Electron localization in rod-shaped triicosahedral gold nanocluster
Meng Zhou, Renxi Jin, Matthew Y. Sfeir, Yuxiang Chen, Yongbo Song, and Rongchao Jin
Understanding the carrier dynamics in ultrasmall (<2-nm) gold nanoclusters is fundamentally important for their applications in solar energy storage and conversion. This work tackles how the electrons of gold nanoclusters flow after photoexcitation. The electron localization/delocalization is commonly observed in polymers and other molecular aggregates, but little is known about this phenomenon in gold nanoclusters. Here, we observed ∼100-ps excited-state electron localization in atomically precise rod-shaped Au37 nanoclusters made up of three icosahedra. The activation energy was further obtained by temperature-dependent experiments. The excitation localization observed in rod-shaped clusters of clusters will advance their applications in nonlinear optics and energy harvesting. (See pp. E4697–E4705.)
Hidden role of intermolecular proton transfer in the anomalously diffuse vibrational spectrum of a trapped hydronium ion
Stephanie M. Craig, Fabian S. Menges, Chinh H. Duong, Joanna K. Denton, Lindsey R. Madison, Anne B. McCoy, and Mark A. Johnson
Understanding the origin of the extremely diffuse vibrational spectrum of an excess proton in water presents a grand challenge for contemporary physical chemistry. Here, we report the key observation that such diffuse bands occur even when the hydronium ion is held in the binding pocket of a rigid crown ether scaffold at 10 K. The broadening is traced to the zero-point vibrational displacements of the ion in the crown. The diffuse spectra are therefore an intrinsic property of the system and mimic the action of thermal fluctuations at elevated temperatures. We treat the mechanics underlying this phenomenon with a vibrationally adiabatic ansatz, from which emerges a qualitative picture that emphasizes the hidden role of vibrationally driven, intermolecular proton transfer. (See pp. E4706–E4713.)
Myosin Va’s adaptor protein melanophilin enforces track selection on the microtubule and actin networks in vitro
Angela Oberhofer, Peter Spieler, Yuliya Rosenfeld, Willi L. Stepp, Augustine Cleetus, Alistair N. Hume, Felix Mueller-Planitz, and Zeynep Ökten
Inner organization of eukaryotic cells intimately depends on the active transport of diverse intracellular cargo on the ubiquitous actin and microtubule networks. The underlying mechanisms of such directional transport processes have been of outstanding interest. We studied a motor complex composed of Rab27a, melanophilin, and myosin Va and found, surprisingly, that the adaptor protein melanophilin toggled the binding preference toward actin or microtubules in vitro. Our results offer unexpected mechanistic insights into biasing the directionality of a moving organelle on the cytoskeleton through phospho-targeting the adaptor protein rather than its motor in vivo. (See pp. E4714–E4723.)
Mechanistic insights on the reduction of glutathione disulfide by protein disulfide isomerase
Rui P. P. Neves, Pedro Alexandrino Fernandes, and Maria João Ramos
Protein disulfide isomerase (PDI) is a ubiquitous enzyme involved in disulfide bond formation during protein folding. It has been related to neurological diseases (Parkinson or Alzheimer’s) because of unfolded protein response phenomena. It also participates in the regulation of the glutathione redox buffer [glutathione/glutathione disulfide (GSH/GSSG)] in the endoplasmic reticulum, also important in protein folding. PDI catalyzes the nucleophilic attack of thiolates to disulfide bonds (thiol-disulfide exchange), enhancing the formation of correct disulfide links that drive protein folding and ensure protein function. This reaction is ubiquitous to disulfide-oxidoreductases across several organisms, and it shows a distinctive chemistry. We present the enzymatic mechanism of PDI in the GSH/GSSG buffer and discuss the chemistry of thiol-disulfide exchange. (See pp. E4724–E4733.)
Superresolution microscopy reveals the three-dimensional organization of meiotic chromosome axes in intact Caenorhabditis elegans tissue
Simone Köhler, Michal Wojcik, Ke Xu, and Abby F. Dernburg
Meiosis is the essential cell division process that generates haploid gametes from diploid precursor cells. It relies on a dramatic reorganization of chromosomes around a central axis, which establishes a platform for homologous pairing and its subsequent stabilization by the formation of a proteinaceous structure, the synaptonemal complex, and by physical linkages resulting from crossover recombination events. Despite their central role in regulating key meiotic events, little is known about the organization of the chromosome axes. Here, we use superresolution microscopy, combined with CRISPR/Cas9 genome editing, to build a three-dimensional model of the synapsed chromosome axis. Our data link axis structure to its functions in synapsis and regulation of partner choice during meiotic double-strand break repair. (See pp. E4734–E4743.)
Evidence for self-organization in determining spatial patterns of stream nutrients, despite primacy of the geomorphic template
Xiaoli Dong, Albert Ruhí, and Nancy B. Grimm
Rivers and streams are open, heterogeneous ecosystems. Stream water chemistry is influenced by organisms and the physical environment, resulting in longitudinal (upstream-downstream) heterogeneity that can be analyzed with time-series methods. Applying statistical techniques to longitudinal nutrient concentration data in a desert stream, we found evidence of substantial internal regulation of surface-water nutrient patterns, realized via spatial feedbacks. The strength of these feedbacks increased over succession. Although inputs from subsurface zones (a feature associated with the physical template) remained a major factor in explaining nutrient patterns, by late succession, the effect size of internal feedbacks was equal to the effect size of major upwelling zones. Our study demonstrates that multiple processes interact in a dynamic way to create ecosystem spatial heterogeneity. (See pp. E4744–E4752.)
RNA-seq reveals conservation of function among the yolk sacs of human, mouse, and chicken
Tereza Cindrova-Davies, Eric Jauniaux, Michael G. Elliot, Sungsam Gong, Graham J. Burton, and D. Stephen Charnock-Jones
The human yolk sac is often considered vestigial. Here, we report RNA-sequencing analysis of the human and murine yolk sacs and compare with that of the chicken. We relate the human RNA-sequencing data to coelomic fluid proteomic data. Conservation of transcripts across the species indicates the human secondary yolk sac likely performs key functions early in development, particularly uptake and processing of macro- and micronutrients, many of which are found in coelomic fluid. More generally, our findings shed light on evolutionary mechanisms giving rise to complex structures such as the placenta. We propose that although a choriovitelline placenta is never established physically in the human, the placental villi, exocoelomic cavity, and secondary yolk sac function together as a physiological equivalent. (See pp. E4753–E4761.)
Estimating the parameters of background selection and selective sweeps in Drosophila in the presence of gene conversion
José Luis Campos, Lei Zhao (赵磊), and Brian Charlesworth
The level of DNA sequence variation at a site in the genome is affected by selection acting on genetically linked sites. We have developed models of selection at linked sites to explain the observed negative relation between the level of nearly neutral variability in Drosophila genes and their protein sequence divergence from a related species. We use fits of these models to polymorphism and divergence data to show that selective sweeps are the main determinants of this pattern. We obtain estimates of the strengths of selection on advantageous mutations and the proportions of new mutations that are selectively advantageous. Gene conversion, a major source of genetic recombination within genes, has a large effect on these parameter estimates. (See pp. E4762–E4771.)
Follistatin is critical for mouse uterine receptivity and decidualization
Paul T. Fullerton Jr., Diana Monsivais, Ramakrishna Kommagani, and Martin M. Matzuk
An estimated 50% of in vitro fertilization attempts fail to achieve implantation, making implantation one of the most significant challenges in the assisted reproductive technologies (ART) clinic. Unfortunately, no effective treatments or biomarkers are available for the receptivity of the uterus to embryo implantation. Understanding the molecular mechanisms underlying implantation will enable future advances in ART to improve success rates and reduce the emotional, physical, and financial toll that ART failure takes on patients. We generated conditional knockout of follistatin in the uterus and demonstrated that follistatin plays a critical role in establishing uterine receptivity. Our results contribute to the understanding of the molecular mechanisms underlying uterine receptivity and offer a useful animal model for studying implantation and decidualization failure. (See pp. E4772–E4781.)
Manipulating DNA damage-response signaling for the treatment of immune-mediated diseases
Jonathan P. McNally, Scott H. Millen, Vandana Chaturvedi, Nora Lakes, Catherine E. Terrell, Eileen E. Elfers, Kaitlin R. Carroll, Simon P. Hogan, Paul R. Andreassen, Julie Kanter, Carl E. Allen, Michael M. Henry, Jay N. Greenberg, Stephan Ladisch, Michelle L. Hermiston, Michael Joyce, David A. Hildeman, Jonathan D. Katz, and Michael B. Jordan
Therapeutic immune suppression is essential for treating a variety of immune conditions, including autoimmune diseases, immunoregulatory disorders, and in transplantation. Reliance on broadly acting drugs carries substantial risks, and even pathway-specific agents are problematic, because most immune pathways have essential functions. The ideal form of immune suppression would be antigen-specific, suppressing an undesired immune response but sparing all others. We describe a unique strategy for therapeutic immune suppression, relying on targeted manipulation of DNA damage-response signaling, that exploits unique aspects of lymphocyte biology. This approach allows for highly selective suppression of recently activated T cells, displays clear therapeutic benefits, and has less off-target toxicity than conventional DNA-damaging drugs. (See pp. E4782–E4791.)
How an alloreactive T-cell receptor achieves peptide and MHC specificity
Yuan Wang, Nishant K. Singh, Timothy T. Spear, Lance M. Hellman, Kurt H. Piepenbrink, Rachel H. McMahan, Hugo R. Rosen, Craig W. Vander Kooi, Michael I. Nishimura, and Brian M. Baker
T-cell alloreactivity drives transplant rejection. Alloreactive recognition is believed to proceed with limited specificity, accounting for the high numbers of alloreactive T cells in humans. Paradoxically, however, many T cells recognize alloantigens with high specificity, and receptors from such T cells are being explored for use in cancer immunotherapy. Here, we explain how a T-cell receptor (TCR) achieves high specificity toward a peptide antigen presented by allo-major histocompatibility complex (MHC). Counter to prevailing theories of alloreactivity, we find that TCR recognition is driven by a cooperative interplay between features unique to both the allo-MHC and the peptide, such that binding is both MHC- and peptide-centric. Our results have broad implications for the determinants of immune recognition and efforts in immunotherapy. (See pp. E4792–E4801.)
Thermal combination therapies for local drug delivery by magnetic resonance-guided high-intensity focused ultrasound
Nicole Hijnen, Esther Kneepkens, Mariska de Smet, Sander Langereis, Edwin Heijman, and Holger Grüll
MRI-guided high-intensity focused ultrasound (MR-HIFU) is noninvasive technology able to focally heat tumor tissue from hyperthermic up to ablative temperatures. Although ablative temperatures can be used to destroy cancerous tissue directly, adequate ablation of tumor margins is often impossible due to the vicinity of vital structures, leaving a potential source for local recurrence. Another therapeutic option in oncology is hyperthermia-triggered local drug delivery using MR-HIFU in combination with temperature-sensitive liposomes (TSLs). In this study, we compare different MR-HIFU treatment schemes comprising ablation and hyperthermia-triggered drug delivery with respect to drug distribution and therapeutic efficacy. We show that a combination protocol of hyperthermia-induced drug delivery followed by ablation resulted in a homogeneous drug distribution and the highest therapeutic effect. (See pp. E4802–E4811.)
Structure-guided evolution of antigenically distinct adeno-associated virus variants for immune evasion
Longping Victor Tse, Kelli A. Klinc, Victoria J. Madigan, Ruth M. Castellanos Rivera, Lindsey F. Wells, L. Patrick Havlik, J. Kennon Smith, Mavis Agbandje-McKenna, and Aravind Asokan
Preexisting neutralizing antibodies (NAbs) against adeno-associated viruses (AAVs) pose a major, unresolved challenge that restricts patient enrollment in gene therapy clinical trials using recombinant AAV vectors. To tackle this problem, we developed a structure-guided approach to evolve AAV variants with altered antigenic footprints that cannot be recognized by preexisting antibodies. These proof-of-principle studies demonstrate that synthetic AAV variants can be evolved to evade neutralizing sera from different species—mice, nonhuman primates, and humans—without compromising yield and transduction efficiency or altering tropism. Our approach provides a roadmap for engineering any AAV strain to evade NAbs in prospective patients for human gene therapy. (See pp. E4812–E4821.)
AraC-like transcriptional activator CuxR binds c-di-GMP by a PilZ-like mechanism to regulate extracellular polysaccharide production
Simon Schäper, Wieland Steinchen, Elizaveta Krol, Florian Altegoer, Dorota Skotnicka, Lotte Søgaard-Andersen, Gert Bange, and Anke Becker
Cyclic dimeric GMP (c-di-GMP) has emerged as ubiquitous bacterial second messenger, regulating multiple cellular functions, such as cell cycle, virulence, and biofilm formation. However, our knowledge on the molecular inventory, diversity, and function of c-di-GMP receptors, and the molecular evolution of c-di-GMP–responsive proteins is still incomplete. We have identified a class of c-di-GMP–responsive transcription factors, strikingly illustrating how a classical transcription factor has acquired the ability to sense this signaling molecule. The mode of c-di-GMP binding to the AraC-like transcription factor CuxR is highly reminiscent to that of the PilZ domain, the prototypic c-di-GMP receptor. PilZ and CuxR provide an example of convergent evolution in which c-di-GMP binding sites of similar topology have evolved independently in two distinct protein families. (See pp. E4822–E4831.)
Rifamycin action on RNA polymerase in antibiotic-tolerant Mycobacterium tuberculosis results in differentially detectable populations
Kohta Saito, Thulasi Warrier, Selin Somersan-Karakaya, Lina Kaminski, Jianjie Mi, Xiuju Jiang, Suna Park, Kristi Shigyo, Ben Gold, Julia Roberts, Elaina Weber, William R. Jacobs Jr., and Carl F. Nathan
Most of the Mycobacterium tuberculosis (Mtb) bacilli in the sputum of most patients with tuberculosis studied to date do not grow on standard agar-based media but rather grow when diluted in liquid media of similar composition. Here, we describe a rigorously standardized and independently replicated method to generate and count these differentially detectable (DD) Mtb in culture. DD Mtb generation required the action of a rifamycin on RNA polymerase after induction of phenotypic tolerance. Generation of these cells in vitro led to identification of one drug that can kill them and should facilitate the discovery of others. (See pp. E4832–E4840.)
Cortical neurons multiplex reward-related signals along with sensory and motor information
Arjun Ramakrishnan, Yoon Woo Byun, Kyle Rand, Christian E. Pedersen, Mikhail A. Lebedev, and Miguel A. L. Nicolelis
The ability to learn highly skilled movements may depend on the dopamine-related plasticity occurring in motor cortex, because the density of dopamine receptors—the reward sensor—increases in this area from rodents to primates. We hypothesized that primary motor (M1) and somatosensory (S1) neurons would encode rewards during operant conditioned motor behaviors. Rhesus monkeys were implanted with cortical multielectrode implants and trained to perform arm-reaching tasks with different reward schedules. Consistent with our hypothesis, M1 and S1 neurons represented reward anticipation and delivery and a mismatch between the quantities of anticipated and actual rewards. These same neurons also represented arm movement parameters. We suggest that this multiplexing of motor and reinforcement information by cortical neurons underlies motor learning. (See pp. E4841–E4850.)
Input timing for spatial processing is precisely tuned via constant synaptic delays and myelination patterns in the auditory brainstem
Annette Stange-Marten, Alisha L. Nabel, James L. Sinclair, Matthew Fischl, Olga Alexandrova, Hilde Wohlfrom, Conny Kopp-Scheinpflug, Michael Pecka, and Benedikt Grothe
Neural computation depends on precisely timed synaptic inputs, but the way that the timing of inputs is tuned to match postsynaptic processing requirements is not well understood. Here, we studied the same brainstem sound localization pathway in two species with dissimilar temporal processing requirements. Two factors that limit precise timing are synaptic delay and axonal conduction time. In gerbils, which depend on precise timing for sound localization, synaptic delays in fast conducting axons are stable across activity level, and axon myelination is adapted to minimize conduction delays. In mice, which do not depend on precise timing, these specializations are absent. Our results suggest that both axonal and synaptic properties are optimized to the specific functional requirements of neural computation, advancing our understanding of the mechanisms that optimize neural circuits. (See pp. E4851–E4858.)
Contacts between the endoplasmic reticulum and other membranes in neurons
Yumei Wu, Christina Whiteus, C. Shan Xu, Kenneth J. Hayworth, Richard J. Weinberg, Harald F. Hess, and Pietro De Camilli
The cytoplasm of eukaryotic cells is compartmentalized by intracellular membranes that define subcellular organelles. One of these organelles, the endoplasmic reticulum, forms a continuous network of tubules and cisternae that extends throughout all cell compartments, including neuronal dendrites and axons. This network communicates with most other organelles by vesicular transport, and also by contacts that do not lead to fusion but allow cross-talk between adjacent bilayers. Though these membrane contacts have previously been observed in neurons, their distribution and abundance has not been systematically analyzed. Here, we have carried out such analysis. Our studies reveal new aspects of the internal structure of neurons and provide a critical complement to information about interorganelle communication emerging from functional and biochemical studies. (See pp. E4859–E4867.)
The POTRA domains of Toc75 exhibit chaperone-like function to facilitate import into chloroplasts
Patrick K. O’Neil, Lynn G. L. Richardson, Yamuna D. Paila, Grzegorz Piszczek, Srinivas Chakravarthy, Nicholas Noinaj, and Danny Schnell
Nearly all proteins found within chloroplasts are synthesized in the cytoplasm as preproteins and then imported and trafficked to their final destination. The initial steps in importation are orchestrated by the TOC complex, which includes Toc75, serving as the translocation channel, and Toc33 and Toc159, both containing GTPase domains, which help drive substrate selection and importation. Aside from the soluble domain of Toc33/34, structural information for the TOC complex is lacking, hindering our ability to form mechanistic models for function. Here we report a structure of Toc75 consisting of three tandem POTRA domains. Our findings indicate that the POTRA domains may help facilitate preprotein import by directly binding preproteins and orchestrating handoff to the TIC complex. (See pp. E4868–E4876.)
AP1G mediates vacuolar acidification during synergid-controlled pollen tube reception
Jia-Gang Wang, Chong Feng, Hai-Hong Liu, Qiang-Nan Feng, Sha Li, and Yan Zhang
Double fertilization of angiosperms is preceded by the death of gametophytic cells: Two synergid cells degenerate to create a microenvironment for fertilization, whereas the pollen tube bursts to discharge sperm cells. This process is called pollen tube reception, in which a few synergid surface proteins have been identified, but intracellular activities involved are obscure. We report here that vacuolar acidification, mediated by V-ATPases and adaptor protein 1, might be an important mechanism for synergid degeneration during pollen tube reception. The study provides insights into a cell-death mechanism specifically adopted by the plant phylum. (See pp. E4877–E4883.)
Auxin steers root cell expansion via apoplastic pH regulation in Arabidopsis thaliana
Elke Barbez, Kai Dünser, Angelika Gaidora, Thomas Lendl, and Wolfgang Busch
Cellular growth in plants is constrained by cell walls; hence, loosening these structures is required for growth. The long-standing acid growth theory links auxin signaling, apoplastic pH homeostasis, and cellular expansion, providing a conceptual framework for cell expansion in plant shoots. Intriguingly, this model remains heavily debated for roots. Here, we present a fluorescent dye that allows for the correlation of cell size and apoplastic pH at a cellular resolution in Arabidopsis thaliana. This enabled us to elucidate a complex involvement of auxin in root apoplastic pH homeostasis, which is important for root cell expansion and gravitropic response. These findings shed light on the poorly understood acid growth mechanism in roots. (See pp. E4884–E4893.)