Christos T. Santis, Yaakov Vilenchik, Naresh Satyan, George Rakuljic, and Amnon Yariv
The semiconductor laser, arguably the most versatile member of the family of lasers, has become a technological staple of a massively interconnected, data-driven world, with its spectral purity (i.e., temporal coherence) an increasingly important figure of merit. The present work describes a conceptually fundamental “recipe” for the enhancement of coherence, predicated on direct control of the coherence-limiting process itself, the field–matter interaction. As such, it is inherently adaptable and technologically scalable. As photonic materials and fabrication techniques continue to improve, the described approach has the potential of serving as a roadmap for major and sustained improvements in coherence. With experimentally demonstrated coherence limited at 1 kHz in this work, we envision “deep” sub-kilohertz-level coherence to be soon within reach. (See pp. E7896–E7904.)
Xingcheng Lin, Jeffrey K. Noel, Qinghua Wang, Jianpeng Ma, and José N. Onuchic
Influenza hemagglutinin (HA) is the viral membrane protein that guides entry of flu viruses into host cells. A global refolding of the stem domain of HA, HA2, is crucial to successful viral membrane fusion, but the molecular mechanism relating this conformational change of HA2 to its fusogenic properties remains unclear. We use molecular dynamics simulations to quantify the thermodynamic importance of the B loop domain for driving the overall HA2 rearrangement. We find that the B loop's loop-to-coiled-coil refolding is not strongly downhill and thus is a possible target for therapeutics. A buried hydrophilic amino acid is implicated in destabilizing the coiled coil, and the sequence divergence at this position may indicate functional differences between group 1 and 2 HAs. (See pp. E7905–E7913.)
Miranda Simon, Cassilde Schwartz, David Hudson, and Shane D. Johnson
Would more restrictive immigration policies stop individuals from migrating? We present an agent-based computational model, calibrated using original survey and experimental data, which represents an important step in estimating the “substitution effect” whereby migrants reorient toward unauthorized channels due to changes in policy. We find that government-imposed restrictions on migrants can decrease total migration. However, some restrictions are highly ineffective while others decrease legal migration only at the cost of driving migrants into unauthorized channels. Restrictions on students and high-skilled workers are least effective in reducing migration, and restrictions on family-based visas are especially counterproductive in diverting migrants to back channels. We also find that increasing enforcement would not effectively eliminate the diversion to unauthorized channels. (See pp. E7914–E7923.)
Matthias Quick, Ara M. Abramyan, Pattama Wiriyasermkul, Harel Weinstein, Lei Shi, and Jonathan A. Javitch
The crystal structure of the human serotonin transporter (hSERT) features a drug bound not only in the primary site, but also simultaneously in a second site, thereby supporting the existence of two binding sites in neurotransmitter:sodium symporters (NSSs) as we demonstrated previously for the bacterial NSS homolog LeuT. Here, we provide evidence that MhsT, another NSS homolog, can simultaneously bind substrate molecules both in its primary substrate (S1) site and in its secondary substrate (S2) site. As in LeuT, substrate binding to both the S1 and S2 sites of MhsT is critical for transport, providing further support for a mechanistic model in which the allosteric interaction between the two substrate sites is a key element of Na+-coupled symport. (See pp. E7924–E7931.)
Victor Ovchinnikov, Tracy A. Stone, Charles M. Deber, and Martin Karplus
Bacterial pathogens are developing resistance to antibiotic compounds at an alarming rate. We use computer simulations to design inhibitors of the Escherichia coli multidrug resistance protein EmrE (efflux-multidrug resistance E) from the small multidrug family. Starting with low-resolution X-ray data, we obtain an atomic structure of EmrE using extensive molecular simulations. Based on the structure, we design hydrocarbon-stapled peptide inhibitors of EmrE, which are synthesized and shown to be effective in vivo. The rational drug design approach described here holds promise for combating efflux-mediated drug resistance in microbes and, more generally, in cancer. (See pp. E7932–E7941.)
Karl Lundquist, Jeremy Bakelar, Nicholas Noinaj, and James C. Gumbart
Nearly all outer-membrane proteins (OMPs) in Gram-negative bacteria are inserted via the β-barrel assembly machinery (BAM) complex. Molecular dynamics simulations have revealed a persistent C-terminal strand kink of the core BAM component, BamA, which permits BamA to open laterally to the membrane. Experiments show that inhibiting kinking makes bacteria more susceptible to antibiotics. Kink-induced lateral gating may catalyze OMP insertion through a local disruption of the membrane, a pathway into the membrane, or both. (See pp. E7942–E7949.)
Aniruddha Mitra, Felix Ruhnow, Salvatore Girardo, and Stefan Diez
Microtubules consist of parallel protofilament lanes for motor-based intracellular transport. While most motors translocate along individual protofilaments, members of the kinesin-8 family have been reported to sidestep. However, the mechanism of sidestepping is currently not understood. Here, we track the 3D motion of single kinesin-8 motors on freely suspended microtubules. We find that individual motors sidestep with a bias to the left, the probability of which is increased with the time taken per forward step. We relate this behavior to a bifurcation in the step cycle of kinesin-8. Experiments on a kinesin-1 with an elongated neck linker suggest that sidestepping is intrinsic to all processive kinesins possessing long neck linkers, potentially helping them to circumnavigate obstacles on the microtubule. (See pp. E7950–E7959.)
Kimberli J. Kamer, Yasemin Sancak, Yevgenia Fomina, Joshua D. Meisel, Dipayan Chaudhuri, Zenon Grabarek, and Vamsi K. Mootha
Mechanisms by which heavy metals contribute to cellular toxicity are not well understood. Here, we report that cellular manganese toxicity is due in part to manganese transport into mitochondria via the calcium uniporter. Genetic loss of the uniporter’s pore-forming subunit in human cells and in worms confers partial resistance against manganese toxicity, while loss of its regulatory subunit, MICU1, sensitizes human cells to manganese toxicity. MICU1 provides an additional checkpoint to discriminate between Ca2+ and Mn2+, enabling the uniporter to evolve high conductance while maintaining selectivity. Our work has implications for understanding mechanisms of manganese toxicity in neurodegenerative diseases such as Parkinson’s disease. (See pp. E7960–E7969.)
Gaëlle J. S. Talhouarne and Joseph G. Gall
Introns are noncoding DNA sequences interspersed among the coding sequences of genes. Shortly after transcription, the intronic sequences are spliced out of the primary RNA transcript as lariat RNAs (circular molecules with a short tail). Most of these lariats are destroyed within minutes in the cell nucleus. We report here that many such intronic RNAs are, in fact, exported to the cytoplasm, where they remain as stable circular molecules. These cytoplasmic introns are derived from hundreds of different genes of widely different functions. We find them in cells of human, mouse, chicken, frog, and zebrafish. The widespread occurrence of so many stable lariat RNAs in the cytoplasm suggests that they play some as-yet unexpected role in cell metabolism. (See pp. E7970–E7977.)
Boryana Petrova, Keke Liu, Caiping Tian, Maiko Kitaoka, Elizaveta Freinkman, Jing Yang, and Terry L. Orr-Weaver
Reactive oxygen species (ROS) increase with age and have been shown to negatively impact age-related diseases. However, the physiological roles they might play in development have not been extensively characterized. Here, we show that ROS have essential functions as oocytes complete meiosis, the specialized cell division that generates haploid eggs, and transition to embryonic development. Meiotic progression and early embryonic divisions are defective when ROS is misregulated. Furthermore, we document the effects of ROS on specific proteins. Our identification of proteins altered in redox state as the oocyte transitions to an embryo provides a valuable resource to guide future exploration of ROS functions in early development. The regulatory system described here has important implications for female fertility. (See pp. E7978–E7986.)
Pierre Mattar, Milanka Stevanovic, Ivana Nad, and Michel Cayouette
Eukaryotic cells depend on precise genome organization within the nucleus to maintain an appropriate gene-expression profile. Critical to this process is the packaging of functional domains of open and closed chromatin to specific regions of the nucleus, but how this is regulated remains unclear. In this study, we show that the zinc finger protein Casz1 regulates higher-order nuclear organization of rod photoreceptors in the mouse retina by repressing nuclear lamina function, which leads to central localization of heterochromatin. Loss of Casz1 in rods leads to an abnormal transcriptional profile followed by degeneration. These results identify Casz1 as a regulator of higher-order genome organization. (See pp. E7987–E7996.)
Michael W. Dorrity, Josh T. Cuperus, Jolie A. Carlisle, Stanley Fields, and Christine Queitsch
Transcription factors have been intensively examined to decipher how they regulate cellular decisions, but there are few in-depth studies of these factors across traits, environments, and genetic backgrounds. Here, we analyze the Saccharomyces cerevisiae Ste12 protein, a transcription factor essential for both mating and invasion in many fungal species. Generating thousands of variants in the Ste12 DNA-binding domain, we scored each variant for its activity in promoting both mating and invasion. We found altered DNA-binding patterns of exceptional variants that result in yeast that lose their mating efficiency, but gain increased competence in invasion. This surprising malleability in transcription factor function has implications for understanding the evolution of pathogenicity in fungi. (See pp. E7997–E8006.)
Alexandre Bolze, Bertrand Boisson, Barbara Bosch, Alexander Antipenko, Matthieu Bouaziz, Paul Sackstein, Malik Chaker-Margot, Vincent Barlogis, Tracy Briggs, Elena Colino, Aurora C. Elmore, Alain Fischer, Ferah Genel, Angela Hewlett, Maher Jedidi, Jadranka Kelecic, Renate Krüger, Cheng-Lung Ku, Dinakantha Kumararatne, Alain Lefevre-Utile, Sam Loughlin, Nizar Mahlaoui, Susanne Markus, Juan-Miguel Garcia, Mathilde Nizon, Matias Oleastro, Malgorzata Pac, Capucine Picard, Andrew J. Pollard, Carlos Rodriguez-Gallego, Caroline Thomas, Horst Von Bernuth, Austen Worth, Isabelle Meyts, Maurizio Risolino, Licia Selleri, Anne Puel, Sebastian Klinge, Laurent Abel, and Jean-Laurent Casanova
Isolated congenital asplenia (ICA) is characterized by the absence of a spleen at birth without any other developmental defect. ICA predisposes individuals to severe bacterial infections early in childhood. In 2013, we showed that very rare deleterious mutations in the protein-coding region of RPSA, which codes for a protein in the ribosome, caused ICA in 8 of 23 kindreds. We have since enrolled 33 more kindreds and identified 11 new ICA-causing RPSA protein-coding mutations, as well as the first two ICA-causing mutations in the 5′-UTR of this gene. A few individuals carrying one of the new RPSA mutations had a spleen, indicating that mutations in RPSA can cause ICA with incomplete penetrance. (See pp. E8007–E8016.)
Marie Liebmann, Stephanie Hucke, Kathrin Koch, Melanie Eschborn, Julia Ghelman, Achmet I. Chasan, Shirin Glander, Martin Schädlich, Meike Kuhlencord, Niklas M. Daber, Maria Eveslage, Marc Beyer, Michael Dietrich, Philipp Albrecht, Monika Stoll, Karin B. Busch, Heinz Wiendl, Johannes Roth, Tanja Kuhlmann, and Luisa Klotz
The role of metabolic processes during T cell activation has been increasingly acknowledged, and recent data suggest an impact of T cell immunometabolism on T cell function and T cell-mediated autoimmunity. The factors regulating metabolic function in T cells are not clear, however. We identify the nuclear receptor Nur77 as central regulator of T cell immunometabolism, controlling oxidative phosphorylation and aerobic glycolysis during T cell activation. Functionally, Nur77 restricts murine and human T cell activation and proliferation and limits inflammation in autoimmune conditions in animal models of CNS autoimmunity, contact dermatitis, and arthritis. These findings identify Nur77 as a central regulator of T cell immunometabolism that restricts T cell-mediated autoimmunity, which might open up new avenues for a more tailored therapeutic approach. (See pp. E8017–E8026.)
Wei-Chan Hsu, Ming-Yu Chen, Shu-Ching Hsu, Li-Rung Huang, Cheng-Yuan Kao, Wen-Hui Cheng, Chien-Hsiung Pan, Ming-Sian Wu, Guann-Yi Yu, Ming-Shiu Hung, Chuen-Miin Leu, Tse-Hua Tan, and Yu-Wen Su
Naive T cells at quiescent state utilize mitochondrial respiration to generate ATP. In response to antigen, activated T cells through T cell receptor (TCR) and CD28 differentiate to TH1, TH2, or TH17 effector cells with an induction of aerobic glycolysis. Here, we demonstrate a role for DUSP6 in the glycolysis commitment during T cell activation. DUSP6 deficiency prompts activated T cells to rely on glucose-independent fuels. We show that DUSP6 fine-tunes TCR-MAPK signaling, which determines follicular helper T (TFH) effector-cell differentiation regardless of a glycolytic defect. Our findings imply that glycolysis is required for survival of activated T cells but optional for IL-21 production in TFH cell differentiation. (See pp. E8027–E8036.)
Charlotte Gistelinck, Ronald Y. Kwon, Fransiska Malfait, Sofie Symoens, Matthew P. Harris, Katrin Henke, Michael B. Hawkins, Shannon Fisher, Patrick Sips, Brecht Guillemyn, Jan Willem Bek, Petra Vermassen, Hanna De Saffel, Paul Eckhard Witten, MaryAnn Weis, Anne De Paepe, David R. Eyre, Andy Willaert, and Paul J. Coucke
Type I collagenopathies are a heterogenous group of connective tissue disorders, caused by genetic defects in type I collagen. Inherent to these disorders is a large clinical variability, of which the underlying molecular basis remains undefined. By systematically analyzing skeletal phenotypes in a large set of type I collagen zebrafish mutants, we show that zebrafish models are able to both genocopy and phenocopy different forms of human type I collagenopathies, arguing for a similar pathogenetic basis. This study illustrates the future potential of zebrafish as a tool to further dissect the molecular basis of phenotypic variability in human type I collagenopathies, to improve diagnostic strategies as well as promote the discovery of new targetable pathways for pharmacological intervention of these disorders. (See pp. E8037–E8046.)
Jennifer-Magdalena Masch, Heinz Steffens, Joachim Fischer, Johann Engelhardt, Jasmine Hubrich, Jan Keller-Findeisen, Elisa D’Este, Nicolai T. Urban, Seth G. N. Grant, Steffen J. Sahl, Dirk Kamin, and Stefan W. Hell
In vivo fluorescence microscopy with resolution well beyond the diffraction limit entails complexities that challenge the attainment of sufficient image brightness and contrast. These challenges have so far hampered investigations of the nanoscale distributions of synaptic proteins in the living mouse. Here, we describe a combination of stimulated emission depletion microscopy and endogenous protein labeling, providing high-quality in vivo data of the key scaffolding protein PSD95 at the postsynaptic membrane, which frequently appeared in extended distributions rather than as isolated nanoclusters. Operating in the far-red to near-IR wavelength range, this combination promises reduced photostress compared with prior in vivo nanoscopy at much shorter wavelengths. (See pp. E8047–E8056.)
Andrew J. Shepherd, Aaron D. Mickle, Judith P. Golden, Madison R. Mack, Carmen M. Halabi, Annette D. de Kloet, Vijay K. Samineni, Brian S. Kim, Eric G. Krause, Robert W. Gereau IV, and Durga P. Mohapatra
Neuropathic pain is a widespread problem that is undermanaged by currently available analgesic drugs. An antagonist of the type II angiotensin II receptor (AT2R) reduces pain behaviors related to neuropathy, suggesting that angiotensin receptor signaling is involved in this pain. We find that AT2R expression is detected not in sensory neurons themselves, but in macrophages that infiltrate the site of nerve injury. Inducible depletion of peripheral macrophages attenuates mechanical and cold pain hypersensitivity related to neuropathy, as does transplantation of AT2R-null bone marrow into an otherwise WT recipient. Our observations provide powerful evidence that neuropathic pain is dependent upon angiotensin signaling, macrophages, and the AT2R-mediated downstream signaling therein. (See pp. E8057–E8066.)
Adam M. Large, Nathan W. Vogler, Martha Canto-Bustos, F. Kathryn Friason, Paul Schick, and Anne-Marie M. Oswald
In most primary sensory cortical areas, sensory information is topographically organized. The spatial mapping of sensory features has proven useful in elucidating circuit mechanisms that underlie sensory representations in the brain. The piriform cortex is postulated to play a prominent role in the formation of odor percepts but lacks a topographic representation for odor information. Consequently, the circuit mechanisms underlying odor processing have remained elusive. We show spatial patterning of inhibition along the rostrocaudal axis of piriform cortex that differs with respect to excitatory and inhibitory neurons. Exploration of the underlying mechanisms revealed that inhibitory circuits differ in rostral vs. caudal piriform cortex. This rostrocaudal spatial organization could provide a scaffold for investigating circuit computations during odor processing. (See pp. E8067–E8076.)
Kay L. Richards, Carol J. Milligan, Robert J. Richardson, Nikola Jancovski, Morten Grunnet, Laura H. Jacobson, Eivind A. B. Undheim, Mehdi Mobli, Chun Yuen Chow, Volker Herzig, Agota Csoti, Gyorgy Panyi, Christopher A. Reid, Glenn F. King, and Steven Petrou
Spider venom is a rich source of peptides, many targeting ion channels. We assessed a venom peptide, Hm1a, as a potential targeted therapy for Dravet syndrome, the genetic epilepsy linked to a mutation in the gene encoding the sodium channel alpha subunit NaV1.1. Cell-based assays showed Hm1a was selective for hNaV1.1 over other sodium and potassium channels. Utilizing a mouse model of Dravet syndrome, Hm1a restored inhibitory neuron function and significantly reduced seizures and mortality in heterozygote mice. Evidence from the structure of Hm1a and modeling suggest Hm1a interacts with NaV1.1 inactivation domains, providing a structural correlate of the functional mechanisms. This proof-of-concept study provides a promising strategy for future drug development in genetic epilepsy and other neurogenetic disorders. (See pp. E8077–E8085.)
Galen E. Flynn and William N. Zagotta
Unlike most other voltage-activated ion channels, hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels open in response to hyperpolarizing membrane voltages. The molecular mechanism that is responsible for this unique voltage dependence in HCN channels is unknown. Here, we show that the covalent linkage between the voltage-sensing domain and the pore domain, the S4-S5 linker, is not required for hyperpolarization-dependent activation or ligand-dependent gating, as previously thought. Instead the voltage-sensing domain is inhibitory on the pore domain, and hyperpolarizing voltages relieve this autoinhibition, allowing the pore to open. This model explains the unique hyperpolarization-dependent activation of HCN channels. (See pp. E8086–E8095.)
Eva Knoch, Satoko Sugawara, Tetsuya Mori, Christian Poulsen, Atsushi Fukushima, Jesper Harholt, Yoshinori Fujimoto, Naoyuki Umemoto, and Kazuki Saito
Withanolides form a major class of plant steroids with unique side-chain modifications. Withanolides are one of the main active components in an Indian Ayurvedic medicinal plant, ashwagandha, which has been used for over 3,000 y. Because of their highly diversified structures, withanolides are promising pharmacological compounds with proven antiinflammatory and anticancer properties. We identified a sterol Δ24-isomerase (24ISO) catalyzing the first committed step in the biosynthesis of withanolides and related compounds. Identification of 24ISO paves the way for targeted manipulations to increase withanolide yields and as a starting point to elucidate the downstream pathway of yet-unknown withanolide biosynthesis. This study also demonstrates how the evolution of enzymes catalyzing double-bond modifications of triterpene side chains lead to diversity in structures and functions. (See pp. E8096–E8103.)
