TY - JOUR AB - De novo heterozygous variants in KCNC2 encoding the voltage-gated potassium (K+) channel subunit Kv3.2 are a recently described cause of developmental and epileptic encephalopathy (DEE). A de novo variant in KCNC2 c.374G > A (p.Cys125Tyr) was identified via exome sequencing in a patient with DEE. Relative to wild-type Kv3.2, Kv3.2-p.Cys125Tyr induces K+ currents exhibiting a large hyperpolarizing shift in the voltage dependence of activation, accelerated activation, and delayed deactivation consistent with a relative stabilization of the open conformation, along with increased current density. Leveraging the cryogenic electron microscopy (cryo-EM) structure of Kv3.1, molecular dynamic simulations suggest that a strong π-π stacking interaction between the variant Tyr125 and Tyr156 in the α-6 helix of the T1 domain promotes a relative stabilization of the open conformation of the channel, which underlies the observed gain of function. A multicompartment computational model of a Kv3-expressing parvalbumin-positive cerebral cortex fast-spiking γ-aminobutyric acidergic (GABAergic) interneuron (PV-IN) demonstrates how the Kv3.2-Cys125Tyr variant impairs neuronal excitability and dysregulates inhibition in cerebral cortex circuits to explain the resulting epilepsy. AU - Clatot, Jerome AU - Currin, Christopher AU - Liang, Qiansheng AU - Pipatpolkai, Tanadet AU - Massey, Shavonne L. AU - Helbig, Ingo AU - Delemotte, Lucie AU - Vogels, Tim P AU - Covarrubias, Manuel AU - Goldberg, Ethan M. ID - 14841 IS - 3 JF - Proceedings of the National Academy of Sciences of the United States of America TI - A structurally precise mechanism links an epilepsy-associated KCNC2 potassium channel mutation to interneuron dysfunction VL - 121 ER - TY - JOUR AB - Self-replication of amyloid fibrils via secondary nucleation is an intriguing physicochemical phenomenon in which existing fibrils catalyze the formation of their own copies. The molecular events behind this fibril surface-mediated process remain largely inaccessible to current structural and imaging techniques. Using statistical mechanics, computer modeling, and chemical kinetics, we show that the catalytic structure of the fibril surface can be inferred from the aggregation behavior in the presence and absence of a fibril-binding inhibitor. We apply our approach to the case of Alzheimer’s A amyloid fibrils formed in the presence of proSP-C Brichos inhibitors. We find that self-replication of A fibrils occurs on small catalytic sites on the fibril surface, which are far apart from each other, and each of which can be covered by a single Brichos inhibitor. AU - Curk, Samo AU - Krausser, Johannes AU - Meisl, Georg AU - Frenkel, Daan AU - Linse, Sara AU - Michaels, Thomas C.T. AU - Knowles, Tuomas P.J. AU - Šarić, Anđela ID - 15001 IS - 7 JF - Proceedings of the National Academy of Sciences of the United States of America TI - Self-replication of Aβ42 aggregates occurs on small and isolated fibril sites VL - 121 ER - TY - JOUR AB - GABAB receptor (GBR) activation inhibits neurotransmitter release in axon terminals in the brain, except in medial habenula (MHb) terminals, which show robust potentiation. However, mechanisms underlying this enigmatic potentiation remain elusive. Here, we report that GBR activation on MHb terminals induces an activity-dependent transition from a facilitating, tonic to a depressing, phasic neurotransmitter release mode. This transition is accompanied by a 4.1-fold increase in readily releasable vesicle pool (RRP) size and a 3.5-fold increase of docked synaptic vesicles (SVs) at the presynaptic active zone (AZ). Strikingly, the depressing phasic release exhibits looser coupling distance than the tonic release. Furthermore, the tonic and phasic release are selectively affected by deletion of synaptoporin (SPO) and Ca 2+ -dependent activator protein for secretion 2 (CAPS2), respectively. SPO modulates augmentation, the short-term plasticity associated with tonic release, and CAPS2 retains the increased RRP for initial responses in phasic response trains. The cytosolic protein CAPS2 showed a SV-associated distribution similar to the vesicular transmembrane protein SPO, and they were colocalized in the same terminals. We developed the “Flash and Freeze-fracture” method, and revealed the release of SPO-associated vesicles in both tonic and phasic modes and activity-dependent recruitment of CAPS2 to the AZ during phasic release, which lasted several minutes. Overall, these results indicate that GBR activation translocates CAPS2 to the AZ along with the fusion of CAPS2-associated SVs, contributing to persistency of the RRP increase. Thus, we identified structural and molecular mechanisms underlying tonic and phasic neurotransmitter release and their transition by GBR activation in MHb terminals. AU - Koppensteiner, Peter AU - Bhandari, Pradeep AU - Önal, Hüseyin C AU - Borges Merjane, Carolina AU - Le Monnier, Elodie AU - Roy, Utsa AU - Nakamura, Yukihiro AU - Sadakata, Tetsushi AU - Sanbo, Makoto AU - Hirabayashi, Masumi AU - Rhee, JeongSeop AU - Brose, Nils AU - Jonas, Peter M AU - Shigemoto, Ryuichi ID - 15084 IS - 8 JF - Proceedings of the National Academy of Sciences SN - 0027-8424 TI - GABAB receptors induce phasic release from medial habenula terminals through activity-dependent recruitment of release-ready vesicles VL - 121 ER - TY - JOUR AB - Direct reciprocity is a powerful mechanism for cooperation in social dilemmas. The very logic of reciprocity, however, seems to require that individuals are symmetric, and that everyone has the same means to influence each others’ payoffs. Yet in many applications, individuals are asymmetric. Herein, we study the effect of asymmetry in linear public good games. Individuals may differ in their endowments (their ability to contribute to a public good) and in their productivities (how effective their contributions are). Given the individuals’ productivities, we ask which allocation of endowments is optimal for cooperation. To this end, we consider two notions of optimality. The first notion focuses on the resilience of cooperation. The respective endowment distribution ensures that full cooperation is feasible even under the most adverse conditions. The second notion focuses on efficiency. The corresponding endowment distribution maximizes group welfare. Using analytical methods, we fully characterize these two endowment distributions. This analysis reveals that both optimality notions favor some endowment inequality: More productive players ought to get higher endowments. Yet the two notions disagree on how unequal endowments are supposed to be. A focus on resilience results in less inequality. With additional simulations, we show that the optimal endowment allocation needs to account for both the resilience and the efficiency of cooperation. AU - Hübner, Valentin AU - Staab, Manuel AU - Hilbe, Christian AU - Chatterjee, Krishnendu AU - Kleshnina, Maria ID - 15083 IS - 10 JF - Proceedings of the National Academy of Sciences SN - 0027-8424 TI - Efficiency and resilience of cooperation in asymmetric social dilemmas VL - 121 ER - TY - JOUR AB - Entire chromosomes are typically only transmitted vertically from one generation to the next. The horizontal transfer of such chromosomes has long been considered improbable, yet gained recent support in several pathogenic fungi where it may affect the fitness or host specificity. To date, it is unknown how these transfers occur, how common they are and whether they can occur between different species. In this study, we show multiple independent instances of horizontal transfers of the same accessory chromosome between two distinct strains of the asexual entomopathogenic fungusMetarhizium robertsiiduring experimental co-infection of its insect host, the Argentine ant. Notably, only the one chromosome – but no other – was transferred from the donor to the recipient strain. The recipient strain, now harboring the accessory chromosome, exhibited a competitive advantage under certain host conditions. By phylogenetic analysis we further demonstrate that the same accessory chromosome was horizontally transferred in a natural environment betweenM. robertsiiand another congeneric insect pathogen,M. guizhouense. Hence horizontal chromosome transfer is not limited to the observed frequent events within species during experimental infections but also occurs naturally across species. The transferred accessory chromosome contains genes that might be involved in its preferential horizontal transfer, encoding putative histones and histone-modifying enzymes, but also putative virulence factors that may support its establishment. Our study reveals that both intra- and interspecies horizontal transfer of entire chromosomes is more frequent than previously assumed, likely representing a not uncommon mechanism for gene exchange.Significance StatementThe enormous success of bacterial pathogens has been attributed to their ability to exchange genetic material between one another. Similarly, in eukaryotes, horizontal transfer of genetic material allowed the spread of virulence factors across species. The horizontal transfer of whole chromosomes could be an important pathway for such exchange of genetic material, but little is known about the origin of transferable chromosomes and how frequently they are exchanged. Here, we show that the transfer of accessory chromosomes - chromosomes that are non-essential but may provide fitness benefits - is common during fungal co-infections and is even possible between distant pathogenic species, highlighting the importance of horizontal gene transfer via chromosome transfer also for the evolution and function of eukaryotic pathogens. AU - Habig, Michael AU - Grasse, Anna V AU - Müller, Judith AU - Stukenbrock, Eva H. AU - Leitner, Hanna AU - Cremer, Sylvia ID - 14478 IS - 11 JF - Proceedings of the National Academy of Sciences of the United States of America SN - 0027-8424 TI - Frequent horizontal chromosome transfer between asexual fungal insect pathogens VL - 121 ER - TY - JOUR AB - Water is known to play an important role in collagen self-assembly, but it is still largely unclear how water–collagen interactions influence the assembly process and determine the fibril network properties. Here, we use the H2O/D2O isotope effect on the hydrogen-bond strength in water to investigate the role of hydration in collagen self-assembly. We dissolve collagen in H2O and D2O and compare the growth kinetics and the structure of the collagen assemblies formed in these water isotopomers. Surprisingly, collagen assembly occurs ten times faster in D2O than in H2O, and collagen in D2O self-assembles into much thinner fibrils, that form a more inhomogeneous and softer network, with a fourfold reduction in elastic modulus when compared to H2O. Combining spectroscopic measurements with atomistic simulations, we show that collagen in D2O is less hydrated than in H2O. This partial dehydration lowers the enthalpic penalty for water removal and reorganization at the collagen–water interface, increasing the self-assembly rate and the number of nucleation centers, leading to thinner fibrils and a softer network. Coarse-grained simulations show that the acceleration in the initial nucleation rate can be reproduced by the enhancement of electrostatic interactions. These results show that water acts as a mediator between collagen monomers, by modulating their interactions so as to optimize the assembly process and, thus, the final network properties. We believe that isotopically modulating the hydration of proteins can be a valuable method to investigate the role of water in protein structural dynamics and protein self-assembly. AU - Giubertoni, Giulia AU - Feng, Liru AU - Klein, Kevin AU - Giannetti, Guido AU - Rutten, Luco AU - Choi, Yeji AU - Van Der Net, Anouk AU - Castro-Linares, Gerard AU - Caporaletti, Federico AU - Micha, Dimitra AU - Hunger, Johannes AU - Deblais, Antoine AU - Bonn, Daniel AU - Sommerdijk, Nico AU - Šarić, Anđela AU - Ilie, Ioana M. AU - Koenderink, Gijsje H. AU - Woutersen, Sander ID - 15116 IS - 11 JF - Proceedings of the National Academy of Sciences of the United States of America SN - 0027-8424 TI - Elucidating the role of water in collagen self-assembly by isotopically modulating collagen hydration VL - 121 ER - TY - JOUR AB - How do statistical dependencies in measurement noise influence high-dimensional inference? To answer this, we study the paradigmatic spiked matrix model of principal components analysis (PCA), where a rank-one matrix is corrupted by additive noise. We go beyond the usual independence assumption on the noise entries, by drawing the noise from a low-order polynomial orthogonal matrix ensemble. The resulting noise correlations make the setting relevant for applications but analytically challenging. We provide characterization of the Bayes optimal limits of inference in this model. If the spike is rotation invariant, we show that standard spectral PCA is optimal. However, for more general priors, both PCA and the existing approximate message-passing algorithm (AMP) fall short of achieving the information-theoretic limits, which we compute using the replica method from statistical physics. We thus propose an AMP, inspired by the theory of adaptive Thouless–Anderson–Palmer equations, which is empirically observed to saturate the conjectured theoretical limit. This AMP comes with a rigorous state evolution analysis tracking its performance. Although we focus on specific noise distributions, our methodology can be generalized to a wide class of trace matrix ensembles at the cost of more involved expressions. Finally, despite the seemingly strong assumption of rotation-invariant noise, our theory empirically predicts algorithmic performance on real data, pointing at strong universality properties. AU - Barbier, Jean AU - Camilli, Francesco AU - Mondelli, Marco AU - Sáenz, Manuel ID - 13315 IS - 30 JF - Proceedings of the National Academy of Sciences of the United States of America TI - Fundamental limits in structured principal component analysis and how to reach them VL - 120 ER - TY - JOUR AB - Traditionally, nuclear spin is not considered to affect biological processes. Recently, this has changed as isotopic fractionation that deviates from classical mass dependence was reported both in vitro and in vivo. In these cases, the isotopic effect correlates with the nuclear magnetic spin. Here, we show nuclear spin effects using stable oxygen isotopes (16O, 17O, and 18O) in two separate setups: an artificial dioxygen production system and biological aquaporin channels in cells. We observe that oxygen dynamics in chiral environments (in particular its transport) depend on nuclear spin, suggesting future applications for controlled isotope separation to be used, for instance, in NMR. To demonstrate the mechanism behind our findings, we formulate theoretical models based on a nuclear-spin-enhanced switch between electronic spin states. Accounting for the role of nuclear spin in biology can provide insights into the role of quantum effects in living systems and help inspire the development of future biotechnology solutions. AU - Vardi, Ofek AU - Maroudas-Sklare, Naama AU - Kolodny, Yuval AU - Volosniev, Artem AU - Saragovi, Amijai AU - Galili, Nir AU - Ferrera, Stav AU - Ghazaryan, Areg AU - Yuran, Nir AU - Affek, Hagit P. AU - Luz, Boaz AU - Goldsmith, Yonaton AU - Keren, Nir AU - Yochelis, Shira AU - Halevy, Itay AU - Lemeshko, Mikhail AU - Paltiel, Yossi ID - 14037 IS - 32 JF - Proceedings of the National Academy of Sciences of the United States of America TI - Nuclear spin effects in biological processes VL - 120 ER - TY - JOUR AB - So-called spontaneous activity is a central hallmark of most nervous systems. Such non-causal firing is contrary to the tenet of spikes as a means of communication, and its purpose remains unclear. We propose that self-initiated firing can serve as a release valve to protect neurons from the toxic conditions arising in mitochondria from lower-than-baseline energy consumption. To demonstrate the viability of our hypothesis, we built a set of models that incorporate recent experimental results indicating homeostatic control of metabolic products—Adenosine triphosphate (ATP), adenosine diphosphate (ADP), and reactive oxygen species (ROS)—by changes in firing. We explore the relationship of metabolic cost of spiking with its effect on the temporal patterning of spikes and reproduce experimentally observed changes in intrinsic firing in the fruitfly dorsal fan-shaped body neuron in a model with ROS-modulated potassium channels. We also show that metabolic spiking homeostasis can produce indefinitely sustained avalanche dynamics in cortical circuits. Our theory can account for key features of neuronal activity observed in many studies ranging from ion channel function all the way to resting state dynamics. We finish with a set of experimental predictions that would confirm an integrated, crucial role for metabolically regulated spiking and firmly link metabolic homeostasis and neuronal function. AU - Chintaluri, Chaitanya AU - Vogels, Tim P ID - 14666 IS - 48 JF - Proceedings of the National Academy of Sciences of the United States of America SN - 0027-8424 TI - Metabolically regulated spiking could serve neuronal energy homeostasis and protect from reactive oxygen species VL - 120 ER - TY - JOUR AB - As a crucial nitrogen source, nitrate (NO3−) is a key nutrient for plants. Accordingly, root systems adapt to maximize NO3− availability, a developmental regulation also involving the phytohormone auxin. Nonetheless, the molecular mechanisms underlying this regulation remain poorly understood. Here, we identify low-nitrate-resistant mutant (lonr) in Arabidopsis (Arabidopsis thaliana), whose root growth fails to adapt to low-NO3− conditions. lonr2 is defective in the high-affinity NO3− transporter NRT2.1. lonr2 (nrt2.1) mutants exhibit defects in polar auxin transport, and their low-NO3−-induced root phenotype depends on the PIN7 auxin exporter activity. NRT2.1 directly associates with PIN7 and antagonizes PIN7-mediated auxin efflux depending on NO3− levels. These results reveal a mechanism by which NRT2.1 in response to NO3− limitation directly regulates auxin transport activity and, thus, root growth. This adaptive mechanism contributes to the root developmental plasticity to help plants cope with changes in NO3− availability. AU - Wang, Yalu AU - Yuan, Zhi AU - Wang, Jinyi AU - Xiao, Huixin AU - Wan, Lu AU - Li, Lanxin AU - Guo, Yan AU - Gong, Zhizhong AU - Friml, Jiří AU - Zhang, Jing ID - 13201 IS - 25 JF - Proceedings of the National Academy of Sciences of the United States of America SN - 0027-8424 TI - The nitrate transporter NRT2.1 directly antagonizes PIN7-mediated auxin transport for root growth adaptation VL - 120 ER - TY - JOUR AB - When Mendel’s work was rediscovered in 1900, and extended to establish classical genetics, it was initially seen in opposition to Darwin’s theory of evolution by natural selection on continuous variation, as represented by the biometric research program that was the foundation of quantitative genetics. As Fisher, Haldane, and Wright established a century ago, Mendelian inheritance is exactly what is needed for natural selection to work efficiently. Yet, the synthesis remains unfinished. We do not understand why sexual reproduction and a fair meiosis predominate in eukaryotes, or how far these are responsible for their diversity and complexity. Moreover, although quantitative geneticists have long known that adaptive variation is highly polygenic, and that this is essential for efficient selection, this is only now becoming appreciated by molecular biologists—and we still do not have a good framework for understanding polygenic variation or diffuse function. AU - Barton, Nicholas H ID - 11702 IS - 30 JF - Proceedings of the National Academy of Sciences of the United States of America SN - 0027-8424 TI - The "New Synthesis" VL - 119 ER - TY - JOUR AB - Glaciers are key components of the mountain water towers of Asia and are vital for downstream domestic, agricultural, and industrial uses. The glacier mass loss rate over the southeastern Tibetan Plateau is among the highest in Asia and has accelerated in recent decades. This acceleration has been attributed to increased warming, but the mechanisms behind these glaciers’ high sensitivity to warming remain unclear, while the influence of changes in precipitation over the past decades is poorly quantified. Here, we reconstruct glacier mass changes and catchment runoff since 1975 at a benchmark glacier, Parlung No. 4, to shed light on the drivers of recent mass losses for the monsoonal, spring-accumulation glaciers of the Tibetan Plateau. Our modeling demonstrates how a temperature increase (mean of 0.39C ⋅dec−1since 1990) has accelerated mass loss rates by altering both the ablation and accumulation regimes in a complex manner. The majority of the post-2000 mass loss occurred during the monsoon months, caused by simultaneous decreases in the solid precipitation ratio (from 0.70 to 0.56) and precipitation amount (–10%), leading to reduced monsoon accumulation (–26%). Higher solid precipitation in spring (+18%) during the last two decades was increasingly important in mitigating glacier mass loss by providing mass to the glacier and protecting it from melting in the early monsoon. With bare ice exposed to warmer temperatures for longer periods, icemelt and catchment discharge have unsustainably intensified since the start of the 21st century, raising concerns for long-term water supply and hazard occurrence in the region. AU - Jouberton, Achille AU - Shaw, Thomas E. AU - Miles, Evan AU - McCarthy, Michael AU - Fugger, Stefan AU - Ren, Shaoting AU - Dehecq, Amaury AU - Yang, Wei AU - Pellicciotti, Francesca ID - 12577 IS - 37 JF - PNAS KW - Multidisciplinary SN - 0027-8424 TI - Warming-induced monsoon precipitation phase change intensifies glacier mass loss in the southeastern Tibetan Plateau VL - 119 ER - TY - JOUR AB - Despite the growing interest in using chemical genetics in plant research, small molecule target identification remains a major challenge. The cellular thermal shift assay coupled with high-resolution mass spectrometry (CETSA MS) that monitors changes in the thermal stability of proteins caused by their interactions with small molecules, other proteins, or posttranslational modifications, allows the discovery of drug targets or the study of protein–metabolite and protein–protein interactions mainly in mammalian cells. To showcase the applicability of this method in plants, we applied CETSA MS to intact Arabidopsis thaliana cells and identified the thermal proteome of the plant-specific glycogen synthase kinase 3 (GSK3) inhibitor, bikinin. A comparison between the thermal and the phosphoproteomes of bikinin revealed the auxin efflux carrier PIN-FORMED1 (PIN1) as a substrate of the Arabidopsis GSK3s that negatively regulate the brassinosteroid signaling. We established that PIN1 phosphorylation by the GSK3s is essential for maintaining its intracellular polarity that is required for auxin-mediated regulation of vascular patterning in the leaf, thus revealing cross-talk between brassinosteroid and auxin signaling. AU - Lu, Qing AU - Zhang, Yonghong AU - Hellner, Joakim AU - Giannini, Caterina AU - Xu, Xiangyu AU - Pauwels, Jarne AU - Ma, Qian AU - Dejonghe, Wim AU - Han, Huibin AU - Van De Cotte, Brigitte AU - Impens, Francis AU - Gevaert, Kris AU - De Smet, Ive AU - Friml, Jiří AU - Molina, Daniel Martinez AU - Russinova, Eugenia ID - 10888 IS - 11 JF - Proceedings of the National Academy of Sciences of the United States of America TI - Proteome-wide cellular thermal shift assay reveals unexpected cross-talk between brassinosteroid and auxin signaling VL - 119 ER - TY - JOUR AB - Mineral nutrition is one of the key environmental factors determining plant development and growth. Nitrate is the major form of macronutrient nitrogen that plants take up from the soil. Fluctuating availability or deficiency of this element severely limits plant growth and negatively affects crop production in the agricultural system. To cope with the heterogeneity of nitrate distribution in soil, plants evolved a complex regulatory mechanism that allows rapid adjustment of physiological and developmental processes to the status of this nutrient. The root, as a major exploitation organ that controls the uptake of nitrate to the plant body, acts as a regulatory hub that, according to nitrate availability, coordinates the growth and development of other plant organs. Here, we identified a regulatory framework, where cytokinin response factors (CRFs) play a central role as a molecular readout of the nitrate status in roots to guide shoot adaptive developmental response. We show that nitrate-driven activation of NLP7, a master regulator of nitrate response in plants, fine tunes biosynthesis of cytokinin in roots and its translocation to shoots where it enhances expression of CRFs. CRFs, through direct transcriptional regulation of PIN auxin transporters, promote the flow of auxin and thereby stimulate the development of shoot organs. AU - Abualia, Rashed AU - Ötvös, Krisztina AU - Novák, Ondřej AU - Bouguyon, Eleonore AU - Domanegg, Kevin AU - Krapp, Anne AU - Nacry, Philip AU - Gojon, Alain AU - Lacombe, Benoit AU - Benková, Eva ID - 11734 IS - 31 JF - Proceedings of the National Academy of Sciences of the United States of America TI - Molecular framework integrating nitrate sensing in root and auxin-guided shoot adaptive responses VL - 119 ER - TY - JOUR AB - Genetically informed, deep-phenotyped biobanks are an important research resource and it is imperative that the most powerful, versatile, and efficient analysis approaches are used. Here, we apply our recently developed Bayesian grouped mixture of regressions model (GMRM) in the UK and Estonian Biobanks and obtain the highest genomic prediction accuracy reported to date across 21 heritable traits. When compared to other approaches, GMRM accuracy was greater than annotation prediction models run in the LDAK or LDPred-funct software by 15% (SE 7%) and 14% (SE 2%), respectively, and was 18% (SE 3%) greater than a baseline BayesR model without single-nucleotide polymorphism (SNP) markers grouped into minor allele frequency–linkage disequilibrium (MAF-LD) annotation categories. For height, the prediction accuracy R2 was 47% in a UK Biobank holdout sample, which was 76% of the estimated h2SNP. We then extend our GMRM prediction model to provide mixed-linear model association (MLMA) SNP marker estimates for genome-wide association (GWAS) discovery, which increased the independent loci detected to 16,162 in unrelated UK Biobank individuals, compared to 10,550 from BoltLMM and 10,095 from Regenie, a 62 and 65% increase, respectively. The average χ2 value of the leading markers increased by 15.24 (SE 0.41) for every 1% increase in prediction accuracy gained over a baseline BayesR model across the traits. Thus, we show that modeling genetic associations accounting for MAF and LD differences among SNP markers, and incorporating prior knowledge of genomic function, is important for both genomic prediction and discovery in large-scale individual-level studies. AU - Orliac, Etienne J. AU - Trejo Banos, Daniel AU - Ojavee, Sven E. AU - Läll, Kristi AU - Mägi, Reedik AU - Visscher, Peter M. AU - Robinson, Matthew Richard ID - 11733 IS - 31 JF - Proceedings of the National Academy of Sciences of the United States of America TI - Improving GWAS discovery and genomic prediction accuracy in biobank data VL - 119 ER - TY - JOUR AB - Plant cell growth responds rapidly to various stimuli, adapting architecture to environmental changes. Two major endogenous signals regulating growth are the phytohormone auxin and the secreted peptides rapid alkalinization factors (RALFs). Both trigger very rapid cellular responses and also exert long-term effects [Du et al., Annu. Rev. Plant Biol. 71, 379–402 (2020); Blackburn et al., Plant Physiol. 182, 1657–1666 (2020)]. However, the way, in which these distinct signaling pathways converge to regulate growth, remains unknown. Here, using vertical confocal microscopy combined with a microfluidic chip, we addressed the mechanism of RALF action on growth. We observed correlation between RALF1-induced rapid Arabidopsis thaliana root growth inhibition and apoplast alkalinization during the initial phase of the response, and revealed that RALF1 reversibly inhibits primary root growth through apoplast alkalinization faster than within 1 min. This rapid apoplast alkalinization was the result of RALF1-induced net H+ influx and was mediated by the receptor FERONIA (FER). Furthermore, we investigated the cross-talk between RALF1 and the auxin signaling pathways during root growth regulation. The results showed that RALF-FER signaling triggered auxin signaling with a delay of approximately 1 h by up-regulating auxin biosynthesis, thus contributing to sustained RALF1-induced growth inhibition. This biphasic RALF1 action on growth allows plants to respond rapidly to environmental stimuli and also reprogram growth and development in the long term. AU - Li, Lanxin AU - Chen, Huihuang AU - Alotaibi, Saqer S. AU - Pěnčík, Aleš AU - Adamowski, Maciek AU - Novák, Ondřej AU - Friml, Jiří ID - 11723 IS - 31 JF - Proceedings of the National Academy of Sciences KW - Multidisciplinary SN - 0027-8424 TI - RALF1 peptide triggers biphasic root growth inhibition upstream of auxin biosynthesis VL - 119 ER - TY - JOUR AB - Primary nucleation is the fundamental event that initiates the conversion of proteins from their normal physiological forms into pathological amyloid aggregates associated with the onset and development of disorders including systemic amyloidosis, as well as the neurodegenerative conditions Alzheimer’s and Parkinson’s diseases. It has become apparent that the presence of surfaces can dramatically modulate nucleation. However, the underlying physicochemical parameters governing this process have been challenging to elucidate, with interfaces in some cases having been found to accelerate aggregation, while in others they can inhibit the kinetics of this process. Here we show through kinetic analysis that for three different fibril-forming proteins, interfaces affect the aggregation reaction mainly through modulating the primary nucleation step. Moreover, we show through direct measurements of the Gibbs free energy of adsorption, combined with theory and coarse-grained computer simulations, that overall nucleation rates are suppressed at high and at low surface interaction strengths but significantly enhanced at intermediate strengths, and we verify these regimes experimentally. Taken together, these results provide a quantitative description of the fundamental process which triggers amyloid formation and shed light on the key factors that control this process. AU - Toprakcioglu, Zenon AU - Kamada, Ayaka AU - Michaels, Thomas C.T. AU - Xie, Mengqi AU - Krausser, Johannes AU - Wei, Jiapeng AU - Šarić, Anđela AU - Vendruscolo, Michele AU - Knowles, Tuomas P.J. ID - 11841 IS - 31 JF - Proceedings of the National Academy of Sciences of the United States of America SN - 0027-8424 TI - Adsorption free energy predicts amyloid protein nucleation rates VL - 119 ER - TY - JOUR AB - Selection accumulates information in the genome—it guides stochastically evolving populations toward states (genotype frequencies) that would be unlikely under neutrality. This can be quantified as the Kullback–Leibler (KL) divergence between the actual distribution of genotype frequencies and the corresponding neutral distribution. First, we show that this population-level information sets an upper bound on the information at the level of genotype and phenotype, limiting how precisely they can be specified by selection. Next, we study how the accumulation and maintenance of information is limited by the cost of selection, measured as the genetic load or the relative fitness variance, both of which we connect to the control-theoretic KL cost of control. The information accumulation rate is upper bounded by the population size times the cost of selection. This bound is very general, and applies across models (Wright–Fisher, Moran, diffusion) and to arbitrary forms of selection, mutation, and recombination. Finally, the cost of maintaining information depends on how it is encoded: Specifying a single allele out of two is expensive, but one bit encoded among many weakly specified loci (as in a polygenic trait) is cheap. AU - Hledik, Michal AU - Barton, Nicholas H AU - Tkačik, Gašper ID - 12081 IS - 36 JF - Proceedings of the National Academy of Sciences SN - 0027-8424 TI - Accumulation and maintenance of information in evolution VL - 119 ER - TY - JOUR AB - Unlike crystalline atomic and ionic solids, texture development due to crystallographically preferred growth in colloidal crystals is less studied. Here we investigate the underlying mechanisms of the texture evolution in an evaporation-induced colloidal assembly process through experiments, modeling, and theoretical analysis. In this widely used approach to obtain large-area colloidal crystals, the colloidal particles are driven to the meniscus via the evaporation of a solvent or matrix precursor solution where they close-pack to form a face-centered cubic colloidal assembly. Via two-dimensional large-area crystallographic mapping, we show that the initial crystal orientation is dominated by the interaction of particles with the meniscus, resulting in the expected coalignment of the close-packed direction with the local meniscus geometry. By combining with crystal structure analysis at a single-particle level, we further reveal that, at the later stage of self-assembly, however, the colloidal crystal undergoes a gradual rotation facilitated by geometrically necessary dislocations (GNDs) and achieves a large-area uniform crystallographic orientation with the close-packed direction perpendicular to the meniscus and parallel to the growth direction. Classical slip analysis, finite element-based mechanical simulation, computational colloidal assembly modeling, and continuum theory unequivocally show that these GNDs result from the tensile stress field along the meniscus direction due to the constrained shrinkage of the colloidal crystal during drying. The generation of GNDs with specific slip systems within individual grains leads to crystallographic rotation to accommodate the mechanical stress. The mechanistic understanding reported here can be utilized to control crystallographic features of colloidal assemblies, and may provide further insights into crystallographically preferred growth in synthetic, biological, and geological crystals. AU - Li, Ling AU - Goodrich, Carl Peter AU - Yang, Haizhao AU - Phillips, Katherine R. AU - Jia, Zian AU - Chen, Hongshun AU - Wang, Lifeng AU - Zhong, Jinjin AU - Liu, Anhua AU - Lu, Jianfeng AU - Shuai, Jianwei AU - Brenner, Michael P. AU - Spaepen, Frans AU - Aizenberg, Joanna ID - 12667 IS - 32 JF - PNAS SN - 0027-8424 TI - Microscopic origins of the crystallographically preferred growth in evaporation-induced colloidal crystals VL - 118 ER - TY - JOUR AB - The inverse problem of designing component interactions to target emergent structure is fundamental to numerous applications in biotechnology, materials science, and statistical physics. Equally important is the inverse problem of designing emergent kinetics, but this has received considerably less attention. Using recent advances in automatic differentiation, we show how kinetic pathways can be precisely designed by directly differentiating through statistical physics models, namely free energy calculations and molecular dynamics simulations. We consider two systems that are crucial to our understanding of structural self-assembly: bulk crystallization and small nanoclusters. In each case, we are able to assemble precise dynamical features. Using gradient information, we manipulate interactions among constituent particles to tune the rate at which these systems yield specific structures of interest. Moreover, we use this approach to learn nontrivial features about the high-dimensional design space, allowing us to accurately predict when multiple kinetic features can be simultaneously and independently controlled. These results provide a concrete and generalizable foundation for studying nonstructural self-assembly, including kinetic properties as well as other complex emergent properties, in a vast array of systems. AU - Goodrich, Carl Peter AU - King, Ella M. AU - Schoenholz, Samuel S. AU - Cubuk, Ekin D. AU - Brenner, Michael P. ID - 9257 IS - 10 JF - Proceedings of the National Academy of Sciences SN - 0027-8424 TI - Designing self-assembling kinetics with differentiable statistical physics models VL - 118 ER - TY - JOUR AB - In nerve cells the genes encoding for α2δ subunits of voltage-gated calcium channels have been linked to synaptic functions and neurological disease. Here we show that α2δ subunits are essential for the formation and organization of glutamatergic synapses. Using a cellular α2δ subunit triple-knockout/knockdown model, we demonstrate a failure in presynaptic differentiation evidenced by defective presynaptic calcium channel clustering and calcium influx, smaller presynaptic active zones, and a strongly reduced accumulation of presynaptic vesicle-associated proteins (synapsin and vGLUT). The presynaptic defect is associated with the downscaling of postsynaptic AMPA receptors and the postsynaptic density. The role of α2δ isoforms as synaptic organizers is highly redundant, as each individual α2δ isoform can rescue presynaptic calcium channel trafficking and expression of synaptic proteins. Moreover, α2δ-2 and α2δ-3 with mutated metal ion-dependent adhesion sites can fully rescue presynaptic synapsin expression but only partially calcium channel trafficking, suggesting that the regulatory role of α2δ subunits is independent from its role as a calcium channel subunit. Our findings influence the current view on excitatory synapse formation. First, our study suggests that postsynaptic differentiation is secondary to presynaptic differentiation. Second, the dependence of presynaptic differentiation on α2δ implicates α2δ subunits as potential nucleation points for the organization of synapses. Finally, our results suggest that α2δ subunits act as transsynaptic organizers of glutamatergic synapses, thereby aligning the synaptic active zone with the postsynaptic density. AU - Schöpf, Clemens L. AU - Ablinger, Cornelia AU - Geisler, Stefanie M. AU - Stanika, Ruslan I. AU - Campiglio, Marta AU - Kaufmann, Walter AU - Nimmervoll, Benedikt AU - Schlick, Bettina AU - Brockhaus, Johannes AU - Missler, Markus AU - Shigemoto, Ryuichi AU - Obermair, Gerald J. ID - 9330 IS - 14 JF - PNAS TI - Presynaptic α2δ subunits are key organizers of glutamatergic synapses VL - 118 ER - TY - JOUR AB - Parent-of-origin–dependent gene expression in mammals and flowering plants results from differing chromatin imprints (genomic imprinting) between maternally and paternally inherited alleles. Imprinted gene expression in the endosperm of seeds is associated with localized hypomethylation of maternally but not paternally inherited DNA, with certain small RNAs also displaying parent-of-origin–specific expression. To understand the evolution of imprinting mechanisms in Oryza sativa (rice), we analyzed imprinting divergence among four cultivars that span both japonica and indica subspecies: Nipponbare, Kitaake, 93-11, and IR64. Most imprinted genes are imprinted across cultivars and enriched for functions in chromatin and transcriptional regulation, development, and signaling. However, 4 to 11% of imprinted genes display divergent imprinting. Analyses of DNA methylation and small RNAs revealed that endosperm-specific 24-nt small RNA–producing loci show weak RNA-directed DNA methylation, frequently overlap genes, and are imprinted four times more often than genes. However, imprinting divergence most often correlated with local DNA methylation epimutations (9 of 17 assessable loci), which were largely stable within subspecies. Small insertion/deletion events and transposable element insertions accompanied 4 of the 9 locally epimutated loci and associated with imprinting divergence at another 4 of the remaining 8 loci. Correlating epigenetic and genetic variation occurred at key regulatory regions—the promoter and transcription start site of maternally biased genes, and the promoter and gene body of paternally biased genes. Our results reinforce models for the role of maternal-specific DNA hypomethylation in imprinting of both maternally and paternally biased genes, and highlight the role of transposition and epimutation in rice imprinting evolution. AU - Rodrigues, Jessica A. AU - Hsieh, Ping-Hung AU - Ruan, Deling AU - Nishimura, Toshiro AU - Sharma, Manoj K. AU - Sharma, Rita AU - Ye, XinYi AU - Nguyen, Nicholas D. AU - Nijjar, Sukhranjan AU - Ronald, Pamela C. AU - Fischer, Robert L. AU - Zilberman, Daniel ID - 9877 IS - 29 JF - Proceedings of the National Academy of Sciences SN - 0027-8424 TI - Divergence among rice cultivars reveals roles for transposition and epimutation in ongoing evolution of genomic imprinting VL - 118 ER - TY - JOUR AB - Turbulence generally arises in shear flows if velocities and hence, inertial forces are sufficiently large. In striking contrast, viscoelastic fluids can exhibit disordered motion even at vanishing inertia. Intermediate between these cases, a state of chaotic motion, “elastoinertial turbulence” (EIT), has been observed in a narrow Reynolds number interval. We here determine the origin of EIT in experiments and show that characteristic EIT structures can be detected across an unexpectedly wide range of parameters. Close to onset, a pattern of chevron-shaped streaks emerges in qualitative agreement with linear and weakly nonlinear theory. However, in experiments, the dynamics remain weakly chaotic, and the instability can be traced to far lower Reynolds numbers than permitted by theory. For increasing inertia, the flow undergoes a transformation to a wall mode composed of inclined near-wall streaks and shear layers. This mode persists to what is known as the “maximum drag reduction limit,” and overall EIT is found to dominate viscoelastic flows across more than three orders of magnitude in Reynolds number. AU - Choueiri, George H AU - Lopez Alonso, Jose M AU - Varshney, Atul AU - Sankar, Sarath AU - Hof, Björn ID - 10299 IS - 45 JF - Proceedings of the National Academy of Sciences KW - multidisciplinary KW - elastoinertial turbulence KW - viscoelastic flows KW - elastic instability KW - drag reduction SN - 0027-8424 TI - Experimental observation of the origin and structure of elastoinertial turbulence VL - 118 ER - TY - JOUR AB - Electrodepositing insulating lithium peroxide (Li2O2) is the key process during discharge of aprotic Li–O2 batteries and determines rate, capacity, and reversibility. Current understanding states that the partition between surface adsorbed and dissolved lithium superoxide governs whether Li2O2 grows as a conformal surface film or larger particles, leading to low or high capacities, respectively. However, better understanding governing factors for Li2O2 packing density and capacity requires structural sensitive in situ metrologies. Here, we establish in situ small- and wide-angle X-ray scattering (SAXS/WAXS) as a suitable method to record the Li2O2 phase evolution with atomic to submicrometer resolution during cycling a custom-built in situ Li–O2 cell. Combined with sophisticated data analysis, SAXS allows retrieving rich quantitative structural information from complex multiphase systems. Surprisingly, we find that features are absent that would point at a Li2O2 surface film formed via two consecutive electron transfers, even in poorly solvating electrolytes thought to be prototypical for surface growth. All scattering data can be modeled by stacks of thin Li2O2 platelets potentially forming large toroidal particles. Li2O2 solution growth is further justified by rotating ring-disk electrode measurements and electron microscopy. Higher discharge overpotentials lead to smaller Li2O2 particles, but there is no transition to an electronically passivating, conformal Li2O2 coating. Hence, mass transport of reactive species rather than electronic transport through a Li2O2 film limits the discharge capacity. Provided that species mobilities and carbon surface areas are high, this allows for high discharge capacities even in weakly solvating electrolytes. The currently accepted Li–O2 reaction mechanism ought to be reconsidered. AU - Prehal, Christian AU - Samojlov, Aleksej AU - Nachtnebel, Manfred AU - Lovicar, Ludek AU - Kriechbaum, Manfred AU - Amenitsch, Heinz AU - Freunberger, Stefan Alexander ID - 9301 IS - 14 JF - Proceedings of the National Academy of Sciences KW - small-angle X-ray scattering KW - oxygen reduction KW - disproportionation KW - Li-air battery SN - 0027-8424 TI - In situ small-angle X-ray scattering reveals solution phase discharge of Li–O2 batteries with weakly solvating electrolytes VL - 118 ER - TY - JOUR AB - Clathrin-mediated endocytosis is the major route of entry of cargos into cells and thus underpins many physiological processes. During endocytosis, an area of flat membrane is remodeled by proteins to create a spherical vesicle against intracellular forces. The protein machinery which mediates this membrane bending in plants is unknown. However, it is known that plant endocytosis is actin independent, thus indicating that plants utilize a unique mechanism to mediate membrane bending against high-turgor pressure compared to other model systems. Here, we investigate the TPLATE complex, a plant-specific endocytosis protein complex. It has been thought to function as a classical adaptor functioning underneath the clathrin coat. However, by using biochemical and advanced live microscopy approaches, we found that TPLATE is peripherally associated with clathrin-coated vesicles and localizes at the rim of endocytosis events. As this localization is more fitting to the protein machinery involved in membrane bending during endocytosis, we examined cells in which the TPLATE complex was disrupted and found that the clathrin structures present as flat patches. This suggests a requirement of the TPLATE complex for membrane bending during plant clathrin–mediated endocytosis. Next, we used in vitro biophysical assays to confirm that the TPLATE complex possesses protein domains with intrinsic membrane remodeling activity. These results redefine the role of the TPLATE complex and implicate it as a key component of the evolutionarily distinct plant endocytosis mechanism, which mediates endocytic membrane bending against the high-turgor pressure in plant cells. AU - Johnson, Alexander J AU - Dahhan, Dana A AU - Gnyliukh, Nataliia AU - Kaufmann, Walter AU - Zheden, Vanessa AU - Costanzo, Tommaso AU - Mahou, Pierre AU - Hrtyan, Mónika AU - Wang, Jie AU - Aguilera Servin, Juan L AU - van Damme, Daniël AU - Beaurepaire, Emmanuel AU - Loose, Martin AU - Bednarek, Sebastian Y AU - Friml, Jiří ID - 9887 IS - 51 JF - Proceedings of the National Academy of Sciences TI - The TPLATE complex mediates membrane bending during plant clathrin-mediated endocytosis VL - 118 ER - TY - JOUR AB - Biological membranes can dramatically accelerate the aggregation of normally soluble protein molecules into amyloid fibrils and alter the fibril morphologies, yet the molecular mechanisms through which this accelerated nucleation takes place are not yet understood. Here, we develop a coarse-grained model to systematically explore the effect that the structural properties of the lipid membrane and the nature of protein–membrane interactions have on the nucleation rates of amyloid fibrils. We identify two physically distinct nucleation pathways—protein-rich and lipid-rich—and quantify how the membrane fluidity and protein–membrane affinity control the relative importance of those molecular pathways. We find that the membrane’s susceptibility to reshaping and being incorporated into the fibrillar aggregates is a key determinant of its ability to promote protein aggregation. We then characterize the rates and the free-energy profile associated with this heterogeneous nucleation process, in which the surface itself participates in the aggregate structure. Finally, we compare quantitatively our data to experiments on membrane-catalyzed amyloid aggregation of α-synuclein, a protein implicated in Parkinson’s disease that predominately nucleates on membranes. More generally, our results provide a framework for understanding macromolecular aggregation on lipid membranes in a broad biological and biotechnological context. AU - Krausser, Johannes AU - Knowles, Tuomas P. J. AU - Šarić, Anđela ID - 10336 IS - 52 JF - Proceedings of the National Academy of Sciences SN - 0027-8424 TI - Physical mechanisms of amyloid nucleation on fluid membranes VL - 117 ER - TY - JOUR AB - Understanding the mechanism of action of compounds capable of inhibiting amyloid-fibril formation is critical to the development of potential therapeutics against protein-misfolding diseases. A fundamental challenge for progress is the range of possible target species and the disparate timescales involved, since the aggregating proteins are simultaneously the reactants, products, intermediates, and catalysts of the reaction. It is a complex problem, therefore, to choose the states of the aggregating proteins that should be bound by the compounds to achieve the most potent inhibition. We present here a comprehensive kinetic theory of amyloid-aggregation inhibition that reveals the fundamental thermodynamic and kinetic signatures characterizing effective inhibitors by identifying quantitative relationships between the aggregation and binding rate constants. These results provide general physical laws to guide the design and optimization of inhibitors of amyloid-fibril formation, revealing in particular the important role of on-rates in the binding of the inhibitors. AU - Michaels, Thomas C. T. AU - Šarić, Anđela AU - Meisl, Georg AU - Heller, Gabriella T. AU - Curk, Samo AU - Arosio, Paolo AU - Linse, Sara AU - Dobson, Christopher M. AU - Vendruscolo, Michele AU - Knowles, Tuomas P. J. ID - 10347 IS - 39 JF - Proceedings of the National Academy of Sciences KW - multidisciplinary SN - 0027-8424 TI - Thermodynamic and kinetic design principles for amyloid-aggregation inhibitors VL - 117 ER - TY - JOUR AB - The eukaryotic endomembrane system is controlled by small GTPases of the Rab family, which are activated at defined times and locations in a switch-like manner. While this switch is well understood for an individual protein, how regulatory networks produce intracellular activity patterns is currently not known. Here, we combine in vitro reconstitution experiments with computational modeling to study a minimal Rab5 activation network. We find that the molecular interactions in this system give rise to a positive feedback and bistable collective switching of Rab5. Furthermore, we find that switching near the critical point is intrinsically stochastic and provide evidence that controlling the inactive population of Rab5 on the membrane can shape the network response. Notably, we demonstrate that collective switching can spread on the membrane surface as a traveling wave of Rab5 activation. Together, our findings reveal how biochemical signaling networks control vesicle trafficking pathways and how their nonequilibrium properties define the spatiotemporal organization of the cell. AU - Bezeljak, Urban AU - Loya, Hrushikesh AU - Kaczmarek, Beata M AU - Saunders, Timothy E. AU - Loose, Martin ID - 7580 IS - 12 JF - Proceedings of the National Academy of Sciences SN - 0027-8424 TI - Stochastic activation and bistability in a Rab GTPase regulatory network VL - 117 ER - TY - JOUR AB - The actin cytoskeleton, a dynamic network of actin filaments and associated F-actin–binding proteins, is fundamentally important in eukaryotes. α-Actinins are major F-actin bundlers that are inhibited by Ca2+ in nonmuscle cells. Here we report the mechanism of Ca2+-mediated regulation of Entamoeba histolytica α-actinin-2 (EhActn2) with features expected for the common ancestor of Entamoeba and higher eukaryotic α-actinins. Crystal structures of Ca2+-free and Ca2+-bound EhActn2 reveal a calmodulin-like domain (CaMD) uniquely inserted within the rod domain. Integrative studies reveal an exceptionally high affinity of the EhActn2 CaMD for Ca2+, binding of which can only be regulated in the presence of physiological concentrations of Mg2+. Ca2+ binding triggers an increase in protein multidomain rigidity, reducing conformational flexibility of F-actin–binding domains via interdomain cross-talk and consequently inhibiting F-actin bundling. In vivo studies uncover that EhActn2 plays an important role in phagocytic cup formation and might constitute a new drug target for amoebic dysentery. AU - Pinotsis, Nikos AU - Zielinska, Karolina AU - Babuta, Mrigya AU - Arolas, Joan L. AU - Kostan, Julius AU - Khan, Muhammad Bashir AU - Schreiner, Claudia AU - Testa Salmazo, Anita P AU - Ciccarelli, Luciano AU - Puchinger, Martin AU - Gkougkoulia, Eirini A. AU - Ribeiro, Euripedes de Almeida AU - Marlovits, Thomas C. AU - Bhattacharya, Alok AU - Djinovic-Carugo, Kristina ID - 15061 IS - 36 JF - Proceedings of the National Academy of Sciences SN - 0027-8424 TI - Calcium modulates the domain flexibility and function of an α-actinin similar to the ancestral α-actinin VL - 117 ER - TY - JOUR AB - Wound healing in plant tissues, consisting of rigid cell wall-encapsulated cells, represents a considerable challenge and occurs through largely unknown mechanisms distinct from those in animals. Owing to their inability to migrate, plant cells rely on targeted cell division and expansion to regenerate wounds. Strict coordination of these wound-induced responses is essential to ensure efficient, spatially restricted wound healing. Single-cell tracking by live imaging allowed us to gain mechanistic insight into the wound perception and coordination of wound responses after laser-based wounding in Arabidopsis root. We revealed a crucial contribution of the collapse of damaged cells in wound perception and detected an auxin increase specific to cells immediately adjacent to the wound. This localized auxin increase balances wound-induced cell expansion and restorative division rates in a dose-dependent manner, leading to tumorous overproliferation when the canonical TIR1 auxin signaling is disrupted. Auxin and wound-induced turgor pressure changes together also spatially define the activation of key components of regeneration, such as the transcription regulator ERF115. Our observations suggest that the wound signaling involves the sensing of collapse of damaged cells and a local auxin signaling activation to coordinate the downstream transcriptional responses in the immediate wound vicinity. AU - Hörmayer, Lukas AU - Montesinos López, Juan C AU - Marhavá, Petra AU - Benková, Eva AU - Yoshida, Saiko AU - Friml, Jiří ID - 8002 IS - 26 JF - Proceedings of the National Academy of Sciences SN - 0027-8424 TI - Wounding-induced changes in cellular pressure and localized auxin signalling spatially coordinate restorative divisions in roots VL - 117 ER - TY - JOUR AB - Epigenetic reprogramming is required for proper regulation of gene expression in eukaryotic organisms. In Arabidopsis, active DNA demethylation is crucial for seed viability, pollen function, and successful reproduction. The DEMETER (DME) DNA glycosylase initiates localized DNA demethylation in vegetative and central cells, so-called companion cells that are adjacent to sperm and egg gametes, respectively. In rice, the central cell genome displays local DNA hypomethylation, suggesting that active DNA demethylation also occurs in rice; however, the enzyme responsible for this process is unknown. One candidate is the rice REPRESSOR OF SILENCING 1a (ROS1a) gene, which is related to DME and is essential for rice seed viability and pollen function. Here, we report genome-wide analyses of DNA methylation in wild-type and ros1a mutant sperm and vegetative cells. We find that the rice vegetative cell genome is locally hypomethylated compared with sperm by a process that requires ROS1a activity. We show that many ROS1a target sequences in the vegetative cell are hypomethylated in the rice central cell, suggesting that ROS1a also demethylates the central cell genome. Similar to Arabidopsis, we show that sperm non-CG methylation is indirectly promoted by DNA demethylation in the vegetative cell. These results reveal that DNA glycosylase-mediated DNA demethylation processes are conserved in Arabidopsis and rice, plant species that diverged 150 million years ago. Finally, although global non-CG methylation levels of sperm and egg differ, the maternal and paternal embryo genomes show similar non-CG methylation levels, suggesting that rice gamete genomes undergo dynamic DNA methylation reprogramming after cell fusion. AU - Kim, M. Yvonne AU - Ono, Akemi AU - Scholten, Stefan AU - Kinoshita, Tetsu AU - Zilberman, Daniel AU - Okamoto, Takashi AU - Fischer, Robert L. ID - 9460 IS - 19 JF - Proceedings of the National Academy of Sciences KW - Multidisciplinary SN - 0027-8424 TI - DNA demethylation by ROS1a in rice vegetative cells promotes methylation in sperm VL - 116 ER - TY - JOUR AB - A central goal of computational physics and chemistry is to predict material properties by using first-principles methods based on the fundamental laws of quantum mechanics. However, the high computational costs of these methods typically prevent rigorous predictions of macroscopic quantities at finite temperatures, such as heat capacity, density, and chemical potential. Here, we enable such predictions by marrying advanced free-energy methods with data-driven machine-learning interatomic potentials. We show that, for the ubiquitous and technologically essential system of water, a first-principles thermodynamic description not only leads to excellent agreement with experiments, but also reveals the crucial role of nuclear quantum fluctuations in modulating the thermodynamic stabilities of different phases of water. AU - Cheng, Bingqing AU - Engel, Edgar A. AU - Behler, Jörg AU - Dellago, Christoph AU - Ceriotti, Michele ID - 9689 IS - 4 JF - Proceedings of the National Academy of Sciences SN - 0027-8424 TI - Ab initio thermodynamics of liquid and solid water VL - 116 ER - TY - JOUR AB - Chiral molecules interact and react differently with other chiral objects, depending on their handedness. Therefore, it is essential to understand and ultimately control the evolution of molecular chirality during chemical reactions. Although highly sophisticated techniques for the controlled synthesis of chiral molecules have been developed, the observation of chirality on the natural femtosecond time scale of a chemical reaction has so far remained out of reach in the gas phase. Here, we demonstrate a general experimental technique, based on high-harmonic generation in tailored laser fields, and apply it to probe the time evolution of molecular chirality during the photodissociation of 2-iodobutane. These measurements show a change in sign and a pronounced increase in the magnitude of the chiral response over the first 100 fs, followed by its decay within less than 500 fs, revealing the photodissociation to achiral products. The observed time evolution is explained in terms of the variation of the electric and magnetic transition-dipole moments between the lowest electronic states of the cation as a function of the reaction coordinate. These results open the path to investigations of the chirality of molecular-reaction pathways, light-induced chirality in chemical processes, and the control of molecular chirality through tailored laser pulses. AU - Baykusheva, Denitsa Rangelova AU - Zindel, Daniel AU - Svoboda, Vít AU - Bommeli, Elias AU - Ochsner, Manuel AU - Tehlar, Andres AU - Wörner, Hans Jakob ID - 14001 IS - 48 JF - Proceedings of the National Academy of Sciences KW - Multidisciplinary SN - 0027-8424 TI - Real-time probing of chirality during a chemical reaction VL - 116 ER - TY - JOUR AB - The abelian sandpile serves as a model to study self-organized criticality, a phenomenon occurring in biological, physical and social processes. The identity of the abelian group is a fractal composed of self-similar patches, and its limit is subject of extensive collaborative research. Here, we analyze the evolution of the sandpile identity under harmonic fields of different orders. We show that this evolution corresponds to periodic cycles through the abelian group characterized by the smooth transformation and apparent conservation of the patches constituting the identity. The dynamics induced by second and third order harmonics resemble smooth stretchings, respectively translations, of the identity, while the ones induced by fourth order harmonics resemble magnifications and rotations. Starting with order three, the dynamics pass through extended regions of seemingly random configurations which spontaneously reassemble into accentuated patterns. We show that the space of harmonic functions projects to the extended analogue of the sandpile group, thus providing a set of universal coordinates identifying configurations between different domains. Since the original sandpile group is a subgroup of the extended one, this directly implies that it admits a natural renormalization. Furthermore, we show that the harmonic fields can be induced by simple Markov processes, and that the corresponding stochastic dynamics show remarkable robustness over hundreds of periods. Finally, we encode information into seemingly random configurations, and decode this information with an algorithm requiring minimal prior knowledge. Our results suggest that harmonic fields might split the sandpile group into sub-sets showing different critical coefficients, and that it might be possible to extend the fractal structure of the identity beyond the boundaries of its domain. AU - Lang, Moritz AU - Shkolnikov, Mikhail ID - 196 IS - 8 JF - Proceedings of the National Academy of Sciences TI - Harmonic dynamics of the Abelian sandpile VL - 116 ER - TY - JOUR AB - Plasmodesmata (PD) are plant-specific membrane-lined channels that create cytoplasmic and membrane continuities between adjacent cells, thereby facilitating cell–cell communication and virus movement. Plant cells have evolved diverse mechanisms to regulate PD plasticity in response to numerous environmental stimuli. In particular, during defense against plant pathogens, the defense hormone, salicylic acid (SA), plays a crucial role in the regulation of PD permeability in a callose-dependent manner. Here, we uncover a mechanism by which plants restrict the spreading of virus and PD cargoes using SA signaling by increasing lipid order and closure of PD. We showed that exogenous SA application triggered the compartmentalization of lipid raft nanodomains through a modulation of the lipid raft-regulatory protein, Remorin (REM). Genetic studies, superresolution imaging, and transmission electron microscopy observation together demonstrated that Arabidopsis REM1.2 and REM1.3 are crucial for plasma membrane nanodomain assembly to control PD aperture and functionality. In addition, we also found that a 14-3-3 epsilon protein modulates REM clustering and membrane nanodomain compartmentalization through its direct interaction with REM proteins. This study unveils a molecular mechanism by which the key plant defense hormone, SA, triggers membrane lipid nanodomain reorganization, thereby regulating PD closure to impede virus spreading. AU - Huang, D AU - Sun, Y AU - Ma, Z AU - Ke, M AU - Cui, Y AU - Chen, Z AU - Chen, C AU - Ji, C AU - Tran, TM AU - Yang, L AU - Lam, SM AU - Han, Y AU - Shu, G AU - Friml, Jiří AU - Miao, Y AU - Jiang, L AU - Chen, X ID - 6999 IS - 42 JF - Proceedings of the National Academy of Sciences of the United States of America SN - 0027-8424 TI - Salicylic acid-mediated plasmodesmal closure via Remorin-dependent lipid organization VL - 116 ER - TY - JOUR AB - The DEMETER (DME) DNA glycosylase catalyzes genome-wide DNA demethylation and is required for endosperm genomic imprinting and embryo viability. Targets of DME-mediated DNA demethylation reside in small, euchromatic, AT-rich transposons and at the boundaries of large transposons, but how DME interacts with these diverse chromatin states is unknown. The STRUCTURE SPECIFIC RECOGNITION PROTEIN 1 (SSRP1) subunit of the chromatin remodeler FACT (facilitates chromatin transactions), was previously shown to be involved in the DME-dependent regulation of genomic imprinting in Arabidopsis endosperm. Therefore, to investigate the interaction between DME and chromatin, we focused on the activity of the two FACT subunits, SSRP1 and SUPPRESSOR of TY16 (SPT16), during reproduction in Arabidopsis. We found that FACT colocalizes with nuclear DME in vivo, and that DME has two classes of target sites, the first being euchromatic and accessible to DME, but the second, representing over half of DME targets, requiring the action of FACT for DME-mediated DNA demethylation genome-wide. Our results show that the FACT-dependent DME targets are GC-rich heterochromatin domains with high nucleosome occupancy enriched with H3K9me2 and H3K27me1. Further, we demonstrate that heterochromatin-associated linker histone H1 specifically mediates the requirement for FACT at a subset of DME-target loci. Overall, our results demonstrate that FACT is required for DME targeting by facilitating its access to heterochromatin. AU - Frost, Jennifer M. AU - Kim, M. Yvonne AU - Park, Guen Tae AU - Hsieh, Ping-Hung AU - Nakamura, Miyuki AU - Lin, Samuel J. H. AU - Yoo, Hyunjin AU - Choi, Jaemyung AU - Ikeda, Yoko AU - Kinoshita, Tetsu AU - Choi, Yeonhee AU - Zilberman, Daniel AU - Fischer, Robert L. ID - 9471 IS - 20 JF - Proceedings of the National Academy of Sciences KW - Multidisciplinary SN - 0027-8424 TI - FACT complex is required for DNA demethylation at heterochromatin during reproduction in Arabidopsis VL - 115 ER - TY - JOUR AB - Supraglacial ice cliffs exist on debris-covered glaciers worldwide, but despite their importance as melt hot spots, their life cycle is little understood. Early field observations had advanced a hypothesis of survival of north-facing and disappearance of south-facing cliffs, which is central for predicting the contribution of cliffs to total glacier mass losses. Their role as windows of energy transfer suggests they may explain the anomalously high mass losses of debris-covered glaciers in High Mountain Asia (HMA) despite the insulating debris, currently at the center of a debated controversy. We use a 3D model of cliff evolution coupled to very high-resolution topographic data to demonstrate that ice cliffs facing south (in the Northern Hemisphere) disappear within a few months due to enhanced solar radiation receipts and that aspect is the key control on cliffs evolution. We reproduce continuous flattening of south-facing cliffs, a result of their vertical gradient of incoming solar radiation and sky view factor. Our results establish that only north-facing cliffs are recurrent features and thus stable contributors to the melting of debris-covered glaciers. Satellite observations and mass balance modeling confirms that few south-facing cliffs of small size exist on the glaciers of Langtang, and their contribution to the glacier volume losses is very small (∼1%). This has major implications for the mass balance of HMA debris-covered glaciers as it provides the basis for new parameterizations of cliff evolution and distribution to constrain volume losses in a region where glaciers are highly relevant as water sources for millions of people. AU - Buri, Pascal AU - Pellicciotti, Francesca ID - 12607 IS - 17 JF - PNAS SN - 0027-8424 TI - Aspect controls the survival of ice cliffs on debris-covered glaciers VL - 115 ER - TY - JOUR AB - Efficient molecular switching in confined spaces is critical for the successful development of artificial molecular machines. However, molecular switching events often entail large structural changes and therefore require conformational freedom, which is typically limited under confinement conditions. Here, we investigated the behavior of azobenzene—the key building block of light-controlled molecular machines—in a confined environment that is flexible and can adapt its shape to that of the bound guest. To this end, we encapsulated several structurally diverse azobenzenes within the cavity of a flexible, water-soluble coordination cage, and investigated their light-responsive behavior. Using UV/Vis absorption spectroscopy and a combination of NMR methods, we showed that each of the encapsulated azobenzenes exhibited distinct switching properties. An azobenzene forming a 1:1 host–guest inclusion complex could be efficiently photoisomerized in a reversible fashion. In contrast, successful switching in inclusion complexes incorporating two azobenzene guests was dependent on the availability of free cages in the system, and it involved reversible trafficking of azobenzene between the cages. In the absence of extra cages, photoswitching was either suppressed or it involved expulsion of azobenzene from the cage and consequently its precipitation from the solution. This finding was utilized to develop an information storage medium in which messages could be written and erased in a reversible fashion using light. AU - Samanta, Dipak AU - Gemen, Julius AU - Chu, Zonglin AU - Diskin-Posner, Yael AU - Shimon, Linda J. W. AU - Klajn, Rafal ID - 13376 IS - 38 JF - Proceedings of the National Academy of Sciences KW - Multidisciplinary SN - 0027-8424 TI - Reversible photoswitching of encapsulated azobenzenes in water VL - 115 ER - TY - JOUR AB - The optic tectum (TeO), or superior colliculus, is a multisensory midbrain center that organizes spatially orienting responses to relevant stimuli. To define the stimulus with the highest priority at each moment, a network of reciprocal connections between the TeO and the isthmi promotes competition between concurrent tectal inputs. In the avian midbrain, the neurons mediating enhancement and suppression of tectal inputs are located in separate isthmic nuclei, facilitating the analysis of the neural processes that mediate competition. A specific subset of radial neurons in the intermediate tectal layers relay retinal inputs to the isthmi, but at present it is unclear whether separate neurons innervate individual nuclei or a single neural type sends a common input to several of them. In this study, we used in vitro neural tracing and cell-filling experiments in chickens to show that single neurons innervate, via axon collaterals, the three nuclei that comprise the isthmotectal network. This demonstrates that the input signals representing the strength of the incoming stimuli are simultaneously relayed to the mechanisms promoting both enhancement and suppression of the input signals. By performing in vivo recordings in anesthetized chicks, we also show that this common input generates synchrony between both antagonistic mechanisms, demonstrating that activity enhancement and suppression are closely coordinated. From a computational point of view, these results suggest that these tectal neurons constitute integrative nodes that combine inputs from different sources to drive in parallel several concurrent neural processes, each performing complementary functions within the network through different firing patterns and connectivity. AU - Garrido-Charad, Florencia AU - Vega Zuniga, Tomas A AU - Gutiérrez-Ibáñez, Cristián AU - Fernandez, Pedro AU - López-Jury, Luciana AU - González-Cabrera, Cristian AU - Karten, Harvey J. AU - Luksch, Harald AU - Marín, Gonzalo J. ID - 6010 IS - 32 JF - Proceedings of the National Academy of Sciences SN - 0027-8424 TI - “Shepherd’s crook” neurons drive and synchronize the enhancing and suppressive mechanisms of the midbrain stimulus selection network VL - 115 ER - TY - JOUR AB - Electric charges are conserved. The same would be expected to hold for magnetic charges, yet magnetic monopoles have never been observed. It is therefore surprising that the laws of nonequilibrium thermodynamics, combined with Maxwell’s equations, suggest that colloidal particles heated or cooled in certain polar or paramagnetic solvents may behave as if they carry an electric/magnetic charge. Here, we present numerical simulations that show that the field distribution around a pair of such heated/cooled colloidal particles agrees quantitatively with the theoretical predictions for a pair of oppositely charged electric or magnetic monopoles. However, in other respects, the nonequilibrium colloidal particles do not behave as monopoles: They cannot be moved by a homogeneous applied field. The numerical evidence for the monopole-like fields around heated/cooled colloidal particles is crucial because the experimental and numerical determination of forces between such colloidal particles would be complicated by the presence of other effects, such as thermophoresis. AU - Wirnsberger, Peter AU - Fijan, Domagoj AU - Lightwood, Roger A. AU - Šarić, Anđela AU - Dellago, Christoph AU - Frenkel, Daan ID - 10373 IS - 19 JF - Proceedings of the National Academy of Sciences KW - multidisciplinary SN - 0027-8424 TI - Numerical evidence for thermally induced monopoles VL - 114 ER - TY - JOUR AB - Nervous systems use excitatory cell assemblies to encode and represent sensory percepts. Similarly, synaptically connected cell assemblies or "engrams" are thought to represent memories of past experience. Multiple lines of recent evidence indicate that brain systems create and use inhibitory replicas of excitatory representations for important cognitive functions. Such matched "inhibitory engrams" can form through homeostatic potentiation of inhibition onto postsynaptic cells that show increased levels of excitation. Inhibitory engrams can reduce behavioral responses to familiar stimuli, thereby resulting in behavioral habituation. In addition, by preventing inappropriate activation of excitatory memory engrams, inhibitory engrams can make memories quiescent, stored in a latent form that is available for context-relevant activation. In neural networks with balanced excitatory and inhibitory engrams, the release of innate responses and recall of associative memories can occur through focused disinhibition. Understanding mechanisms that regulate the formation and expression of inhibitory engrams in vivo may help not only to explain key features of cognition but also to provide insight into transdiagnostic traits associated with psychiatric conditions such as autism, schizophrenia, and posttraumatic stress disorder. AU - Barron, Helen C. AU - Vogels, Tim P AU - Behrens, Timothy E. AU - Ramaswami, Mani ID - 8018 IS - 26 JF - Proceedings of the National Academy of Sciences SN - 0027-8424 TI - Inhibitory engrams in perception and memory VL - 114 ER - TY - JOUR AB - The basic helix–loop–helix PAS domain (bHLH-PAS) transcription factor CLOCK:BMAL1 (brain and muscle Arnt-like protein 1) sits at the core of the mammalian circadian transcription/translation feedback loop. Precise control of CLOCK:BMAL1 activity by coactivators and repressors establishes the ∼24-h periodicity of gene expression. Formation of a repressive complex, defined by the core clock proteins cryptochrome 1 (CRY1):CLOCK:BMAL1, plays an important role controlling the switch from repression to activation each day. Here we show that CRY1 binds directly to the PAS domain core of CLOCK:BMAL1, driven primarily by interaction with the CLOCK PAS-B domain. Integrative modeling and solution X-ray scattering studies unambiguously position a key loop of the CLOCK PAS-B domain in the secondary pocket of CRY1, analogous to the antenna chromophore-binding pocket of photolyase. CRY1 docks onto the transcription factor alongside the PAS domains, extending above the DNA-binding bHLH domain. Single point mutations at the interface on either CRY1 or CLOCK disrupt formation of the ternary complex, highlighting the importance of this interface for direct regulation of CLOCK:BMAL1 activity by CRY1. AU - Michael, Alicia Kathleen AU - Fribourgh, Jennifer L. AU - Chelliah, Yogarany AU - Sandate, Colby R. AU - Hura, Greg L. AU - Schneidman-Duhovny, Dina AU - Tripathi, Sarvind M. AU - Takahashi, Joseph S. AU - Partch, Carrie L. ID - 15157 IS - 7 JF - Proceedings of the National Academy of Sciences KW - Multidisciplinary SN - 0027-8424 TI - Formation of a repressive complex in the mammalian circadian clock is mediated by the secondary pocket of CRY1 VL - 114 ER - TY - JOUR AB - Mountain ranges are the world’s natural water towers and provide water resources for millions of people. However, their hydrological balance and possible future changes in river flow remain poorly understood because of high meteorological variability, physical inaccessibility, and the complex interplay between climate, cryosphere, and hydrological processes. Here, we use a state-of-the art glacio-hydrological model informed by data from high-altitude observations and the latest climate change scenarios to quantify the climate change impact on water resources of two contrasting catchments vulnerable to changes in the cryosphere. The two study catchments are located in the Central Andes of Chile and in the Nepalese Himalaya in close vicinity of densely populated areas. Although both sites reveal a strong decrease in glacier area, they show a remarkably different hydrological response to projected climate change. In the Juncal catchment in Chile, runoff is likely to sharply decrease in the future and the runoff seasonality is sensitive to projected climatic changes. In the Langtang catchment in Nepal, future water availability is on the rise for decades to come with limited shifts between seasons. Owing to the high spatiotemporal resolution of the simulations and process complexity included in the modeling, the response times and the mechanisms underlying the variations in glacier area and river flow can be well constrained. The projections indicate that climate change adaptation in Central Chile should focus on dealing with a reduction in water availability, whereas in Nepal preparedness for flood extremes should be the policy priority. AU - Ragettli, Silvan AU - Immerzeel, Walter W. AU - Pellicciotti, Francesca ID - 12618 IS - 33 JF - PNAS KW - Multidisciplinary SN - 0027-8424 TI - Contrasting climate change impact on river flows from high-altitude catchments in the Himalayan and Andes Mountains VL - 113 ER - TY - JOUR AB - Cytosine methylation is a DNA modification with important regulatory functions in eukaryotes. In flowering plants, sexual reproduction is accompanied by extensive DNA demethylation, which is required for proper gene expression in the endosperm, a nutritive extraembryonic seed tissue. Endosperm arises from a fusion of a sperm cell carried in the pollen and a female central cell. Endosperm DNA demethylation is observed specifically on the chromosomes inherited from the central cell in Arabidopsis thaliana, rice, and maize, and requires the DEMETER DNA demethylase in Arabidopsis. DEMETER is expressed in the central cell before fertilization, suggesting that endosperm demethylation patterns are inherited from the central cell. Down-regulation of the MET1 DNA methyltransferase has also been proposed to contribute to central cell demethylation. However, with the exception of three maize genes, central cell DNA methylation has not been directly measured, leaving the origin and mechanism of endosperm demethylation uncertain. Here, we report genome-wide analysis of DNA methylation in the central cells of Arabidopsis and rice—species that diverged 150 million years ago—as well as in rice egg cells. We find that DNA demethylation in both species is initiated in central cells, which requires DEMETER in Arabidopsis. However, we do not observe a global reduction of CG methylation that would be indicative of lowered MET1 activity; on the contrary, CG methylation efficiency is elevated in female gametes compared with nonsexual tissues. Our results demonstrate that locus-specific, active DNA demethylation in the central cell is the origin of maternal chromosome hypomethylation in the endosperm. AU - Park, Kyunghyuk AU - Kim, M. Yvonne AU - Vickers, Martin AU - Park, Jin-Sup AU - Hyun, Youbong AU - Okamoto, Takashi AU - Zilberman, Daniel AU - Fischer, Robert L. AU - Feng, Xiaoqi AU - Choi, Yeonhee AU - Scholten, Stefan ID - 9477 IS - 52 JF - Proceedings of the National Academy of Sciences KW - Multidisciplinary SN - 0027-8424 TI - DNA demethylation is initiated in the central cells of Arabidopsis and rice VL - 113 ER - TY - JOUR AB - Cytosine DNA methylation regulates the expression of eukaryotic genes and transposons. Methylation is copied by methyltransferases after DNA replication, which results in faithful transmission of methylation patterns during cell division and, at least in flowering plants, across generations. Transgenerational inheritance is mediated by a small group of cells that includes gametes and their progenitors. However, methylation is usually analyzed in somatic tissues that do not contribute to the next generation, and the mechanisms of transgenerational inheritance are inferred from such studies. To gain a better understanding of how DNA methylation is inherited, we analyzed purified Arabidopsis thaliana sperm and vegetative cells-the cell types that comprise pollen-with mutations in the DRM, CMT2, and CMT3 methyltransferases. We find that DNA methylation dependency on these enzymes is similar in sperm, vegetative cells, and somatic tissues, although DRM activity extends into heterochromatin in vegetative cells, likely reflecting transcription of heterochromatic transposons in this cell type. We also show that lack of histone H1, which elevates heterochromatic DNA methylation in somatic tissues, does not have this effect in pollen. Instead, levels of CG methylation in wild-type sperm and vegetative cells, as well as in wild-type microspores from which both pollen cell types originate, are substantially higher than in wild-type somatic tissues and similar to those of H1-depleted roots. Our results demonstrate that the mechanisms of methylation maintenance are similar between pollen and somatic cells, but the efficiency of CG methylation is higher in pollen, allowing methylation patterns to be accurately inherited across generations. AU - Hsieh, Ping-Hung AU - He, Shengbo AU - Buttress, Toby AU - Gao, Hongbo AU - Couchman, Matthew AU - Fischer, Robert L. AU - Zilberman, Daniel AU - Feng, Xiaoqi ID - 9473 IS - 52 JF - Proceedings of the National Academy of Sciences SN - 0027-8424 TI - Arabidopsis male sexual lineage exhibits more robust maintenance of CG methylation than somatic tissues VL - 113 ER - TY - JOUR AB - Despite the recent rapid progress in cryo-electron microscopy (cryo-EM), there still exist ample opportunities for improvement in sample preparation. Macromolecular complexes may disassociate or adopt nonrandom orientations against the extended air–water interface that exists for a short time before the sample is frozen. We designed a hollow support structure using 3D DNA origami to protect complexes from the detrimental effects of cryo-EM sample preparation. For a first proof-of-principle, we concentrated on the transcription factor p53, which binds to specific DNA sequences on double-stranded DNA. The support structures spontaneously form monolayers of preoriented particles in a thin film of water, and offer advantages in particle picking and sorting. By controlling the position of the binding sequence on a single helix that spans the hollow support structure, we also sought to control the orientation of individual p53 complexes. Although the latter did not yet yield the desired results, the support structures did provide partial information about the relative orientations of individual p53 complexes. We used this information to calculate a tomographic 3D reconstruction, and refined this structure to a final resolution of ∼15 Å. This structure settles an ongoing debate about the symmetry of the p53 tetramer bound to DNA. AU - Martin, Thomas G. AU - Bharat, Tanmay A. M. AU - Joerger, Andreas C. AU - Bai, Xiao-chen AU - Praetorius, Florian M AU - Fersht, Alan R. AU - Dietz, Hendrik AU - Scheres, Sjors H. W. ID - 14304 IS - 47 JF - PNAS SN - 0027-8424 TI - Design of a molecular support for cryo-EM structure determination VL - 113 ER - TY - JOUR AB - Protein oligomers have been implicated as toxic agents in a wide range of amyloid-related diseases. However, it has remained unsolved whether the oligomers are a necessary step in the formation of amyloid fibrils or just a dangerous byproduct. Analogously, it has not been resolved if the amyloid nucleation process is a classical one-step nucleation process or a two-step process involving prenucleation clusters. We use coarse-grained computer simulations to study the effect of nonspecific attractions between peptides on the primary nucleation process underlying amyloid fibrillization. We find that, for peptides that do not attract, the classical one-step nucleation mechanism is possible but only at nonphysiologically high peptide concentrations. At low peptide concentrations, which mimic the physiologically relevant regime, attractive interpeptide interactions are essential for fibril formation. Nucleation then inevitably takes place through a two-step mechanism involving prefibrillar oligomers. We show that oligomers not only help peptides meet each other but also, create an environment that facilitates the conversion of monomers into the β-sheet–rich form characteristic of fibrils. Nucleation typically does not proceed through the most prevalent oligomers but through an oligomer size that is only observed in rare fluctuations, which is why such aggregates might be hard to capture experimentally. Finally, we find that the nucleation of amyloid fibrils cannot be described by classical nucleation theory: in the two-step mechanism, the critical nucleus size increases with increases in both concentration and interpeptide interactions, which is in direct contrast with predictions from classical nucleation theory. AU - Šarić, Anđela AU - Chebaro, Yassmine C. AU - Knowles, Tuomas P. J. AU - Frenkel, Daan ID - 10382 IS - 50 JF - Proceedings of the National Academy of Sciences KW - multidisciplinary SN - 0027-8424 TI - Crucial role of nonspecific interactions in amyloid nucleation VL - 111 ER - TY - JOUR AB - Most excitatory inputs in the mammalian brain are made on dendritic spines, rather than on dendritic shafts. Spines compartmentalize calcium, and this biochemical isolation can underlie input-specific synaptic plasticity, providing a raison d'etre for spines. However, recent results indicate that the spine can experience a membrane potential different from that in the parent dendrite, as though the spine neck electrically isolated the spine. Here we use two-photon calcium imaging of mouse neocortical pyramidal neurons to analyze the correlation between the morphologies of spines activated under minimal synaptic stimulation and the excitatory postsynaptic potentials they generate. We find that excitatory postsynaptic potential amplitudes are inversely correlated with spine neck lengths. Furthermore, a spike timing-dependent plasticity protocol, in which two-photon glutamate uncaging over a spine is paired with postsynaptic spikes, produces rapid shrinkage of the spine neck and concomitant increases in the amplitude of the evoked spine potentials. Using numerical simulations, we explore the parameter regimes for the spine neck resistance and synaptic conductance changes necessary to explain our observations. Our data, directly correlating synaptic and morphological plasticity, imply that long-necked spines have small or negligible somatic voltage contributions, but that, upon synaptic stimulation paired with postsynaptic activity, they can shorten their necks and increase synaptic efficacy, thus changing the input/output gain of pyramidal neurons. AU - Araya, R. AU - Vogels, Tim P AU - Yuste, R. ID - 8021 IS - 28 JF - Proceedings of the National Academy of Sciences SN - 0027-8424 TI - Activity-dependent dendritic spine neck changes are correlated with synaptic strength VL - 111 ER - TY - JOUR AB - Centromeres mediate chromosome segregation and are defined by the centromere-specific histone H3 variant (CenH3)/centromere protein A (CENP-A). Removal of CenH3 from centromeres is a general property of terminally differentiated cells, and the persistence of CenH3 increases the risk of diseases such as cancer. However, active mechanisms of centromere disassembly are unknown. Nondividing Arabidopsis pollen vegetative cells, which transport engulfed sperm by extended tip growth, undergo loss of CenH3; centromeric heterochromatin decondensation; and bulk activation of silent rRNA genes, accompanied by their translocation into the nucleolus. Here, we show that these processes are blocked by mutations in the evolutionarily conserved AAA-ATPase molecular chaperone, CDC48A, homologous to yeast Cdc48 and human p97 proteins, both of which are implicated in ubiquitin/small ubiquitin-like modifier (SUMO)-targeted protein degradation. We demonstrate that CDC48A physically associates with its heterodimeric cofactor UFD1-NPL4, known to bind ubiquitin and SUMO, as well as with SUMO1-modified CenH3 and mutations in NPL4 phenocopy cdc48a mutations. In WT vegetative cell nuclei, genetically unlinked ribosomal DNA (rDNA) loci are uniquely clustered together within the nucleolus and all major rRNA gene variants, including those rDNA variants silenced in leaves, are transcribed. In cdc48a mutant vegetative cell nuclei, however, these rDNA loci frequently colocalized with condensed centromeric heterochromatin at the external periphery of the nucleolus. Our results indicate that the CDC48ANPL4 complex actively removes sumoylated CenH3 from centromeres and disrupts centromeric heterochromatin to release bulk rRNA genes into the nucleolus for ribosome production, which fuels single nucleus-driven pollen tube growth and is essential for plant reproduction. AU - Mérai, Zsuzsanna AU - Chumak, Nina AU - García-Aguilar, Marcelina AU - Hsieh, Tzung-Fu AU - Nishimura, Toshiro AU - Schoft, Vera K. AU - Bindics, János AU - Ślusarz, Lucyna AU - Arnoux, Stéphanie AU - Opravil, Susanne AU - Mechtler, Karl AU - Zilberman, Daniel AU - Fischer, Robert L. AU - Tamaru, Hisashi ID - 9479 IS - 45 JF - Proceedings of the National Academy of Sciences SN - 0027-8424 TI - The AAA-ATPase molecular chaperone Cdc48/p97 disassembles sumoylated centromeres, decondenses heterochromatin, and activates ribosomal RNA genes VL - 111 ER - TY - JOUR AB - Arabidopsis thaliana endosperm, a transient tissue that nourishes the embryo, exhibits extensive localized DNA demethylation on maternally inherited chromosomes. Demethylation mediates parent-of-origin–specific (imprinted) gene expression but is apparently unnecessary for the extensive accumulation of maternally biased small RNA (sRNA) molecules detected in seeds. Endosperm DNA in the distantly related monocots rice and maize is likewise locally hypomethylated, but whether this hypomethylation is generally parent-of-origin specific is unknown. Imprinted expression of sRNA also remains uninvestigated in monocot seeds. Here, we report high-coverage sequencing of the Kitaake rice cultivar that enabled us to show that localized hypomethylation in rice endosperm occurs solely on the maternal genome, preferring regions of high DNA accessibility. Maternally expressed imprinted genes are enriched for hypomethylation at putative promoter regions and transcriptional termini and paternally expressed genes at promoters and gene bodies, mirroring our recent results in A. thaliana. However, unlike in A. thaliana, rice endosperm sRNA populations are dominated by specific strong sRNA-producing loci, and imprinted 24-nt sRNAs are expressed from both parental genomes and correlate with hypomethylation. Overlaps between imprinted sRNA loci and imprinted genes expressed from opposite alleles suggest that sRNAs may regulate genomic imprinting. Whereas sRNAs in seedling tissues primarily originate from small class II (cut-and-paste) transposable elements, those in endosperm are more uniformly derived, including sequences from other transposon classes, as well as genic and intergenic regions. Our data indicate that the endosperm exhibits a unique pattern of sRNA expression and suggest that localized hypomethylation of maternal endosperm DNA is conserved in flowering plants. AU - Rodrigues, Jessica A. AU - Ruan, Randy AU - Nishimura, Toshiro AU - Sharma, Manoj K. AU - Sharma, Rita AU - Ronald, Pamela C AU - Fischer, Robert L. AU - Zilberman, Daniel ID - 9481 IS - 19 JF - Proceedings of the National Academy of Sciences KW - Multidisciplinary SN - 0027-8424 TI - Imprinted expression of genes and small RNA is associated with localized hypomethylation of the maternal genome in rice endosperm VL - 110 ER - TY - JOUR AB - Imprinted genes are expressed primarily or exclusively from either the maternal or paternal allele, a phenomenon that occurs in flowering plants and mammals. Flowering plant imprinted gene expression has been described primarily in endosperm, a terminal nutritive tissue consumed by the embryo during seed development or after germination. Imprinted expression in Arabidopsis thaliana endosperm is orchestrated by differences in cytosine DNA methylation between the paternal and maternal genomes as well as by Polycomb group proteins. Currently, only 11 imprinted A. thaliana genes are known. Here, we use extensive sequencing of cDNA libraries to identify 9 paternally expressed and 34 maternally expressed imprinted genes in A. thaliana endosperm that are regulated by the DNA-demethylating glycosylase DEMETER, the DNA methyltransferase MET1, and/or the core Polycomb group protein FIE. These genes encode transcription factors, proteins involved in hormone signaling, components of the ubiquitin protein degradation pathway, regulators of histone and DNA methylation, and small RNA pathway proteins. We also identify maternally expressed genes that may be regulated by unknown mechanisms or deposited from maternal tissues. We did not detect any imprinted genes in the embryo. Our results show that imprinted gene expression is an extensive mechanistically complex phenomenon that likely affects multiple aspects of seed development. AU - Hsieh, Tzung-Fu AU - Shin, Juhyun AU - Uzawa, Rie AU - Silva, Pedro AU - Cohen, Stephanie AU - Bauer, Matthew J. AU - Hashimoto, Meryl AU - Kirkbride, Ryan C. AU - Harada, John J. AU - Zilberman, Daniel AU - Fischer, Robert L. ID - 9483 IS - 5 JF - Proceedings of the National Academy of Sciences SN - 0027-8424 TI - Regulation of imprinted gene expression in Arabidopsis endosperm VL - 108 ER - TY - JOUR AB - Understanding the mechanism of protein folding requires a detailed knowledge of the structural properties of the barriers separating unfolded from native conformations. The S-peptide from ribonuclease S forms its α-helical structure only upon binding to the folded S-protein. We characterized the transition state for this binding-induced folding reaction at high resolution by determining the effect of site-specific backbone thioxylation and side-chain modifications on the kinetics and thermodynamics of the reaction, which allows us to monitor formation of backbone hydrogen bonds and side-chain interactions in the transition state. The experiments reveal that α-helical structure in the S-peptide is absent in the transition state of binding. Recognition between the unfolded S-peptide and the S-protein is mediated by loosely packed hydrophobic side-chain interactions in two well defined regions on the S-peptide. Close packing and helix formation occurs rapidly after binding. Introducing hydrophobic residues at positions outside the recognition region can drastically slow down association. AU - Bachmann, Annett AU - Wildemann, Dirk AU - Praetorius, Florian M AU - Fischer, Gunter AU - Kiefhaber, Thomas ID - 14305 IS - 10 JF - PNAS KW - Multidisciplinary SN - 0027-8424 TI - Mapping backbone and side-chain interactions in the transition state of a coupled protein folding and binding reaction VL - 108 ER - TY - JOUR AB - Cytosine methylation silences transposable elements in plants, vertebrates, and fungi but also regulates gene expression. Plant methylation is catalyzed by three families of enzymes, each with a preferred sequence context: CG, CHG (H = A, C, or T), and CHH, with CHH methylation targeted by the RNAi pathway. Arabidopsis thaliana endosperm, a placenta-like tissue that nourishes the embryo, is globally hypomethylated in the CG context while retaining high non-CG methylation. Global methylation dynamics in seeds of cereal crops that provide the bulk of human nutrition remain unknown. Here, we show that rice endosperm DNA is hypomethylated in all sequence contexts. Non-CG methylation is reduced evenly across the genome, whereas CG hypomethylation is localized. CHH methylation of small transposable elements is increased in embryos, suggesting that endosperm demethylation enhances transposon silencing. Genes preferentially expressed in endosperm, including those coding for major storage proteins and starch synthesizing enzymes, are frequently hypomethylated in endosperm, indicating that DNA methylation is a crucial regulator of rice endosperm biogenesis. Our data show that genome-wide reshaping of seed DNA methylation is conserved among angiosperms and has a profound effect on gene expression in cereal crops. AU - Zemach, Assaf AU - Kim, M. Yvonne AU - Silva, Pedro AU - Rodrigues, Jessica A. AU - Dotson, Bradley AU - Brooks, Matthew D. AU - Zilberman, Daniel ID - 9485 IS - 43 JF - Proceedings of the National Academy of Sciences SN - 0027-8424 TI - Local DNA hypomethylation activates genes in rice endosperm VL - 107 ER - TY - JOUR AB - Atom-resolved real-time studies of kinetic processes in proteins have been hampered in the past by the lack of experimental techniques that yield sufficient temporal and atomic resolution. Here we present band-selective optimized flip-angle short transient (SOFAST) real-time 2D NMR spectroscopy, a method that allows simultaneous observation of reaction kinetics for a large number of nuclear sites along the polypeptide chain of a protein with an unprecedented time resolution of a few seconds. SOFAST real-time 2D NMR spectroscopy combines fast NMR data acquisition techniques with rapid sample mixing inside the NMR magnet to initiate the kinetic event. We demonstrate the use of SOFAST real-time 2D NMR to monitor the conformational transition of α-lactalbumin from a molten globular to the native state for a large number of amide sites along the polypeptide chain. The kinetic behavior observed for the disappearance of the molten globule and the appearance of the native state is monoexponential and uniform along the polypeptide chain. This observation confirms previous findings that a single transition state ensemble controls folding of α-lactalbumin from the molten globule to the native state. In a second application, the spontaneous unfolding of native ubiquitin under nondenaturing conditions is characterized by amide hydrogen exchange rate constants measured at high pH by using SOFAST real-time 2D NMR. Our data reveal that ubiquitin unfolds in a gradual manner with distinct unfolding regimes. AU - Schanda, Paul AU - Forge, V. AU - Brutscher, B. ID - 8483 IS - 27 JF - Proceedings of the National Academy of Sciences KW - Multidisciplinary SN - 0027-8424 TI - Protein folding and unfolding studied at atomic resolution by fast two-dimensional NMR spectroscopy VL - 104 ER - TY - JOUR AB - Cytosine DNA methylation is considered to be a stable epigenetic mark, but active demethylation has been observed in both plants and animals. In Arabidopsis thaliana, DNA glycosylases of the DEMETER (DME) family remove methylcytosines from DNA. Demethylation by DME is necessary for genomic imprinting, and demethylation by a related protein, REPRESSOR OF SILENCING1, prevents gene silencing in a transgenic background. However, the extent and function of demethylation by DEMETER-LIKE (DML) proteins in WT plants is not known. Using genome-tiling microarrays, we mapped DNA methylation in mutant and WT plants and identified 179 loci actively demethylated by DML enzymes. Mutations in DML genes lead to locus-specific DNA hypermethylation. Reintroducing WT DML genes restores most loci to the normal pattern of methylation, although at some loci, hypermethylated epialleles persist. Of loci demethylated by DML enzymes, >80% are near or overlap genes. Genic demethylation by DML enzymes primarily occurs at the 5′ and 3′ ends, a pattern opposite to the overall distribution of WT DNA methylation. Our results show that demethylation by DML DNA glycosylases edits the patterns of DNA methylation within the Arabidopsis genome to protect genes from potentially deleterious methylation. AU - Penterman, Jon AU - Zilberman, Daniel AU - Huh, Jin Hoe AU - Ballinger, Tracy AU - Henikoff, Steven AU - Fischer, Robert L. ID - 9487 IS - 16 JF - Proceedings of the National Academy of Sciences SN - 0027-8424 TI - DNA demethylation in the Arabidopsis genome VL - 104 ER - TY - JOUR AB - Nanoparticles (NPs) decorated with ligands combining photoswitchable dipoles and covalent cross-linkers can be assembled by light into organized, three-dimensional suprastructures of various types and sizes. NPs covered with only few photoactive ligands form metastable crystals that can be assembled and disassembled “on demand” by using light of different wavelengths. For higher surface concentrations, self-assembly is irreversible, and the NPs organize into permanently cross-linked structures including robust supracrystals and plastic spherical aggregates. AU - Klajn, Rafal AU - Bishop, Kyle J. M. AU - Grzybowski, Bartosz A. ID - 13425 IS - 25 JF - Proceedings of the National Academy of Sciences KW - Multidisciplinary SN - 0027-8424 TI - Light-controlled self-assembly of reversible and irreversible nanoparticle suprastructures VL - 104 ER - TY - JOUR AB - The World Wide Web provides a unprecedented opportunity to automatically analyze a large sample of interests and activity in the world. We discuss methods for extracting knowledge from the web by randomly sampling and analyzing hosts and pages, and by analyzing the link structure of the web and how links accumulate over time. A variety of interesting and valuable information can be extracted, such as the distribution of web pages over domains, the distribution of interest in different areas, communities related to different topics, the nature of competition in different categories of sites, and the degree of communication between different communities or countries. AU - Henzinger, Monika H AU - Lawrence, Steve ID - 11877 IS - suppl_1 JF - Proceedings of the National Academy of Sciences SN - 0027-8424 TI - Extracting knowledge from the World Wide Web VL - 101 ER - TY - JOUR AB - Amphibian myelinated nerve fibers were treated with collagenase and protease. Axons with retraction of the myelin sheath were patch-clamped in the nodal and paranodal region. One type of Na channel was found. It has a single-channel conductance of 11 pS (15 degrees C) and is blocked by tetrodotoxin. Averaged events show the typical activation and inactivation kinetics of macroscopic Na current. Three potential-dependent K channels were identified (I, F, and S channel). The I channel, being the most frequent type, has a single-channel conductance of 23 pS (inward current, 105 mM K on both sides of the membrane), activates between -60 and -30 mV, deactivates with intermediate kinetics, and is sensitive to dendrotoxin. The F channel has a conductance of 30 pS, activates between -40 and 60 mV, and deactivates with fast kinetics. The former inactivates within tens of seconds; the latter inactivates within seconds. The third type, the S channel, has a conductance of 7 pS and deactivates slowly. All three channels can be blocked by external tetraethylammonium chloride. We suggest that these distinct K channel types form the basis for the different components of macroscopic K current described previously. AU - Jonas, Peter M AU - Bräu, Michael AU - Hermsteiner, Markus AU - Vogel, Werner ID - 3466 IS - 18 JF - PNAS SN - 0027-8424 TI - Single-channel recording in myelinated nerve fibers reveals one type of Na channel but different K channels VL - 86 ER -