@misc{13126, abstract = {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.}, author = {Danzl, Johann G}, publisher = {Institute of Science and Technology Austria}, title = {{Research data for the publication "Imaging brain tissue architecture across millimeter to nanometer scales"}}, doi = {10.15479/AT:ISTA:13126}, year = {2023}, } @article{14257, abstract = {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.}, author = {Michalska, Julia M and Lyudchik, Julia and Velicky, Philipp and Korinkova, Hana and Watson, Jake and Cenameri, Alban and Sommer, Christoph M and Amberg, Nicole and Venturino, Alessandro and Roessler, Karl and Czech, Thomas and Höftberger, Romana and Siegert, Sandra and Novarino, Gaia and Jonas, Peter M and Danzl, Johann G}, issn = {1546-1696}, journal = {Nature Biotechnology}, publisher = {Springer Nature}, title = {{Imaging brain tissue architecture across millimeter to nanometer scales}}, doi = {10.1038/s41587-023-01911-8}, year = {2023}, } @article{13044, abstract = {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.}, author = {Mondal, Soumyadip and Jethwa, Rajesh B and Pant, Bhargavi and Hauschild, Robert and Freunberger, Stefan Alexander}, issn = {1364-5498}, journal = {Faraday Discussions}, keywords = {Physical and Theoretical Chemistry}, publisher = {Royal Society of Chemistry}, title = {{Singlet oxygen in non-aqueous oxygen redox: Direct spectroscopic evidence for formation pathways and reliability of chemical probes}}, doi = {10.1039/d3fd00088e}, year = {2023}, } @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{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{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{12122, abstract = {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.}, author = {Weier, Ann-Kathrin and Homrich, Mirka and Ebbinghaus, Stephanie and Juda, Pavel and Miková, Eliška and Hauschild, Robert and Zhang, Lili and Quast, Thomas and Mass, Elvira and Schlitzer, Andreas and Kolanus, Waldemar and Burgdorf, Sven and Gruß, Oliver J. and Hons, Miroslav and Wieser, Stefan and Kiermaier, Eva}, issn = {1540-8140}, journal = {Journal of Cell Biology}, keywords = {Cell Biology}, number = {12}, publisher = {Rockefeller University Press}, title = {{Multiple centrosomes enhance migration and immune cell effector functions of mature dendritic cells}}, doi = {10.1083/jcb.202107134}, volume = {221}, year = {2022}, } @article{8582, abstract = {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.}, author = {Li, Hongjiang and von Wangenheim, Daniel and Zhang, Xixi and Tan, Shutang and Darwish-Miranda, Nasser and Naramoto, Satoshi and Wabnik, Krzysztof T and de Rycke, Riet and Kaufmann, Walter and Gütl, Daniel J and Tejos, Ricardo and Grones, Peter and Ke, Meiyu and Chen, Xu and Dettmer, Jan and Friml, Jiří}, issn = {14698137}, journal = {New Phytologist}, number = {1}, pages = {351--369}, publisher = {Wiley}, title = {{Cellular requirements for PIN polar cargo clustering in Arabidopsis thaliana}}, doi = {10.1111/nph.16887}, volume = {229}, year = {2021}, } @article{9259, abstract = {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.}, author = {Vaahtomeri, Kari and Moussion, Christine and Hauschild, Robert and Sixt, Michael K}, issn = {1664-3224}, journal = {Frontiers in Immunology}, publisher = {Frontiers}, title = {{Shape and function of interstitial chemokine CCL21 gradients are independent of heparan sulfates produced by lymphatic endothelium}}, doi = {10.3389/fimmu.2021.630002}, volume = {12}, year = {2021}, } @article{9361, abstract = {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.}, author = {Gast, Matthieu and Kadzioch, Nicole P. and Milius, Doreen and Origgi, Francesco and Plattet, Philippe}, issn = {23795042}, journal = {mSphere}, number = {2}, publisher = {American Society for Microbiology}, title = {{Oligomerization and cell egress controlled by two microdomains of canine distemper virus matrix protein}}, doi = {10.1128/mSphere.01024-20}, volume = {6}, year = {2021}, }