TY - JOUR AB - The plant-signaling molecule auxin triggers fast and slow cellular responses across land plants and algae. The nuclear auxin pathway mediates gene expression and controls growth and development in land plants, but this pathway is absent from algal sister groups. Several components of rapid responses have been identified in Arabidopsis, but it is unknown if these are part of a conserved mechanism. We recently identified a fast, proteome-wide phosphorylation response to auxin. Here, we show that this response occurs across 5 land plant and algal species and converges on a core group of shared targets. We found conserved rapid physiological responses to auxin in the same species and identified rapidly accelerated fibrosarcoma (RAF)-like protein kinases as central mediators of auxin-triggered phosphorylation across species. Genetic analysis connects this kinase to both auxin-triggered protein phosphorylation and rapid cellular response, thus identifying an ancient mechanism for fast auxin responses in the green lineage. AU - Kuhn, Andre AU - Roosjen, Mark AU - Mutte, Sumanth AU - Dubey, Shiv Mani AU - Carrillo Carrasco, Vanessa Polet AU - Boeren, Sjef AU - Monzer, Aline AU - Koehorst, Jasper AU - Kohchi, Takayuki AU - Nishihama, Ryuichi AU - Fendrych, Matyas AU - Sprakel, Joris AU - Friml, Jiří AU - Weijers, Dolf ID - 14826 IS - 1 JF - Cell KW - General Biochemistry KW - Genetics and Molecular Biology SN - 0092-8674 TI - RAF-like protein kinases mediate a deeply conserved, rapid auxin response VL - 187 ER - TY - JOUR AB - The GNOM (GN) Guanine nucleotide Exchange Factor for ARF small GTPases (ARF-GEF) is among the best studied trafficking regulators in plants, playing crucial and unique developmental roles in patterning and polarity. The current models place GN at the Golgi apparatus (GA), where it mediates secretion/recycling, and at the plasma membrane (PM) presumably contributing to clathrin-mediated endocytosis (CME). The mechanistic basis of the developmental function of GN, distinct from the other ARF-GEFs including its closest homologue GNOM-LIKE1 (GNL1), remains elusive. Insights from this study largely extend the current notions of GN function. We show that GN, but not GNL1, localizes to the cell periphery at long-lived structures distinct from clathrin-coated pits, while CME and secretion proceed normally in gn knockouts. The functional GN mutant variant GNfewerroots, absent from the GA, suggests that the cell periphery is the major site of GN action responsible for its developmental function. Following inhibition by Brefeldin A, GN, but not GNL1, relocates to the PM likely on exocytic vesicles, suggesting selective molecular associations en route to the cell periphery. A study of GN-GNL1 chimeric ARF-GEFs indicates that all GN domains contribute to the specific GN function in a partially redundant manner. Together, this study offers significant steps toward the elucidation of the mechanism underlying unique cellular and development functions of GNOM. AU - Adamowski, Maciek AU - Matijevic, Ivana AU - Friml, Jiří ID - 15033 JF - eLife KW - General Immunology and Microbiology KW - General Biochemistry KW - Genetics and Molecular Biology KW - General Medicine KW - General Neuroscience SN - 2050-084X TI - Developmental patterning function of GNOM ARF-GEF mediated from the cell periphery VL - 13 ER - TY - JOUR AB - Characterizing and controlling entanglement in quantum materials is crucial for the development of next-generation quantum technologies. However, defining a quantifiable figure of merit for entanglement in macroscopic solids is theoretically and experimentally challenging. At equilibrium the presence of entanglement can be diagnosed by extracting entanglement witnesses from spectroscopic observables and a nonequilibrium extension of this method could lead to the discovery of novel dynamical phenomena. Here, we propose a systematic approach to quantify the time-dependent quantum Fisher information and entanglement depth of transient states of quantum materials with time-resolved resonant inelastic x-ray scattering. Using a quarter-filled extended Hubbard model as an example, we benchmark the efficiency of this approach and predict a light-enhanced many-body entanglement due to the proximity to a phase boundary. Our work sets the stage for experimentally witnessing and controlling entanglement in light-driven quantum materials via ultrafast spectroscopic measurements. AU - Hales, Jordyn AU - Bajpai, Utkarsh AU - Liu, Tongtong AU - Baykusheva, Denitsa Rangelova AU - Li, Mingda AU - Mitrano, Matteo AU - Wang, Yao ID - 13989 JF - Nature Communications KW - General Physics and Astronomy KW - General Biochemistry KW - Genetics and Molecular Biology KW - General Chemistry KW - Multidisciplinary TI - Witnessing light-driven entanglement using time-resolved resonant inelastic X-ray scattering VL - 14 ER - TY - JOUR AB - Mosaic analysis with double markers (MADM) technology enables the generation of genetic mosaic tissue in mice and high-resolution phenotyping at the individual cell level. Here, we present a protocol for isolating MADM-labeled cells with high yield for downstream molecular analyses using fluorescence-activated cell sorting (FACS). We describe steps for generating MADM-labeled mice, perfusion, single-cell suspension, and debris removal. We then detail procedures for cell sorting by FACS and downstream analysis. This protocol is suitable for embryonic to adult mice. For complete details on the use and execution of this protocol, please refer to Contreras et al. (2021).1 AU - Amberg, Nicole AU - Cheung, Giselle T AU - Hippenmeyer, Simon ID - 14683 IS - 1 JF - STAR Protocols KW - General Immunology and Microbiology KW - General Biochemistry KW - Genetics and Molecular Biology KW - General Neuroscience SN - 2666-1667 TI - Protocol for sorting cells from mouse brains labeled with mosaic analysis with double markers by flow cytometry VL - 5 ER - TY - JOUR AB - Chromosomal rearrangements (CRs) have been known since almost the beginning of genetics. While an important role for CRs in speciation has been suggested, evidence primarily stems from theoretical and empirical studies focusing on the microevolutionary level (i.e., on taxon pairs where speciation is often incomplete). Although the role of CRs in eukaryotic speciation at a macroevolutionary level has been supported by associations between species diversity and rates of evolution of CRs across phylogenies, these findings are limited to a restricted range of CRs and taxa. Now that more broadly applicable and precise CR detection approaches have become available, we address the challenges in filling some of the conceptual and empirical gaps between micro- and macroevolutionary studies on the role of CRs in speciation. We synthesize what is known about the macroevolutionary impact of CRs and suggest new research avenues to overcome the pitfalls of previous studies to gain a more comprehensive understanding of the evolutionary significance of CRs in speciation across the tree of life. AU - Lucek, Kay AU - Giménez, Mabel D. AU - Joron, Mathieu AU - Rafajlović, Marina AU - Searle, Jeremy B. AU - Walden, Nora AU - Westram, Anja M AU - Faria, Rui ID - 14742 IS - 11 JF - Cold Spring Harbor Perspectives in Biology KW - General Biochemistry KW - Genetics and Molecular Biology SN - 1943-0264 TI - The impact of chromosomal rearrangements in speciation: From micro- to macroevolution VL - 15 ER - TY - JOUR AB - Germ granules, condensates of phase-separated RNA and protein, are organelles that are essential for germline development in different organisms. The patterning of the granules and their relevance for germ cell fate are not fully understood. Combining three-dimensional in vivo structural and functional analyses, we study the dynamic spatial organization of molecules within zebrafish germ granules. We find that the localization of RNA molecules to the periphery of the granules, where ribosomes are localized, depends on translational activity at this location. In addition, we find that the vertebrate-specific Dead end (Dnd1) protein is essential for nanos3 RNA localization at the condensates’ periphery. Accordingly, in the absence of Dnd1, or when translation is inhibited, nanos3 RNA translocates into the granule interior, away from the ribosomes, a process that is correlated with the loss of germ cell fate. These findings highlight the relevance of sub-granule compartmentalization for post-transcriptional control and its importance for preserving germ cell totipotency. AU - Westerich, Kim Joana AU - Tarbashevich, Katsiaryna AU - Schick, Jan AU - Gupta, Antra AU - Zhu, Mingzhao AU - Hull, Kenneth AU - Romo, Daniel AU - Zeuschner, Dagmar AU - Goudarzi, Mohammad AU - Gross-Thebing, Theresa AU - Raz, Erez ID - 14781 IS - 17 JF - Developmental Cell KW - Developmental Biology KW - Cell Biology KW - General Biochemistry KW - Genetics and Molecular Biology KW - Molecular Biology SN - 1534-5807 TI - Spatial organization and function of RNA molecules within phase-separated condensates in zebrafish are controlled by Dnd1 VL - 58 ER - TY - JOUR AB - Little is known about the critical metabolic changes that neural cells have to undergo during development and how temporary shifts in this program can influence brain circuitries and behavior. Inspired by the discovery that mutations in SLC7A5, a transporter of metabolically essential large neutral amino acids (LNAAs), lead to autism, we employed metabolomic profiling to study the metabolic states of the cerebral cortex across different developmental stages. We found that the forebrain undergoes significant metabolic remodeling throughout development, with certain groups of metabolites showing stage-specific changes, but what are the consequences of perturbing this metabolic program? By manipulating Slc7a5 expression in neural cells, we found that the metabolism of LNAAs and lipids are interconnected in the cortex. Deletion of Slc7a5 in neurons affects the postnatal metabolic state, leading to a shift in lipid metabolism. Additionally, it causes stage- and cell-type-specific alterations in neuronal activity patterns, resulting in a long-term circuit dysfunction. AU - Knaus, Lisa AU - Basilico, Bernadette AU - Malzl, Daniel AU - Gerykova Bujalkova, Maria AU - Smogavec, Mateja AU - Schwarz, Lena A. AU - Gorkiewicz, Sarah AU - Amberg, Nicole AU - Pauler, Florian AU - Knittl-Frank, Christian AU - Tassinari, Marianna AU - Maulide, Nuno AU - Rülicke, Thomas AU - Menche, Jörg AU - Hippenmeyer, Simon AU - Novarino, Gaia ID - 12802 IS - 9 JF - Cell KW - General Biochemistry KW - Genetics and Molecular Biology SN - 0092-8674 TI - Large neutral amino acid levels tune perinatal neuronal excitability and survival VL - 186 ER - TY - JOUR AB - Objective: MazF is a sequence-specific endoribonuclease-toxin of the MazEF toxin–antitoxin system. MazF cleaves single-stranded ribonucleic acid (RNA) regions at adenine–cytosine–adenine (ACA) sequences in the bacterium Escherichia coli. The MazEF system has been used in various biotechnology and synthetic biology applications. In this study, we infer how ectopic mazF overexpression affects production of heterologous proteins. To this end, we quantified the levels of fluorescent proteins expressed in E. coli from reporters translated from the ACA-containing or ACA-less messenger RNAs (mRNAs). Additionally, we addressed the impact of the 5′-untranslated region of these reporter mRNAs under the same conditions by comparing expression from mRNAs that comprise (canonical mRNA) or lack this region (leaderless mRNA). Results: Flow cytometry analysis indicates that during mazF overexpression, fluorescent proteins are translated from the canonical as well as leaderless mRNAs. Our analysis further indicates that longer mazF overexpression generally increases the concentration of fluorescent proteins translated from ACA-less mRNAs, however it also substantially increases bacterial population heterogeneity. Finally, our results suggest that the strength and duration of mazF overexpression should be optimized for each experimental setup, to maximize the heterologous protein production and minimize the amount of phenotypic heterogeneity in bacterial populations, which is unfavorable in biotechnological processes. AU - Nikolic, Nela AU - Sauert, Martina AU - Albanese, Tanino G. AU - Moll, Isabella ID - 11713 JF - BMC Research Notes KW - General Biochemistry KW - Genetics and Molecular Biology KW - General Medicine SN - 1756-0500 TI - Quantifying heterologous gene expression during ectopic MazF production in Escherichia coli VL - 15 ER - TY - JOUR AB - Models of transcriptional regulation that assume equilibrium binding of transcription factors have been less successful at predicting gene expression from sequence in eukaryotes than in bacteria. This could be due to the non-equilibrium nature of eukaryotic regulation. Unfortunately, the space of possible non-equilibrium mechanisms is vast and predominantly uninteresting. The key question is therefore how this space can be navigated efficiently, to focus on mechanisms and models that are biologically relevant. In this review, we advocate for the normative role of theory—theory that prescribes rather than just describes—in providing such a focus. Theory should expand its remit beyond inferring mechanistic models from data, towards identifying non-equilibrium gene regulatory schemes that may have been evolutionarily selected, despite their energy consumption, because they are precise, reliable, fast, or otherwise outperform regulation at equilibrium. We illustrate our reasoning by toy examples for which we provide simulation code. AU - Zoller, Benjamin AU - Gregor, Thomas AU - Tkačik, Gašper ID - 12156 IS - 9 JF - Current Opinion in Systems Biology KW - Applied Mathematics KW - Computer Science Applications KW - Drug Discovery KW - General Biochemistry KW - Genetics and Molecular Biology KW - Modeling and Simulation SN - 2452-3100 TI - Eukaryotic gene regulation at equilibrium, or non? VL - 31 ER - TY - JOUR AB - DNA methylation plays essential homeostatic functions in eukaryotic genomes. In animals, DNA methylation is also developmentally regulated and, in turn, regulates development. In the past two decades, huge research effort has endorsed the understanding that DNA methylation plays a similar role in plant development, especially during sexual reproduction. The power of whole-genome sequencing and cell isolation techniques, as well as bioinformatics tools, have enabled recent studies to reveal dynamic changes in DNA methylation during germline development. Furthermore, the combination of these technological advances with genetics, developmental biology and cell biology tools has revealed functional methylation reprogramming events that control gene and transposon activities in flowering plant germlines. In this review, we discuss the major advances in our knowledge of DNA methylation dynamics during male and female germline development in flowering plants. AU - He, Shengbo AU - Feng, Xiaoqi ID - 12670 IS - 12 JF - Journal of Integrative Plant Biology KW - Plant Science KW - General Biochemistry KW - Genetics and Molecular Biology KW - Biochemistry SN - 1672-9072 TI - DNA methylation dynamics during germline development VL - 64 ER - TY - JOUR AB - One hallmark of plant cells is their cell wall. They protect cells against the environment and high turgor and mediate morphogenesis through the dynamics of their mechanical and chemical properties. The walls are a complex polysaccharidic structure. Although their biochemical composition is well known, how the different components organize in the volume of the cell wall and interact with each other is not well understood and yet is key to the wall’s mechanical properties. To investigate the ultrastructure of the plant cell wall, we imaged the walls of onion (Allium cepa) bulbs in a near-native state via cryo-focused ion beam milling (cryo-FIB milling) and cryo-electron tomography (cryo-ET). This allowed the high-resolution visualization of cellulose fibers in situ. We reveal the coexistence of dense fiber fields bathed in a reticulated matrix we termed “meshing,” which is more abundant at the inner surface of the cell wall. The fibers adopted a regular bimodal angular distribution at all depths in the cell wall and bundled according to their orientation, creating layers within the cell wall. Concomitantly, employing homogalacturonan (HG)-specific enzymatic digestion, we observed changes in the meshing, suggesting that it is—at least in part—composed of HG pectins. We propose the following model for the construction of the abaxial epidermal primary cell wall: the cell deposits successive layers of cellulose fibers at −45° and +45° relative to the cell’s long axis and secretes the surrounding HG-rich meshing proximal to the plasma membrane, which then migrates to more distal regions of the cell wall. AU - Nicolas, William J. AU - Fäßler, Florian AU - Dutka, Przemysław AU - Schur, Florian KM AU - Jensen, Grant AU - Meyerowitz, Elliot ID - 11351 IS - 11 JF - Current Biology KW - General Agricultural and Biological Sciences KW - General Biochemistry KW - Genetics and Molecular Biology SN - 0960-9822 TI - Cryo-electron tomography of the onion cell wall shows bimodally oriented cellulose fibers and reticulated homogalacturonan networks VL - 32 ER - TY - JOUR AB - Studies of protein fitness landscapes reveal biophysical constraints guiding protein evolution and empower prediction of functional proteins. However, generalisation of these findings is limited due to scarceness of systematic data on fitness landscapes of proteins with a defined evolutionary relationship. We characterized the fitness peaks of four orthologous fluorescent proteins with a broad range of sequence divergence. While two of the four studied fitness peaks were sharp, the other two were considerably flatter, being almost entirely free of epistatic interactions. Mutationally robust proteins, characterized by a flat fitness peak, were not optimal templates for machine-learning-driven protein design – instead, predictions were more accurate for fragile proteins with epistatic landscapes. Our work paves insights for practical application of fitness landscape heterogeneity in protein engineering. AU - Gonzalez Somermeyer, Louisa AU - Fleiss, Aubin AU - Mishin, Alexander S AU - Bozhanova, Nina G AU - Igolkina, Anna A AU - Meiler, Jens AU - Alaball Pujol, Maria-Elisenda AU - Putintseva, Ekaterina V AU - Sarkisyan, Karen S AU - Kondrashov, Fyodor ID - 11448 JF - eLife KW - General Immunology and Microbiology KW - General Biochemistry KW - Genetics and Molecular Biology KW - General Medicine KW - General Neuroscience SN - 2050-084X TI - Heterogeneity of the GFP fitness landscape and data-driven protein design VL - 11 ER - TY - JOUR AB - Empirical essays of fitness landscapes suggest that they may be rugged, that is having multiple fitness peaks. Such fitness landscapes, those that have multiple peaks, necessarily have special local structures, called reciprocal sign epistasis (Poelwijk et al. in J Theor Biol 272:141–144, 2011). Here, we investigate the quantitative relationship between the number of fitness peaks and the number of reciprocal sign epistatic interactions. Previously, it has been shown (Poelwijk et al. in J Theor Biol 272:141–144, 2011) that pairwise reciprocal sign epistasis is a necessary but not sufficient condition for the existence of multiple peaks. Applying discrete Morse theory, which to our knowledge has never been used in this context, we extend this result by giving the minimal number of reciprocal sign epistatic interactions required to create a given number of peaks. AU - Saona Urmeneta, Raimundo J AU - Kondrashov, Fyodor AU - Khudiakova, Kseniia ID - 11447 IS - 8 JF - Bulletin of Mathematical Biology KW - Computational Theory and Mathematics KW - General Agricultural and Biological Sciences KW - Pharmacology KW - General Environmental Science KW - General Biochemistry KW - Genetics and Molecular Biology KW - General Mathematics KW - Immunology KW - General Neuroscience SN - 0092-8240 TI - Relation between the number of peaks and the number of reciprocal sign epistatic interactions VL - 84 ER - TY - JOUR AB - Local adaptation leads to differences between populations within a species. In many systems, similar environmental contrasts occur repeatedly, sometimes driving parallel phenotypic evolution. Understanding the genomic basis of local adaptation and parallel evolution is a major goal of evolutionary genomics. It is now known that by preventing the break-up of favourable combinations of alleles across multiple loci, genetic architectures that reduce recombination, like chromosomal inversions, can make an important contribution to local adaptation. However, little is known about whether inversions also contribute disproportionately to parallel evolution. Our aim here is to highlight this knowledge gap, to showcase existing studies, and to illustrate the differences between genomic architectures with and without inversions using simple models. We predict that by generating stronger effective selection, inversions can sometimes speed up the parallel adaptive process or enable parallel adaptation where it would be impossible otherwise, but this is highly dependent on the spatial setting. We highlight that further empirical work is needed, in particular to cover a broader taxonomic range and to understand the relative importance of inversions compared to genomic regions without inversions. AU - Westram, Anja M AU - Faria, Rui AU - Johannesson, Kerstin AU - Butlin, Roger AU - Barton, Nicholas H ID - 11546 IS - 1856 JF - Philosophical Transactions of the Royal Society B: Biological Sciences KW - General Agricultural and Biological Sciences KW - General Biochemistry KW - Genetics and Molecular Biology SN - 0962-8436 TI - Inversions and parallel evolution VL - 377 ER - TY - JOUR AB - The mammalian hippocampal formation (HF) plays a key role in several higher brain functions, such as spatial coding, learning and memory. Its simple circuit architecture is often viewed as a trisynaptic loop, processing input originating from the superficial layers of the entorhinal cortex (EC) and sending it back to its deeper layers. Here, we show that excitatory neurons in layer 6b of the mouse EC project to all sub-regions comprising the HF and receive input from the CA1, thalamus and claustrum. Furthermore, their output is characterized by unique slow-decaying excitatory postsynaptic currents capable of driving plateau-like potentials in their postsynaptic targets. Optogenetic inhibition of the EC-6b pathway affects spatial coding in CA1 pyramidal neurons, while cell ablation impairs not only acquisition of new spatial memories, but also degradation of previously acquired ones. Our results provide evidence of a functional role for cortical layer 6b neurons in the adult brain. AU - Ben Simon, Yoav AU - Käfer, Karola AU - Velicky, Philipp AU - Csicsvari, Jozsef L AU - Danzl, Johann G AU - Jonas, Peter M ID - 11951 JF - Nature Communications KW - General Physics and Astronomy KW - General Biochemistry KW - Genetics and Molecular Biology KW - General Chemistry KW - Multidisciplinary SN - 2041-1723 TI - A direct excitatory projection from entorhinal layer 6b neurons to the hippocampus contributes to spatial coding and memory VL - 13 ER - TY - JOUR AB - Transcription of the ribosomal RNA precursor by RNA polymerase (Pol) I is a major determinant of cellular growth, and dysregulation is observed in many cancer types. Here, we present the purification of human Pol I from cells carrying a genomic GFP fusion on the largest subunit allowing the structural and functional analysis of the enzyme across species. In contrast to yeast, human Pol I carries a single-subunit stalk, and in vitro transcription indicates a reduced proofreading activity. Determination of the human Pol I cryo-EM reconstruction in a close-to-native state rationalizes the effects of disease-associated mutations and uncovers an additional domain that is built into the sequence of Pol I subunit RPA1. This “dock II” domain resembles a truncated HMG box incapable of DNA binding which may serve as a downstream transcription factor–binding platform in metazoans. Biochemical analysis, in situ modelling, and ChIP data indicate that Topoisomerase 2a can be recruited to Pol I via the domain and cooperates with the HMG box domain–containing factor UBF. These adaptations of the metazoan Pol I transcription system may allow efficient release of positive DNA supercoils accumulating downstream of the transcription bubble. AU - Daiß, Julia L AU - Pilsl, Michael AU - Straub, Kristina AU - Bleckmann, Andrea AU - Höcherl, Mona AU - Heiss, Florian B AU - Abascal-Palacios, Guillermo AU - Ramsay, Ewan P AU - Tluckova, Katarina AU - Mars, Jean-Clement AU - Fürtges, Torben AU - Bruckmann, Astrid AU - Rudack, Till AU - Bernecky, Carrie A AU - Lamour, Valérie AU - Panov, Konstantin AU - Vannini, Alessandro AU - Moss, Tom AU - Engel, Christoph ID - 12051 IS - 11 JF - Life Science Alliance KW - Health KW - Toxicology and Mutagenesis KW - Plant Science KW - Biochemistry KW - Genetics and Molecular Biology (miscellaneous) KW - Ecology SN - 2575-1077 TI - The human RNA polymerase I structure reveals an HMG-like docking domain specific to metazoans VL - 5 ER - TY - JOUR AB - Germline determination is essential for species survival and evolution in multicellular organisms. In most flowering plants, formation of the female germline is initiated with specification of one megaspore mother cell (MMC) in each ovule; however, the molecular mechanism underlying this key event remains unclear. Here we report that spatially restricted auxin signaling promotes MMC fate in Arabidopsis. Our results show that the microRNA160 (miR160) targeted gene ARF17 (AUXIN RESPONSE FACTOR17) is required for promoting MMC specification by genetically interacting with the SPL/NZZ (SPOROCYTELESS/NOZZLE) gene. Alterations of auxin signaling cause formation of supernumerary MMCs in an ARF17- and SPL/NZZ-dependent manner. Furthermore, miR160 and ARF17 are indispensable for attaining a normal auxin maximum at the ovule apex via modulating the expression domain of PIN1 (PIN-FORMED1) auxin transporter. Our findings elucidate the mechanism by which auxin signaling promotes the acquisition of female germline cell fate in plants. AU - Huang, Jian AU - Zhao, Lei AU - Malik, Shikha AU - Gentile, Benjamin R. AU - Xiong, Va AU - Arazi, Tzahi AU - Owen, Heather A. AU - Friml, Jiří AU - Zhao, Dazhong ID - 12130 JF - Nature Communications KW - General Physics and Astronomy KW - General Biochemistry KW - Genetics and Molecular Biology KW - General Chemistry KW - Multidisciplinary SN - 2041-1723 TI - Specification of female germline by microRNA orchestrated auxin signaling in Arabidopsis VL - 13 ER - TY - JOUR AB - Polygenic adaptation is thought to be ubiquitous, yet remains poorly understood. Here, we model this process analytically, in the plausible setting of a highly polygenic, quantitative trait that experiences a sudden shift in the fitness optimum. We show how the mean phenotype changes over time, depending on the effect sizes of loci that contribute to variance in the trait, and characterize the allele dynamics at these loci. Notably, we describe the two phases of the allele dynamics: The first is a rapid phase, in which directional selection introduces small frequency differences between alleles whose effects are aligned with or opposed to the shift, ultimately leading to small differences in their probability of fixation during a second, longer phase, governed by stabilizing selection. As we discuss, key results should hold in more general settings and have important implications for efforts to identify the genetic basis of adaptation in humans and other species. AU - Hayward, Laura AU - Sella, Guy ID - 12157 JF - eLife KW - General Immunology and Microbiology KW - General Biochemistry KW - Genetics and Molecular Biology KW - General Medicine KW - General Neuroscience TI - Polygenic adaptation after a sudden change in environment VL - 11 ER - TY - JOUR AB - The inadequate understanding of the mechanisms that reversibly convert molecular sulfur (S) into lithium sulfide (Li2S) via soluble polysulfides (PSs) formation impedes the development of high-performance lithium-sulfur (Li-S) batteries with non-aqueous electrolyte solutions. Here, we use operando small and wide angle X-ray scattering and operando small angle neutron scattering (SANS) measurements to track the nucleation, growth and dissolution of solid deposits from atomic to sub-micron scales during real-time Li-S cell operation. In particular, stochastic modelling based on the SANS data allows quantifying the nanoscale phase evolution during battery cycling. We show that next to nano-crystalline Li2S the deposit comprises solid short-chain PSs particles. The analysis of the experimental data suggests that initially, Li2S2 precipitates from the solution and then is partially converted via solid-state electroreduction to Li2S. We further demonstrate that mass transport, rather than electron transport through a thin passivating film, limits the discharge capacity and rate performance in Li-S cells. AU - Prehal, Christian AU - von Mentlen, Jean-Marc AU - Drvarič Talian, Sara AU - Vizintin, Alen AU - Dominko, Robert AU - Amenitsch, Heinz AU - Porcar, Lionel AU - Freunberger, Stefan Alexander AU - Wood, Vanessa ID - 12208 JF - Nature Communications KW - General Physics and Astronomy KW - General Biochemistry KW - Genetics and Molecular Biology KW - General Chemistry KW - Multidisciplinary SN - 2041-1723 TI - On the nanoscale structural evolution of solid discharge products in lithium-sulfur batteries using operando scattering VL - 13 ER - TY - JOUR AB - The development dynamics and self-organization of glandular branched epithelia is of utmost importance for our understanding of diverse processes ranging from normal tissue growth to the growth of cancerous tissues. Using single primary murine pancreatic ductal adenocarcinoma (PDAC) cells embedded in a collagen matrix and adapted media supplementation, we generate organoids that self-organize into highly branched structures displaying a seamless lumen connecting terminal end buds, replicating in vivo PDAC architecture. We identify distinct morphogenesis phases, each characterized by a unique pattern of cell invasion, matrix deformation, protein expression, and respective molecular dependencies. We propose a minimal theoretical model of a branching and proliferating tissue, capturing the dynamics of the first phases. Observing the interaction of morphogenesis, mechanical environment and gene expression in vitro sets a benchmark for the understanding of self-organization processes governing complex organoid structure formation processes and branching morphogenesis. AU - Randriamanantsoa, S. AU - Papargyriou, A. AU - Maurer, H. C. AU - Peschke, K. AU - Schuster, M. AU - Zecchin, G. AU - Steiger, K. AU - Öllinger, R. AU - Saur, D. AU - Scheel, C. AU - Rad, R. AU - Hannezo, Edouard B AU - Reichert, M. AU - Bausch, A. R. ID - 12217 JF - Nature Communications KW - General Physics and Astronomy KW - General Biochemistry KW - Genetics and Molecular Biology KW - General Chemistry KW - Multidisciplinary SN - 2041-1723 TI - Spatiotemporal dynamics of self-organized branching in pancreas-derived organoids VL - 13 ER - TY - JOUR AB - Muskelin (Mkln1) is implicated in neuronal function, regulating plasma membrane receptor trafficking. However, its influence on intrinsic brain activity and corresponding behavioral processes remains unclear. Here we show that murine Mkln1 knockout causes non-habituating locomotor activity, increased exploratory drive, and decreased locomotor response to amphetamine. Muskelin deficiency impairs social novelty detection while promoting the retention of spatial reference memory and fear extinction recall. This is strongly mirrored in either weaker or stronger resting-state functional connectivity between critical circuits mediating locomotor exploration and cognition. We show that Mkln1 deletion alters dendrite branching and spine structure, coinciding with enhanced AMPAR-mediated synaptic transmission but selective impairment in synaptic potentiation maintenance. We identify muskelin at excitatory synapses and highlight its role in regulating dendritic spine actin stability. Our findings point to aberrant spine actin modulation and changes in glutamatergic synaptic function as critical mechanisms that contribute to the neurobehavioral phenotype arising from Mkln1 ablation. AU - Muhia, Mary W AU - YuanXiang, PingAn AU - Sedlacik, Jan AU - Schwarz, Jürgen R. AU - Heisler, Frank F. AU - Gromova, Kira V. AU - Thies, Edda AU - Breiden, Petra AU - Pechmann, Yvonne AU - Kreutz, Michael R. AU - Kneussel, Matthias ID - 12224 JF - Communications Biology KW - General Agricultural and Biological Sciences KW - General Biochemistry KW - Genetics and Molecular Biology KW - Medicine (miscellaneous) SN - 2399-3642 TI - Muskelin regulates actin-dependent synaptic changes and intrinsic brain activity relevant to behavioral and cognitive processes VL - 5 ER - TY - JOUR AB - Upon the initiation of collective cell migration, the cells at the free edge are specified as leader cells; however, the mechanism underlying the leader cell specification remains elusive. Here, we show that lamellipodial extension after the release from mechanical confinement causes sustained extracellular signal-regulated kinase (ERK) activation and underlies the leader cell specification. Live-imaging of Madin-Darby canine kidney (MDCK) cells and mouse epidermis through the use of Förster resonance energy transfer (FRET)-based biosensors showed that leader cells exhibit sustained ERK activation in a hepatocyte growth factor (HGF)-dependent manner. Meanwhile, follower cells exhibit oscillatory ERK activation waves in an epidermal growth factor (EGF) signaling-dependent manner. Lamellipodial extension at the free edge increases the cellular sensitivity to HGF. The HGF-dependent ERK activation, in turn, promotes lamellipodial extension, thereby forming a positive feedback loop between cell extension and ERK activation and specifying the cells at the free edge as the leader cells. Our findings show that the integration of physical and biochemical cues underlies the leader cell specification during collective cell migration. AU - Hino, Naoya AU - Matsuda, Kimiya AU - Jikko, Yuya AU - Maryu, Gembu AU - Sakai, Katsuya AU - Imamura, Ryu AU - Tsukiji, Shinya AU - Aoki, Kazuhiro AU - Terai, Kenta AU - Hirashima, Tsuyoshi AU - Trepat, Xavier AU - Matsuda, Michiyuki ID - 12238 IS - 19 JF - Developmental Cell KW - Developmental Biology KW - Cell Biology KW - General Biochemistry KW - Genetics and Molecular Biology KW - Molecular Biology SN - 1534-5807 TI - A feedback loop between lamellipodial extension and HGF-ERK signaling specifies leader cells during collective cell migration VL - 57 ER - TY - JOUR AB - Dose–response relationships are a general concept for quantitatively describing biological systems across multiple scales, from the molecular to the whole-cell level. A clinically relevant example is the bacterial growth response to antibiotics, which is routinely characterized by dose–response curves. The shape of the dose–response curve varies drastically between antibiotics and plays a key role in treatment, drug interactions, and resistance evolution. However, the mechanisms shaping the dose–response curve remain largely unclear. Here, we show in Escherichia coli that the distinctively shallow dose–response curve of the antibiotic trimethoprim is caused by a negative growth-mediated feedback loop: Trimethoprim slows growth, which in turn weakens the effect of this antibiotic. At the molecular level, this feedback is caused by the upregulation of the drug target dihydrofolate reductase (FolA/DHFR). We show that this upregulation is not a specific response to trimethoprim but follows a universal trend line that depends primarily on the growth rate, irrespective of its cause. Rewiring the feedback loop alters the dose–response curve in a predictable manner, which we corroborate using a mathematical model of cellular resource allocation and growth. Our results indicate that growth-mediated feedback loops may shape drug responses more generally and could be exploited to design evolutionary traps that enable selection against drug resistance. AU - Angermayr, Andreas AU - Pang, Tin Yau AU - Chevereau, Guillaume AU - Mitosch, Karin AU - Lercher, Martin J AU - Bollenbach, Mark Tobias ID - 12261 IS - 9 JF - Molecular Systems Biology KW - Applied Mathematics KW - Computational Theory and Mathematics KW - General Agricultural and Biological Sciences KW - General Immunology and Microbiology KW - General Biochemistry KW - Genetics and Molecular Biology KW - Information Systems TI - Growth‐mediated negative feedback shapes quantitative antibiotic response VL - 18 ER - TY - JOUR AB - To understand the function of neuronal circuits, it is crucial to disentangle the connectivity patterns within the network. However, most tools currently used to explore connectivity have low throughput, low selectivity, or limited accessibility. Here, we report the development of an improved packaging system for the production of the highly neurotropic RVdGenvA-CVS-N2c rabies viral vectors, yielding titers orders of magnitude higher with no background contamination, at a fraction of the production time, while preserving the efficiency of transsynaptic labeling. Along with the production pipeline, we developed suites of ‘starter’ AAV and bicistronic RVdG-CVS-N2c vectors, enabling retrograde labeling from a wide range of neuronal populations, tailored for diverse experimental requirements. We demonstrate the power and flexibility of the new system by uncovering hidden local and distal inhibitory connections in the mouse hippocampal formation and by imaging the functional properties of a cortical microcircuit across weeks. Our novel production pipeline provides a convenient approach to generate new rabies vectors, while our toolkit flexibly and efficiently expands the current capacity to label, manipulate and image the neuronal activity of interconnected neuronal circuits in vitro and in vivo. AU - Sumser, Anton L AU - Jösch, Maximilian A AU - Jonas, Peter M AU - Ben Simon, Yoav ID - 12288 JF - eLife KW - General Immunology and Microbiology KW - General Biochemistry KW - Genetics and Molecular Biology KW - General Medicine KW - General Neuroscience TI - Fast, high-throughput production of improved rabies viral vectors for specific, efficient and versatile transsynaptic retrograde labeling VL - 11 ER - TY - JOUR AB - Plant root architecture flexibly adapts to changing nitrate (NO3−) availability in the soil; however, the underlying molecular mechanism of this adaptive development remains under-studied. To explore the regulation of NO3−-mediated root growth, we screened for low-nitrate-resistant mutant (lonr) and identified mutants that were defective in the NAC transcription factor NAC075 (lonr1) as being less sensitive to low NO3− in terms of primary root growth. We show that NAC075 is a mobile transcription factor relocating from the root stele tissues to the endodermis based on NO3− availability. Under low-NO3− availability, the kinase CBL-interacting protein kinase 1 (CIPK1) is activated, and it phosphorylates NAC075, restricting its movement from the stele, which leads to the transcriptional regulation of downstream target WRKY53, consequently leading to adapted root architecture. Our work thus identifies an adaptive mechanism involving translocation of transcription factor based on nutrient availability and leading to cell-specific reprogramming of plant root growth. AU - Xiao, Huixin AU - Hu, Yumei AU - Wang, Yaping AU - Cheng, Jinkui AU - Wang, Jinyi AU - Chen, Guojingwei AU - Li, Qian AU - Wang, Shuwei AU - Wang, Yalu AU - Wang, Shao-Shuai AU - Wang, Yi AU - Xuan, Wei AU - Li, Zhen AU - Guo, Yan AU - Gong, Zhizhong AU - Friml, Jiří AU - Zhang, Jing ID - 12120 IS - 23 JF - Developmental Cell KW - Developmental Biology KW - Cell Biology KW - General Biochemistry KW - Genetics and Molecular Biology KW - Molecular Biology SN - 1534-5807 TI - Nitrate availability controls translocation of the transcription factor NAC075 for cell-type-specific reprogramming of root growth VL - 57 ER - TY - JOUR AB - To understand how potential gene manipulations affect in vitro microglia, we provide a set of short protocols to evaluate microglia identity and function. We detail steps for immunostaining to determine microglia identity. We describe three functional assays for microglia: phagocytosis, calcium response following ATP stimulation, and cytokine expression upon inflammatory stimuli. We apply these protocols to human induced-pluripotent-stem-cell (hiPSC)-derived microglia, but they can be also applied to other in vitro microglial models including primary mouse microglia. For complete details on the use and execution of this protocol, please refer to Bartalska et al. (2022).1 AU - Hübschmann, Verena AU - Korkut, Medina AU - Siegert, Sandra ID - 12117 IS - 4 JF - STAR Protocols KW - General Immunology and Microbiology KW - General Biochemistry KW - Genetics and Molecular Biology KW - General Neuroscience SN - 2666-1667 TI - Assessing human iPSC-derived microglia identity and function by immunostaining, phagocytosis, calcium activity, and inflammation assay VL - 3 ER - TY - JOUR AB - A species distributed across diverse environments may adapt to local conditions. We ask how quickly such a species changes its range in response to changed conditions. Szép et al. (Szép E, Sachdeva H, Barton NH. 2021 Polygenic local adaptation in metapopulations: a stochastic eco-evolutionary model. Evolution75, 1030–1045 (doi:10.1111/evo.14210)) used the infinite island model to find the stationary distribution of allele frequencies and deme sizes. We extend this to find how a metapopulation responds to changes in carrying capacity, selection strength, or migration rate when deme sizes are fixed. We further develop a ‘fixed-state’ approximation. Under this approximation, polymorphism is only possible for a narrow range of habitat proportions when selection is weak compared to drift, but for a much wider range otherwise. When rates of selection or migration relative to drift change in a single deme of the metapopulation, the population takes a time of order m−1 to reach the new equilibrium. However, even with many loci, there can be substantial fluctuations in net adaptation, because at each locus, alleles randomly get lost or fixed. Thus, in a finite metapopulation, variation may gradually be lost by chance, even if it would persist in an infinite metapopulation. When conditions change across the whole metapopulation, there can be rapid change, which is predicted well by the fixed-state approximation. This work helps towards an understanding of how metapopulations extend their range across diverse environments. This article is part of the theme issue ‘Species’ ranges in the face of changing environments (Part II)’. AU - Barton, Nicholas H AU - Olusanya, Oluwafunmilola O ID - 10787 IS - 1848 JF - Philosophical Transactions of the Royal Society B: Biological Sciences KW - General Agricultural and Biological Sciences KW - General Biochemistry KW - Genetics and Molecular Biology SN - 0962-8436 TI - The response of a metapopulation to a changing environment VL - 377 ER - TY - JOUR AB - The actin-homologue FtsA is essential for E. coli cell division, as it links FtsZ filaments in the Z-ring to transmembrane proteins. FtsA is thought to initiate cell constriction by switching from an inactive polymeric to an active monomeric conformation, which recruits downstream proteins and stabilizes the Z-ring. However, direct biochemical evidence for this mechanism is missing. Here, we use reconstitution experiments and quantitative fluorescence microscopy to study divisome activation in vitro. By comparing wild-type FtsA with FtsA R286W, we find that this hyperactive mutant outperforms FtsA WT in replicating FtsZ treadmilling dynamics, FtsZ filament stabilization and recruitment of FtsN. We could attribute these differences to a faster exchange and denser packing of FtsA R286W below FtsZ filaments. Using FRET microscopy, we also find that FtsN binding promotes FtsA self-interaction. We propose that in the active divisome FtsA and FtsN exist as a dynamic copolymer that follows treadmilling filaments of FtsZ. AU - Radler, Philipp AU - Baranova, Natalia S. AU - Dos Santos Caldas, Paulo R AU - Sommer, Christoph M AU - Lopez Pelegrin, Maria D AU - Michalik, David AU - Loose, Martin ID - 11373 JF - Nature Communications KW - General Physics and Astronomy KW - General Biochemistry KW - Genetics and Molecular Biology KW - General Chemistry SN - 2041-1723 TI - In vitro reconstitution of Escherichia coli divisome activation VL - 13 ER - TY - JOUR AB - Mutations in the chromodomain helicase DNA-binding 8 (CHD8) gene are a frequent cause of autism spectrum disorder (ASD). While its phenotypic spectrum often encompasses macrocephaly, implicating cortical abnormalities, how CHD8 haploinsufficiency affects neurodevelopmental is unclear. Here, employing human cerebral organoids, we find that CHD8 haploinsufficiency disrupted neurodevelopmental trajectories with an accelerated and delayed generation of, respectively, inhibitory and excitatory neurons that yields, at days 60 and 120, symmetrically opposite expansions in their proportions. This imbalance is consistent with an enlargement of cerebral organoids as an in vitro correlate of patients’ macrocephaly. Through an isogenic design of patient-specific mutations and mosaic organoids, we define genotype-phenotype relationships and uncover their cell-autonomous nature. Our results define cell-type-specific CHD8-dependent molecular defects related to an abnormal program of proliferation and alternative splicing. By identifying cell-type-specific effects of CHD8 mutations, our study uncovers reproducible developmental alterations that may be employed for neurodevelopmental disease modeling. AU - Villa, Carlo Emanuele AU - Cheroni, Cristina AU - Dotter, Christoph AU - López-Tóbon, Alejandro AU - Oliveira, Bárbara AU - Sacco, Roberto AU - Yahya, Aysan Çerağ AU - Morandell, Jasmin AU - Gabriele, Michele AU - Tavakoli, Mojtaba AU - Lyudchik, Julia AU - Sommer, Christoph M AU - Gabitto, Mariano AU - Danzl, Johann G AU - Testa, Giuseppe AU - Novarino, Gaia ID - 11160 IS - 1 JF - Cell Reports KW - General Biochemistry KW - Genetics and Molecular Biology SN - 2211-1247 TI - CHD8 haploinsufficiency links autism to transient alterations in excitatory and inhibitory trajectories VL - 39 ER - TY - JOUR AB - In order to combat molecular damage, most cellular proteins undergo rapid turnover. We have previously identified large nuclear protein assemblies that can persist for years in post-mitotic tissues and are subject to age-related decline. Here, we report that mitochondria can be long lived in the mouse brain and reveal that specific mitochondrial proteins have half-lives longer than the average proteome. These mitochondrial long-lived proteins (mitoLLPs) are core components of the electron transport chain (ETC) and display increased longevity in respiratory supercomplexes. We find that COX7C, a mitoLLP that forms a stable contact site between complexes I and IV, is required for complex IV and supercomplex assembly. Remarkably, even upon depletion of COX7C transcripts, ETC function is maintained for days, effectively uncoupling mitochondrial function from ongoing transcription of its mitoLLPs. Our results suggest that modulating protein longevity within the ETC is critical for mitochondrial proteome maintenance and the robustness of mitochondrial function. AU - Krishna, Shefali AU - Arrojo e Drigo, Rafael AU - Capitanio, Juliana S. AU - Ramachandra, Ranjan AU - Ellisman, Mark AU - HETZER, Martin W ID - 11052 IS - 21 JF - Developmental Cell KW - Developmental Biology KW - Cell Biology KW - General Biochemistry KW - Genetics and Molecular Biology KW - Molecular Biology SN - 1534-5807 TI - Identification of long-lived proteins in the mitochondria reveals increased stability of the electron transport chain VL - 56 ER - TY - JOUR AB - Glaciers in High Mountain Asia generate meltwater that supports the water needs of 250 million people, but current knowledge of annual accumulation and ablation is limited to sparse field measurements biased in location and glacier size. Here, we present altitudinally-resolved specific mass balances (surface, internal, and basal combined) for 5527 glaciers in High Mountain Asia for 2000–2016, derived by correcting observed glacier thinning patterns for mass redistribution due to ice flow. We find that 41% of glaciers accumulated mass over less than 20% of their area, and only 60% ± 10% of regional annual ablation was compensated by accumulation. Even without 21st century warming, 21% ± 1% of ice volume will be lost by 2100 due to current climatic-geometric imbalance, representing a reduction in glacier ablation into rivers of 28% ± 1%. The ablation of glaciers in the Himalayas and Tien Shan was mostly unsustainable and ice volume in these regions will reduce by at least 30% by 2100. The most important and vulnerable glacier-fed river basins (Amu Darya, Indus, Syr Darya, Tarim Interior) were supplied with >50% sustainable glacier ablation but will see long-term reductions in ice mass and glacier meltwater supply regardless of the Karakoram Anomaly. AU - Miles, Evan AU - McCarthy, Michael AU - Dehecq, Amaury AU - Kneib, Marin AU - Fugger, Stefan AU - Pellicciotti, Francesca ID - 12585 JF - Nature Communications KW - General Physics and Astronomy KW - General Biochemistry KW - Genetics and Molecular Biology KW - General Chemistry KW - Multidisciplinary SN - 2041-1723 TI - Health and sustainability of glaciers in High Mountain Asia VL - 12 ER - TY - JOUR AB - Self-assembly of nanoparticles can be mediated by polymers, but has so far led almost exclusively to nanoparticle aggregates that are amorphous. Here, we employed Coulombic interactions to generate a range of composite materials from mixtures of charged nanoparticles and oppositely charged polymers. The assembly behavior of these nanoparticle/polymer composites depends on their order of addition: polymers added to nanoparticles give rise to stable aggregates, but nanoparticles added to polymers disassemble the initially formed aggregates. The amorphous aggregates were transformed into crystalline ones by transiently increasing the ionic strength of the solution. The morphology of the resulting crystals depended on the length of the polymer: short polymer chains mediated the self-assembly of nanoparticles into strongly faceted crystals, whereas long chains led to pseudospherical nanoparticle/polymer assemblies, within which the crystalline order of nanoparticles was retained. AU - Bian, Tong AU - Klajn, Rafal ID - 13356 IS - 1 JF - Annals of the New York Academy of Sciences KW - History and Philosophy of Science KW - General Biochemistry KW - Genetics and Molecular Biology KW - General Neuroscience SN - 0077-8923 TI - Morphology control in crystalline nanoparticle–polymer aggregates VL - 1505 ER - TY - JOUR AB - We report the complete analysis of a deterministic model of deleterious mutations and negative selection against them at two haploid loci without recombination. As long as mutation is a weaker force than selection, mutant alleles remain rare at the only stable equilibrium, and otherwise, a variety of dynamics are possible. If the mutation-free genotype is absent, generally the only stable equilibrium is the one that corresponds to fixation of the mutant allele at the locus where it is less deleterious. This result suggests that fixation of a deleterious allele that follows a click of the Muller’s ratchet is governed by natural selection, instead of random drift. AU - Khudiakova, Kseniia AU - Neretina, Tatiana Yu. AU - Kondrashov, Alexey S. ID - 9387 JF - Journal of Theoretical Biology KW - General Biochemistry KW - Genetics and Molecular Biology KW - Modelling and Simulation KW - Statistics and Probability KW - General Immunology and Microbiology KW - Applied Mathematics KW - General Agricultural and Biological Sciences KW - General Medicine SN - 0022-5193 TI - Two linked loci under mutation-selection balance and Muller’s ratchet VL - 524 ER - TY - JOUR AB - Inositol hexakisphosphate (IP6) is an assembly cofactor for HIV-1. We report here that IP6 is also used for assembly of Rous sarcoma virus (RSV), a retrovirus from a different genus. IP6 is ~100-fold more potent at promoting RSV mature capsid protein (CA) assembly than observed for HIV-1 and removal of IP6 in cells reduces infectivity by 100-fold. Here, visualized by cryo-electron tomography and subtomogram averaging, mature capsid-like particles show an IP6-like density in the CA hexamer, coordinated by rings of six lysines and six arginines. Phosphate and IP6 have opposing effects on CA in vitro assembly, inducing formation of T = 1 icosahedrons and tubes, respectively, implying that phosphate promotes pentamer and IP6 hexamer formation. Subtomogram averaging and classification optimized for analysis of pleomorphic retrovirus particles reveal that the heterogeneity of mature RSV CA polyhedrons results from an unexpected, intrinsic CA hexamer flexibility. In contrast, the CA pentamer forms rigid units organizing the local architecture. These different features of hexamers and pentamers determine the structural mechanism to form CA polyhedrons of variable shape in mature RSV particles. AU - Obr, Martin AU - Ricana, Clifton L. AU - Nikulin, Nadia AU - Feathers, Jon-Philip R. AU - Klanschnig, Marco AU - Thader, Andreas AU - Johnson, Marc C. AU - Vogt, Volker M. AU - Schur, Florian KM AU - Dick, Robert A. ID - 9431 IS - 1 JF - Nature Communications KW - General Biochemistry KW - Genetics and Molecular Biology KW - General Physics and Astronomy KW - General Chemistry TI - Structure of the mature Rous sarcoma virus lattice reveals a role for IP6 in the formation of the capsid hexamer VL - 12 ER - TY - JOUR AB - The hexameric AAA-ATPase Drg1 is a key factor in eukaryotic ribosome biogenesis and initiates cytoplasmic maturation of the large ribosomal subunit by releasing the shuttling maturation factor Rlp24. Drg1 monomers contain two AAA-domains (D1 and D2) that act in a concerted manner. Rlp24 release is inhibited by the drug diazaborine which blocks ATP hydrolysis in D2. The mode of inhibition was unknown. Here we show the first cryo-EM structure of Drg1 revealing the inhibitory mechanism. Diazaborine forms a covalent bond to the 2′-OH of the nucleotide in D2, explaining its specificity for this site. As a consequence, the D2 domain is locked in a rigid, inactive state, stalling the whole Drg1 hexamer. Resistance mechanisms identified include abolished drug binding and altered positioning of the nucleotide. Our results suggest nucleotide-modifying compounds as potential novel inhibitors for AAA-ATPases. AU - Prattes, Michael AU - Grishkovskaya, Irina AU - Hodirnau, Victor-Valentin AU - Rössler, Ingrid AU - Klein, Isabella AU - Hetzmannseder, Christina AU - Zisser, Gertrude AU - Gruber, Christian C. AU - Gruber, Karl AU - Haselbach, David AU - Bergler, Helmut ID - 9540 IS - 1 JF - Nature Communications KW - General Biochemistry KW - Genetics and Molecular Biology KW - General Physics and Astronomy KW - General Chemistry TI - Structural basis for inhibition of the AAA-ATPase Drg1 by diazaborine VL - 12 ER - TY - JOUR AB - The hippocampal mossy fiber synapse is a key synapse of the trisynaptic circuit. Post-tetanic potentiation (PTP) is the most powerful form of plasticity at this synaptic connection. It is widely believed that mossy fiber PTP is an entirely presynaptic phenomenon, implying that PTP induction is input-specific, and requires neither activity of multiple inputs nor stimulation of postsynaptic neurons. To directly test cooperativity and associativity, we made paired recordings between single mossy fiber terminals and postsynaptic CA3 pyramidal neurons in rat brain slices. By stimulating non-overlapping mossy fiber inputs converging onto single CA3 neurons, we confirm that PTP is input-specific and non-cooperative. Unexpectedly, mossy fiber PTP exhibits anti-associative induction properties. EPSCs show only minimal PTP after combined pre- and postsynaptic high-frequency stimulation with intact postsynaptic Ca2+ signaling, but marked PTP in the absence of postsynaptic spiking and after suppression of postsynaptic Ca2+ signaling (10 mM EGTA). PTP is largely recovered by inhibitors of voltage-gated R- and L-type Ca2+ channels, group II mGluRs, and vacuolar-type H+-ATPase, suggesting the involvement of retrograde vesicular glutamate signaling. Transsynaptic regulation of PTP extends the repertoire of synaptic computations, implementing a brake on mossy fiber detonation and a “smart teacher” function of hippocampal mossy fiber synapses. AU - Vandael, David H AU - Okamoto, Yuji AU - Jonas, Peter M ID - 9778 IS - 1 JF - Nature Communications KW - general physics and astronomy KW - general biochemistry KW - genetics and molecular biology KW - general chemistry SN - 2041-1723 TI - Transsynaptic modulation of presynaptic short-term plasticity in hippocampal mossy fiber synapses VL - 12 ER - TY - JOUR AB - The C-terminal domain (CTD) of the largest subunit of RNA polymerase II (Pol II) is a regulatory hub for transcription and RNA processing. Here, we identify PHD-finger protein 3 (PHF3) as a regulator of transcription and mRNA stability that docks onto Pol II CTD through its SPOC domain. We characterize SPOC as a CTD reader domain that preferentially binds two phosphorylated Serine-2 marks in adjacent CTD repeats. PHF3 drives liquid-liquid phase separation of phosphorylated Pol II, colocalizes with Pol II clusters and tracks with Pol II across the length of genes. PHF3 knock-out or SPOC deletion in human cells results in increased Pol II stalling, reduced elongation rate and an increase in mRNA stability, with marked derepression of neuronal genes. Key neuronal genes are aberrantly expressed in Phf3 knock-out mouse embryonic stem cells, resulting in impaired neuronal differentiation. Our data suggest that PHF3 acts as a prominent effector of neuronal gene regulation by bridging transcription with mRNA decay. AU - Appel, Lisa-Marie AU - Franke, Vedran AU - Bruno, Melania AU - Grishkovskaya, Irina AU - Kasiliauskaite, Aiste AU - Kaufmann, Tanja AU - Schoeberl, Ursula E. AU - Puchinger, Martin G. AU - Kostrhon, Sebastian AU - Ebenwaldner, Carmen AU - Sebesta, Marek AU - Beltzung, Etienne AU - Mechtler, Karl AU - Lin, Gen AU - Vlasova, Anna AU - Leeb, Martin AU - Pavri, Rushad AU - Stark, Alexander AU - Akalin, Altuna AU - Stefl, Richard AU - Bernecky, Carrie A AU - Djinovic-Carugo, Kristina AU - Slade, Dea ID - 10163 IS - 1 JF - Nature Communications KW - general physics and astronomy KW - general biochemistry KW - genetics and molecular biology KW - general chemistry TI - PHF3 regulates neuronal gene expression through the Pol II CTD reader domain SPOC VL - 12 ER - TY - JOUR AB - De novo protein synthesis is required for synapse modifications underlying stable memory encoding. Yet neurons are highly compartmentalized cells and how protein synthesis can be regulated at the synapse level is unknown. Here, we characterize neuronal signaling complexes formed by the postsynaptic scaffold GIT1, the mechanistic target of rapamycin (mTOR) kinase, and Raptor that couple synaptic stimuli to mTOR-dependent protein synthesis; and identify NMDA receptors containing GluN3A subunits as key negative regulators of GIT1 binding to mTOR. Disruption of GIT1/mTOR complexes by enhancing GluN3A expression or silencing GIT1 inhibits synaptic mTOR activation and restricts the mTOR-dependent translation of specific activity-regulated mRNAs. Conversely, GluN3A removal enables complex formation, potentiates mTOR-dependent protein synthesis, and facilitates the consolidation of associative and spatial memories in mice. The memory enhancement becomes evident with light or spaced training, can be achieved by selectively deleting GluN3A from excitatory neurons during adulthood, and does not compromise other aspects of cognition such as memory flexibility or extinction. Our findings provide mechanistic insight into synaptic translational control and reveal a potentially selective target for cognitive enhancement. AU - Conde-Dusman, María J AU - Dey, Partha N AU - Elía-Zudaire, Óscar AU - Garcia Rabaneda, Luis E AU - García-Lira, Carmen AU - Grand, Teddy AU - Briz, Victor AU - Velasco, Eric R AU - Andero Galí, Raül AU - Niñerola, Sergio AU - Barco, Angel AU - Paoletti, Pierre AU - Wesseling, John F AU - Gardoni, Fabrizio AU - Tavalin, Steven J AU - Perez-Otaño, Isabel ID - 10301 JF - eLife KW - general immunology and microbiology KW - general biochemistry KW - genetics and molecular biology KW - general medicine KW - general neuroscience SN - 2050-084X TI - Control of protein synthesis and memory by GluN3A-NMDA receptors through inhibition of GIT1/mTORC1 assembly VL - 10 ER - TY - JOUR AB - A high-resolution structure of trimeric cyanobacterial Photosystem I (PSI) from Thermosynechococcus elongatus was reported as the first atomic model of PSI almost 20 years ago. However, the monomeric PSI structure has not yet been reported despite long-standing interest in its structure and extensive spectroscopic characterization of the loss of red chlorophylls upon monomerization. Here, we describe the structure of monomeric PSI from Thermosynechococcus elongatus BP-1. Comparison with the trimer structure gave detailed insights into monomerization-induced changes in both the central trimerization domain and the peripheral regions of the complex. Monomerization-induced loss of red chlorophylls is assigned to a cluster of chlorophylls adjacent to PsaX. Based on our findings, we propose a role of PsaX in the stabilization of red chlorophylls and that lipids of the surrounding membrane present a major source of thermal energy for uphill excitation energy transfer from red chlorophylls to P700. AU - Çoruh, Mehmet Orkun AU - Frank, Anna AU - Tanaka, Hideaki AU - Kawamoto, Akihiro AU - El-Mohsnawy, Eithar AU - Kato, Takayuki AU - Namba, Keiichi AU - Gerle, Christoph AU - Nowaczyk, Marc M. AU - Kurisu, Genji ID - 10310 IS - 1 JF - Communications Biology KW - general agricultural and biological Sciences KW - general biochemistry KW - genetics and molecular biology KW - medicine (miscellaneous) SN - 2399-3642 TI - Cryo-EM structure of a functional monomeric Photosystem I from Thermosynechococcus elongatus reveals red chlorophyll cluster VL - 4 ER - TY - JOUR AB - Flowering plants utilize small RNA molecules to guide DNA methyltransferases to genomic sequences. This RNA-directed DNA methylation (RdDM) pathway preferentially targets euchromatic transposable elements. However, RdDM is thought to be recruited by methylation of histone H3 at lysine 9 (H3K9me), a hallmark of heterochromatin. How RdDM is targeted to euchromatin despite an affinity for H3K9me is unclear. Here we show that loss of histone H1 enhances heterochromatic RdDM, preferentially at nucleosome linker DNA. Surprisingly, this does not require SHH1, the RdDM component that binds H3K9me. Furthermore, H3K9me is dispensable for RdDM, as is CG DNA methylation. Instead, we find that non-CG methylation is specifically associated with small RNA biogenesis, and without H1 small RNA production quantitatively expands to non-CG methylated loci. Our results demonstrate that H1 enforces the separation of euchromatic and heterochromatic DNA methylation pathways by excluding the small RNA-generating branch of RdDM from non-CG methylated heterochromatin. AU - Choi, Jaemyung AU - Lyons, David B AU - Zilberman, Daniel ID - 10533 JF - eLife KW - genetics and molecular biology SN - 2050-084X TI - Histone H1 prevents non-CG methylation-mediated small RNA biogenesis in Arabidopsis heterochromatin VL - 10 ER - TY - JOUR AB - Hematopoietic-specific protein 1 (Hem1) is an essential subunit of the WAVE regulatory complex (WRC) in immune cells. WRC is crucial for Arp2/3 complex activation and the protrusion of branched actin filament networks. Moreover, Hem1 loss of function in immune cells causes autoimmune diseases in humans. Here, we show that genetic removal of Hem1 in macrophages diminishes frequency and efficacy of phagocytosis as well as phagocytic cup formation in addition to defects in lamellipodial protrusion and migration. Moreover, Hem1-null macrophages displayed strong defects in cell adhesion despite unaltered podosome formation and concomitant extracellular matrix degradation. Specifically, dynamics of both adhesion and de-adhesion as well as concomitant phosphorylation of paxillin and focal adhesion kinase (FAK) were significantly compromised. Accordingly, disruption of WRC function in non-hematopoietic cells coincided with both defects in adhesion turnover and altered FAK and paxillin phosphorylation. Consistently, platelets exhibited reduced adhesion and diminished integrin αIIbβ3 activation upon WRC removal. Interestingly, adhesion phenotypes, but not lamellipodia formation, were partially rescued by small molecule activation of FAK. A full rescue of the phenotype, including lamellipodia formation, required not only the presence of WRCs but also their binding to and activation by Rac. Collectively, our results uncover that WRC impacts on integrin-dependent processes in a FAK-dependent manner, controlling formation and dismantling of adhesions, relevant for properly grabbing onto extracellular surfaces and particles during cell edge expansion, like in migration or phagocytosis. AU - Stahnke, Stephanie AU - Döring, Hermann AU - Kusch, Charly AU - de Gorter, David J.J. AU - Dütting, Sebastian AU - Guledani, Aleks AU - Pleines, Irina AU - Schnoor, Michael AU - Sixt, Michael K AU - Geffers, Robert AU - Rohde, Manfred AU - Müsken, Mathias AU - Kage, Frieda AU - Steffen, Anika AU - Faix, Jan AU - Nieswandt, Bernhard AU - Rottner, Klemens AU - Stradal, Theresia E.B. ID - 10834 IS - 10 JF - Current Biology KW - General Agricultural and Biological Sciences KW - General Biochemistry KW - Genetics and Molecular Biology SN - 0960-9822 TI - Loss of Hem1 disrupts macrophage function and impacts migration, phagocytosis, and integrin-mediated adhesion VL - 31 ER - TY - JOUR AB - Gene expression levels are influenced by multiple coexisting molecular mechanisms. Some of these interactions such as those of transcription factors and promoters have been studied extensively. However, predicting phenotypes of gene regulatory networks (GRNs) remains a major challenge. Here, we use a well-defined synthetic GRN to study in Escherichia coli how network phenotypes depend on local genetic context, i.e. the genetic neighborhood of a transcription factor and its relative position. We show that one GRN with fixed topology can display not only quantitatively but also qualitatively different phenotypes, depending solely on the local genetic context of its components. Transcriptional read-through is the main molecular mechanism that places one transcriptional unit (TU) within two separate regulons without the need for complex regulatory sequences. We propose that relative order of individual TUs, with its potential for combinatorial complexity, plays an important role in shaping phenotypes of GRNs. AU - Nagy-Staron, Anna A AU - Tomasek, Kathrin AU - Caruso Carter, Caroline AU - Sonnleitner, Elisabeth AU - Kavcic, Bor AU - Paixão, Tiago AU - Guet, Calin C ID - 9283 JF - eLife KW - Genetics and Molecular Biology SN - 2050-084X TI - Local genetic context shapes the function of a gene regulatory network VL - 10 ER - TY - JOUR AB - Eukaryotic DNA-binding proteins operate in the context of chromatin, where nucleosomes are the elementary building blocks. Nucleosomal DNA is wrapped around a histone core, thereby rendering a large fraction of the DNA surface inaccessible to DNA-binding proteins. Nevertheless, first responders in DNA repair and sequence-specific transcription factors bind DNA target sites obstructed by chromatin. While early studies examined protein binding to histone-free DNA, it is only now beginning to emerge how DNA sequences are interrogated on nucleosomes. These readout strategies range from the release of nucleosomal DNA from histones, to rotational/translation register shifts of the DNA motif, and nucleosome-specific DNA binding modes that differ from those observed on naked DNA. Since DNA motif engagement on nucleosomes strongly depends on position and orientation, we argue that motif location and nucleosome positioning co-determine protein access to DNA in transcription and DNA repair. AU - Michael, Alicia AU - Thomä, Nicolas H. ID - 15151 IS - 14 JF - Cell KW - General Biochemistry KW - Genetics and Molecular Biology SN - 0092-8674 TI - Reading the chromatinized genome VL - 184 ER - TY - JOUR AB - De novo loss of function mutations in the ubiquitin ligase-encoding gene Cullin3 lead to autism spectrum disorder (ASD). In mouse, constitutive haploinsufficiency leads to motor coordination deficits as well as ASD-relevant social and cognitive impairments. However, induction of Cul3 haploinsufficiency later in life does not lead to ASD-relevant behaviors, pointing to an important role of Cul3 during a critical developmental window. Here we show that Cul3 is essential to regulate neuronal migration and, therefore, constitutive Cul3 heterozygous mutant mice display cortical lamination abnormalities. At the molecular level, we found that Cul3 controls neuronal migration by tightly regulating the amount of Plastin3 (Pls3), a previously unrecognized player of neural migration. Furthermore, we found that Pls3 cell-autonomously regulates cell migration by regulating actin cytoskeleton organization, and its levels are inversely proportional to neural migration speed. Finally, we provide evidence that cellular phenotypes associated with autism-linked gene haploinsufficiency can be rescued by transcriptional activation of the intact allele in vitro, offering a proof of concept for a potential therapeutic approach for ASDs. AU - Morandell, Jasmin AU - Schwarz, Lena A AU - Basilico, Bernadette AU - Tasciyan, Saren AU - Dimchev, Georgi A AU - Nicolas, Armel AU - Sommer, Christoph M AU - Kreuzinger, Caroline AU - Dotter, Christoph AU - Knaus, Lisa AU - Dobler, Zoe AU - Cacci, Emanuele AU - Schur, Florian KM AU - Danzl, Johann G AU - Novarino, Gaia ID - 9429 IS - 1 JF - Nature Communications KW - General Biochemistry KW - Genetics and Molecular Biology TI - Cul3 regulates cytoskeleton protein homeostasis and cell migration during a critical window of brain development VL - 12 ER - TY - JOUR AB - The endosomal sorting complex required for transport-III (ESCRT-III) catalyzes membrane fission from within membrane necks, a process that is essential for many cellular functions, from cell division to lysosome degradation and autophagy. How it breaks membranes, though, remains unknown. Here, we characterize a sequential polymerization of ESCRT-III subunits that, driven by a recruitment cascade and by continuous subunit-turnover powered by the ATPase Vps4, induces membrane deformation and fission. During this process, the exchange of Vps24 for Did2 induces a tilt in the polymer-membrane interface, which triggers transition from flat spiral polymers to helical filament to drive the formation of membrane protrusions, and ends with the formation of a highly constricted Did2-Ist1 co-polymer that we show is competent to promote fission when bound on the inside of membrane necks. Overall, our results suggest a mechanism of stepwise changes in ESCRT-III filament structure and mechanical properties via exchange of the filament subunits to catalyze ESCRT-III activity. AU - Pfitzner, Anna-Katharina AU - Mercier, Vincent AU - Jiang, Xiuyun AU - Moser von Filseck, Joachim AU - Baum, Buzz AU - Šarić, Anđela AU - Roux, Aurélien ID - 10348 IS - 5 JF - Cell KW - general biochemistry KW - genetics and molecular biology SN - 0092-8674 TI - An ESCRT-III polymerization sequence drives membrane deformation and fission VL - 182 ER - TY - JOUR AB - Aging of the circulatory system correlates with the pathogenesis of a large spectrum of diseases. However, it is largely unknown which factors drive the age-dependent or pathological decline of the vasculature and how vascular defects relate to tissue aging. The goal of the study is to design a multianalytical approach to identify how the cellular microenvironment (i.e., fibroblasts) and serum from healthy donors of different ages or Alzheimer disease (AD) patients can modulate the functionality of organ-specific vascular endothelial cells (VECs). Long-living human microvascular networks embedding VECs and fibroblasts from skin biopsies are generated. RNA-seq, secretome analyses, and microfluidic assays demonstrate that fibroblasts from young donors restore the functionality of aged endothelial cells, an effect also achieved by serum from young donors. New biomarkers of vascular aging are validated in human biopsies and it is shown that young serum induces angiopoietin-like-4, which can restore compromised vascular barriers. This strategy is then employed to characterize transcriptional/functional changes induced on the blood–brain barrier by AD serum, demonstrating the importance of PTP4A3 in the regulation of permeability. Features of vascular degeneration during aging and AD are recapitulated, and a tool to identify novel biomarkers that can be exploited to develop future therapeutics modulating vascular function is established. AU - Bersini, Simone AU - Arrojo e Drigo, Rafael AU - Huang, Ling AU - Shokhirev, Maxim N. AU - HETZER, Martin W ID - 11056 IS - 5 JF - Advanced Biosystems KW - General Biochemistry KW - Genetics and Molecular Biology KW - Biomedical Engineering KW - Biomaterials SN - 2366-7478 TI - Transcriptional and functional changes of the human microvasculature during physiological aging and Alzheimer disease VL - 4 ER - TY - JOUR AB - Vascular dysfunctions are a common feature of multiple age-related diseases. However, modeling healthy and pathological aging of the human vasculature represents an unresolved experimental challenge. Here, we generated induced vascular endothelial cells (iVECs) and smooth muscle cells (iSMCs) by direct reprogramming of healthy human fibroblasts from donors of different ages and Hutchinson-Gilford Progeria Syndrome (HGPS) patients. iVECs induced from old donors revealed upregulation of GSTM1 and PALD1, genes linked to oxidative stress, inflammation and endothelial junction stability, as vascular aging markers. A functional assay performed on PALD1 KD VECs demonstrated a recovery in vascular permeability. We found that iSMCs from HGPS donors overexpressed bone morphogenetic protein (BMP)−4, which plays a key role in both vascular calcification and endothelial barrier damage observed in HGPS. Strikingly, BMP4 concentrations are higher in serum from HGPS vs. age-matched mice. Furthermore, targeting BMP4 with blocking antibody recovered the functionality of the vascular barrier in vitro, hence representing a potential future therapeutic strategy to limit cardiovascular dysfunction in HGPS. These results show that iVECs and iSMCs retain disease-related signatures, allowing modeling of vascular aging and HGPS in vitro. AU - Bersini, Simone AU - Schulte, Roberta AU - Huang, Ling AU - Tsai, Hannah AU - HETZER, Martin W ID - 11055 JF - eLife KW - General Immunology and Microbiology KW - General Biochemistry KW - Genetics and Molecular Biology KW - General Medicine KW - General Neuroscience SN - 2050-084X TI - Direct reprogramming of human smooth muscle and vascular endothelial cells reveals defects associated with aging and Hutchinson-Gilford progeria syndrome VL - 9 ER - TY - JOUR AB - Nucleoporin 93 (Nup93) expression inversely correlates with the survival of triple-negative breast cancer patients. However, our knowledge of Nup93 function in breast cancer besides its role as structural component of the nuclear pore complex is not understood. Combination of functional assays and genetic analyses suggested that chromatin interaction of Nup93 partially modulates the expression of genes associated with actin cytoskeleton remodeling and epithelial to mesenchymal transition, resulting in impaired invasion of triple-negative, claudin-low breast cancer cells. Nup93 depletion induced stress fiber formation associated with reduced cell migration/proliferation and impaired expression of mesenchymal-like genes. Silencing LIMCH1, a gene responsible for actin cytoskeleton remodeling and up-regulated upon Nup93 depletion, partially restored the invasive phenotype of cancer cells. Loss of Nup93 led to significant defects in tumor establishment/propagation in vivo, whereas patient samples revealed that high Nup93 and low LIMCH1 expression correlate with late tumor stage. Our approach identified Nup93 as contributor of triple-negative, claudin-low breast cancer cell invasion and paves the way to study the role of nuclear envelope proteins during breast cancer tumorigenesis. AU - Bersini, Simone AU - Lytle, Nikki K AU - Schulte, Roberta AU - Huang, Ling AU - Wahl, Geoffrey M AU - HETZER, Martin W ID - 11058 IS - 1 JF - Life Science Alliance KW - Health KW - Toxicology and Mutagenesis KW - Plant Science KW - Biochemistry KW - Genetics and Molecular Biology (miscellaneous) KW - Ecology SN - 2575-1077 TI - Nup93 regulates breast tumor growth by modulating cell proliferation and actin cytoskeleton remodeling VL - 3 ER - TY - JOUR AB - Background: The mitochondrial pyruvate carrier (MPC) plays a central role in energy metabolism by transporting pyruvate across the inner mitochondrial membrane. Its heterodimeric composition and homology to SWEET and semiSWEET transporters set the MPC apart from the canonical mitochondrial carrier family (named MCF or SLC25). The import of the canonical carriers is mediated by the carrier translocase of the inner membrane (TIM22) pathway and is dependent on their structure, which features an even number of transmembrane segments and both termini in the intermembrane space. The import pathway of MPC proteins has not been elucidated. The odd number of transmembrane segments and positioning of the N-terminus in the matrix argues against an import via the TIM22 carrier pathway but favors an import via the flexible presequence pathway. Results: Here, we systematically analyzed the import pathways of Mpc2 and Mpc3 and report that, contrary to an expected import via the flexible presequence pathway, yeast MPC proteins with an odd number of transmembrane segments and matrix-exposed N-terminus are imported by the carrier pathway, using the receptor Tom70, small TIM chaperones, and the TIM22 complex. The TIM9·10 complex chaperones MPC proteins through the mitochondrial intermembrane space using conserved hydrophobic motifs that are also required for the interaction with canonical carrier proteins. Conclusions: The carrier pathway can import paired and non-paired transmembrane helices and translocate N-termini to either side of the mitochondrial inner membrane, revealing an unexpected versatility of the mitochondrial import pathway for non-cleavable inner membrane proteins. AU - Rampelt, Heike AU - Sucec, Iva AU - Bersch, Beate AU - Horten, Patrick AU - Perschil, Inge AU - Martinou, Jean-Claude AU - van der Laan, Martin AU - Wiedemann, Nils AU - Schanda, Paul AU - Pfanner, Nikolaus ID - 8402 JF - BMC Biology KW - Biotechnology KW - Plant Science KW - General Biochemistry KW - Genetics and Molecular Biology KW - Developmental Biology KW - Cell Biology KW - Physiology KW - Ecology KW - Evolution KW - Behavior and Systematics KW - Structural Biology KW - General Agricultural and Biological Sciences SN - 1741-7007 TI - The mitochondrial carrier pathway transports non-canonical substrates with an odd number of transmembrane segments VL - 18 ER - TY - JOUR AB - Practical quantum networks require low-loss and noise-resilient optical interconnects as well as non-Gaussian resources for entanglement distillation and distributed quantum computation. The latter could be provided by superconducting circuits but existing solutions to interface the microwave and optical domains lack either scalability or efficiency, and in most cases the conversion noise is not known. In this work we utilize the unique opportunities of silicon photonics, cavity optomechanics and superconducting circuits to demonstrate a fully integrated, coherent transducer interfacing the microwave X and the telecom S bands with a total (internal) bidirectional transduction efficiency of 1.2% (135%) at millikelvin temperatures. The coupling relies solely on the radiation pressure interaction mediated by the femtometer-scale motion of two silicon nanobeams reaching a Vπ as low as 16 μV for sub-nanowatt pump powers. Without the associated optomechanical gain, we achieve a total (internal) pure conversion efficiency of up to 0.019% (1.6%), relevant for future noise-free operation on this qubit-compatible platform. AU - Arnold, Georg M AU - Wulf, Matthias AU - Barzanjeh, Shabir AU - Redchenko, Elena AU - Rueda Sanchez, Alfredo R AU - Hease, William J AU - Hassani, Farid AU - Fink, Johannes M ID - 8529 JF - Nature Communications KW - General Biochemistry KW - Genetics and Molecular Biology KW - General Physics and Astronomy KW - General Chemistry SN - 2041-1723 TI - Converting microwave and telecom photons with a silicon photonic nanomechanical interface VL - 11 ER - TY - JOUR AB - Glioblastoma is the most malignant cancer in the brain and currently incurable. It is urgent to identify effective targets for this lethal disease. Inhibition of such targets should suppress the growth of cancer cells and, ideally also precancerous cells for early prevention, but minimally affect their normal counterparts. Using genetic mouse models with neural stem cells (NSCs) or oligodendrocyte precursor cells (OPCs) as the cells‐of‐origin/mutation, it is shown that the susceptibility of cells within the development hierarchy of glioma to the knockout of insulin‐like growth factor I receptor (IGF1R) is determined not only by their oncogenic states, but also by their cell identities/states. Knockout of IGF1R selectively disrupts the growth of mutant and transformed, but not normal OPCs, or NSCs. The desirable outcome of IGF1R knockout on cell growth requires the mutant cells to commit to the OPC identity regardless of its development hierarchical status. At the molecular level, oncogenic mutations reprogram the cellular network of OPCs and force them to depend more on IGF1R for their growth. A new‐generation brain‐penetrable, orally available IGF1R inhibitor harnessing tumor OPCs in the brain is also developed. The findings reveal the cellular window of IGF1R targeting and establish IGF1R as an effective target for the prevention and treatment of glioblastoma. AU - Tian, Anhao AU - Kang, Bo AU - Li, Baizhou AU - Qiu, Biying AU - Jiang, Wenhong AU - Shao, Fangjie AU - Gao, Qingqing AU - Liu, Rui AU - Cai, Chengwei AU - Jing, Rui AU - Wang, Wei AU - Chen, Pengxiang AU - Liang, Qinghui AU - Bao, Lili AU - Man, Jianghong AU - Wang, Yan AU - Shi, Yu AU - Li, Jin AU - Yang, Minmin AU - Wang, Lisha AU - Zhang, Jianmin AU - Hippenmeyer, Simon AU - Zhu, Junming AU - Bian, Xiuwu AU - Wang, Ying‐Jie AU - Liu, Chong ID - 8592 IS - 21 JF - Advanced Science KW - General Engineering KW - General Physics and Astronomy KW - General Materials Science KW - Medicine (miscellaneous) KW - General Chemical Engineering KW - Biochemistry KW - Genetics and Molecular Biology (miscellaneous) SN - 2198-3844 TI - Oncogenic state and cell identity combinatorially dictate the susceptibility of cells within glioma development hierarchy to IGF1R targeting VL - 7 ER - TY - JOUR AB - Aqueous iodine based electrochemical energy storage is considered a potential candidate to improve sustainability and performance of current battery and supercapacitor technology. It harnesses the redox activity of iodide, iodine, and polyiodide species in the confined geometry of nanoporous carbon electrodes. However, current descriptions of the electrochemical reaction mechanism to interconvert these species are elusive. Here we show that electrochemical oxidation of iodide in nanoporous carbons forms persistent solid iodine deposits. Confinement slows down dissolution into triiodide and pentaiodide, responsible for otherwise significant self-discharge via shuttling. The main tools for these insights are in situ Raman spectroscopy and in situ small and wide-angle X-ray scattering (in situ SAXS/WAXS). In situ Raman confirms the reversible formation of triiodide and pentaiodide. In situ SAXS/WAXS indicates remarkable amounts of solid iodine deposited in the carbon nanopores. Combined with stochastic modeling, in situ SAXS allows quantifying the solid iodine volume fraction and visualizing the iodine structure on 3D lattice models at the sub-nanometer scale. Based on the derived mechanism, we demonstrate strategies for improved iodine pore filling capacity and prevention of self-discharge, applicable to hybrid supercapacitors and batteries. AU - Prehal, Christian AU - Fitzek, Harald AU - Kothleitner, Gerald AU - Presser, Volker AU - Gollas, Bernhard AU - Freunberger, Stefan Alexander AU - Abbas, Qamar ID - 8568 JF - Nature Communications KW - General Biochemistry KW - Genetics and Molecular Biology KW - General Physics and Astronomy KW - General Chemistry SN - 2041-1723 TI - Persistent and reversible solid iodine electrodeposition in nanoporous carbons VL - 11 ER - TY - JOUR AB - Understanding the conformational sampling of translation-arrested ribosome nascent chain complexes is key to understand co-translational folding. Up to now, coupling of cysteine oxidation, disulfide bond formation and structure formation in nascent chains has remained elusive. Here, we investigate the eye-lens protein γB-crystallin in the ribosomal exit tunnel. Using mass spectrometry, theoretical simulations, dynamic nuclear polarization-enhanced solid-state nuclear magnetic resonance and cryo-electron microscopy, we show that thiol groups of cysteine residues undergo S-glutathionylation and S-nitrosylation and form non-native disulfide bonds. Thus, covalent modification chemistry occurs already prior to nascent chain release as the ribosome exit tunnel provides sufficient space even for disulfide bond formation which can guide protein folding. AU - Schulte, Linda AU - Mao, Jiafei AU - Reitz, Julian AU - Sreeramulu, Sridhar AU - Kudlinzki, Denis AU - Hodirnau, Victor-Valentin AU - Meier-Credo, Jakob AU - Saxena, Krishna AU - Buhr, Florian AU - Langer, Julian D. AU - Blackledge, Martin AU - Frangakis, Achilleas S. AU - Glaubitz, Clemens AU - Schwalbe, Harald ID - 8744 JF - Nature Communications KW - General Biochemistry KW - Genetics and Molecular Biology KW - General Physics and Astronomy KW - General Chemistry SN - 2041-1723 TI - Cysteine oxidation and disulfide formation in the ribosomal exit tunnel VL - 11 ER - TY - JOUR AB - The actin-related protein (Arp)2/3 complex nucleates branched actin filament networks pivotal for cell migration, endocytosis and pathogen infection. Its activation is tightly regulated and involves complex structural rearrangements and actin filament binding, which are yet to be understood. Here, we report a 9.0 Å resolution structure of the actin filament Arp2/3 complex branch junction in cells using cryo-electron tomography and subtomogram averaging. This allows us to generate an accurate model of the active Arp2/3 complex in the branch junction and its interaction with actin filaments. Notably, our model reveals a previously undescribed set of interactions of the Arp2/3 complex with the mother filament, significantly different to the previous branch junction model. Our structure also indicates a central role for the ArpC3 subunit in stabilizing the active conformation. AU - Fäßler, Florian AU - Dimchev, Georgi A AU - Hodirnau, Victor-Valentin AU - Wan, William AU - Schur, Florian KM ID - 8971 JF - Nature Communications KW - General Biochemistry KW - Genetics and Molecular Biology KW - General Physics and Astronomy KW - General Chemistry SN - 2041-1723 TI - Cryo-electron tomography structure of Arp2/3 complex in cells reveals new insights into the branch junction VL - 11 ER - TY - JOUR AB - Mammalian circadian rhythms are generated by a transcription-based feedback loop in which CLOCK:BMAL1 drives transcription of its repressors (PER1/2, CRY1/2), which ultimately interact with CLOCK:BMAL1 to close the feedback loop with ~24 hr periodicity. Here we pinpoint a key difference between CRY1 and CRY2 that underlies their differential strengths as transcriptional repressors. Both cryptochromes bind the BMAL1 transactivation domain similarly to sequester it from coactivators and repress CLOCK:BMAL1 activity. However, we find that CRY1 is recruited with much higher affinity to the PAS domain core of CLOCK:BMAL1, allowing it to serve as a stronger repressor that lengthens circadian period. We discovered a dynamic serine-rich loop adjacent to the secondary pocket in the photolyase homology region (PHR) domain that regulates differential binding of cryptochromes to the PAS domain core of CLOCK:BMAL1. Notably, binding of the co-repressor PER2 remodels the serine loop of CRY2, making it more CRY1-like and enhancing its affinity for CLOCK:BMAL1. AU - Fribourgh, Jennifer L AU - Srivastava, Ashutosh AU - Sandate, Colby R AU - Michael, Alicia Kathleen AU - Hsu, Peter L AU - Rakers, Christin AU - Nguyen, Leslee T AU - Torgrimson, Megan R AU - Parico, Gian Carlo G AU - Tripathi, Sarvind AU - Zheng, Ning AU - Lander, Gabriel C AU - Hirota, Tsuyoshi AU - Tama, Florence AU - Partch, Carrie L ID - 15153 JF - eLife KW - General Immunology and Microbiology KW - General Biochemistry KW - Genetics and Molecular Biology KW - General Medicine KW - General Neuroscience SN - 2050-084X TI - Dynamics at the serine loop underlie differential affinity of cryptochromes for CLOCK:BMAL1 to control circadian timing VL - 9 ER - TY - JOUR AB - Atomic-resolution structure determination is crucial for understanding protein function. Cryo-EM and NMR spectroscopy both provide structural information, but currently cryo-EM does not routinely give access to atomic-level structural data, and, generally, NMR structure determination is restricted to small (<30 kDa) proteins. We introduce an integrated structure determination approach that simultaneously uses NMR and EM data to overcome the limits of each of these methods. The approach enables structure determination of the 468 kDa large dodecameric aminopeptidase TET2 to a precision and accuracy below 1 Å by combining secondary-structure information obtained from near-complete magic-angle-spinning NMR assignments of the 39 kDa-large subunits, distance restraints from backbone amides and ILV methyl groups, and a 4.1 Å resolution EM map. The resulting structure exceeds current standards of NMR and EM structure determination in terms of molecular weight and precision. Importantly, the approach is successful even in cases where only medium-resolution cryo-EM data are available. AU - Gauto, Diego F. AU - Estrozi, Leandro F. AU - Schwieters, Charles D. AU - Effantin, Gregory AU - Macek, Pavel AU - Sounier, Remy AU - Sivertsen, Astrid C. AU - Schmidt, Elena AU - Kerfah, Rime AU - Mas, Guillaume AU - Colletier, Jacques-Philippe AU - Güntert, Peter AU - Favier, Adrien AU - Schoehn, Guy AU - Schanda, Paul AU - Boisbouvier, Jerome ID - 8405 JF - Nature Communications KW - General Biochemistry KW - Genetics and Molecular Biology KW - General Physics and Astronomy KW - General Chemistry SN - 2041-1723 TI - Integrated NMR and cryo-EM atomic-resolution structure determination of a half-megadalton enzyme complex VL - 10 ER - TY - JOUR AB - Molecular motors are essential to the living, generating fluctuations that boost transport and assist assembly. Active colloids, that consume energy to move, hold similar potential for man-made materials controlled by forces generated from within. Yet, their use as a powerhouse in materials science lacks. Here we show a massive acceleration of the annealing of a monolayer of passive beads by moderate addition of self-propelled microparticles. We rationalize our observations with a model of collisions that drive active fluctuations and activate the annealing. The experiment is quantitatively compared with Brownian dynamic simulations that further unveil a dynamical transition in the mechanism of annealing. Active dopants travel uniformly in the system or co-localize at the grain boundaries as a result of the persistence of their motion. Our findings uncover the potential of internal activity to control materials and lay the groundwork for the rise of materials science beyond equilibrium. AU - Ramananarivo, Sophie AU - Ducrot, Etienne AU - Palacci, Jérémie A ID - 9060 IS - 1 JF - Nature Communications KW - General Biochemistry KW - Genetics and Molecular Biology KW - General Physics and Astronomy KW - General Chemistry SN - 2041-1723 TI - Activity-controlled annealing of colloidal monolayers VL - 10 ER - TY - JOUR AB - Transposable elements (TEs), the movement of which can damage the genome, are epigenetically silenced in eukaryotes. Intriguingly, TEs are activated in the sperm companion cell – vegetative cell (VC) – of the flowering plant Arabidopsis thaliana. However, the extent and mechanism of this activation are unknown. Here we show that about 100 heterochromatic TEs are activated in VCs, mostly by DEMETER-catalyzed DNA demethylation. We further demonstrate that DEMETER access to some of these TEs is permitted by the natural depletion of linker histone H1 in VCs. Ectopically expressed H1 suppresses TEs in VCs by reducing DNA demethylation and via a methylation-independent mechanism. We demonstrate that H1 is required for heterochromatin condensation in plant cells and show that H1 overexpression creates heterochromatic foci in the VC progenitor cell. Taken together, our results demonstrate that the natural depletion of H1 during male gametogenesis facilitates DEMETER-directed DNA demethylation, heterochromatin relaxation, and TE activation. AU - He, Shengbo AU - Vickers, Martin AU - Zhang, Jingyi AU - Feng, Xiaoqi ID - 12192 JF - eLife KW - General Immunology and Microbiology KW - General Biochemistry KW - Genetics and Molecular Biology KW - General Medicine KW - General Neuroscience SN - 2050-084X TI - Natural depletion of histone H1 in sex cells causes DNA demethylation, heterochromatin decondensation and transposon activation VL - 8 ER - TY - JOUR AB - Meiotic crossover frequency varies within genomes, which influences genetic diversity and adaptation. In turn, genetic variation within populations can act to modify crossover frequency in cis and trans. To identify genetic variation that controls meiotic crossover frequency, we screened Arabidopsis accessions using fluorescent recombination reporters. We mapped a genetic modifier of crossover frequency in Col × Bur populations of Arabidopsis to a premature stop codon within TBP-ASSOCIATED FACTOR 4b (TAF4b), which encodes a subunit of the RNA polymerase II general transcription factor TFIID. The Arabidopsis taf4b mutation is a rare variant found in the British Isles, originating in South-West Ireland. Using genetics, genomics, and immunocytology, we demonstrate a genome-wide decrease in taf4b crossovers, with strongest reduction in the sub-telomeric regions. Using RNA sequencing (RNA-seq) from purified meiocytes, we show that TAF4b expression is meiocyte enriched, whereas its paralog TAF4 is broadly expressed. Consistent with the role of TFIID in promoting gene expression, RNA-seq of wild-type and taf4b meiocytes identified widespread transcriptional changes, including in genes that regulate the meiotic cell cycle and recombination. Therefore, TAF4b duplication is associated with acquisition of meiocyte-specific expression and promotion of germline transcription, which act directly or indirectly to elevate crossovers. This identifies a novel mode of meiotic recombination control via a general transcription factor. AU - Lawrence, Emma J. AU - Gao, Hongbo AU - Tock, Andrew J. AU - Lambing, Christophe AU - Blackwell, Alexander R. AU - Feng, Xiaoqi AU - Henderson, Ian R. ID - 12190 IS - 16 JF - Current Biology KW - General Agricultural and Biological Sciences KW - General Biochemistry KW - Genetics and Molecular Biology SN - 0960-9822 TI - Natural variation in TBP-ASSOCIATED FACTOR 4b controls meiotic crossover and germline transcription in Arabidopsis VL - 29 ER - TY - JOUR AB - The inner nuclear membrane (INM) is a subdomain of the endoplasmic reticulum (ER) that is gated by the nuclear pore complex. It is unknown whether proteins of the INM and ER are degraded through shared or distinct pathways in mammalian cells. We applied dynamic proteomics to profile protein half-lives and report that INM and ER residents turn over at similar rates, indicating that the INM’s unique topology is not a barrier to turnover. Using a microscopy approach, we observed that the proteasome can degrade INM proteins in situ. However, we also uncovered evidence for selective, vesicular transport-mediated turnover of a single INM protein, emerin, that is potentiated by ER stress. Emerin is rapidly cleared from the INM by a mechanism that requires emerin’s LEM domain to mediate vesicular trafficking to lysosomes. This work demonstrates that the INM can be dynamically remodeled in response to environmental inputs. AU - Buchwalter, Abigail AU - Schulte, Roberta AU - Tsai, Hsiao AU - Capitanio, Juliana AU - HETZER, Martin W ID - 11060 JF - eLife KW - General Immunology and Microbiology KW - General Biochemistry KW - Genetics and Molecular Biology KW - General Medicine KW - General Neuroscience SN - 2050-084X TI - Selective clearance of the inner nuclear membrane protein emerin by vesicular transport during ER stress VL - 8 ER - TY - JOUR AB - The exchange of metabolites between the mitochondrial matrix and the cytosol depends on β-barrel channels in the outer membrane and α-helical carrier proteins in the inner membrane. The essential translocase of the inner membrane (TIM) chaperones escort these proteins through the intermembrane space, but the structural and mechanistic details remain elusive. We have used an integrated structural biology approach to reveal the functional principle of TIM chaperones. Multiple clamp-like binding sites hold the mitochondrial membrane proteins in a translocation-competent elongated form, thus mimicking characteristics of co-translational membrane insertion. The bound preprotein undergoes conformational dynamics within the chaperone binding clefts, pointing to a multitude of dynamic local binding events. Mutations in these binding sites cause cell death or growth defects associated with impairment of carrier and β-barrel protein biogenesis. Our work reveals how a single mitochondrial “transfer-chaperone” system is able to guide α-helical and β-barrel membrane proteins in a “nascent chain-like” conformation through a ribosome-free compartment. AU - Weinhäupl, Katharina AU - Lindau, Caroline AU - Hessel, Audrey AU - Wang, Yong AU - Schütze, Conny AU - Jores, Tobias AU - Melchionda, Laura AU - Schönfisch, Birgit AU - Kalbacher, Hubert AU - Bersch, Beate AU - Rapaport, Doron AU - Brennich, Martha AU - Lindorff-Larsen, Kresten AU - Wiedemann, Nils AU - Schanda, Paul ID - 8436 IS - 5 JF - Cell KW - General Biochemistry KW - Genetics and Molecular Biology SN - 0092-8674 TI - Structural basis of membrane protein chaperoning through the mitochondrial intermembrane space VL - 175 ER - TY - JOUR AB - Confining molecules to volumes only slightly larger than the molecules themselves can profoundly alter their properties. Molecular switches—entities that can be toggled between two or more forms upon exposure to an external stimulus—often require conformational freedom to isomerize. Therefore, placing these switches in confined spaces can render them non-operational. To preserve the switchability of these species under confinement, we work with a water-soluble coordination cage that is flexible enough to adapt its shape to the conformation of the encapsulated guest. We show that owing to its flexibility, the cage is not only capable of accommodating—and solubilizing in water—several light-responsive spiropyran-based molecular switches, but, more importantly, it also provides an environment suitable for the efficient, reversible photoisomerization of the bound guests. Our findings pave the way towards studying various molecular switching processes in confined environments. AU - Samanta, Dipak AU - Galaktionova, Daria AU - Gemen, Julius AU - Shimon, Linda J. W. AU - Diskin-Posner, Yael AU - Avram, Liat AU - Král, Petr AU - Klajn, Rafal ID - 13374 JF - Nature Communications KW - General Physics and Astronomy KW - General Biochemistry KW - Genetics and Molecular Biology KW - General Chemistry KW - Multidisciplinary TI - Reversible chromism of spiropyran in the cavity of a flexible coordination cage VL - 9 ER - TY - JOUR AB - Pore-forming toxins (PFT) are virulence factors that transform from soluble to membrane-bound states. The Yersinia YaxAB system represents a family of binary α-PFTs with orthologues in human, insect, and plant pathogens, with unknown structures. YaxAB was shown to be cytotoxic and likely involved in pathogenesis, though the molecular basis for its two-component lytic mechanism remains elusive. Here, we present crystal structures of YaxA and YaxB, together with a cryo-electron microscopy map of the YaxAB complex. Our structures reveal a pore predominantly composed of decamers of YaxA–YaxB heterodimers. Both subunits bear membrane-active moieties, but only YaxA is capable of binding to membranes by itself. YaxB can subsequently be recruited to membrane-associated YaxA and induced to present its lytic transmembrane helices. Pore formation can progress by further oligomerization of YaxA–YaxB dimers. Our results allow for a comparison between pore assemblies belonging to the wider ClyA-like family of α-PFTs, highlighting diverse pore architectures. AU - Bräuning, Bastian AU - Bertosin, Eva AU - Praetorius, Florian M AU - Ihling, Christian AU - Schatt, Alexandra AU - Adler, Agnes AU - Richter, Klaus AU - Sinz, Andrea AU - Dietz, Hendrik AU - Groll, Michael ID - 14284 JF - Nature Communications KW - General Physics and Astronomy KW - General Biochemistry KW - Genetics and Molecular Biology KW - General Chemistry KW - Multidisciplinary SN - 2041-1723 TI - Structure and mechanism of the two-component α-helical pore-forming toxin YaxAB VL - 9 ER - TY - JOUR AB - Eukaryotic cells are densely packed with macromolecular complexes and intertwining organelles, continually transported and reshaped. Intriguingly, organelles avoid clashing and entangling with each other in such limited space. Mitochondria form extensive networks constantly remodeled by fission and fusion. Here, we show that mitochondrial fission is triggered by mechanical forces. Mechano-stimulation of mitochondria – via encounter with motile intracellular pathogens, via external pressure applied by an atomic force microscope, or via cell migration across uneven microsurfaces – results in the recruitment of the mitochondrial fission machinery, and subsequent division. We propose that MFF, owing to affinity for narrow mitochondria, acts as a membrane-bound force sensor to recruit the fission machinery to mechanically strained sites. Thus, mitochondria adapt to the environment by sensing and responding to biomechanical cues. Our findings that mechanical triggers can be coupled to biochemical responses in membrane dynamics may explain how organelles orderly cohabit in the crowded cytoplasm. AU - Helle, Sebastian Carsten Johannes AU - Feng, Qian AU - Aebersold, Mathias J AU - Hirt, Luca AU - Grüter, Raphael R AU - Vahid, Afshin AU - Sirianni, Andrea AU - Mostowy, Serge AU - Snedeker, Jess G AU - Šarić, Anđela AU - Idema, Timon AU - Zambelli, Tomaso AU - Kornmann, Benoît ID - 10370 JF - eLife KW - general immunology and microbiology KW - general biochemistry KW - genetics and molecular biology KW - general medicine KW - general neuroscience SN - 2050-084X TI - Mechanical force induces mitochondrial fission VL - 6 ER - TY - JOUR AB - Premature aging disorders provide an opportunity to study the mechanisms that drive aging. In Hutchinson-Gilford progeria syndrome (HGPS), a mutant form of the nuclear scaffold protein lamin A distorts nuclei and sequesters nuclear proteins. We sought to investigate protein homeostasis in this disease. Here, we report a widespread increase in protein turnover in HGPS-derived cells compared to normal cells. We determine that global protein synthesis is elevated as a consequence of activated nucleoli and enhanced ribosome biogenesis in HGPS-derived fibroblasts. Depleting normal lamin A or inducing mutant lamin A expression are each sufficient to drive nucleolar expansion. We further show that nucleolar size correlates with donor age in primary fibroblasts derived from healthy individuals and that ribosomal RNA production increases with age, indicating that nucleolar size and activity can serve as aging biomarkers. While limiting ribosome biogenesis extends lifespan in several systems, we show that increased ribosome biogenesis and activity are a hallmark of premature aging. AU - Buchwalter, Abigail AU - HETZER, Martin W ID - 11065 JF - Nature Communications KW - General Physics and Astronomy KW - General Biochemistry KW - Genetics and Molecular Biology KW - General Chemistry SN - 2041-1723 TI - Nucleolar expansion and elevated protein translation in premature aging VL - 8 ER - TY - JOUR AB - Strong-field photoelectron holography and laser-induced electron diffraction (LIED) are two powerful emerging methods for probing the ultrafast dynamics of molecules. However, both of them have remained restricted to static systems and to nuclear dynamics induced by strong-field ionization. Here we extend these promising methods to image purely electronic valence-shell dynamics in molecules using photoelectron holography. In the same experiment, we use LIED and photoelectron holography simultaneously, to observe coupled electronic-rotational dynamics taking place on similar timescales. These results offer perspectives for imaging ultrafast dynamics of molecules on femtosecond to attosecond timescales. AU - Walt, Samuel G. AU - Bhargava Ram, Niraghatam AU - Atala, Marcos AU - Shvetsov-Shilovski, Nikolay I AU - von Conta, Aaron AU - Baykusheva, Denitsa Rangelova AU - Lein, Manfred AU - Wörner, Hans Jakob ID - 14005 JF - Nature Communications KW - General Physics and Astronomy KW - General Biochemistry KW - Genetics and Molecular Biology KW - General Chemistry KW - Multidisciplinary TI - Dynamics of valence-shell electrons and nuclei probed by strong-field holography and rescattering VL - 8 ER - TY - JOUR AB - Biofilm formation is critical for the infection cycle of Vibrio cholerae. Vibrio exopolysaccharides (VPS) and the matrix proteins RbmA, Bap1 and RbmC are required for the development of biofilm architecture. We demonstrate that RbmA binds VPS directly and uses a binary structural switch within its first fibronectin type III (FnIII-1) domain to control RbmA structural dynamics and the formation of VPS-dependent higher-order structures. The structural switch in FnIII-1 regulates interactions in trans with the FnIII-2 domain, leading to open (monomeric) or closed (dimeric) interfaces. The ability of RbmA to switch between open and closed states is important for V. cholerae biofilm formation, as RbmA variants with switches that are locked in either of the two states lead to biofilms with altered architecture and structural integrity. AU - Fong, Jiunn CN AU - Rogers, Andrew AU - Michael, Alicia Kathleen AU - Parsley, Nicole C AU - Cornell, William-Cole AU - Lin, Yu-Cheng AU - Singh, Praveen K AU - Hartmann, Raimo AU - Drescher, Knut AU - Vinogradov, Evgeny AU - Dietrich, Lars EP AU - Partch, Carrie L AU - Yildiz, Fitnat H ID - 15154 JF - eLife KW - General Immunology and Microbiology KW - General Biochemistry KW - Genetics and Molecular Biology KW - General Medicine KW - General Neuroscience SN - 2050-084X TI - Structural dynamics of RbmA governs plasticity of Vibrio cholerae biofilms VL - 6 ER - TY - JOUR AB - Spatiotemporal activation of RhoA and actomyosin contraction underpins cellular adhesion and division. Loss of cell–cell adhesion and chromosomal instability are cardinal events that drive tumour progression. Here, we show that p120-catenin (p120) not only controls cell–cell adhesion, but also acts as a critical regulator of cytokinesis. We find that p120 regulates actomyosin contractility through concomitant binding to RhoA and the centralspindlin component MKLP1, independent of cadherin association. In anaphase, p120 is enriched at the cleavage furrow where it binds MKLP1 to spatially control RhoA GTPase cycling. Binding of p120 to MKLP1 during cytokinesis depends on the N-terminal coiled-coil domain of p120 isoform 1A. Importantly, clinical data show that loss of p120 expression is a common event in breast cancer that strongly correlates with multinucleation and adverse patient survival. In summary, our study identifies p120 loss as a driver event of chromosomal instability in cancer. AU - van de Ven, Robert A.H. AU - de Groot, Jolien S. AU - Park, Danielle AU - van Domselaar, Robert AU - de Jong, Danielle AU - Szuhai, Karoly AU - van der Wall, Elsken AU - Rueda, Oscar M. AU - Ali, H. Raza AU - Caldas, Carlos AU - van Diest, Paul J. AU - HETZER, Martin W AU - Sahai, Erik AU - Derksen, Patrick W.B. ID - 11072 JF - Nature Communications KW - General Physics and Astronomy KW - General Biochemistry KW - Genetics and Molecular Biology KW - General Chemistry SN - 2041-1723 TI - p120-catenin prevents multinucleation through control of MKLP1-dependent RhoA activity during cytokinesis VL - 7 ER - TY - JOUR AB - Human cancer cells bear complex chromosome rearrangements that can be potential drivers of cancer development. However, the molecular mechanisms underlying these rearrangements have been unclear. Zhang et al. use a new technique combining live-cell imaging and single-cell sequencing to demonstrate that chromosomes mis-segregated to micronuclei frequently undergo chromothripsis-like rearrangements in the subsequent cell cycle. AU - Hatch, Emily M. AU - HETZER, Martin W ID - 11073 IS - 7 JF - Cell KW - General Biochemistry KW - Genetics and Molecular Biology SN - 0092-8674 TI - Linking micronuclei to chromosome fragmentation VL - 161 ER - TY - JOUR AU - Hatch, Emily M. AU - HETZER, Martin W ID - 11074 IS - 10 JF - Current Biology KW - General Agricultural and Biological Sciences KW - General Biochemistry KW - Genetics and Molecular Biology SN - 0960-9822 TI - Chromothripsis VL - 25 ER - TY - JOUR AB - The large majority of three-dimensional structures of biological macromolecules have been determined by X-ray diffraction of crystalline samples. High-resolution structure determination crucially depends on the homogeneity of the protein crystal. Overall ‘rocking’ motion of molecules in the crystal is expected to influence diffraction quality, and such motion may therefore affect the process of solving crystal structures. Yet, so far overall molecular motion has not directly been observed in protein crystals, and the timescale of such dynamics remains unclear. Here we use solid-state NMR, X-ray diffraction methods and μs-long molecular dynamics simulations to directly characterize the rigid-body motion of a protein in different crystal forms. For ubiquitin crystals investigated in this study we determine the range of possible correlation times of rocking motion, 0.1–100 μs. The amplitude of rocking varies from one crystal form to another and is correlated with the resolution obtainable in X-ray diffraction experiments. AU - Ma, Peixiang AU - Xue, Yi AU - Coquelle, Nicolas AU - Haller, Jens D. AU - Yuwen, Tairan AU - Ayala, Isabel AU - Mikhailovskii, Oleg AU - Willbold, Dieter AU - Colletier, Jacques-Philippe AU - Skrynnikov, Nikolai R. AU - Schanda, Paul ID - 8456 JF - Nature Communications KW - General Biochemistry KW - Genetics and Molecular Biology KW - General Physics and Astronomy KW - General Chemistry SN - 2041-1723 TI - Observing the overall rocking motion of a protein in a crystal VL - 6 ER - TY - JOUR AB - All attosecond time-resolved measurements have so far relied on the use of intense near-infrared laser pulses. In particular, attosecond streaking, laser-induced electron diffraction and high-harmonic generation all make use of non-perturbative light–matter interactions. Remarkably, the effect of the strong laser field on the studied sample has often been neglected in previous studies. Here we use high-harmonic spectroscopy to measure laser-induced modifications of the electronic structure of molecules. We study high-harmonic spectra of spatially oriented CH3F and CH3Br as generic examples of polar polyatomic molecules. We accurately measure intensity ratios of even and odd-harmonic orders, and of the emission from aligned and unaligned molecules. We show that these robust observables reveal a substantial modification of the molecular electronic structure by the external laser field. Our insights offer new challenges and opportunities for a range of emerging strong-field attosecond spectroscopies. AU - Kraus, P. M. AU - Tolstikhin, O. I. AU - Baykusheva, Denitsa Rangelova AU - Rupenyan, A. AU - Schneider, J. AU - Bisgaard, C. Z. AU - Morishita, T. AU - Jensen, F. AU - Madsen, L. B. AU - Wörner, H. J. ID - 14016 JF - Nature Communications KW - General Physics and Astronomy KW - General Biochemistry KW - Genetics and Molecular Biology KW - General Chemistry KW - Multidisciplinary TI - Observation of laser-induced electronic structure in oriented polyatomic molecules VL - 6 ER - TY - JOUR AB - The spindle assembly checkpoint prevents separation of sister chromatids until each kinetochore is attached to the mitotic spindle. Rodriguez-Bravo et al. report that the nuclear pore complex scaffolds spindle assembly checkpoint signaling in interphase, providing a store of inhibitory signals that limits the speed of the subsequent mitosis. AU - Buchwalter, Abigail AU - HETZER, Martin W ID - 11080 IS - 5 JF - Cell KW - General Biochemistry KW - Genetics and Molecular Biology SN - 0092-8674 TI - Nuclear pores set the speed limit for mitosis VL - 156 ER - TY - JOUR AB - Nanoporous frameworks are polymeric materials built from rigid molecules, which give rise to their nanoporous structures with applications in gas sorption and storage, catalysis and others. Conceptually new applications could emerge, should these beneficial properties be manipulated by external stimuli in a reversible manner. One approach to render nanoporous frameworks responsive to external signals would be to immobilize molecular switches within their nanopores. Although the majority of molecular switches require conformational freedom to isomerize, and switching in the solid state is prohibited, the nanopores may provide enough room for the switches to efficiently isomerize. Here we describe two families of nanoporous materials incorporating the spiropyran molecular switch. These materials exhibit a variety of interesting properties, including reversible photochromism and acidochromism under solvent-free conditions, light-controlled capture and release of metal ions, as well reversible chromism induced by solvation/desolvation. AU - Kundu, Pintu K. AU - Olsen, Gregory L. AU - Kiss, Vladimir AU - Klajn, Rafal ID - 13402 JF - Nature Communications KW - General Physics and Astronomy KW - General Biochemistry KW - Genetics and Molecular Biology KW - General Chemistry KW - Multidisciplinary TI - Nanoporous frameworks exhibiting multiple stimuli responsiveness VL - 5 ER - TY - JOUR AB - Intracellular proteins with long lifespans have recently been linked to age-dependent defects, ranging from decreased fertility to the functional decline of neurons. Why long-lived proteins exist in metabolically active cellular environments and how they are maintained over time remains poorly understood. Here, we provide a system-wide identification of proteins with exceptional lifespans in the rat brain. These proteins are inefficiently replenished despite being translated robustly throughout adulthood. Using nucleoporins as a paradigm for long-term protein persistence, we found that nuclear pore complexes (NPCs) are maintained over a cell’s life through slow but finite exchange of even its most stable subcomplexes. This maintenance is limited, however, as some nucleoporin levels decrease during aging, providing a rationale for the previously observed age-dependent deterioration of NPC function. Our identification of a long-lived proteome reveals cellular components that are at increased risk for damage accumulation, linking long-term protein persistence to the cellular aging process. AU - Toyama, Brandon H. AU - Savas, Jeffrey N. AU - Park, Sung Kyu AU - Harris, Michael S. AU - Ingolia, Nicholas T. AU - Yates, John R. AU - HETZER, Martin W ID - 11087 IS - 5 JF - Cell KW - General Biochemistry KW - Genetics and Molecular Biology SN - 0092-8674 TI - Identification of long-lived proteins reveals exceptional stability of essential cellular structures VL - 154 ER - TY - JOUR AB - During mitotic exit, missegregated chromosomes can recruit their own nuclear envelope (NE) to form micronuclei (MN). MN have reduced functioning compared to primary nuclei in the same cell, although the two compartments appear to be structurally comparable. Here we show that over 60% of MN undergo an irreversible loss of compartmentalization during interphase due to NE collapse. This disruption of the MN, which is induced by defects in nuclear lamina assembly, drastically reduces nuclear functions and can trigger massive DNA damage. MN disruption is associated with chromatin compaction and invasion of endoplasmic reticulum (ER) tubules into the chromatin. We identified disrupted MN in both major subtypes of human non-small-cell lung cancer, suggesting that disrupted MN could be a useful objective biomarker for genomic instability in solid tumors. Our study shows that NE collapse is a key event underlying MN dysfunction and establishes a link between aberrant NE organization and aneuploidy. AU - Hatch, Emily M. AU - Fischer, Andrew H. AU - Deerinck, Thomas J. AU - HETZER, Martin W ID - 11085 IS - 1 JF - Cell KW - General Biochemistry KW - Genetics and Molecular Biology SN - 0092-8674 TI - Catastrophic nuclear envelope collapse in cancer cell micronuclei VL - 154 ER - TY - JOUR AB - Nuclear pore complexes (NPCs) are built from ∼30 different proteins called nucleoporins or Nups. Previous studies have shown that several Nups exhibit cell-type-specific expression and that mutations in NPC components result in tissue-specific diseases. Here we show that a specific change in NPC composition is required for both myogenic and neuronal differentiation. The transmembrane nucleoporin Nup210 is absent in proliferating myoblasts and embryonic stem cells (ESCs) but becomes expressed and incorporated into NPCs during cell differentiation. Preventing Nup210 production by RNAi blocks myogenesis and the differentiation of ESCs into neuroprogenitors. We found that the addition of Nup210 to NPCs does not affect nuclear transport but is required for the induction of genes that are essential for cell differentiation. Our results identify a single change in NPC composition as an essential step in cell differentiation and establish a role for Nup210 in gene expression regulation and cell fate determination. AU - D'Angelo, Maximiliano A. AU - Gomez-Cavazos, J. Sebastian AU - Mei, Arianna AU - Lackner, Daniel H. AU - HETZER, Martin W ID - 11093 IS - 2 JF - Developmental Cell KW - Developmental Biology KW - Cell Biology KW - General Biochemistry KW - Genetics and Molecular Biology KW - Molecular Biology SN - 1534-5807 TI - A change in nuclear pore complex composition regulates cell differentiation VL - 22 ER - TY - JOUR AB - Nuclear export of mRNAs is thought to occur exclusively through nuclear pore complexes. In this issue of Cell, Speese et al. identify an alternate pathway for mRNA export in muscle cells where ribonucleoprotein complexes involved in forming neuromuscular junctions transit the nuclear envelope by fusing with and budding through the nuclear membrane. AU - Hatch, Emily M. AU - HETZER, Martin W ID - 11090 IS - 4 JF - Cell KW - General Biochemistry KW - Genetics and Molecular Biology SN - 0092-8674 TI - RNP export by nuclear envelope budding VL - 149 ER - TY - JOUR AB - The nuclear envelope (NE) is a highly regulated membrane barrier that separates the nucleus from the cytoplasm in eukaryotic cells. It contains a large number of different proteins that have been implicated in chromatin organization and gene regulation. Although the nuclear membrane enables complex levels of gene expression, it also poses a challenge when it comes to cell division. To allow access of the mitotic spindle to chromatin, the nucleus of metazoans must completely disassemble during mitosis, generating the need to re-establish the nuclear compartment at the end of each cell division. Here, I summarize our current understanding of the dynamic remodeling of the NE during the cell cycle. AU - HETZER, Martin W ID - 11097 IS - 3 JF - Cold Spring Harbor Perspectives in Biology KW - General Biochemistry KW - Genetics and Molecular Biology SN - 1943-0264 TI - The nuclear envelope VL - 2 ER - TY - JOUR AB - Nuclear pore complexes have recently been shown to play roles in gene activation; however their potential involvement in metazoan transcription remains unclear. Here we show that the nucleoporins Sec13, Nup98, and Nup88, as well as a group of FG-repeat nucleoporins, bind to the Drosophila genome at functionally distinct loci that often do not represent nuclear envelope contact sites. Whereas Nup88 localizes to silent loci, Sec13, Nup98, and a subset of FG-repeat nucleoporins bind to developmentally regulated genes undergoing transcription induction. Strikingly, RNAi-mediated knockdown of intranuclear Sec13 and Nup98 specifically inhibits transcription of their target genes and prevents efficient reactivation of transcription after heat shock, suggesting an essential role of NPC components in regulating complex gene expression programs of multicellular organisms. AU - Capelson, Maya AU - Liang, Yun AU - Schulte, Roberta AU - Mair, William AU - Wagner, Ulrich AU - HETZER, Martin W ID - 11102 IS - 3 JF - Cell KW - General Biochemistry KW - Genetics and Molecular Biology SN - 0092-8674 TI - Chromatin-bound nuclear pore components regulate gene expression in higher eukaryotes VL - 140 ER - TY - JOUR AB - In metazoa, nuclear pore complexes (NPCs) assemble from disassembled precursors into a reforming nuclear envelope (NE) at the end of mitosis and into growing intact NEs during interphase. Here, we show via RNAi-mediated knockdown that ELYS, a nucleoporin critical for the recruitment of the essential Nup107/160 complex to chromatin, is required for NPC assembly at the end of mitosis but not during interphase. Conversely, the transmembrane nucleoporin POM121 is critical for the incorporation of the Nup107/160 complex into new assembly sites specifically during interphase. Strikingly, recruitment of the Nup107/160 complex to an intact NE involves a membrane curvature-sensing domain of its constituent Nup133, which is not required for postmitotic NPC formation. Our results suggest that in organisms with open mitosis, NPCs assemble via two distinct mechanisms to accommodate cell cycle-dependent differences in NE topology. AU - Doucet, Christine M. AU - Talamas, Jessica A. AU - HETZER, Martin W ID - 11101 IS - 6 JF - Cell KW - General Biochemistry KW - Genetics and Molecular Biology SN - 0092-8674 TI - Cell cycle-dependent differences in nuclear pore complex assembly in metazoa VL - 141 ER - TY - JOUR AB - Over the last decade, the nuclear envelope (NE) has emerged as a key component in the organization and function of the nuclear genome. As many as 100 different proteins are thought to specifically localize to this double membrane that separates the cytoplasm and the nucleoplasm of eukaryotic cells. Selective portals through the NE are formed at sites where the inner and outer nuclear membranes are fused, and the coincident assembly of ∼30 proteins into nuclear pore complexes occurs. These nuclear pore complexes are essential for the control of nucleocytoplasmic exchange. Many of the NE and nuclear pore proteins are thought to play crucial roles in gene regulation and thus are increasingly linked to human diseases. AU - HETZER, Martin W AU - Wente, Susan R. ID - 11103 IS - 5 JF - Developmental Cell KW - Developmental Biology KW - Cell Biology KW - General Biochemistry KW - Genetics and Molecular Biology KW - Molecular Biology SN - 1534-5807 TI - Border control at the nucleus: Biogenesis and organization of the nuclear membrane and pore complexes VL - 17 ER - TY - JOUR AB - In dividing cells, nuclear pore complexes (NPCs) disassemble during mitosis and reassemble into the newly forming nuclei. However, the fate of nuclear pores in postmitotic cells is unknown. Here, we show that NPCs, unlike other nuclear structures, do not turn over in differentiated cells. While a subset of NPC components, like Nup153 and Nup50, are continuously exchanged, scaffold nucleoporins, like the Nup107/160 complex, are extremely long-lived and remain incorporated in the nuclear membrane during the entire cellular life span. Besides the lack of nucleoporin expression and NPC turnover, we discovered an age-related deterioration of NPCs, leading to an increase in nuclear permeability and the leaking of cytoplasmic proteins into the nucleus. Our finding that nuclear “leakiness” is dramatically accelerated during aging and that a subset of nucleoporins is oxidatively damaged in old cells suggests that the accumulation of damage at the NPC might be a crucial aging event. AU - D'Angelo, Maximiliano A. AU - Raices, Marcela AU - Panowski, Siler H. AU - HETZER, Martin W ID - 11108 IS - 2 JF - Cell KW - General Biochemistry KW - Genetics and Molecular Biology SN - 0092-8674 TI - Age-dependent deterioration of nuclear pore complexes causes a loss of nuclear integrity in postmitotic cells VL - 136 ER - TY - JOUR AB - Nuclear pore complexes (NPCs) are large multiprotein assemblies that allow traffic between the cytoplasm and the nucleus. During mitosis in higher eukaryotes, the Nuclear Envelope (NE) breaks down and NPCs disassemble. How NPCs reassemble and incorporate into the NE upon mitotic exit is poorly understood. We demonstrate a function for the conserved Nup107-160 complex in this process. Partial in vivo depletion of Nup133 or Nup107 via RNAi in HeLa cells resulted in reduced levels of multiple nucleoporins and decreased NPC density in the NE. Immunodepletion of the entire Nup107-160 complex from in vitro nuclear assembly reactions produced nuclei with a continuous NE but no NPCs. This phenotype was reversible only if Nup107-160 complex was readded before closed NE formation. Depletion also prevented association of FG-repeat nucleoporins with chromatin. We propose a stepwise model in which postmitotic NPC assembly initiates on chromatin via early recruitment of the Nup107-160 complex. AU - Walther, Tobias C. AU - Alves, Annabelle AU - Pickersgill, Helen AU - Loı̈odice, Isabelle AU - HETZER, Martin W AU - Galy, Vincent AU - Hülsmann, Bastian B. AU - Köcher, Thomas AU - Wilm, Matthias AU - Allen, Terry AU - Mattaj, Iain W. AU - Doye, Valérie ID - 11122 IS - 2 JF - Cell KW - General Biochemistry KW - Genetics and Molecular Biology SN - 0092-8674 TI - The conserved Nup107-160 complex is critical for nuclear pore complex assembly VL - 113 ER - TY - JOUR AB - Ran GTPase plays important roles in nucleocytoplasmic transport in interphase [1, 2] and in both spindle formation and nuclear envelope (NE) assembly during mitosis [3, 4, 5]. The latter functions rely on the presence of high local concentrations of GTP-bound Ran near mitotic chromatin [3, 4, 5]. RanGTP localization has been proposed to result from the association of Ran's GDP/GTP exchange factor, RCC1, with chromatin [6, 7, 8, 9], but Ran is shown here to bind directly to chromatin in two modes, either dependent or independent of RCC1, and, where bound, to increase the affinity of chromatin for NE membranes. We propose that the Ran binding capacity of chromatin contributes to localized spindle and NE assembly. AU - Bilbao-Cortés, Daniel AU - HETZER, Martin W AU - Längst, Gernot AU - Becker, Peter B. AU - Mattaj, Iain W. ID - 11124 IS - 13 JF - Current Biology KW - General Agricultural and Biological Sciences KW - General Biochemistry KW - Genetics and Molecular Biology SN - 0960-9822 TI - Ran binds to chromatin by two distinct mechanisms VL - 12 ER -