--- _id: '8350' abstract: - lang: eng text: "Cytoplasm is a gel-like crowded environment composed of tens of thousands of macromolecules, organelles, cytoskeletal networks and cytosol. The structure of the cytoplasm is thought to be highly organized and heterogeneous due to the crowding of its constituents and their effective compartmentalization. In such an environment, the diffusive dynamics of the molecules is very restricted, an effect that is further amplified by clustering and anchoring of molecules. Despite the jammed nature of the cytoplasm at the microscopic scale, large-scale reorganization of cytoplasm is essential for important cellular functions, such as nuclear positioning and cell division. How such mesoscale reorganization of the cytoplasm is achieved, especially for very large cells such as oocytes or syncytial tissues that can span hundreds of micrometers in size, has only begun to be understood.\r\nIn this thesis, I focus on the recent advances in elucidating the molecular, cellular and biophysical principles underlying cytoplasmic organization across different scales, structures and species. First, I outline which of these principles have been identified by reductionist approaches, such as in vitro reconstitution assays, where boundary conditions and components can be modulated at ease. I then describe how the theoretical and experimental framework established in these reduced systems have been applied to their more complex in vivo counterparts, in particular oocytes and embryonic syncytial structures, and discuss how such complex biological systems can initiate symmetry breaking and establish patterning.\r\nSpecifically, I examine an example of large-scale reorganizations taking place in zebrafish embryos, where extensive cytoplasmic streaming leads to the segregation of cytoplasm from yolk granules along the animal-vegetal axis of the embryo. Using biophysical experimentation and theory, I investigate the forces underlying this process, to show that this process does not rely on cortical actin reorganization, as previously thought, but instead on a cell-cycle-dependent bulk actin polymerization wave traveling from the animal to the vegetal pole of the embryo. This wave functions in segregation by both pulling cytoplasm animally and pushing yolk granules vegetally. Cytoplasm pulling is mediated by bulk actin network flows exerting friction forces on the cytoplasm, while yolk granule pushing is achieved by a mechanism closely resembling actin comet formation on yolk granules. This study defines a novel role of bulk actin polymerization waves in embryo polarization via cytoplasmic segregation. Lastly, I describe the cytoplasmic reorganizations taking place during zebrafish oocyte maturation, where the initial segregation of the cytoplasm and yolk granules occurs. Here, I demonstrate a previously uncharacterized wave of microtubule aster formation, traveling the oocyte along the animal-vegetal axis. Further research is required to determine the role of such microtubule structures in cytoplasmic reorganizations therein.\r\nCollectively, these studies provide further evidence for the coupling between cell cytoskeleton and cell cycle machinery, which can underlie a core self-organizing mechanism for orchestrating large-scale reorganizations in a cell-cycle-tunable manner, where the modulations of the force-generating machinery and cytoplasmic mechanics can be harbored to fulfill cellular functions." acknowledged_ssus: - _id: PreCl - _id: Bio - _id: EM-Fac acknowledgement: "I would have had no fish and hence no results without our wonderful fish facility crew, Verena Mayer, Eva Schlegl, Andreas Mlak and Matthias Nowak. Special thanks to Verena for being always happy to help and dealing with our chaotic schedules in the lab. Danke auch, Verena, für deine Geduld, mit mir auf Deutsch zu sprechen. Das hat mir sehr geholfen.\r\nSpecial thanks to the Bioimaging and EM facilities at IST Austria for supporting us every day. Very special thanks would go to Robert Hauschild for his continuous support on data analysis and also to Jack Merrin for designing and building microfabricated chambers for the project and for the various discussions on making zebrafish extracts." alternative_title: - ISTA Thesis article_processing_charge: No author: - first_name: Shayan full_name: Shamipour, Shayan id: 40B34FE2-F248-11E8-B48F-1D18A9856A87 last_name: Shamipour citation: ama: Shamipour S. Bulk actin dynamics drive phase segregation in zebrafish oocytes . 2020. doi:10.15479/AT:ISTA:8350 apa: Shamipour, S. (2020). Bulk actin dynamics drive phase segregation in zebrafish oocytes . Institute of Science and Technology Austria. https://doi.org/10.15479/AT:ISTA:8350 chicago: Shamipour, Shayan. “Bulk Actin Dynamics Drive Phase Segregation in Zebrafish Oocytes .” Institute of Science and Technology Austria, 2020. https://doi.org/10.15479/AT:ISTA:8350. ieee: S. Shamipour, “Bulk actin dynamics drive phase segregation in zebrafish oocytes ,” Institute of Science and Technology Austria, 2020. ista: Shamipour S. 2020. Bulk actin dynamics drive phase segregation in zebrafish oocytes . Institute of Science and Technology Austria. mla: Shamipour, Shayan. Bulk Actin Dynamics Drive Phase Segregation in Zebrafish Oocytes . Institute of Science and Technology Austria, 2020, doi:10.15479/AT:ISTA:8350. short: S. Shamipour, Bulk Actin Dynamics Drive Phase Segregation in Zebrafish Oocytes , Institute of Science and Technology Austria, 2020. date_created: 2020-09-09T11:12:10Z date_published: 2020-09-09T00:00:00Z date_updated: 2023-09-27T14:16:45Z day: '09' ddc: - '570' degree_awarded: PhD department: - _id: BjHo - _id: CaHe doi: 10.15479/AT:ISTA:8350 file: - access_level: closed checksum: 6e47871c74f85008b9876112eb3fcfa1 content_type: application/vnd.openxmlformats-officedocument.wordprocessingml.document creator: sshamip date_created: 2020-09-09T11:06:27Z date_updated: 2021-09-11T22:30:05Z embargo_to: open_access file_id: '8351' file_name: Shayan-Thesis-Final.docx file_size: 65194814 relation: source_file - access_level: open_access checksum: 1b44c57f04d7e8a6fe41b1c9c55a52a3 content_type: application/pdf creator: sshamip date_created: 2020-09-09T11:06:13Z date_updated: 2021-09-11T22:30:05Z embargo: 2021-09-10 file_id: '8352' file_name: Shayan-Thesis-Final.pdf file_size: 23729605 relation: main_file file_date_updated: 2021-09-11T22:30:05Z has_accepted_license: '1' language: - iso: eng month: '09' oa: 1 oa_version: None page: '107' publication_identifier: issn: - 2663-337X publication_status: published publisher: Institute of Science and Technology Austria related_material: record: - id: '661' relation: part_of_dissertation status: public - id: '6508' relation: part_of_dissertation status: public - id: '7001' relation: part_of_dissertation status: public - id: '735' relation: part_of_dissertation status: public status: public supervisor: - first_name: Carl-Philipp J full_name: Heisenberg, Carl-Philipp J id: 39427864-F248-11E8-B48F-1D18A9856A87 last_name: Heisenberg orcid: 0000-0002-0912-4566 - first_name: Björn full_name: Hof, Björn id: 3A374330-F248-11E8-B48F-1D18A9856A87 last_name: Hof orcid: 0000-0003-2057-2754 title: 'Bulk actin dynamics drive phase segregation in zebrafish oocytes ' type: dissertation user_id: c635000d-4b10-11ee-a964-aac5a93f6ac1 year: '2020' ... --- _id: '8353' abstract: - lang: eng text: "Mrp (Multi resistance and pH adaptation) are broadly distributed secondary active antiporters that catalyze the transport of monovalent ions such as sodium and potassium outside of the cell coupled to the inward translocation of protons. Mrp antiporters are unique in a way that they are composed of seven subunits (MrpABCDEFG) encoded in a single operon, whereas other antiporters catalyzing the same reaction are mostly encoded by a single gene. Mrp exchangers are crucial for intracellular pH homeostasis and Na+ efflux, essential mechanisms for H+ uptake under alkaline environments and for reduction of the intracellular concentration of toxic cations. Mrp displays no homology to any other monovalent Na+(K+)/H+ antiporters but Mrp subunits have primary sequence similarity to essential redox-driven proton pumps, such as respiratory complex I and membrane-bound hydrogenases. This similarity reinforces the hypothesis that these present day redox-driven proton pumps are descended from the Mrp antiporter. The Mrp structure serves as a model to understand the yet obscure coupling mechanism between ion or electron transfer and proton translocation in this large group of proteins. In the thesis, I am presenting the purification, biochemical analysis, cryo-EM analysis and molecular structure of the Mrp complex from Anoxybacillus flavithermus solved by cryo-EM at 3.0 Å resolution. Numerous conditions were screened to purify Mrp to high homogeneity and to obtain an appropriate distribution of single particles on cryo-EM grids covered with a continuous layer of ultrathin carbon. A preferred particle orientation problem was solved by performing a tilted data collection. The activity assays showed the specific pH-dependent\r\nprofile of secondary active antiporters. The molecular structure shows that Mrp is a dimer of seven-subunit protomers with 50 trans-membrane helices each. The dimer interface is built by many short and tilted transmembrane helices, probably causing a thinning of the bacterial membrane. The surface charge distribution shows an extraordinary asymmetry within each monomer, revealing presumable proton and sodium translocation pathways. The two largest\r\nand homologous Mrp subunits MrpA and MrpD probably translocate one proton each into the cell. The sodium ion is likely being translocated in the opposite direction within the small subunits along a ladder of charged and conserved residues. Based on the structure, we propose a mechanism were the antiport activity is accomplished via electrostatic interactions between the charged cations and key charged residues. The flexible key TM helices coordinate these\r\nelectrostatic interactions, while the membrane thinning between the monomers enables the translocation of sodium across the charged membrane. The entire family of redox-driven proton pumps is likely to perform their mechanism in a likewise manner." acknowledged_ssus: - _id: LifeSc - _id: EM-Fac - _id: ScienComp acknowledgement: "I acknowledge the scientific service units of the IST Austria for providing resources by the Life Science Facility, the Electron Microscopy Facility and the high-performance computer cluster. Special thanks to the cryo-EM specialists Valentin Hodirnau and Daniel Johann Gütl for spending many hours with me in front of the microscope and for supporting me to collect the data presented here. I also want to thank Professor Masahiro Ito for providing plasmid DNA\r\nencoding Mrp from Anoxybacillus flavithermus WK1. I am a recipient of a DOC Fellowship of the Austrian Academy of Sciences." alternative_title: - ISTA Thesis article_processing_charge: No author: - first_name: Julia full_name: Steiner, Julia id: 3BB67EB0-F248-11E8-B48F-1D18A9856A87 last_name: Steiner orcid: 0000-0003-0493-3775 citation: ama: Steiner J. Biochemical and structural investigation of the Mrp antiporter, an ancestor of complex I. 2020. doi:10.15479/AT:ISTA:8353 apa: Steiner, J. (2020). Biochemical and structural investigation of the Mrp antiporter, an ancestor of complex I. Institute of Science and Technology Austria. https://doi.org/10.15479/AT:ISTA:8353 chicago: Steiner, Julia. “Biochemical and Structural Investigation of the Mrp Antiporter, an Ancestor of Complex I.” Institute of Science and Technology Austria, 2020. https://doi.org/10.15479/AT:ISTA:8353. ieee: J. Steiner, “Biochemical and structural investigation of the Mrp antiporter, an ancestor of complex I,” Institute of Science and Technology Austria, 2020. ista: Steiner J. 2020. Biochemical and structural investigation of the Mrp antiporter, an ancestor of complex I. Institute of Science and Technology Austria. mla: Steiner, Julia. Biochemical and Structural Investigation of the Mrp Antiporter, an Ancestor of Complex I. Institute of Science and Technology Austria, 2020, doi:10.15479/AT:ISTA:8353. short: J. Steiner, Biochemical and Structural Investigation of the Mrp Antiporter, an Ancestor of Complex I, Institute of Science and Technology Austria, 2020. date_created: 2020-09-09T14:27:01Z date_published: 2020-09-09T00:00:00Z date_updated: 2023-09-07T13:14:09Z day: '09' ddc: - '572' degree_awarded: PhD department: - _id: LeSa doi: 10.15479/AT:ISTA:8353 file: - access_level: open_access checksum: 2388d7e6e7a4d364c096fa89f305c3de content_type: application/pdf creator: jsteiner date_created: 2020-09-09T14:22:35Z date_updated: 2021-09-16T12:40:56Z file_id: '8354' file_name: Thesis_Julia_Steiner_pdfA.pdf file_size: 117547589 relation: main_file - access_level: closed checksum: ba112f957b7145462d0ab79044873ee9 content_type: application/vnd.openxmlformats-officedocument.wordprocessingml.document creator: jsteiner date_created: 2020-09-09T14:23:25Z date_updated: 2020-09-15T08:48:37Z file_id: '8355' file_name: Thesis_Julia_Steiner.docx file_size: 223328668 relation: source_file file_date_updated: 2021-09-16T12:40:56Z has_accepted_license: '1' language: - iso: eng month: '09' oa: 1 oa_version: None page: '191' project: - _id: 26169496-B435-11E9-9278-68D0E5697425 grant_number: '24741' name: Revealing the functional mechanism of Mrp antiporter, an ancestor of complex I publication_identifier: issn: - 2663-337X publication_status: published publisher: Institute of Science and Technology Austria related_material: record: - id: '8284' relation: part_of_dissertation status: public status: public supervisor: - first_name: Leonid A full_name: Sazanov, Leonid A id: 338D39FE-F248-11E8-B48F-1D18A9856A87 last_name: Sazanov orcid: 0000-0002-0977-7989 title: Biochemical and structural investigation of the Mrp antiporter, an ancestor of complex I type: dissertation user_id: c635000d-4b10-11ee-a964-aac5a93f6ac1 year: '2020' ... --- _id: '8581' abstract: - lang: eng text: The majority of adenosine triphosphate (ATP) powering cellular processes in eukaryotes is produced by the mitochondrial F1Fo ATP synthase. Here, we present the atomic models of the membrane Fo domain and the entire mammalian (ovine) F1Fo, determined by cryo-electron microscopy. Subunits in the membrane domain are arranged in the ‘proton translocation cluster’ attached to the c-ring and a more distant ‘hook apparatus’ holding subunit e. Unexpectedly, this subunit is anchored to a lipid ‘plug’ capping the c-ring. We present a detailed proton translocation pathway in mammalian Fo and key inter-monomer contacts in F1Fo multimers. Cryo-EM maps of F1Fo exposed to calcium reveal a retracted subunit e and a disassembled c-ring, suggesting permeability transition pore opening. We propose a model for the permeability transition pore opening, whereby subunit e pulls the lipid plug out of the c-ring. Our structure will allow the design of drugs for many emerging applications in medicine. acknowledged_ssus: - _id: EM-Fac - _id: ScienComp acknowledgement: We thank J. Novacek from CEITEC (Brno, Czech Republic) for assistance with collecting the FEI Krios dataset and iNEXT for providing access to CEITEC. We thank the IST Austria EM facility for access and assistance with collecting the FEI Glacios dataset. Data processing was performed at the IST high-performance computing cluster. This work has been supported by iNEXT EM HEDC (proposal 4506), funded by the Horizon 2020 Programme of the European Commission. article_processing_charge: No article_type: original author: - first_name: Gergely full_name: Pinke, Gergely id: 4D5303E6-F248-11E8-B48F-1D18A9856A87 last_name: Pinke - first_name: Long full_name: Zhou, Long id: 3E751364-F248-11E8-B48F-1D18A9856A87 last_name: Zhou orcid: 0000-0002-1864-8951 - first_name: Leonid A full_name: Sazanov, Leonid A id: 338D39FE-F248-11E8-B48F-1D18A9856A87 last_name: Sazanov orcid: 0000-0002-0977-7989 citation: ama: Pinke G, Zhou L, Sazanov LA. Cryo-EM structure of the entire mammalian F-type ATP synthase. Nature Structural and Molecular Biology. 2020;27(11):1077-1085. doi:10.1038/s41594-020-0503-8 apa: Pinke, G., Zhou, L., & Sazanov, L. A. (2020). Cryo-EM structure of the entire mammalian F-type ATP synthase. Nature Structural and Molecular Biology. Springer Nature. https://doi.org/10.1038/s41594-020-0503-8 chicago: Pinke, Gergely, Long Zhou, and Leonid A Sazanov. “Cryo-EM Structure of the Entire Mammalian F-Type ATP Synthase.” Nature Structural and Molecular Biology. Springer Nature, 2020. https://doi.org/10.1038/s41594-020-0503-8. ieee: G. Pinke, L. Zhou, and L. A. Sazanov, “Cryo-EM structure of the entire mammalian F-type ATP synthase,” Nature Structural and Molecular Biology, vol. 27, no. 11. Springer Nature, pp. 1077–1085, 2020. ista: Pinke G, Zhou L, Sazanov LA. 2020. Cryo-EM structure of the entire mammalian F-type ATP synthase. Nature Structural and Molecular Biology. 27(11), 1077–1085. mla: Pinke, Gergely, et al. “Cryo-EM Structure of the Entire Mammalian F-Type ATP Synthase.” Nature Structural and Molecular Biology, vol. 27, no. 11, Springer Nature, 2020, pp. 1077–85, doi:10.1038/s41594-020-0503-8. short: G. Pinke, L. Zhou, L.A. Sazanov, Nature Structural and Molecular Biology 27 (2020) 1077–1085. date_created: 2020-09-28T08:59:27Z date_published: 2020-11-01T00:00:00Z date_updated: 2023-08-22T09:33:09Z day: '01' department: - _id: LeSa doi: 10.1038/s41594-020-0503-8 external_id: isi: - '000569299400004' pmid: - '32929284' intvolume: ' 27' isi: 1 issue: '11' language: - iso: eng month: '11' oa_version: None page: 1077-1085 pmid: 1 publication: Nature Structural and Molecular Biology publication_identifier: eissn: - '15459985' issn: - '15459993' publication_status: published publisher: Springer Nature quality_controlled: '1' related_material: link: - description: News on IST Homepage relation: press_release url: https://ist.ac.at/en/news/structure-of-atpase-solved/ scopus_import: '1' status: public title: Cryo-EM structure of the entire mammalian F-type ATP synthase type: journal_article user_id: 4359f0d1-fa6c-11eb-b949-802e58b17ae8 volume: 27 year: '2020' ... --- _id: '8586' abstract: - lang: eng text: Cryo-electron microscopy (cryo-EM) of cellular specimens provides insights into biological processes and structures within a native context. However, a major challenge still lies in the efficient and reproducible preparation of adherent cells for subsequent cryo-EM analysis. This is due to the sensitivity of many cellular specimens to the varying seeding and culturing conditions required for EM experiments, the often limited amount of cellular material and also the fragility of EM grids and their substrate. Here, we present low-cost and reusable 3D printed grid holders, designed to improve specimen preparation when culturing challenging cellular samples directly on grids. The described grid holders increase cell culture reproducibility and throughput, and reduce the resources required for cell culturing. We show that grid holders can be integrated into various cryo-EM workflows, including micro-patterning approaches to control cell seeding on grids, and for generating samples for cryo-focused ion beam milling and cryo-electron tomography experiments. Their adaptable design allows for the generation of specialized grid holders customized to a large variety of applications. acknowledged_ssus: - _id: ScienComp - _id: LifeSc - _id: Bio - _id: EM-Fac acknowledgement: This work was supported by the Austrian Science Fund (FWF, P33367) to FKMS. BZ acknowledges support by the Niederösterreich Fond. This research was also supported by the Scientific Service Units (SSU) of IST Austria through resources provided by Scientific Computing (SciComp), the Life Science Facility (LSF), the BioImaging Facility (BIF) and the Electron Microscopy Facility (EMF). We thank Georgi Dimchev (IST Austria) and Sonja Jacob (Vienna Biocenter Core Facilities) for testing our grid holders in different experimental setups and Daniel Gütl and the Kondrashov group (IST Austria) for granting us repeated access to their 3D printers. We also thank Jonna Alanko and the Sixt lab (IST Austria) for providing us HeLa cells, primary BL6 mouse tail fibroblasts, NIH 3T3 fibroblasts and human telomerase immortalised foreskin fibroblasts for our experiments. We are thankful to Ori Avinoam and William Wan for helpful comments on the manuscript and also thank Dorotea Fracchiolla (Art&Science) for illustrating the graphical abstract. article_number: '107633' article_processing_charge: Yes (via OA deal) article_type: original author: - first_name: Florian full_name: Fäßler, Florian id: 404F5528-F248-11E8-B48F-1D18A9856A87 last_name: Fäßler orcid: 0000-0001-7149-769X - first_name: Bettina full_name: Zens, Bettina id: 45FD126C-F248-11E8-B48F-1D18A9856A87 last_name: Zens - first_name: Robert full_name: Hauschild, Robert id: 4E01D6B4-F248-11E8-B48F-1D18A9856A87 last_name: Hauschild orcid: 0000-0001-9843-3522 - first_name: Florian KM full_name: Schur, Florian KM id: 48AD8942-F248-11E8-B48F-1D18A9856A87 last_name: Schur orcid: 0000-0003-4790-8078 citation: ama: Fäßler F, Zens B, Hauschild R, Schur FK. 3D printed cell culture grid holders for improved cellular specimen preparation in cryo-electron microscopy. Journal of Structural Biology. 2020;212(3). doi:10.1016/j.jsb.2020.107633 apa: Fäßler, F., Zens, B., Hauschild, R., & Schur, F. K. (2020). 3D printed cell culture grid holders for improved cellular specimen preparation in cryo-electron microscopy. Journal of Structural Biology. Elsevier. https://doi.org/10.1016/j.jsb.2020.107633 chicago: Fäßler, Florian, Bettina Zens, Robert Hauschild, and Florian KM Schur. “3D Printed Cell Culture Grid Holders for Improved Cellular Specimen Preparation in Cryo-Electron Microscopy.” Journal of Structural Biology. Elsevier, 2020. https://doi.org/10.1016/j.jsb.2020.107633. ieee: F. Fäßler, B. Zens, R. Hauschild, and F. K. Schur, “3D printed cell culture grid holders for improved cellular specimen preparation in cryo-electron microscopy,” Journal of Structural Biology, vol. 212, no. 3. Elsevier, 2020. ista: Fäßler F, Zens B, Hauschild R, Schur FK. 2020. 3D printed cell culture grid holders for improved cellular specimen preparation in cryo-electron microscopy. Journal of Structural Biology. 212(3), 107633. mla: Fäßler, Florian, et al. “3D Printed Cell Culture Grid Holders for Improved Cellular Specimen Preparation in Cryo-Electron Microscopy.” Journal of Structural Biology, vol. 212, no. 3, 107633, Elsevier, 2020, doi:10.1016/j.jsb.2020.107633. short: F. Fäßler, B. Zens, R. Hauschild, F.K. Schur, Journal of Structural Biology 212 (2020). date_created: 2020-09-29T13:24:06Z date_published: 2020-12-01T00:00:00Z date_updated: 2024-03-25T23:30:04Z day: '01' ddc: - '570' department: - _id: FlSc doi: 10.1016/j.jsb.2020.107633 external_id: isi: - '000600997800008' file: - access_level: open_access checksum: c48cbf594e84fc2f91966ffaafc0918c content_type: application/pdf creator: dernst date_created: 2020-12-10T14:01:10Z date_updated: 2020-12-10T14:01:10Z file_id: '8937' file_name: 2020_JourStrucBiology_Faessler.pdf file_size: 7076870 relation: main_file success: 1 file_date_updated: 2020-12-10T14:01:10Z has_accepted_license: '1' intvolume: ' 212' isi: 1 issue: '3' keyword: - electron microscopy - cryo-EM - EM sample preparation - 3D printing - cell culture language: - iso: eng month: '12' oa: 1 oa_version: Published Version project: - _id: 9B954C5C-BA93-11EA-9121-9846C619BF3A grant_number: P33367 name: Structure and isoform diversity of the Arp2/3 complex - _id: 059B463C-7A3F-11EA-A408-12923DDC885E name: NÖ-Fonds Preis für die Jungforscherin des Jahres am IST Austria publication: Journal of Structural Biology publication_identifier: issn: - 1047-8477 publication_status: published publisher: Elsevier quality_controlled: '1' related_material: record: - id: '14592' relation: used_in_publication status: public - id: '12491' relation: dissertation_contains status: public scopus_import: '1' status: public title: 3D printed cell culture grid holders for improved cellular specimen preparation in cryo-electron microscopy tmp: image: /images/cc_by.png legal_code_url: https://creativecommons.org/licenses/by/4.0/legalcode name: Creative Commons Attribution 4.0 International Public License (CC-BY 4.0) short: CC BY (4.0) type: journal_article user_id: 4359f0d1-fa6c-11eb-b949-802e58b17ae8 volume: 212 year: '2020' ... --- _id: '8737' abstract: - lang: eng text: Mitochondrial complex I couples NADH:ubiquinone oxidoreduction to proton pumping by an unknown mechanism. Here, we present cryo-electron microscopy structures of ovine complex I in five different conditions, including turnover, at resolutions up to 2.3 to 2.5 angstroms. Resolved water molecules allowed us to experimentally define the proton translocation pathways. Quinone binds at three positions along the quinone cavity, as does the inhibitor rotenone that also binds within subunit ND4. Dramatic conformational changes around the quinone cavity couple the redox reaction to proton translocation during open-to-closed state transitions of the enzyme. In the induced deactive state, the open conformation is arrested by the ND6 subunit. We propose a detailed molecular coupling mechanism of complex I, which is an unexpected combination of conformational changes and electrostatic interactions. acknowledged_ssus: - _id: LifeSc - _id: EM-Fac acknowledgement: We thank J. Novacek (CEITEC Brno) and V.-V. Hodirnau (IST Austria) for their help with collecting cryo-EM datasets. We thank the IST Life Science and Electron Microscopy Facilities for providing equipment. This work has been supported by iNEXT,project number 653706, funded by the Horizon 2020 program of the European Union. This article reflects only the authors’view,and the European Commission is not responsible for any use that may be made of the information it contains. CIISB research infrastructure project LM2015043 funded by MEYS CR is gratefully acknowledged for the financial support of the measurements at the CF Cryo-electron Microscopy and Tomography CEITEC MU.This project has received funding from the European Union’s Horizon 2020 research and innovation program under the Marie Skłodowska-Curie Grant Agreement no. 665385 article_number: eabc4209 article_processing_charge: No article_type: original author: - first_name: Domen full_name: Kampjut, Domen id: 37233050-F248-11E8-B48F-1D18A9856A87 last_name: Kampjut - first_name: Leonid A full_name: Sazanov, Leonid A id: 338D39FE-F248-11E8-B48F-1D18A9856A87 last_name: Sazanov orcid: 0000-0002-0977-7989 citation: ama: Kampjut D, Sazanov LA. The coupling mechanism of mammalian respiratory complex I. Science. 2020;370(6516). doi:10.1126/science.abc4209 apa: Kampjut, D., & Sazanov, L. A. (2020). The coupling mechanism of mammalian respiratory complex I. Science. American Association for the Advancement of Science. https://doi.org/10.1126/science.abc4209 chicago: Kampjut, Domen, and Leonid A Sazanov. “The Coupling Mechanism of Mammalian Respiratory Complex I.” Science. American Association for the Advancement of Science, 2020. https://doi.org/10.1126/science.abc4209. ieee: D. Kampjut and L. A. Sazanov, “The coupling mechanism of mammalian respiratory complex I,” Science, vol. 370, no. 6516. American Association for the Advancement of Science, 2020. ista: Kampjut D, Sazanov LA. 2020. The coupling mechanism of mammalian respiratory complex I. Science. 370(6516), eabc4209. mla: Kampjut, Domen, and Leonid A. Sazanov. “The Coupling Mechanism of Mammalian Respiratory Complex I.” Science, vol. 370, no. 6516, eabc4209, American Association for the Advancement of Science, 2020, doi:10.1126/science.abc4209. short: D. Kampjut, L.A. Sazanov, Science 370 (2020). date_created: 2020-11-08T23:01:23Z date_published: 2020-10-30T00:00:00Z date_updated: 2023-08-22T12:35:38Z day: '30' ddc: - '572' department: - _id: LeSa doi: 10.1126/science.abc4209 ec_funded: 1 external_id: isi: - '000583031800004' pmid: - '32972993' file: - access_level: open_access checksum: 658ba90979ca9528a2efdfac8547047a content_type: application/pdf creator: lsazanov date_created: 2020-11-26T18:47:58Z date_updated: 2020-11-26T18:47:58Z file_id: '8820' file_name: Full_manuscript_with_SI_opt_red.pdf file_size: 7618987 relation: main_file success: 1 file_date_updated: 2020-11-26T18:47:58Z has_accepted_license: '1' intvolume: ' 370' isi: 1 issue: '6516' language: - iso: eng month: '10' oa: 1 oa_version: Submitted Version pmid: 1 project: - _id: 2564DBCA-B435-11E9-9278-68D0E5697425 call_identifier: H2020 grant_number: '665385' name: International IST Doctoral Program publication: Science publication_identifier: eissn: - '10959203' publication_status: published publisher: American Association for the Advancement of Science quality_controlled: '1' scopus_import: '1' status: public title: The coupling mechanism of mammalian respiratory complex I type: journal_article user_id: 4359f0d1-fa6c-11eb-b949-802e58b17ae8 volume: 370 year: '2020' ... --- _id: '8971' abstract: - lang: eng text: The actin-related protein (Arp)2/3 complex nucleates branched actin filament networks pivotal for cell migration, endocytosis and pathogen infection. Its activation is tightly regulated and involves complex structural rearrangements and actin filament binding, which are yet to be understood. Here, we report a 9.0 Å resolution structure of the actin filament Arp2/3 complex branch junction in cells using cryo-electron tomography and subtomogram averaging. This allows us to generate an accurate model of the active Arp2/3 complex in the branch junction and its interaction with actin filaments. Notably, our model reveals a previously undescribed set of interactions of the Arp2/3 complex with the mother filament, significantly different to the previous branch junction model. Our structure also indicates a central role for the ArpC3 subunit in stabilizing the active conformation. acknowledged_ssus: - _id: ScienComp - _id: LifeSc - _id: Bio - _id: EM-Fac acknowledgement: "This research was supported by the Scientific Service Units (SSUs) of IST Austria through resources provided by Scientific Computing (SciComp), the Life Science Facility (LSF), the BioImaging Facility (BIF), and the Electron Microscopy Facility (EMF). We also thank Dimitry Tegunov (MPI for Biophysical Chemistry) for helpful discussions\r\nabout the M software, and Michael Sixt (IST Austria) and Klemens Rottner (Technical University Braunschweig, HZI Braunschweig) for critical reading of the manuscript. We also thank Gregory Voth (University of Chicago) for providing us the MD-derived branch junction model for comparison. The authors acknowledge support from IST Austria and from the Austrian Science Fund (FWF): M02495 to G.D. and Austrian Science Fund (FWF): P33367 to F.K.M.S. " article_number: '6437' article_processing_charge: No article_type: original author: - first_name: Florian full_name: Fäßler, Florian id: 404F5528-F248-11E8-B48F-1D18A9856A87 last_name: Fäßler orcid: 0000-0001-7149-769X - first_name: Georgi A full_name: Dimchev, Georgi A id: 38C393BE-F248-11E8-B48F-1D18A9856A87 last_name: Dimchev orcid: 0000-0001-8370-6161 - first_name: Victor-Valentin full_name: Hodirnau, Victor-Valentin id: 3661B498-F248-11E8-B48F-1D18A9856A87 last_name: Hodirnau - first_name: William full_name: Wan, William last_name: Wan - first_name: Florian KM full_name: Schur, Florian KM id: 48AD8942-F248-11E8-B48F-1D18A9856A87 last_name: Schur orcid: 0000-0003-4790-8078 citation: ama: Fäßler F, Dimchev GA, Hodirnau V-V, Wan W, Schur FK. Cryo-electron tomography structure of Arp2/3 complex in cells reveals new insights into the branch junction. Nature Communications. 2020;11. doi:10.1038/s41467-020-20286-x apa: Fäßler, F., Dimchev, G. A., Hodirnau, V.-V., Wan, W., & Schur, F. K. (2020). Cryo-electron tomography structure of Arp2/3 complex in cells reveals new insights into the branch junction. Nature Communications. Springer Nature. https://doi.org/10.1038/s41467-020-20286-x chicago: Fäßler, Florian, Georgi A Dimchev, Victor-Valentin Hodirnau, William Wan, and Florian KM Schur. “Cryo-Electron Tomography Structure of Arp2/3 Complex in Cells Reveals New Insights into the Branch Junction.” Nature Communications. Springer Nature, 2020. https://doi.org/10.1038/s41467-020-20286-x. ieee: F. Fäßler, G. A. Dimchev, V.-V. Hodirnau, W. Wan, and F. K. Schur, “Cryo-electron tomography structure of Arp2/3 complex in cells reveals new insights into the branch junction,” Nature Communications, vol. 11. Springer Nature, 2020. ista: Fäßler F, Dimchev GA, Hodirnau V-V, Wan W, Schur FK. 2020. Cryo-electron tomography structure of Arp2/3 complex in cells reveals new insights into the branch junction. Nature Communications. 11, 6437. mla: Fäßler, Florian, et al. “Cryo-Electron Tomography Structure of Arp2/3 Complex in Cells Reveals New Insights into the Branch Junction.” Nature Communications, vol. 11, 6437, Springer Nature, 2020, doi:10.1038/s41467-020-20286-x. short: F. Fäßler, G.A. Dimchev, V.-V. Hodirnau, W. Wan, F.K. Schur, Nature Communications 11 (2020). date_created: 2020-12-23T08:25:45Z date_published: 2020-12-22T00:00:00Z date_updated: 2023-08-24T11:01:50Z day: '22' ddc: - '570' department: - _id: FlSc - _id: EM-Fac doi: 10.1038/s41467-020-20286-x external_id: isi: - '000603078000003' file: - access_level: open_access checksum: 55d43ea0061cc4027ba45e966e1db8cc content_type: application/pdf creator: dernst date_created: 2020-12-28T08:16:10Z date_updated: 2020-12-28T08:16:10Z file_id: '8975' file_name: 2020_NatureComm_Faessler.pdf file_size: 3958727 relation: main_file success: 1 file_date_updated: 2020-12-28T08:16:10Z has_accepted_license: '1' intvolume: ' 11' isi: 1 keyword: - General Biochemistry - Genetics and Molecular Biology - General Physics and Astronomy - General Chemistry language: - iso: eng month: '12' oa: 1 oa_version: Published Version project: - _id: 9B954C5C-BA93-11EA-9121-9846C619BF3A grant_number: P33367 name: Structure and isoform diversity of the Arp2/3 complex - _id: 2674F658-B435-11E9-9278-68D0E5697425 call_identifier: FWF grant_number: M02495 name: Protein structure and function in filopodia across scales publication: Nature Communications publication_identifier: issn: - 2041-1723 publication_status: published publisher: Springer Nature quality_controlled: '1' related_material: link: - description: News on IST Homepage relation: press_release url: https://ist.ac.at/en/news/cutting-edge-technology-reveals-structures-within-cells/ scopus_import: '1' status: public title: Cryo-electron tomography structure of Arp2/3 complex in cells reveals new insights into the branch junction tmp: image: /images/cc_by.png legal_code_url: https://creativecommons.org/licenses/by/4.0/legalcode name: Creative Commons Attribution 4.0 International Public License (CC-BY 4.0) short: CC BY (4.0) type: journal_article user_id: 4359f0d1-fa6c-11eb-b949-802e58b17ae8 volume: 11 year: '2020' ... --- _id: '7490' abstract: - lang: eng text: In plants, clathrin mediated endocytosis (CME) represents the major route for cargo internalisation from the cell surface. It has been assumed to operate in an evolutionary conserved manner as in yeast and animals. Here we report characterisation of ultrastructure, dynamics and mechanisms of plant CME as allowed by our advancement in electron microscopy and quantitative live imaging techniques. Arabidopsis CME appears to follow the constant curvature model and the bona fide CME population generates vesicles of a predominantly hexagonal-basket type; larger and with faster kinetics than in other models. Contrary to the existing paradigm, actin is dispensable for CME events at the plasma membrane but plays a unique role in collecting endocytic vesicles, sorting of internalised cargos and directional endosome movement that itself actively promote CME events. Internalized vesicles display a strongly delayed and sequential uncoating. These unique features highlight the independent evolution of the plant CME mechanism during the autonomous rise of multicellularity in eukaryotes. acknowledged_ssus: - _id: LifeSc - _id: Bio - _id: EM-Fac article_number: e52067 article_processing_charge: No article_type: original author: - first_name: Madhumitha full_name: Narasimhan, Madhumitha id: 44BF24D0-F248-11E8-B48F-1D18A9856A87 last_name: Narasimhan orcid: 0000-0002-8600-0671 - first_name: Alexander J full_name: Johnson, Alexander J id: 46A62C3A-F248-11E8-B48F-1D18A9856A87 last_name: Johnson orcid: 0000-0002-2739-8843 - first_name: Roshan full_name: Prizak, Roshan id: 4456104E-F248-11E8-B48F-1D18A9856A87 last_name: Prizak - first_name: Walter full_name: Kaufmann, Walter id: 3F99E422-F248-11E8-B48F-1D18A9856A87 last_name: Kaufmann orcid: 0000-0001-9735-5315 - first_name: Shutang full_name: Tan, Shutang id: 2DE75584-F248-11E8-B48F-1D18A9856A87 last_name: Tan orcid: 0000-0002-0471-8285 - first_name: Barbara E full_name: Casillas Perez, Barbara E id: 351ED2AA-F248-11E8-B48F-1D18A9856A87 last_name: Casillas Perez - first_name: Jiří full_name: Friml, Jiří id: 4159519E-F248-11E8-B48F-1D18A9856A87 last_name: Friml orcid: 0000-0002-8302-7596 citation: ama: Narasimhan M, Johnson AJ, Prizak R, et al. Evolutionarily unique mechanistic framework of clathrin-mediated endocytosis in plants. eLife. 2020;9. doi:10.7554/eLife.52067 apa: Narasimhan, M., Johnson, A. J., Prizak, R., Kaufmann, W., Tan, S., Casillas Perez, B. E., & Friml, J. (2020). Evolutionarily unique mechanistic framework of clathrin-mediated endocytosis in plants. ELife. eLife Sciences Publications. https://doi.org/10.7554/eLife.52067 chicago: Narasimhan, Madhumitha, Alexander J Johnson, Roshan Prizak, Walter Kaufmann, Shutang Tan, Barbara E Casillas Perez, and Jiří Friml. “Evolutionarily Unique Mechanistic Framework of Clathrin-Mediated Endocytosis in Plants.” ELife. eLife Sciences Publications, 2020. https://doi.org/10.7554/eLife.52067. ieee: M. Narasimhan et al., “Evolutionarily unique mechanistic framework of clathrin-mediated endocytosis in plants,” eLife, vol. 9. eLife Sciences Publications, 2020. ista: Narasimhan M, Johnson AJ, Prizak R, Kaufmann W, Tan S, Casillas Perez BE, Friml J. 2020. Evolutionarily unique mechanistic framework of clathrin-mediated endocytosis in plants. eLife. 9, e52067. mla: Narasimhan, Madhumitha, et al. “Evolutionarily Unique Mechanistic Framework of Clathrin-Mediated Endocytosis in Plants.” ELife, vol. 9, e52067, eLife Sciences Publications, 2020, doi:10.7554/eLife.52067. short: M. Narasimhan, A.J. Johnson, R. Prizak, W. Kaufmann, S. Tan, B.E. Casillas Perez, J. Friml, ELife 9 (2020). date_created: 2020-02-16T23:00:50Z date_published: 2020-01-23T00:00:00Z date_updated: 2023-08-18T06:33:07Z day: '23' ddc: - '570' - '580' department: - _id: JiFr - _id: GaTk - _id: EM-Fac - _id: SyCr doi: 10.7554/eLife.52067 ec_funded: 1 external_id: isi: - '000514104100001' pmid: - '31971511' file: - access_level: open_access checksum: 2052daa4be5019534f3a42f200a09f32 content_type: application/pdf creator: dernst date_created: 2020-02-18T07:21:16Z date_updated: 2020-07-14T12:47:59Z file_id: '7494' file_name: 2020_eLife_Narasimhan.pdf file_size: 7247468 relation: main_file file_date_updated: 2020-07-14T12:47:59Z has_accepted_license: '1' intvolume: ' 9' isi: 1 language: - iso: eng month: '01' oa: 1 oa_version: Published Version pmid: 1 project: - _id: 261099A6-B435-11E9-9278-68D0E5697425 call_identifier: H2020 grant_number: '742985' name: Tracing Evolution of Auxin Transport and Polarity in Plants - _id: 26538374-B435-11E9-9278-68D0E5697425 call_identifier: FWF grant_number: I03630 name: Molecular mechanisms of endocytic cargo recognition in plants publication: eLife publication_identifier: eissn: - 2050-084X publication_status: published publisher: eLife Sciences Publications quality_controlled: '1' scopus_import: '1' status: public title: Evolutionarily unique mechanistic framework of clathrin-mediated endocytosis in plants tmp: image: /images/cc_by.png legal_code_url: https://creativecommons.org/licenses/by/4.0/legalcode name: Creative Commons Attribution 4.0 International Public License (CC-BY 4.0) short: CC BY (4.0) type: journal_article user_id: 4359f0d1-fa6c-11eb-b949-802e58b17ae8 volume: 9 year: '2020' ... --- _id: '7525' abstract: - lang: eng text: "The medial habenula (MHb) is an evolutionary conserved epithalamic structure important for the modulation of emotional memory. It is involved in regulation of anxiety, compulsive behavior, addiction (nicotinic and opioid), sexual and feeding behavior. MHb receives inputs from septal regions and projects exclusively to the interpeduncular nucleus (IPN). Distinct sub-regions of the septum project to different subnuclei of MHb: the bed nucleus of anterior commissure projects to dorsal MHb and the triangular septum projects to ventral MHb. Furthermore, the dorsal and ventral MHb project to the lateral and rostral/central IPN, respectively. Importantly, these projections have unique features of prominent co-release of different neurotransmitters and requirement of a peculiar type of calcium channel for release. In general, synaptic neurotransmission requires an activity-dependent influx of Ca2+ into the presynaptic terminal through voltage-gated calcium channels. The calcium channel family most commonly involved in neurotransmitter release comprises three members, P/Q-, N- and R-type with Cav2.1, Cav2.2 and Cav2.3 subunits, respectively. In contrast to most CNS synapses that mainly express Cav2.1 and/or Cav2.2, MHb terminals in the IPN exclusively express Cav2.3. In other parts of the brain, such as the hippocampus, Cav2.3 is mostly located to postsynaptic elements. This unusual presynaptic location of Cav2.3 in the MHb-IPN pathway implies unique mechanisms of glutamate release in this pathway. One potential example of such uniqueness is the facilitation of release by GABAB receptor (GBR) activation. Presynaptic GBRs usually inhibit the release of neurotransmitters by inhibiting presynaptic calcium channels. MHb shows the highest expression levels of GBR in the brain. GBRs comprise two subunits, GABAB1 (GB1) and GABAB2 (GB2), and are associated with auxiliary subunits, called potassium channel tetramerization domain containing proteins (KCTD) 8, 12, 12b and 16. Among these four subunits, KCTD12b is exclusively expressed in ventral MHb, and KCTD8 shows the strongest expression in the whole MHb among other brain regions, indicating that KCTD8 and KCTD12b may be involved in the unique mechanisms of neurotransmitter release mediated by Cav2.3 and regulated by GBRs in this pathway. \r\nIn the present study, we first verified that neurotransmission in both dorsal and ventral MHb-IPN pathways is mainly mediated by Cav2.3 using a selective blocker of R-type channels, SNX-482. We next found that baclofen, a GBR agonist, has facilitatory effects on release from ventral MHb terminal in rostral IPN, whereas it has inhibitory effects on release from dorsal MHb terminals in lateral IPN, indicating that KCTD12b expressed exclusively in ventral MHb may have a role in the facilitatory effects of GBR activation. In a heterologous expression system using HEK cells, we found that KCTD8 and KCTD12b but not KCTD12 directly bind with Cav2.3. Pre-embedding immunogold electron microscopy data show that Cav2.3 and KCTD12b are distributed most densely in presynaptic active zone in IPN with KCTD12b being present only in rostral/central but not lateral IPN, whereas GABAB, KCTD8 and KCTD12 are distributed most densely in perisynaptic sites with KCTD12 present more frequently in postsynaptic elements and only in rostral/central IPN. In freeze-fracture replica labelling, Cav2.3, KCTD8 and KCTD12b are co-localized with each other in the same active zone indicating that they may form complexes regulating vesicle release in rostral IPN. \r\nOn electrophysiological studies of wild type (WT) mice, we found that paired-pulse ratio in rostral IPN of KCTD12b knock-out (KO) mice is lower than those of WT and KCTD8 KO mice. Consistent with this finding, in mean variance analysis, release probability in rostral IPN of KCTD12b KO mice is higher than that of WT and KCTD8 KO mice. Although paired-pulse ratios are not different between WT and KCTD8 KO mice, the mean variance analysis revealed significantly lower release probability in rostral IPN of KCTD8 KO than WT mice. These results demonstrate bidirectional regulation of Cav2.3-mediated release by KCTD8 and KCTD12b without GBR activation in rostral IPN. Finally, we examined the baclofen effects in rostral IPN of KCTD8 and KCTD12b KO mice, and found the facilitation of release remained in both KO mice, indicating that the peculiar effects of the GBR activation in this pathway do not depend on the selective expression of these KCTD subunits in ventral MHb. However, we found that presynaptic potentiation of evoked EPSC amplitude by baclofen falls to baseline after washout faster in KCTD12b KO mice than WT, KCTD8 KO and KCTD8/12b double KO mice. This result indicates that KCTD12b is involved in sustained potentiation of vesicle release by GBR activation, whereas KCTD8 is involved in its termination in the absence of KCTD12b. Consistent with these functional findings, replica labelling revealed an increase in density of KCTD8, but not Cav2.3 or GBR at active zone in rostral IPN of KCTD12b KO mice compared with that of WT mice, suggesting that increased association of KCTD8 with Cav2.3 facilitates the release probability and termination of the GBR effect in the absence of KCTD12b.\r\nIn summary, our study provided new insights into the physiological roles of presynaptic Cav2.3, GBRs and their auxiliary subunits KCTDs at an evolutionary conserved neuronal circuit. Future studies will be required to identify the exact molecular mechanism underlying the GBR-mediated presynaptic potentiation on ventral MHb terminals. It remains to be determined whether the prominent presence of presynaptic KCTDs at active zone could exert similar neuromodulatory functions in different pathways of the brain.\r\n" acknowledged_ssus: - _id: EM-Fac alternative_title: - ISTA Thesis article_processing_charge: No author: - first_name: Pradeep full_name: Bhandari, Pradeep id: 45EDD1BC-F248-11E8-B48F-1D18A9856A87 last_name: Bhandari orcid: 0000-0003-0863-4481 citation: ama: Bhandari P. Localization and functional role of Cav2.3 in the medial habenula to interpeduncular nucleus pathway. 2020. doi:10.15479/AT:ISTA:7525 apa: Bhandari, P. (2020). Localization and functional role of Cav2.3 in the medial habenula to interpeduncular nucleus pathway. Institute of Science and Technology Austria. https://doi.org/10.15479/AT:ISTA:7525 chicago: Bhandari, Pradeep. “Localization and Functional Role of Cav2.3 in the Medial Habenula to Interpeduncular Nucleus Pathway.” Institute of Science and Technology Austria, 2020. https://doi.org/10.15479/AT:ISTA:7525. ieee: P. Bhandari, “Localization and functional role of Cav2.3 in the medial habenula to interpeduncular nucleus pathway,” Institute of Science and Technology Austria, 2020. ista: Bhandari P. 2020. Localization and functional role of Cav2.3 in the medial habenula to interpeduncular nucleus pathway. Institute of Science and Technology Austria. mla: Bhandari, Pradeep. Localization and Functional Role of Cav2.3 in the Medial Habenula to Interpeduncular Nucleus Pathway. Institute of Science and Technology Austria, 2020, doi:10.15479/AT:ISTA:7525. short: P. Bhandari, Localization and Functional Role of Cav2.3 in the Medial Habenula to Interpeduncular Nucleus Pathway, Institute of Science and Technology Austria, 2020. date_created: 2020-02-26T10:56:37Z date_published: 2020-02-28T00:00:00Z date_updated: 2023-09-07T13:20:03Z day: '28' ddc: - '570' degree_awarded: PhD department: - _id: RySh doi: 10.15479/AT:ISTA:7525 file: - access_level: open_access checksum: 4589234fdb12b4ad72273b311723a7b4 content_type: application/pdf creator: pbhandari date_created: 2020-02-28T08:37:53Z date_updated: 2021-03-01T23:30:04Z embargo: 2021-02-28 file_id: '7538' file_name: Pradeep Bhandari Thesis.pdf file_size: 9646346 relation: main_file title: Localization and functional role of Cav2.3 in the medial habenula to interpeduncular nucleus pathway - access_level: closed checksum: aa79490553ca0a5c9b6fbcd152e93928 content_type: application/vnd.openxmlformats-officedocument.wordprocessingml.document creator: pbhandari date_created: 2020-02-28T08:47:14Z date_updated: 2021-03-01T23:30:04Z embargo_to: open_access file_id: '7539' file_name: Pradeep Bhandari Thesis.docx file_size: 35252164 relation: source_file title: Localization and functional role of Cav2.3 in the medial habenula to interpeduncular nucleus pathway file_date_updated: 2021-03-01T23:30:04Z has_accepted_license: '1' keyword: - Cav2.3 - medial habenula (MHb) - interpeduncular nucleus (IPN) language: - iso: eng month: '02' oa: 1 oa_version: Published Version page: '79' publication_identifier: issn: - 2663-337X publication_status: published publisher: Institute of Science and Technology Austria status: public supervisor: - first_name: Ryuichi full_name: Shigemoto, Ryuichi id: 499F3ABC-F248-11E8-B48F-1D18A9856A87 last_name: Shigemoto orcid: 0000-0001-8761-9444 title: Localization and functional role of Cav2.3 in the medial habenula to interpeduncular nucleus pathway type: dissertation user_id: c635000d-4b10-11ee-a964-aac5a93f6ac1 year: '2020' ... --- _id: '9750' abstract: - lang: eng text: 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 grow1,2. 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. Once 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. acknowledged_ssus: - _id: Bio - _id: EM-Fac - _id: SSU acknowledgement: We would like to thank Edouard Hannezo for discussions, Shayan Shami Pour and Daniel Capek for help with data analysis, Vanessa Barone and other members of the Heisenberg laboratory for thoughtful discussions and comments on the manuscript. We also thank Jack Merrin for preparing the microwells, and the Scientific Service Units at IST Austria, specifically Bioimaging and Electron Microscopy, and the Zebrafish Facility for continuous support. We acknowledge Hitoshi Morita for the kind gift of VinculinB-GFP plasmid. This research was supported by an ERC Advanced Grant (MECSPEC) to C.-P.H, EMBO Long Term grant (ALTF 187-2013) to M.S and IST Fellow Marie-Curie COFUND No. P_IST_EU01 to J.S. article_processing_charge: No author: - first_name: Jana full_name: Slovakova, Jana id: 30F3F2F0-F248-11E8-B48F-1D18A9856A87 last_name: Slovakova - first_name: Mateusz K full_name: Sikora, Mateusz K id: 2F74BCDE-F248-11E8-B48F-1D18A9856A87 last_name: Sikora - first_name: Silvia full_name: Caballero Mancebo, Silvia id: 2F1E1758-F248-11E8-B48F-1D18A9856A87 last_name: Caballero Mancebo orcid: 0000-0002-5223-3346 - first_name: Gabriel full_name: Krens, Gabriel id: 2B819732-F248-11E8-B48F-1D18A9856A87 last_name: Krens orcid: 0000-0003-4761-5996 - first_name: Walter full_name: Kaufmann, Walter id: 3F99E422-F248-11E8-B48F-1D18A9856A87 last_name: Kaufmann orcid: 0000-0001-9735-5315 - first_name: Karla full_name: Huljev, Karla id: 44C6F6A6-F248-11E8-B48F-1D18A9856A87 last_name: Huljev - first_name: Carl-Philipp J full_name: Heisenberg, Carl-Philipp J id: 39427864-F248-11E8-B48F-1D18A9856A87 last_name: Heisenberg orcid: 0000-0002-0912-4566 citation: ama: Slovakova J, Sikora MK, Caballero Mancebo S, et al. Tension-dependent stabilization of E-cadherin limits cell-cell contact expansion. bioRxiv. 2020. doi:10.1101/2020.11.20.391284 apa: Slovakova, J., Sikora, M. K., Caballero Mancebo, S., Krens, G., Kaufmann, W., Huljev, K., & Heisenberg, C.-P. J. (2020). Tension-dependent stabilization of E-cadherin limits cell-cell contact expansion. bioRxiv. Cold Spring Harbor Laboratory. https://doi.org/10.1101/2020.11.20.391284 chicago: Slovakova, Jana, Mateusz K Sikora, Silvia Caballero Mancebo, Gabriel Krens, Walter Kaufmann, Karla Huljev, and Carl-Philipp J Heisenberg. “Tension-Dependent Stabilization of E-Cadherin Limits Cell-Cell Contact Expansion.” BioRxiv. Cold Spring Harbor Laboratory, 2020. https://doi.org/10.1101/2020.11.20.391284. ieee: J. Slovakova et al., “Tension-dependent stabilization of E-cadherin limits cell-cell contact expansion,” bioRxiv. Cold Spring Harbor Laboratory, 2020. ista: Slovakova J, Sikora MK, Caballero Mancebo S, Krens G, Kaufmann W, Huljev K, Heisenberg C-PJ. 2020. Tension-dependent stabilization of E-cadherin limits cell-cell contact expansion. bioRxiv, 10.1101/2020.11.20.391284. mla: Slovakova, Jana, et al. “Tension-Dependent Stabilization of E-Cadherin Limits Cell-Cell Contact Expansion.” BioRxiv, Cold Spring Harbor Laboratory, 2020, doi:10.1101/2020.11.20.391284. short: J. Slovakova, M.K. Sikora, S. Caballero Mancebo, G. Krens, W. Kaufmann, K. Huljev, C.-P.J. Heisenberg, BioRxiv (2020). date_created: 2021-07-29T11:29:50Z date_published: 2020-11-20T00:00:00Z date_updated: 2024-03-25T23:30:10Z day: '20' department: - _id: CaHe - _id: EM-Fac - _id: Bio doi: 10.1101/2020.11.20.391284 ec_funded: 1 language: - iso: eng main_file_link: - open_access: '1' url: https://doi.org/10.1101/2020.11.20.391284 month: '11' oa: 1 oa_version: Preprint page: '41' project: - _id: 25681D80-B435-11E9-9278-68D0E5697425 call_identifier: FP7 grant_number: '291734' name: International IST Postdoc Fellowship Programme - _id: 260F1432-B435-11E9-9278-68D0E5697425 call_identifier: H2020 grant_number: '742573' name: Interaction and feedback between cell mechanics and fate specification in vertebrate gastrulation - _id: 2521E28E-B435-11E9-9278-68D0E5697425 grant_number: 187-2013 name: Modulation of adhesion function in cell-cell contact formation by cortical tension publication: bioRxiv publication_status: published publisher: Cold Spring Harbor Laboratory related_material: record: - id: '10766' relation: later_version status: public - id: '9623' relation: dissertation_contains status: public status: public title: Tension-dependent stabilization of E-cadherin limits cell-cell contact expansion type: preprint user_id: 8b945eb4-e2f2-11eb-945a-df72226e66a9 year: '2020' ... --- _id: '6947' abstract: - lang: eng text: Lymph nodes are es s ential organs of the immune s ys tem where adaptive immune responses originate, and consist of various leukocyte populations and a stromal backbone. Fibroblastic reticular cells (FRCs) are the main stromal cells and form a sponge-like extracellular matrix network, called conduits , which they thems elves enwrap and contract. Lymph, containing s oluble antigens , arrive in lymph nodes via afferent lymphatic vessels that connect to the s ubcaps ular s inus and conduit network. According to the current paradigm, the conduit network dis tributes afferent lymph through lymph nodes and thus provides acces s for immune cells to lymph-borne antigens. An elas tic caps ule s urrounds the organ and confines the immune cells and FRC network. Lymph nodes are completely packed with lymphocytes and lymphocyte numbers directly dictates the size of the organ. Although lymphocytes cons tantly enter and leave the lymph node, its s ize remains remarkedly s table under homeostatic conditions. It is only partly known how the cellularity and s ize of the lymph node is regulated and how the lymph node is able to swell in inflammation. The role of the FRC network in lymph node s welling and trans fer of fluids are inves tigated in this thes is. Furthermore, we s tudied what trafficking routes are us ed by cancer cells in lymph nodes to form distal metastases.We examined the role of a mechanical feedback in regulation of lymph node swelling. Using parallel plate compression and UV-las er cutting experiments we dis s ected the mechanical force dynamics of the whole lymph node, and individually for FRCs and the caps ule. Physical forces generated by packed lymphocytes directly affect the tens ion on the FRC network and capsule, which increases its resistance to swelling. This implies a feedback mechanism between tis s ue pres s ure and ability of lymphocytes to enter the organ. Following inflammation, the lymph node swells ∼10 fold in two weeks . Yet, what is the role for tens ion on the FRC network and caps ule, and how are lymphocytes able to enter in conditions that resist swelling remain open ques tions . We s how that tens ion on the FRC network is important to limit the swelling rate of the organ so that the FRC network can grow in a coordinated fashion. This is illustrated by interfering with FRC contractility, which leads to faster swelling rates and a dis organized FRC network in the inflamed lymph node. Growth of the FRC network in turn is expected to releas e tens ion on thes e s tructures and lowers the res is tance to swelling, thereby allowing more lymphocytes to enter the organ and drive more swelling. Halt of swelling coincides with a thickening of the caps ule, which forms a thick res is tant band around the organ and lowers tens ion on the FRC network to form a new force equilibrium.The FRC and conduit network are further believed to be a privileged s ite of s oluble information within the lymph node, although many details remain uns olved. We s how by 3D ultra-recons truction that FRCs and antigen pres enting cells cover the s urface of conduit s ys tem for more than 99% and we dis cus s the implications for s oluble information exchangeat the conduit level.Finally, there is an ongoing debate in the cancer field whether and how cancer cells in lymph nodes s eed dis tal metas tas es . We s how that cancer cells infus ed into the lymph node can utilize trafficking routes of immune cells and rapidly migrate to blood vessels. Once in the blood circulation, these cells are able to form metastases in distal tissues. acknowledged_ssus: - _id: Bio - _id: PreCl - _id: EM-Fac alternative_title: - ISTA Thesis article_processing_charge: No author: - first_name: Frank P full_name: Assen, Frank P id: 3A8E7F24-F248-11E8-B48F-1D18A9856A87 last_name: Assen orcid: 0000-0003-3470-6119 citation: ama: 'Assen FP. Lymph node mechanics: Deciphering the interplay between stroma contractility, morphology and lymphocyte trafficking. 2019. doi:10.15479/AT:ISTA:6947' apa: 'Assen, F. P. (2019). Lymph node mechanics: Deciphering the interplay between stroma contractility, morphology and lymphocyte trafficking. Institute of Science and Technology Austria. https://doi.org/10.15479/AT:ISTA:6947' chicago: 'Assen, Frank P. “Lymph Node Mechanics: Deciphering the Interplay between Stroma Contractility, Morphology and Lymphocyte Trafficking.” Institute of Science and Technology Austria, 2019. https://doi.org/10.15479/AT:ISTA:6947.' ieee: 'F. P. Assen, “Lymph node mechanics: Deciphering the interplay between stroma contractility, morphology and lymphocyte trafficking,” Institute of Science and Technology Austria, 2019.' ista: 'Assen FP. 2019. Lymph node mechanics: Deciphering the interplay between stroma contractility, morphology and lymphocyte trafficking. Institute of Science and Technology Austria.' mla: 'Assen, Frank P. Lymph Node Mechanics: Deciphering the Interplay between Stroma Contractility, Morphology and Lymphocyte Trafficking. Institute of Science and Technology Austria, 2019, doi:10.15479/AT:ISTA:6947.' short: 'F.P. Assen, Lymph Node Mechanics: Deciphering the Interplay between Stroma Contractility, Morphology and Lymphocyte Trafficking, Institute of Science and Technology Austria, 2019.' date_created: 2019-10-14T16:54:52Z date_published: 2019-10-09T00:00:00Z date_updated: 2023-09-13T08:50:57Z day: '9' ddc: - '570' degree_awarded: PhD department: - _id: MiSi doi: 10.15479/AT:ISTA:6947 file: - access_level: closed checksum: 53a739752a500f84d0f8ec953cbbd0b6 content_type: application/vnd.openxmlformats-officedocument.wordprocessingml.document creator: fassen date_created: 2019-11-06T12:30:02Z date_updated: 2020-11-07T23:30:03Z embargo_to: open_access file_id: '6990' file_name: PhDthesis_FrankAssen_revised2.docx file_size: 214172667 relation: source_file - access_level: open_access checksum: 8c156b65d9347bb599623a4b09f15d15 content_type: application/pdf creator: fassen date_created: 2019-11-06T12:30:57Z date_updated: 2020-11-07T23:30:03Z embargo: 2020-11-06 file_id: '6991' file_name: PhDthesis_FrankAssen_revised2.pdf file_size: 83637532 relation: main_file file_date_updated: 2020-11-07T23:30:03Z has_accepted_license: '1' language: - iso: eng month: '10' oa: 1 oa_version: Published Version page: '142' publication_identifier: issn: - 2663-337X publication_status: published publisher: Institute of Science and Technology Austria related_material: record: - id: '664' relation: part_of_dissertation status: public - id: '402' relation: part_of_dissertation status: public status: public supervisor: - first_name: Michael K full_name: Sixt, Michael K id: 41E9FBEA-F248-11E8-B48F-1D18A9856A87 last_name: Sixt orcid: 0000-0002-6620-9179 title: 'Lymph node mechanics: Deciphering the interplay between stroma contractility, morphology and lymphocyte trafficking' type: dissertation user_id: c635000d-4b10-11ee-a964-aac5a93f6ac1 year: '2019' ... --- _id: '7186' abstract: - lang: eng text: "Tissue morphogenesis in developmental or physiological processes is regulated by molecular\r\nand mechanical signals. While the molecular signaling cascades are increasingly well\r\ndescribed, the mechanical signals affecting tissue shape changes have only recently been\r\nstudied in greater detail. To gain more insight into the mechanochemical and biophysical\r\nbasis of an epithelial spreading process (epiboly) in early zebrafish development, we studied\r\ncell-cell junction formation and actomyosin network dynamics at the boundary between\r\nsurface layer epithelial cells (EVL) and the yolk syncytial layer (YSL). During zebrafish epiboly,\r\nthe cell mass sitting on top of the yolk cell spreads to engulf the yolk cell by the end of\r\ngastrulation. It has been previously shown that an actomyosin ring residing within the YSL\r\npulls on the EVL tissue through a cable-constriction and a flow-friction motor, thereby\r\ndragging the tissue vegetal wards. Pulling forces are likely transmitted from the YSL\r\nactomyosin ring to EVL cells; however, the nature and formation of the junctional structure\r\nmediating this process has not been well described so far. Therefore, our main aim was to\r\ndetermine the nature, dynamics and potential function of the EVL-YSL junction during this\r\nepithelial tissue spreading. Specifically, we show that the EVL-YSL junction is a\r\nmechanosensitive structure, predominantly made of tight junction (TJ) proteins. The process\r\nof TJ mechanosensation depends on the retrograde flow of non-junctional, phase-separated\r\nZonula Occludens-1 (ZO-1) protein clusters towards the EVL-YSL boundary. Interestingly, we\r\ncould demonstrate that ZO-1 is present in a non-junctional pool on the surface of the yolk\r\ncell, and ZO-1 undergoes a phase separation process that likely renders the protein\r\nresponsive to flows. These flows are directed towards the junction and mediate proper\r\ntension-dependent recruitment of ZO-1. Upon reaching the EVL-YSL junction ZO-1 gets\r\nincorporated into the junctional pool mediated through its direct actin-binding domain.\r\nWhen the non-junctional pool and/or ZO-1 direct actin binding is absent, TJs fail in their\r\nproper mechanosensitive responses resulting in slower tissue spreading. We could further\r\ndemonstrate that depletion of ZO proteins within the YSL results in diminished actomyosin\r\nring formation. This suggests that a mechanochemical feedback loop is at work during\r\nzebrafish epiboly: ZO proteins help in proper actomyosin ring formation and actomyosin\r\ncontractility and flows positively influence ZO-1 junctional recruitment. Finally, such a\r\nmesoscale polarization process mediated through the flow of phase-separated protein\r\nclusters might have implications for other processes such as immunological synapse\r\nformation, C. elegans zygote polarization and wound healing." acknowledged_ssus: - _id: Bio - _id: LifeSc - _id: EM-Fac - _id: SSU alternative_title: - ISTA Thesis article_processing_charge: No author: - first_name: Cornelia full_name: Schwayer, Cornelia id: 3436488C-F248-11E8-B48F-1D18A9856A87 last_name: Schwayer orcid: 0000-0001-5130-2226 citation: ama: Schwayer C. Mechanosensation of tight junctions depends on ZO-1 phase separation and flow. 2019. doi:10.15479/AT:ISTA:7186 apa: Schwayer, C. (2019). Mechanosensation of tight junctions depends on ZO-1 phase separation and flow. Institute of Science and Technology Austria. https://doi.org/10.15479/AT:ISTA:7186 chicago: Schwayer, Cornelia. “Mechanosensation of Tight Junctions Depends on ZO-1 Phase Separation and Flow.” Institute of Science and Technology Austria, 2019. https://doi.org/10.15479/AT:ISTA:7186. ieee: C. Schwayer, “Mechanosensation of tight junctions depends on ZO-1 phase separation and flow,” Institute of Science and Technology Austria, 2019. ista: Schwayer C. 2019. Mechanosensation of tight junctions depends on ZO-1 phase separation and flow. Institute of Science and Technology Austria. mla: Schwayer, Cornelia. Mechanosensation of Tight Junctions Depends on ZO-1 Phase Separation and Flow. Institute of Science and Technology Austria, 2019, doi:10.15479/AT:ISTA:7186. short: C. Schwayer, Mechanosensation of Tight Junctions Depends on ZO-1 Phase Separation and Flow, Institute of Science and Technology Austria, 2019. date_created: 2019-12-16T14:26:14Z date_published: 2019-12-16T00:00:00Z date_updated: 2023-09-07T12:56:42Z day: '16' ddc: - '570' degree_awarded: PhD department: - _id: CaHe doi: 10.15479/AT:ISTA:7186 file: - access_level: closed checksum: 585583c1c875c5d9525703a539668a7c content_type: application/zip creator: cschwayer date_created: 2019-12-19T15:18:11Z date_updated: 2020-07-14T12:47:52Z file_id: '7194' file_name: DocumentSourceFiles.zip file_size: 19431292 relation: source_file - access_level: open_access checksum: 9b9b24351514948d27cec659e632e2cd content_type: application/pdf creator: cschwayer date_created: 2019-12-19T15:19:21Z date_updated: 2020-07-14T12:47:52Z file_id: '7195' file_name: Thesis_CS_final.pdf file_size: 19226428 relation: main_file file_date_updated: 2020-07-14T12:47:52Z has_accepted_license: '1' language: - iso: eng month: '12' oa: 1 oa_version: Published Version page: '107' publication_identifier: issn: - 2663-337X publication_status: published publisher: Institute of Science and Technology Austria related_material: record: - id: '1096' relation: dissertation_contains status: public - id: '7001' relation: part_of_dissertation status: public status: public supervisor: - first_name: Carl-Philipp J full_name: Heisenberg, Carl-Philipp J id: 39427864-F248-11E8-B48F-1D18A9856A87 last_name: Heisenberg orcid: 0000-0002-0912-4566 title: Mechanosensation of tight junctions depends on ZO-1 phase separation and flow type: dissertation user_id: c635000d-4b10-11ee-a964-aac5a93f6ac1 year: '2019' ... --- _id: '7406' abstract: - lang: eng text: "Background\r\nSynaptic vesicles (SVs) are an integral part of the neurotransmission machinery, and isolation of SVs from their host neuron is necessary to reveal their most fundamental biochemical and functional properties in in vitro assays. Isolated SVs from neurons that have been genetically engineered, e.g. to introduce genetically encoded indicators, are not readily available but would permit new insights into SV structure and function. Furthermore, it is unclear if cultured neurons can provide sufficient starting material for SV isolation procedures.\r\n\r\nNew method\r\nHere, we demonstrate an efficient ex vivo procedure to obtain functional SVs from cultured rat cortical neurons after genetic engineering with a lentivirus.\r\n\r\nResults\r\nWe show that ∼108 plated cortical neurons allow isolation of suitable SV amounts for functional analysis and imaging. We found that SVs isolated from cultured neurons have neurotransmitter uptake comparable to that of SVs isolated from intact cortex. Using total internal reflection fluorescence (TIRF) microscopy, we visualized an exogenous SV-targeted marker protein and demonstrated the high efficiency of SV modification.\r\n\r\nComparison with existing methods\r\nObtaining SVs from genetically engineered neurons currently generally requires the availability of transgenic animals, which is constrained by technical (e.g. cost and time) and biological (e.g. developmental defects and lethality) limitations.\r\n\r\nConclusions\r\nThese results demonstrate the modification and isolation of functional SVs using cultured neurons and viral transduction. The ability to readily obtain SVs from genetically engineered neurons will permit linking in situ studies to in vitro experiments in a variety of genetic contexts." acknowledged_ssus: - _id: Bio - _id: EM-Fac article_processing_charge: No article_type: original author: - first_name: Catherine full_name: Mckenzie, Catherine id: 3EEDE19A-F248-11E8-B48F-1D18A9856A87 last_name: Mckenzie - first_name: Miroslava full_name: Spanova, Miroslava id: 44A924DC-F248-11E8-B48F-1D18A9856A87 last_name: Spanova - first_name: Alexander J full_name: Johnson, Alexander J id: 46A62C3A-F248-11E8-B48F-1D18A9856A87 last_name: Johnson orcid: 0000-0002-2739-8843 - first_name: Stephanie full_name: Kainrath, Stephanie id: 32CFBA64-F248-11E8-B48F-1D18A9856A87 last_name: Kainrath - first_name: Vanessa full_name: Zheden, Vanessa id: 39C5A68A-F248-11E8-B48F-1D18A9856A87 last_name: Zheden orcid: 0000-0002-9438-4783 - first_name: Harald H. full_name: Sitte, Harald H. last_name: Sitte - first_name: Harald L full_name: Janovjak, Harald L id: 33BA6C30-F248-11E8-B48F-1D18A9856A87 last_name: Janovjak orcid: 0000-0002-8023-9315 citation: ama: Mckenzie C, Spanova M, Johnson AJ, et al. Isolation of synaptic vesicles from genetically engineered cultured neurons. Journal of Neuroscience Methods. 2019;312:114-121. doi:10.1016/j.jneumeth.2018.11.018 apa: Mckenzie, C., Spanova, M., Johnson, A. J., Kainrath, S., Zheden, V., Sitte, H. H., & Janovjak, H. L. (2019). Isolation of synaptic vesicles from genetically engineered cultured neurons. Journal of Neuroscience Methods. Elsevier. https://doi.org/10.1016/j.jneumeth.2018.11.018 chicago: Mckenzie, Catherine, Miroslava Spanova, Alexander J Johnson, Stephanie Kainrath, Vanessa Zheden, Harald H. Sitte, and Harald L Janovjak. “Isolation of Synaptic Vesicles from Genetically Engineered Cultured Neurons.” Journal of Neuroscience Methods. Elsevier, 2019. https://doi.org/10.1016/j.jneumeth.2018.11.018. ieee: C. Mckenzie et al., “Isolation of synaptic vesicles from genetically engineered cultured neurons,” Journal of Neuroscience Methods, vol. 312. Elsevier, pp. 114–121, 2019. ista: Mckenzie C, Spanova M, Johnson AJ, Kainrath S, Zheden V, Sitte HH, Janovjak HL. 2019. Isolation of synaptic vesicles from genetically engineered cultured neurons. Journal of Neuroscience Methods. 312, 114–121. mla: Mckenzie, Catherine, et al. “Isolation of Synaptic Vesicles from Genetically Engineered Cultured Neurons.” Journal of Neuroscience Methods, vol. 312, Elsevier, 2019, pp. 114–21, doi:10.1016/j.jneumeth.2018.11.018. short: C. Mckenzie, M. Spanova, A.J. Johnson, S. Kainrath, V. Zheden, H.H. Sitte, H.L. Janovjak, Journal of Neuroscience Methods 312 (2019) 114–121. date_created: 2020-01-30T09:12:19Z date_published: 2019-01-15T00:00:00Z date_updated: 2023-09-06T15:27:29Z day: '15' department: - _id: HaJa - _id: Bio doi: 10.1016/j.jneumeth.2018.11.018 ec_funded: 1 external_id: isi: - '000456220900013' pmid: - '30496761' intvolume: ' 312' isi: 1 language: - iso: eng month: '01' oa_version: None page: 114-121 pmid: 1 project: - _id: 25548C20-B435-11E9-9278-68D0E5697425 call_identifier: FP7 grant_number: '303564' name: Microbial Ion Channels for Synthetic Neurobiology - _id: 26538374-B435-11E9-9278-68D0E5697425 call_identifier: FWF grant_number: I03630 name: Molecular mechanisms of endocytic cargo recognition in plants - _id: 2548AE96-B435-11E9-9278-68D0E5697425 call_identifier: FWF grant_number: W1232-B24 name: Molecular Drug Targets publication: Journal of Neuroscience Methods publication_identifier: issn: - 0165-0270 publication_status: published publisher: Elsevier quality_controlled: '1' scopus_import: '1' status: public title: Isolation of synaptic vesicles from genetically engineered cultured neurons type: journal_article user_id: c635000d-4b10-11ee-a964-aac5a93f6ac1 volume: 312 year: '2019' ... --- _id: '6087' abstract: - lang: eng text: Cell fate specification by lateral inhibition typically involves contact signaling through the Delta-Notch signaling pathway. However, whether this is the only signaling mode mediating lateral inhibition remains unclear. Here we show that in zebrafish oogenesis, a group of cells within the granulosa cell layer at the oocyte animal pole acquire elevated levels of the transcriptional coactivator TAZ in their nuclei. One of these cells, the future micropyle precursor cell (MPC), accumulates increasingly high levels of nuclear TAZ and grows faster than its surrounding cells, mechanically compressing those cells, which ultimately lose TAZ from their nuclei. Strikingly, relieving neighbor-cell compression by MPC ablation or aspiration restores nuclear TAZ accumulation in neighboring cells, eventually leading to MPC re-specification from these cells. Conversely, MPC specification is defective in taz−/− follicles. These findings uncover a novel mode of lateral inhibition in cell fate specification based on mechanical signals controlling TAZ activity. acknowledged_ssus: - _id: Bio - _id: EM-Fac - _id: LifeSc acknowledgement: We thank Roland Dosch, Makoto Furutani-Seiki, Brian Link, Mary Mullins, and Masazumi Tada for providing transgenic and/or mutant zebrafish lines; Alexandra Schauer, Shayan Shami-Pour, and the rest of the Heisenberg lab for technical assistance and feedback on the manuscript; and the Bioimaging, Electron Microscopy, and Zebrafish facilities of IST Austria for continuous support. This work was supported by an ERC advanced grant ( MECSPEC to C.-P.H.). article_processing_charge: No article_type: original author: - first_name: Peng full_name: Xia, Peng id: 4AB6C7D0-F248-11E8-B48F-1D18A9856A87 last_name: Xia orcid: 0000-0002-5419-7756 - first_name: Daniel J full_name: Gütl, Daniel J id: 381929CE-F248-11E8-B48F-1D18A9856A87 last_name: Gütl - first_name: Vanessa full_name: Zheden, Vanessa id: 39C5A68A-F248-11E8-B48F-1D18A9856A87 last_name: Zheden orcid: 0000-0002-9438-4783 - first_name: Carl-Philipp J full_name: Heisenberg, Carl-Philipp J id: 39427864-F248-11E8-B48F-1D18A9856A87 last_name: Heisenberg orcid: 0000-0002-0912-4566 citation: ama: Xia P, Gütl DJ, Zheden V, Heisenberg C-PJ. Lateral inhibition in cell specification mediated by mechanical signals modulating TAZ activity. Cell. 2019;176(6):1379-1392.e14. doi:10.1016/j.cell.2019.01.019 apa: Xia, P., Gütl, D. J., Zheden, V., & Heisenberg, C.-P. J. (2019). Lateral inhibition in cell specification mediated by mechanical signals modulating TAZ activity. Cell. Elsevier. https://doi.org/10.1016/j.cell.2019.01.019 chicago: Xia, Peng, Daniel J Gütl, Vanessa Zheden, and Carl-Philipp J Heisenberg. “Lateral Inhibition in Cell Specification Mediated by Mechanical Signals Modulating TAZ Activity.” Cell. Elsevier, 2019. https://doi.org/10.1016/j.cell.2019.01.019. ieee: P. Xia, D. J. Gütl, V. Zheden, and C.-P. J. Heisenberg, “Lateral inhibition in cell specification mediated by mechanical signals modulating TAZ activity,” Cell, vol. 176, no. 6. Elsevier, p. 1379–1392.e14, 2019. ista: Xia P, Gütl DJ, Zheden V, Heisenberg C-PJ. 2019. Lateral inhibition in cell specification mediated by mechanical signals modulating TAZ activity. Cell. 176(6), 1379–1392.e14. mla: Xia, Peng, et al. “Lateral Inhibition in Cell Specification Mediated by Mechanical Signals Modulating TAZ Activity.” Cell, vol. 176, no. 6, Elsevier, 2019, p. 1379–1392.e14, doi:10.1016/j.cell.2019.01.019. short: P. Xia, D.J. Gütl, V. Zheden, C.-P.J. Heisenberg, Cell 176 (2019) 1379–1392.e14. date_created: 2019-03-10T22:59:19Z date_published: 2019-03-07T00:00:00Z date_updated: 2023-08-25T08:02:23Z day: '07' department: - _id: CaHe - _id: EM-Fac doi: 10.1016/j.cell.2019.01.019 ec_funded: 1 external_id: isi: - '000460509600013' pmid: - '30773315' intvolume: ' 176' isi: 1 issue: '6' language: - iso: eng main_file_link: - open_access: '1' url: https://doi.org/10.1016/j.cell.2019.01.019 month: '03' oa: 1 oa_version: Published Version page: 1379-1392.e14 pmid: 1 project: - _id: 260F1432-B435-11E9-9278-68D0E5697425 call_identifier: H2020 grant_number: '742573' name: Interaction and feedback between cell mechanics and fate specification in vertebrate gastrulation publication: Cell publication_status: published publisher: Elsevier quality_controlled: '1' related_material: link: - description: News on IST Homepage relation: press_release url: https://ist.ac.at/en/news/in-zebrafish-eggs-most-rapidly-growing-cell-inhibits-its-neighbours-through-mechanical-signals/ scopus_import: '1' status: public title: Lateral inhibition in cell specification mediated by mechanical signals modulating TAZ activity type: journal_article user_id: 4359f0d1-fa6c-11eb-b949-802e58b17ae8 volume: 176 year: '2019' ... --- _id: '6269' abstract: - lang: eng text: 'Clathrin-Mediated Endocytosis (CME) is an aspect of cellular trafficking that is constantly regulated for mediating developmental and physiological responses. The main aim of my thesis is to decipher the basic mechanisms of CME and post-endocytic trafficking in the whole multicellular organ systems of Arabidopsis. The first chapter of my thesis describes the search for new components involved in CME. Tandem affinity purification was conducted using CLC and its interacting partners were identified. Amongst the identified proteins were the Auxilin-likes1 and 2 (Axl1/2), putative uncoating factors, for which we made a full functional analysis. Over-expression of Axl1/2 causes extreme modifications in the dynamics of the machinery proteins and inhibition of endocytosis altogether. However the loss of function of the axl1/2 did not present any cellular or physiological phenotype, meaning Auxilin-likes do not form the major uncoating machinery. The second chapter of my thesis describes the establishment/utilisation of techniques to capture the dynamicity and the complexity of CME and post-endocytic trafficking. We have studied the development of endocytic pits at the PM – specifically, the mode of membrane remodeling during pit development and the role of actin in it, given plant cells possess high turgor pressure. Utilizing the improved z-resolution of TIRF and VAEM techniques, we captured the time-lapse of the endocytic events at the plasma membrane; and using particle detection software, we quantitatively analysed all the endocytic trajectories in an unbiased way to obtain the endocytic rate of the system. This together with the direct analysis of cargo internalisation from the PM provided an estimate on the endocytic potential of the cell. We also developed a methodology for ultrastructural analysis of different populations of Clathrin-Coated Structures (CCSs) in both PM and endomembranes in unroofed protoplasts. Structural analysis, together with the intensity profile of CCSs at the PM show that the mode of CCP development at the PM follows ‘Constant curvature model’; meaning that clathrin polymerisation energy is a major contributing factor of membrane remodeling. In addition, other analyses clearly show that actin is not required for membrane remodeling during invagination or any other step of CCP development, despite the prevalent high turgor pressure. However, actin is essential in orchestrating the post-endocytic trafficking of CCVs facilitating the EE formation. We also observed that the uncoating process post-endocytosis is not immediate; an alternative mechanism of uncoating – Sequential multi-step process – functions in the cell. Finally we also looked at one of the important physiological stimuli modulating the process – hormone, auxin. auxin has been known to influence CME before. We have made a detailed study on the concentration-time based effect of auxin on the machinery proteins, CCP development, and the specificity of cargoes endocytosed. To this end, we saw no general effect of auxin on CME at earlier time points. However, very low concentration of IAA, such as 50nM, accelerates endocytosis of specifically PIN2 through CME. Such a tight regulatory control with high specificity to PIN2 could be essential in modulating its polarity. ' acknowledged_ssus: - _id: Bio - _id: EM-Fac alternative_title: - ISTA Thesis article_processing_charge: No author: - first_name: Madhumitha full_name: Narasimhan, Madhumitha id: 44BF24D0-F248-11E8-B48F-1D18A9856A87 last_name: Narasimhan orcid: 0000-0002-8600-0671 citation: ama: Narasimhan M. Clathrin-Mediated endocytosis, post-endocytic trafficking and their regulatory controls in plants . 2019. doi:10.15479/at:ista:th1075 apa: Narasimhan, M. (2019). Clathrin-Mediated endocytosis, post-endocytic trafficking and their regulatory controls in plants . Institute of Science and Technology Austria. https://doi.org/10.15479/at:ista:th1075 chicago: Narasimhan, Madhumitha. “Clathrin-Mediated Endocytosis, Post-Endocytic Trafficking and Their Regulatory Controls in Plants .” Institute of Science and Technology Austria, 2019. https://doi.org/10.15479/at:ista:th1075. ieee: M. Narasimhan, “Clathrin-Mediated endocytosis, post-endocytic trafficking and their regulatory controls in plants ,” Institute of Science and Technology Austria, 2019. ista: Narasimhan M. 2019. Clathrin-Mediated endocytosis, post-endocytic trafficking and their regulatory controls in plants . Institute of Science and Technology Austria. mla: Narasimhan, Madhumitha. Clathrin-Mediated Endocytosis, Post-Endocytic Trafficking and Their Regulatory Controls in Plants . Institute of Science and Technology Austria, 2019, doi:10.15479/at:ista:th1075. short: M. Narasimhan, Clathrin-Mediated Endocytosis, Post-Endocytic Trafficking and Their Regulatory Controls in Plants , Institute of Science and Technology Austria, 2019. date_created: 2019-04-09T14:37:06Z date_published: 2019-02-04T00:00:00Z date_updated: 2025-05-07T11:12:27Z day: '04' ddc: - '575' degree_awarded: PhD department: - _id: JiFr doi: 10.15479/at:ista:th1075 file: - access_level: open_access checksum: c958f27dd752712886e7e2638b847a3c content_type: video/x-msvideo creator: dernst date_created: 2019-04-09T14:35:18Z date_updated: 2021-02-11T23:30:15Z embargo: 2020-02-11 file_id: '6270' file_name: Supplementary_movie_1.avi file_size: 5402078 relation: main_file - access_level: open_access checksum: 8786fdc29c62987c0aad3c866a4d3691 content_type: video/x-msvideo creator: dernst date_created: 2019-04-09T14:35:18Z date_updated: 2021-02-11T23:30:15Z embargo: 2020-02-11 file_id: '6271' file_name: 3.7_supplementary_movie_10.avi file_size: 5927736 relation: main_file - access_level: open_access checksum: 25f784c5159d6f4d966b2f9b371ebaf6 content_type: video/x-msvideo creator: dernst date_created: 2019-04-09T14:35:18Z date_updated: 2021-02-11T23:30:15Z embargo: 2020-02-11 file_id: '6272' file_name: 3.7_supplementary_movie_9.avi file_size: 9570210 relation: main_file - access_level: open_access checksum: 917069272a7a08d1f38224d5e12765d6 content_type: video/x-msvideo creator: dernst date_created: 2019-04-09T14:35:18Z date_updated: 2021-02-11T23:30:15Z embargo: 2020-02-11 file_id: '6273' file_name: 3.7_supplementary_movie_8.avi file_size: 2827360 relation: main_file - access_level: open_access checksum: 81e74f5ca0ad70050504f18192236dc0 content_type: video/x-msvideo creator: dernst date_created: 2019-04-09T14:35:18Z date_updated: 2021-02-11T23:30:15Z embargo: 2020-02-11 file_id: '6274' file_name: 3.7_supplementary_movie_7.avi file_size: 5771410 relation: main_file - access_level: open_access checksum: 47eb37b27a2930252713924307ea8c6f content_type: video/x-msvideo creator: dernst date_created: 2019-04-09T14:35:18Z date_updated: 2021-02-11T23:30:15Z embargo: 2020-02-11 file_id: '6275' file_name: 3.7_supplementary_movie_6.avi file_size: 1113486 relation: main_file - access_level: open_access checksum: f68f66721041ce84e331959c9a5779c3 content_type: video/x-msvideo creator: dernst date_created: 2019-04-09T14:35:18Z date_updated: 2021-02-11T23:30:15Z embargo: 2020-02-11 file_id: '6276' file_name: 3.7_supplementary_movie_5.avi file_size: 1057232 relation: main_file - access_level: open_access checksum: 67c01cefab51b363c5e214fe4cd671f3 content_type: video/x-msvideo creator: dernst date_created: 2019-04-09T14:35:23Z date_updated: 2021-02-11T23:30:15Z embargo: 2020-02-11 file_id: '6277' file_name: 3.7_supplementary_movie_3.avi file_size: 127472916 relation: main_file - access_level: open_access checksum: e5a397edbee05b8821e2b19b3c1a9260 content_type: video/x-msvideo creator: dernst date_created: 2019-04-09T14:35:19Z date_updated: 2021-02-11T23:30:15Z embargo: 2020-02-11 file_id: '6278' file_name: 3.7_supplementary_movie_4.avi file_size: 3181238 relation: main_file - access_level: open_access checksum: 32d92b2a9277f956fdb0b42351d07c0b content_type: video/x-msvideo creator: dernst date_created: 2019-04-09T14:35:19Z date_updated: 2021-02-11T23:30:15Z embargo: 2020-02-11 file_id: '6279' file_name: 3.7_supplementary_movie_2.avi file_size: 5970952 relation: main_file - access_level: open_access checksum: efe7001f5d9a8c61e631e12d5f324ade content_type: video/x-msvideo creator: dernst date_created: 2019-04-09T14:35:21Z date_updated: 2021-02-11T23:30:15Z embargo: 2020-02-11 file_id: '6280' file_name: 3.7_Supplementary_movie_1.avi file_size: 39835236 relation: main_file - access_level: open_access checksum: eeb0a5603c6449c5f34eacd5ff0b3a16 content_type: video/x-msvideo creator: dernst date_created: 2019-04-09T14:35:21Z date_updated: 2021-02-11T23:30:15Z embargo: 2020-02-11 file_id: '6281' file_name: 2.5_Suppl_Movie_4_AP2A1_TagRFP.avi file_size: 3696740 relation: main_file - access_level: open_access checksum: 8e7c00ef6223bf0e177deb168338af13 content_type: video/x-msvideo creator: dernst date_created: 2019-04-09T14:35:21Z date_updated: 2021-02-11T23:30:15Z embargo: 2020-02-11 file_id: '6282' file_name: 2.5_Suppl_Movie_3_TPLATE_GFP.avi file_size: 6741232 relation: main_file - access_level: open_access checksum: 3636006a7cb709a7543d6581e359b28d content_type: video/x-msvideo creator: dernst date_created: 2019-04-09T14:35:22Z date_updated: 2021-02-11T23:30:15Z embargo: 2020-02-11 file_id: '6283' file_name: 2.5_Suppl_Movie_2_CLC_GFP.avi file_size: 2445946 relation: main_file - access_level: open_access checksum: 39ca5519a6e9a38356e7b3704004fea7 content_type: video/x-msvideo creator: dernst date_created: 2019-04-09T14:35:22Z date_updated: 2021-02-11T23:30:15Z embargo: 2020-02-11 file_id: '6284' file_name: 2.5_Suppl_Movie_1_CLC_GFPxAxl1_mcherry.avi file_size: 58594 relation: main_file - access_level: open_access checksum: 4fcdaa3a6c645514a3b3205f0f69dc76 content_type: application/pdf creator: dernst date_created: 2019-04-09T14:35:33Z date_updated: 2021-02-11T11:17:15Z embargo: 2020-02-11 file_id: '6285' file_name: 2019_Thesis_Narasimhan.pdf file_size: 10553937 relation: main_file - access_level: closed checksum: 268f0b6bad21d5f0d671e5d4b88104a7 content_type: application/vnd.openxmlformats-officedocument.wordprocessingml.document creator: dernst date_created: 2019-04-09T14:35:36Z date_updated: 2020-07-14T12:47:26Z embargo_to: open_access file_id: '6286' file_name: 2019_Thesis_Narasimhan_source.docx file_size: 135291990 relation: source_file file_date_updated: 2021-02-11T23:30:15Z has_accepted_license: '1' language: - iso: eng month: '02' oa: 1 oa_version: Published Version page: '138' publication_identifier: issn: - 2663-337X publication_status: published publisher: Institute of Science and Technology Austria related_material: record: - id: '412' relation: part_of_dissertation status: public status: public supervisor: - first_name: Jiří full_name: Friml, Jiří id: 4159519E-F248-11E8-B48F-1D18A9856A87 last_name: Friml orcid: 0000-0002-8302-7596 title: 'Clathrin-Mediated endocytosis, post-endocytic trafficking and their regulatory controls in plants ' tmp: image: /images/cc_by.png legal_code_url: https://creativecommons.org/licenses/by/4.0/legalcode name: Creative Commons Attribution 4.0 International Public License (CC-BY 4.0) short: CC BY (4.0) type: dissertation user_id: c635000d-4b10-11ee-a964-aac5a93f6ac1 year: '2019' ... --- _id: '323' abstract: - lang: eng text: 'In the here presented thesis, we explore the role of branched actin networks in cell migration and antigen presentation, the two most relevant processes in dendritic cell biology. Branched actin networks construct lamellipodial protrusions at the leading edge of migrating cells. These are typically seen as adhesive structures, which mediate force transduction to the extracellular matrix that leads to forward locomotion. We ablated Arp2/3 nucleation promoting factor WAVE in DCs and found that the resulting cells lack lamellipodial protrusions. Instead, depending on the maturation state, one or multiple filopodia were formed. By challenging these cells in a variety of migration assays we found that lamellipodial protrusions are dispensable for the locomotion of leukocytes and actually dampen the speed of migration. However, lamellipodia are critically required to negotiate complex environments that DCs experience while they travel to the next draining lymph node. Taken together our results suggest that leukocyte lamellipodia have rather a sensory- than a force transducing function. Furthermore, we show for the first time structure and dynamics of dendritic cell F-actin at the immunological synapse with naïve T cells. Dendritic cell F-actin appears as dynamic foci that are nucleated by the Arp2/3 complex. WAVE ablated dendritic cells show increased membrane tension, leading to an altered ultrastructure of the immunological synapse and severe T cell priming defects. These results point towards a previously unappreciated role of the cellular mechanics of dendritic cells in T cell activation. Additionally, we present a novel cell culture based system for the differentiation of dendritic cells from conditionally immortalized hematopoietic precursors. These precursor cells are genetically tractable via the CRISPR/Cas9 system while they retain their ability to differentiate into highly migratory dendritic cells and other immune cells. This will foster the study of all aspects of dendritic cell biology and beyond. ' acknowledged_ssus: - _id: NanoFab - _id: Bio - _id: PreCl - _id: EM-Fac acknowledgement: "First of all I would like to thank Michael Sixt for giving me the opportunity to work in \r\nhis group and for his support throughout the years. He is a truly inspiring person and \r\nthe best boss one can imagine. I would \ also like to thank all current and past \r\nmembers of the Sixt group for their help and the great working atmosphere in the lab. \r\nIt is a true privilege to work with such a bright, funny and friendly group of people and \r\nI’m proud \ that I could be part of it. Furthermore, I would like to say ‘thank \ you’ to Daria Siekhaus for all the meetings and discussion we had throughout the years \r\nand to Federica Benvenuti for being part of my committee. \ I am also grateful to Jack \r\nMerrin in the nanofabrication facility \ and all the people working in the bioimaging-\r\n, the electron microscopy- and the preclinical facilities." alternative_title: - ISTA Thesis article_processing_charge: No author: - first_name: Alexander F full_name: Leithner, Alexander F id: 3B1B77E4-F248-11E8-B48F-1D18A9856A87 last_name: Leithner orcid: 0000-0002-1073-744X citation: ama: Leithner AF. Branched actin networks in dendritic cell biology. 2018. doi:10.15479/AT:ISTA:th_998 apa: Leithner, A. F. (2018). Branched actin networks in dendritic cell biology. Institute of Science and Technology Austria. https://doi.org/10.15479/AT:ISTA:th_998 chicago: Leithner, Alexander F. “Branched Actin Networks in Dendritic Cell Biology.” Institute of Science and Technology Austria, 2018. https://doi.org/10.15479/AT:ISTA:th_998. ieee: A. F. Leithner, “Branched actin networks in dendritic cell biology,” Institute of Science and Technology Austria, 2018. ista: Leithner AF. 2018. Branched actin networks in dendritic cell biology. Institute of Science and Technology Austria. mla: Leithner, Alexander F. Branched Actin Networks in Dendritic Cell Biology. Institute of Science and Technology Austria, 2018, doi:10.15479/AT:ISTA:th_998. short: A.F. Leithner, Branched Actin Networks in Dendritic Cell Biology, Institute of Science and Technology Austria, 2018. date_created: 2018-12-11T11:45:49Z date_published: 2018-04-12T00:00:00Z date_updated: 2023-09-07T12:39:44Z day: '12' ddc: - '571' - '599' - '610' degree_awarded: PhD department: - _id: MiSi doi: 10.15479/AT:ISTA:th_998 file: - access_level: closed checksum: d5e3edbac548c26c1fa43a4b37a54a4c content_type: application/vnd.openxmlformats-officedocument.wordprocessingml.document creator: dernst date_created: 2019-04-05T09:23:11Z date_updated: 2021-02-11T23:30:17Z embargo_to: open_access file_id: '6219' file_name: PhD_thesis_AlexLeithner_final_version.docx file_size: 29027671 relation: source_file - access_level: open_access checksum: 071f7476db29e41146824ebd0697cb10 content_type: application/pdf creator: dernst date_created: 2019-04-05T09:23:11Z date_updated: 2021-02-11T11:17:16Z embargo: 2019-04-15 file_id: '6220' file_name: PhD_thesis_AlexLeithner.pdf file_size: 66045341 relation: main_file file_date_updated: 2021-02-11T23:30:17Z has_accepted_license: '1' language: - iso: eng month: '04' oa: 1 oa_version: Published Version page: '99' publication_identifier: issn: - 2663-337X publication_status: published publisher: Institute of Science and Technology Austria publist_id: '7542' pubrep_id: '998' related_material: record: - id: '1321' relation: part_of_dissertation status: public status: public supervisor: - first_name: Michael K full_name: Sixt, Michael K id: 41E9FBEA-F248-11E8-B48F-1D18A9856A87 last_name: Sixt orcid: 0000-0002-6620-9179 title: Branched actin networks in dendritic cell biology tmp: image: /images/cc_by.png legal_code_url: https://creativecommons.org/licenses/by/4.0/legalcode name: Creative Commons Attribution 4.0 International Public License (CC-BY 4.0) short: CC BY (4.0) type: dissertation user_id: c635000d-4b10-11ee-a964-aac5a93f6ac1 year: '2018' ... --- _id: '326' abstract: - lang: eng text: Three-dimensional (3D) super-resolution microscopy technique structured illumination microscopy (SIM) imaging of dendritic spines along the dendrite has not been previously performed in fixed tissues, mainly due to deterioration of the stripe pattern of the excitation laser induced by light scattering and optical aberrations. To address this issue and solve these optical problems, we applied a novel clearing reagent, LUCID, to fixed brains. In SIM imaging, the penetration depth and the spatial resolution were improved in LUCID-treated slices, and 160-nm spatial resolution was obtained in a large portion of the imaging volume on a single apical dendrite. Furthermore, in a morphological analysis of spine heads of layer V pyramidal neurons (L5PNs) in the medial prefrontal cortex (mPFC) of chronic dexamethasone (Dex)-treated mice, SIM imaging revealed an altered distribution of spine forms that could not be detected by high-NA confocal imaging. Thus, super-resolution SIM imaging represents a promising high-throughput method for revealing spine morphologies in single dendrites. acknowledged_ssus: - _id: EM-Fac article_processing_charge: No author: - first_name: Kazuaki full_name: Sawada, Kazuaki last_name: Sawada - first_name: Ryosuke full_name: Kawakami, Ryosuke last_name: Kawakami - first_name: Ryuichi full_name: Shigemoto, Ryuichi id: 499F3ABC-F248-11E8-B48F-1D18A9856A87 last_name: Shigemoto orcid: 0000-0001-8761-9444 - first_name: Tomomi full_name: Nemoto, Tomomi last_name: Nemoto citation: ama: Sawada K, Kawakami R, Shigemoto R, Nemoto T. Super resolution structural analysis of dendritic spines using three-dimensional structured illumination microscopy in cleared mouse brain slices. European Journal of Neuroscience. 2018;47(9):1033-1042. doi:10.1111/ejn.13901 apa: Sawada, K., Kawakami, R., Shigemoto, R., & Nemoto, T. (2018). Super resolution structural analysis of dendritic spines using three-dimensional structured illumination microscopy in cleared mouse brain slices. European Journal of Neuroscience. Wiley. https://doi.org/10.1111/ejn.13901 chicago: Sawada, Kazuaki, Ryosuke Kawakami, Ryuichi Shigemoto, and Tomomi Nemoto. “Super Resolution Structural Analysis of Dendritic Spines Using Three-Dimensional Structured Illumination Microscopy in Cleared Mouse Brain Slices.” European Journal of Neuroscience. Wiley, 2018. https://doi.org/10.1111/ejn.13901. ieee: K. Sawada, R. Kawakami, R. Shigemoto, and T. Nemoto, “Super resolution structural analysis of dendritic spines using three-dimensional structured illumination microscopy in cleared mouse brain slices,” European Journal of Neuroscience, vol. 47, no. 9. Wiley, pp. 1033–1042, 2018. ista: Sawada K, Kawakami R, Shigemoto R, Nemoto T. 2018. Super resolution structural analysis of dendritic spines using three-dimensional structured illumination microscopy in cleared mouse brain slices. European Journal of Neuroscience. 47(9), 1033–1042. mla: Sawada, Kazuaki, et al. “Super Resolution Structural Analysis of Dendritic Spines Using Three-Dimensional Structured Illumination Microscopy in Cleared Mouse Brain Slices.” European Journal of Neuroscience, vol. 47, no. 9, Wiley, 2018, pp. 1033–42, doi:10.1111/ejn.13901. short: K. Sawada, R. Kawakami, R. Shigemoto, T. Nemoto, European Journal of Neuroscience 47 (2018) 1033–1042. date_created: 2018-12-11T11:45:50Z date_published: 2018-03-07T00:00:00Z date_updated: 2023-09-19T09:58:40Z day: '07' ddc: - '570' department: - _id: RySh doi: 10.1111/ejn.13901 external_id: isi: - '000431496400001' file: - access_level: open_access checksum: 98e901d8229e44aa8f3b51d248dedd09 content_type: application/pdf creator: dernst date_created: 2018-12-17T16:16:50Z date_updated: 2020-07-14T12:46:06Z file_id: '5721' file_name: 2018_EJN_Sawada.pdf file_size: 4850261 relation: main_file file_date_updated: 2020-07-14T12:46:06Z has_accepted_license: '1' intvolume: ' 47' isi: 1 issue: '9' language: - iso: eng license: https://creativecommons.org/licenses/by-nc/4.0/ month: '03' oa: 1 oa_version: Published Version page: 1033 - 1042 publication: European Journal of Neuroscience publication_status: published publisher: Wiley publist_id: '7539' quality_controlled: '1' scopus_import: '1' status: public title: Super resolution structural analysis of dendritic spines using three-dimensional structured illumination microscopy in cleared mouse brain slices tmp: image: /images/cc_by_nc.png legal_code_url: https://creativecommons.org/licenses/by-nc/4.0/legalcode name: Creative Commons Attribution-NonCommercial 4.0 International (CC BY-NC 4.0) short: CC BY-NC (4.0) type: journal_article user_id: c635000d-4b10-11ee-a964-aac5a93f6ac1 volume: 47 year: '2018' ... --- _id: '395' abstract: - lang: eng text: 'Autism spectrum disorders (ASD) are a group of genetic disorders often overlapping with other neurological conditions. Despite the remarkable number of scientific breakthroughs of the last 100 years, the treatment of neurodevelopmental disorders (e.g. autism spectrum disorder, intellectual disability, epilepsy) remains a great challenge. Recent advancements in geno mics, like whole-exome or whole-genome sequencing, have enabled scientists to identify numerous mutations underlying neurodevelopmental disorders. Given the few hundred risk genes that were discovered, the etiological variability and the heterogeneous phenotypic outcomes, the need for genotype -along with phenotype- based diagnosis of individual patients becomes a requisite. Driven by this rationale, in a previous study our group described mutations, identified via whole - exome sequencing, in the gene BCKDK – encoding for a key regulator of branched chain amin o acid (BCAA) catabolism - as a cause of ASD. Following up on the role of BCAAs, in the study described here we show that the solute carrier transporter 7a5 (SLC7A5), a large neutral amino acid transporter localized mainly at the blood brain barrier (BBB), has an essential role in maintaining normal levels of brain BCAAs. In mice, deletion of Slc7a5 from the endothelial cells of the BBB leads to atypical brain amino acid profile, abnormal mRNA translation and severe neurolo gical abnormalities. Additionally, deletion of Slc7a5 from the neural progenitor cell population leads to microcephaly. Interestingly, we demonstrate that BCAA intracerebroventricular administration ameliorates abnormal behaviors in adult mutant mice. Furthermore, whole - exome sequencing of patients diagnosed with neurological dis o r ders helped us identify several patients with autistic traits, microcephaly and motor delay carrying deleterious homozygous mutations in the SLC7A5 gene. In conclusion, our data elucidate a neurological syndrome defined by SLC7A5 mutations and support an essential role for t he BCAA s in human bra in function. Together with r ecent studies (described in chapter two) that have successfully made the transition into clinical practice, our findings on the role of B CAAs might have a crucial impact on the development of novel individualized therapeutic strategies for ASD. ' acknowledged_ssus: - _id: PreCl - _id: EM-Fac - _id: Bio alternative_title: - ISTA Thesis article_processing_charge: No author: - first_name: Dora-Clara full_name: Tarlungeanu, Dora-Clara id: 2ABCE612-F248-11E8-B48F-1D18A9856A87 last_name: Tarlungeanu citation: ama: Tarlungeanu D-C. The branched chain amino acids in autism spectrum disorders . 2018. doi:10.15479/AT:ISTA:th_992 apa: Tarlungeanu, D.-C. (2018). The branched chain amino acids in autism spectrum disorders . Institute of Science and Technology Austria. https://doi.org/10.15479/AT:ISTA:th_992 chicago: Tarlungeanu, Dora-Clara. “The Branched Chain Amino Acids in Autism Spectrum Disorders .” Institute of Science and Technology Austria, 2018. https://doi.org/10.15479/AT:ISTA:th_992. ieee: D.-C. Tarlungeanu, “The branched chain amino acids in autism spectrum disorders ,” Institute of Science and Technology Austria, 2018. ista: Tarlungeanu D-C. 2018. The branched chain amino acids in autism spectrum disorders . Institute of Science and Technology Austria. mla: Tarlungeanu, Dora-Clara. The Branched Chain Amino Acids in Autism Spectrum Disorders . Institute of Science and Technology Austria, 2018, doi:10.15479/AT:ISTA:th_992. short: D.-C. Tarlungeanu, The Branched Chain Amino Acids in Autism Spectrum Disorders , Institute of Science and Technology Austria, 2018. date_created: 2018-12-11T11:46:14Z date_published: 2018-03-01T00:00:00Z date_updated: 2023-09-07T12:38:59Z day: '01' ddc: - '570' - '616' degree_awarded: PhD department: - _id: GaNo doi: 10.15479/AT:ISTA:th_992 file: - access_level: closed checksum: 9f5231c96e0ad945040841a8630232da content_type: application/vnd.openxmlformats-officedocument.wordprocessingml.document creator: dernst date_created: 2019-04-05T09:19:17Z date_updated: 2021-02-11T23:30:15Z embargo_to: open_access file_id: '6217' file_name: 2018_Thesis_Tarlungeanu_source.docx file_size: 43684035 relation: source_file - access_level: open_access checksum: 0c33c370aa2010df5c552db57a6d01e9 content_type: application/pdf creator: dernst date_created: 2019-04-05T09:19:17Z date_updated: 2021-02-11T11:17:16Z embargo: 2018-03-15 file_id: '6218' file_name: 2018_Thesis_Tarlungeanu.pdf file_size: 30511532 relation: main_file file_date_updated: 2021-02-11T23:30:15Z has_accepted_license: '1' language: - iso: eng month: '03' oa: 1 oa_version: Published Version page: '88' project: - _id: 25473368-B435-11E9-9278-68D0E5697425 call_identifier: FWF grant_number: F03523 name: Transmembrane Transporters in Health and Disease publication_identifier: issn: - 2663-337X publication_status: published publisher: Institute of Science and Technology Austria publist_id: '7434' pubrep_id: '992' related_material: record: - id: '1183' relation: part_of_dissertation status: public status: public supervisor: - first_name: Gaia full_name: Novarino, Gaia id: 3E57A680-F248-11E8-B48F-1D18A9856A87 last_name: Novarino orcid: 0000-0002-7673-7178 title: 'The branched chain amino acids in autism spectrum disorders ' tmp: image: /images/cc_by.png legal_code_url: https://creativecommons.org/licenses/by/4.0/legalcode name: Creative Commons Attribution 4.0 International Public License (CC-BY 4.0) short: CC BY (4.0) type: dissertation user_id: c635000d-4b10-11ee-a964-aac5a93f6ac1 year: '2018' ... --- _id: '1094' abstract: - lang: eng text: Immunogold labeling of freeze-fracture replicas has recently been used for high-resolution visualization of protein localization in electron microscopy. This method has higher labeling efficiency than conventional immunogold methods for membrane molecules allowing precise quantitative measurements. However, one of the limitations of freeze-fracture replica immunolabeling is difficulty in keeping structural orientation and identifying labeled profiles in complex tissues like brain. The difficulty is partly due to fragmentation of freeze-fracture replica preparations during labeling procedures and limited morphological clues on the replica surface. To overcome these issues, we introduce here a grid-glued replica method combined with SEM observation. This method allows histological staining before dissolving the tissue and easy handling of replicas during immunogold labeling, and keeps the whole replica surface intact without fragmentation. The procedure described here is also useful for matched double-replica analysis allowing further identification of labeled profiles in corresponding P-face and E-face. acknowledged_ssus: - _id: EM-Fac acknowledgement: 'We thank Prof. Elek Molnár for providing us a pan-AMPAR anti-body used in Fig.2 and Dr. Ludek Lovicar for technical assistance in scanning electron microscope imaging. This work was supported by the European Union (HBP—Project Ref. 604102). ' alternative_title: - Methods in Molecular Biology article_processing_charge: No author: - first_name: Harumi full_name: Harada, Harumi id: 2E55CDF2-F248-11E8-B48F-1D18A9856A87 last_name: Harada orcid: 0000-0001-7429-7896 - first_name: Ryuichi full_name: Shigemoto, Ryuichi id: 499F3ABC-F248-11E8-B48F-1D18A9856A87 last_name: Shigemoto orcid: 0000-0001-8761-9444 citation: ama: 'Harada H, Shigemoto R. Immunogold protein localization on grid-glued freeze-fracture replicas. In: High-Resolution Imaging of Cellular Proteins. Vol 1474. Springer; 2016:203-216. doi:10.1007/978-1-4939-6352-2_12' apa: Harada, H., & Shigemoto, R. (2016). Immunogold protein localization on grid-glued freeze-fracture replicas. In High-Resolution Imaging of Cellular Proteins (Vol. 1474, pp. 203–216). Springer. https://doi.org/10.1007/978-1-4939-6352-2_12 chicago: Harada, Harumi, and Ryuichi Shigemoto. “Immunogold Protein Localization on Grid-Glued Freeze-Fracture Replicas.” In High-Resolution Imaging of Cellular Proteins, 1474:203–16. Springer, 2016. https://doi.org/10.1007/978-1-4939-6352-2_12. ieee: H. Harada and R. Shigemoto, “Immunogold protein localization on grid-glued freeze-fracture replicas,” in High-Resolution Imaging of Cellular Proteins, vol. 1474, Springer, 2016, pp. 203–216. ista: 'Harada H, Shigemoto R. 2016.Immunogold protein localization on grid-glued freeze-fracture replicas. In: High-Resolution Imaging of Cellular Proteins. Methods in Molecular Biology, vol. 1474, 203–216.' mla: Harada, Harumi, and Ryuichi Shigemoto. “Immunogold Protein Localization on Grid-Glued Freeze-Fracture Replicas.” High-Resolution Imaging of Cellular Proteins, vol. 1474, Springer, 2016, pp. 203–16, doi:10.1007/978-1-4939-6352-2_12. short: H. Harada, R. Shigemoto, in:, High-Resolution Imaging of Cellular Proteins, Springer, 2016, pp. 203–216. date_created: 2018-12-11T11:50:06Z date_published: 2016-08-12T00:00:00Z date_updated: 2023-09-05T14:09:01Z day: '12' department: - _id: RySh doi: 10.1007/978-1-4939-6352-2_12 ec_funded: 1 intvolume: ' 1474' language: - iso: eng month: '08' oa_version: None page: 203 - 216 project: - _id: 25CD3DD2-B435-11E9-9278-68D0E5697425 call_identifier: FP7 grant_number: '604102' name: Localization of ion channels and receptors by two and three-dimensional immunoelectron microscopic approaches publication: High-Resolution Imaging of Cellular Proteins publication_identifier: eissn: - 1611-3349 issn: - 0302-9743 publication_status: published publisher: Springer publist_id: '6281' quality_controlled: '1' status: public title: Immunogold protein localization on grid-glued freeze-fracture replicas type: book_chapter user_id: c635000d-4b10-11ee-a964-aac5a93f6ac1 volume: 1474 year: '2016' ... --- _id: '1993' abstract: - lang: eng text: 'The fitness effects of symbionts on their hosts can be context-dependent, with usually benign symbionts causing detrimental effects when their hosts are stressed, or typically parasitic symbionts providing protection towards their hosts (e.g. against pathogen infection). Here, we studied the novel association between the invasive garden ant Lasius neglectus and its fungal ectosymbiont Laboulbenia formicarum for potential costs and benefits. We tested ants with different Laboulbenia levels for their survival and immunity under resource limitation and exposure to the obligate killing entomopathogen Metarhizium brunneum. While survival of L. neglectus workers under starvation was significantly decreased with increasing Laboulbenia levels, host survival under Metarhizium exposure increased with higher levels of the ectosymbiont, suggesting a symbiont-mediated anti-pathogen protection, which seems to be driven mechanistically by both improved sanitary behaviours and an upregulated immune system. Ants with high Laboulbenia levels showed significantly longer self-grooming and elevated expression of immune genes relevant for wound repair and antifungal responses (β-1,3-glucan binding protein, Prophenoloxidase), compared with ants carrying low Laboulbenia levels. This suggests that the ectosymbiont Laboulbenia formicarum weakens its ant host by either direct resource exploitation or the costs of an upregulated behavioural and immunological response, which, however, provides a prophylactic protection upon later exposure to pathogens. ' acknowledged_ssus: - _id: EM-Fac acknowledgement: "Funding was obtained by the German Research Foundation (CR 118–2) and an ERC StG (243071) by the European Research Council (both to S.C.).\r\nWe thank Line V. Ugelvig for help with ant collection and statistical discussion, Xavier Espadaler for detailed information on the ant collection site, Birgit Lautenschläger for the electron microscopy images and Eva Sixt for ant drawings. We further thank Jørgen Eilenberg for the fungal strain, Meghan L. Vyleta for genetic strain characterization and immune gene primer development, Paul Schmid-Hempel for discussion, and Line V. Ugelvig, Xavier Espadaler and Christopher D. Pull for comments on the manuscript. S.C., M.K. and S.T. conceived the study; M.K. and A.V.G. performed the experiments; M.K. performed the statistical analysis; S.C. and M.K. wrote the manuscript with intense contributions of A.V.G. and S.T.; all authors approved the manuscript." article_number: '20141976' article_processing_charge: No article_type: original author: - first_name: Matthias full_name: Konrad, Matthias id: 46528076-F248-11E8-B48F-1D18A9856A87 last_name: Konrad - first_name: Anna V full_name: Grasse, Anna V id: 406F989C-F248-11E8-B48F-1D18A9856A87 last_name: Grasse - first_name: Simon full_name: Tragust, Simon id: 35A7A418-F248-11E8-B48F-1D18A9856A87 last_name: Tragust - first_name: Sylvia full_name: Cremer, Sylvia id: 2F64EC8C-F248-11E8-B48F-1D18A9856A87 last_name: Cremer orcid: 0000-0002-2193-3868 citation: ama: Konrad M, Grasse AV, Tragust S, Cremer S. Anti-pathogen protection versus survival costs mediated by an ectosymbiont in an ant host. Proceedings of the Royal Society of London Series B Biological Sciences. 2015;282(1799). doi:10.1098/rspb.2014.1976 apa: Konrad, M., Grasse, A. V., Tragust, S., & Cremer, S. (2015). Anti-pathogen protection versus survival costs mediated by an ectosymbiont in an ant host. Proceedings of the Royal Society of London Series B Biological Sciences. The Royal Society. https://doi.org/10.1098/rspb.2014.1976 chicago: Konrad, Matthias, Anna V Grasse, Simon Tragust, and Sylvia Cremer. “Anti-Pathogen Protection versus Survival Costs Mediated by an Ectosymbiont in an Ant Host.” Proceedings of the Royal Society of London Series B Biological Sciences. The Royal Society, 2015. https://doi.org/10.1098/rspb.2014.1976. ieee: M. Konrad, A. V. Grasse, S. Tragust, and S. Cremer, “Anti-pathogen protection versus survival costs mediated by an ectosymbiont in an ant host,” Proceedings of the Royal Society of London Series B Biological Sciences, vol. 282, no. 1799. The Royal Society, 2015. ista: Konrad M, Grasse AV, Tragust S, Cremer S. 2015. Anti-pathogen protection versus survival costs mediated by an ectosymbiont in an ant host. Proceedings of the Royal Society of London Series B Biological Sciences. 282(1799), 20141976. mla: Konrad, Matthias, et al. “Anti-Pathogen Protection versus Survival Costs Mediated by an Ectosymbiont in an Ant Host.” Proceedings of the Royal Society of London Series B Biological Sciences, vol. 282, no. 1799, 20141976, The Royal Society, 2015, doi:10.1098/rspb.2014.1976. short: M. Konrad, A.V. Grasse, S. Tragust, S. Cremer, Proceedings of the Royal Society of London Series B Biological Sciences 282 (2015). date_created: 2018-12-11T11:55:06Z date_published: 2015-01-22T00:00:00Z date_updated: 2023-02-23T14:06:41Z day: '22' department: - _id: SyCr doi: 10.1098/rspb.2014.1976 ec_funded: 1 external_id: pmid: - '25473011' intvolume: ' 282' issue: '1799' language: - iso: eng main_file_link: - open_access: '1' url: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4286035/ month: '01' oa: 1 oa_version: Submitted Version pmid: 1 project: - _id: 25DC711C-B435-11E9-9278-68D0E5697425 call_identifier: FP7 grant_number: '243071' name: 'Social Vaccination in Ant Colonies: from Individual Mechanisms to Society Effects' - _id: 25DAF0B2-B435-11E9-9278-68D0E5697425 grant_number: CR-118/3-1 name: Host-Parasite Coevolution publication: Proceedings of the Royal Society of London Series B Biological Sciences publication_identifier: eissn: - 1471-2954 issn: - 0962-8452 publication_status: published publisher: The Royal Society publist_id: '5090' quality_controlled: '1' related_material: record: - id: '9740' relation: research_data status: public scopus_import: '1' status: public title: Anti-pathogen protection versus survival costs mediated by an ectosymbiont in an ant host type: journal_article user_id: 2DF688A6-F248-11E8-B48F-1D18A9856A87 volume: 282 year: '2015' ...