[{"keyword":["electron microscopy","cryo-EM","EM sample preparation","3D printing","cell culture"],"citation":{"short":"F. Fäßler, B. Zens, R. Hauschild, F.K. Schur, Journal of Structural Biology 212 (2020).","ama":"Fäßler F, Zens B, Hauschild R, Schur FK. 3D printed cell culture grid holders for improved cellular specimen preparation in cryo-electron microscopy. <i>Journal of Structural Biology</i>. 2020;212(3). doi:<a href=\"https://doi.org/10.1016/j.jsb.2020.107633\">10.1016/j.jsb.2020.107633</a>","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.","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.” <i>Journal of Structural Biology</i>. Elsevier, 2020. <a href=\"https://doi.org/10.1016/j.jsb.2020.107633\">https://doi.org/10.1016/j.jsb.2020.107633</a>.","apa":"Fäßler, F., Zens, B., Hauschild, R., &#38; Schur, F. K. (2020). 3D printed cell culture grid holders for improved cellular specimen preparation in cryo-electron microscopy. <i>Journal of Structural Biology</i>. Elsevier. <a href=\"https://doi.org/10.1016/j.jsb.2020.107633\">https://doi.org/10.1016/j.jsb.2020.107633</a>","mla":"Fäßler, Florian, et al. “3D Printed Cell Culture Grid Holders for Improved Cellular Specimen Preparation in Cryo-Electron Microscopy.” <i>Journal of Structural Biology</i>, vol. 212, no. 3, 107633, Elsevier, 2020, doi:<a href=\"https://doi.org/10.1016/j.jsb.2020.107633\">10.1016/j.jsb.2020.107633</a>.","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,” <i>Journal of Structural Biology</i>, vol. 212, no. 3. Elsevier, 2020."},"intvolume":"       212","day":"01","has_accepted_license":"1","status":"public","article_number":"107633","abstract":[{"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.","lang":"eng"}],"department":[{"_id":"FlSc"}],"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.","publication_status":"published","related_material":{"record":[{"status":"public","relation":"used_in_publication","id":"14592"},{"id":"12491","relation":"dissertation_contains","status":"public"}]},"oa":1,"title":"3D printed cell culture grid holders for improved cellular specimen preparation in cryo-electron microscopy","article_type":"original","month":"12","_id":"8586","user_id":"4359f0d1-fa6c-11eb-b949-802e58b17ae8","publication_identifier":{"issn":["1047-8477"]},"scopus_import":"1","article_processing_charge":"Yes (via OA deal)","tmp":{"legal_code_url":"https://creativecommons.org/licenses/by/4.0/legalcode","name":"Creative Commons Attribution 4.0 International Public License (CC-BY 4.0)","image":"/images/cc_by.png","short":"CC BY (4.0)"},"file":[{"relation":"main_file","checksum":"c48cbf594e84fc2f91966ffaafc0918c","file_size":7076870,"creator":"dernst","file_name":"2020_JourStrucBiology_Faessler.pdf","date_created":"2020-12-10T14:01:10Z","content_type":"application/pdf","file_id":"8937","success":1,"access_level":"open_access","date_updated":"2020-12-10T14:01:10Z"}],"isi":1,"volume":212,"year":"2020","date_created":"2020-09-29T13:24:06Z","quality_controlled":"1","project":[{"_id":"9B954C5C-BA93-11EA-9121-9846C619BF3A","grant_number":"P33367","name":"Structure and isoform diversity of the Arp2/3 complex"},{"name":"NÖ-Fonds Preis für die Jungforscherin des Jahres am IST Austria","_id":"059B463C-7A3F-11EA-A408-12923DDC885E"}],"acknowledged_ssus":[{"_id":"ScienComp"},{"_id":"LifeSc"},{"_id":"Bio"},{"_id":"EM-Fac"}],"date_updated":"2024-03-25T23:30:04Z","issue":"3","publication":"Journal of Structural Biology","author":[{"id":"404F5528-F248-11E8-B48F-1D18A9856A87","orcid":"0000-0001-7149-769X","first_name":"Florian","full_name":"Fäßler, Florian","last_name":"Fäßler"},{"id":"45FD126C-F248-11E8-B48F-1D18A9856A87","full_name":"Zens, Bettina","first_name":"Bettina","last_name":"Zens"},{"last_name":"Hauschild","first_name":"Robert","full_name":"Hauschild, Robert","id":"4E01D6B4-F248-11E8-B48F-1D18A9856A87","orcid":"0000-0001-9843-3522"},{"first_name":"Florian KM","full_name":"Schur, Florian KM","last_name":"Schur","orcid":"0000-0003-4790-8078","id":"48AD8942-F248-11E8-B48F-1D18A9856A87"}],"ddc":["570"],"oa_version":"Published Version","external_id":{"isi":["000600997800008"]},"date_published":"2020-12-01T00:00:00Z","language":[{"iso":"eng"}],"type":"journal_article","file_date_updated":"2020-12-10T14:01:10Z","doi":"10.1016/j.jsb.2020.107633","publisher":"Elsevier"},{"oa":1,"title":"The coupling mechanism of mammalian respiratory complex I","publication_status":"published","article_type":"original","user_id":"4359f0d1-fa6c-11eb-b949-802e58b17ae8","_id":"8737","month":"10","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","department":[{"_id":"LeSa"}],"article_number":"eabc4209","status":"public","abstract":[{"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.","lang":"eng"}],"ec_funded":1,"citation":{"short":"D. Kampjut, L.A. Sazanov, Science 370 (2020).","ama":"Kampjut D, Sazanov LA. The coupling mechanism of mammalian respiratory complex I. <i>Science</i>. 2020;370(6516). doi:<a href=\"https://doi.org/10.1126/science.abc4209\">10.1126/science.abc4209</a>","apa":"Kampjut, D., &#38; Sazanov, L. A. (2020). The coupling mechanism of mammalian respiratory complex I. <i>Science</i>. American Association for the Advancement of Science. <a href=\"https://doi.org/10.1126/science.abc4209\">https://doi.org/10.1126/science.abc4209</a>","ista":"Kampjut D, Sazanov LA. 2020. The coupling mechanism of mammalian respiratory complex I. Science. 370(6516), eabc4209.","chicago":"Kampjut, Domen, and Leonid A Sazanov. “The Coupling Mechanism of Mammalian Respiratory Complex I.” <i>Science</i>. American Association for the Advancement of Science, 2020. <a href=\"https://doi.org/10.1126/science.abc4209\">https://doi.org/10.1126/science.abc4209</a>.","ieee":"D. Kampjut and L. A. Sazanov, “The coupling mechanism of mammalian respiratory complex I,” <i>Science</i>, vol. 370, no. 6516. American Association for the Advancement of Science, 2020.","mla":"Kampjut, Domen, and Leonid A. Sazanov. “The Coupling Mechanism of Mammalian Respiratory Complex I.” <i>Science</i>, vol. 370, no. 6516, eabc4209, American Association for the Advancement of Science, 2020, doi:<a href=\"https://doi.org/10.1126/science.abc4209\">10.1126/science.abc4209</a>."},"intvolume":"       370","day":"30","has_accepted_license":"1","language":[{"iso":"eng"}],"file_date_updated":"2020-11-26T18:47:58Z","type":"journal_article","doi":"10.1126/science.abc4209","publisher":"American Association for the Advancement of Science","issue":"6516","publication":"Science","ddc":["572"],"author":[{"id":"37233050-F248-11E8-B48F-1D18A9856A87","full_name":"Kampjut, Domen","first_name":"Domen","last_name":"Kampjut"},{"id":"338D39FE-F248-11E8-B48F-1D18A9856A87","orcid":"0000-0002-0977-7989","full_name":"Sazanov, Leonid A","first_name":"Leonid A","last_name":"Sazanov"}],"external_id":{"pmid":["32972993"],"isi":["000583031800004"]},"oa_version":"Submitted Version","date_published":"2020-10-30T00:00:00Z","volume":370,"isi":1,"file":[{"checksum":"658ba90979ca9528a2efdfac8547047a","relation":"main_file","creator":"lsazanov","file_size":7618987,"file_name":"Full_manuscript_with_SI_opt_red.pdf","access_level":"open_access","success":1,"date_updated":"2020-11-26T18:47:58Z","date_created":"2020-11-26T18:47:58Z","content_type":"application/pdf","file_id":"8820"}],"quality_controlled":"1","year":"2020","date_created":"2020-11-08T23:01:23Z","date_updated":"2023-08-22T12:35:38Z","acknowledged_ssus":[{"_id":"LifeSc"},{"_id":"EM-Fac"}],"project":[{"name":"International IST Doctoral Program","grant_number":"665385","_id":"2564DBCA-B435-11E9-9278-68D0E5697425","call_identifier":"H2020"}],"publication_identifier":{"eissn":["10959203"]},"scopus_import":"1","pmid":1,"article_processing_charge":"No"},{"has_accepted_license":"1","day":"22","citation":{"apa":"Fäßler, F., Dimchev, G. A., Hodirnau, V.-V., Wan, W., &#38; Schur, F. K. (2020). Cryo-electron tomography structure of Arp2/3 complex in cells reveals new insights into the branch junction. <i>Nature Communications</i>. Springer Nature. <a href=\"https://doi.org/10.1038/s41467-020-20286-x\">https://doi.org/10.1038/s41467-020-20286-x</a>","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.","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.” <i>Nature Communications</i>. Springer Nature, 2020. <a href=\"https://doi.org/10.1038/s41467-020-20286-x\">https://doi.org/10.1038/s41467-020-20286-x</a>.","mla":"Fäßler, Florian, et al. “Cryo-Electron Tomography Structure of Arp2/3 Complex in Cells Reveals New Insights into the Branch Junction.” <i>Nature Communications</i>, vol. 11, 6437, Springer Nature, 2020, doi:<a href=\"https://doi.org/10.1038/s41467-020-20286-x\">10.1038/s41467-020-20286-x</a>.","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,” <i>Nature Communications</i>, vol. 11. Springer Nature, 2020.","short":"F. Fäßler, G.A. Dimchev, V.-V. Hodirnau, W. Wan, F.K. Schur, Nature Communications 11 (2020).","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. <i>Nature Communications</i>. 2020;11. doi:<a href=\"https://doi.org/10.1038/s41467-020-20286-x\">10.1038/s41467-020-20286-x</a>"},"intvolume":"        11","keyword":["General Biochemistry","Genetics and Molecular Biology","General Physics and Astronomy","General Chemistry"],"abstract":[{"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.","lang":"eng"}],"status":"public","article_number":"6437","department":[{"_id":"FlSc"},{"_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. ","month":"12","_id":"8971","user_id":"4359f0d1-fa6c-11eb-b949-802e58b17ae8","article_type":"original","publication_status":"published","title":"Cryo-electron tomography structure of Arp2/3 complex in cells reveals new insights into the branch junction","related_material":{"link":[{"description":"News on IST Homepage","url":"https://ist.ac.at/en/news/cutting-edge-technology-reveals-structures-within-cells/","relation":"press_release"}]},"oa":1,"tmp":{"legal_code_url":"https://creativecommons.org/licenses/by/4.0/legalcode","name":"Creative Commons Attribution 4.0 International Public License (CC-BY 4.0)","image":"/images/cc_by.png","short":"CC BY (4.0)"},"article_processing_charge":"No","scopus_import":"1","publication_identifier":{"issn":["2041-1723"]},"project":[{"grant_number":"P33367","name":"Structure and isoform diversity of the Arp2/3 complex","_id":"9B954C5C-BA93-11EA-9121-9846C619BF3A"},{"call_identifier":"FWF","name":"Protein structure and function in filopodia across scales","grant_number":"M02495","_id":"2674F658-B435-11E9-9278-68D0E5697425"}],"acknowledged_ssus":[{"_id":"ScienComp"},{"_id":"LifeSc"},{"_id":"Bio"},{"_id":"EM-Fac"}],"date_updated":"2023-08-24T11:01:50Z","date_created":"2020-12-23T08:25:45Z","year":"2020","quality_controlled":"1","isi":1,"file":[{"checksum":"55d43ea0061cc4027ba45e966e1db8cc","relation":"main_file","creator":"dernst","file_size":3958727,"file_name":"2020_NatureComm_Faessler.pdf","success":1,"access_level":"open_access","date_updated":"2020-12-28T08:16:10Z","date_created":"2020-12-28T08:16:10Z","content_type":"application/pdf","file_id":"8975"}],"volume":11,"date_published":"2020-12-22T00:00:00Z","oa_version":"Published Version","external_id":{"isi":["000603078000003"]},"author":[{"last_name":"Fäßler","full_name":"Fäßler, Florian","first_name":"Florian","orcid":"0000-0001-7149-769X","id":"404F5528-F248-11E8-B48F-1D18A9856A87"},{"orcid":"0000-0001-8370-6161","id":"38C393BE-F248-11E8-B48F-1D18A9856A87","last_name":"Dimchev","first_name":"Georgi A","full_name":"Dimchev, Georgi A"},{"first_name":"Victor-Valentin","full_name":"Hodirnau, Victor-Valentin","last_name":"Hodirnau","id":"3661B498-F248-11E8-B48F-1D18A9856A87"},{"last_name":"Wan","first_name":"William","full_name":"Wan, William"},{"id":"48AD8942-F248-11E8-B48F-1D18A9856A87","orcid":"0000-0003-4790-8078","full_name":"Schur, Florian KM","first_name":"Florian KM","last_name":"Schur"}],"ddc":["570"],"publication":"Nature Communications","publisher":"Springer Nature","doi":"10.1038/s41467-020-20286-x","type":"journal_article","file_date_updated":"2020-12-28T08:16:10Z","language":[{"iso":"eng"}]},{"citation":{"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.” <i>ELife</i>. eLife Sciences Publications, 2020. <a href=\"https://doi.org/10.7554/eLife.52067\">https://doi.org/10.7554/eLife.52067</a>.","apa":"Narasimhan, M., Johnson, A. J., Prizak, R., Kaufmann, W., Tan, S., Casillas Perez, B. E., &#38; Friml, J. (2020). Evolutionarily unique mechanistic framework of clathrin-mediated endocytosis in plants. <i>ELife</i>. eLife Sciences Publications. <a href=\"https://doi.org/10.7554/eLife.52067\">https://doi.org/10.7554/eLife.52067</a>","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.","ieee":"M. Narasimhan <i>et al.</i>, “Evolutionarily unique mechanistic framework of clathrin-mediated endocytosis in plants,” <i>eLife</i>, vol. 9. eLife Sciences Publications, 2020.","mla":"Narasimhan, Madhumitha, et al. “Evolutionarily Unique Mechanistic Framework of Clathrin-Mediated Endocytosis in Plants.” <i>ELife</i>, vol. 9, e52067, eLife Sciences Publications, 2020, doi:<a href=\"https://doi.org/10.7554/eLife.52067\">10.7554/eLife.52067</a>.","short":"M. Narasimhan, A.J. Johnson, R. Prizak, W. Kaufmann, S. Tan, B.E. Casillas Perez, J. Friml, ELife 9 (2020).","ama":"Narasimhan M, Johnson AJ, Prizak R, et al. Evolutionarily unique mechanistic framework of clathrin-mediated endocytosis in plants. <i>eLife</i>. 2020;9. doi:<a href=\"https://doi.org/10.7554/eLife.52067\">10.7554/eLife.52067</a>"},"intvolume":"         9","day":"23","has_accepted_license":"1","status":"public","article_number":"e52067","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."}],"ec_funded":1,"department":[{"_id":"JiFr"},{"_id":"GaTk"},{"_id":"EM-Fac"},{"_id":"SyCr"}],"publication_status":"published","oa":1,"title":"Evolutionarily unique mechanistic framework of clathrin-mediated endocytosis in plants","article_type":"original","month":"01","_id":"7490","user_id":"4359f0d1-fa6c-11eb-b949-802e58b17ae8","publication_identifier":{"eissn":["2050-084X"]},"pmid":1,"scopus_import":"1","article_processing_charge":"No","tmp":{"legal_code_url":"https://creativecommons.org/licenses/by/4.0/legalcode","name":"Creative Commons Attribution 4.0 International Public License (CC-BY 4.0)","image":"/images/cc_by.png","short":"CC BY (4.0)"},"isi":1,"file":[{"date_updated":"2020-07-14T12:47:59Z","access_level":"open_access","content_type":"application/pdf","date_created":"2020-02-18T07:21:16Z","file_id":"7494","file_name":"2020_eLife_Narasimhan.pdf","creator":"dernst","file_size":7247468,"checksum":"2052daa4be5019534f3a42f200a09f32","relation":"main_file"}],"volume":9,"year":"2020","date_created":"2020-02-16T23:00:50Z","quality_controlled":"1","project":[{"call_identifier":"H2020","_id":"261099A6-B435-11E9-9278-68D0E5697425","name":"Tracing Evolution of Auxin Transport and Polarity in Plants","grant_number":"742985"},{"grant_number":"I03630","name":"Molecular mechanisms of endocytic cargo recognition in plants","_id":"26538374-B435-11E9-9278-68D0E5697425","call_identifier":"FWF"}],"acknowledged_ssus":[{"_id":"LifeSc"},{"_id":"Bio"},{"_id":"EM-Fac"}],"date_updated":"2023-08-18T06:33:07Z","publication":"eLife","author":[{"orcid":"0000-0002-8600-0671","id":"44BF24D0-F248-11E8-B48F-1D18A9856A87","full_name":"Narasimhan, Madhumitha","first_name":"Madhumitha","last_name":"Narasimhan"},{"full_name":"Johnson, Alexander J","first_name":"Alexander J","last_name":"Johnson","id":"46A62C3A-F248-11E8-B48F-1D18A9856A87","orcid":"0000-0002-2739-8843"},{"last_name":"Prizak","full_name":"Prizak, Roshan","first_name":"Roshan","id":"4456104E-F248-11E8-B48F-1D18A9856A87"},{"first_name":"Walter","full_name":"Kaufmann, Walter","last_name":"Kaufmann","id":"3F99E422-F248-11E8-B48F-1D18A9856A87","orcid":"0000-0001-9735-5315"},{"first_name":"Shutang","full_name":"Tan, Shutang","last_name":"Tan","orcid":"0000-0002-0471-8285","id":"2DE75584-F248-11E8-B48F-1D18A9856A87"},{"id":"351ED2AA-F248-11E8-B48F-1D18A9856A87","full_name":"Casillas Perez, Barbara E","first_name":"Barbara E","last_name":"Casillas Perez"},{"last_name":"Friml","full_name":"Friml, Jiří","first_name":"Jiří","orcid":"0000-0002-8302-7596","id":"4159519E-F248-11E8-B48F-1D18A9856A87"}],"ddc":["570","580"],"oa_version":"Published Version","external_id":{"isi":["000514104100001"],"pmid":["31971511"]},"date_published":"2020-01-23T00:00:00Z","language":[{"iso":"eng"}],"type":"journal_article","file_date_updated":"2020-07-14T12:47:59Z","doi":"10.7554/eLife.52067","publisher":"eLife Sciences Publications"},{"user_id":"c635000d-4b10-11ee-a964-aac5a93f6ac1","month":"02","_id":"7525","title":"Localization and functional role of Cav2.3 in the medial habenula to interpeduncular nucleus pathway","oa":1,"publication_status":"published","department":[{"_id":"RySh"}],"alternative_title":["ISTA Thesis"],"degree_awarded":"PhD","status":"public","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"}],"day":"28","has_accepted_license":"1","supervisor":[{"orcid":"0000-0001-8761-9444","id":"499F3ABC-F248-11E8-B48F-1D18A9856A87","last_name":"Shigemoto","first_name":"Ryuichi","full_name":"Shigemoto, Ryuichi"}],"keyword":["Cav2.3","medial habenula (MHb)","interpeduncular nucleus (IPN)"],"citation":{"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.","ama":"Bhandari P. Localization and functional role of Cav2.3 in the medial habenula to interpeduncular nucleus pathway. 2020. doi:<a href=\"https://doi.org/10.15479/AT:ISTA:7525\">10.15479/AT:ISTA:7525</a>","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. <a href=\"https://doi.org/10.15479/AT:ISTA:7525\">https://doi.org/10.15479/AT:ISTA:7525</a>.","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.","apa":"Bhandari, P. (2020). <i>Localization and functional role of Cav2.3 in the medial habenula to interpeduncular nucleus pathway</i>. Institute of Science and Technology Austria. <a href=\"https://doi.org/10.15479/AT:ISTA:7525\">https://doi.org/10.15479/AT:ISTA:7525</a>","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.","mla":"Bhandari, Pradeep. <i>Localization and Functional Role of Cav2.3 in the Medial Habenula to Interpeduncular Nucleus Pathway</i>. Institute of Science and Technology Austria, 2020, doi:<a href=\"https://doi.org/10.15479/AT:ISTA:7525\">10.15479/AT:ISTA:7525</a>."},"doi":"10.15479/AT:ISTA:7525","publisher":"Institute of Science and Technology Austria","language":[{"iso":"eng"}],"file_date_updated":"2021-03-01T23:30:04Z","type":"dissertation","oa_version":"Published Version","date_published":"2020-02-28T00:00:00Z","ddc":["570"],"author":[{"id":"45EDD1BC-F248-11E8-B48F-1D18A9856A87","orcid":"0000-0003-0863-4481","full_name":"Bhandari, Pradeep","first_name":"Pradeep","last_name":"Bhandari"}],"page":"79","year":"2020","date_created":"2020-02-26T10:56:37Z","date_updated":"2023-09-07T13:20:03Z","acknowledged_ssus":[{"_id":"EM-Fac"}],"file":[{"relation":"main_file","checksum":"4589234fdb12b4ad72273b311723a7b4","file_size":9646346,"creator":"pbhandari","title":"Localization and functional role of Cav2.3 in the medial habenula to interpeduncular nucleus pathway","file_name":"Pradeep Bhandari Thesis.pdf","embargo":"2021-02-28","content_type":"application/pdf","file_id":"7538","date_created":"2020-02-28T08:37:53Z","date_updated":"2021-03-01T23:30:04Z","access_level":"open_access"},{"file_id":"7539","date_created":"2020-02-28T08:47:14Z","content_type":"application/vnd.openxmlformats-officedocument.wordprocessingml.document","embargo_to":"open_access","date_updated":"2021-03-01T23:30:04Z","access_level":"closed","file_name":"Pradeep Bhandari Thesis.docx","title":"Localization and functional role of Cav2.3 in the medial habenula to interpeduncular nucleus pathway","file_size":35252164,"creator":"pbhandari","relation":"source_file","checksum":"aa79490553ca0a5c9b6fbcd152e93928"}],"article_processing_charge":"No","publication_identifier":{"issn":["2663-337X"]}},{"file":[{"date_updated":"2020-07-14T12:48:03Z","access_level":"open_access","content_type":"application/pdf","date_created":"2020-05-11T11:34:08Z","file_id":"7818","file_name":"2020_FrontiersEduc_Beattie.pdf","creator":"dernst","file_size":1402146,"checksum":"a24ec24e38d843341ae620ec76c53688","relation":"main_file"}],"volume":5,"acknowledged_ssus":[{"_id":"Bio"},{"_id":"LifeSc"},{"_id":"PreCl"},{"_id":"EM-Fac"}],"project":[{"grant_number":"M02416","name":"Molecular Mechanisms Regulating Gliogenesis in the Cerebral Cortex","_id":"264E56E2-B435-11E9-9278-68D0E5697425","call_identifier":"FWF"},{"call_identifier":"H2020","grant_number":"725780","name":"Principles of Neural Stem Cell Lineage Progression in Cerebral Cortex Development","_id":"260018B0-B435-11E9-9278-68D0E5697425"}],"date_updated":"2021-01-12T08:15:42Z","date_created":"2020-05-11T08:18:48Z","year":"2020","quality_controlled":"1","publication_identifier":{"issn":["2504-284X"]},"tmp":{"legal_code_url":"https://creativecommons.org/licenses/by/4.0/legalcode","name":"Creative Commons Attribution 4.0 International Public License (CC-BY 4.0)","image":"/images/cc_by.png","short":"CC BY (4.0)"},"article_processing_charge":"No","type":"journal_article","file_date_updated":"2020-07-14T12:48:03Z","language":[{"iso":"eng"}],"publisher":"Frontiers Media","doi":"10.3389/feduc.2020.00048","author":[{"first_name":"Robert J","full_name":"Beattie, Robert J","last_name":"Beattie","orcid":"0000-0002-8483-8753","id":"2E26DF60-F248-11E8-B48F-1D18A9856A87"},{"id":"37B36620-F248-11E8-B48F-1D18A9856A87","orcid":"0000-0003-2279-1061","full_name":"Hippenmeyer, Simon","first_name":"Simon","last_name":"Hippenmeyer"},{"first_name":"Florian","full_name":"Pauler, Florian","last_name":"Pauler","id":"48EA0138-F248-11E8-B48F-1D18A9856A87"}],"ddc":["570"],"publication":"Frontiers in Education","date_published":"2020-05-08T00:00:00Z","oa_version":"Published Version","abstract":[{"text":"Scientific research is to date largely restricted to wealthy laboratories in developed nations due to the necessity of complex and expensive equipment. This inequality limits the capacity of science to be used as a diplomatic channel. Maker movements use open-source technologies including additive manufacturing (3D printing) and laser cutting, together with low-cost computers for developing novel products. This movement is setting the groundwork for a revolution, allowing scientific equipment to be sourced at a fraction of the cost and has the potential to increase the availability of equipment for scientists around the world. Science education is increasingly recognized as another channel for science diplomacy. In this perspective, we introduce the idea that the Maker movement and open-source technologies have the potential to revolutionize science, technology, engineering and mathematics (STEM) education worldwide. We present an open-source STEM didactic tool called SCOPES (Sparking Curiosity through Open-source Platforms in Education and Science). SCOPES is self-contained, independent of local resources, and cost-effective. SCOPES can be adapted to communicate complex subjects from genetics to neurobiology, perform real-world biological experiments and explore digitized scientific samples. We envision such platforms will enhance science diplomacy by providing a means for scientists to share their findings with classrooms and for educators to incorporate didactic concepts into STEM lessons. By providing students the opportunity to design, perform, and share scientific experiments, students also experience firsthand the benefits of a multinational scientific community. We provide instructions on how to build and use SCOPES on our webpage: http://scopeseducation.org.","lang":"eng"}],"status":"public","article_number":"48","ec_funded":1,"citation":{"ieee":"R. J. Beattie, S. Hippenmeyer, and F. Pauler, “SCOPES: Sparking curiosity through Open-Source platforms in education and science,” <i>Frontiers in Education</i>, vol. 5. Frontiers Media, 2020.","mla":"Beattie, Robert J., et al. “SCOPES: Sparking Curiosity through Open-Source Platforms in Education and Science.” <i>Frontiers in Education</i>, vol. 5, 48, Frontiers Media, 2020, doi:<a href=\"https://doi.org/10.3389/feduc.2020.00048\">10.3389/feduc.2020.00048</a>.","ista":"Beattie RJ, Hippenmeyer S, Pauler F. 2020. SCOPES: Sparking curiosity through Open-Source platforms in education and science. Frontiers in Education. 5, 48.","apa":"Beattie, R. J., Hippenmeyer, S., &#38; Pauler, F. (2020). SCOPES: Sparking curiosity through Open-Source platforms in education and science. <i>Frontiers in Education</i>. Frontiers Media. <a href=\"https://doi.org/10.3389/feduc.2020.00048\">https://doi.org/10.3389/feduc.2020.00048</a>","chicago":"Beattie, Robert J, Simon Hippenmeyer, and Florian Pauler. “SCOPES: Sparking Curiosity through Open-Source Platforms in Education and Science.” <i>Frontiers in Education</i>. Frontiers Media, 2020. <a href=\"https://doi.org/10.3389/feduc.2020.00048\">https://doi.org/10.3389/feduc.2020.00048</a>.","ama":"Beattie RJ, Hippenmeyer S, Pauler F. SCOPES: Sparking curiosity through Open-Source platforms in education and science. <i>Frontiers in Education</i>. 2020;5. doi:<a href=\"https://doi.org/10.3389/feduc.2020.00048\">10.3389/feduc.2020.00048</a>","short":"R.J. Beattie, S. Hippenmeyer, F. Pauler, Frontiers in Education 5 (2020)."},"intvolume":"         5","has_accepted_license":"1","day":"08","article_type":"original","publication_status":"published","oa":1,"title":"SCOPES: Sparking curiosity through Open-Source platforms in education and science","_id":"7814","month":"05","user_id":"2DF688A6-F248-11E8-B48F-1D18A9856A87","department":[{"_id":"SiHi"}]},{"day":"06","has_accepted_license":"1","citation":{"ieee":"A. J. Johnson <i>et al.</i>, “Experimental toolbox for quantitative evaluation of clathrin-mediated endocytosis in the plant model Arabidopsis,” <i>Journal of Cell Science</i>, vol. 133, no. 15. The Company of Biologists, 2020.","mla":"Johnson, Alexander J., et al. “Experimental Toolbox for Quantitative Evaluation of Clathrin-Mediated Endocytosis in the Plant Model Arabidopsis.” <i>Journal of Cell Science</i>, vol. 133, no. 15, jcs248062, The Company of Biologists, 2020, doi:<a href=\"https://doi.org/10.1242/jcs.248062\">10.1242/jcs.248062</a>.","ista":"Johnson AJ, Gnyliukh N, Kaufmann W, Narasimhan M, Vert G, Bednarek S, Friml J. 2020. Experimental toolbox for quantitative evaluation of clathrin-mediated endocytosis in the plant model Arabidopsis. Journal of Cell Science. 133(15), jcs248062.","apa":"Johnson, A. J., Gnyliukh, N., Kaufmann, W., Narasimhan, M., Vert, G., Bednarek, S., &#38; Friml, J. (2020). Experimental toolbox for quantitative evaluation of clathrin-mediated endocytosis in the plant model Arabidopsis. <i>Journal of Cell Science</i>. The Company of Biologists. <a href=\"https://doi.org/10.1242/jcs.248062\">https://doi.org/10.1242/jcs.248062</a>","chicago":"Johnson, Alexander J, Nataliia Gnyliukh, Walter Kaufmann, Madhumitha Narasimhan, G Vert, SY Bednarek, and Jiří Friml. “Experimental Toolbox for Quantitative Evaluation of Clathrin-Mediated Endocytosis in the Plant Model Arabidopsis.” <i>Journal of Cell Science</i>. The Company of Biologists, 2020. <a href=\"https://doi.org/10.1242/jcs.248062\">https://doi.org/10.1242/jcs.248062</a>.","ama":"Johnson AJ, Gnyliukh N, Kaufmann W, et al. Experimental toolbox for quantitative evaluation of clathrin-mediated endocytosis in the plant model Arabidopsis. <i>Journal of Cell Science</i>. 2020;133(15). doi:<a href=\"https://doi.org/10.1242/jcs.248062\">10.1242/jcs.248062</a>","short":"A.J. Johnson, N. Gnyliukh, W. Kaufmann, M. Narasimhan, G. Vert, S. Bednarek, J. Friml, Journal of Cell Science 133 (2020)."},"intvolume":"       133","ec_funded":1,"article_number":"jcs248062","status":"public","abstract":[{"text":"Clathrin-mediated endocytosis (CME) is a crucial cellular process implicated in many aspects of plant growth, development, intra- and inter-cellular signaling, nutrient uptake and pathogen defense. Despite these significant roles, little is known about the precise molecular details of how it functions in planta. In order to facilitate the direct quantitative study of plant CME, here we review current routinely used methods and present refined, standardized quantitative imaging protocols which allow the detailed characterization of CME at multiple scales in plant tissues. These include: (i) an efficient electron microscopy protocol for the imaging of Arabidopsis CME vesicles in situ, thus providing a method for the detailed characterization of the ultra-structure of clathrin-coated vesicles; (ii) a detailed protocol and analysis for quantitative live-cell fluorescence microscopy to precisely examine the temporal interplay of endocytosis components during single CME events; (iii) a semi-automated analysis to allow the quantitative characterization of global internalization of cargos in whole plant tissues; and (iv) an overview and validation of useful genetic and pharmacological tools to interrogate the molecular mechanisms and function of CME in intact plant samples.","lang":"eng"}],"acknowledgement":"This paper is dedicated to the memory of Christien Merrifield. He pioneered quantitative\r\nimaging approaches in mammalian CME and his mentorship inspired the development of all\r\nthe analysis methods presented here. His joy in research, pure scientific curiosity and\r\nmicroscopy excellence remain a constant inspiration. We thank Daniel Van Damme for gifting\r\nus the CLC2-GFP x TPLATE-TagRFP plants used in this manuscript. We further thank the\r\nScientific Service Units at IST Austria; specifically, the Electron Microscopy Facility for\r\ntechnical assistance (in particular Vanessa Zheden) and the BioImaging Facility BioImaging\r\nFacility for access to equipment. ","department":[{"_id":"JiFr"},{"_id":"EM-Fac"}],"user_id":"c635000d-4b10-11ee-a964-aac5a93f6ac1","month":"08","_id":"8139","oa":1,"related_material":{"record":[{"status":"public","relation":"dissertation_contains","id":"14510"}]},"title":"Experimental toolbox for quantitative evaluation of clathrin-mediated endocytosis in the plant model Arabidopsis","publication_status":"published","article_type":"original","article_processing_charge":"No","publication_identifier":{"issn":["0021-9533"],"eissn":["1477-9137"]},"scopus_import":"1","pmid":1,"quality_controlled":"1","date_created":"2020-07-21T08:58:19Z","year":"2020","date_updated":"2023-12-01T13:51:07Z","acknowledged_ssus":[{"_id":"EM-Fac"},{"_id":"Bio"}],"project":[{"grant_number":"I03630","name":"Molecular mechanisms of endocytic cargo recognition in plants","_id":"26538374-B435-11E9-9278-68D0E5697425","call_identifier":"FWF"},{"name":"International IST Doctoral Program","grant_number":"665385","_id":"2564DBCA-B435-11E9-9278-68D0E5697425","call_identifier":"H2020"}],"volume":133,"file":[{"file_name":"2020 - Johnson - JSC - plant CME toolbox.pdf","embargo":"2021-08-07","date_created":"2020-11-26T17:12:51Z","file_id":"8815","content_type":"application/pdf","date_updated":"2021-08-08T22:30:03Z","access_level":"open_access","relation":"main_file","checksum":"2d11f79a0b4e0a380fb002b933da331a","file_size":15150403,"creator":"ajohnson"}],"isi":1,"oa_version":"Published Version","external_id":{"pmid":["32616560"],"isi":["000561047900021"]},"date_published":"2020-08-06T00:00:00Z","publication":"Journal of Cell Science","issue":"15","ddc":["575"],"author":[{"id":"46A62C3A-F248-11E8-B48F-1D18A9856A87","orcid":"0000-0002-2739-8843","last_name":"Johnson","first_name":"Alexander J","full_name":"Johnson, Alexander J"},{"orcid":"0000-0002-2198-0509","id":"390C1120-F248-11E8-B48F-1D18A9856A87","last_name":"Gnyliukh","full_name":"Gnyliukh, Nataliia","first_name":"Nataliia"},{"last_name":"Kaufmann","full_name":"Kaufmann, Walter","first_name":"Walter","orcid":"0000-0001-9735-5315","id":"3F99E422-F248-11E8-B48F-1D18A9856A87"},{"last_name":"Narasimhan","first_name":"Madhumitha","full_name":"Narasimhan, Madhumitha","id":"44BF24D0-F248-11E8-B48F-1D18A9856A87","orcid":"0000-0002-8600-0671"},{"last_name":"Vert","first_name":"G","full_name":"Vert, G"},{"last_name":"Bednarek","first_name":"SY","full_name":"Bednarek, SY"},{"orcid":"0000-0002-8302-7596","id":"4159519E-F248-11E8-B48F-1D18A9856A87","last_name":"Friml","full_name":"Friml, Jiří","first_name":"Jiří"}],"doi":"10.1242/jcs.248062","publisher":"The Company of Biologists","language":[{"iso":"eng"}],"file_date_updated":"2021-08-08T22:30:03Z","type":"journal_article"},{"publication":"bioRxiv","author":[{"last_name":"Slovakova","first_name":"Jana","full_name":"Slovakova, Jana","id":"30F3F2F0-F248-11E8-B48F-1D18A9856A87"},{"id":"2F74BCDE-F248-11E8-B48F-1D18A9856A87","first_name":"Mateusz K","full_name":"Sikora, Mateusz K","last_name":"Sikora"},{"id":"2F1E1758-F248-11E8-B48F-1D18A9856A87","orcid":"0000-0002-5223-3346","last_name":"Caballero Mancebo","first_name":"Silvia","full_name":"Caballero Mancebo, Silvia"},{"id":"2B819732-F248-11E8-B48F-1D18A9856A87","orcid":"0000-0003-4761-5996","first_name":"Gabriel","full_name":"Krens, Gabriel","last_name":"Krens"},{"id":"3F99E422-F248-11E8-B48F-1D18A9856A87","orcid":"0000-0001-9735-5315","last_name":"Kaufmann","full_name":"Kaufmann, Walter","first_name":"Walter"},{"last_name":"Huljev","first_name":"Karla","full_name":"Huljev, Karla","id":"44C6F6A6-F248-11E8-B48F-1D18A9856A87"},{"last_name":"Heisenberg","full_name":"Heisenberg, Carl-Philipp J","first_name":"Carl-Philipp J","orcid":"0000-0002-0912-4566","id":"39427864-F248-11E8-B48F-1D18A9856A87"}],"oa_version":"Preprint","date_published":"2020-11-20T00:00:00Z","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.","department":[{"_id":"CaHe"},{"_id":"EM-Fac"},{"_id":"Bio"}],"oa":1,"related_material":{"record":[{"id":"10766","relation":"later_version","status":"public"},{"relation":"dissertation_contains","status":"public","id":"9623"}]},"title":"Tension-dependent stabilization of E-cadherin limits cell-cell contact expansion","language":[{"iso":"eng"}],"publication_status":"published","type":"preprint","user_id":"8b945eb4-e2f2-11eb-945a-df72226e66a9","month":"11","_id":"9750","doi":"10.1101/2020.11.20.391284","publisher":"Cold Spring Harbor Laboratory","citation":{"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, <a href=\"https://doi.org/10.1101/2020.11.20.391284\">10.1101/2020.11.20.391284</a>.","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.” <i>BioRxiv</i>. Cold Spring Harbor Laboratory, 2020. <a href=\"https://doi.org/10.1101/2020.11.20.391284\">https://doi.org/10.1101/2020.11.20.391284</a>.","apa":"Slovakova, J., Sikora, M. K., Caballero Mancebo, S., Krens, G., Kaufmann, W., Huljev, K., &#38; Heisenberg, C.-P. J. (2020). Tension-dependent stabilization of E-cadherin limits cell-cell contact expansion. <i>bioRxiv</i>. Cold Spring Harbor Laboratory. <a href=\"https://doi.org/10.1101/2020.11.20.391284\">https://doi.org/10.1101/2020.11.20.391284</a>","mla":"Slovakova, Jana, et al. “Tension-Dependent Stabilization of E-Cadherin Limits Cell-Cell Contact Expansion.” <i>BioRxiv</i>, Cold Spring Harbor Laboratory, 2020, doi:<a href=\"https://doi.org/10.1101/2020.11.20.391284\">10.1101/2020.11.20.391284</a>.","ieee":"J. Slovakova <i>et al.</i>, “Tension-dependent stabilization of E-cadherin limits cell-cell contact expansion,” <i>bioRxiv</i>. Cold Spring Harbor Laboratory, 2020.","short":"J. Slovakova, M.K. Sikora, S. Caballero Mancebo, G. Krens, W. Kaufmann, K. Huljev, C.-P.J. Heisenberg, BioRxiv (2020).","ama":"Slovakova J, Sikora MK, Caballero Mancebo S, et al. Tension-dependent stabilization of E-cadherin limits cell-cell contact expansion. <i>bioRxiv</i>. 2020. doi:<a href=\"https://doi.org/10.1101/2020.11.20.391284\">10.1101/2020.11.20.391284</a>"},"main_file_link":[{"open_access":"1","url":"https://doi.org/10.1101/2020.11.20.391284"}],"day":"20","article_processing_charge":"No","status":"public","abstract":[{"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.","lang":"eng"}],"page":"41","date_created":"2021-07-29T11:29:50Z","year":"2020","date_updated":"2024-03-25T23:30:10Z","ec_funded":1,"acknowledged_ssus":[{"_id":"Bio"},{"_id":"EM-Fac"},{"_id":"SSU"}],"project":[{"call_identifier":"FP7","grant_number":"291734","name":"International IST Postdoc Fellowship Programme","_id":"25681D80-B435-11E9-9278-68D0E5697425"},{"call_identifier":"H2020","name":"Interaction and feedback between cell mechanics and fate specification in vertebrate gastrulation","grant_number":"742573","_id":"260F1432-B435-11E9-9278-68D0E5697425"},{"grant_number":"187-2013","name":"Modulation of adhesion function in cell-cell contact formation by cortical tension","_id":"2521E28E-B435-11E9-9278-68D0E5697425"}]},{"date_updated":"2023-09-13T08:50:57Z","acknowledged_ssus":[{"_id":"Bio"},{"_id":"PreCl"},{"_id":"EM-Fac"}],"page":"142","year":"2019","date_created":"2019-10-14T16:54:52Z","file":[{"file_name":"PhDthesis_FrankAssen_revised2.docx","date_updated":"2020-11-07T23:30:03Z","access_level":"closed","embargo_to":"open_access","content_type":"application/vnd.openxmlformats-officedocument.wordprocessingml.document","date_created":"2019-11-06T12:30:02Z","file_id":"6990","checksum":"53a739752a500f84d0f8ec953cbbd0b6","relation":"source_file","creator":"fassen","file_size":214172667},{"file_size":83637532,"creator":"fassen","relation":"main_file","checksum":"8c156b65d9347bb599623a4b09f15d15","file_id":"6991","content_type":"application/pdf","date_created":"2019-11-06T12:30:57Z","date_updated":"2020-11-07T23:30:03Z","access_level":"open_access","file_name":"PhDthesis_FrankAssen_revised2.pdf","embargo":"2020-11-06"}],"article_processing_charge":"No","publication_identifier":{"issn":["2663-337X"]},"publisher":"Institute of Science and Technology Austria","doi":"10.15479/AT:ISTA:6947","file_date_updated":"2020-11-07T23:30:03Z","type":"dissertation","language":[{"iso":"eng"}],"date_published":"2019-10-09T00:00:00Z","oa_version":"Published Version","ddc":["570"],"author":[{"last_name":"Assen","full_name":"Assen, Frank P","first_name":"Frank P","orcid":"0000-0003-3470-6119","id":"3A8E7F24-F248-11E8-B48F-1D18A9856A87"}],"degree_awarded":"PhD","abstract":[{"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.","lang":"eng"}],"status":"public","has_accepted_license":"1","day":"9","citation":{"ieee":"F. P. Assen, “Lymph node mechanics: Deciphering the interplay between stroma contractility, morphology and lymphocyte trafficking,” Institute of Science and Technology Austria, 2019.","mla":"Assen, Frank P. <i>Lymph Node Mechanics: Deciphering the Interplay between Stroma Contractility, Morphology and Lymphocyte Trafficking</i>. Institute of Science and Technology Austria, 2019, doi:<a href=\"https://doi.org/10.15479/AT:ISTA:6947\">10.15479/AT:ISTA:6947</a>.","ista":"Assen FP. 2019. Lymph node mechanics: Deciphering the interplay between stroma contractility, morphology and lymphocyte trafficking. Institute of Science and Technology Austria.","apa":"Assen, F. P. (2019). <i>Lymph node mechanics: Deciphering the interplay between stroma contractility, morphology and lymphocyte trafficking</i>. Institute of Science and Technology Austria. <a href=\"https://doi.org/10.15479/AT:ISTA:6947\">https://doi.org/10.15479/AT:ISTA:6947</a>","chicago":"Assen, Frank P. “Lymph Node Mechanics: Deciphering the Interplay between Stroma Contractility, Morphology and Lymphocyte Trafficking.” Institute of Science and Technology Austria, 2019. <a href=\"https://doi.org/10.15479/AT:ISTA:6947\">https://doi.org/10.15479/AT:ISTA:6947</a>.","ama":"Assen FP. Lymph node mechanics: Deciphering the interplay between stroma contractility, morphology and lymphocyte trafficking. 2019. doi:<a href=\"https://doi.org/10.15479/AT:ISTA:6947\">10.15479/AT:ISTA:6947</a>","short":"F.P. Assen, Lymph Node Mechanics: Deciphering the Interplay between Stroma Contractility, Morphology and Lymphocyte Trafficking, Institute of Science and Technology Austria, 2019."},"supervisor":[{"full_name":"Sixt, Michael K","first_name":"Michael K","last_name":"Sixt","orcid":"0000-0002-6620-9179","id":"41E9FBEA-F248-11E8-B48F-1D18A9856A87"}],"user_id":"c635000d-4b10-11ee-a964-aac5a93f6ac1","_id":"6947","month":"10","title":"Lymph node mechanics: Deciphering the interplay between stroma contractility, morphology and lymphocyte trafficking","oa":1,"related_material":{"record":[{"id":"664","status":"public","relation":"part_of_dissertation"},{"id":"402","relation":"part_of_dissertation","status":"public"}]},"publication_status":"published","department":[{"_id":"MiSi"}],"alternative_title":["ISTA Thesis"]},{"doi":"10.15479/AT:ISTA:7186","publisher":"Institute of Science and Technology Austria","language":[{"iso":"eng"}],"file_date_updated":"2020-07-14T12:47:52Z","type":"dissertation","oa_version":"Published Version","date_published":"2019-12-16T00:00:00Z","ddc":["570"],"author":[{"last_name":"Schwayer","full_name":"Schwayer, Cornelia","first_name":"Cornelia","orcid":"0000-0001-5130-2226","id":"3436488C-F248-11E8-B48F-1D18A9856A87"}],"page":"107","year":"2019","date_created":"2019-12-16T14:26:14Z","date_updated":"2023-09-07T12:56:42Z","acknowledged_ssus":[{"_id":"Bio"},{"_id":"LifeSc"},{"_id":"EM-Fac"},{"_id":"SSU"}],"file":[{"access_level":"closed","date_updated":"2020-07-14T12:47:52Z","content_type":"application/zip","date_created":"2019-12-19T15:18:11Z","file_id":"7194","file_name":"DocumentSourceFiles.zip","creator":"cschwayer","file_size":19431292,"checksum":"585583c1c875c5d9525703a539668a7c","relation":"source_file"},{"checksum":"9b9b24351514948d27cec659e632e2cd","relation":"main_file","creator":"cschwayer","file_size":19226428,"file_name":"Thesis_CS_final.pdf","access_level":"open_access","date_updated":"2020-07-14T12:47:52Z","content_type":"application/pdf","date_created":"2019-12-19T15:19:21Z","file_id":"7195"}],"article_processing_charge":"No","publication_identifier":{"issn":["2663-337X"]},"user_id":"c635000d-4b10-11ee-a964-aac5a93f6ac1","month":"12","_id":"7186","title":"Mechanosensation of tight junctions depends on ZO-1 phase separation and flow","related_material":{"record":[{"id":"1096","status":"public","relation":"dissertation_contains"},{"id":"7001","relation":"part_of_dissertation","status":"public"}]},"oa":1,"publication_status":"published","department":[{"_id":"CaHe"}],"alternative_title":["ISTA Thesis"],"degree_awarded":"PhD","status":"public","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."}],"day":"16","has_accepted_license":"1","supervisor":[{"first_name":"Carl-Philipp J","full_name":"Heisenberg, Carl-Philipp J","last_name":"Heisenberg","orcid":"0000-0002-0912-4566","id":"39427864-F248-11E8-B48F-1D18A9856A87"}],"citation":{"ista":"Schwayer C. 2019. Mechanosensation of tight junctions depends on ZO-1 phase separation and flow. Institute of Science and Technology Austria.","chicago":"Schwayer, Cornelia. “Mechanosensation of Tight Junctions Depends on ZO-1 Phase Separation and Flow.” Institute of Science and Technology Austria, 2019. <a href=\"https://doi.org/10.15479/AT:ISTA:7186\">https://doi.org/10.15479/AT:ISTA:7186</a>.","apa":"Schwayer, C. (2019). <i>Mechanosensation of tight junctions depends on ZO-1 phase separation and flow</i>. Institute of Science and Technology Austria. <a href=\"https://doi.org/10.15479/AT:ISTA:7186\">https://doi.org/10.15479/AT:ISTA:7186</a>","mla":"Schwayer, Cornelia. <i>Mechanosensation of Tight Junctions Depends on ZO-1 Phase Separation and Flow</i>. Institute of Science and Technology Austria, 2019, doi:<a href=\"https://doi.org/10.15479/AT:ISTA:7186\">10.15479/AT:ISTA:7186</a>.","ieee":"C. Schwayer, “Mechanosensation of tight junctions depends on ZO-1 phase separation and flow,” Institute of Science and Technology Austria, 2019.","short":"C. Schwayer, Mechanosensation of Tight Junctions Depends on ZO-1 Phase Separation and Flow, Institute of Science and Technology Austria, 2019.","ama":"Schwayer C. Mechanosensation of tight junctions depends on ZO-1 phase separation and flow. 2019. doi:<a href=\"https://doi.org/10.15479/AT:ISTA:7186\">10.15479/AT:ISTA:7186</a>"}},{"author":[{"id":"3EEDE19A-F248-11E8-B48F-1D18A9856A87","last_name":"Mckenzie","full_name":"Mckenzie, Catherine","first_name":"Catherine"},{"id":"44A924DC-F248-11E8-B48F-1D18A9856A87","last_name":"Spanova","first_name":"Miroslava","full_name":"Spanova, Miroslava"},{"full_name":"Johnson, Alexander J","first_name":"Alexander J","last_name":"Johnson","orcid":"0000-0002-2739-8843","id":"46A62C3A-F248-11E8-B48F-1D18A9856A87"},{"first_name":"Stephanie","full_name":"Kainrath, Stephanie","last_name":"Kainrath","id":"32CFBA64-F248-11E8-B48F-1D18A9856A87"},{"last_name":"Zheden","full_name":"Zheden, Vanessa","first_name":"Vanessa","id":"39C5A68A-F248-11E8-B48F-1D18A9856A87","orcid":"0000-0002-9438-4783"},{"last_name":"Sitte","first_name":"Harald H.","full_name":"Sitte, Harald H."},{"orcid":"0000-0002-8023-9315","id":"33BA6C30-F248-11E8-B48F-1D18A9856A87","first_name":"Harald L","full_name":"Janovjak, Harald L","last_name":"Janovjak"}],"publication":"Journal of Neuroscience Methods","date_published":"2019-01-15T00:00:00Z","external_id":{"pmid":["30496761"],"isi":["000456220900013"]},"oa_version":"None","type":"journal_article","language":[{"iso":"eng"}],"publisher":"Elsevier","doi":"10.1016/j.jneumeth.2018.11.018","pmid":1,"scopus_import":"1","publication_identifier":{"issn":["0165-0270"]},"article_processing_charge":"No","isi":1,"volume":312,"acknowledged_ssus":[{"_id":"Bio"},{"_id":"EM-Fac"}],"project":[{"call_identifier":"FP7","name":"Microbial Ion Channels for Synthetic Neurobiology","grant_number":"303564","_id":"25548C20-B435-11E9-9278-68D0E5697425"},{"_id":"26538374-B435-11E9-9278-68D0E5697425","grant_number":"I03630","name":"Molecular mechanisms of endocytic cargo recognition in plants","call_identifier":"FWF"},{"call_identifier":"FWF","_id":"2548AE96-B435-11E9-9278-68D0E5697425","grant_number":"W1232-B24","name":"Molecular Drug Targets"}],"date_updated":"2023-09-06T15:27:29Z","year":"2019","date_created":"2020-01-30T09:12:19Z","page":"114-121","quality_controlled":"1","department":[{"_id":"HaJa"},{"_id":"Bio"}],"article_type":"original","publication_status":"published","title":"Isolation of synaptic vesicles from genetically engineered cultured neurons","_id":"7406","month":"01","user_id":"c635000d-4b10-11ee-a964-aac5a93f6ac1","citation":{"mla":"Mckenzie, Catherine, et al. “Isolation of Synaptic Vesicles from Genetically Engineered Cultured Neurons.” <i>Journal of Neuroscience Methods</i>, vol. 312, Elsevier, 2019, pp. 114–21, doi:<a href=\"https://doi.org/10.1016/j.jneumeth.2018.11.018\">10.1016/j.jneumeth.2018.11.018</a>.","ieee":"C. Mckenzie <i>et al.</i>, “Isolation of synaptic vesicles from genetically engineered cultured neurons,” <i>Journal of Neuroscience Methods</i>, 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.","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.” <i>Journal of Neuroscience Methods</i>. Elsevier, 2019. <a href=\"https://doi.org/10.1016/j.jneumeth.2018.11.018\">https://doi.org/10.1016/j.jneumeth.2018.11.018</a>.","apa":"Mckenzie, C., Spanova, M., Johnson, A. J., Kainrath, S., Zheden, V., Sitte, H. H., &#38; Janovjak, H. L. (2019). Isolation of synaptic vesicles from genetically engineered cultured neurons. <i>Journal of Neuroscience Methods</i>. Elsevier. <a href=\"https://doi.org/10.1016/j.jneumeth.2018.11.018\">https://doi.org/10.1016/j.jneumeth.2018.11.018</a>","ama":"Mckenzie C, Spanova M, Johnson AJ, et al. Isolation of synaptic vesicles from genetically engineered cultured neurons. <i>Journal of Neuroscience Methods</i>. 2019;312:114-121. doi:<a href=\"https://doi.org/10.1016/j.jneumeth.2018.11.018\">10.1016/j.jneumeth.2018.11.018</a>","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."},"intvolume":"       312","day":"15","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."}],"status":"public","ec_funded":1},{"external_id":{"pmid":["30773315"],"isi":["000460509600013"]},"oa_version":"Published Version","date_published":"2019-03-07T00:00:00Z","publication":"Cell","issue":"6","author":[{"id":"4AB6C7D0-F248-11E8-B48F-1D18A9856A87","orcid":"0000-0002-5419-7756","last_name":"Xia","full_name":"Xia, Peng","first_name":"Peng"},{"id":"381929CE-F248-11E8-B48F-1D18A9856A87","full_name":"Gütl, Daniel J","first_name":"Daniel J","last_name":"Gütl"},{"full_name":"Zheden, Vanessa","first_name":"Vanessa","last_name":"Zheden","orcid":"0000-0002-9438-4783","id":"39C5A68A-F248-11E8-B48F-1D18A9856A87"},{"full_name":"Heisenberg, Carl-Philipp J","first_name":"Carl-Philipp J","last_name":"Heisenberg","id":"39427864-F248-11E8-B48F-1D18A9856A87","orcid":"0000-0002-0912-4566"}],"doi":"10.1016/j.cell.2019.01.019","publisher":"Elsevier","language":[{"iso":"eng"}],"type":"journal_article","article_processing_charge":"No","scopus_import":"1","main_file_link":[{"open_access":"1","url":"https://doi.org/10.1016/j.cell.2019.01.019"}],"pmid":1,"quality_controlled":"1","page":"1379-1392.e14","year":"2019","date_created":"2019-03-10T22:59:19Z","date_updated":"2023-08-25T08:02:23Z","acknowledged_ssus":[{"_id":"Bio"},{"_id":"EM-Fac"},{"_id":"LifeSc"}],"project":[{"grant_number":"742573","name":"Interaction and feedback between cell mechanics and fate specification in vertebrate gastrulation","_id":"260F1432-B435-11E9-9278-68D0E5697425","call_identifier":"H2020"}],"volume":176,"isi":1,"department":[{"_id":"CaHe"},{"_id":"EM-Fac"}],"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.).","user_id":"4359f0d1-fa6c-11eb-b949-802e58b17ae8","month":"03","_id":"6087","related_material":{"link":[{"relation":"press_release","description":"News on IST Homepage","url":"https://ist.ac.at/en/news/in-zebrafish-eggs-most-rapidly-growing-cell-inhibits-its-neighbours-through-mechanical-signals/"}]},"oa":1,"title":"Lateral inhibition in cell specification mediated by mechanical signals modulating TAZ activity","publication_status":"published","article_type":"original","day":"07","citation":{"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.","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.” <i>Cell</i>. Elsevier, 2019. <a href=\"https://doi.org/10.1016/j.cell.2019.01.019\">https://doi.org/10.1016/j.cell.2019.01.019</a>.","apa":"Xia, P., Gütl, D. J., Zheden, V., &#38; Heisenberg, C.-P. J. (2019). Lateral inhibition in cell specification mediated by mechanical signals modulating TAZ activity. <i>Cell</i>. Elsevier. <a href=\"https://doi.org/10.1016/j.cell.2019.01.019\">https://doi.org/10.1016/j.cell.2019.01.019</a>","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,” <i>Cell</i>, vol. 176, no. 6. Elsevier, p. 1379–1392.e14, 2019.","mla":"Xia, Peng, et al. “Lateral Inhibition in Cell Specification Mediated by Mechanical Signals Modulating TAZ Activity.” <i>Cell</i>, vol. 176, no. 6, Elsevier, 2019, p. 1379–1392.e14, doi:<a href=\"https://doi.org/10.1016/j.cell.2019.01.019\">10.1016/j.cell.2019.01.019</a>.","short":"P. Xia, D.J. Gütl, V. Zheden, C.-P.J. Heisenberg, Cell 176 (2019) 1379–1392.e14.","ama":"Xia P, Gütl DJ, Zheden V, Heisenberg C-PJ. Lateral inhibition in cell specification mediated by mechanical signals modulating TAZ activity. <i>Cell</i>. 2019;176(6):1379-1392.e14. doi:<a href=\"https://doi.org/10.1016/j.cell.2019.01.019\">10.1016/j.cell.2019.01.019</a>"},"intvolume":"       176","ec_funded":1,"status":"public","abstract":[{"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.","lang":"eng"}]},{"tmp":{"legal_code_url":"https://creativecommons.org/licenses/by/4.0/legalcode","name":"Creative Commons Attribution 4.0 International Public License (CC-BY 4.0)","image":"/images/cc_by.png","short":"CC BY 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Version","ddc":["575"],"author":[{"id":"44BF24D0-F248-11E8-B48F-1D18A9856A87","orcid":"0000-0002-8600-0671","last_name":"Narasimhan","full_name":"Narasimhan, Madhumitha","first_name":"Madhumitha"}],"publisher":"Institute of Science and Technology Austria","doi":"10.15479/at:ista:th1075","file_date_updated":"2021-02-11T23:30:15Z","type":"dissertation","language":[{"iso":"eng"}],"has_accepted_license":"1","day":"04","citation":{"ieee":"M. Narasimhan, “Clathrin-Mediated endocytosis, post-endocytic trafficking and their regulatory controls in plants ,” Institute of Science and Technology Austria, 2019.","mla":"Narasimhan, Madhumitha. <i>Clathrin-Mediated Endocytosis, Post-Endocytic Trafficking and Their Regulatory Controls in Plants </i>. Institute of Science and Technology Austria, 2019, doi:<a href=\"https://doi.org/10.15479/at:ista:th1075\">10.15479/at:ista:th1075</a>.","apa":"Narasimhan, M. (2019). <i>Clathrin-Mediated endocytosis, post-endocytic trafficking and their regulatory controls in plants </i>. Institute of Science and Technology Austria. <a href=\"https://doi.org/10.15479/at:ista:th1075\">https://doi.org/10.15479/at:ista:th1075</a>","ista":"Narasimhan M. 2019. Clathrin-Mediated endocytosis, post-endocytic trafficking and their regulatory controls in plants . Institute of Science and Technology Austria.","chicago":"Narasimhan, Madhumitha. “Clathrin-Mediated Endocytosis, Post-Endocytic Trafficking and Their Regulatory Controls in Plants .” Institute of Science and Technology Austria, 2019. <a href=\"https://doi.org/10.15479/at:ista:th1075\">https://doi.org/10.15479/at:ista:th1075</a>.","ama":"Narasimhan M. Clathrin-Mediated endocytosis, post-endocytic trafficking and their regulatory controls in plants . 2019. doi:<a href=\"https://doi.org/10.15479/at:ista:th1075\">10.15479/at:ista:th1075</a>","short":"M. Narasimhan, Clathrin-Mediated Endocytosis, Post-Endocytic Trafficking and Their Regulatory Controls in Plants , Institute of Science and Technology Austria, 2019."},"supervisor":[{"full_name":"Friml, Jiří","first_name":"Jiří","last_name":"Friml","id":"4159519E-F248-11E8-B48F-1D18A9856A87","orcid":"0000-0002-8302-7596"}],"degree_awarded":"PhD","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. "}],"status":"public","department":[{"_id":"JiFr"}],"alternative_title":["ISTA Thesis"],"user_id":"c635000d-4b10-11ee-a964-aac5a93f6ac1","_id":"6269","month":"02","title":"Clathrin-Mediated endocytosis, post-endocytic trafficking and their regulatory controls in plants ","oa":1,"related_material":{"record":[{"relation":"part_of_dissertation","status":"public","id":"412"}]},"publication_status":"published"},{"department":[{"_id":"MiSi"}],"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"],"pubrep_id":"998","user_id":"c635000d-4b10-11ee-a964-aac5a93f6ac1","month":"04","_id":"323","title":"Branched actin networks in dendritic cell biology","oa":1,"related_material":{"record":[{"id":"1321","relation":"part_of_dissertation","status":"public"}]},"publication_status":"published","has_accepted_license":"1","day":"12","citation":{"short":"A.F. Leithner, Branched Actin Networks in Dendritic Cell Biology, Institute of Science and Technology Austria, 2018.","ama":"Leithner AF. Branched actin networks in dendritic cell biology. 2018. doi:<a href=\"https://doi.org/10.15479/AT:ISTA:th_998\">10.15479/AT:ISTA:th_998</a>","chicago":"Leithner, Alexander F. “Branched Actin Networks in Dendritic Cell Biology.” Institute of Science and Technology Austria, 2018. <a href=\"https://doi.org/10.15479/AT:ISTA:th_998\">https://doi.org/10.15479/AT:ISTA:th_998</a>.","ista":"Leithner AF. 2018. Branched actin networks in dendritic cell biology. Institute of Science and Technology Austria.","apa":"Leithner, A. F. (2018). <i>Branched actin networks in dendritic cell biology</i>. Institute of Science and Technology Austria. <a href=\"https://doi.org/10.15479/AT:ISTA:th_998\">https://doi.org/10.15479/AT:ISTA:th_998</a>","mla":"Leithner, Alexander F. <i>Branched Actin Networks in Dendritic Cell Biology</i>. Institute of Science and Technology Austria, 2018, doi:<a href=\"https://doi.org/10.15479/AT:ISTA:th_998\">10.15479/AT:ISTA:th_998</a>.","ieee":"A. F. Leithner, “Branched actin networks in dendritic cell biology,” Institute of Science and Technology Austria, 2018."},"supervisor":[{"id":"41E9FBEA-F248-11E8-B48F-1D18A9856A87","orcid":"0000-0002-6620-9179","last_name":"Sixt","full_name":"Sixt, Michael K","first_name":"Michael K"}],"degree_awarded":"PhD","abstract":[{"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. ","lang":"eng"}],"status":"public","date_published":"2018-04-12T00:00:00Z","oa_version":"Published Version","ddc":["571","599","610"],"author":[{"full_name":"Leithner, Alexander F","first_name":"Alexander F","last_name":"Leithner","orcid":"0000-0002-1073-744X","id":"3B1B77E4-F248-11E8-B48F-1D18A9856A87"}],"publist_id":"7542","publisher":"Institute of Science and Technology Austria","doi":"10.15479/AT:ISTA:th_998","file_date_updated":"2021-02-11T23:30:17Z","type":"dissertation","language":[{"iso":"eng"}],"article_processing_charge":"No","tmp":{"legal_code_url":"https://creativecommons.org/licenses/by/4.0/legalcode","name":"Creative Commons Attribution 4.0 International Public License (CC-BY 4.0)","image":"/images/cc_by.png","short":"CC BY (4.0)"},"publication_identifier":{"issn":["2663-337X"]},"date_updated":"2023-09-07T12:39:44Z","acknowledged_ssus":[{"_id":"NanoFab"},{"_id":"Bio"},{"_id":"PreCl"},{"_id":"EM-Fac"}],"page":"99","date_created":"2018-12-11T11:45:49Z","year":"2018","file":[{"creator":"dernst","file_size":29027671,"checksum":"d5e3edbac548c26c1fa43a4b37a54a4c","relation":"source_file","date_updated":"2021-02-11T23:30:17Z","access_level":"closed","file_id":"6219","embargo_to":"open_access","date_created":"2019-04-05T09:23:11Z","content_type":"application/vnd.openxmlformats-officedocument.wordprocessingml.document","file_name":"PhD_thesis_AlexLeithner_final_version.docx"},{"creator":"dernst","file_size":66045341,"checksum":"071f7476db29e41146824ebd0697cb10","relation":"main_file","access_level":"open_access","date_updated":"2021-02-11T11:17:16Z","content_type":"application/pdf","date_created":"2019-04-05T09:23:11Z","file_id":"6220","embargo":"2019-04-15","file_name":"PhD_thesis_AlexLeithner.pdf"}]},{"day":"07","has_accepted_license":"1","intvolume":"        47","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. <i>European Journal of Neuroscience</i>. 2018;47(9):1033-1042. doi:<a href=\"https://doi.org/10.1111/ejn.13901\">10.1111/ejn.13901</a>","short":"K. Sawada, R. Kawakami, R. Shigemoto, T. Nemoto, European Journal of Neuroscience 47 (2018) 1033–1042.","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,” <i>European Journal of Neuroscience</i>, vol. 47, no. 9. Wiley, pp. 1033–1042, 2018.","mla":"Sawada, Kazuaki, et al. “Super Resolution Structural Analysis of Dendritic Spines Using Three-Dimensional Structured Illumination Microscopy in Cleared Mouse Brain Slices.” <i>European Journal of Neuroscience</i>, vol. 47, no. 9, Wiley, 2018, pp. 1033–42, doi:<a href=\"https://doi.org/10.1111/ejn.13901\">10.1111/ejn.13901</a>.","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.","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.” <i>European Journal of Neuroscience</i>. Wiley, 2018. <a href=\"https://doi.org/10.1111/ejn.13901\">https://doi.org/10.1111/ejn.13901</a>.","apa":"Sawada, K., Kawakami, R., Shigemoto, R., &#38; Nemoto, T. (2018). Super resolution structural analysis of dendritic spines using three-dimensional structured illumination microscopy in cleared mouse brain slices. <i>European Journal of Neuroscience</i>. Wiley. <a href=\"https://doi.org/10.1111/ejn.13901\">https://doi.org/10.1111/ejn.13901</a>"},"status":"public","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."}],"department":[{"_id":"RySh"}],"month":"03","_id":"326","user_id":"c635000d-4b10-11ee-a964-aac5a93f6ac1","publication_status":"published","oa":1,"title":"Super resolution structural analysis of dendritic spines using three-dimensional structured illumination microscopy in cleared mouse brain slices","tmp":{"short":"CC BY-NC (4.0)","name":"Creative Commons Attribution-NonCommercial 4.0 International (CC BY-NC 4.0)","legal_code_url":"https://creativecommons.org/licenses/by-nc/4.0/legalcode","image":"/images/cc_by_nc.png"},"article_processing_charge":"No","scopus_import":"1","date_created":"2018-12-11T11:45:50Z","year":"2018","quality_controlled":"1","page":"1033 - 1042","acknowledged_ssus":[{"_id":"EM-Fac"}],"date_updated":"2023-09-19T09:58:40Z","isi":1,"file":[{"date_created":"2018-12-17T16:16:50Z","content_type":"application/pdf","file_id":"5721","date_updated":"2020-07-14T12:46:06Z","access_level":"open_access","file_name":"2018_EJN_Sawada.pdf","file_size":4850261,"creator":"dernst","relation":"main_file","checksum":"98e901d8229e44aa8f3b51d248dedd09"}],"volume":47,"external_id":{"isi":["000431496400001"]},"license":"https://creativecommons.org/licenses/by-nc/4.0/","oa_version":"Published Version","date_published":"2018-03-07T00:00:00Z","publication":"European Journal of Neuroscience","publist_id":"7539","issue":"9","author":[{"last_name":"Sawada","full_name":"Sawada, Kazuaki","first_name":"Kazuaki"},{"last_name":"Kawakami","first_name":"Ryosuke","full_name":"Kawakami, Ryosuke"},{"full_name":"Shigemoto, Ryuichi","first_name":"Ryuichi","last_name":"Shigemoto","orcid":"0000-0001-8761-9444","id":"499F3ABC-F248-11E8-B48F-1D18A9856A87"},{"last_name":"Nemoto","full_name":"Nemoto, Tomomi","first_name":"Tomomi"}],"ddc":["570"],"doi":"10.1111/ejn.13901","publisher":"Wiley","language":[{"iso":"eng"}],"type":"journal_article","file_date_updated":"2020-07-14T12:46:06Z"},{"department":[{"_id":"GaNo"}],"alternative_title":["ISTA Thesis"],"pubrep_id":"992","user_id":"c635000d-4b10-11ee-a964-aac5a93f6ac1","_id":"395","month":"03","related_material":{"record":[{"relation":"part_of_dissertation","status":"public","id":"1183"}]},"oa":1,"title":"The branched chain amino acids in autism spectrum disorders ","publication_status":"published","has_accepted_license":"1","day":"01","citation":{"short":"D.-C. Tarlungeanu, The Branched Chain Amino Acids in Autism Spectrum Disorders , Institute of Science and Technology Austria, 2018.","ama":"Tarlungeanu D-C. The branched chain amino acids in autism spectrum disorders . 2018. doi:<a href=\"https://doi.org/10.15479/AT:ISTA:th_992\">10.15479/AT:ISTA:th_992</a>","apa":"Tarlungeanu, D.-C. (2018). <i>The branched chain amino acids in autism spectrum disorders </i>. Institute of Science and Technology Austria. <a href=\"https://doi.org/10.15479/AT:ISTA:th_992\">https://doi.org/10.15479/AT:ISTA:th_992</a>","ista":"Tarlungeanu D-C. 2018. The branched chain amino acids in autism spectrum disorders . Institute of Science and Technology Austria.","chicago":"Tarlungeanu, Dora-Clara. “The Branched Chain Amino Acids in Autism Spectrum Disorders .” Institute of Science and Technology Austria, 2018. <a href=\"https://doi.org/10.15479/AT:ISTA:th_992\">https://doi.org/10.15479/AT:ISTA:th_992</a>.","mla":"Tarlungeanu, Dora-Clara. <i>The Branched Chain Amino Acids in Autism Spectrum Disorders </i>. Institute of Science and Technology Austria, 2018, doi:<a href=\"https://doi.org/10.15479/AT:ISTA:th_992\">10.15479/AT:ISTA:th_992</a>.","ieee":"D.-C. Tarlungeanu, “The branched chain amino acids in autism spectrum disorders ,” Institute of Science and Technology Austria, 2018."},"supervisor":[{"full_name":"Novarino, Gaia","first_name":"Gaia","last_name":"Novarino","id":"3E57A680-F248-11E8-B48F-1D18A9856A87","orcid":"0000-0002-7673-7178"}],"degree_awarded":"PhD","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. "}],"status":"public","date_published":"2018-03-01T00:00:00Z","oa_version":"Published Version","ddc":["570","616"],"author":[{"id":"2ABCE612-F248-11E8-B48F-1D18A9856A87","first_name":"Dora-Clara","full_name":"Tarlungeanu, Dora-Clara","last_name":"Tarlungeanu"}],"publist_id":"7434","publisher":"Institute of Science and Technology Austria","doi":"10.15479/AT:ISTA:th_992","file_date_updated":"2021-02-11T23:30:15Z","type":"dissertation","language":[{"iso":"eng"}],"tmp":{"legal_code_url":"https://creativecommons.org/licenses/by/4.0/legalcode","name":"Creative Commons Attribution 4.0 International Public License (CC-BY 4.0)","image":"/images/cc_by.png","short":"CC BY (4.0)"},"article_processing_charge":"No","publication_identifier":{"issn":["2663-337X"]},"date_updated":"2023-09-07T12:38:59Z","project":[{"grant_number":"F03523","name":"Transmembrane Transporters in Health and Disease","_id":"25473368-B435-11E9-9278-68D0E5697425","call_identifier":"FWF"}],"acknowledged_ssus":[{"_id":"PreCl"},{"_id":"EM-Fac"},{"_id":"Bio"}],"page":"88","date_created":"2018-12-11T11:46:14Z","year":"2018","file":[{"date_updated":"2021-02-11T23:30:15Z","access_level":"closed","embargo_to":"open_access","content_type":"application/vnd.openxmlformats-officedocument.wordprocessingml.document","file_id":"6217","date_created":"2019-04-05T09:19:17Z","file_name":"2018_Thesis_Tarlungeanu_source.docx","creator":"dernst","file_size":43684035,"checksum":"9f5231c96e0ad945040841a8630232da","relation":"source_file"},{"checksum":"0c33c370aa2010df5c552db57a6d01e9","relation":"main_file","creator":"dernst","file_size":30511532,"embargo":"2018-03-15","file_name":"2018_Thesis_Tarlungeanu.pdf","date_updated":"2021-02-11T11:17:16Z","access_level":"open_access","content_type":"application/pdf","file_id":"6218","date_created":"2019-04-05T09:19:17Z"}]},{"article_processing_charge":"No","publication_identifier":{"eissn":["1611-3349"],"issn":["0302-9743"]},"page":"203 - 216","quality_controlled":"1","year":"2016","date_created":"2018-12-11T11:50:06Z","date_updated":"2023-09-05T14:09:01Z","project":[{"call_identifier":"FP7","_id":"25CD3DD2-B435-11E9-9278-68D0E5697425","name":"Localization of ion channels and receptors by two and three-dimensional immunoelectron microscopic approaches","grant_number":"604102"}],"acknowledged_ssus":[{"_id":"EM-Fac"}],"volume":1474,"oa_version":"None","date_published":"2016-08-12T00:00:00Z","publist_id":"6281","publication":"High-Resolution Imaging of Cellular Proteins","author":[{"last_name":"Harada","full_name":"Harada, Harumi","first_name":"Harumi","orcid":"0000-0001-7429-7896","id":"2E55CDF2-F248-11E8-B48F-1D18A9856A87"},{"last_name":"Shigemoto","first_name":"Ryuichi","full_name":"Shigemoto, Ryuichi","id":"499F3ABC-F248-11E8-B48F-1D18A9856A87","orcid":"0000-0001-8761-9444"}],"doi":"10.1007/978-1-4939-6352-2_12","publisher":"Springer","language":[{"iso":"eng"}],"type":"book_chapter","day":"12","citation":{"mla":"Harada, Harumi, and Ryuichi Shigemoto. “Immunogold Protein Localization on Grid-Glued Freeze-Fracture Replicas.” <i>High-Resolution Imaging of Cellular Proteins</i>, vol. 1474, Springer, 2016, pp. 203–16, doi:<a href=\"https://doi.org/10.1007/978-1-4939-6352-2_12\">10.1007/978-1-4939-6352-2_12</a>.","ieee":"H. Harada and R. Shigemoto, “Immunogold protein localization on grid-glued freeze-fracture replicas,” in <i>High-Resolution Imaging of Cellular Proteins</i>, vol. 1474, Springer, 2016, pp. 203–216.","chicago":"Harada, Harumi, and Ryuichi Shigemoto. “Immunogold Protein Localization on Grid-Glued Freeze-Fracture Replicas.” In <i>High-Resolution Imaging of Cellular Proteins</i>, 1474:203–16. Springer, 2016. <a href=\"https://doi.org/10.1007/978-1-4939-6352-2_12\">https://doi.org/10.1007/978-1-4939-6352-2_12</a>.","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.","apa":"Harada, H., &#38; Shigemoto, R. (2016). Immunogold protein localization on grid-glued freeze-fracture replicas. In <i>High-Resolution Imaging of Cellular Proteins</i> (Vol. 1474, pp. 203–216). Springer. <a href=\"https://doi.org/10.1007/978-1-4939-6352-2_12\">https://doi.org/10.1007/978-1-4939-6352-2_12</a>","ama":"Harada H, Shigemoto R. Immunogold protein localization on grid-glued freeze-fracture replicas. In: <i>High-Resolution Imaging of Cellular Proteins</i>. Vol 1474. Springer; 2016:203-216. doi:<a href=\"https://doi.org/10.1007/978-1-4939-6352-2_12\">10.1007/978-1-4939-6352-2_12</a>","short":"H. Harada, R. Shigemoto, in:, High-Resolution Imaging of Cellular Proteins, Springer, 2016, pp. 203–216."},"intvolume":"      1474","ec_funded":1,"status":"public","abstract":[{"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.","lang":"eng"}],"department":[{"_id":"RySh"}],"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"],"user_id":"c635000d-4b10-11ee-a964-aac5a93f6ac1","_id":"1094","month":"08","title":"Immunogold protein localization on grid-glued freeze-fracture replicas","publication_status":"published"},{"external_id":{"pmid":["25473011"]},"oa_version":"Submitted Version","date_published":"2015-01-22T00:00:00Z","issue":"1799","publication":"Proceedings of the Royal Society of London Series B Biological Sciences","publist_id":"5090","author":[{"first_name":"Matthias","full_name":"Konrad, Matthias","last_name":"Konrad","id":"46528076-F248-11E8-B48F-1D18A9856A87"},{"last_name":"Grasse","first_name":"Anna V","full_name":"Grasse, Anna V","id":"406F989C-F248-11E8-B48F-1D18A9856A87"},{"first_name":"Simon","full_name":"Tragust, Simon","last_name":"Tragust","id":"35A7A418-F248-11E8-B48F-1D18A9856A87"},{"full_name":"Cremer, Sylvia","first_name":"Sylvia","last_name":"Cremer","orcid":"0000-0002-2193-3868","id":"2F64EC8C-F248-11E8-B48F-1D18A9856A87"}],"doi":"10.1098/rspb.2014.1976","publisher":"The Royal Society","language":[{"iso":"eng"}],"type":"journal_article","article_processing_charge":"No","publication_identifier":{"eissn":["1471-2954"],"issn":["0962-8452"]},"scopus_import":"1","pmid":1,"main_file_link":[{"url":"http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4286035/","open_access":"1"}],"quality_controlled":"1","date_created":"2018-12-11T11:55:06Z","year":"2015","date_updated":"2023-02-23T14:06:41Z","project":[{"call_identifier":"FP7","_id":"25DC711C-B435-11E9-9278-68D0E5697425","name":"Social Vaccination in Ant Colonies: from Individual Mechanisms to Society Effects","grant_number":"243071"},{"_id":"25DAF0B2-B435-11E9-9278-68D0E5697425","grant_number":"CR-118/3-1","name":"Host-Parasite Coevolution"}],"acknowledged_ssus":[{"_id":"EM-Fac"}],"volume":282,"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.","department":[{"_id":"SyCr"}],"user_id":"2DF688A6-F248-11E8-B48F-1D18A9856A87","month":"01","_id":"1993","related_material":{"record":[{"id":"9740","status":"public","relation":"research_data"}]},"title":"Anti-pathogen protection versus survival costs mediated by an ectosymbiont in an ant host","oa":1,"publication_status":"published","article_type":"original","day":"22","citation":{"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.” <i>Proceedings of the Royal Society of London Series B Biological Sciences</i>. The Royal Society, 2015. <a href=\"https://doi.org/10.1098/rspb.2014.1976\">https://doi.org/10.1098/rspb.2014.1976</a>.","apa":"Konrad, M., Grasse, A. V., Tragust, S., &#38; Cremer, S. (2015). Anti-pathogen protection versus survival costs mediated by an ectosymbiont in an ant host. <i>Proceedings of the Royal Society of London Series B Biological Sciences</i>. The Royal Society. <a href=\"https://doi.org/10.1098/rspb.2014.1976\">https://doi.org/10.1098/rspb.2014.1976</a>","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.","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,” <i>Proceedings of the Royal Society of London Series B Biological Sciences</i>, vol. 282, no. 1799. The Royal Society, 2015.","mla":"Konrad, Matthias, et al. “Anti-Pathogen Protection versus Survival Costs Mediated by an Ectosymbiont in an Ant Host.” <i>Proceedings of the Royal Society of London Series B Biological Sciences</i>, vol. 282, no. 1799, 20141976, The Royal Society, 2015, doi:<a href=\"https://doi.org/10.1098/rspb.2014.1976\">10.1098/rspb.2014.1976</a>.","short":"M. Konrad, A.V. Grasse, S. Tragust, S. Cremer, Proceedings of the Royal Society of London Series B Biological Sciences 282 (2015).","ama":"Konrad M, Grasse AV, Tragust S, Cremer S. Anti-pathogen protection versus survival costs mediated by an ectosymbiont in an ant host. <i>Proceedings of the Royal Society of London Series B Biological Sciences</i>. 2015;282(1799). doi:<a href=\"https://doi.org/10.1098/rspb.2014.1976\">10.1098/rspb.2014.1976</a>"},"intvolume":"       282","ec_funded":1,"article_number":"20141976","status":"public","abstract":[{"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. ","lang":"eng"}]}]