@unpublished{14644, abstract = {Transcription by RNA polymerase II (Pol II) can be repressed by noncoding RNA, including the human RNA Alu. However, the mechanism by which endogenous RNAs repress transcription remains unclear. Here we present cryo-electron microscopy structures of Pol II bound to Alu RNA, which reveal that Alu RNA mimics how DNA and RNA bind to Pol II during transcription elongation. Further, we show how domains of the general transcription factor TFIIF affect complex dynamics and control repressive activity. Together, we reveal how a non-coding RNA can regulate mammalian gene expression.}, author = {Tluckova, Katarina and Testa Salmazo, Anita P and Bernecky, Carrie A}, publisher = {Institute of Science and Technology Austria}, title = {{Mechanism of mammalian transcriptional repression by noncoding RNA}}, doi = {10.15479/AT:ISTA:14644}, year = {2023}, } @article{14719, abstract = {Lithium–sulfur batteries are regarded as an advantageous option for meeting the growing demand for high-energy-density storage, but their commercialization relies on solving the current limitations of both sulfur cathodes and lithium metal anodes. In this scenario, the implementation of lithium sulfide (Li2S) cathodes compatible with alternative anode materials such as silicon has the potential to alleviate the safety concerns associated with lithium metal. In this direction, here, we report a sulfur cathode based on Li2S nanocrystals grown on a catalytic host consisting of CoFeP nanoparticles supported on tubular carbon nitride. Nanosized Li2S is incorporated into the host by a scalable liquid infiltration–evaporation method. Theoretical calculations and experimental results demonstrate that the CoFeP–CN composite can boost the polysulfide adsorption/conversion reaction kinetics and strongly reduce the initial overpotential activation barrier by stretching the Li–S bonds of Li2S. Besides, the ultrasmall size of the Li2S particles in the Li2S–CoFeP–CN composite cathode facilitates the initial activation. Overall, the Li2S–CoFeP–CN electrodes exhibit a low activation barrier of 2.56 V, a high initial capacity of 991 mA h gLi2S–1, and outstanding cyclability with a small fading rate of 0.029% per cycle over 800 cycles. Moreover, Si/Li2S full cells are assembled using the nanostructured Li2S–CoFeP–CN cathode and a prelithiated anode based on graphite-supported silicon nanowires. These Si/Li2S cells demonstrate high initial discharge capacities above 900 mA h gLi2S–1 and good cyclability with a capacity fading rate of 0.28% per cycle over 150 cycles.}, author = {Mollania, Hamid and Zhang, Chaoqi and Du, Ruifeng and Qi, Xueqiang and Li, Junshan and Horta, Sharona and Ibáñez, Maria and Keller, Caroline and Chenevier, Pascale and Oloomi-Buygi, Majid and Cabot, Andreu}, issn = {1944-8252}, journal = {ACS Applied Materials and Interfaces}, number = {50}, pages = {58462–58475}, publisher = {American Chemical Society}, title = {{Nanostructured Li₂S cathodes for silicon-sulfur batteries}}, doi = {10.1021/acsami.3c14072}, volume = {15}, year = {2023}, } @article{12174, abstract = {Vacuolar-type H+-ATPase (V-ATPase) is a multimeric complex present in a variety of cellular membranes that acts as an ATP-dependent proton pump and plays a key role in pH homeostasis and intracellular signalling pathways. In humans, 22 autosomal genes encode for a redundant set of subunits allowing the composition of diverse V-ATPase complexes with specific properties and expression. Sixteen subunits have been linked to human disease. Here we describe 26 patients harbouring 20 distinct pathogenic de novo missense ATP6V1A variants, mainly clustering within the ATP synthase α/β family-nucleotide-binding domain. At a mean age of 7 years (extremes: 6 weeks, youngest deceased patient to 22 years, oldest patient) clinical pictures included early lethal encephalopathies with rapidly progressive massive brain atrophy, severe developmental epileptic encephalopathies and static intellectual disability with epilepsy. The first clinical manifestation was early hypotonia, in 70%; 81% developed epilepsy, manifested as developmental epileptic encephalopathies in 58% of the cohort and with infantile spasms in 62%; 63% of developmental epileptic encephalopathies failed to achieve any developmental, communicative or motor skills. Less severe outcomes were observed in 23% of patients who, at a mean age of 10 years and 6 months, exhibited moderate intellectual disability, with independent walking and variable epilepsy. None of the patients developed communicative language. Microcephaly (38%) and amelogenesis imperfecta/enamel dysplasia (42%) were additional clinical features. Brain MRI demonstrated hypomyelination and generalized atrophy in 68%. Atrophy was progressive in all eight individuals undergoing repeated MRIs. Fibroblasts of two patients with developmental epileptic encephalopathies showed decreased LAMP1 expression, Lysotracker staining and increased organelle pH, consistent with lysosomal impairment and loss of V-ATPase function. Fibroblasts of two patients with milder disease, exhibited a different phenotype with increased Lysotracker staining, decreased organelle pH and no significant modification in LAMP1 expression. Quantification of substrates for lysosomal enzymes in cellular extracts from four patients revealed discrete accumulation. Transmission electron microscopy of fibroblasts of four patients with variable severity and of induced pluripotent stem cell-derived neurons from two patients with developmental epileptic encephalopathies showed electron-dense inclusions, lipid droplets, osmiophilic material and lamellated membrane structures resembling phospholipids. Quantitative assessment in induced pluripotent stem cell-derived neurons identified significantly smaller lysosomes. ATP6V1A-related encephalopathy represents a new paradigm among lysosomal disorders. It results from a dysfunctional endo-lysosomal membrane protein causing altered pH homeostasis. Its pathophysiology implies intracellular accumulation of substrates whose composition remains unclear, and a combination of developmental brain abnormalities and neurodegenerative changes established during prenatal and early postanal development, whose severity is variably determined by specific pathogenic variants.}, author = {Guerrini, Renzo and Mei, Davide and Szigeti, Margit Katalin and Pepe, Sara and Koenig, Mary Kay and Von Allmen, Gretchen and Cho, Megan T and McDonald, Kimberly and Baker, Janice and Bhambhani, Vikas and Powis, Zöe and Rodan, Lance and Nabbout, Rima and Barcia, Giulia and Rosenfeld, Jill A and Bacino, Carlos A and Mignot, Cyril and Power, Lillian H and Harris, Catharine J and Marjanovic, Dragan and Møller, Rikke S and Hammer, Trine B and Keski Filppula, Riikka and Vieira, Päivi and Hildebrandt, Clara and Sacharow, Stephanie and Maragliano, Luca and Benfenati, Fabio and Lachlan, Katherine and Benneche, Andreas and Petit, Florence and de Sainte Agathe, Jean Madeleine and Hallinan, Barbara and Si, Yue and Wentzensen, Ingrid M and Zou, Fanggeng and Narayanan, Vinodh and Matsumoto, Naomichi and Boncristiano, Alessandra and la Marca, Giancarlo and Kato, Mitsuhiro and Anderson, Kristin and Barba, Carmen and Sturiale, Luisa and Garozzo, Domenico and Bei, Roberto and Masuelli, Laura and Conti, Valerio and Novarino, Gaia and Fassio, Anna}, issn = {1460-2156}, journal = {Brain}, keywords = {Neurology (clinical)}, number = {8}, pages = {2687--2703}, publisher = {Oxford University Press}, title = {{Phenotypic and genetic spectrum of ATP6V1A encephalopathy: A disorder of lysosomal homeostasis}}, doi = {10.1093/brain/awac145}, volume = {145}, year = {2022}, } @article{12239, abstract = {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.}, author = {Johnson, Alexander J and Kaufmann, Walter and Sommer, Christoph M and Costanzo, Tommaso and Dahhan, Dana A. and Bednarek, Sebastian Y. and Friml, Jiří}, issn = {1674-2052}, journal = {Molecular Plant}, keywords = {Plant Science, Molecular Biology}, number = {10}, pages = {1533--1542}, publisher = {Elsevier}, title = {{Three-dimensional visualization of planta clathrin-coated vesicles at ultrastructural resolution}}, doi = {10.1016/j.molp.2022.09.003}, volume = {15}, year = {2022}, } @article{12262, abstract = {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.}, author = {Prattes, Michael and Grishkovskaya, Irina and Hodirnau, Victor-Valentin and Hetzmannseder, Christina and Zisser, Gertrude and Sailer, Carolin and Kargas, Vasileios and Loibl, Mathias and Gerhalter, Magdalena and Kofler, Lisa and Warren, Alan J. and Stengel, Florian and Haselbach, David and Bergler, Helmut}, issn = {1545-9985}, journal = {Nature Structural & Molecular Biology}, keywords = {Molecular Biology, Structural Biology}, number = {9}, pages = {942--953}, publisher = {Springer Nature}, title = {{Visualizing maturation factor extraction from the nascent ribosome by the AAA-ATPase Drg1}}, doi = {10.1038/s41594-022-00832-5}, volume = {29}, year = {2022}, } @article{12291, abstract = {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.}, author = {Friml, Jiří and Gallei, Michelle C and Gelová, Zuzana and Johnson, Alexander J and Mazur, Ewa and Monzer, Aline and Rodriguez Solovey, Lesia and Roosjen, Mark and Verstraeten, Inge and Živanović, Branka D. and Zou, Minxia and Fiedler, Lukas and Giannini, Caterina and Grones, Peter and Hrtyan, Mónika and Kaufmann, Walter and Kuhn, Andre and Narasimhan, Madhumitha and Randuch, Marek and Rýdza, Nikola and Takahashi, Koji and Tan, Shutang and Teplova, Anastasiia and Kinoshita, Toshinori and Weijers, Dolf and Rakusová, Hana}, issn = {1476-4687}, journal = {Nature}, number = {7927}, pages = {575--581}, publisher = {Springer Nature}, title = {{ABP1–TMK auxin perception for global phosphorylation and auxin canalization}}, doi = {10.1038/s41586-022-05187-x}, volume = {609}, year = {2022}, } @phdthesis{12366, abstract = {Recent substantial advances in the feld of superconducting circuits have shown its potential as a leading platform for future quantum computing. In contrast to classical computers based on bits that are represented by a single binary value, 0 or 1, quantum bits (or qubits) can be in a superposition of both. Thus, quantum computers can store and handle more information at the same time and a quantum advantage has already been demonstrated for two types of computational tasks. Rapid progress in academic and industry labs accelerates the development of superconducting processors which may soon fnd applications in complex computations, chemical simulations, cryptography, and optimization. Now that these machines are scaled up to tackle such problems the questions of qubit interconnects and networks becomes very relevant. How to route signals on-chip between diferent processor components? What is the most efcient way to entangle qubits? And how to then send and process entangled signals between distant cryostats hosting superconducting processors? In this thesis, we are looking for solutions to these problems by studying the collective behavior of superconducting qubit ensembles. We frst demonstrate on-demand tunable directional scattering of microwave photons from a pair of qubits in a waveguide. Such a device can route microwave photons on-chip with a high diode efciency. Then we focus on studying ultra-strong coupling regimes between light (microwave photons) and matter (superconducting qubits), a regime that could be promising for extremely fast multi-qubit entanglement generation. Finally, we show coherent pulse storage and periodic revivals in a fve qubit ensemble strongly coupled to a resonator. Such a reconfgurable storage device could be used as part of a quantum repeater that is needed for longer-distance quantum communication. The achieved high degree of control over multi-qubit ensembles highlights not only the beautiful physics of circuit quantum electrodynamics, it also represents the frst step toward new quantum simulation and communication methods, and certain techniques may also fnd applications in future superconducting quantum computing hardware. }, author = {Redchenko, Elena}, isbn = {978-3-99078-024-4}, issn = {2663-337X}, pages = {168}, publisher = {Institute of Science and Technology Austria}, title = {{Controllable states of superconducting Qubit ensembles}}, doi = {10.15479/at:ista:12132}, year = {2022}, } @article{10639, abstract = {With more than 80 members worldwide, the Orthobunyavirus genus in the Peribunyaviridae family is a large genus of enveloped RNA viruses, many of which are emerging pathogens in humans and livestock. How orthobunyaviruses (OBVs) penetrate and infect mammalian host cells remains poorly characterized. Here, we investigated the entry mechanisms of the OBV Germiston (GERV). Viral particles were visualized by cryo-electron microscopy and appeared roughly spherical with an average diameter of 98 nm. Labeling of the virus with fluorescent dyes did not adversely affect its infectivity and allowed the monitoring of single particles in fixed and live cells. Using this approach, we found that endocytic internalization of bound viruses was asynchronous and occurred within 30-40 min. The virus entered Rab5a+ early endosomes and, subsequently, late endosomal vacuoles containing Rab7a but not LAMP-1. Infectious entry did not require proteolytic cleavage, and endosomal acidification was sufficient and necessary for viral fusion. Acid-activated penetration began 15-25 min after initiation of virus internalization and relied on maturation of early endosomes to late endosomes. The optimal pH for viral membrane fusion was slightly below 6.0, and penetration was hampered when the potassium influx was abolished. Overall, our study provides real-time visualization of GERV entry into host cells and demonstrates the importance of late endosomal maturation in facilitating OBV penetration.}, author = {Windhaber, Stefan and Xin, Qilin and Uckeley, Zina M. and Koch, Jana and Obr, Martin and Garnier, Céline and Luengo-Guyonnot, Catherine and Duboeuf, Maëva and Schur, Florian KM and Lozach, Pierre-Yves}, issn = {1098-5514}, journal = {Journal of Virology}, keywords = {virology, insect science, immunology, microbiology}, number = {5}, publisher = {American Society for Microbiology}, title = {{The Orthobunyavirus Germiston enters host cells from late endosomes}}, doi = {10.1128/jvi.02146-21}, volume = {96}, year = {2022}, } @article{10703, abstract = {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.}, author = {Gaertner, Florian and Reis-Rodrigues, Patricia and De Vries, Ingrid and Hons, Miroslav and Aguilera, Juan and Riedl, Michael and Leithner, Alexander F and Tasciyan, Saren and Kopf, Aglaja and Merrin, Jack and Zheden, Vanessa and Kaufmann, Walter and Hauschild, Robert and Sixt, Michael K}, issn = {1878-1551}, journal = {Developmental Cell}, number = {1}, pages = {47--62.e9}, publisher = {Cell Press ; Elsevier}, title = {{WASp triggers mechanosensitive actin patches to facilitate immune cell migration in dense tissues}}, doi = {10.1016/j.devcel.2021.11.024}, volume = {57}, year = {2022}, } @article{10766, abstract = {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.}, author = {Slovakova, Jana and Sikora, Mateusz K and Arslan, Feyza N and Caballero Mancebo, Silvia and Krens, Gabriel and Kaufmann, Walter and Merrin, Jack and Heisenberg, Carl-Philipp J}, issn = {10916490}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, number = {8}, publisher = {Proceedings of the National Academy of Sciences}, title = {{Tension-dependent stabilization of E-cadherin limits cell-cell contact expansion in zebrafish germ-layer progenitor cells}}, doi = {10.1073/pnas.2122030119}, volume = {119}, year = {2022}, } @article{9794, abstract = {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.}, author = {Assen, Frank P and Abe, Jun and Hons, Miroslav and Hauschild, Robert and Shamipour, Shayan and Kaufmann, Walter and Costanzo, Tommaso and Krens, Gabriel and Brown, Markus and Ludewig, Burkhard and Hippenmeyer, Simon and Heisenberg, Carl-Philipp J and Weninger, Wolfgang and Hannezo, Edouard B and Luther, Sanjiv A. and Stein, Jens V. and Sixt, Michael K}, issn = {1529-2916}, journal = {Nature Immunology}, pages = {1246--1255}, publisher = {Springer Nature}, title = {{Multitier mechanics control stromal adaptations in swelling lymph nodes}}, doi = {10.1038/s41590-022-01257-4}, volume = {23}, year = {2022}, } @article{10841, abstract = {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.}, author = {Dahhan, DA and Reynolds, GD and Cárdenas, JJ and Eeckhout, D and Johnson, Alexander J and Yperman, K and Kaufmann, Walter and Vang, N and Yan, X and Hwang, I and Heese, A and De Jaeger, G and Friml, Jiří and Van Damme, D and Pan, J and Bednarek, SY}, issn = {1532-298x}, journal = {Plant Cell}, number = {6}, pages = {2150--2173}, publisher = {Oxford Academic}, title = {{Proteomic characterization of isolated Arabidopsis clathrin-coated vesicles reveals evolutionarily conserved and plant-specific components}}, doi = {10.1093/plcell/koac071}, volume = {34}, year = {2022}, } @article{11155, abstract = {The potential of energy filtering and direct electron detection for cryo-electron microscopy (cryo-EM) has been well documented. Here, we assess the performance of recently introduced hardware for cryo-electron tomography (cryo-ET) and subtomogram averaging (STA), an increasingly popular structural determination method for complex 3D specimens. We acquired cryo-ET datasets of EIAV virus-like particles (VLPs) on two contemporary cryo-EM systems equipped with different energy filters and direct electron detectors (DED), specifically a Krios G4, equipped with a cold field emission gun (CFEG), Thermo Fisher Scientific Selectris X energy filter, and a Falcon 4 DED; and a Krios G3i, with a Schottky field emission gun (XFEG), a Gatan Bioquantum energy filter, and a K3 DED. We performed constrained cross-correlation-based STA on equally sized datasets acquired on the respective systems. The resulting EIAV CA hexamer reconstructions show that both systems perform comparably in the 4–6 Å resolution range based on Fourier-Shell correlation (FSC). In addition, by employing a recently introduced multiparticle refinement approach, we obtained a reconstruction of the EIAV CA hexamer at 2.9 Å. Our results demonstrate the potential of the new generation of energy filters and DEDs for STA, and the effects of using different processing pipelines on their STA outcomes.}, author = {Obr, Martin and Hagen, Wim J.H. and Dick, Robert A. and Yu, Lingbo and Kotecha, Abhay and Schur, Florian KM}, issn = {1047-8477}, journal = {Journal of Structural Biology}, keywords = {Structural Biology}, number = {2}, publisher = {Elsevier}, title = {{Exploring high-resolution cryo-ET and subtomogram averaging capabilities of contemporary DEDs}}, doi = {10.1016/j.jsb.2022.107852}, volume = {214}, year = {2022}, } @phdthesis{11196, abstract = {One of the fundamental questions in Neuroscience is how the structure of synapses and their physiological properties are related. While synaptic transmission remains a dynamic process, electron microscopy provides images with comparably low temporal resolution (Studer et al., 2014). The current work overcomes this challenge and describes an improved “Flash and Freeze” technique (Watanabe et al., 2013a; Watanabe et al., 2013b) to study synaptic transmission at the hippocampal mossy fiber-CA3 pyramidal neuron synapses, using mouse acute brain slices and organotypic slices culture. The improved method allowed for selective stimulation of presynaptic mossy fiber boutons and the observation of synaptic vesicle pool dynamics at the active zones. Our results uncovered several intriguing morphological features of mossy fiber boutons. First, the docked vesicle pool was largely depleted (more than 70%) after stimulation, implying that the docked synaptic vesicles pool and readily releasable pool are vastly overlapping in mossy fiber boutons. Second, the synaptic vesicles are skewed towards larger diameters, displaying a wide range of sizes. An increase in the mean diameter of synaptic vesicles, after single and repetitive stimulation, suggests that smaller vesicles have a higher release probability. Third, we observed putative endocytotic structures after moderate light stimulation, matching the timing of previously described ultrafast endocytosis (Watanabe et al., 2013a; Delvendahl et al., 2016). In addition, synaptic transmission depends on a sophisticated system of protein machinery and calcium channels (Südhof, 2013b), which amplifies the challenge in studying synaptic communication as these interactions can be potentially modified during synaptic plasticity. And although recent study elucidated the potential correlation between physiological and morphological properties of synapses during synaptic plasticity (Vandael et al., 2020), the molecular underpinning of it remains unknown. Thus, the presented work tries to overcome this challenge and aims to pinpoint changes in the molecular architecture at hippocampal mossy fiber bouton synapses during short- and long-term potentiation (STP and LTP), we combined chemical potentiation, with the application of a cyclic adenosine monophosphate agonist (i.e. forskolin) and freeze-fracture replica immunolabelling. This method allowed the localization of membrane-bound proteins with nanometer precision within the active zone, in particular, P/Q-type calcium channels and synaptic vesicle priming proteins Munc13-1/2. First, we found that the number of clusters of Munc13-1 in the mossy fiber bouton active zone increased significantly during STP, but decreased to lower than the control value during LTP. Secondly, although the distance between the calcium channels and Munc13-1s did not change after induction of STP, it shortened during the LTP phase. Additionally, forskolin did not affect Munc13-2 distribution during STP and LTP. These results indicate the existence of two distinct mechanisms that govern STP and LTP at mossy fiber bouton synapses: an increase in the readily realizable pool in the case of STP and a potential increase in release probability during LTP. “Flash and freeze” and functional electron microscopy, are versatile methods that can be successfully applied to intact brain circuits to study synaptic transmission even at the molecular level. }, author = {Kim, Olena}, issn = {2663-337X}, pages = {132}, publisher = {Institute of Science and Technology Austria}, title = {{Nanoarchitecture of hippocampal mossy fiber-CA3 pyramidal neuron synapses}}, doi = {10.15479/at:ista:11196}, year = {2022}, } @phdthesis{11393, abstract = {AMPA receptors (AMPARs) mediate fast excitatory neurotransmission and their role is implicated in complex processes such as learning and memory and various neurological diseases. These receptors are composed of different subunits and the subunit composition can affect channel properties, receptor trafficking and interaction with other associated proteins. Using the high sensitivity SDS-digested freeze-fracture replica labeling (SDS-FRL) for electron microscopy I investigated the number, density, and localization of AMPAR subunits, GluA1, GluA2, GluA3, and GluA1-3 (panAMPA) in pyramidal cells in the CA1 area of mouse hippocampus. I have found that the immunogold labeling for all of these subunits in the postsynaptic sites was highest in stratum radiatum and lowest in stratum lacunosummoleculare. The labeling density for the all subunits in the extrasynaptic sites showed a gradual increase from the pyramidal cell soma towards the distal part of stratum radiatum. The densities of extrasynaptic GluA1, GluA2 and panAMPA labeling reached 10-15% of synaptic densities, while the ratio of extrasynaptic labeling for GluA3 was significantly lower compared than those for other subunits. The labeling patterns for GluA1, GluA2 and GluA1-3 are similar and their densities were higher in the periphery than center of synapses. In contrast, the GluA3- containing receptors were more centrally localized compared to the GluA1- and GluA2- containing receptors. The hippocampus plays a central role in learning and memory. Contextual learning has been shown to require the delivery of AMPA receptors to CA1 synapses in the dorsal hippocampus. However, proximodistal heterogeneity of this plasticity and particular contribution of different AMPA receptor subunits are not fully understood. By combining inhibitory avoidance task, a hippocampus-dependent contextual fear-learning paradigm, with SDS-FRL, I have revealed an increase in synaptic density specific to GluA1-containing AMPA receptors in the CA1 area. The intrasynaptic distribution of GluA1 also changed from the periphery to center-preferred pattern. Furthermore, this synaptic plasticity was evident selectively in stratum radiatum but not stratum oriens, and in the CA1 subregion proximal but not distal to CA2. These findings further contribute to our understanding of how specific hippocampal subregions and AMPA receptor subunits are involved in physiological learning. Although the immunolabeling results above shed light on subunit-specific plasticity in AMPAR distribution, no tools to visualize and study the subunit composition at the single channel level in situ have been available. Electron microscopy with conventional immunogold labeling approaches has limitations in the single channel analysis because of the large size of antibodies and steric hindrance hampering multiple subunit labeling of single channels. I managed to develop a new chemical labeling system using a short peptide tag and small synthetic probes, which form specific covalent bond with a cysteine residue in the tag fused to proteins of interest (reactive tag system). I additionally made substantial progress into adapting this system for AMPA receptor subunits.}, author = {Jevtic, Marijo}, issn = {2663-337X}, pages = {108}, publisher = {Institute of Science and Technology Austria}, title = {{Contextual fear learning induced changes in AMPA receptor subtypes along the proximodistal axis in dorsal hippocampus}}, doi = {10.15479/at:ista:11393}, year = {2022}, } @article{11705, abstract = {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.}, author = {Chang, Cheng and Liu, Yu and Lee, Seungho and Spadaro, Maria and Koskela, Kristopher M. and Kleinhanns, Tobias and Costanzo, Tommaso and Arbiol, Jordi and Brutchey, Richard L. and Ibáñez, Maria}, issn = {1521-3773}, journal = {Angewandte Chemie - International Edition}, number = {35}, publisher = {Wiley}, title = {{Surface functionalization of surfactant-free particles: A strategy to tailor the properties of nanocomposites for enhanced thermoelectric performance}}, doi = {10.1002/anie.202207002}, volume = {61}, year = {2022}, } @article{11843, abstract = {A key attribute of persistent or recurring bacterial infections is the ability of the pathogen to evade the host’s immune response. Many Enterobacteriaceae express type 1 pili, a pre-adapted virulence trait, to invade host epithelial cells and establish persistent infections. However, the molecular mechanisms and strategies by which bacteria actively circumvent the immune response of the host remain poorly understood. Here, we identified CD14, the major co-receptor for lipopolysaccharide detection, on mouse dendritic cells (DCs) as a binding partner of FimH, the protein located at the tip of the type 1 pilus of Escherichia coli. The FimH amino acids involved in CD14 binding are highly conserved across pathogenic and non-pathogenic strains. Binding of the pathogenic strain CFT073 to CD14 reduced DC migration by overactivation of integrins and blunted expression of co-stimulatory molecules by overactivating the NFAT (nuclear factor of activated T-cells) pathway, both rate-limiting factors of T cell activation. This response was binary at the single-cell level, but averaged in larger populations exposed to both piliated and non-piliated pathogens, presumably via the exchange of immunomodulatory cytokines. While defining an active molecular mechanism of immune evasion by pathogens, the interaction between FimH and CD14 represents a potential target to interfere with persistent and recurrent infections, such as urinary tract infections or Crohn’s disease.}, author = {Tomasek, Kathrin and Leithner, Alexander F and Glatzová, Ivana and Lukesch, Michael S. and Guet, Calin C and Sixt, Michael K}, issn = {2050-084X}, journal = {eLife}, publisher = {eLife Sciences Publications}, title = {{Type 1 piliated uropathogenic Escherichia coli hijack the host immune response by binding to CD14}}, doi = {10.7554/eLife.78995}, volume = {11}, year = {2022}, } @article{12065, abstract = {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.}, author = {Prehal, Christian and Mondal, Soumyadip and Lovicar, Ludek and Freunberger, Stefan Alexander}, issn = {2380-8195}, journal = {ACS Energy Letters}, number = {9}, pages = {3112--3119}, publisher = {American Chemical Society}, title = {{Exclusive solution discharge in Li-O₂ batteries?}}, doi = {10.1021/acsenergylett.2c01711}, volume = {7}, year = {2022}, } @article{12138, abstract = {Complex I is the first enzyme in the respiratory chain, which is responsible for energy production in mitochondria and bacteria1. Complex I couples the transfer of two electrons from NADH to quinone and the translocation of four protons across the membrane2, but the coupling mechanism remains contentious. Here we present cryo-electron microscopy structures of Escherichia coli complex I (EcCI) in different redox states, including catalytic turnover. EcCI exists mostly in the open state, in which the quinone cavity is exposed to the cytosol, allowing access for water molecules, which enable quinone movements. Unlike the mammalian paralogues3, EcCI can convert to the closed state only during turnover, showing that closed and open states are genuine turnover intermediates. The open-to-closed transition results in the tightly engulfed quinone cavity being connected to the central axis of the membrane arm, a source of substrate protons. Consistently, the proportion of the closed state increases with increasing pH. We propose a detailed but straightforward and robust mechanism comprising a ‘domino effect’ series of proton transfers and electrostatic interactions: the forward wave (‘dominoes stacking’) primes the pump, and the reverse wave (‘dominoes falling’) results in the ejection of all pumped protons from the distal subunit NuoL. This mechanism explains why protons exit exclusively from the NuoL subunit and is supported by our mutagenesis data. We contend that this is a universal coupling mechanism of complex I and related enzymes.}, author = {Kravchuk, Vladyslav and Petrova, Olga and Kampjut, Domen and Wojciechowska-Bason, Anna and Breese, Zara and Sazanov, Leonid A}, issn = {1476-4687}, journal = {Nature}, keywords = {Multidisciplinary}, number = {7928}, pages = {808--814}, publisher = {Springer Nature}, title = {{A universal coupling mechanism of respiratory complex I}}, doi = {10.1038/s41586-022-05199-7}, volume = {609}, year = {2022}, } @article{12143, abstract = {MicroRNA (miRNA) and RNA interference (RNAi) pathways rely on small RNAs produced by Dicer endonucleases. Mammalian Dicer primarily supports the essential gene-regulating miRNA pathway, but how it is specifically adapted to miRNA biogenesis is unknown. We show that the adaptation entails a unique structural role of Dicer’s DExD/H helicase domain. Although mice tolerate loss of its putative ATPase function, the complete absence of the domain is lethal because it assures high-fidelity miRNA biogenesis. Structures of murine Dicer⋅miRNA precursor complexes revealed that the DExD/H domain has a helicase-unrelated structural function. It locks Dicer in a closed state, which facilitates miRNA precursor selection. Transition to a cleavage-competent open state is stimulated by Dicer-binding protein TARBP2. Absence of the DExD/H domain or its mutations unlocks the closed state, reduces substrate selectivity, and activates RNAi. Thus, the DExD/H domain structurally contributes to mammalian miRNA biogenesis and underlies mechanistical partitioning of miRNA and RNAi pathways.}, author = {Zapletal, David and Taborska, Eliska and Pasulka, Josef and Malik, Radek and Kubicek, Karel and Zanova, Martina and Much, Christian and Sebesta, Marek and Buccheri, Valeria and Horvat, Filip and Jenickova, Irena and Prochazkova, Michaela and Prochazka, Jan and Pinkas, Matyas and Novacek, Jiri and Joseph, Diego F. and Sedlacek, Radislav and Bernecky, Carrie A and O’Carroll, Dónal and Stefl, Richard and Svoboda, Petr}, issn = {1097-2765}, journal = {Molecular Cell}, keywords = {Cell Biology, Molecular Biology}, number = {21}, pages = {4064--4079.e13}, publisher = {Elsevier}, title = {{Structural and functional basis of mammalian microRNA biogenesis by Dicer}}, doi = {10.1016/j.molcel.2022.10.010}, volume = {82}, year = {2022}, }