Telomere crisis occurs during tumorigenesis when depletion of the telomere reserve leads to frequent telomere fusions. The resulting dicentric chromosomes have been proposed to drive genome instability. Here we examine the fate of dicentric human chromosomes in telomere crisis. We observed that dicentric chromosomes invariably persisted through mitosis and developed into 50-200 μm chromatin bridges connecting the daughter cells. Before their resolution at 3-20 h after anaphase, the chromatin bridges induced nuclear envelope rupture in interphase, accumulated the cytoplasmic 3' nuclease TREX1, and developed RPA-coated single stranded (ss) DNA. CRISPR knockouts showed that TREX1 contributed to the generation of the ssDNA and the resolution of the chromatin bridges. Post-crisis clones showed chromothripsis and kataegis, presumably resulting from DNA repair and APOBEC editing of the fragmented chromatin bridge DNA. We propose that chromothripsis in human cancer may arise through TREX1-mediated fragmentation of dicentric chromosomes formed in telomere crisis.
telomere crisis; chromothripsis; kataegis; NERDI; TREX1; APOBEC; dicentric chromosome
We have defined a network of interacting Drosophila cell surface proteins in which a 21-member IgSF subfamily, the Dprs, binds to a 9-member subfamily, the DIPs. The structural basis of the Dpr-DIP interaction code appears to be dictated by shape complementarity within the Dpr-DIP binding interface. Each of the 6 dpr and DIP genes examined here is expressed by a unique subset of larval and pupal neurons. In the neuromuscular system, interactions between Dpr11 and DIP-γ affect presynaptic terminal development, trophic factor responses, and neurotransmission. In the visual system, dpr11 is selectively expressed by R7 photoreceptors that use Rh4 opsin (yR7s). Their primary synaptic targets, Dm8 amacrine neurons, express DIP-γ. In dpr11 or DIP-γ mutants, yR7 terminals extend beyond their normal termination zones in layer M6 of the medulla. DIP-γ is also required for Dm8 survival or differentiation. Our findings suggest that Dpr-DIP interactions are important determinants of synaptic connectivity.
synaptogenesis; cell adhesion; MiMIC; GFP tagging; optic lobe; immunoglobulin
Learned and adaptive behaviors rely on neural circuits that flexibly couple the same sensory input to alternative output pathways. Here, we show that the Drosophila mushroom body functions like a switchboard in which neuromodulation reroutes the same odor signal to different behavioral circuits, depending on the state and experience of the fly. Using functional synaptic imaging and electrophysiology, we reveal that dopaminergic inputs to the mushroom body modulate synaptic transmission with exquisite spatial specificity, allowing individual neurons to differentially convey olfactory signals to each of their postsynaptic targets. Moreover, we show that the dopaminergic neurons function as an interconnected network, encoding information about both an animal's external context and internal state to coordinate synaptic plasticity throughout the mushroom body. Our data suggest a general circuit mechanism for behavioral flexibility in which neuromodulatory networks act with synaptic precision to transform a single sensory input into different patterns of output activity.
Spatial genome organization and its effect on transcription remains a fundamental question. We applied an advanced ChIA-PET strategy to comprehensively map higher-order chromosome folding and specific chromatin interactions mediated by CTCF and RNAPII with haplotype specificity and nucleotide resolution in different human cell lineages. We find that CTCF/cohesin-mediated interaction anchors serve as structural foci for spatial organization of constitutive genes concordant with CTCF-motif orientation, whereas RNAPII interacts within these structures by selectively drawing cell-type-specific genes towards CTCF-foci for coordinated transcription. Furthermore, we show that haplotype-variants and allelic-interactions have differential effects on chromosome configuration influencing gene expression and may provide mechanistic insights into functions associated with disease susceptibility. 3D-genome simulation suggests a model of chromatin folding around chromosomal axes, where CTCF is involved in defining the interface between condensed and open compartments for structural regulation. Our 3D-genome strategy thus provides unique insights in the topological mechanism of human variations and diseases.
The goal of understanding living nervous systems has driven interest in high-speed and large field-of-view volumetric imaging at cellular resolution. Light-sheet microscopy approaches have emerged for cellular-resolution functional brain imaging in small organisms such as larval zebrafish, but remain fundamentally limited in speed. Here we have developed SPED light sheet microscopy, which combines large volumetric field-of-view via an extended depth of field with the optical sectioning of light sheet microscopy, thereby eliminating the need to physically scan detection objectives for volumetric imaging. SPED enables scanning of thousands of volumes-per-second, limited only by camera acquisition rate, through the harnessing of optical mechanisms that normally result in unwanted spherical aberrations. We demonstrate capabilities of SPED microscopy by performing fast sub-cellular resolution imaging of CLARITY mouse brains and cellular-resolution volumetric Ca2+ imaging of entire zebrafish nervous systems. Together, SPED light sheet methods enable high-speed cellular-resolution volumetric mapping of biological system structure and function.
Cellular lipid requirements are achieved through a combination of biosynthesis and import programs. Using isotope tracer analysis, we show that type I interferon (IFN) signaling shifts the balance of these programs by decreasing synthesis and increasing import of cholesterol and long chain fatty acids. Genetically enforcing this metabolic shift in macrophages is sufficient to render mice resistant to viral challenge, demonstrating the importance of reprogramming the balance of these two metabolic pathways in vivo. Unexpectedly, mechanistic studies reveal that limiting flux through the cholesterol biosynthetic pathway spontaneously engages a type I IFN response in a STING-dependent manner. The upregulation of type I IFNs was traced to a decrease in the pool size of synthesized cholesterol, and could be inhibited by replenishing cells with free cholesterol. Taken together, these studies delineate a metabolic-inflammatory circuit that links perturbations in cholesterol biosynthesis with activation of innate immunity.
Trimethylamine N-oxide (TMAO), a gut microbiota dependent metabolite, both enhances atherosclerosis in animal models and is associated with cardiovascular risks in clinical studies. Here we investigate the impact of targeted inhibition of the first step in TMAO generation, commensal microbial trimethylamine (TMA) production, on diet-induced atherosclerosis. A structural analogue of choline, 3,3-dimethyl-1-butanol (DMB), is shown to non-lethally inhibit TMA formation from cultured microbes, to inhibit distinct microbial TMA lyases, and to both inhibit TMA production from physiologic polymicrobial cultures (eg intestinal contents, human feces) and reduce TMAO levels in mice fed a high choline or carnitine diet. DMB inhibited choline diet-enhanced endogenous macrophage foam cell formation and atherosclerotic lesion development in apolipoprotein e−/− mice without alterations in circulating cholesterol levels. The present studies suggest gut microbial production of TMA specifically, and non-lethal microbial inhibitors in general, may serve as a potential therapeutic approach for the treatment of cardiometabolic diseases.
How stochastic is gene expression in mammalian cells? Not very, according to
Battich et al. (2015), who report that single-cell
variability in cytoplasmic mRNAs is remarkably predictable given measurements of a
cell’s phenotypic state and microenvironment. The noise from transcriptional bursts
is buffered by a hallmark of eukaryotes—the nucleus.
Activation of Agouti-related peptide (AgRP) neurons potently promotes feeding, and chronically altering their activity also affects peripheral glucose homeostasis. We demonstrate that acute activation of AgRP neurons causes insulin resistance through impairment of insulin-stimulated glucose uptake into brown adipose tissue (BAT). AgRP neuron activation acutely reprograms gene expression in BAT toward a myogenic signature, including increased expression of myostatin. Interference with myostatin activity improves insulin sensitivity that was impaired by AgRP neurons activation. Optogenetic circuitry mapping reveals that feeding and insulin sensitivity are controlled by both distinct and overlapping projections. Stimulation of AgRP → LHA projections impairs insulin sensitivity and promotes feeding while activation of AgRP → anterior bed nucleus of the stria terminalis (aBNST)vl projections, distinct from AgRP → aBNSTdm projections controlling feeding, mediate the effect of AgRP neuron activation on BAT-myostatin expression and insulin sensitivity. Collectively, our results suggest that AgRP neurons in mice induce not only eating, but also insulin resistance by stimulating expression of muscle-related genes in BAT, revealing a mechanism by which these neurons rapidly coordinate hunger states with glucose homeostasis.
Neural circuits containing AgRP neurons coordinate hunger states with glucose homeostasis by inducing, in addition to eating, insulin resistance via stimulating expression of muscle-related genes in brown adipose tissue that inhibit glucose uptake.
Sexual reproduction culminates in a totipotent zygote with the potential to produce a whole organism. Sperm chromatin reorganization and epigenetic reprogramming that alter DNA and histone modifications generate a totipotent embryo. Active DNA demethylation of the paternal genome has been proposed to involve base excision and DNA repair-based mechanisms. The nature and consequence of DNA lesions generated during reprogramming are not known. Using mouse genetics and chemical biology, we discovered that Tet3-dependent zygotic reprogramming generates paternal DNA lesions that are monitored by a surveillance mechanism. In vivo structure-function rescue assays revealed that cohesin-dependent repair of paternal DNA lesions prevents activation of a Chk1-dependent checkpoint that delays mitotic entry. Culturing conditions affect checkpoint stringency, which has implications for human in vitro fertilization. We propose the zygotic checkpoint senses DNA lesions generated during paternal DNA demethylation and ensures reprogrammed loci are repaired before mitosis to prevent chromosome fragmentation, embryo loss, and infertility.
•Active DNA demethylation during zygotic reprogramming produces paternal DNA lesions•Paternal DNA lesions depend on Tet3 activity and require Scc1-cohesin for repair•Inefficient repair of paternal DNA lesions activates a Chk1-mediated checkpoint•A zygotic checkpoint coordinates repair with mitosis to protect genome stability
A surveillance mechanism that facilitates the repair of DNA lesions in the paternal genome during zygotic reprogramming has implications for in vitro fertilization.
zygote; reprogramming; checkpoint; cohesin; DNA damage repair
S phase and mitotic onset are brought about by the action of multiple different cyclin-CDK complexes. However, it has been suggested that changes in the total level of CDK kinase activity, rather than substrate specificity, drive the temporal ordering of S phase and mitosis. Here, we present a phosphoproteomics-based systems analysis of CDK substrates in fission yeast and demonstrate that the phosphorylation of different CDK substrates can be temporally ordered during the cell cycle by a single cyclin-CDK. This is achieved by rising CDK activity and the differential sensitivity of substrates to CDK activity over a wide dynamic range. This is combined with rapid phosphorylation turnover to generate clearly resolved substrate-specific activity thresholds, which in turn ensures the appropriate ordering of downstream cell-cycle events. Comparative analysis with wild-type cells expressing multiple cyclin-CDK complexes reveals how cyclin-substrate specificity works alongside activity thresholds to fine-tune the patterns of substrate phosphorylation.
•Global analysis of CDK substrates and their phosphorylation dynamics in fission yeast•CDK substrate phosphorylation can be temporally ordered by a single cyclin-CDK•Substrate-specific activity thresholds set phosphorylation timing and cell-cycle order•Cyclin-substrate specificity provides a secondary, more minor, level of regulation
A systems analysis of CDK substrates in yeast shows that the phosphorylation of different CDK substrates can be temporally ordered during the cell cycle by a single cyclin-CDK via rising CDK activity and the differential sensitivity of substrates to CDK activity.
CDK; cyclin-dependent kinase; cell cycle; S phase; mitosis; phosphorylation; kinase; phosphoproteomics
Stress granules are membrane-less organelles composed of RNA-binding proteins (RBPs) and RNA. Functional impairment of stress granules has been implicated in amyotrophic lateral sclerosis, frontotemporal dementia and multisystem proteinopathy - diseases that are characterized by fibrillar inclusions of RBPs. Genetic evidence suggests a link between persistent stress granules and the accumulation of pathological inclusions. Here we demonstrate that the RBP hnRNPA1 undergoes liquid-liquid phase separation (LLPS) into protein-rich droplets mediated by a low complexity sequence domain (LCD). While the LCD of hnRNPA1 is sufficient to mediate LLPS, the RNA recognition motifs contribute to LLPS in the presence of RNA, giving rise to several mechanisms for regulating assembly. Importantly, while not required for LLPS, fibrillization is enhanced in protein-rich droplets. We suggest that LCD-mediated LLPS contributes to the assembly of stress granules and their liquid properties, and provides a mechanistic link between persistent stress granules and fibrillar protein pathology in disease.
Liquid-liquid phase separation by RNA-binding proteins harboring low complexity sequence domains is the molecular basis for stress granule assembly, and persistent stress granules promote pathological protein fibrillization.
phase transition; liquid-liquid demixing; RNA-binding protein; ALS; RNA granules; amyloid
In this issue of Cell, Langen et al. use time-lapse multiphoton
microscopy to show how Drosophila photoreceptor growth cones find their
targets. Based on the observed dynamics, they develop a simple developmental algorithm
recapitulating the highly complex connectivity pattern of these neurons, suggesting a
basic framework for establishing wiring specificity.
The type-1 ryanodine receptor (RyR1) is an intracellular calcium (Ca2+) release channel required for skeletal muscle contraction. Here we present cryo-EM reconstructions of RyR1 in multiple functional states revealing the structural basis of channel gating and ligand-dependent activation. Binding sites for the channel activators Ca2+, ATP and caffeine were identified at interdomain interfaces of the C-terminal domain. Either ATP or Ca2+ alone induce conformational changes in the cytoplasmic assembly (‘priming’), without pore dilation. In contrast, in the presence of all three activating ligands, high-resolution reconstructions of open and closed states of RyR1 were obtained from the same sample, enabling analyses of conformational changes associated with gating. Gating involves global conformational changes in the cytosolic assembly accompanied by local changes in the transmembrane domain, which include bending of the S6 transmembrane segment and consequent pore dilation, displacement and deformation of the S4-S5 linker, and conformational changes in the pseudo-voltage-sensor domain.
Th17 cells play a critical role in host defense against extracellular pathogens and tissue homeostasis, but can induce autoimmunity. The mechanisms implicated in balancing ‘pathogenic’ and ‘non-pathogenic’ Th17 cell states remain largely unknown. We used single-cell RNA-seq to identify CD5L/AIM as a regulator expressed in ‘non-pathogenic’ but not in ‘pathogenic’ Th17 cells. Although CD5L does not affect Th17 differentiation, it is a functional switch that regulates the pathogenicity of Th17 cells. Loss of CD5L converts ‘non-pathogenic’ Th17 cells into ‘pathogenic’ cells that induce autoimmunity. CD5L mediates this effect by modulating the intracellular lipidome, altering fatty acid composition, and restricting cholesterol biosynthesis, and thus ligand availability for Rorγt, the master transcription factor of Th17 cells. Our study identifies CD5L as a critical regulator of the Th17 cell functional state and highlights the importance of lipid metabolism in balancing immune protection and disease induced by T cells.
Extensive cellular heterogeneity exists within specific immune-cell subtypes classified as a single lineage, but its molecular underpinnings are rarely characterized at a genomic scale. Here, we use single-cell RNA-seq to investigate the molecular mechanisms governing heterogeneity and pathogenicity of Th17 cells isolated from the central nervous system (CNS) and lymph nodes (LN) at the peak of autoimmune encephalomyelitis (EAE) or differentiated in vitro under either pathogenic or non-pathogenic polarization conditions. Computational analysis relates a spectrum of cellular states in vivo to in vitro differentiated Th17 cells, and unveils genes governing pathogenicity and disease susceptibility. Using knockout mice, we validate four new genes: Gpr65, Plzp, Toso and Cd5l (in a companion paper). Cellular heterogeneity thus informs Th17 function in autoimmunity, and can identify targets for selective suppression of pathogenic Th17 cells while potentially sparing non-pathogenic tissue-protective ones.
Spontaneous electrical activity of neurons in developing sensory systems promotes their maturation and proper connectivity. In the auditory system, spontaneous activity of cochlear inner hair cells (IHCs) is initiated by the release of ATP from glia-like inner supporting cells (ISCs), facilitating maturation of central pathways before hearing onset. Here, we find that ATP stimulates purinergic autoreceptors in ISCs, triggering Cl− efflux and osmotic cell shrinkage by opening TMEM16A Ca2+-activated Cl− channels. Release of Cl− from ISCs also forces K+ efflux, causing transient depolarization of IHCs near ATP release sites. Genetic deletion of TMEM16A markedly reduces the spontaneous activity of IHCs and spiral ganglion neurons in the developing cochlea, and prevents ATP-dependent shrinkage of supporting cells. These results indicate that support cells in the developing cochlea have adapted a pathway used for fluid secretion in other organs to induce periodic excitation of hair cells.
One of the clearest functions of the gut microbiota in humans is resistance to colonization by enteric bacterial pathogens. Reconstitution of the microbiota offers an exciting therapeutic approach, but great challenges must be overcome.
Memories are thought to be formed in response to transient experiences in part through changes in local protein synthesis at synapses. In Drosophila, the amyloidogenic (prion-like) state of the RNA binding protein Orb2 has been implicated in long-term memory, but how conformational conversion of Orb2 promotes memory formation is unclear. Combining in vitro and in vivo studies, we find that the monomeric form of Orb2 represses translation and removes mRNA polyA tails, while the oligomeric form enhances translation and elongates the polyA tails, and imparts its translational state to the monomer. The CG13928 protein, which binds only to monomeric Orb2, promotes deadenylation, whereas the putative polyA binding protein CG4612 promotes oligomeric Orb2-dependent translation. Our data support a model in which monomeric Orb2 keeps target mRNA in a translationally dormant state, and experience-dependent conversion to the amyloidogenic state activates translation, resulting in persistent alteration of synaptic activity and stabilization of memory.
Lifespan is a remarkably diverse trait ranging from a few days to several hundred years in nature, but the mechanisms underlying the evolution of lifespan differences remain elusive. Here we de novo assemble a reference genome for the naturally short-lived African turquoise killifish, providing a unique resource for comparative and experimental genomics. The identification of genes under positive selection in this fish reveals potential candidates to explain its compressed lifespan. Several aging genes are under positive selection in this short-lived fish and long-lived species, raising the intriguing possibility that the same gene could underlie evolution of both compressed and extended lifespans. Comparative genomics and linkage analysis identify candidate genes associated with lifespan differences between various turquoise killifish strains. Remarkably, these genes are clustered on the sex chromosome, suggesting that short lifespan might have co-evolved with sex determination. Our study provides insights into the evolutionary forces that shape lifespan in nature.
The 2015 Nobel Prize in Physiology or Medicine has been awarded to William C. Campbell and Satoshi Omura, and Youyou Tu for the discovery of avermectins and artemisinin, respectively, therapies that revolutionized the treatment of devastating parasite diseases. With the recent technological advances, a New Golden Age of natural products drug discovery is dawning.
Centrioles coordinate the primary microtubule organizing center of the cell and template the formation of cilia, thereby operating at a nexus of critical cellular functions. Here we use proximity-dependent biotinylation (BioID) to map the centrosome-cilium interface; with 58 bait proteins we generate a protein topology network comprising >7000 interactions. Analysis of interaction profiles coupled with high resolution phenotypic profiling implicates a number of new protein modules in centriole duplication, ciliogenesis and centriolar satellite biogenesis, and highlights extensive interplay between these processes. By monitoring dynamic changes in the centrosome-cilium protein interaction landscape during ciliogenesis, we also identify satellite proteins that support cilia formation. Systematic profiling of proximity interactions combined with functional analysis thus provides a rich resource for better understanding human centrosome and cilia biology. Similar strategies may be applied to other complex biological structures or pathways.
We use in vivo proximity-dependent biotinylation(BioID) to generate a protein interaction map of the human centrosome-cilium interface. Avast and functionally rich interaction space is characterized, allowing us to uncover protein modules critical for centrosome and cilium biogenesis. We demonstrate pervasive interplay between centriole duplication, centriolar satellite biogenesis and ciliogenesis, anddiscover pronounced dynamic modulation of the protein interaction landscape during the ciologenesis program. Our work thus provides a rich resource for better understanding human centrosome and cilia biology.
A three-dimensional culture of cortical tissues derived from pluripotent
stem cells offers an opportunity to model human brain development and disorders.
In a recent issue of Nature, Lancaster et al. describe a new
method for generating cerebral organoids in a dish and use it to model
Structural and functional brain connectivity, synaptic activity and information processing require highly coordinated signal transduction between different cell types within the neurovascular unit and intact blood-brain barrier (BBB) functions. Here, we examine the mechanisms regulating the formation and maintenance of the BBB and functions of BBB-associated cell types. Furthermore, we discuss the growing evidence associating BBB breakdown with the pathogenesis of inherited monogenic neurological disorders and complex multifactorial diseases including Alzheimer’s disease.