Epigenetic modification of OXT and human sociability
Brian W. Haas, Megan M. Filkowski, R. Nick Cochran, Lydia Denison, Alexandra Ishak, Shota Nishitani, and Alicia K. Smith
Elucidating the genetic and biological substrates of social behavior serves to advance the way basic human nature is understood and improves the way genetic and biological markers can be used to prevent, diagnose, and treat people with impairments in social cognition and behavior. This study shows that epigenetic modification of the structural gene for oxytocin (OXT) is an important factor associated with individual differences in social processing, including self-report, behavior, and brain function and structure in humans. (See pp. E3816–E3823.)
Cardiac myosin light chain is phosphorylated by Ca2+/calmodulin-dependent and -independent kinase activities
Audrey N. Chang, Pravin Mahajan, Stefan Knapp, Hannah Barton, H. Lee Sweeney, Kristine E. Kamm, and James T. Stull
Chronic heart failure is associated with decreased cardiac myosin light chain kinase (MLCK; cMLCK) expression and myosin regulatory light chain (RLC) phosphorylation, similar to heart failure associated with mutations in numerous sarcomeric proteins. Although ablation of cMLCK expression reduces RLC phosphorylation sufficiently to cause heart failure, the residual phosphorylation indicates that another kinase also phosphorylates RLC. We find that MLCK4 is also expressed abundantly in cardiac muscle, and structural analyses indicate that it is a Ca2+/calmodulin (CaM)-independent kinase, in contrast to Ca2+/CaM-stimulated cMLCK. Biochemical kinetic analyses confirmed these structural predictions. These studies define distinct regulation of cMLCK and MLCK4 activities to affect RLC phosphorylation, and lay the foundation for RLC phosphorylation as a therapeutic target for heart failure. (See pp. E3824–E3833.)
Role and structural mechanism of WASP-triggered conformational changes in branched actin filament nucleation by Arp2/3 complex
Max Rodnick-Smith, Qing Luan, Su-Ling Liu, and Brad J. Nolen
Assembly of actin filaments is tightly regulated to orchestrate basic cellular processes such as motility, division, and differentiation. To control when and where actin filaments assemble, cells rely on actin filament nucleators including the Arp2/3 (Actin-related proteins 2/3) complex, a seven-subunit protein assembly that nucleates branched actin filaments to create dendritic actin networks. Activity of the Arp2/3 complex is controlled by WASP (Wiskott–Aldrich syndrome protein) proteins, which bind directly to it to activate nucleation. To understand how WASP proteins activate the complex, we used chemical cross-linking to engineer an Arp2/3 complex that is locked into an “on” state without WASP. In addition, we used biochemical experiments to determine how WASP proteins stimulate the on state. These results have important implications for understanding how cells control the actin cytoskeleton. (See pp. E3834–E3843.)
Immobilization of the N-terminal helix stabilizes prefusion paramyxovirus fusion proteins
Albert S. Song, Taylor A. Poor, Luciano A. Abriata, Theodore S. Jardetzky, Matteo Dal Peraro, and Robert A. Lamb
Paramyxovirus fusion proteins (F), critical for viral entry and infection, initially fold into a metastable prefusion state and, upon triggering, refold irreversibly to a stable postfusion state to physically mediate membrane fusion. The large-scale conformational changes that occur in the F-refolding pathway are understood, but a detailed structural understanding of F-protein metastability remains elusive. Here, stabilizing and destabilizing mutations of the parainfluenza virus 5 fusion protein were examined to reveal that the immobilization of the N-terminal helix stabilizes paramyxovirus prefusion F proteins. The N-terminal helix, the interaction of which with domain II appears to be a critical early step in the F-protein refolding pathway, presents a novel alternative target for structure-based antiviral therapeutics. (See pp. E3844–E3851.)
Directly measuring single-molecule heterogeneity using force spectroscopy
Michael Hinczewski, Changbong Hyeon, and D. Thirumalai
The relationship between structure and function is the heart of modern cell biology. Technological innovations in manipulating single molecules of proteins, DNA/RNA, and their complexes, are beginning to reveal the surprising intricacies of this relationship. In certain cases, the same molecule randomly switches between various long-lived structures, each with different functional properties. We present a theory to extract the extent and dynamics of these structural fluctuations from single-molecule experimental data. We find large heterogeneity in DNA and RNA complexes, supporting the notion that energy landscapes involving nucleic acids are rugged. Our work shows that functional heterogeneity is far more common than previously thought and suggests experimental approaches for estimating the timescales of these fluctuations with unprecedented accuracy. (See pp. E3852–E3861.)
Evolution of domain–peptide interactions to coadapt specificity and affinity to functional diversity
Abdellali Kelil, Emmanuel D. Levy, and Stephen W. Michnick
Today, we understand how short linear peptides bind to distinct recognition domains. However, at the cellular level, we still do not grasp why certain binding peptides are highly specific, whereas others are highly cross-reactive, and also what is the relationship with the functions of domain–peptide interactions. We revealed remarkable relationships among binding peptides, between their binding specificity, binding affinity, structural properties, and evolution. Surprisingly, we found that these properties are also related to functional specificity of domain–peptide interactions. Our results suggest that the structural properties and sequences of binding peptides have coevolved to achieve the levels of binding specificity and binding affinity that are required for the different levels of functional specificity of domain–peptide interactions. (See pp. E3862–E3871.)
Relating conformation to function in integrin α5β1
Yang Su, Wei Xia, Jing Li, Thomas Walz, Martin J. Humphries, Dietmar Vestweber, Carlos Cabañas, Chafen Lu, and Timothy A. Springer
β1 integrins form the largest and most diverse integrin subfamily, with 12 distinct αβ heterodimers. Together, the seven other integrin β-subunits form an equal number of 12 heterodimers. Despite speculation that different integrin subfamilies may differ in activation mechanism or conformation, we show that α5β1, similar to β2 and β3 integrins, can adopt the bent-closed, extended-closed, and extended-open overall global conformational states. We characterize nine function-perturbing antibodies for the subunit and domain they bind and the conformation they stabilize. Only the extended-open α5β1 conformation mediates adhesion to fibronectin. Our results enable many cell biological studies on the 12 β1 integrins to be interpreted in terms of the integrin conformational states on cell surfaces that are stabilized. (See pp. E3872–E3881.)
Human pluripotent stem cells as a model of trophoblast differentiation in both normal development and disease
Mariko Horii, Yingchun Li, Anna K. Wakeland, Donald P. Pizzo, Katharine K. Nelson, Karen Sabatini, Louise Chang Laurent, Ying Liu, and Mana M. Parast
Human pluripotent stem cells (hPSCs) continue to be underappreciated as a model for studying trophoblast differentiation. In this study, we provide a reproducible, two-step protocol by which hPSCs can be differentiated into bipotential cytotrophoblast (CTB) stem-like cells and subsequently into functional, terminally differentiated trophoblasts. In addition, we provide evidence that the response of hPSC-derived CTBs to low oxygen is similar to that of primary CTBs. Finally, using trisomy 21-affected hPSCs, we show, for the first time to our knowledge, that hPSCs can model a trophoblast differentiation defect. We propose that hPSCs are superior to other currently available models for studying human trophoblast differentiation. (See pp. E3882–E3891.)
Versatile in vivo regulation of tumor phenotypes by dCas9-mediated transcriptional perturbation
Christian J. Braun, Peter M. Bruno, Max A. Horlbeck, Luke A. Gilbert, Jonathan S. Weissman, and Michael T. Hemann
Tumor development is accompanied by widespread genomic and transcriptional changes. The mere acquisition of this information has greatly outpaced our capability to functionally study the biological roles of altered genes. This dilemma highlights the necessity to develop technologies that facilitate a rapid functional prioritization among lists of altered genes. Here, we use catalytically dead Cas9 to specifically activate or inactivate the transcription of genes in mouse models of cancer. This approach allows us to study the impact of gene-level changes in vivo and to systematically screen for novel genetic mediators of treatment relapse. We expect that this approach can be used to systematically dissect the biological role of cancer-related genes, a process critical to identifying new cancer drug targets. (See pp. E3892–E3900.)
OX40L blockade protects against inflammation-driven fibrosis
Muriel Elhai, Jérôme Avouac, Anna Maria Hoffmann-Vold, Nadira Ruzehaji, Olivia Amiar, Barbara Ruiz, Hassina Brahiti, Matthieu Ponsoye, Maxime Fréchet, Anne Burgevin, Sonia Pezet, Jérémy Sadoine, Thomas Guilbert, Carole Nicco, Hisaya Akiba, Vigo Heissmeyer, Arun Subramaniam, Robert Resnick, Øyvind Molberg, André Kahan, Gilles Chiocchia, and Yannick Allanore
Fibrosis is a leading cause of death in industrialized countries. Until now, there has been no effective therapy to prevent or counteract the fibrotic process. This article describes the effect of the blockade of a late costimulatory molecule to prevent inflammation-driven skin, lung, and vessel fibrosis and to induce regression of established dermal fibrosis in vivo in complementary murine models of systemic sclerosis, a prototypic autoimmune fibrotic disease. This article also reveals an unexpected role of this protein as a biomarker of worsening fibrosis that might help delineate the prognosis of patients in clinical practice more accurately. (See pp. E3901–E3910.)
YY1 plays an essential role at all stages of B-cell differentiation
Eden Kleiman, Haiqun Jia, Salvatore Loguercio, Andrew I. Su, and Ann J. Feeney
Ying Yang 1 (YY1) is a ubiquitously expressed transcription factor that has been demonstrated to be essential for pro–B-cell development as well as lymphoma. It has recently been proposed that YY1 regulates the germinal center B-cell transcriptional program. We confirm this hypothesis and additionally show that YY1 is equally essential for all stages of B-cell differentiation. Through ChIP-sequencing analysis of YY1 binding, and analysis of differentially expressed genes from RNA-sequencing, our data show that, in addition to the regulation of several B-cell–specific genes, YY1 regulates many genes and pathways important in basic cellular functions, such as mitochondrial bioenergetics, transcription, ribosomal function, and cellular proliferation, thus explaining the requirement for YY1 at all stages of B-cell differentiation. (See pp. E3911–E3920.)
HIV-1 and HIV-2 exhibit divergent interactions with HLTF and UNG2 DNA repair proteins
Kasia Hrecka, Caili Hao, Ming-Chieh Shun, Sarabpreet Kaur, Selene K. Swanson, Laurence Florens, Michael P. Washburn, and Jacek Skowronski
In nondividing host cells, HIV is targeted by intrinsic antiviral defense mechanisms that introduce marks of damage into viral cDNA, thereby tagging it for processing by cellular DNA repair machinery. Surprisingly, our findings reveal that the two main types of HIV exhibit very different interactions with enzymes involved in DNA repair. HIV-1, but not HIV-2, efficiently removes select DNA repair enzymes, whereas HIV-2 increases dNTP supply in infected cells by removing SAMHD1 (a cell cycle-regulated dNTP triphosphohydrolase) dNTPase. Our findings imply that increasing dNTP supply during viral cDNA synthesis or repair, or blocking cDNA processing by DNA repair enzymes, are alternative strategies used by HIV-2 and HIV-1 to guard their DNA genomes and facilitate their replication/persistence in the host. (See pp. E3921–E3930.)
VgrG C terminus confers the type VI effector transport specificity and is required for binding with PAAR and adaptor–effector complex
Devanand D. Bondage, Jer-Sheng Lin, Lay-Sun Ma, Chih-Horng Kuo, and Erh-Min Lai
The type VI secretion system involves multiple strategies for effector delivery via fusion or interaction of effectors to structural components of the phage tail-like structure, the tube component Hcp or spike protein VgrG. However, the detailed mechanisms underlying how diverse VgrG proteins govern effector delivery remains unclear. Here, we report that the divergent C-terminus of VgrG protein is the molecular determinant governing specific effector delivery and is required for interacting with a specific adaptor/chaperone protein that stabilizes and binds directly with the cognate effector. The striking conservation of genetic modules encoding homologous VgrG, a distinct set of potential adaptor/chaperone, and a specific effector in various Proteobacteria strongly suggest a conserved mechanism in type VI effector delivery. (See pp. E3931–E3940.)
Caenorhabditis elegans responses to bacteria from its natural habitats
Buck S. Samuel, Holli Rowedder, Christian Braendle, Marie-Anne Félix, and Gary Ruvkun
Caenorhabditis elegans is a major model organism, both from the pathogenesis dimension and also for metabolism, aging, and developmental biology perspectives. And yet, its natural ecology, most especially, its natural microbiome, is almost untouched. Here we establish the natural microbial community of C. elegans. Using extensive culture collections, we categorize its spectrum of responses (from antagonistic to beneficial) to a wide breadth of wild bacteria with nearly 80% of isolates supporting robust growth. In the wild, specific microbes correlate with the population state of the animals, which is supported by reconstruction experiments in the lab. Thus, a simplified natural community related to that found in the wild can now be studied in the laboratory for its impact on C. elegans physiology. (See pp. E3941–E3949.)
Structural rearrangement of the intracellular domains during AMPA receptor activation
Linda G. Zachariassen, Ljudmila Katchan, Anna G. Jensen, Darryl S. Pickering, Andrew J. R. Plested, and Anders S. Kristensen
α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) are the main excitatory receptors in the brain. Although neurotransmitter binding to AMPARs is well understood, knowledge regarding their activation mechanisms remains incomplete. We exploited genetic insertion of fluorescent proteins to create AMPARs displaying fluorescence resonance energy transfer and allow optical monitoring of conformational changes in the receptor. Ligand-driven conformational changes are observed to occur in two intracellular domains of the receptor that are distant from the extracellular site where glutamate binds. The C terminus extends laterally from the receptor and remains relatively close to the plasma membrane. By measuring the activation of receptors electrically and optically at the same time, this study reveals AMPAR conformational changes occurring during receptor state transitions for a region not resolved in structural studies. (See pp. E3950–E3959.)
Stochastic steps in secondary active sugar transport
Joshua L. Adelman, Chiara Ghezzi, Paola Bisignano, Donald D. F. Loo, Seungho Choe, Jeff Abramson, John M. Rosenberg, Ernest M. Wright, and Michael Grabe
The potential energy stored in ion gradients across cell membranes drives nutrients in and out of cells by cotransport proteins, e.g., uphill glucose accumulation in cells by sodium cotransporters. Insight into the mechanism of cotransport has been obtained from high-resolution atomic structures of the transporters, but further progress requires dynamic information about ion and substrate movements through the proteins. We have used multiple long molecular-dynamic simulations and electrophysiological assays to explore the dynamics of the transport cycle. Ligands bound to sodium-dependent glucose transporters are released to the cytoplasm stochastically, whereas release to the external solution is ordered with sugar first. The order of events is intimately tied to how the protein converts the energy stored in an ion gradient into a sugar gradient. (See pp. E3960–E3966.)
Two distinct redox cascades cooperatively regulate chloroplast functions and sustain plant viability
Keisuke Yoshida and Toru Hisabori
A fundamental challenge for the plant life cycle is how to manage fluctuating light conditions. The thiol-based redox regulation in chloroplasts is a significant system for controlling chloroplast functions in response to light signals. Although chloroplasts likely possess a complex redox network supported by multiple redox-mediator proteins and target enzymes, the whole organization and biological significance have not been fully resolved. We performed biochemical and reverse-genetic studies and concluded that (i) two major redox systems via the ferredoxin-thioredoxin reductase/thioredoxin and NADPH-thioredoxin reductase C pathways differentially but cooperatively drive chloroplast redox regulation and (ii) their concerted action is critical for plant autotrophic growth. The regulatory circuits presented in this work contribute to the understanding of how plants survive in continuously changing environments. (See pp. E3967–E3976.)
Budgerigars and zebra finches differ in how they generalize in an artificial grammar learning experiment
Michelle J. Spierings and Carel ten Cate
Human language learning is based on learning abstract grammatical structures. Whether any nonhuman animal can detect such structures is a contentious issue. We tested zebra finches and budgerigars on whether experience with auditory strings with different grammatical structures resulted in learning these structures. Both species were able to distinguish the differently structured strings but did so very differently. Zebra finches attended to the ordinal positions of specific items in a string. In contrast, budgerigars detected the underlying structure and used this structure to classify correctly strings consisting of fully novel items. This ability to perceive the abstract relation between items is comparable to analogical reasoning, something long thought to be unique for humans and known from only a few species. (See pp. E3977–E3984.)