Muscles not only generate force. They may act as springs, providing energy storage to drive locomotion. Although extensible myofilaments are implicated as sites of energy storage, we show that intramuscular temperature gradients may enable molecular motors (cross-bridges) to store elastic strain energy. Using time-resolved small-angle X-ray diffraction paired with in situ measurements of mechanical energy exchange in flight muscle of Manduca sexta we produced high-speed movies of X-ray equatorial reflections indicating cross-bridge association with myofilaments. A temperature gradient within the flight muscle leads to lower cross-bridge cycling in the cooler regions. Those cross-bridges could elastically return energy at the extrema of muscle lengthening and shortening, helping drive cyclic wing motions. These results suggest cross-bridges can perform functions other than contraction, acting as molecular links for elastic energy storage.
The mouse germ-line presents a unique opportunity to study epigenetic reprogramming in vivo1. Recently we showed that genome-wide active DNA demethylation in primordial germ cells (PGCs) is linked to changes in nuclear architecture and extensive loss of histone modifications brought about by widespread histone replacement2. Notably, this chromatin remodelling follows the onset of genome-wide DNA demethylation, which raises a possibility that DNA demethylation may be linked to a DNA repair process2. Here we show the activation of components of the base excision repair (BER) pathway and the presence of single-strand DNA (ssDNA) breaks at the time of genome-wide DNA demethylation in PGCs. We found high levels of expression of critical BER components as well as chromatin-bound XRCC1 together with nuclear poly-ADP-ribosylation (PAR), specifically at the time when PGCs are undergoing DNA demethylation. A similar wave of genome-wide DNA demethylation occurs in the zygote affecting only the paternal genome3-5 where we observed a strikingly similar activation of BER components. Notably, maternally inherited Stella promotes this epigenetic asymmetry, since in zygotes lacking this protein, DNA demethylation is detected in both pronuclei. Crucially, zygotes lacking Stella exhibit aberrant targeting of active BER to both pronuclei. Finally we demonstrate that small molecule inhibitors of diverse BER components administered during in vitro fertilisation interfere with the progress of DNA demethylation.
Our combined observations demonstrate that DNA repair through BER represents a core component of genome-wide DNA demethylation and provides a vital mechanistic link to the extensive chromatin remodelling in developing PGCs2.
Some nascent proteins that fold within the endoplasmic reticulum (ER) never reach their native state. Misfolded proteins are removed from the folding machinery, dislocated from the ER into the cytosol, and degraded in a series of pathways collectively referred to as ER-associated degradation (ERAD). Distinct ERAD pathways centered on different E3 ubiquitin ligases survey the range of potential substrates. We now know many of the components of the ERAD machinery and pathways used to detect substrates and target them for degradation. Much less is known about the features used to identify terminally misfolded conformations and the broader role of these pathways in regulating protein half-lives.
Direction-selective responses to motion can be to the onset (On) or cessation (Off) of illumination. Here, we show that the transmembrane protein semaphorin 6A and its receptor plexin A2 are critical for achieving radially symmetric arborization of On starburst amacrine cell (SAC) dendrites and normal SAC stratification in the mouse retina. Plexin A2 is expressed in both On and Off SACs; however, semaphorin 6A is expressed in On SACs. Specific On-Off bistratified direction-selective ganglion cells in semaphorin 6A−/− mutants exhibit decreased tuning of On directional motion responses. These results correlate the elaboration of symmetric SAC dendritic morphology and asymmetric responses to motion, shedding light on the development of visual pathways that use the same cell types for divergent outputs.
Our prevailing view of vertebrate host defense is strongly shaped by the notion of a specialized set of immune cells as sole guardians of antimicrobial resistance. Yet this view greatly underestimates a capacity for most cell lineages—the majority of which fall outside the traditional province of the immune system—to defend themselves against infection. This ancient and ubiquitous form of host protection is termed cell-autonomous immunity and operates across all three domains of life. Here, we discuss the organizing principles that govern cellular self-defense and how intracellular compartmentalization has shaped its activities to provide effective protection against a wide variety of microbial pathogens.
The presence of DNA in the cytoplasm of mammalian cells is a danger signal that triggers the host immune responses such as the production of type-I interferons (IFN). Cytosolic DNA induces IFN through the production of cyclic-GMP-AMP (cGAMP), which binds to and activates the adaptor protein STING. Through biochemical fractionation and quantitative mass spectrometry, we identified a cGAMP synthase (cGAS), which belongs to the nucleotidyltransferase family. Overexpression of cGAS activated the transcription factor IRF3 and induced IFNβ in a STING-dependent manner. Knockdown of cGAS inhibited IRF3 activation and IFNβ induction by DNA transfection or DNA virus infection. cGAS bound to DNA in the cytoplasm and catalyzed cGAMP synthesis. These results indicate that cGAS is a cytosolic DNA sensor that induces interferons by producing the second messenger cGAMP.
Progress in the development of asymmetric Heck couplings of arenes and acyclic olefins has been limited by a tenuous understanding of the factors that dictate selectivity in migratory insertion and β-hydride elimination. On the basis of key mechanistic insight recently garnered in the exploration of selective Heck reactions, we report here an enantioselective variant that delivers β-, γ-, or δ-aryl carbonyl products from acyclic alkenol substrates. The catalyst system imparts notable regioselectivity during migratory insertion and promotes the migration of the alkene's unsaturation toward the alcohol to ultimately form the ketone product. The reaction uses aryldiazonium salts as the arene source, yields enantiomeric products from opposite starting alkene configurations, and uses a readily accessible ligand. The racemic nature of the alkenol substrate does not bias the enantioselection.
Human infants face the formidable challenge of learning the structure of their social environment. Previous research indicates that infants have early-developing representations of intentional agents, and of cooperative social interactions, that help meet that challenge. Here we report five studies with 144 infant participants showing that 10-to 13-month-old, but not 8-month-old, infants recognize when two novel agents have conflicting goals, and that they use the agents’ relative size to predict the outcome of the very first dominance contests between them. These results suggest that preverbal infants mentally represent social dominance and use a cue that covaries with it phylogenetically, and marks it metaphorically across human cultures and languages, to predict which of two agents is likely to prevail in a conflict of goals.
The restart of a stalled replication fork is a major challenge for DNA replication. Depending upon the nature of the damage, different repair processes might be triggered; one is template switching that is bypass of a leading strand lesion via fork regression. Using magnetic tweezers to study the T4 bacteriophage enzymes, we have reproduced in vitro the complete process of template switching. We show that the UvsW DNA helicase in cooperation with the T4 holoenzyme can overcome leading strand lesion damage by a pseudo stochastic process periodically forming and migrating a four ways Holliday junction. The initiation of the repair process requires partial replisome disassembly via the departure of the replicative helicase. The results support the role of fork regression pathways in DNA repair.
Bacterial type III protein secretion systems deliver effector proteins into eukaryotic cells in order to modulate cellular processes. Central to the function of these protein delivery machines is their ability to recognize and secrete substrates in a defined order. Here, we describe a mechanism by which a type III secretion system from the bacterial enteropathogen Salmonella enterica serovar Typhimurium can sort its substrates prior to secretion. This mechanism involves a cytoplasmic sorting platform that is sequentially loaded with the appropriate secreted proteins. The sequential loading of this platform, facilitated by customized chaperones, ensures the hierarchy in type III protein secretion. Given the presence of these machines in many important pathogens, these findings can serve as the bases for the development of novel antimicrobial strategies.
Circadian rhythms in mammals are generated by a feedback loop in which the three PERIOD (PER) proteins, acting in a large complex, inhibit the transcriptional activity of the CLOCK-BMAL1 dimer, repressing their own expression. Although fundamental, the mechanism of negative feedback in the mammalian clock, or any eukaryotic clock, is unknown. We analyzed protein constituents of PER complexes purified from mouse tissues and identified PSF (polypyrimidine tract binding protein-associated splicing factor). Our analysis indicates that PSF within the PER complex recruits SIN3A, a scaffold for assembly of transcriptional inhibitory complexes, and that the PER complex thereby rhythmically delivers histone deacetylases to the Per1 promoter, repressing Per1 transcription. These findings provide a function for the PER complex and a molecular mechanism for circadian clock negative feedback.
Death is a vital developmental cell fate. In Caenorhabditis elegans, programmed death of the linker cell, which leads gonadal elongation, proceeds independently of caspases and apoptotic effectors. To identify genes promoting linker-cell death, we performed a genome-wide RNA interference screen. We show that linker-cell death requires the gene pqn-41, encoding an endogenous polyglutamine-repeat protein. pqn-41 functions cell autonomously, and is expressed at the onset of linker-cell death. pqn-41 expression is controlled by the MAP kinase kinase SEK-1, which functions in parallel to the Zn-finger protein LIN-29 to promote cellular demise. Linker-cell death is morphologically similar to cell death associated with normal vertebrate development and polyglutamine-induced neurodegeneration. Our results may, therefore, provide molecular in-roads to understanding non-apoptotic cell death in metazoan development and disease.
A fundamental aspect of the adaptive immune system is the generation and maintenance of a diverse and self-tolerant T cell repertoire. Through its regulation of T cell development, homeostasis, tolerance, and differentiation, the highly evolutionarily conserved cytokine transforming growth factor-β (TGF-β) critically supports a functional T cell pool. The pleiotropic nature of this regulation is likely due to the elaborate control of TGF-β production and activation in the immune system, and the intricacy of TGF-β signaling pathways. This review discusses our current understanding of TGF-β regulation of T cells.
Genome scale network reconstruction has enabled predictive modeling of metabolism for many systems. Traditionally, protein structural information has not been represented in such reconstructions. Expanding a genome-scale model of Escherichia coli metabolism by including experimental and predicted protein structures enabled the analysis of protein thermostability in a network context, allowing prediction of protein activities that limit network function at super-optimal temperature and mechanistic interpretations of mutations found in strains adapted to heat. Predicted growth-limiting factors for thermotolerance were validated through nutrient supplementation experiments and defined metabolic sensitivities to heat stress, providing evidence that metabolic enzyme thermostability is rate limiting at super-optimal temperature. Inclusion of structural information expanded the content and predictive capability of genome-scale metabolic networks enabling structural systems biology of metabolism.
Cytosolic DNA induces type-I interferons and other cytokines that are important for antimicrobial defense but can also result in autoimmunity. This DNA signaling pathway requires the adaptor protein STING and the transcription factor IRF3, but the mechanism of DNA sensing is unclear. Here we showed that mammalian cytosolic extracts synthesized cyclic-GMP-AMP (cGAMP) in vitro from ATP and GTP in the presence of DNA but not RNA. DNA transfection or DNA virus infection of mammalian cells also triggered cGAMP production. cGAMP bound to STING, leading to the activation of IRF3 and induction of interferon-β. Thus, cGAMP represents the first cyclic di-nucleotide in metazoa and it functions as an endogenous second messenger that triggers interferon production in response to cytosolic DNA.
More detailed sequence standards that keep up with revolutionary sequencing technologies will aid the research community in evaluating data.
Although the visual cortex is organized retinotopically, it is not clear whether the cortical representation of position necessarily reflects perceived position. Using functional magnetic resonance imaging (fMRI), we show that the retinotopic representation of a stationary object in the cortex was systematically shifted when visual motion was present in the scene. Whereas the object could appear shifted in the direction of the visual motion, the representation of the object in the visual cortex was always shifted in the opposite direction. The results show that the representation of position in the primary visual cortex, as revealed by fMRI, can be dissociated from perceived location.
Mouse primordial germ cells (PGC) undergo sequential epigenetic changes and genome-wide DNA demethylation to reset the epigenome for totipotency. Here, we demonstrate that erasure of CpG methylation (5mC) in PGCs occurs via conversion to 5-hydroxymethylcytosine (5hmC), driven by high levels of TET1 and TET2. Global conversion to 5hmC initiates asynchronously among PGCs at embryonic day (E) 9.5-E10.5 and accounts for the unique process of imprint erasure. Mechanistically, 5hmC enrichment is followed by its protracted decline thereafter at a rate consistent with replication-coupled dilution. The conversion to 5hmC is a significant component of parallel redundant systems that drive comprehensive reprogramming in PGCs. Nonetheless, we identify rare regulatory elements that escape systematic DNA demethylation in PGCs, providing a potential mechanistic basis for transgenerational epigenetic inheritance.
Phosphorylated O-mannosyl trisaccharide [N-acetylgalactosamine-β3-N-acetylglucosamine-β4-(phosphate-6-)mannose] is required for dystroglycan to bind laminin-G domain-containing extracellular proteins with high affinity in muscle and brain. However, the enzymes that produce this structure have not been fully elucidated. Here we found that glycosyltransferase-like domain containing 2 (GTDC2) is a protein O-linked mannose β 1,4-N-acetylglucosaminyltransferase whose product could be extended by β 1,3-N-acetylgalactosaminyltransferase2 (B3GALNT2) to form the O-mannosyl trisaccharide. Furthermore, we identified SGK196 as an atypical kinase that phosphorylated the 6-position of O-mannose, specifically after the mannose had been modified by both GTDC2 and B3GALNT2. These findings suggest how mutations in GTDC2, B3GALNT2, and SGK196 disrupt dystroglycan receptor function and lead to congenital muscular dystrophy.
A strain of Halomonas bacteria, GFAJ-1, has been reported to be able to use arsenate as a nutrient when phosphate is limiting, and to specifically incorporate arsenic into its DNA in place of phosphorus. However, we have found that arsenate does not contribute to growth of GFAJ-1 when phosphate is limiting and that DNA purified from cells grown with limiting phosphate and abundant arsenate does not exhibit the spontaneous hydrolysis expected of arsenate ester bonds. Furthermore, mass spectrometry showed that this DNA contains only trace amounts of free arsenate and no detectable covalently bound arsenate.
Chronic infections strain the regenerative capacity of antiviral T lymphocyte populations, leading to failure in long-term immunity. The cellular and molecular events controlling this regenerative capacity, however, are unknown. We found that two distinct states of virus-specific CD8+ T cells exist in chronically infected mice and humans. Differential expression of the T-box transcription factors T-bet and Eomesodermin (Eomes) facilitated the cooperative maintenance of the pool of antiviral CD8+ T cells during chronic viral infection. T-bethi cells displayed low intrinsic turnover but proliferated in response to persisting antigen, giving rise to Eomeshi terminal progeny. Genetic elimination of either subset resulted in failure to control chronic infection, which suggests that an imbalance in differentiation and renewal could underlie the collapse of immunity in humans with chronic infections.
In spite of the centrality of informed consent in clinical genetics and genomics, new recommendations from the American College of Medical Genetics and Genomics (ACMG) call for laboratories and clinicians to test for and report specific genetic incidental findings, even when the patient does not consent to the testing or disclosure and even when the patient is a child.
Mitochondrial fission is fundamentally important to cellular physiology. The dynamin-related protein Drp1 mediates fission, and interaction between mitochondrion and endoplasmic reticulum (ER) enhances fission. However, the mechanism for Drp1 recruitment to mitochondria is unclear, although previous results implicate actin involvement. Here, we found that actin polymerization through ER-localized inverted formin 2 (INF2) was required for efficient mitochondrial fission in mammalian cells. INF2 functioned upstream of Drp1. Actin filaments appeared to accumulate between mitochondria and INF2-enriched ER membranes at constriction sites. Thus, INF2-induced actin filaments may drive initial mitochondrial constriction, which allows Drp1-driven secondary constriction. Because INF2 mutations can lead to Charcot-Marie-Tooth disease, our results provide a potential cellular mechanism for this disease state.
We describe a simple and robust method to construct complex three-dimensional (3D) structures using short synthetic DNA strands that we call “DNA bricks”. In one-step annealing reactions, bricks with hundreds of distinct sequences self-assemble into prescribed 3D shapes. Each 32-nucleotide brick is a modular component; it binds to four local neighbors and can be removed or added independently. Each 8-base-pair interaction between bricks defines a voxel with dimensions 2.5 nanometers by 2.5 nanometers by 2.7 nanometers, and a master brick collection defines a “molecular canvas” with dimensions of 10 by 10 by 10 voxels. By selecting subsets of bricks from this canvas, we constructed a panel of 102 distinct shapes exhibiting sophisticated surface features as well as intricate interior cavities and tunnels.
Epithelial cells respond to physico-chemical damage with up-regulation of major histocompatbility complex–like ligands that can activate the cytolytic potential of neighboring intraepithelial T cells by binding the activating receptor, NKG2D. The systemic implications of this lymphoid stress-surveillance response, however, are unknown. We found that antigens encountered at the same time as cutaneous epithelial stress induced strong primary and secondary systemic, T helper 2 (TH2)–associated atopic responses in mice. These responses required NKG2D-dependent communication between dysregulated epithelial cells and tissue-associated lymphoid cells. These data are germane to uncertainty over the afferent induction of TH2 responses and provide a molecular framework for considering atopy as an important component of the response to tissue damage and carcinogenesis.