TY - JOUR AB - Regulation of the Arp2/3 complex is required for productive nucleation of branched actin networks. An emerging aspect of regulation is the incorporation of subunit isoforms into the Arp2/3 complex. Specifically, both ArpC5 subunit isoforms, ArpC5 and ArpC5L, have been reported to fine-tune nucleation activity and branch junction stability. We have combined reverse genetics and cellular structural biology to describe how ArpC5 and ArpC5L differentially affect cell migration. Both define the structural stability of ArpC1 in branch junctions and, in turn, by determining protrusion characteristics, affect protein dynamics and actin network ultrastructure. ArpC5 isoforms also affect the positioning of members of the Ena/Vasodilator-stimulated phosphoprotein (VASP) family of actin filament elongators, which mediate ArpC5 isoform–specific effects on the actin assembly level. Our results suggest that ArpC5 and Ena/VASP proteins are part of a signaling pathway enhancing cell migration. AU - Fäßler, Florian AU - Javoor, Manjunath AU - Datler, Julia AU - Döring, Hermann AU - Hofer, Florian AU - Dimchev, Georgi A AU - Hodirnau, Victor-Valentin AU - Faix, Jan AU - Rottner, Klemens AU - Schur, Florian KM ID - 12334 IS - 3 JF - Science Advances KW - Multidisciplinary SN - 2375-2548 TI - ArpC5 isoforms regulate Arp2/3 complex–dependent protrusion through differential Ena/VASP positioning VL - 9 ER - TY - JOUR AB - Motile cells moving in multicellular organisms encounter microenvironments of locally heterogeneous mechanochemical composition. Individual compositional parameters like chemotactic signals, adhesiveness, and pore sizes are well known to be sensed by motile cells, providing individual guidance cues for cellular pathfinding. However, motile cells encounter diverse mechanochemical signals at the same time, raising the question of how cells respond to locally diverse and potentially competing signals on their migration routes. Here, we reveal that motile amoeboid cells require nuclear repositioning, termed nucleokinesis, for adaptive pathfinding in heterogeneous mechanochemical microenvironments. Using mammalian immune cells and the amoebaDictyostelium discoideum, we discover that frequent, rapid and long-distance nucleokinesis is a basic component of amoeboid pathfinding, enabling cells to reorientate quickly between locally competing cues. Amoeboid nucleokinesis comprises a two-step cell polarity switch and is driven by myosin II-forces, sliding the nucleus from a ‘losing’ to the ‘winning’ leading edge to re-adjust the nuclear to the cellular path. Impaired nucleokinesis distorts fast path adaptions and causes cellular arrest in the microenvironment. Our findings establish that nucleokinesis is required for amoeboid cell navigation. Given that motile single-cell amoebae, many immune cells, and some cancer cells utilize an amoeboid migration strategy, these results suggest that amoeboid nucleokinesis underlies cellular navigation during unicellular biology, immunity, and disease. AU - Kroll, Janina AU - Hauschild, Robert AU - Kuznetcov, Arthur AU - Stefanowski, Kasia AU - Hermann, Monika D. AU - Merrin, Jack AU - Shafeek, Lubuna B AU - Müller-Taubenberger, Annette AU - Renkawitz, Jörg ID - 13342 JF - EMBO Journal SN - 0261-4189 TI - Adaptive pathfinding by nucleokinesis during amoeboid migration ER - TY - JOUR AB - Muscle degeneration is the most prevalent cause for frailty and dependency in inherited diseases and ageing. Elucidation of pathophysiological mechanisms, as well as effective treatments for muscle diseases, represents an important goal in improving human health. Here, we show that the lipid synthesis enzyme phosphatidylethanolamine cytidyltransferase (PCYT2/ECT) is critical to muscle health. Human deficiency in PCYT2 causes a severe disease with failure to thrive and progressive weakness. pcyt2-mutant zebrafish and muscle-specific Pcyt2-knockout mice recapitulate the participant phenotypes, with failure to thrive, progressive muscle weakness and accelerated ageing. Mechanistically, muscle Pcyt2 deficiency affects cellular bioenergetics and membrane lipid bilayer structure and stability. PCYT2 activity declines in ageing muscles of mice and humans, and adeno-associated virus-based delivery of PCYT2 ameliorates muscle weakness in Pcyt2-knockout and old mice, offering a therapy for individuals with a rare disease and muscle ageing. Thus, PCYT2 plays a fundamental and conserved role in vertebrate muscle health, linking PCYT2 and PCYT2-synthesized lipids to severe muscle dystrophy and ageing. AU - Cikes, Domagoj AU - Elsayad, Kareem AU - Sezgin, Erdinc AU - Koitai, Erika AU - Ferenc, Torma AU - Orthofer, Michael AU - Yarwood, Rebecca AU - Heinz, Leonhard X. AU - Sedlyarov, Vitaly AU - Darwish-Miranda, Nasser AU - Taylor, Adrian AU - Grapentine, Sophie AU - al-Murshedi, Fathiya AU - Abot, Anne AU - Weidinger, Adelheid AU - Kutchukian, Candice AU - Sanchez, Colline AU - Cronin, Shane J. F. AU - Novatchkova, Maria AU - Kavirayani, Anoop AU - Schuetz, Thomas AU - Haubner, Bernhard AU - Haas, Lisa AU - Hagelkruys, Astrid AU - Jackowski, Suzanne AU - Kozlov, Andrey AU - Jacquemond, Vincent AU - Knauf, Claude AU - Superti-Furga, Giulio AU - Rullman, Eric AU - Gustafsson, Thomas AU - McDermot, John AU - Lowe, Martin AU - Radak, Zsolt AU - Chamberlain, Jeffrey S. AU - Bakovic, Marica AU - Banka, Siddharth AU - Penninger, Josef M. ID - 12747 JF - Nature Metabolism KW - Cell Biology KW - Physiology (medical) KW - Endocrinology KW - Diabetes and Metabolism KW - Internal Medicine SN - 2522-5812 TI - PCYT2-regulated lipid biosynthesis is critical to muscle health and ageing VL - 5 ER - TY - JOUR AB - Tissue morphogenesis and patterning during development involve the segregation of cell types. Segregation is driven by differential tissue surface tensions generated by cell types through controlling cell-cell contact formation by regulating adhesion and actomyosin contractility-based cellular cortical tensions. We use vertebrate tissue cell types and zebrafish germ layer progenitors as in vitro models of 3-dimensional heterotypic segregation and developed a quantitative analysis of their dynamics based on 3D time-lapse microscopy. We show that general inhibition of actomyosin contractility by the Rho kinase inhibitor Y27632 delays segregation. Cell type-specific inhibition of non-muscle myosin2 activity by overexpression of myosin assembly inhibitor S100A4 reduces tissue surface tension, manifested in decreased compaction during aggregation and inverted geometry observed during segregation. The same is observed when we express a constitutively active Rho kinase isoform to ubiquitously keep actomyosin contractility high at cell-cell and cell-medium interfaces and thus overriding the interface-specific regulation of cortical tensions. Tissue surface tension regulation can become an effective tool in tissue engineering. AU - Méhes, Elod AU - Mones, Enys AU - Varga, Máté AU - Zsigmond, Áron AU - Biri-Kovács, Beáta AU - Nyitray, László AU - Barone, Vanessa AU - Krens, Gabriel AU - Heisenberg, Carl-Philipp J AU - Vicsek, Tamás ID - 14041 JF - Communications Biology TI - 3D cell segregation geometry and dynamics are governed by tissue surface tension regulation VL - 6 ER - TY - JOUR AB - Whether one considers swarming insects, flocking birds, or bacterial colonies, collective motion arises from the coordination of individuals and entails the adjustment of their respective velocities. In particular, in close confinements, such as those encountered by dense cell populations during development or regeneration, collective migration can only arise coordinately. Yet, how individuals unify their velocities is often not understood. Focusing on a finite number of cells in circular confinements, we identify waves of polymerizing actin that function as a pacemaker governing the speed of individual cells. We show that the onset of collective motion coincides with the synchronization of the wave nucleation frequencies across the population. Employing a simpler and more readily accessible mechanical model system of active spheres, we identify the synchronization of the individuals’ internal oscillators as one of the essential requirements to reach the corresponding collective state. The mechanical ‘toy’ experiment illustrates that the global synchronous state is achieved by nearest neighbor coupling. We suggest by analogy that local coupling and the synchronization of actin waves are essential for the emergent, self-organized motion of cell collectives. AU - Riedl, Michael AU - Mayer, Isabelle D AU - Merrin, Jack AU - Sixt, Michael K AU - Hof, Björn ID - 14361 JF - Nature Communications TI - Synchronization in collectively moving inanimate and living active matter VL - 14 ER - TY - JOUR AB - The rapid development of machine learning (ML) techniques has opened up the data-dense field of microbiome research for novel therapeutic, diagnostic, and prognostic applications targeting a wide range of disorders, which could substantially improve healthcare practices in the era of precision medicine. However, several challenges must be addressed to exploit the benefits of ML in this field fully. In particular, there is a need to establish “gold standard” protocols for conducting ML analysis experiments and improve interactions between microbiome researchers and ML experts. The Machine Learning Techniques in Human Microbiome Studies (ML4Microbiome) COST Action CA18131 is a European network established in 2019 to promote collaboration between discovery-oriented microbiome researchers and data-driven ML experts to optimize and standardize ML approaches for microbiome analysis. This perspective paper presents the key achievements of ML4Microbiome, which include identifying predictive and discriminatory ‘omics’ features, improving repeatability and comparability, developing automation procedures, and defining priority areas for the novel development of ML methods targeting the microbiome. The insights gained from ML4Microbiome will help to maximize the potential of ML in microbiome research and pave the way for new and improved healthcare practices. AU - D’Elia, Domenica AU - Truu, Jaak AU - Lahti, Leo AU - Berland, Magali AU - Papoutsoglou, Georgios AU - Ceci, Michelangelo AU - Zomer, Aldert AU - Lopes, Marta B. AU - Ibrahimi, Eliana AU - Gruca, Aleksandra AU - Nechyporenko, Alina AU - Frohme, Marcus AU - Klammsteiner, Thomas AU - Pau, Enrique Carrillo De Santa AU - Marcos-Zambrano, Laura Judith AU - Hron, Karel AU - Pio, Gianvito AU - Simeon, Andrea AU - Suharoschi, Ramona AU - Moreno-Indias, Isabel AU - Temko, Andriy AU - Nedyalkova, Miroslava AU - Apostol, Elena Simona AU - Truică, Ciprian Octavian AU - Shigdel, Rajesh AU - Telalović, Jasminka Hasić AU - Bongcam-Rudloff, Erik AU - Przymus, Piotr AU - Jordamović, Naida Babić AU - Falquet, Laurent AU - Tarazona, Sonia AU - Sampri, Alexia AU - Isola, Gaetano AU - Pérez-Serrano, David AU - Trajkovik, Vladimir AU - Klucar, Lubos AU - Loncar-Turukalo, Tatjana AU - Havulinna, Aki S. AU - Jansen, Christian AU - Bertelsen, Randi J. AU - Claesson, Marcus Joakim ID - 14449 JF - Frontiers in Microbiology TI - Advancing microbiome research with machine learning: Key findings from the ML4Microbiome COST action VL - 14 ER - TY - JOUR AB - Immune responses rely on the rapid and coordinated migration of leukocytes. Whereas it is well established that single-cell migration is often guided by gradients of chemokines and other chemoattractants, it remains poorly understood how these gradients are generated, maintained, and modulated. By combining experimental data with theory on leukocyte chemotaxis guided by the G protein–coupled receptor (GPCR) CCR7, we demonstrate that in addition to its role as the sensory receptor that steers migration, CCR7 also acts as a generator and a modulator of chemotactic gradients. Upon exposure to the CCR7 ligand CCL19, dendritic cells (DCs) effectively internalize the receptor and ligand as part of the canonical GPCR desensitization response. We show that CCR7 internalization also acts as an effective sink for the chemoattractant, dynamically shaping the spatiotemporal distribution of the chemokine. This mechanism drives complex collective migration patterns, enabling DCs to create or sharpen chemotactic gradients. We further show that these self-generated gradients can sustain the long-range guidance of DCs, adapt collective migration patterns to the size and geometry of the environment, and provide a guidance cue for other comigrating cells. Such a dual role of CCR7 as a GPCR that both senses and consumes its ligand can thus provide a novel mode of cellular self-organization. AU - Alanko, Jonna H AU - Ucar, Mehmet C AU - Canigova, Nikola AU - Stopp, Julian A AU - Schwarz, Jan AU - Merrin, Jack AU - Hannezo, Edouard B AU - Sixt, Michael K ID - 14274 IS - 87 JF - Science Immunology KW - General Medicine KW - Immunology SN - 2470-9468 TI - CCR7 acts as both a sensor and a sink for CCL19 to coordinate collective leukocyte migration VL - 8 ER - TY - JOUR AB - Three-dimensional (3D) reconstruction of living brain tissue down to an individual synapse level would create opportunities for decoding the dynamics and structure–function relationships of the brain’s complex and dense information processing network; however, this has been hindered by insufficient 3D resolution, inadequate signal-to-noise ratio and prohibitive light burden in optical imaging, whereas electron microscopy is inherently static. Here we solved these challenges by developing an integrated optical/machine-learning technology, LIONESS (live information-optimized nanoscopy enabling saturated segmentation). This leverages optical modifications to stimulated emission depletion microscopy in comprehensively, extracellularly labeled tissue and previous information on sample structure via machine learning to simultaneously achieve isotropic super-resolution, high signal-to-noise ratio and compatibility with living tissue. This allows dense deep-learning-based instance segmentation and 3D reconstruction at a synapse level, incorporating molecular, activity and morphodynamic information. LIONESS opens up avenues for studying the dynamic functional (nano-)architecture of living brain tissue. AU - Velicky, Philipp AU - Miguel Villalba, Eder AU - Michalska, Julia M AU - Lyudchik, Julia AU - Wei, Donglai AU - Lin, Zudi AU - Watson, Jake AU - Troidl, Jakob AU - Beyer, Johanna AU - Ben Simon, Yoav AU - Sommer, Christoph M AU - Jahr, Wiebke AU - Cenameri, Alban AU - Broichhagen, Johannes AU - Grant, Seth G.N. AU - Jonas, Peter M AU - Novarino, Gaia AU - Pfister, Hanspeter AU - Bickel, Bernd AU - Danzl, Johann G ID - 13267 JF - Nature Methods SN - 1548-7091 TI - Dense 4D nanoscale reconstruction of living brain tissue VL - 20 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 - Acanthocephalans, intestinal parasites of vertebrates, are characterised by orders of magnitude higher metal accumulation than free-living organisms, but the mechanism of such effective metal accumulation is still unknown. The aim of our study was to gain new insights into the high-resolution localization of elements in the bodies of acanthocephalans, thus taking an initial step towards elucidating metal uptake and accumulation in organisms under real environmental conditions. For the first time, nanoscale secondary ion mass spectrometry (NanoSIMS) was used for high-resolution mapping of 12 elements (C, Ca, Cu, Fe, N, Na, O, P, Pb, S, Se, and Tl) in three selected body parts (trunk spines, inner part of the proboscis receptacle and inner surface of the tegument) of Dentitruncus truttae, a parasite of brown trout (Salmo trutta) from the Krka River in Croatia. In addition, the same body parts were examined using transmission electron microscopy (TEM) and correlated with NanoSIMS images. Metal concentrations determined using HR ICP-MS confirmed higher accumulation in D. truttae than in the fish intestine. The chemical composition of the acanthocephalan body showed the highest density of C, Ca, N, Na, O, S, as important and constitutive elements in living cells in all studied structures, while Fe was predominant among trace elements. In general, higher element density was found in trunk spines and tegument, as body structures responsible for substance absorption in parasites. The results obtained with NanoSIMS and TEM-NanoSIMS correlative imaging represent pilot data for mapping of elements at nanoscale resolution in the ultrastructure of various body parts of acanthocephalans and generally provide a contribution for further application of this technique in all parasite species. AU - Filipović Marijić, Vlatka AU - Subirana, Maria Angels AU - Schaumlöffel, Dirk AU - Barišić, Josip AU - Gontier, Etienne AU - Krasnici, Nesrete AU - Mijošek, Tatjana AU - Hernández-Orts, Jesús S. AU - Scholz, Tomáš AU - Erk, Marijana ID - 14786 JF - Science of The Total Environment KW - Pollution KW - Waste Management and Disposal KW - Environmental Chemistry KW - Environmental Engineering SN - 0048-9697 TI - First insight in element localisation in different body parts of the acanthocephalan Dentitruncus truttae using TEM and NanoSIMS VL - 887 ER - TY - JOUR AB - A round-robin study has been carried out to estimate the impact of the human element in small-angle scattering data analysis. Four corrected datasets were provided to participants ready for analysis. All datasets were measured on samples containing spherical scatterers, with two datasets in dilute dispersions and two from powders. Most of the 46 participants correctly identified the number of populations in the dilute dispersions, with half of the population mean entries within 1.5% and half of the population width entries within 40%. Due to the added complexity of the structure factor, far fewer people submitted answers on the powder datasets. For those that did, half of the entries for the means and widths were within 44 and 86%, respectively. This round-robin experiment highlights several causes for the discrepancies, for which solutions are proposed. AU - Pauw, Brian R. AU - Smales, Glen J. AU - Anker, Andy S. AU - Annadurai, Venkatasamy AU - Balazs, Daniel AU - Bienert, Ralf AU - Bouwman, Wim G. AU - Breßler, Ingo AU - Breternitz, Joachim AU - Brok, Erik S. AU - Bryant, Gary AU - Clulow, Andrew J. AU - Crater, Erin R. AU - De Geuser, Frédéric AU - Giudice, Alessandra Del AU - Deumer, Jérôme AU - Disch, Sabrina AU - Dutt, Shankar AU - Frank, Kilian AU - Fratini, Emiliano AU - Garcia, Paulo R.A.F. AU - Gilbert, Elliot P. AU - Hahn, Marc B. AU - Hallett, James AU - Hohenschutz, Max AU - Hollamby, Martin AU - Huband, Steven AU - Ilavsky, Jan AU - Jochum, Johanna K. AU - Juelsholt, Mikkel AU - Mansel, Bradley W. AU - Penttilä, Paavo AU - Pittkowski, Rebecca K. AU - Portale, Giuseppe AU - Pozzo, Lilo D. AU - Rochels, Leonhard AU - Rosalie, Julian M. AU - Saloga, Patrick E.J. AU - Seibt, Susanne AU - Smith, Andrew J. AU - Smith, Gregory N. AU - Spiering, Glenn A. AU - Stawski, Tomasz M. AU - Taché, Olivier AU - Thünemann, Andreas F. AU - Toth, Kristof AU - Whitten, Andrew E. AU - Wuttke, Joachim ID - 14799 IS - 6 JF - Journal of Applied Crystallography SN - 0021-8898 TI - The human factor: Results of a small-angle scattering data analysis round robin VL - 56 ER - TY - GEN AB - Superconductor/semiconductor hybrid devices have attracted increasing interest in the past years. Superconducting electronics aims to complement semiconductor technology, while hybrid architectures are at the forefront of new ideas such as topological superconductivity and protected qubits. In this work, we engineer the induced superconductivity in two-dimensional germanium hole gas by varying the distance between the quantum well and the aluminum. We demonstrate a hard superconducting gap and realize an electrically and flux tunable superconducting diode using a superconducting quantum interference device (SQUID). This allows to tune the current phase relation (CPR), to a regime where single Cooper pair tunneling is suppressed, creating a $ \sin \left( 2 \varphi \right)$ CPR. Shapiro experiments complement this interpretation and the microwave drive allows to create a diode with $ \approx 100 \%$ efficiency. The reported results open up the path towards monolithic integration of spin qubit devices, microwave resonators and (protected) superconducting qubits on a silicon technology compatible platform. AU - Valentini, Marco AU - Sagi, Oliver AU - Baghumyan, Levon AU - Gijsel, Thijs de AU - Jung, Jason AU - Calcaterra, Stefano AU - Ballabio, Andrea AU - Servin, Juan Aguilera AU - Aggarwal, Kushagra AU - Janik, Marian AU - Adletzberger, Thomas AU - Souto, Rubén Seoane AU - Leijnse, Martin AU - Danon, Jeroen AU - Schrade, Constantin AU - Bakkers, Erik AU - Chrastina, Daniel AU - Isella, Giovanni AU - Katsaros, Georgios ID - 13312 KW - Mesoscale and Nanoscale Physics T2 - arXiv TI - Radio frequency driven superconducting diode and parity conserving Cooper pair transport in a two-dimensional germanium hole gas ER - TY - DATA AB - Mapping the complex and dense arrangement of cells and their connectivity in brain tissue demands nanoscale spatial resolution imaging. Super-resolution optical microscopy excels at visualizing specific molecules and individual cells but fails to provide tissue context. Here, we developed Comprehensive Analysis of Tissues across Scales (CATS), a technology to densely map brain tissue architecture from millimeter regional to nanometer synaptic scales in diverse chemically fixed brain preparations, including rodent and human. CATS uses fixation-compatible extracellular labeling and optical imaging, including stimulated emission depletion or expansion microscopy, to comprehensively delineate cellular structures. It enables three-dimensional reconstruction of single synapses and mapping of synaptic connectivity by identification and analysis of putative synaptic cleft regions. Applying CATS to the mouse hippocampal mossy fiber circuitry, we reconstructed and quantified the synaptic input and output structure of identified neurons. We furthermore demonstrate applicability to clinically derived human tissue samples, including formalin-fixed paraffin-embedded routine diagnostic specimens, for visualizing the cellular architecture of brain tissue in health and disease. AU - Danzl, Johann G ID - 13126 TI - Research data for the publication "Imaging brain tissue architecture across millimeter to nanometer scales" ER - TY - JOUR AB - Mapping the complex and dense arrangement of cells and their connectivity in brain tissue demands nanoscale spatial resolution imaging. Super-resolution optical microscopy excels at visualizing specific molecules and individual cells but fails to provide tissue context. Here we developed Comprehensive Analysis of Tissues across Scales (CATS), a technology to densely map brain tissue architecture from millimeter regional to nanometer synaptic scales in diverse chemically fixed brain preparations, including rodent and human. CATS uses fixation-compatible extracellular labeling and optical imaging, including stimulated emission depletion or expansion microscopy, to comprehensively delineate cellular structures. It enables three-dimensional reconstruction of single synapses and mapping of synaptic connectivity by identification and analysis of putative synaptic cleft regions. Applying CATS to the mouse hippocampal mossy fiber circuitry, we reconstructed and quantified the synaptic input and output structure of identified neurons. We furthermore demonstrate applicability to clinically derived human tissue samples, including formalin-fixed paraffin-embedded routine diagnostic specimens, for visualizing the cellular architecture of brain tissue in health and disease. AU - Michalska, Julia M AU - Lyudchik, Julia AU - Velicky, Philipp AU - Korinkova, Hana AU - Watson, Jake AU - Cenameri, Alban AU - Sommer, Christoph M AU - Amberg, Nicole AU - Venturino, Alessandro AU - Roessler, Karl AU - Czech, Thomas AU - Höftberger, Romana AU - Siegert, Sandra AU - Novarino, Gaia AU - Jonas, Peter M AU - Danzl, Johann G ID - 14257 JF - Nature Biotechnology SN - 1087-0156 TI - Imaging brain tissue architecture across millimeter to nanometer scales ER - TY - JOUR AB - Singlet oxygen (1O2) formation is now recognised as a key aspect of non-aqueous oxygen redox chemistry. For identifying 1O2, chemical trapping via 9,10-dimethylanthracene (DMA) to form the endoperoxide (DMA-O2) has become the mainstay method due to its sensitivity, selectivity, and ease of use. While DMA has been shown to be selective for 1O2, rather than forming DMA-O2 with a wide variety of potentially reactive O-containing species, false positives might hypothetically be obtained in the presence of previously overlooked species. Here, we first give unequivocal direct spectroscopic proof by the 1O2-specific near infrared (NIR) emission at 1270 nm for the previously proposed 1O2 formation pathways, which centre around superoxide disproportionation. We then show that peroxocarbonates, common intermediates in metal-O2 and metal carbonate electrochemistry, do not produce false-positive DMA-O2. Moreover, we identify a previously unreported 1O2-forming pathway through the reaction of CO2 with superoxide. Overall, we give unequivocal proof for 1O2 formation in non-aqueous oxygen redox and show that chemical trapping with DMA is a reliable method to assess 1O2 formation. AU - Mondal, Soumyadip AU - Jethwa, Rajesh B AU - Pant, Bhargavi AU - Hauschild, Robert AU - Freunberger, Stefan Alexander ID - 13044 JF - Faraday Discussions KW - Physical and Theoretical Chemistry SN - 1359-6640 TI - Singlet oxygen in non-aqueous oxygen redox: Direct spectroscopic evidence for formation pathways and reliability of chemical probes ER - TY - JOUR AB - 5-Carboxycytosine (5caC) is a rare epigenetic modification found in nucleic acids of all domains of life. Despite its sparse genomic abundance, 5caC is presumed to play essential regulatory roles in transcription, maintenance and base-excision processes in DNA. In this work, we utilize nuclear magnetic resonance (NMR) spectroscopy to address the effects of 5caC incorporation into canonical DNA strands at multiple pH and temperature conditions. Our results demonstrate that 5caC has a pH-dependent global destabilizing and a base-pair mobility enhancing local impact on dsDNA, albeit without any detectable influence on the ground-state B-DNA structure. Measurement of hybridization thermodynamics and kinetics of 5caC-bearing DNA duplexes highlighted how acidic environment (pH 5.8 and 4.7) destabilizes the double-stranded structure by ∼10–20 kJ mol–1 at 37 °C when compared to the same sample at neutral pH. Protonation of 5caC results in a lower activation energy for the dissociation process and a higher barrier for annealing. Studies on conformational exchange on the microsecond time scale regime revealed a sharply localized base-pair motion involving exclusively the modified site and its immediate surroundings. By direct comparison with canonical and 5-formylcytosine (5fC)-edited strands, we were able to address the impact of the two most oxidized naturally occurring cytosine derivatives in the genome. These insights on 5caC’s subtle sensitivity to acidic pH contribute to the long-standing questions of its capacity as a substrate in base excision repair processes and its purpose as an independent, stable epigenetic mark. AU - Dubini, Romeo C. A. AU - Korytiaková, Eva AU - Schinkel, Thea AU - Heinrichs, Pia AU - Carell, Thomas AU - Rovo, Petra ID - 10758 IS - 3 JF - ACS Physical Chemistry Au TI - 1H NMR chemical exchange techniques reveal local and global effects of oxidized cytosine derivatives VL - 2 ER - TY - JOUR AB - Immune cells are constantly on the move through multicellular organisms to explore and respond to pathogens and other harmful insults. While moving, immune cells efficiently traverse microenvironments composed of tissue cells and extracellular fibers, which together form complex environments of various porosity, stiffness, topography, and chemical composition. In this protocol we describe experimental procedures to investigate immune cell migration through microenvironments of heterogeneous porosity. In particular, we describe micro-channels, micro-pillars, and collagen networks as cell migration paths with alternative pore size choices. Employing micro-channels or micro-pillars that divide at junctions into alternative paths with initially differentially sized pores allows us to precisely (1) measure the cellular translocation time through these porous path junctions, (2) quantify the cellular preference for individual pore sizes, and (3) image cellular components like the nucleus and the cytoskeleton. This reductionistic experimental setup thus can elucidate how immune cells perform decisions in complex microenvironments of various porosity like the interstitium. The setup further allows investigation of the underlying forces of cellular squeezing and the consequences of cellular deformation on the integrity of the cell and its organelles. As a complementary approach that does not require any micro-engineering expertise, we describe the usage of three-dimensional collagen networks with different pore sizes. Whereas we here focus on dendritic cells as a model for motile immune cells, the described protocols are versatile as they are also applicable for other immune cell types like neutrophils and non-immune cell types such as mesenchymal and cancer cells. In summary, we here describe protocols to identify the mechanisms and principles of cellular probing, decision making, and squeezing during cellular movement through microenvironments of heterogeneous porosity. AU - Kroll, Janina AU - Ruiz-Fernandez, Mauricio J.A. AU - Braun, Malte B. AU - Merrin, Jack AU - Renkawitz, Jörg ID - 11182 IS - 4 JF - Current Protocols TI - Quantifying the probing and selection of microenvironmental pores by motile immune cells VL - 2 ER - TY - JOUR AB - This article investigates library-related documents written by Gerard van Swieten (1700–72) during his tenure as Library Prefect in the Imperial Library of Vienna (1745–72). Van Swieten’s time as Library Prefect is considered through a textual analysis. Handwritten letters were deconstructed in terms of their appearance, layout, and tone in order to mine them for meaning. Furthermore, the contents were examined for library matters such as censorship, catalogues, and collection development. The Imperial Court Library held a prominent role as a repository for rare and valuable works, later becoming the National Library of Austria. Gerard van Swieten’s work as a librarian tends to be overlooked, perhaps because he is better known as the private physician of Maria Theresia, as well as a medical reformer. Nevertheless, he was a hard-working chief librarian deeply involved in all aspects of librarianship. Van Swieten endorsed modern scientific works, which were otherwise banned officially by the censorship commission, for the use of scholars in the library, expanded the collection by acquiring books through his network of scholars and publishers, and reissued library catalogues. He also provided for the comfort of users in the library reading room, at a time when such considerations were unusual. In conclusion, a proposal is made that van Swieten viewed his role as librarian with some importance and pride. AU - Chlebak, Clara A AU - Reid, Peter H. ID - 11444 IS - 1 JF - Library and Information History SN - 1758-3489 TI - From the prefect’s desk: Gerard van Swieten’s library correspondence VL - 38 ER - TY - GEN AU - Schlögl, Alois AU - Hornoiu, Andrei AU - Elefante, Stefano AU - Stadlbauer, Stephan ID - 12894 SN - 978-3-200-08499-5 T2 - ASHPC22 - Austrian-Slovenian HPC Meeting 2022 TI - Where is the sweet spot? A procurement story of general purpose compute nodes ER - TY - JOUR AB - Lymph nodes (LNs) comprise two main structural elements: fibroblastic reticular cells that form dedicated niches for immune cell interaction and capsular fibroblasts that build a shell around the organ. Immunological challenge causes LNs to increase more than tenfold in size within a few days. Here, we characterized the biomechanics of LN swelling on the cellular and organ scale. We identified lymphocyte trapping by influx and proliferation as drivers of an outward pressure force, causing fibroblastic reticular cells of the T-zone (TRCs) and their associated conduits to stretch. After an initial phase of relaxation, TRCs sensed the resulting strain through cell matrix adhesions, which coordinated local growth and remodeling of the stromal network. While the expanded TRC network readopted its typical configuration, a massive fibrotic reaction of the organ capsule set in and countered further organ expansion. Thus, different fibroblast populations mechanically control LN swelling in a multitier fashion. AU - Assen, Frank P AU - Abe, Jun AU - Hons, Miroslav AU - Hauschild, Robert AU - Shamipour, Shayan AU - Kaufmann, Walter AU - Costanzo, Tommaso AU - Krens, Gabriel AU - Brown, Markus AU - Ludewig, Burkhard AU - Hippenmeyer, Simon AU - Heisenberg, Carl-Philipp J AU - Weninger, Wolfgang AU - Hannezo, Edouard B AU - Luther, Sanjiv A. AU - Stein, Jens V. AU - Sixt, Michael K ID - 9794 JF - Nature Immunology SN - 1529-2908 TI - Multitier mechanics control stromal adaptations in swelling lymph nodes VL - 23 ER - TY - JOUR AB - Tension of the actomyosin cell cortex plays a key role in determining cell–cell contact growth and size. The level of cortical tension outside of the cell–cell contact, when pulling at the contact edge, scales with the total size to which a cell–cell contact can grow [J.-L. Maître et al., Science 338, 253–256 (2012)]. Here, we show in zebrafish primary germ-layer progenitor cells that this monotonic relationship only applies to a narrow range of cortical tension increase and that above a critical threshold, contact size inversely scales with cortical tension. This switch from cortical tension increasing to decreasing progenitor cell–cell contact size is caused by cortical tension promoting E-cadherin anchoring to the actomyosin cytoskeleton, thereby increasing clustering and stability of E-cadherin at the contact. After tension-mediated E-cadherin stabilization at the contact exceeds a critical threshold level, the rate by which the contact expands in response to pulling forces from the cortex sharply drops, leading to smaller contacts at physiologically relevant timescales of contact formation. Thus, the activity of cortical tension in expanding cell–cell contact size is limited by tension-stabilizing E-cadherin–actin complexes at the contact. AU - Slovakova, Jana AU - Sikora, Mateusz K AU - Arslan, Feyza N AU - Caballero Mancebo, Silvia AU - Krens, Gabriel AU - Kaufmann, Walter AU - Merrin, Jack AU - Heisenberg, Carl-Philipp J ID - 10766 IS - 8 JF - Proceedings of the National Academy of Sciences of the United States of America TI - Tension-dependent stabilization of E-cadherin limits cell-cell contact expansion in zebrafish germ-layer progenitor cells VL - 119 ER - TY - JOUR AB - In eukaryotes, clathrin-coated vesicles (CCVs) facilitate the internalization of material from the cell surface as well as the movement of cargo in post-Golgi trafficking pathways. This diversity of functions is partially provided by multiple monomeric and multimeric clathrin adaptor complexes that provide compartment and cargo selectivity. The adaptor-protein assembly polypeptide-1 (AP-1) complex operates as part of the secretory pathway at the trans-Golgi network (TGN), while the AP-2 complex and the TPLATE complex jointly operate at the plasma membrane to execute clathrin-mediated endocytosis. Key to our further understanding of clathrin-mediated trafficking in plants will be the comprehensive identification and characterization of the network of evolutionarily conserved and plant-specific core and accessory machinery involved in the formation and targeting of CCVs. To facilitate these studies, we have analyzed the proteome of enriched TGN/early endosome-derived and endocytic CCVs isolated from dividing and expanding suspension-cultured Arabidopsis (Arabidopsis thaliana) cells. Tandem mass spectrometry analysis results were validated by differential chemical labeling experiments to identify proteins co-enriching with CCVs. Proteins enriched in CCVs included previously characterized CCV components and cargos such as the vacuolar sorting receptors in addition to conserved and plant-specific components whose function in clathrin-mediated trafficking has not been previously defined. Notably, in addition to AP-1 and AP-2, all subunits of the AP-4 complex, but not AP-3 or AP-5, were found to be in high abundance in the CCV proteome. The association of AP-4 with suspension-cultured Arabidopsis CCVs is further supported via additional biochemical data. AU - Dahhan, DA AU - Reynolds, GD AU - Cárdenas, JJ AU - Eeckhout, D AU - Johnson, Alexander J AU - Yperman, K AU - Kaufmann, Walter AU - Vang, N AU - Yan, X AU - Hwang, I AU - Heese, A AU - De Jaeger, G AU - Friml, Jiří AU - Van Damme, D AU - Pan, J AU - Bednarek, SY ID - 10841 IS - 6 JF - Plant Cell SN - 1040-4651 TI - Proteomic characterization of isolated Arabidopsis clathrin-coated vesicles reveals evolutionarily conserved and plant-specific components VL - 34 ER - TY - JOUR AB - The broad implementation of thermoelectricity requires high-performance and low-cost materials. One possibility is employing surfactant-free solution synthesis to produce nanopowders. We propose the strategy of functionalizing “naked” particles’ surface by inorganic molecules to control the nanostructure and, consequently, thermoelectric performance. In particular, we use bismuth thiolates to functionalize surfactant-free SnTe particles’ surfaces. Upon thermal processing, bismuth thiolates decomposition renders SnTe-Bi2S3 nanocomposites with synergistic functions: 1) carrier concentration optimization by Bi doping; 2) Seebeck coefficient enhancement and bipolar effect suppression by energy filtering; and 3) lattice thermal conductivity reduction by small grain domains, grain boundaries and nanostructuration. Overall, the SnTe-Bi2S3 nanocomposites exhibit peak z T up to 1.3 at 873 K and an average z T of ≈0.6 at 300–873 K, which is among the highest reported for solution-processed SnTe. AU - Chang, Cheng AU - Liu, Yu AU - Lee, Seungho AU - Spadaro, Maria AU - Koskela, Kristopher M. AU - Kleinhanns, Tobias AU - Costanzo, Tommaso AU - Arbiol, Jordi AU - Brutchey, Richard L. AU - Ibáñez, Maria ID - 11705 IS - 35 JF - Angewandte Chemie - International Edition SN - 1433-7851 TI - Surface functionalization of surfactant-free particles: A strategy to tailor the properties of nanocomposites for enhanced thermoelectric performance VL - 61 ER - TY - JOUR AB - Capacity, rate performance, and cycle life of aprotic Li–O2 batteries critically depend on reversible electrodeposition of Li2O2. Current understanding states surface-adsorbed versus solvated LiO2 controls Li2O2 growth as surface film or as large particles. Herein, we show that Li2O2 forms across a wide range of electrolytes, carbons, and current densities as particles via solution-mediated LiO2 disproportionation, bringing into question the prevalence of any surface growth under practical conditions. We describe a unified O2 reduction mechanism, which can explain all found capacity relations and Li2O2 morphologies with exclusive solution discharge. Determining particle morphology and achievable capacities are species mobilities, true areal rate, and the degree of LiO2 association in solution. Capacity is conclusively limited by mass transport through the tortuous Li2O2 rather than electron transport through a passivating Li2O2 film. Provided that species mobilities and surface growth are high, high capacities are also achieved with weakly solvating electrolytes, which were previously considered prototypical for low capacity via surface growth. AU - Prehal, Christian AU - Mondal, Soumyadip AU - Lovicar, Ludek AU - Freunberger, Stefan Alexander ID - 12065 IS - 9 JF - ACS Energy Letters TI - Exclusive solution discharge in Li-O₂ batteries? VL - 7 ER - TY - JOUR AB - Kelvin probe force microscopy (KPFM) is a powerful tool for studying contact electrification (CE) at the nanoscale, but converting KPFM voltage maps to charge density maps is nontrivial due to long-range forces and complex system geometry. Here we present a strategy using finite-element method (FEM) simulations to determine the Green's function of the KPFM probe/insulator/ground system, which allows us to quantitatively extract surface charge. Testing our approach with synthetic data, we find that accounting for the atomic force microscope (AFM) tip, cone, and cantilever is necessary to recover a known input and that existing methods lead to gross miscalculation or even the incorrect sign of the underlying charge. Applying it to experimental data, we demonstrate its capacity to extract realistic surface charge densities and fine details from contact-charged surfaces. Our method gives a straightforward recipe to convert qualitative KPFM voltage data into quantitative charge data over a range of experimental conditions, enabling quantitative CE at the nanoscale. AU - Pertl, Felix AU - Sobarzo Ponce, Juan Carlos A AU - Shafeek, Lubuna B AU - Cramer, Tobias AU - Waitukaitis, Scott R ID - 12109 IS - 12 JF - Physical Review Materials TI - Quantifying nanoscale charge density features of contact-charged surfaces with an FEM/KPFM-hybrid approach VL - 6 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 - The question of how RNA, as the principal carrier of genetic information evolved is fundamentally important for our understanding of the origin of life. The RNA molecule is far too complex to have formed in one evolutionary step, suggesting that ancestral proto-RNAs (first ancestor of RNA) may have existed, which evolved over time into the RNA of today. Here we show that isoxazole nucleosides, which are quickly formed from hydroxylamine, cyanoacetylene, urea and ribose, are plausible precursors for RNA. The isoxazole nucleoside can rearrange within an RNA-strand to give cytidine, which leads to an increase of pairing stability. If the proto-RNA contains a canonical seed-nucleoside with defined stereochemistry, the seed-nucleoside can control the configuration of the anomeric center that forms during the in-RNA transformation. The results demonstrate that RNA could have emerged from evolutionarily primitive precursor isoxazole ribosides after strand formation. AU - Xu, Felix AU - Crisp, Antony AU - Schinkel, Thea AU - Dubini, Romeo C. A. AU - Hübner, Sarah AU - Becker, Sidney AU - Schelter, Florian AU - Rovo, Petra AU - Carell, Thomas ID - 12228 IS - 45 JF - Angewandte Chemie International Edition KW - General Chemistry KW - Catalysis SN - 1433-7851 TI - Isoxazole nucleosides as building blocks for a plausible proto‐RNA VL - 61 ER - TY - JOUR AB - Biological systems are the sum of their dynamic three-dimensional (3D) parts. Therefore, it is critical to study biological structures in 3D and at high resolution to gain insights into their physiological functions. Electron microscopy of metal replicas of unroofed cells and isolated organelles has been a key technique to visualize intracellular structures at nanometer resolution. However, many of these methods require specialized equipment and personnel to complete them. Here, we present novel accessible methods to analyze biological structures in unroofed cells and biochemically isolated organelles in 3D and at nanometer resolution, focusing on Arabidopsis clathrin-coated vesicles (CCVs). While CCVs are essential trafficking organelles, their detailed structural information is lacking due to their poor preservation when observed via classical electron microscopy protocols experiments. First, we establish a method to visualize CCVs in unroofed cells using scanning transmission electron microscopy tomography, providing sufficient resolution to define the clathrin coat arrangements. Critically, the samples are prepared directly on electron microscopy grids, removing the requirement to use extremely corrosive acids, thereby enabling the use of this method in any electron microscopy lab. Secondly, we demonstrate that this standardized sample preparation allows the direct comparison of isolated CCV samples with those visualized in cells. Finally, to facilitate the high-throughput and robust screening of metal replicated samples, we provide a deep learning analysis method to screen the “pseudo 3D” morphologies of CCVs imaged with 2D modalities. Collectively, our work establishes accessible ways to examine the 3D structure of biological samples and provide novel insights into the structure of plant CCVs. AU - Johnson, Alexander J AU - Kaufmann, Walter AU - Sommer, Christoph M AU - Costanzo, Tommaso AU - Dahhan, Dana A. AU - Bednarek, Sebastian Y. AU - Friml, Jiří ID - 12239 IS - 10 JF - Molecular Plant KW - Plant Science KW - Molecular Biology SN - 1674-2052 TI - Three-dimensional visualization of planta clathrin-coated vesicles at ultrastructural resolution VL - 15 ER - TY - JOUR AB - Theoretical foundations of chaos have been predominantly laid out for finite-dimensional dynamical systems, such as the three-body problem in classical mechanics and the Lorenz model in dissipative systems. In contrast, many real-world chaotic phenomena, e.g., weather, arise in systems with many (formally infinite) degrees of freedom, which limits direct quantitative analysis of such systems using chaos theory. In the present work, we demonstrate that the hydrodynamic pilot-wave systems offer a bridge between low- and high-dimensional chaotic phenomena by allowing for a systematic study of how the former connects to the latter. Specifically, we present experimental results, which show the formation of low-dimensional chaotic attractors upon destabilization of regular dynamics and a final transition to high-dimensional chaos via the merging of distinct chaotic regions through a crisis bifurcation. Moreover, we show that the post-crisis dynamics of the system can be rationalized as consecutive scatterings from the nonattracting chaotic sets with lifetimes following exponential distributions. AU - Choueiri, George H AU - Suri, Balachandra AU - Merrin, Jack AU - Serbyn, Maksym AU - Hof, Björn AU - Budanur, Nazmi B ID - 12259 IS - 9 JF - Chaos: An Interdisciplinary Journal of Nonlinear Science KW - Applied Mathematics KW - General Physics and Astronomy KW - Mathematical Physics KW - Statistical and Nonlinear Physics SN - 1054-1500 TI - Crises and chaotic scattering in hydrodynamic pilot-wave experiments VL - 32 ER - TY - JOUR AB - The AAA-ATPase Drg1 is a key factor in eukaryotic ribosome biogenesis that initiates cytoplasmic maturation of the large ribosomal subunit. Drg1 releases the shuttling maturation factor Rlp24 from pre-60S particles shortly after nuclear export, a strict requirement for downstream maturation. The molecular mechanism of release remained elusive. Here, we report a series of cryo-EM structures that captured the extraction of Rlp24 from pre-60S particles by Saccharomyces cerevisiae Drg1. These structures reveal that Arx1 and the eukaryote-specific rRNA expansion segment ES27 form a joint docking platform that positions Drg1 for efficient extraction of Rlp24 from the pre-ribosome. The tips of the Drg1 N domains thereby guide the Rlp24 C terminus into the central pore of the Drg1 hexamer, enabling extraction by a hand-over-hand translocation mechanism. Our results uncover substrate recognition and processing by Drg1 step by step and provide a comprehensive mechanistic picture of the conserved modus operandi of AAA-ATPases. AU - Prattes, Michael AU - Grishkovskaya, Irina AU - Hodirnau, Victor-Valentin AU - Hetzmannseder, Christina AU - Zisser, Gertrude AU - Sailer, Carolin AU - Kargas, Vasileios AU - Loibl, Mathias AU - Gerhalter, Magdalena AU - Kofler, Lisa AU - Warren, Alan J. AU - Stengel, Florian AU - Haselbach, David AU - Bergler, Helmut ID - 12262 IS - 9 JF - Nature Structural & Molecular Biology KW - Molecular Biology KW - Structural Biology SN - 1545-9993 TI - Visualizing maturation factor extraction from the nascent ribosome by the AAA-ATPase Drg1 VL - 29 ER - TY - JOUR AB - Centrosomes play a crucial role during immune cell interactions and initiation of the immune response. In proliferating cells, centrosome numbers are tightly controlled and generally limited to one in G1 and two prior to mitosis. Defects in regulating centrosome numbers have been associated with cell transformation and tumorigenesis. Here, we report the emergence of extra centrosomes in leukocytes during immune activation. Upon antigen encounter, dendritic cells pass through incomplete mitosis and arrest in the subsequent G1 phase leading to tetraploid cells with accumulated centrosomes. In addition, cell stimulation increases expression of polo-like kinase 2, resulting in diploid cells with two centrosomes in G1-arrested cells. During cell migration, centrosomes tightly cluster and act as functional microtubule-organizing centers allowing for increased persistent locomotion along gradients of chemotactic cues. Moreover, dendritic cells with extra centrosomes display enhanced secretion of inflammatory cytokines and optimized T cell responses. Together, these results demonstrate a previously unappreciated role of extra centrosomes for regular cell and tissue homeostasis. AU - Weier, Ann-Kathrin AU - Homrich, Mirka AU - Ebbinghaus, Stephanie AU - Juda, Pavel AU - Miková, Eliška AU - Hauschild, Robert AU - Zhang, Lili AU - Quast, Thomas AU - Mass, Elvira AU - Schlitzer, Andreas AU - Kolanus, Waldemar AU - Burgdorf, Sven AU - Gruß, Oliver J. AU - Hons, Miroslav AU - Wieser, Stefan AU - Kiermaier, Eva ID - 12122 IS - 12 JF - Journal of Cell Biology KW - Cell Biology SN - 0021-9525 TI - Multiple centrosomes enhance migration and immune cell effector functions of mature dendritic cells VL - 221 ER - TY - JOUR AB - The phytohormone auxin triggers transcriptional reprogramming through a well-characterized perception machinery in the nucleus. By contrast, mechanisms that underlie fast effects of auxin, such as the regulation of ion fluxes, rapid phosphorylation of proteins or auxin feedback on its transport, remain unclear1,2,3. Whether auxin-binding protein 1 (ABP1) is an auxin receptor has been a source of debate for decades1,4. Here we show that a fraction of Arabidopsis thaliana ABP1 is secreted and binds auxin specifically at an acidic pH that is typical of the apoplast. ABP1 and its plasma-membrane-localized partner, transmembrane kinase 1 (TMK1), are required for the auxin-induced ultrafast global phospho-response and for downstream processes that include the activation of H+-ATPase and accelerated cytoplasmic streaming. abp1 and tmk mutants cannot establish auxin-transporting channels and show defective auxin-induced vasculature formation and regeneration. An ABP1(M2X) variant that lacks the capacity to bind auxin is unable to complement these defects in abp1 mutants. These data indicate that ABP1 is the auxin receptor for TMK1-based cell-surface signalling, which mediates the global phospho-response and auxin canalization. AU - Friml, Jiří AU - Gallei, Michelle C AU - Gelová, Zuzana AU - Johnson, Alexander J AU - Mazur, Ewa AU - Monzer, Aline AU - Rodriguez Solovey, Lesia AU - Roosjen, Mark AU - Verstraeten, Inge AU - Živanović, Branka D. AU - Zou, Minxia AU - Fiedler, Lukas AU - Giannini, Caterina AU - Grones, Peter AU - Hrtyan, Mónika AU - Kaufmann, Walter AU - Kuhn, Andre AU - Narasimhan, Madhumitha AU - Randuch, Marek AU - Rýdza, Nikola AU - Takahashi, Koji AU - Tan, Shutang AU - Teplova, Anastasiia AU - Kinoshita, Toshinori AU - Weijers, Dolf AU - Rakusová, Hana ID - 12291 IS - 7927 JF - Nature SN - 0028-0836 TI - ABP1–TMK auxin perception for global phosphorylation and auxin canalization VL - 609 ER - TY - JOUR AB - The mammalian neocortex is composed of diverse neuronal and glial cell classes that broadly arrange in six distinct laminae. Cortical layers emerge during development and defects in the developmental programs that orchestrate cortical lamination are associated with neurodevelopmental diseases. The developmental principle of cortical layer formation depends on concerted radial projection neuron migration, from their birthplace to their final target position. Radial migration occurs in defined sequential steps, regulated by a large array of signaling pathways. However, based on genetic loss-of-function experiments, most studies have thus far focused on the role of cell-autonomous gene function. Yet, cortical neuron migration in situ is a complex process and migrating neurons traverse along diverse cellular compartments and environments. The role of tissue-wide properties and genetic state in radial neuron migration is however not clear. Here we utilized mosaic analysis with double markers (MADM) technology to either sparsely or globally delete gene function, followed by quantitative single-cell phenotyping. The MADM-based gene ablation paradigms in combination with computational modeling demonstrated that global tissue-wide effects predominate cell-autonomous gene function albeit in a gene-specific manner. Our results thus suggest that the genetic landscape in a tissue critically affects the overall migration phenotype of individual cortical projection neurons. In a broader context, our findings imply that global tissue-wide effects represent an essential component of the underlying etiology associated with focal malformations of cortical development in particular, and neurological diseases in general. AU - Hansen, Andi H AU - Pauler, Florian AU - Riedl, Michael AU - Streicher, Carmen AU - Heger, Anna-Magdalena AU - Laukoter, Susanne AU - Sommer, Christoph M AU - Nicolas, Armel AU - Hof, Björn AU - Tsai, Li Huei AU - Rülicke, Thomas AU - Hippenmeyer, Simon ID - 10791 IS - 1 JF - Oxford Open Neuroscience TI - Tissue-wide effects override cell-intrinsic gene function in radial neuron migration VL - 1 ER - TY - JOUR AB - When crawling through the body, leukocytes often traverse tissues that are densely packed with extracellular matrix and other cells, and this raises the question: How do leukocytes overcome compressive mechanical loads? Here, we show that the actin cortex of leukocytes is mechanoresponsive and that this responsiveness requires neither force sensing via the nucleus nor adhesive interactions with a substrate. Upon global compression of the cell body as well as local indentation of the plasma membrane, Wiskott-Aldrich syndrome protein (WASp) assembles into dot-like structures, providing activation platforms for Arp2/3 nucleated actin patches. These patches locally push against the external load, which can be obstructing collagen fibers or other cells, and thereby create space to facilitate forward locomotion. We show in vitro and in vivo that this WASp function is rate limiting for ameboid leukocyte migration in dense but not in loose environments and is required for trafficking through diverse tissues such as skin and lymph nodes. AU - Gaertner, Florian AU - Reis-Rodrigues, Patricia AU - De Vries, Ingrid AU - Hons, Miroslav AU - Aguilera, Juan AU - Riedl, Michael AU - Leithner, Alexander F AU - Tasciyan, Saren AU - Kopf, Aglaja AU - Merrin, Jack AU - Zheden, Vanessa AU - Kaufmann, Walter AU - Hauschild, Robert AU - Sixt, Michael K ID - 10703 IS - 1 JF - Developmental Cell SN - 1534-5807 TI - WASp triggers mechanosensitive actin patches to facilitate immune cell migration in dense tissues VL - 57 ER - TY - GEN AU - Schlögl, Alois AU - Elefante, Stefano AU - Hornoiu, Andrei AU - Stadlbauer, Stephan ID - 12909 SN - 978-961-6980-77-7 T2 - ASHPC21 – Austrian-Slovenian HPC Meeting 2021 TI - Managing software on a heterogenous HPC cluster ER - TY - JOUR AB - Cell and tissue polarization is fundamental for plant growth and morphogenesis. The polar, cellular localization of Arabidopsis PIN‐FORMED (PIN) proteins is crucial for their function in directional auxin transport. The clustering of PIN polar cargoes within the plasma membrane has been proposed to be important for the maintenance of their polar distribution. However, the more detailed features of PIN clusters and the cellular requirements of cargo clustering remain unclear. Here, we characterized PIN clusters in detail by means of multiple advanced microscopy and quantification methods, such as 3D quantitative imaging or freeze‐fracture replica labeling. The size and aggregation types of PIN clusters were determined by electron microscopy at the nanometer level at different polar domains and at different developmental stages, revealing a strong preference for clustering at the polar domains. Pharmacological and genetic studies revealed that PIN clusters depend on phosphoinositol pathways, cytoskeletal structures and specific cell‐wall components as well as connections between the cell wall and the plasma membrane. This study identifies the role of different cellular processes and structures in polar cargo clustering and provides initial mechanistic insight into the maintenance of polarity in plants and other systems. AU - Li, Hongjiang AU - von Wangenheim, Daniel AU - Zhang, Xixi AU - Tan, Shutang AU - Darwish-Miranda, Nasser AU - Naramoto, Satoshi AU - Wabnik, Krzysztof T AU - de Rycke, Riet AU - Kaufmann, Walter AU - Gütl, Daniel J AU - Tejos, Ricardo AU - Grones, Peter AU - Ke, Meiyu AU - Chen, Xu AU - Dettmer, Jan AU - Friml, Jiří ID - 8582 IS - 1 JF - New Phytologist SN - 0028646X TI - Cellular requirements for PIN polar cargo clustering in Arabidopsis thaliana VL - 229 ER - TY - JOUR AB - The recent outbreak of coronavirus disease 2019 (COVID‐19), caused by the Severe Acute Respiratory Syndrome Coronavirus‐2 (SARS‐CoV‐2) has resulted in a world‐wide pandemic. Disseminated lung injury with the development of acute respiratory distress syndrome (ARDS) is the main cause of mortality in COVID‐19. Although liver failure does not seem to occur in the absence of pre‐existing liver disease, hepatic involvement in COVID‐19 may correlate with overall disease severity and serve as a prognostic factor for the development of ARDS. The spectrum of liver injury in COVID‐19 may range from direct infection by SARS‐CoV‐2, indirect involvement by systemic inflammation, hypoxic changes, iatrogenic causes such as drugs and ventilation to exacerbation of underlying liver disease. This concise review discusses the potential pathophysiological mechanisms for SARS‐CoV‐2 hepatic tropism as well as acute and possibly long‐term liver injury in COVID‐19. AU - Nardo, Alexander D. AU - Schneeweiss-Gleixner, Mathias AU - Bakail, May M AU - Dixon, Emmanuel D. AU - Lax, Sigurd F. AU - Trauner, Michael ID - 8927 IS - 1 JF - Liver International SN - 14783223 TI - Pathophysiological mechanisms of liver injury in COVID-19 VL - 41 ER - TY - JOUR AB - Layered materials in which individual atomic layers are bonded by weak van der Waals forces (vdW materials) constitute one of the most prominent platforms for materials research. Particularly, polar vdW crystals, such as hexagonal boron nitride (h-BN), alpha-molybdenum trioxide (α-MoO3) or alpha-vanadium pentoxide (α-V2O5), have received significant attention in nano-optics, since they support phonon polaritons (PhPs)―light coupled to lattice vibrations― with strong electromagnetic confinement and low optical losses. Recently, correlative far- and near-field studies of α-MoO3 have been demonstrated as an effective strategy to accurately extract the permittivity of this material. Here, we use this accurately characterized and low-loss polaritonic material to sense its local dielectric environment, namely silica (SiO2), one of the most widespread substrates in nanotechnology. By studying the propagation of PhPs on α-MoO3 flakes with different thicknesses laying on SiO2 substrates via near-field microscopy (s-SNOM), we extract locally the infrared permittivity of SiO2. Our work reveals PhPs nanoimaging as a versatile method for the quantitative characterization of the local optical properties of dielectric substrates, crucial for understanding and predicting the response of nanomaterials and for the future scalability of integrated nanophotonic devices. AU - Aguilar-Merino, Patricia AU - Álvarez-Pérez, Gonzalo AU - Taboada-Gutiérrez, Javier AU - Duan, Jiahua AU - Prieto Gonzalez, Ivan AU - Álvarez-Prado, Luis Manuel AU - Nikitin, Alexey Y. AU - Martín-Sánchez, Javier AU - Alonso-González, Pablo ID - 9038 IS - 1 JF - Nanomaterials TI - Extracting the infrared permittivity of SiO2 substrates locally by near-field imaging of phonon polaritons in a van der Waals crystal VL - 11 ER - TY - JOUR AB - Sequence-specific oligomers with predictable folding patterns, i.e., foldamers, provide new opportunities to mimic α-helical peptides and design inhibitors of protein-protein interactions. One major hurdle of this strategy is to retain the correct orientation of key side chains involved in protein surface recognition. Here, we show that the structural plasticity of a foldamer backbone may notably contribute to the required spatial adjustment for optimal interaction with the protein surface. By using oligoureas as α helix mimics, we designed a foldamer/peptide hybrid inhibitor of histone chaperone ASF1, a key regulator of chromatin dynamics. The crystal structure of its complex with ASF1 reveals a notable plasticity of the urea backbone, which adapts to the ASF1 surface to maintain the same binding interface. One additional benefit of generating ASF1 ligands with nonpeptide oligourea segments is the resistance to proteolysis in human plasma, which was highly improved compared to the cognate α-helical peptide. AU - Mbianda, Johanne AU - Bakail, May M AU - André, Christophe AU - Moal, Gwenaëlle AU - Perrin, Marie E. AU - Pinna, Guillaume AU - Guerois, Raphaël AU - Becher, Francois AU - Legrand, Pierre AU - Traoré, Seydou AU - Douat, Céline AU - Guichard, Gilles AU - Ochsenbein, Françoise ID - 9262 IS - 12 JF - Science Advances SN - 2375-2548 TI - Optimal anchoring of a foldamer inhibitor of ASF1 histone chaperone through backbone plasticity VL - 7 ER - TY - JOUR AB - Gradients of chemokines and growth factors guide migrating cells and morphogenetic processes. Migration of antigen-presenting dendritic cells from the interstitium into the lymphatic system is dependent on chemokine CCL21, which is secreted by endothelial cells of the lymphatic capillary, binds heparan sulfates and forms gradients decaying into the interstitium. Despite the importance of CCL21 gradients, and chemokine gradients in general, the mechanisms of gradient formation are unclear. Studies on fibroblast growth factors have shown that limited diffusion is crucial for gradient formation. Here, we used the mouse dermis as a model tissue to address the necessity of CCL21 anchoring to lymphatic capillary heparan sulfates in the formation of interstitial CCL21 gradients. Surprisingly, the absence of lymphatic endothelial heparan sulfates resulted only in a modest decrease of CCL21 levels at the lymphatic capillaries and did neither affect interstitial CCL21 gradient shape nor dendritic cell migration toward lymphatic capillaries. Thus, heparan sulfates at the level of the lymphatic endothelium are dispensable for the formation of a functional CCL21 gradient. AU - Vaahtomeri, Kari AU - Moussion, Christine AU - Hauschild, Robert AU - Sixt, Michael K ID - 9259 JF - Frontiers in Immunology TI - Shape and function of interstitial chemokine CCL21 gradients are independent of heparan sulfates produced by lymphatic endothelium VL - 12 ER - TY - JOUR AB - Background: To understand information coding in single neurons, it is necessary to analyze subthreshold synaptic events, action potentials (APs), and their interrelation in different behavioral states. However, detecting excitatory postsynaptic potentials (EPSPs) or currents (EPSCs) in behaving animals remains challenging, because of unfavorable signal-to-noise ratio, high frequency, fluctuating amplitude, and variable time course of synaptic events. New method: We developed a method for synaptic event detection, termed MOD (Machine-learning Optimal-filtering Detection-procedure), which combines concepts of supervised machine learning and optimal Wiener filtering. Experts were asked to manually score short epochs of data. The algorithm was trained to obtain the optimal filter coefficients of a Wiener filter and the optimal detection threshold. Scored and unscored data were then processed with the optimal filter, and events were detected as peaks above threshold. Results: We challenged MOD with EPSP traces in vivo in mice during spatial navigation and EPSC traces in vitro in slices under conditions of enhanced transmitter release. The area under the curve (AUC) of the receiver operating characteristics (ROC) curve was, on average, 0.894 for in vivo and 0.969 for in vitro data sets, indicating high detection accuracy and efficiency. Comparison with existing methods: When benchmarked using a (1 − AUC)−1 metric, MOD outperformed previous methods (template-fit, deconvolution, and Bayesian methods) by an average factor of 3.13 for in vivo data sets, but showed comparable (template-fit, deconvolution) or higher (Bayesian) computational efficacy. Conclusions: MOD may become an important new tool for large-scale, real-time analysis of synaptic activity. AU - Zhang, Xiaomin AU - Schlögl, Alois AU - Vandael, David H AU - Jonas, Peter M ID - 9329 IS - 6 JF - Journal of Neuroscience Methods SN - 0165-0270 TI - MOD: A novel machine-learning optimal-filtering method for accurate and efficient detection of subthreshold synaptic events in vivo VL - 357 ER - TY - JOUR AB - In nerve cells the genes encoding for α2δ subunits of voltage-gated calcium channels have been linked to synaptic functions and neurological disease. Here we show that α2δ subunits are essential for the formation and organization of glutamatergic synapses. Using a cellular α2δ subunit triple-knockout/knockdown model, we demonstrate a failure in presynaptic differentiation evidenced by defective presynaptic calcium channel clustering and calcium influx, smaller presynaptic active zones, and a strongly reduced accumulation of presynaptic vesicle-associated proteins (synapsin and vGLUT). The presynaptic defect is associated with the downscaling of postsynaptic AMPA receptors and the postsynaptic density. The role of α2δ isoforms as synaptic organizers is highly redundant, as each individual α2δ isoform can rescue presynaptic calcium channel trafficking and expression of synaptic proteins. Moreover, α2δ-2 and α2δ-3 with mutated metal ion-dependent adhesion sites can fully rescue presynaptic synapsin expression but only partially calcium channel trafficking, suggesting that the regulatory role of α2δ subunits is independent from its role as a calcium channel subunit. Our findings influence the current view on excitatory synapse formation. First, our study suggests that postsynaptic differentiation is secondary to presynaptic differentiation. Second, the dependence of presynaptic differentiation on α2δ implicates α2δ subunits as potential nucleation points for the organization of synapses. Finally, our results suggest that α2δ subunits act as transsynaptic organizers of glutamatergic synapses, thereby aligning the synaptic active zone with the postsynaptic density. AU - Schöpf, Clemens L. AU - Ablinger, Cornelia AU - Geisler, Stefanie M. AU - Stanika, Ruslan I. AU - Campiglio, Marta AU - Kaufmann, Walter AU - Nimmervoll, Benedikt AU - Schlick, Bettina AU - Brockhaus, Johannes AU - Missler, Markus AU - Shigemoto, Ryuichi AU - Obermair, Gerald J. ID - 9330 IS - 14 JF - PNAS TI - Presynaptic α2δ subunits are key organizers of glutamatergic synapses VL - 118 ER - TY - JOUR AB - Polaritons with directional in-plane propagation and ultralow losses in van der Waals (vdW) crystals promise unprecedented manipulation of light at the nanoscale. However, these polaritons present a crucial limitation: their directional propagation is intrinsically determined by the crystal structure of the host material, imposing forbidden directions of propagation. Here, we demonstrate that directional polaritons (in-plane hyperbolic phonon polaritons) in a vdW crystal (α-phase molybdenum trioxide) can be directed along forbidden directions by inducing an optical topological transition, which emerges when the slab is placed on a substrate with a given negative permittivity (4H–silicon carbide). By visualizing the transition in real space, we observe exotic polaritonic states between mutually orthogonal hyperbolic regimes, which unveil the topological origin of the transition: a gap opening in the dispersion. This work provides insights into optical topological transitions in vdW crystals, which introduce a route to direct light at the nanoscale. AU - Duan, J. AU - Álvarez-Pérez, G. AU - Voronin, K. V. AU - Prieto Gonzalez, Ivan AU - Taboada-Gutiérrez, J. AU - Volkov, V. S. AU - Martín-Sánchez, J. AU - Nikitin, A. Y. AU - Alonso-González, P. ID - 9334 IS - 14 JF - Science Advances TI - Enabling propagation of anisotropic polaritons along forbidden directions via a topological transition VL - 7 ER - TY - JOUR AB - Optogenetics has been harnessed to shed new mechanistic light on current and future therapeutic strategies. This has been to date achieved by the regulation of ion flow and electrical signals in neuronal cells and neural circuits that are known to be affected by disease. In contrast, the optogenetic delivery of trophic biochemical signals, which support cell survival and are implicated in degenerative disorders, has never been demonstrated in an animal model of disease. Here, we reengineered the human and Drosophila melanogaster REarranged during Transfection (hRET and dRET) receptors to be activated by light, creating one-component optogenetic tools termed Opto-hRET and Opto-dRET. Upon blue light stimulation, these receptors robustly induced the MAPK/ERK proliferative signaling pathway in cultured cells. In PINK1B9 flies that exhibit loss of PTEN-induced putative kinase 1 (PINK1), a kinase associated with familial Parkinson’s disease (PD), light activation of Opto-dRET suppressed mitochondrial defects, tissue degeneration and behavioral deficits. In human cells with PINK1 loss-of-function, mitochondrial fragmentation was rescued using Opto-dRET via the PI3K/NF-кB pathway. Our results demonstrate that a light-activated receptor can ameliorate disease hallmarks in a genetic model of PD. The optogenetic delivery of trophic signals is cell type-specific and reversible and thus has the potential to inspire novel strategies towards a spatio-temporal regulation of tissue repair. AU - Inglés Prieto, Álvaro AU - Furthmann, Nikolas AU - Crossman, Samuel H. AU - Tichy, Alexandra Madelaine AU - Hoyer, Nina AU - Petersen, Meike AU - Zheden, Vanessa AU - Bicher, Julia AU - Gschaider-Reichhart, Eva AU - György, Attila AU - Siekhaus, Daria E AU - Soba, Peter AU - Winklhofer, Konstanze F. AU - Janovjak, Harald L ID - 9363 IS - 4 JF - PLoS genetics TI - Optogenetic delivery of trophic signals in a genetic model of Parkinson's disease VL - 17 ER - TY - JOUR AB - The multimeric matrix (M) protein of clinically relevant paramyxoviruses orchestrates assembly and budding activity of viral particles at the plasma membrane (PM). We identified within the canine distemper virus (CDV) M protein two microdomains, potentially assuming α-helix structures, which are essential for membrane budding activity. Remarkably, while two rationally designed microdomain M mutants (E89R, microdomain 1 and L239D, microdomain 2) preserved proper folding, dimerization, interaction with the nucleocapsid protein, localization at and deformation of the PM, the virus-like particle formation, as well as production of infectious virions (as monitored using a membrane budding-complementation system), were, in sharp contrast, strongly impaired. Of major importance, raster image correlation spectroscopy (RICS) revealed that both microdomains contributed to finely tune M protein mobility specifically at the PM. Collectively, our data highlighted the cornerstone membrane budding-priming activity of two spatially discrete M microdomains, potentially by coordinating the assembly of productive higher oligomers at the PM. AU - Gast, Matthieu AU - Kadzioch, Nicole P. AU - Milius, Doreen AU - Origgi, Francesco AU - Plattet, Philippe ID - 9361 IS - 2 JF - mSphere TI - Oligomerization and cell egress controlled by two microdomains of canine distemper virus matrix protein VL - 6 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 - 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 -