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 - While high risk of failure is an inherent part of developing innovative therapies, it can be reduced by adherence to evidence-based rigorous research practices. Numerous analyses conducted to date have clearly identified measures that need to be taken to improve research rigor. Supported through the European Union's Innovative Medicines Initiative, the EQIPD consortium has developed a novel preclinical research quality system that can be applied in both public and private sectors and is free for anyone to use. The EQIPD Quality System was designed to be suited to boost innovation by ensuring the generation of robust and reliable preclinical data while being lean, effective and not becoming a burden that could negatively impact the freedom to explore scientific questions. EQIPD defines research quality as the extent to which research data are fit for their intended use. Fitness, in this context, is defined by the stakeholders, who are the scientists directly involved in the research, but also their funders, sponsors, publishers, research tool manufacturers and collaboration partners such as peers in a multi-site research project. The essence of the EQIPD Quality System is the set of 18 core requirements that can be addressed flexibly, according to user-specific needs and following a user-defined trajectory. The EQIPD Quality System proposes guidance on expectations for quality-related measures, defines criteria for adequate processes (i.e., performance standards) and provides examples of how such measures can be developed and implemented. However, it does not prescribe any pre-determined solutions. EQIPD has also developed tools (for optional use) to support users in implementing the system and assessment services for those research units that successfully implement the quality system and seek formal accreditation. Building upon the feedback from users and continuous improvement, a sustainable EQIPD Quality System will ultimately serve the entire community of scientists conducting non-regulated preclinical research, by helping them generate reliable data that are fit for their intended use. AU - Bespalov, Anton AU - Bernard, René AU - Gilis, Anja AU - Gerlach, Björn AU - Guillén, Javier AU - Castagné, Vincent AU - Lefevre, Isabel A. AU - Ducrey, Fiona AU - Monk, Lee AU - Bongiovanni, Sandrine AU - Altevogt, Bruce AU - Arroyo-Araujo, María AU - Bikovski, Lior AU - De Bruin, Natasja AU - Castaños-Vélez, Esmeralda AU - Dityatev, Alexander AU - Emmerich, Christoph H. AU - Fares, Raafat AU - Ferland-Beckham, Chantelle AU - Froger-Colléaux, Christelle AU - Gailus-Durner, Valerie AU - Hölter, Sabine M. AU - Hofmann, Martine Cj AU - Kabitzke, Patricia AU - Kas, Martien Jh AU - Kurreck, Claudia AU - Moser, Paul AU - Pietraszek, Malgorzata AU - Popik, Piotr AU - Potschka, Heidrun AU - Prado Montes De Oca, Ernesto AU - Restivo, Leonardo AU - Riedel, Gernot AU - Ritskes-Hoitinga, Merel AU - Samardzic, Janko AU - Schunn, Michael AU - Stöger, Claudia AU - Voikar, Vootele AU - Vollert, Jan AU - Wever, Kimberley E. AU - Wuyts, Kathleen AU - Macleod, Malcolm R. AU - Dirnagl, Ulrich AU - Steckler, Thomas ID - 9607 JF - eLife TI - Introduction to the EQIPD quality system VL - 10 ER - TY - JOUR AB - Mosaic analysis with double markers (MADM) offers one approach to visualize and concomitantly manipulate genetically defined cells in mice with single-cell resolution. MADM applications include the analysis of lineage, single-cell morphology and physiology, genomic imprinting phenotypes, and dissection of cell-autonomous gene functions in vivo in health and disease. Yet, MADM can only be applied to <25% of all mouse genes on select chromosomes to date. To overcome this limitation, we generate transgenic mice with knocked-in MADM cassettes near the centromeres of all 19 autosomes and validate their use across organs. With this resource, >96% of the entire mouse genome can now be subjected to single-cell genetic mosaic analysis. Beyond a proof of principle, we apply our MADM library to systematically trace sister chromatid segregation in distinct mitotic cell lineages. We find striking chromosome-specific biases in segregation patterns, reflecting a putative mechanism for the asymmetric segregation of genetic determinants in somatic stem cell division. AU - Contreras, Ximena AU - Amberg, Nicole AU - Davaatseren, Amarbayasgalan AU - Hansen, Andi H AU - Sonntag, Johanna AU - Andersen, Lill AU - Bernthaler, Tina AU - Streicher, Carmen AU - Heger, Anna-Magdalena AU - Johnson, Randy L. AU - Schwarz, Lindsay A. AU - Luo, Liqun AU - Rülicke, Thomas AU - Hippenmeyer, Simon ID - 9603 IS - 12 JF - Cell Reports TI - A genome-wide library of MADM mice for single-cell genetic mosaic analysis VL - 35 ER - TY - JOUR AB - Attachment of adhesive molecules on cell culture surfaces to restrict cell adhesion to defined areas and shapes has been vital for the progress of in vitro research. In currently existing patterning methods, a combination of pattern properties such as stability, precision, specificity, high-throughput outcome, and spatiotemporal control is highly desirable but challenging to achieve. Here, we introduce a versatile and high-throughput covalent photoimmobilization technique, comprising a light-dose-dependent patterning step and a subsequent functionalization of the pattern via click chemistry. This two-step process is feasible on arbitrary surfaces and allows for generation of sustainable patterns and gradients. The method is validated in different biological systems by patterning adhesive ligands on cell-repellent surfaces, thereby constraining the growth and migration of cells to the designated areas. We then implement a sequential photopatterning approach by adding a second switchable patterning step, allowing for spatiotemporal control over two distinct surface patterns. As a proof of concept, we reconstruct the dynamics of the tip/stalk cell switch during angiogenesis. Our results show that the spatiotemporal control provided by our “sequential photopatterning” system is essential for mimicking dynamic biological processes and that our innovative approach has great potential for further applications in cell science. AU - Zisis, Themistoklis AU - Schwarz, Jan AU - Balles, Miriam AU - Kretschmer, Maibritt AU - Nemethova, Maria AU - Chait, Remy P AU - Hauschild, Robert AU - Lange, Janina AU - Guet, Calin C AU - Sixt, Michael K AU - Zahler, Stefan ID - 9822 IS - 30 JF - ACS Applied Materials and Interfaces SN - 19448244 TI - Sequential and switchable patterning for studying cellular processes under spatiotemporal control VL - 13 ER - TY - JOUR AB - A modern day light microscope has evolved from a tool devoted to making primarily empirical observations to what is now a sophisticated , quantitative device that is an integral part of both physical and life science research. Nowadays, microscopes are found in nearly every experimental laboratory. However, despite their prevalent use in capturing and quantifying scientific phenomena, neither a thorough understanding of the principles underlying quantitative imaging techniques nor appropriate knowledge of how to calibrate, operate and maintain microscopes can be taken for granted. This is clearly demonstrated by the well-documented and widespread difficulties that are routinely encountered in evaluating acquired data and reproducing scientific experiments. Indeed, studies have shown that more than 70% of researchers have tried and failed to repeat another scientist's experiments, while more than half have even failed to reproduce their own experiments. One factor behind the reproducibility crisis of experiments published in scientific journals is the frequent underreporting of imaging methods caused by a lack of awareness and/or a lack of knowledge of the applied technique. Whereas quality control procedures for some methods used in biomedical research, such as genomics (e.g. DNA sequencing, RNA-seq) or cytometry, have been introduced (e.g. ENCODE), this issue has not been tackled for optical microscopy instrumentation and images. Although many calibration standards and protocols have been published, there is a lack of awareness and agreement on common standards and guidelines for quality assessment and reproducibility. In April 2020, the QUality Assessment and REProducibility for instruments and images in Light Microscopy (QUAREP-LiMi) initiative was formed. This initiative comprises imaging scientists from academia and industry who share a common interest in achieving a better understanding of the performance and limitations of microscopes and improved quality control (QC) in light microscopy. The ultimate goal of the QUAREP-LiMi initiative is to establish a set of common QC standards, guidelines, metadata models and tools, including detailed protocols, with the ultimate aim of improving reproducible advances in scientific research. This White Paper (1) summarizes the major obstacles identified in the field that motivated the launch of the QUAREP-LiMi initiative; (2) identifies the urgent need to address these obstacles in a grassroots manner, through a community of stakeholders including, researchers, imaging scientists, bioimage analysts, bioimage informatics developers, corporate partners, funding agencies, standards organizations, scientific publishers and observers of such; (3) outlines the current actions of the QUAREP-LiMi initiative and (4) proposes future steps that can be taken to improve the dissemination and acceptance of the proposed guidelines to manage QC. To summarize, the principal goal of the QUAREP-LiMi initiative is to improve the overall quality and reproducibility of light microscope image data by introducing broadly accepted standard practices and accurately captured image data metrics. AU - Nelson, Glyn AU - Boehm, Ulrike AU - Bagley, Steve AU - Bajcsy, Peter AU - Bischof, Johanna AU - Brown, Claire M. AU - Dauphin, Aurélien AU - Dobbie, Ian M. AU - Eriksson, John E. AU - Faklaris, Orestis AU - Fernandez-Rodriguez, Julia AU - Ferrand, Alexia AU - Gelman, Laurent AU - Gheisari, Ali AU - Hartmann, Hella AU - Kukat, Christian AU - Laude, Alex AU - Mitkovski, Miso AU - Munck, Sebastian AU - North, Alison J. AU - Rasse, Tobias M. AU - Resch-Genger, Ute AU - Schuetz, Lucas C. AU - Seitz, Arne AU - Strambio-De-Castillia, Caterina AU - Swedlow, Jason R. AU - Alexopoulos, Ioannis AU - Aumayr, Karin AU - Avilov, Sergiy AU - Bakker, Gert Jan AU - Bammann, Rodrigo R. AU - Bassi, Andrea AU - Beckert, Hannes AU - Beer, Sebastian AU - Belyaev, Yury AU - Bierwagen, Jakob AU - Birngruber, Konstantin A. AU - Bosch, Manel AU - Breitlow, Juergen AU - Cameron, Lisa A. AU - Chalfoun, Joe AU - Chambers, James J. AU - Chen, Chieh Li AU - Conde-Sousa, Eduardo AU - Corbett, Alexander D. AU - Cordelieres, Fabrice P. AU - Nery, Elaine Del AU - Dietzel, Ralf AU - Eismann, Frank AU - Fazeli, Elnaz AU - Felscher, Andreas AU - Fried, Hans AU - Gaudreault, Nathalie AU - Goh, Wah Ing AU - Guilbert, Thomas AU - Hadleigh, Roland AU - Hemmerich, Peter AU - Holst, Gerhard A. AU - Itano, Michelle S. AU - Jaffe, Claudia B. AU - Jambor, Helena K. AU - Jarvis, Stuart C. AU - Keppler, Antje AU - Kirchenbuechler, David AU - Kirchner, Marcel AU - Kobayashi, Norio AU - Krens, Gabriel AU - Kunis, Susanne AU - Lacoste, Judith AU - Marcello, Marco AU - Martins, Gabriel G. AU - Metcalf, Daniel J. AU - Mitchell, Claire A. AU - Moore, Joshua AU - Mueller, Tobias AU - Nelson, Michael S. AU - Ogg, Stephen AU - Onami, Shuichi AU - Palmer, Alexandra L. AU - Paul-Gilloteaux, Perrine AU - Pimentel, Jaime A. AU - Plantard, Laure AU - Podder, Santosh AU - Rexhepaj, Elton AU - Royon, Arnaud AU - Saari, Markku A. AU - Schapman, Damien AU - Schoonderwoert, Vincent AU - Schroth-Diez, Britta AU - Schwartz, Stanley AU - Shaw, Michael AU - Spitaler, Martin AU - Stoeckl, Martin T. AU - Sudar, Damir AU - Teillon, Jeremie AU - Terjung, Stefan AU - Thuenauer, Roland AU - Wilms, Christian D. AU - Wright, Graham D. AU - Nitschke, Roland ID - 9911 IS - 1 JF - Journal of Microscopy SN - 0022-2720 TI - QUAREP-LiMi: A community-driven initiative to establish guidelines for quality assessment and reproducibility for instruments and images in light microscopy VL - 284 ER - TY - JOUR AB - Solution synthesis of particles emerged as an alternative to prepare thermoelectric materials with less demanding processing conditions than conventional solid-state synthetic methods. However, solution synthesis generally involves the presence of additional molecules or ions belonging to the precursors or added to enable solubility and/or regulate nucleation and growth. These molecules or ions can end up in the particles as surface adsorbates and interfere in the material properties. This work demonstrates that ionic adsorbates, in particular Na⁺ ions, are electrostatically adsorbed in SnSe particles synthesized in water and play a crucial role not only in directing the material nano/microstructure but also in determining the transport properties of the consolidated material. In dense pellets prepared by sintering SnSe particles, Na remains within the crystal lattice as dopant, in dislocations, precipitates, and forming grain boundary complexions. These results highlight the importance of considering all the possible unintentional impurities to establish proper structure-property relationships and control material properties in solution-processed thermoelectric materials. AU - Liu, Yu AU - Calcabrini, Mariano AU - Yu, Yuan AU - Genç, Aziz AU - Chang, Cheng AU - Costanzo, Tommaso AU - Kleinhanns, Tobias AU - Lee, Seungho AU - Llorca, Jordi AU - Cojocaru‐Mirédin, Oana AU - Ibáñez, Maria ID - 10123 IS - 52 JF - Advanced Materials KW - mechanical engineering KW - mechanics of materials KW - general materials science SN - 0935-9648 TI - The importance of surface adsorbates in solution‐processed thermoelectric materials: The case of SnSe VL - 33 ER - TY - JOUR AB - Proximity labeling provides a powerful in vivo tool to characterize the proteome of subcellular structures and the interactome of specific proteins. The nematode Caenorhabditis elegans is one of the most intensely studied organisms in biology, offering many advantages for biochemistry. Using the highly active biotin ligase TurboID, we optimize here a proximity labeling protocol for C. elegans. An advantage of TurboID is that biotin's high affinity for streptavidin means biotin-labeled proteins can be affinity-purified under harsh denaturing conditions. By combining extensive sonication with aggressive denaturation using SDS and urea, we achieved near-complete solubilization of worm proteins. We then used this protocol to characterize the proteomes of the worm gut, muscle, skin, and nervous system. Neurons are among the smallest C. elegans cells. To probe the method's sensitivity, we expressed TurboID exclusively in the two AFD neurons and showed that the protocol could identify known and previously unknown proteins expressed selectively in AFD. The active zones of synapses are composed of a protein matrix that is difficult to solubilize and purify. To test if our protocol could solubilize active zone proteins, we knocked TurboID into the endogenous elks-1 gene, which encodes a presynaptic active zone protein. We identified many known ELKS-1-interacting active zone proteins, as well as previously uncharacterized synaptic proteins. Versatile vectors and the inherent advantages of using C. elegans, including fast growth and the ability to rapidly make and functionally test knock-ins, make proximity labeling a valuable addition to the armory of this model organism. AU - Artan, Murat AU - Barratt, Stephen AU - Flynn, Sean M. AU - Begum, Farida AU - Skehel, Mark AU - Nicolas, Armel AU - De Bono, Mario ID - 10117 IS - 3 JF - Journal of Biological Chemistry SN - 0021-9258 TI - Interactome analysis of Caenorhabditis elegans synapses by TurboID-based proximity labeling VL - 297 ER - TY - JOUR AB - Phonon polaritons (PhPs)—light coupled to lattice vibrations—with in-plane hyperbolic dispersion exhibit ray-like propagation with large wave vectors and enhanced density of optical states along certain directions on a surface. As such, they have raised a surge of interest, promising unprecedented manipulation of infrared light at the nanoscale in a planar circuitry. Here, we demonstrate focusing of in-plane hyperbolic PhPs propagating along thin slabs of α-MoO3. To that end, we developed metallic nanoantennas of convex geometries for both efficient launching and focusing of the polaritons. The foci obtained exhibit enhanced near-field confinement and absorption compared to foci produced by in-plane isotropic PhPs. Foci sizes as small as λp/4.5 = λ0/50 were achieved (λp is the polariton wavelength and λ0 is the photon wavelength). Focusing of in-plane hyperbolic polaritons introduces a first and most basic building block developing planar polariton optics using in-plane anisotropic van der Waals materials. AU - Martín-Sánchez, Javier AU - Duan, Jiahua AU - Taboada-Gutiérrez, Javier AU - Álvarez-Pérez, Gonzalo AU - Voronin, Kirill V. AU - Prieto Gonzalez, Ivan AU - Ma, Weiliang AU - Bao, Qiaoliang AU - Volkov, Valentyn S. AU - Hillenbrand, Rainer AU - Nikitin, Alexey Y. AU - Alonso-González, Pablo ID - 10177 IS - 41 JF - Science Advances TI - Focusing of in-plane hyperbolic polaritons in van der Waals crystals with tailored infrared nanoantennas VL - 7 ER - TY - JOUR AB - Inhibitory GABAergic interneurons migrate over long distances from their extracortical origin into the developing cortex. In humans, this process is uniquely slow and prolonged, and it is unclear whether guidance cues unique to humans govern the various phases of this complex developmental process. Here, we use fused cerebral organoids to identify key roles of neurotransmitter signaling pathways in guiding the migratory behavior of human cortical interneurons. We use scRNAseq to reveal expression of GABA, glutamate, glycine, and serotonin receptors along distinct maturation trajectories across interneuron migration. We develop an image analysis software package, TrackPal, to simultaneously assess 48 parameters for entire migration tracks of individual cells. By chemical screening, we show that different modes of interneuron migration depend on distinct neurotransmitter signaling pathways, linking transcriptional maturation of interneurons with their migratory behavior. Altogether, our study provides a comprehensive quantitative analysis of human interneuron migration and its functional modulation by neurotransmitter signaling. AU - Bajaj, Sunanjay AU - Bagley, Joshua A. AU - Sommer, Christoph M AU - Vertesy, Abel AU - Nagumo Wong, Sakurako AU - Krenn, Veronica AU - Lévi-Strauss, Julie AU - Knoblich, Juergen A. ID - 10179 IS - 23 JF - EMBO Journal SN - 0261-4189 TI - Neurotransmitter signaling regulates distinct phases of multimodal human interneuron migration VL - 40 ER - TY - JOUR AB - During the past decade, the scientific community and outside observers have noted a concerning lack of rigor and transparency in preclinical research that led to talk of a “reproducibility crisis” in the life sciences (Baker, 2016; Bespalov & Steckler, 2018; Heddleston et al, 2021). Various measures have been proposed to address the problem: from better training of scientists to more oversight to expanded publishing practices such as preregistration of studies. The recently published EQIPD (Enhancing Quality in Preclinical Data) System is, to date, the largest initiative that aims to establish a systematic approach for increasing the robustness and reliability of biomedical research (Bespalov et al, 2021). However, promoting a cultural change in research practices warrants a broad adoption of the Quality System and its underlying philosophy. It is here that academic Core Facilities (CF), research service providers at universities and research institutions, can make a difference. It is fair to assume that a significant fraction of published data originated from experiments that were designed, run, or analyzed in CFs. These academic services play an important role in the research ecosystem by offering access to cutting-edge equipment and by developing and testing novel techniques and methods that impact research in the academic and private sectors alike (Bikovski et al, 2020). Equipment and infrastructure are not the only value: CFs employ competent personnel with profound knowledge and practical experience of the specific field of interest: animal behavior, imaging, crystallography, genomics, and so on. Thus, CFs are optimally positioned to address concerns about the quality and robustness of preclinical research. AU - Restivo, Leonardo AU - Gerlach, Björn AU - Tsoory, Michael AU - Bikovski, Lior AU - Badurek, Sylvia AU - Pitzer, Claudia AU - Kos-Braun, Isabelle C. AU - Mausset-Bonnefont, Anne Laure Mj AU - Ward, Jonathan AU - Schunn, Michael AU - Noldus, Lucas P.J.J. AU - Bespalov, Anton AU - Voikar, Vootele ID - 10283 JF - EMBO Reports SN - 1469-221X TI - Towards best practices in research: Role of academic core facilities VL - 22 ER -