ALDH1A3-acetaldehyde metabolism potentiates transcriptional heterogeneity in melanoma.

Cancer cellular heterogeneity and therapy resistance arise substantially from metabolic and transcriptional adaptations, but how these are interconnected is poorly understood. Here, we show that, in melanoma, the cancer stem cell marker aldehyde dehydrogenase 1A3 (ALDH1A3) forms an enzymatic partnership with acetyl-coenzyme A (CoA) synthetase 2 (ACSS2) in the nucleus to couple high glucose metabolic flux with acetyl-histone H3 modification of neural crest (NC) lineage and glucose metabolism genes. Importantly, we show that acetaldehyde is a metabolite source for acetyl-histone H3 modification in an ALDH1A3-dependent manner, providing a physiologic function for this highly volatile and toxic metabolite. In a zebrafish melanoma residual disease model, an ALDH1-high subpopulation emerges following BRAF inhibitor treatment, and targeting these with an ALDH1 suicide inhibitor, nifuroxazide, delays or prevents BRAF inhibitor drug-resistant relapse. Our work reveals that the ALDH1A3-ACSS2 couple directly coordinates nuclear acetaldehyde-acetyl-CoA metabolism with specific chromatin-based gene regulation and represents a potential therapeutic vulnerability in melanoma.

Ciliary tip actin dynamics regulate photoreceptor outer segment integrity.

As signalling organelles, cilia regulate their G protein-coupled receptor content by ectocytosis, a process requiring localised actin dynamics to alter membrane shape. Photoreceptor outer segments comprise an expanse of folded membranes (discs) at the tip of highly-specialised connecting cilia, into which photosensitive GPCRs are concentrated. Discs are shed and remade daily. Defects in this process, due to mutations, cause retinitis pigmentosa (RP). Whilst fundamental for vision, the mechanism of photoreceptor disc generation is poorly understood. Here, we show membrane deformation required for disc genesis is driven by dynamic actin changes in a process akin to ectocytosis. We show RPGR, a leading RP gene, regulates actin-binding protein activity central to this process. Actin dynamics, required for disc formation, are perturbed in Rpgr mouse models, leading to aborted membrane shedding as ectosome-like vesicles, photoreceptor death and visual loss. Actin manipulation partially rescues this, suggesting the pathway could be targeted therapeutically. These findings help define how actin-mediated dynamics control outer segment turnover.

DNMT3B PWWP mutations cause hypermethylation of heterochromatin.

The correct establishment of DNA methylation patterns is vital for mammalian development and is achieved by the de novo DNA methyltransferases DNMT3A and DNMT3B. DNMT3B localises to H3K36me3 at actively transcribing gene bodies via its PWWP domain. It also functions at heterochromatin through an unknown recruitment mechanism. Here, we find that knockout of DNMT3B causes loss of methylation predominantly at H3K9me3-marked heterochromatin and that DNMT3B PWWP domain mutations or deletion result in striking increases of methylation in H3K9me3-marked heterochromatin. Removal of the N-terminal region of DNMT3B affects its ability to methylate H3K9me3-marked regions. This region of DNMT3B directly interacts with HP1α and facilitates the bridging of DNMT3B with H3K9me3-marked nucleosomes in vitro. Our results suggest that DNMT3B is recruited to H3K9me3-marked heterochromatin in a PWWP-independent manner that is facilitated by the protein's N-terminal region through an interaction with a key heterochromatin protein. More generally, we suggest that DNMT3B plays a role in DNA methylation homeostasis at heterochromatin, a process which is disrupted in cancer, aging and Immunodeficiency, Centromeric Instability and Facial Anomalies (ICF) syndrome.

Signal-Retaining Autophagy Indicator as a Quantitative Imaging Method for ER-Phagy.

Autophagy is an intracellular lysosomal degradation pathway by which cytoplasmic cargoes are removed to maintain cellular homeostasis. Monitoring autophagy flux is crucial to understand the autophagy process and its biological significance. However, assays to measure autophagy flux are either complex, low throughput or not sensitive enough for reliable quantitative results. Recently, ER-phagy has emerged as a physiologically relevant pathway to maintain ER homeostasis but the process is poorly understood, highlighting the need for tools to monitor ER-phagy flux. In this study, we validate the use of the signal-retaining autophagy indicator (SRAI), a fixable fluorescent probe recently generated and described to detect mitophagy, as a versatile, sensitive and convenient probe for monitoring ER-phagy. This includes the study of either general selective degradation of the endoplasmic reticulum (ER-phagy) or individual forms of ER-phagy involving specific cargo receptors (e.g., FAM134B, FAM134C, TEX264 and CCPG1). Crucially, we present a detailed protocol for the quantification of autophagic flux using automated microscopy and high throughput analysis. Overall, this probe provides a reliable and convenient tool for the measurement of ER-phagy.

Centriolar satellites expedite mother centriole remodeling to promote ciliogenesis.

Centrosomes are orbited by centriolar satellites, dynamic multiprotein assemblies nucleated by Pericentriolar material 1 (PCM1). To study the requirement for centriolar satellites, we generated mice lacking PCM1, a crucial component of satellites. Pcm1-/- mice display partially penetrant perinatal lethality with survivors exhibiting hydrocephalus, oligospermia, and cerebellar hypoplasia, and variably expressive phenotypes such as hydronephrosis. As many of these phenotypes have been observed in human ciliopathies and satellites are implicated in cilia biology, we investigated whether cilia were affected. PCM1 was dispensable for ciliogenesis in many cell types, whereas Pcm1-/- multiciliated ependymal cells and human PCM1-/- retinal pigmented epithelial 1 (RPE1) cells showed reduced ciliogenesis. PCM1-/- RPE1 cells displayed reduced docking of the mother centriole to the ciliary vesicle and removal of CP110 and CEP97 from the distal mother centriole, indicating compromised early ciliogenesis. Similarly, Pcm1-/- ependymal cells exhibited reduced removal of CP110 from basal bodies in vivo. We propose that PCM1 and centriolar satellites facilitate efficient trafficking of proteins to and from centrioles, including the departure of CP110 and CEP97 to initiate ciliogenesis, and that the threshold to trigger ciliogenesis differs between cell types.

Non-fitting FLIM-FRET facilitates analysis of protein interactions in live zebrafish embryos.

Molecular interactions are key to all cellular processes, and particularly interesting to investigate in the context of gene regulation. Protein-protein interactions are challenging to examine in vivo as they are dynamic, and require spatially and temporally resolved studies to interrogate them. Foerster Resonance Energy Transfer (FRET) is a highly sensitive imaging method, which can interrogate molecular interactions. FRET can be detected by Fluorescence Lifetime Imaging Microscopy (FLIM-FRET), which is more robust to concentration variations and photobleaching than intensity-based FRET but typically needs long acquisition times to achieve high photon counts. New variants of non-fitting lifetime-based FRET perform well in samples with lower signal and require less intensive instrument calibration and analysis, making these methods ideal for probing protein-protein interactions in more complex live 3D samples. Here we show that a non-fitting FLIM-FRET variant, based on the Average Arrival Time of photons per pixel (AAT- FRET), is a sensitive and simple way to detect and measure protein-protein interactions in live early stage zebrafish embryos.

A WDR35-dependent coat protein complex transports ciliary membrane cargo vesicles to cilia.

Intraflagellar transport (IFT) is a highly conserved mechanism for motor-driven transport of cargo within cilia, but how this cargo is selectively transported to cilia is unclear. WDR35/IFT121 is a component of the IFT-A complex best known for its role in ciliary retrograde transport. In the absence of WDR35, small mutant cilia form but fail to enrich in diverse classes of ciliary membrane proteins. In Wdr35 mouse mutants, the non-core IFT-A components are degraded and core components accumulate at the ciliary base. We reveal deep sequence homology of WDR35 and other IFT-A subunits to α and ß' COPI coatomer subunits and demonstrate an accumulation of 'coat-less' vesicles that fail to fuse with Wdr35 mutant cilia. We determine that recombinant non-core IFT-As can bind directly to lipids and provide the first in situ evidence of a novel coat function for WDR35, likely with other IFT-A proteins, in delivering ciliary membrane cargo necessary for cilia elongation.

Most human cells have at least one small hair-like structure on their surface called a cilium. These structures can act as antennae and allow the cell to sense signals from the rest of the body. To do this, they contain proteins that differ from the rest of the cell. The content of cilia depends on regulated delivery of these proteins in and out of cilia by a process called the intraflagellar transport or IFT, which involves a large complex made of several proteins. This complex shuttles the cargo proteins back and forth between the base and the tip of the cilia. However, ciliary proteins are not produced in the cilia; instead, they are made in a different part of the cell and then they are transported to the ciliary base. At the point where they enter the cilia, they were thought to bind to the assembling IFT 'trains' and be transported across the ciliary gate to the positions where they are needed in cilia. One of the components of the IFT machinery is a protein called WDR35, also known as IFT121. If the gene that codes for this protein is faulty or missing, it results in severe disorders in both humans and mice including a range of potentially lethal skeletal dysplasias. Interestingly, without WDR35, cells cannot build functional cilia. The absence of this protein not only disrupts IFT, stopping certain ciliary proteins and their associated membranes from entering cilia; it also causes a 'traffic jam' with a pile-up of transport intermediates from the place in cell where they are made to the cilia. It is unclear why a mutation in one of the components of the IFT would have this effect, raising the question of whether WDR35, or IFTs a whole, has another role in bringing the cargo proteins into the cilia. To understand this phenomenon, Quidwai et al. analysed the structure of WDR35 and other IFT proteins and found that they are very similar to a protein complex called COPI, which is involved in transporting membrane proteins around the cell. When certain proteins are newly made, they are stored in small lipid bubbles ­ called vesicles ­ that then selectively move to where the proteins are needed. COPI coats these vesicles, helping them get to where they need to go in a process called vesicular transport. Quidwai et al. found that WDR35 and other IFT proteins are able to bind to specific types of lipid molecules, suggesting that they might be assisting in a form of vesicle transport too. Indeed, when mouse cells grown in the lab were genetically engineered so they could not produce WDR35, coatless vesicles accumulated around the base of the cilia. Adding back WDR35 to these mutant cells rescued these defects in vesicle transport to cilia as well as allowed functional cilia to be formed. These results provide evidence that WDR35, likely with other IFT proteins, acts as a COPI-like complex to deliver proteins to growing cilia. Further research will investigate the composition of these vesicles that transport proteins to cilia, and help pinpoint where they originate. Quidwai et al.'s findings not only shed light on how different genetic mutations found in patients with cilia dysfunction affect different steps of transporting proteins to and within cilia. They also increase our understanding of the cellular roadmap by which cells shuttle building blocks around in order to assemble these important 'antennae'.

Identification and functional modelling of plausibly causative cis-regulatory variants in a highly-selected cohort with X-linked intellectual disability.

Identifying causative variants in cis-regulatory elements (CRE) in neurodevelopmental disorders has proven challenging. We have used in vivo functional analyses to categorize rigorously filtered CRE variants in a clinical cohort that is plausibly enriched for causative CRE mutations: 48 unrelated males with a family history consistent with X-linked intellectual disability (XLID) in whom no detectable cause could be identified in the coding regions of the X chromosome (chrX). Targeted sequencing of all chrX CRE identified six rare variants in five affected individuals that altered conserved bases in CRE targeting known XLID genes and segregated appropriately in families. Two of these variants, FMR1CRE and TENM1CRE, showed consistent site- and stage-specific differences of enhancer function in the developing zebrafish brain using dual-color fluorescent reporter assay. Mouse models were created for both variants. In male mice Fmr1CRE induced alterations in neurodevelopmental Fmr1 expression, olfactory behavior and neurophysiological indicators of FMRP function. The absence of another likely causative variant on whole genome sequencing further supported FMR1CRE as the likely basis of the XLID in this family. Tenm1CRE mice showed no phenotypic anomalies. Following the release of gnomAD 2.1, reanalysis showed that TENM1CRE exceeded the maximum plausible population frequency of a XLID causative allele. Assigning causative status to any ultra-rare CRE variant remains problematic and requires disease-relevant in vivo functional data from multiple sources. The sequential and bespoke nature of such analyses renders them time-consuming and challenging to scale for routine clinical use.

Identification of a localized nonsense-mediated decay pathway at the endoplasmic reticulum.

Nonsense-mediated decay (NMD) is a translation-dependent RNA quality control mechanism that occurs in the cytoplasm. However, it is unknown how NMD regulates the stability of RNAs translated at the endoplasmic reticulum (ER). Here, we identify a localized NMD pathway dedicated to ER-translated mRNAs. We previously identified NBAS, a component of the Syntaxin 18 complex involved in Golgi-to-ER trafficking, as a novel NMD factor. Furthermore, we show that NBAS fulfills an independent function in NMD. This ER-NMD pathway requires the interaction of NBAS with the core NMD factor UPF1, which is partially localized at the ER in the proximity of the translocon. NBAS and UPF1 coregulate the stability of ER-associated transcripts, in particular those associated with the cellular stress response. We propose a model where NBAS recruits UPF1 to the membrane of the ER and activates an ER-dedicated NMD pathway, thus providing an ER-protective function by ensuring quality control of ER-translated mRNAs.

DISC1 regulates N-methyl-D-aspartate receptor dynamics: abnormalities induced by a Disc1 mutation modelling a translocation linked to major mental illness.

The neuromodulatory gene DISC1 is disrupted by a t(1;11) translocation that is highly penetrant for schizophrenia and affective disorders, but how this translocation affects DISC1 function is incompletely understood. N-methyl-D-aspartate receptors (NMDAR) play a central role in synaptic plasticity and cognition, and are implicated in the pathophysiology of schizophrenia through genetic and functional studies. We show that the NMDAR subunit GluN2B complexes with DISC1-associated trafficking factor TRAK1, while DISC1 interacts with the GluN1 subunit and regulates dendritic NMDAR motility in cultured mouse neurons. Moreover, in the first mutant mouse that models DISC1 disruption by the translocation, the pool of NMDAR transport vesicles and surface/synaptic NMDAR expression are increased. Since NMDAR cell surface/synaptic expression is tightly regulated to ensure correct function, these changes in the mutant mouse are likely to affect NMDAR signalling and synaptic plasticity. Consistent with these observations, RNASeq analysis of the translocation carrier-derived human neurons indicates abnormalities of excitatory synapses and vesicle dynamics. RNASeq analysis of the human neurons also identifies many differentially expressed genes previously highlighted as putative schizophrenia and/or depression risk factors through large-scale genome-wide association and copy number variant studies, indicating that the translocation triggers common disease pathways that are shared with unrelated psychiatric patients. Altogether, our findings suggest that translocation-induced disease mechanisms are likely to be relevant to mental illness in general, and that such disease mechanisms include altered NMDAR dynamics and excitatory synapse function. This could contribute to the cognitive disorders displayed by translocation carriers.

Regulation of mitochondrial dynamics by DISC1, a putative risk factor for major mental illness.

Mitochondria are dynamic organelles that are essential to power the process of neurotransmission. Neurons must therefore ensure that mitochondria maintain their functional integrity and are efficiently transported along the full extent of the axons and dendrites, from soma to synapses. Mitochondrial dynamics (trafficking, fission and fusion) co-ordinately regulate mitochondrial quality control and function. DISC1 is a component of the mitochondrial transport machinery and regulates mitochondrial dynamics. DISC1's role in this is adversely affected by sequence variants connected to brain structure/function and disease risk, and by mutant truncation. The DISC1 interactors NDE1 and GSK3ß are also involved, indicating a convergence of putative risk factors for psychiatric illness upon mitochondrial dynamics.

NDE1 and GSK3ß Associate with TRAK1 and Regulate Axonal Mitochondrial Motility: Identification of Cyclic AMP as a Novel Modulator of Axonal Mitochondrial Trafficking.

Mitochondria are essential for neuronal function, providing the energy required to power neurotransmission, and fulfilling many important additional roles. In neurons, mitochondria must be efficiently transported to sites, including synapses, where their functions are required. Neurons, with their highly elongated morphology, are consequently extremely sensitive to defective mitochondrial trafficking which can lead to neuronal ill-health/death. We recently demonstrated that DISC1 associates with mitochondrial trafficking complexes where it associates with the core kinesin and dynein adaptor molecule TRAK1. We now show that the DISC1 interactors NDE1 and GSK3ß also associate robustly with TRAK1 and demonstrate that NDE1 promotes retrograde axonal mitochondrial movement. GSK3ß is known to modulate axonal mitochondrial motility, although reports of its actual effect are conflicting. We show that, in our system, GSK3ß promotes anterograde mitochondrial transport. Finally, we investigated the influence of cAMP elevation upon mitochondrial motility, and found a striking increase in mitochondrial motility and retrograde movement. DISC1, NDE1, and GSK3ß are implicated as risk factors for major mental illness. Our demonstration that they function together within mitochondrial trafficking complexes suggests that defective mitochondrial transport may be a contributory disease mechanism in some cases of psychiatric disorder.

Variation in the autism candidate gene GABRB3 modulates tactile sensitivity in typically developing children.

BACKGROUND: Autism spectrum conditions have a strong genetic component. Atypical sensory sensitivities are one of the core but neglected features of autism spectrum conditions. GABRB3 is a well-characterised candidate gene for autism spectrum conditions. In mice, heterozygous Gabrb3 deletion is associated with increased tactile sensitivity. However, no study has examined if tactile sensitivity is associated with GABRB3 genetic variation in humans. To test this, we conducted two pilot genetic association studies in the general population, analysing two phenotypic measures of tactile sensitivity (a parent-report and a behavioural measure) for association with 43 SNPs in GABRB3. FINDINGS: Across both tactile sensitivity measures, three SNPs (rs11636966, rs8023959 and rs2162241) were nominally associated with both phenotypes, providing a measure of internal validation. Parent-report scores were nominally associated with six SNPs (P <0.05). Behaviourally measured tactile sensitivity was nominally associated with 10 SNPs (three after Bonferroni correction). CONCLUSIONS: This is the first human study to show an association between GABRB3 variation and tactile sensitivity. This provides support for the evidence from animal models implicating the role of GABRB3 variation in the atypical sensory sensitivity in autism spectrum conditions. Future research is underway to directly test this association in cases of autism spectrum conditions.