; for functional de novo protein catalysts in particular, this can provide more enzyme-like catalysts without further rational engineering, computational redesign or directed evolution.

biochemistry↗

bioRxiv2021

Transgenic mice for in vivo epigenome editing with CRISPR-based systems

The discovery, characterization, and adaptation of the RNA-guided clustered regularly interspersed short palindromic repeat (CRISPR)-Cas9 system has greatly increased the ease with which genome and epigenome editing can be performed. Fusion of chromatin-modifying domains to the nuclease-deactivated form of Cas9 (dCas9) has enabled targeted gene activation or repression in both cultured cells and in vivo in animal models. However, delivery of the large dCas9 fusion proteins to target cell types and tissues is an obstacle to widespread adoption of these tools for in vivo studies. Here we describe the generation and validation of two conditional transgenic mouse lines for targeted gene regulation, Rosa26:LSL-dCas9-p300 for gene activation and Rosa26:LSL-dCas9-KRAB for gene repression. Using the dCas9p300 and dCas9KRAB transgenic mice we demonstrate activation or repression of genes in both the brain and liver in vivo, and T cells and fibroblasts ex vivo. We show gene regulation and targeted epigenetic modification with gRNAs targeting either transcriptional start sites (TSS) or distal enhancer elements, as well as corresponding changes to downstream phenotypes. These mouse lines are convenient and valuable tools for facile, temporally controlled, and tissue-restricted epigenome editing and manipulation of gene expression in vivo.

genomics↗

bioRxiv2021

Recurrent Vulvovaginal Candidiasis; a dynamic interkingdom biofilm disease of Candida and Lactobacillus

Vulvovaginal Candidiasis (VVC) is the most prevalent Candida infection in humans affecting 75% of women at least once throughout their lifetime. In its debilitating recurrent form, RVVC is estimated to affect 140 million women annually. Despite this strikingly high prevalence, treatment options for RVVC remain limited with many women experiencing failed clinical treatment with frontline azoles. Further, the cause of onset and recurrence of disease is largely unknown with few studies identifying potential mechanisms of failed treatment. This study aimed to assess a panel of clinical samples from healthy women and those with RVVC to investigate the influence of Candida, vaginal microbiome and antagonism between Candida and Lactobacillus on disease pathology. 16S rRNA sequencing characterised disease by a reduction in specific health-associated Lactobacillus such as L. crispatus, coupled with an increase in L. iners. In vitro analysis showed Candida albicans clinical isolates are capable of heterogeneous biofilm formation and show the presence of hyphae and C. albicans aggregates in vaginal lavage. Additionally, the ability of Lactobacillus to inhibit C. albicans biofilm formation and biofilm-related gene expression was demonstrated. Using RNA sequencing technology, we were able to exploit a possible mechanism by which L. crispatus may aim to re-establish a healthy vaginal environment through amino-acid acquisition from C. albicans. This study suggests RVVC is not entirely due to an arbitrary switch in C. albicans from commensal to pathogen and understanding interactions between the yeast and vaginal Lactobacillus species may be more crucial to elucidating the cause of RVVC and developing appropriate therapies.

microbiology↗

bioRxiv2021

Molecular mechanism for kinesin-1 direct membrane recognition

The cargo-binding capabilities of cytoskeletal motor proteins have expanded during evolution through both gene duplication and alternative splicing. For the light chains of the kinesin-1 family of microtubule motors, this has resulted in an array of carboxy-terminal domain sequences of unknown molecular function. Here, combining phylogenetic analyses with biophysical, biochemical and cell biology approaches we identify a highly conserved membrane-induced curvature-sensitive amphipathic helix within this region of a newly defined subset of long kinesin light chain paralogues and splice isoforms. This helix mediates the direct binding of kinesin-1 to lipid membranes. Membrane binding requires specific anionic phospholipids and is important for kinesin-1 dependent lysosome positioning, a canonical activity that until now has been attributed exclusively the recognition of organelle-associated cargo adaptor proteins. This leads us to propose a new protein-lipid coincidence detection framework for kinesin-1 mediated organelle transport.

cell biology↗

bioRxiv2020

Making the invisible enemy visible

During the COVID-19 pandemic, structural biologists rushed to solve the structures of the 28 proteins encoded by the SARS-CoV-2 genome in order to understand the viral life cycle and enable structure-based drug design. In addition to the 204 previously solved structures from SARS-CoV-1, 548 structures covering 16 of the SARS-CoV-2 viral proteins have been released in a span of only 6 months. These structural models serve as the basis for research to understand how the virus hijacks human cells, for structure-based drug design, and to aid in the development of vaccines. However, errors often occur in even the most careful structure determination - and may be even more common among these structures, which were solved quickly and under immense pressure. The Coronavirus Structural Task Force has responded to this challenge by rapidly categorizing, evaluating and reviewing all of these experimental protein structures in order to help downstream users and original authors. In addition, the Task Force provided improved models for key structures online, which have been used by Folding@Home, OpenPandemics, the EU JEDI COVID-19 challenge and others.

molecular biology↗

bioRxiv2020

Regulation of gene transcription by thyroid hormone receptor β agonists in clinical development for the treatment of non-alcoholic steatohepatitis (NASH)

Thyroid hormones are important modulators of metabolic activity in mammals and alter cholesterol and fatty acid levels through activation of the nuclear thyroid hormone receptor (THR). Currently, there are several THR{beta} agonists in clinical trials for the treatment of non-alcoholic steatohepatitis (NASH) that have demonstrated the potential to reduce liver fat and restore liver function. In this study, we tested three THR{beta}-agonism-based NASH treatment candidates, GC-1 (sobetirome), MGL-3196 (resmetirom), and VK2809, and compared their selectivity for THR{beta} and their ability to modulate the expression of genes specific to cholesterol and fatty acid biosynthesis and metabolism in vitro using human hepatic cells and in vivo using a rat model. Treatment with GC-1 upregulated the transcription of CPT1A in the human hepatocyte-derived Huh-7 cell line with a dose-response comparable to that of the native THR ligand, triiodothyronine (T3). VK2809A (active parent of VK2809), MGL-3196, and VK2809 were approximately 30-fold, 1,000-fold, and 2,000-fold less potent than T3, respectively. Additionally, these relative potencies were confirmed by quantification of other direct gene targets of THR, namely, ANGPTL4 and DIO1. In primary human hepatocytes, potencies were conserved for every compound except for VK2809, which showed significantly increased potency that was comparable to that of its active counterpart, VK2809A. In high-fat diet fed rats, a single dose of T3 significantly reduced total cholesterol levels and concurrently increased liver Dio1 and Me1 RNA expression. MGL-3196 treatment resulted in concentration-dependent decreases in total and low-density lipoprotein cholesterol with corresponding increases in liver gene expression, but the compound was significantly less potent than T3. In conclusion, we have implemented a strategy to rank the efficacy of THR{beta} agonists by quantifying changes in the transcription of genes that lead to metabolic alterations, an effect that is directly downstream of THR binding and activation.

pharmacology and toxicology↗

bioRxiv2020

Precision design of single and multi-heme de novo proteins

The de novo design of simplified porphyrin-binding helical bundles is a versatile approach for the construction of valuable biomolecular tools to both understand and enhance protein functions such as electron transfer, oxygen binding and catalysis. However, the methods utilised to design such proteins by packing hydrophobic side chains into a buried binding pocket for ligands such as heme have typically created highly flexible, molten globule-like structures, which are not amenable to structural determination, hindering precise engineering of subsequent designs. Here we report the crystal structure of a de novo two-heme binding "maquette" protein, 4D2, derived from the previously designed D2 peptide, offering new opportunities for computational design and re-engineering. The 4D2 structure was used as a basis to create a range of heme binding proteins which retain the architecture and stability of the initial crystal structure. A well-structured single-heme binding variant was constructed by computational sequence redesign of the hydrophobic protein core, assessed by NMR, and utilised for experimental validation of computational redox prediction and design. The structure was also extended into a four-heme binding helical bundle resembling a molecular wire. Despite a molecular weight of only 24kDa, imaging by CryoEM illustrated a remarkable level of detail in this structure, indicating the positioning of both the secondary structure and the heme cofactors. The design and determination of atomic-level resolution in such de novo proteins is an invaluable resource for the continued development of novel and functional protein tools.

synthetic biology↗

bioRxiv2020

Inhibition of Streptococcus pneumoniae autolysins highlight distinct differences between chemical and genetic inactivation

Despite renewed interest, development of chemical biology methods to study peptidoglycan metabolism has lagged in comparison to the glycobiology field in general. To address this, a panel of diamides were screened against the Gram-positive pathogen Streptococcus pneumoniae to identify inhibitors of bacterial growth. The screen identified the diamide fgkc as a narrow spectrum bacteriostatic inhibitor of S. pneumoniae growth with an MIC of 7.8 M. The diamide inhibited detergent-induced autolysis in a concentration dependent manner indicating peptidoglycan degradation as the mode-of-action. Genetic screening of autolysin mutants suggested LytB, an endo-N-acetylglucosaminidase, involved in cell division as the potential target. Surprisingly, biochemical, and phenotypic analysis contradicted the genetic screen results. Phenotypic studies with the{Delta} lytb strain illustrate the difference between genetic and chemical inactivation of autolysins. These findings suggest that meta-phenotypes including autolytic activity, cell morphology, and genetic screening can be the result of the complex interaction of one or more possible pathways that are connected to cell wall metabolism.

biochemistry↗

bioRxiv2020

Extracellular matrix and cyclic stretch alter fetal cardiomyocyte proliferation and maturation in a rodent model of heart hypoplasia

Birth defects, particularly those that affect development of the heart, are a leading cause of morbidity and mortality in infants and young children. Babies born with heart hypoplasia (HH) disorders often have a poor prognosis. It remains unclear whether cardiomyocytes from hypoplastic hearts retain the potential to recover growth, although this knowledge would be beneficial for developing therapies for HH disorders. The objective of this study was to determine the proliferation and maturation potential of cardiomyocytes from hypoplastic hearts and whether these behaviors are influenced by biophysical signaling from the extracellular matrix (ECM) and cyclic mechanical stretch. Congenital diaphragmatic hernia (CDH)-associated HH was induced in rat fetuses by maternal exposure to nitrofen. Hearts were isolated from embryonic day 21 nitrofen-treated fetuses positive for CDH (CDH+) and from fetuses without nitrofen administration during gestation. CDH+ hearts were smaller and had decreased myocardial proliferation, along with evidence of decreased maturity compared to healthy hearts. In culture, CDH+ cardiomyocytes remained immature and demonstrated increased proliferative capacity compared to their healthy counterparts. Culture on ECM derived from CDH+ hearts led to a significant reduction in proliferation for both CDH+ and healthy cardiomyocytes. Healthy cardiomyocytes were dosed with exogenous nitrofen to examine whether nitrofen may have an abhorrent effect on the proliferative ability of cardiomyocyte, yet no significant change in proliferation was observed. When subjected to stretch, CDH+ cardiomyocytes underwent lengthening of sarcomeres while healthy cardiomyocyte sarcomeres were unaffected. Taken together, our results suggest that alterations to environmental cues such as ECM and stretch may be important factors in the pathological progression of HH.

cell biology↗

bioRxiv2020

Tsetse salivary glycoproteins are modified with paucimannosidic N-glycans, are recognised by C-type lectins and bind to trypanosomes

African sleeping sickness is caused by Trypanosoma brucei, a parasite transmitted by the bite of a tsetse fly. Trypanosome infection induces a severe transcriptional downregulation of tsetse genes encoding for salivary proteins, which reduces its anti-hemostatic and anti-clotting properties. To better understand trypanosome transmission and the possible role of glycans in insect bloodfeeding, we characterized the N-glycome of tsetse saliva glycoproteins. Tsetse salivary N-glycans were enzymatically released, tagged with either 2-aminobenzamide (2-AB) or procainamide, and analyzed by HILIC-UHPLC-FLR coupled online with positive-ion ESI-LC-MS/MS. We found that the N-glycan profiles of T. brucei-infected and naive tsetse salivary glycoproteins are almost identical, consisting mainly (>50%) of highly processed Man3GlcNAc2 in addition to several other paucimannose, high mannose, and few hybrid-type glycans. In overlay assays, these sugars were differentially recognized by the C-type lectins mannose receptor and DC-SIGN. We also show that salivary glycoproteins bind strongly to the surface of transmissible metacyclic trypanosomes. We suggest that although the repertoire of tsetse salivary N-glycans does not change during a trypanosome infection, the interactions with mannosylated glycoproteins may influence parasite transmission into the vertebrate host.

biochemistry↗

bioRxiv2020

High-fat diet impacts the colon and its transcriptome in a sex-dependent manner that is modifiable by estrogens

Epidemiological studies highlight a strong association between obesity and colorectal cancer (CRC), especially in men. Estrogen, on the other hand, is associated with protection against both the metabolic syndrome and CRC. The colon is the first organ to respond to a high-fat diet (HFD), and estrogen receptor beta (ER{beta}) in the intestine appears to prevent CRC. How estrogen impacts the colon under HFD condition has, however, not been investigated. Estrogen can act through three different receptors (ER, ER{beta}, GPER1) which all may impact metabolism. In an effort to dissect this, we fed mice a control diet or a high-fat diet (HFD) for 13 weeks and administered receptor-selective estrogenic ligands for the last three weeks. We recorded corresponding physiological impact on fat distribution, fasting glucose, colon crypt proliferation and immune cell infiltration, and the colon transcriptome response. We identify clear sex-differences at the transcriptome level, both at base line and after HFD and ligand treatments. An unexpected observation was the significant sex-differences and impact by HFD and estrogens on circadian clock gene expression, such as Npas2 and Arntl (Bmal1), in the colon. Both sexes also exhibited an increased infiltration of F4/80+ macrophages as a result of HFD. In males, but not females, this was accompanied by changes in colonic epithelial cell proliferation. ER-selective PPT treatment had significant systemic effects, reducing body weight in both sexes, whereas ER{beta}-selective DPN treatment did not impact body weight, but reduced infiltration of F4/80+ macrophages in colon of both sexes and attenuated HFD-induced proliferation of male colon crypts. Both ER and ER{beta} activation contributed to circadian clock gene regulations. We detail for the first time how HFD and estrogens modulate the colon transcriptome and physiology in a sex and ER-specific manner.

cell biology↗

bioRxiv2020

Candida auris phenotypic heterogeneity determines pathogenicity in vitro

Candida auris is an enigmatic yeast that provides substantial global risk in healthcare facilities and intensive care units. A unique phenotype exhibited by certain isolates of C. auris is their ability to form small clusters of cells known as aggregates, which have been to a limited extent described in the context of pathogenic traits. In this study, we screened several non-aggregative and aggregative C. auris isolates for biofilm formation, where we observed a level of heterogeneity amongst the different phenotypes. Next, we utilised an RNA-sequencing approach to investigate the transcriptional responses during biofilm formation of a non-aggregative and aggregative isolate of the initial pool. Observations from these analyses indicate unique transcriptional profiles in the two isolates, with several genes identified relating to proteins involved in adhesion and invasion of the host in other fungal species. From these findings we investigated for the first time the fungal recognition and inflammatory responses of a three-dimensional skin epithelial model to these isolates. In these models, a wound was induced to mimic a portal of entry for C. auris. We show both phenotypes elicited minimal response in the model minus induction of the wound, yet in the wounded tissue both phenotypes induced a greater response, with the aggregative isolate more pro-inflammatory. This capacity of aggregative C. auris biofilms to generate such responses in the wounded skin highlights how this opportunistic yeast is a high risk within the intensive care environment where susceptible patients have multiple indwelling lines. ImportanceCandida auris has recently emerged as an important cause of concern within healthcare environments due to its ability to persist and tolerate commonly used antiseptics and disinfectants, particularly when surface attached (biofilms). This yeast is able to colonise and subsequently infect patients, particularly those that are critically ill or immunosuppressed, which may result in death. We have undertaken analysis on two different types of this yeast, using molecular and immunological tools to determine whether either of these has a greater ability to cause serious infections. We describe that both isolates exhibit largely different transcriptional profiles during biofilm development. Finally, we show that the inability to form small aggregates (or clusters) of cells has an adverse effect on the organisms immuno-stimulatory properties, suggestive the non-aggregative phenotype may exhibit a certain level of immune evasion.

microbiology↗

bioRxiv2020

An in silico map of the SARS-CoV-2 RNA Structurome

SARS-CoV-2 is a positive-sense single-stranded RNA virus that has exploded throughout the global human population. This pandemic coronavirus strain has taken scientists and public health researchers by surprise and knowledge of its basic biology (e.g. structure/function relationships in its genomic, messenger and template RNAs) and modes for therapeutic intervention lag behind that of other human pathogens. In this report we used a recently-developed bioinformatics approach, ScanFold, to deduce the RNA structural landscape of the SARS-CoV-2 transcriptome. We recapitulate known elements of RNA structure and provide a model for the folding of an essential frameshift signal. Our results find that the SARS-CoV-2 is greatly enriched in unusually stable and likely evolutionarily ordered RNA structure, which provides a huge reservoir of potential drug targets for RNA-binding small molecules. Our results also predict regions that are accessible for intermolecular interactions, which can aid in the design of antisense therapeutics. All results are made available via a public database (the RNAStructuromeDB) where they may hopefully drive drug discovery efforts to inhibit SARS-CoV-2 pathogenesis.

bioinformatics↗

bioRxiv2020

Potentiating TMEM16A channel function has no effect on airway goblet cells or bronchial and pulmonary vascular smooth muscle function

The calcium-activated chloride channel TMEM16A enables chloride secretion across several transporting epithelia, including in the airway where it represents a therapeutic target for the treatment of cystic fibrosis. Additional roles for TMEM16A have also been proposed, including enhancing goblet cell exocytosis, increasing goblet cell numbers and stimulating smooth muscle contraction. The aim of the present study was to test whether the pharmacological regulation of TMEM16A channel function, both potentiation and inhibition, could affect any of these proposed biological roles. In vitro, a recently described potent and selective TMEM16A potentiator (ETX001) failed to stimulate mucin release from primary human bronchial epithelial (HBE) cells over a 24h exposure period using both biochemical and imaging endpoints. In addition, treatment of HBE cells with ETX001 or a potent and selective TMEM16A inhibitor (Ani9) for 4 days did not influence mucin release or goblet cell formation. In vivo, a TMEM16A potentiator was without effect on goblet cell emptying in an IL-13 driven goblet cell metaplasia model. Using freshly isolated human bronchi and pulmonary arteries, neither ETX001 or Ani9 had any effect on the contractile or relaxant responses of the tissues. In vivo, ETX001 also failed to influence either lung or cardiovascular function when delivered directly into the airways of telemetered rats. Together, these studies do not support a role for TMEM16A in the regulation of goblet cell numbers or mucin release, or on the regulation of airway or pulmonary artery smooth muscle contraction.

physiology↗

bioRxiv2020

Coordinated assembly and release of adhesions builds apical junctional belts during de novo polarisation of an epithelial tube

Using the zebrafish neural tube as a model, we uncover the in vivo mechanisms allowing the generation of two opposing apical epithelial surfaces within the centre of an initially unpolarised, solid organ. We show that NOK/Pals1/Mpp5a and Rab11a play a dual role in coordinating the generation of ipsilateral junctional belts whilst simultaneously releasing contralateral adhesions across the centre of the tissue. We show that Nok and Rab11a mediated resolution of cell-cell adhesions are both necessary for midline lumen opening and contribute to later maintenance of epithelial organisation. We propose these roles for both Nok and Rab11a operate through the transmembrane protein Crumbs. In light of a recent conflicting publication, we also clarify that the junction remodelling role of Nok is not specific to dividing cells.

neuroscience↗

bioRxiv2019

Ankylosis homologue (ANKH) controls extracellular citrate and pyrophosphate homeostasis and affects bone mechanical performance

The membrane protein Ankylosis homologue (ANKH, mouse orthologue: ANK) prevents mineralization of joint-space and articular cartilage. The accepted view is that ANKH mediates cellular release of inorganic pyrophosphate (PPi), a strong physiological inhibitor of mineralization. Using global metabolite profiling, we identified citrate as the most prominent metabolite leaving HEK293 cells in an ANKH-dependent manner. Although PPi levels were increased in culture medium of HEK293-ANKH cells, PPi was formed extracellularly after release of ATP and other nucleoside triphosphates. Ankank/ank mice, which lack functional ANK, had substantially reduced concentrations of citrate in plasma and urine, while citrate was undetectable in urine of a human patient lacking functional ANKH. Bone hydroxyapatite of Ankank/ank mice also contained markedly reduced levels of citrate and PPi and displayed diminished strength. Together, our data show that ANKH is a crucial factor in extracellular citrate and PPi homeostasis that is essential for normal bone development.

developmental biology↗

bioRxiv2019

Wnt Regulation: Exploring Axin-Disheveled interactions and defining mechanisms by which the SCF E3 ubiquitin ligase is recruited to the destruction complex

Wnt signaling plays key roles in embryonic development and adult stem cell homeostasis, and is altered in human cancer. Signaling is turned on and off by regulating stability of the effector {beta}-catenin. The multiprotein destruction complex binds and phosphorylates {beta}-catenin, and transfers it to the SCF-TrCP E3-ubiquitin ligase, for ubiquitination and destruction. Wnt signals act though Dishevelled to turn down the destruction complex, stabilizing {beta}-catenin. Recent work clarified underlying mechanisms, but important questions remain. We explore {beta}-catenin transfer from the destruction complex to the E3 ligase, and test models suggesting Dishevelled and APC2 compete for association with Axin. We find that Slimb/TrCP is a dynamic component of the destruction complex biomolecular condensate, while other E3 proteins are not. Recruitment requires Axin and not APC, and Axins RGS domain plays an important role. We find that elevating Dishevelled levels in Drosophila embryos has paradoxical effects, promoting the ability of limiting levels of Axin to turn off Wnt signaling. When we elevate Dishevelled levels, it forms its own cytoplasmic puncta, but these do not recruit Axin. SIM imaging in mammalian cells suggests that this may result by promoting Dishevelled: Dishevelled interactions at the expense of Dishevelled:Axin interactions when Dishevelled levels are high.

cell biology↗