@article{14843,
abstract = {The coupling between Ca2+ channels and release sensors is a key factor defining the signaling properties of a synapse. However, the coupling nanotopography at many synapses remains unknown, and it is unclear how it changes during development. To address these questions, we examined coupling at the cerebellar inhibitory basket cell (BC)-Purkinje cell (PC) synapse. Biophysical analysis of transmission by paired recording and intracellular pipette perfusion revealed that the effects of exogenous Ca2+ chelators decreased during development, despite constant reliance of release on P/Q-type Ca2+ channels. Structural analysis by freeze-fracture replica labeling (FRL) and transmission electron microscopy (EM) indicated that presynaptic P/Q-type Ca2+ channels formed nanoclusters throughout development, whereas docked vesicles were only clustered at later developmental stages. Modeling suggested a developmental transformation from a more random to a more clustered coupling nanotopography. Thus, presynaptic signaling developmentally approaches a point-to-point configuration, optimizing speed, reliability, and energy efficiency of synaptic transmission.},
author = {Chen, JingJing and Kaufmann, Walter and Chen, Chong and Arai, Itaru and Kim, Olena and Shigemoto, Ryuichi and Jonas, Peter M},
issn = {1097-4199},
journal = {Neuron},
publisher = {Elsevier},
title = {{Developmental transformation of Ca2+ channel-vesicle nanotopography at a central GABAergic synapse}},
doi = {10.1016/j.neuron.2023.12.002},
year = {2024},
}
@article{14846,
abstract = {Contraction and flow of the actin cell cortex have emerged as a common principle by which cells reorganize their cytoplasm and take shape. However, how these cortical flows interact with adjacent cytoplasmic components, changing their form and localization, and how this affects cytoplasmic organization and cell shape remains unclear. Here we show that in ascidian oocytes, the cooperative activities of cortical actomyosin flows and deformation of the adjacent mitochondria-rich myoplasm drive oocyte cytoplasmic reorganization and shape changes following fertilization. We show that vegetal-directed cortical actomyosin flows, established upon oocyte fertilization, lead to both the accumulation of cortical actin at the vegetal pole of the zygote and compression and local buckling of the adjacent elastic solid-like myoplasm layer due to friction forces generated at their interface. Once cortical flows have ceased, the multiple myoplasm buckles resolve into one larger buckle, which again drives the formation of the contraction pole—a protuberance of the zygote’s vegetal pole where maternal mRNAs accumulate. Thus, our findings reveal a mechanism where cortical actomyosin network flows determine cytoplasmic reorganization and cell shape by deforming adjacent cytoplasmic components through friction forces.},
author = {Caballero Mancebo, Silvia and Shinde, Rushikesh and Bolger-Munro, Madison and Peruzzo, Matilda and Szep, Gregory and Steccari, Irene and Labrousse Arias, David and Zheden, Vanessa and Merrin, Jack and Callan-Jones, Andrew and Voituriez, Raphaël and Heisenberg, Carl-Philipp J},
issn = {1745-2481},
journal = {Nature Physics},
publisher = {Springer Nature},
title = {{Friction forces determine cytoplasmic reorganization and shape changes of ascidian oocytes upon fertilization}},
doi = {10.1038/s41567-023-02302-1},
year = {2024},
}
@article{14979,
abstract = {Poxviruses are among the largest double-stranded DNA viruses, with members such as variola virus, monkeypox virus and the vaccination strain vaccinia virus (VACV). Knowledge about the structural proteins that form the viral core has remained sparse. While major core proteins have been annotated via indirect experimental evidence, their structures have remained elusive and they could not be assigned to individual core features. Hence, which proteins constitute which layers of the core, such as the palisade layer and the inner core wall, has remained enigmatic. Here we show, using a multi-modal cryo-electron microscopy (cryo-EM) approach in combination with AlphaFold molecular modeling, that trimers formed by the cleavage product of VACV protein A10 are the key component of the palisade layer. This allows us to place previously obtained descriptions of protein interactions within the core wall into perspective and to provide a detailed model of poxvirus core architecture. Importantly, we show that interactions within A10 trimers are likely generalizable over members of orthopox- and parapoxviruses.},
author = {Datler, Julia and Hansen, Jesse and Thader, Andreas and Schlögl, Alois and Bauer, Lukas W and Hodirnau, Victor-Valentin and Schur, Florian KM},
issn = {1545-9985},
journal = {Nature Structural & Molecular Biology},
keywords = {Molecular Biology, Structural Biology},
publisher = {Springer Nature},
title = {{Multi-modal cryo-EM reveals trimers of protein A10 to form the palisade layer in poxvirus cores}},
doi = {10.1038/s41594-023-01201-6},
year = {2024},
}
@article{14434,
abstract = {High entropy alloys (HEAs) are highly suitable candidate catalysts for oxygen evolution and reduction reactions (OER/ORR) as they offer numerous parameters for optimizing the electronic structure and catalytic sites. Herein, FeCoNiMoW HEA nanoparticles are synthesized using a solution‐based low‐temperature approach. Such FeCoNiMoW nanoparticles show high entropy properties, subtle lattice distortions, and modulated electronic structure, leading to superior OER performance with an overpotential of 233 mV at 10 mA cm−2 and 276 mV at 100 mA cm−2. Density functional theory calculations reveal the electronic structures of the FeCoNiMoW active sites with an optimized d‐band center position that enables suitable adsorption of OOH* intermediates and reduces the Gibbs free energy barrier in the OER process. Aqueous zinc–air batteries (ZABs) based on this HEA demonstrate a high open circuit potential of 1.59 V, a peak power density of 116.9 mW cm−2, a specific capacity of 857 mAh gZn−1, and excellent stability for over 660 h of continuous charge–discharge cycles. Flexible and solid ZABs are also assembled and tested, displaying excellent charge–discharge performance at different bending angles. This work shows the significance of 4d/5d metal‐modulated electronic structure and optimized adsorption ability to improve the performance of OER/ORR, ZABs, and beyond.},
author = {He, Ren and Yang, Linlin and Zhang, Yu and Jiang, Daochuan and Lee, Seungho and Horta, Sharona and Liang, Zhifu and Lu, Xuan and Ostovari Moghaddam, Ahmad and Li, Junshan and Ibáñez, Maria and Xu, Ying and Zhou, Yingtang and Cabot, Andreu},
issn = {0935-9648},
journal = {Advanced Materials},
keywords = {Mechanical Engineering, Mechanics of Materials, General Materials Science},
publisher = {Wiley},
title = {{A 3d‐4d‐5d high entropy alloy as a bifunctional oxygen catalyst for robust aqueous zinc–air batteries}},
doi = {10.1002/adma.202303719},
year = {2023},
}
@phdthesis{14510,
author = {Gnyliukh, Nataliia},
isbn = {978-3-99078-037-4},
issn = {2663-337X},
keywords = {Clathrin-Mediated Endocytosis, vesicle scission, Dynamin-Related Protein 2, SH3P2, TPLATE complex, Total internal reflection fluorescence microscopy, Arabidopsis thaliana},
pages = {180},
publisher = {Institute of Science and Technology Austria},
title = {{Mechanism of clathrin-coated vesicle formation during endocytosis in plants}},
doi = {10.15479/at:ista:14510},
year = {2023},
}
@misc{14562,
abstract = {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.
},
author = {Schur, Florian KM},
publisher = {Institute of Science and Technology Austria},
title = {{Research data of the publication "ArpC5 isoforms regulate Arp2/3 complex-dependent protrusion through differential Ena/VASP positioning"}},
doi = {10.15479/AT:ISTA:14562},
year = {2023},
}
@unpublished{14591,
abstract = {Clathrin-mediated endocytosis (CME) is vital for the regulation of plant growth and development by controlling plasma membrane protein composition and cargo uptake. CME relies on the precise recruitment of regulators for vesicle maturation and release. Homologues of components of mammalian vesicle scission are strong candidates to be part of the scissin machinery in plants, but the precise roles of these proteins in this process is not fully understood. Here, we characterised the roles of Plant Dynamin-Related Proteins 2 (DRP2s) and SH3-domain containing protein 2 (SH3P2), the plant homologue to Dynamins’ recruiters, like Endophilin and Amphiphysin, in the CME by combining high-resolution imaging of endocytic events in vivo and characterisation of the purified proteins in vitro. Although DRP2s and SH3P2 arrive similarly late during CME and physically interact, genetic analysis of the Dsh3p1,2,3 triple-mutant and complementation assays with non-SH3P2-interacting DRP2 variants suggests that SH3P2 does not directly recruit DRP2s to the site of endocytosis. These observations imply that despite the presence of many well-conserved endocytic components, plants have acquired a distinct mechanism for CME. One Sentence Summary In contrast to predictions based on mammalian systems, plant Dynamin-related proteins 2 are recruited to the site of Clathrin-mediated endocytosis independently of BAR-SH3 proteins.},
author = {Gnyliukh, Nataliia and Johnson, Alexander J and Nagel, Marie-Kristin and Monzer, Aline and Hlavata, Annamaria and Isono, Erika and Loose, Martin and Friml, Jiří},
booktitle = {bioRxiv},
title = {{Role of dynamin-related proteins 2 and SH3P2 in clathrin-mediated endocytosis in plants}},
doi = {10.1101/2023.10.09.561523},
year = {2023},
}
@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},
}
@phdthesis{13107,
abstract = {Within the human body, the brain exhibits the highest rate of energy consumption amongst all organs, with the majority of generated ATP being utilized to sustain neuronal activity. Therefore, the metabolism of the mature cerebral cortex is geared towards preserving metabolic homeostasis whilst generating significant amounts of energy. This requires a precise interplay between diverse metabolic pathways, spanning from a tissue-wide scale to the level of individual neurons. Disturbances to this delicate metabolic equilibrium, such as those resulting from maternal malnutrition
or mutations affecting metabolic enzymes, often result in neuropathological variants of neurodevelopment. For instance, mutations in SLC7A5, a transporter of metabolically essential large neutral amino acids (LNAAs), have been associated with autism and microcephaly. However, despite recent progress in the field, the extent of metabolic restructuring that occurs within the developing brain and the corresponding alterations in nutrient demands during various critical periods remain largely unknown. To investigate this, we performed metabolomic profiling of the murine cerebral cortex to characterize the metabolic state of the forebrain at different developmental stages. We found that the developing cortex undergoes substantial metabolic reprogramming, with specific sets of metabolites displaying stage-specific changes. According to our observations, we determined a distinct temporal period in postnatal development during which the cortex displays heightened reliance on LNAAs. Hence, using a conditional knock-out mouse model, we deleted Slc7a5 in neural cells, allowing us to monitor the impact of a perturbed neuronal metabolic state across multiple developmental stages of corticogenesis. We found that manipulating the levels of essential LNAAs in cortical neurons in vivo affects one particular perinatal developmental period critical for cortical network refinement. Abnormally low intracellular LNAA levels result in cell-autonomous alterations in neuronal lipid metabolism, excitability, and survival during this particular time window. Although most of the effects of Slc7a5 deletion on neuronal physiology are transient, derailment of these processes during this brief but crucial window leads to long-term circuit dysfunction in mice. In conclusion, out data indicate that the cerebral cortex undergoes significant metabolic reorganization during development. This process involves the intricate integration of multiple metabolic pathways to ensure optimal neuronal function throughout different developmental stages. Our findings offer a paradigm for understanding how neurons synchronize the expression of nutrient-related genes with their activity to allow proper brain maturation. Further, our results demonstrate that disruptions in these precisely calibrated metabolic processes during critical periods of brain development may result in neuropathological outcomes in mice and in humans.},
author = {Knaus, Lisa},
issn = {2663 - 337X},
pages = {147},
publisher = {Institute of Science and Technology Austria},
title = {{The metabolism of the developing brain : How large neutral amino acids modulate perinatal neuronal excitability and survival}},
doi = {10.15479/at:ista:13107},
year = {2023},
}
@article{13202,
abstract = {Phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) plays an essential role in neuronal activities through interaction with various proteins involved in signaling at membranes. However, the distribution pattern of PI(4,5)P2 and the association with these proteins on the neuronal cell membranes remain elusive. In this study, we established a method for visualizing PI(4,5)P2 by SDS-digested freeze-fracture replica labeling (SDS-FRL) to investigate the quantitative nanoscale distribution of PI(4,5)P2 in cryo-fixed brain. We demonstrate that PI(4,5)P2 forms tiny clusters with a mean size of ∼1000 nm2 rather than randomly distributed in cerebellar neuronal membranes in male C57BL/6J mice. These clusters show preferential accumulation in specific membrane compartments of different cell types, in particular, in Purkinje cell (PC) spines and granule cell (GC) presynaptic active zones. Furthermore, we revealed extensive association of PI(4,5)P2 with CaV2.1 and GIRK3 across different membrane compartments, whereas its association with mGluR1α was compartment specific. These results suggest that our SDS-FRL method provides valuable insights into the physiological functions of PI(4,5)P2 in neurons.},
author = {Eguchi, Kohgaku and Le Monnier, Elodie and Shigemoto, Ryuichi},
issn = {1529-2401},
journal = {The Journal of Neuroscience},
number = {23},
pages = {4197--4216},
publisher = {Society for Neuroscience},
title = {{Nanoscale phosphoinositide distribution on cell membranes of mouse cerebellar neurons}},
doi = {10.1523/JNEUROSCI.1514-22.2023},
volume = {43},
year = {2023},
}
@article{13968,
abstract = {The use of multimodal readout mechanisms next to label-free real-time monitoring of biomolecular interactions can provide valuable insight into surface-based reaction mechanisms. To this end, the combination of an electrolyte-gated field-effect transistor (EG-FET) with a fiber optic-coupled surface plasmon resonance (FO-SPR) probe serving as gate electrode has been investigated to deconvolute surface mass and charge density variations associated to surface reactions. However, applying an electrochemical potential on such gold-coated FO-SPR gate electrodes can induce gradual morphological changes of the thin gold film, leading to an irreversible blue-shift of the SPR wavelength and a substantial signal drift. We show that mild annealing leads to optical and electronic signal stabilization (20-fold lower signal drift than as-sputtered fiber optic gates) and improved overall analytical performance characteristics. The thermal treatment prevents morphological changes of the thin gold-film occurring during operation, hence providing reliable and stable data immediately upon gate voltage application. Thus, the readout output of both transducing principles, the optical FO-SPR and electronic EG-FET, stays constant throughout the whole sensing time-window and the long-term effect of thermal treatment is also improved, providing stable signals even after 1 year of storage. Annealing should therefore be considered a necessary modification for applying fiber optic gate electrodes in real-time multimodal investigations of surface reactions at the solid-liquid interface.},
author = {Hasler, Roger and Steger-Polt, Marie Helene and Reiner-Rozman, Ciril and Fossati, Stefan and Lee, Seungho and Aspermair, Patrik and Kleber, Christoph and Ibáñez, Maria and Dostalek, Jakub and Knoll, Wolfgang},
issn = {2296-424X},
journal = {Frontiers in Physics},
publisher = {Frontiers},
title = {{Optical and electronic signal stabilization of plasmonic fiber optic gate electrodes: Towards improved real-time dual-mode biosensing}},
doi = {10.3389/fphy.2023.1202132},
volume = {11},
year = {2023},
}
@article{14040,
abstract = {Robust oxygenic photosynthesis requires a suite of accessory factors to ensure efficient assembly and repair of the oxygen-evolving photosystem two (PSII) complex. The highly conserved Ycf48 assembly factor binds to the newly synthesized D1 reaction center polypeptide and promotes the initial steps of PSII assembly, but its binding site is unclear. Here we use cryo-electron microscopy to determine the structure of a cyanobacterial PSII D1/D2 reaction center assembly complex with Ycf48 attached. Ycf48, a 7-bladed beta propeller, binds to the amino-acid residues of D1 that ultimately ligate the water-oxidising Mn4CaO5 cluster, thereby preventing the premature binding of Mn2+ and Ca2+ ions and protecting the site from damage. Interactions with D2 help explain how Ycf48 promotes assembly of the D1/D2 complex. Overall, our work provides valuable insights into the early stages of PSII assembly and the structural changes that create the binding site for the Mn4CaO5 cluster.},
author = {Zhao, Ziyu and Vercellino, Irene and Knoppová, Jana and Sobotka, Roman and Murray, James W. and Nixon, Peter J. and Sazanov, Leonid A and Komenda, Josef},
issn = {2041-1723},
journal = {Nature Communications},
publisher = {Springer Nature},
title = {{The Ycf48 accessory factor occupies the site of the oxygen-evolving manganese cluster during photosystem II biogenesis}},
doi = {10.1038/s41467-023-40388-6},
volume = {14},
year = {2023},
}
@article{14255,
abstract = {Toscana virus is a major cause of arboviral disease in humans in the Mediterranean basin during summer. However, early virus-host cell interactions and entry mechanisms remain poorly characterized. Investigating iPSC-derived human neurons and cell lines, we found that virus binding to the cell surface was specific, and 50% of bound virions were endocytosed within 10 min. Virions entered Rab5a+ early endosomes and, subsequently, Rab7a+ and LAMP-1+ late endosomal compartments. Penetration required intact late endosomes and occurred within 30 min following internalization. Virus entry relied on vacuolar acidification, with an optimal pH for viral membrane fusion at pH 5.5. The pH threshold increased to 5.8 with longer pre-exposure of virions to the slightly acidic pH in early endosomes. Strikingly, the particles remained infectious after entering late endosomes with a pH below the fusion threshold. Overall, our study establishes Toscana virus as a late-penetrating virus and reveals an atypical use of vacuolar acidity by this virus to enter host cells.},
author = {Koch, Jana and Xin, Qilin and Obr, Martin and Schäfer, Alicia and Rolfs, Nina and Anagho, Holda A. and Kudulyte, Aiste and Woltereck, Lea and Kummer, Susann and Campos, Joaquin and Uckeley, Zina M. and Bell-Sakyi, Lesley and Kräusslich, Hans Georg and Schur, Florian Km and Acuna, Claudio and Lozach, Pierre Yves},
issn = {1553-7374},
journal = {PLoS Pathogens},
number = {8},
publisher = {Public Library of Science},
title = {{The phenuivirus Toscana virus makes an atypical use of vacuolar acidity to enter host cells}},
doi = {10.1371/journal.ppat.1011562},
volume = {19},
year = {2023},
}
@article{12334,
abstract = {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.},
author = {Fäßler, Florian and Javoor, Manjunath and Datler, Julia and Döring, Hermann and Hofer, Florian and Dimchev, Georgi A and Hodirnau, Victor-Valentin and Faix, Jan and Rottner, Klemens and Schur, Florian KM},
issn = {2375-2548},
journal = {Science Advances},
keywords = {Multidisciplinary},
number = {3},
publisher = {American Association for the Advancement of Science},
title = {{ArpC5 isoforms regulate Arp2/3 complex–dependent protrusion through differential Ena/VASP positioning}},
doi = {10.1126/sciadv.add6495},
volume = {9},
year = {2023},
}
@phdthesis{12470,
abstract = {The brain is an exceptionally sophisticated organ consisting of billions of cells and trillions of
connections that orchestrate our cognition and behavior. To decode its complex connectivity, it is
pivotal to disentangle its intricate architecture spanning from cm-sized circuits down to tens of
nm-small synapses.
To achieve this goal, I developed CATS – Comprehensive Analysis of nervous Tissue across
Scales, a versatile toolbox for obtaining a holistic view of nervous tissue context with (superresolution) fluorescence microscopy. CATS combines comprehensive labeling of the extracellular
space, that is compatible with chemical fixation, with information on molecular markers, superresolved data acquisition and machine-learning based data analysis for segmentation and synapse
identification.
I used CATS to analyze key features of nervous tissue connectivity, ranging from whole tissue
architecture, neuronal in- and output-fields, down to synapse morphology.
Focusing on the hippocampal circuitry, I quantified synaptic transmission properties of mossy
fiber boutons and analyzed the connectivity pattern of dentate gyrus granule cells with CA3
pyramidal neurons. This shows that CATS is a viable tool to study hallmarks of neuronal
connectivity with light microscopy.},
author = {Michalska, Julia M},
isbn = { 978-3-99078-026-8},
issn = {2663-337X},
pages = {201},
publisher = {Institute of Science and Technology Austria},
title = {{A versatile toolbox for the comprehensive analysis of nervous tissue organization with light microscopy}},
doi = {10.15479/at:ista:12470},
year = {2023},
}
@phdthesis{12491,
abstract = {The extracellular matrix (ECM) is a hydrated and complex three-dimensional network consisting of proteins, polysaccharides, and water. It provides structural scaffolding for the cells embedded within it and is essential in regulating numerous physiological processes, including cell migration and proliferation, wound healing, and stem cell fate.
Despite extensive study, detailed structural knowledge of ECM components in physiologically relevant conditions is still rudimentary. This is due to methodological limitations in specimen preparation protocols which are incompatible with keeping large samples, such as the ECM, in their native state for subsequent imaging. Conventional electron microscopy (EM) techniques rely on fixation, dehydration, contrasting, and sectioning. This results in the alteration of a highly hydrated environment and the potential introduction of artifacts. Other structural biology techniques, such as nuclear magnetic resonance (NMR) spectroscopy and X-ray crystallography, allow high-resolution analysis of protein structures but only work on homogenous and purified samples, hence lacking contextual information. Currently, no approach exists for the ultrastructural and structural study of extracellular components under native conditions in a physiological, 3D environment.
In this thesis, I have developed a workflow that allows for the ultrastructural analysis of the ECM in near-native conditions at molecular resolution. The developments I introduced include implementing a novel specimen preparation workflow for cell-derived matrices (CDMs) to render them compatible with ion-beam milling and subsequent high-resolution cryo-electron tomography (ET).
To this end, I have established protocols to generate CDMs grown over several weeks on EM grids that are compatible with downstream cryo-EM sample preparation and imaging techniques. Characterization of these ECMs confirmed that they contain essential ECM components such as collagen I, collagen VI, and fibronectin I in high abundance and hence represent a bona fide biologically-relevant sample. I successfully optimized vitrification of these specimens by testing various vitrification techniques and cryoprotectants.
In order to obtain high-resolution molecular insights into the ultrastructure and organization of CDMs, I established cryo-focused ion beam scanning electron microscopy (FIBSEM) on these challenging and complex specimens. I explored different approaches for the creation of thin cryo-lamellae by FIB milling and succeeded in optimizing the cryo-lift-out technique, resulting in high-quality lamellae of approximately 200 nm thickness.
High-resolution Cryo-ET of these lamellae revealed for the first time the architecture of native CDM in the context of matrix-secreting cells. This allowed for the in situ visualization of fibrillar matrix proteins such as collagen, laying the foundation for future structural and ultrastructural characterization of these proteins in their near-native environment.
In summary, in this thesis, I present a novel workflow that combines state-of-the-art cryo-EM specimen preparation and imaging technologies to permit characterization of the ECM, an important tissue component in higher organisms. This innovative and highly versatile workflow will enable addressing far-reaching questions on ECM architecture, composition, and reciprocal ECM-cell interactions.},
author = {Zens, Bettina},
isbn = {978-3-99078-027-5},
issn = {2663-337X},
keywords = {cryo-EM, cryo-ET, FIB milling, method development, FIBSEM, extracellular matrix, ECM, cell-derived matrices, CDMs, cell culture, high pressure freezing, HPF, structural biology, tomography, collagen},
pages = {187},
publisher = {Institute of Science and Technology Austria},
title = {{Ultrastructural characterization of natively preserved extracellular matrix by cryo-electron tomography}},
doi = {10.15479/at:ista:12491},
year = {2023},
}
@article{12759,
abstract = {Stereological methods for estimating the 3D particle size and density from 2D projections are essential to many research fields. These methods are, however, prone to errors arising from undetected particle profiles due to sectioning and limited resolution, known as ‘lost caps’. A potential solution developed by Keiding, Jensen, and Ranek in 1972, which we refer to as the Keiding model, accounts for lost caps by quantifying the smallest detectable profile in terms of its limiting ‘cap angle’ (ϕ), a size-independent measure of a particle’s distance from the section surface. However, this simple solution has not been widely adopted nor tested. Rather, model-independent design-based stereological methods, which do not explicitly account for lost caps, have come to the fore. Here, we provide the first experimental validation of the Keiding model by comparing the size and density of particles estimated from 2D projections with direct measurement from 3D EM reconstructions of the same tissue. We applied the Keiding model to estimate the size and density of somata, nuclei and vesicles in the cerebellum of mice and rats, where high packing density can be problematic for design-based methods. Our analysis reveals a Gaussian distribution for ϕ rather than a single value. Nevertheless, curve fits of the Keiding model to the 2D diameter distribution accurately estimate the mean ϕ and 3D diameter distribution. While systematic testing using simulations revealed an upper limit to determining ϕ, our analysis shows that estimated ϕ can be used to determine the 3D particle density from the 2D density under a wide range of conditions, and this method is potentially more accurate than minimum-size-based lost-cap corrections and disector methods. Our results show the Keiding model provides an efficient means of accurately estimating the size and density of particles from 2D projections even under conditions of a high density.},
author = {Rothman, Jason Seth and Borges Merjane, Carolina and Holderith, Noemi and Jonas, Peter M and Angus Silver, R.},
issn = {1932-6203},
journal = {PLoS ONE},
number = {3 March},
publisher = {Public Library of Science},
title = {{Validation of a stereological method for estimating particle size and density from 2D projections with high accuracy}},
doi = {10.1371/journal.pone.0277148},
volume = {18},
year = {2023},
}
@phdthesis{12781,
abstract = {Most energy in humans is produced in form of ATP by the mitochondrial respiratory chain consisting of several protein assemblies embedded into lipid membrane (complexes I-V). Complex I is the first and the largest enzyme of the respiratory chain which is essential for energy production. It couples the transfer of two electrons from NADH to ubiquinone with proton translocation across bacterial or inner mitochondrial membrane. The coupling mechanism between electron transfer and proton translocation is one of the biggest enigma in bioenergetics and structural biology. Even though the enzyme has been studied for decades, only recent technological advances in cryo-EM allowed its extensive structural investigation.
Complex I from E.coli appears to be of special importance because it is a perfect model system with a rich mutant library, however the structure of the entire complex was unknown. In this thesis I have resolved structures of the minimal complex I version from E. coli in different states including reduced, inhibited, under reaction turnover and several others. Extensive structural analyses of these structures and comparison to structures from other species allowed to derive general features of conformational dynamics and propose a universal coupling mechanism. The mechanism is straightforward, robust and consistent with decades of experimental data available for complex I from different species.
Cyanobacterial NDH (cyanobacterial complex I) is a part of broad complex I superfamily and was studied as well in this thesis. It plays an important role in cyclic electron transfer (CET), during which electrons are cycled within PSI through ferredoxin and plastoquinone to generate proton gradient without NADPH production. Here, I solved structure of NDH and revealed additional state, which was not observed before. The novel “resting” state allowed to propose the mechanism of CET regulation. Moreover, conformational dynamics of NDH resembles one in complex I which suggest more broad universality of the proposed coupling mechanism.
In summary, results presented here helped to interpret decades of experimental data for complex I and contributed to fundamental mechanistic understanding of protein function.
},
author = {Kravchuk, Vladyslav},
isbn = {978-3-99078-029-9},
issn = {2663-337X},
pages = {127},
publisher = {Institute of Science and Technology Austria},
title = {{Structural and mechanistic study of bacterial complex I and its cyanobacterial ortholog}},
doi = {10.15479/at:ista:12781},
year = {2023},
}
@article{12802,
abstract = {Little is known about the critical metabolic changes that neural cells have to undergo during development and how temporary shifts in this program can influence brain circuitries and behavior. Inspired by the discovery that mutations in SLC7A5, a transporter of metabolically essential large neutral amino acids (LNAAs), lead to autism, we employed metabolomic profiling to study the metabolic states of the cerebral cortex across different developmental stages. We found that the forebrain undergoes significant metabolic remodeling throughout development, with certain groups of metabolites showing stage-specific changes, but what are the consequences of perturbing this metabolic program? By manipulating Slc7a5 expression in neural cells, we found that the metabolism of LNAAs and lipids are interconnected in the cortex. Deletion of Slc7a5 in neurons affects the postnatal metabolic state, leading to a shift in lipid metabolism. Additionally, it causes stage- and cell-type-specific alterations in neuronal activity patterns, resulting in a long-term circuit dysfunction.},
author = {Knaus, Lisa and Basilico, Bernadette and Malzl, Daniel and Gerykova Bujalkova, Maria and Smogavec, Mateja and Schwarz, Lena A. and Gorkiewicz, Sarah and Amberg, Nicole and Pauler, Florian and Knittl-Frank, Christian and Tassinari, Marianna and Maulide, Nuno and Rülicke, Thomas and Menche, Jörg and Hippenmeyer, Simon and Novarino, Gaia},
issn = {0092-8674},
journal = {Cell},
keywords = {General Biochemistry, Genetics and Molecular Biology},
number = {9},
pages = {1950--1967.e25},
publisher = {Elsevier},
title = {{Large neutral amino acid levels tune perinatal neuronal excitability and survival}},
doi = {10.1016/j.cell.2023.02.037},
volume = {186},
year = {2023},
}