[{"publisher":"Society for Neuroscience","doi":"10.1523/JNEUROSCI.1514-22.2023","article_processing_charge":"No","type":"journal_article","date_updated":"2023-10-18T07:12:47Z","_id":"13202","ddc":["570"],"page":"4197-4216","quality_controlled":"1","external_id":{"pmid":["37160366"],"isi":["001020132100005"]},"year":"2023","isi":1,"project":[{"name":"Ultrastructural analysis of phosphoinositides in nerve terminals: distribution, dynamics and physiological roles in synaptic transmission","grant_number":"793482","call_identifier":"H2020","_id":"2659CC84-B435-11E9-9278-68D0E5697425"},{"_id":"25CA28EA-B435-11E9-9278-68D0E5697425","call_identifier":"H2020","name":"In situ analysis of single channel subunit composition in neurons: physiological implication in synaptic plasticity and behaviour","grant_number":"694539"}],"status":"public","publication":"The Journal of Neuroscience","date_published":"2023-06-07T00:00:00Z","acknowledgement":"This work was supported by The Institute of Science and Technology (IST) Austria, the European Union's Horizon 2020 Research and Innovation Program under the Marie Skłodowska-Curie Grant Agreement No. 793482 (to K.E.) and by the European Research Council (ERC) Grant Agreement No. 694539 (to R.S.). We thank Nicoleta Condruz (IST Austria, Klosterneuburg, Austria) for technical assistance with sample preparation, the Electron Microscopy Facility of IST Austria (Klosterneuburg, Austria) for technical support with EM works, Natalia Baranova (University of Vienna, Vienna, Austria) and Martin Loose (IST Austria, Klosterneuburg, Austria) for advice on liposome preparation, and Yugo Fukazawa (University of Fukui, Fukui, Japan) for comments.","pmid":1,"ec_funded":1,"title":"Nanoscale phosphoinositide distribution on cell membranes of mouse cerebellar neurons","oa_version":"Published Version","author":[{"full_name":"Eguchi, Kohgaku","id":"2B7846DC-F248-11E8-B48F-1D18A9856A87","last_name":"Eguchi","orcid":"0000-0002-6170-2546","first_name":"Kohgaku"},{"last_name":"Le Monnier","id":"3B59276A-F248-11E8-B48F-1D18A9856A87","full_name":"Le Monnier, Elodie","first_name":"Elodie"},{"first_name":"Ryuichi","orcid":"0000-0001-8761-9444","full_name":"Shigemoto, Ryuichi","id":"499F3ABC-F248-11E8-B48F-1D18A9856A87","last_name":"Shigemoto"}],"day":"07","scopus_import":"1","article_type":"original","date_created":"2023-07-09T22:01:12Z","volume":43,"abstract":[{"lang":"eng","text":"Phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) plays an essential role in neuronal activities through interaction with various proteins involved in signaling at membranes. However, the distribution pattern of PI(4,5)P2 and the association with these proteins on the neuronal cell membranes remain elusive. In this study, we established a method for visualizing PI(4,5)P2 by SDS-digested freeze-fracture replica labeling (SDS-FRL) to investigate the quantitative nanoscale distribution of PI(4,5)P2 in cryo-fixed brain. We demonstrate that PI(4,5)P2 forms tiny clusters with a mean size of ∼1000 nm2 rather than randomly distributed in cerebellar neuronal membranes in male C57BL/6J mice. These clusters show preferential accumulation in specific membrane compartments of different cell types, in particular, in Purkinje cell (PC) spines and granule cell (GC) presynaptic active zones. Furthermore, we revealed extensive association of PI(4,5)P2 with CaV2.1 and GIRK3 across different membrane compartments, whereas its association with mGluR1α was compartment specific. These results suggest that our SDS-FRL method provides valuable insights into the physiological functions of PI(4,5)P2 in neurons."}],"tmp":{"name":"Creative Commons Attribution 4.0 International Public License (CC-BY 4.0)","legal_code_url":"https://creativecommons.org/licenses/by/4.0/legalcode","image":"/images/cc_by.png","short":"CC BY (4.0)"},"intvolume":"        43","acknowledged_ssus":[{"_id":"EM-Fac"}],"has_accepted_license":"1","publication_status":"published","publication_identifier":{"issn":["0270-6474"],"eissn":["1529-2401"]},"file_date_updated":"2023-07-10T09:04:58Z","month":"06","file":[{"creator":"alisjak","date_updated":"2023-07-10T09:04:58Z","file_size":7794425,"date_created":"2023-07-10T09:04:58Z","file_id":"13205","access_level":"open_access","content_type":"application/pdf","success":1,"file_name":"2023_JN_Eguchi.pdf","checksum":"70b2141870e0bf1c94fd343e18fdbc32","relation":"main_file"}],"department":[{"_id":"RySh"}],"language":[{"iso":"eng"}],"oa":1,"user_id":"2DF688A6-F248-11E8-B48F-1D18A9856A87","issue":"23","citation":{"ama":"Eguchi K, Le Monnier E, Shigemoto R. Nanoscale phosphoinositide distribution on cell membranes of mouse cerebellar neurons. <i>The Journal of Neuroscience</i>. 2023;43(23):4197-4216. doi:<a href=\"https://doi.org/10.1523/JNEUROSCI.1514-22.2023\">10.1523/JNEUROSCI.1514-22.2023</a>","short":"K. Eguchi, E. Le Monnier, R. Shigemoto, The Journal of Neuroscience 43 (2023) 4197–4216.","ieee":"K. Eguchi, E. Le Monnier, and R. Shigemoto, “Nanoscale phosphoinositide distribution on cell membranes of mouse cerebellar neurons,” <i>The Journal of Neuroscience</i>, vol. 43, no. 23. Society for Neuroscience, pp. 4197–4216, 2023.","chicago":"Eguchi, Kohgaku, Elodie Le Monnier, and Ryuichi Shigemoto. “Nanoscale Phosphoinositide Distribution on Cell Membranes of Mouse Cerebellar Neurons.” <i>The Journal of Neuroscience</i>. Society for Neuroscience, 2023. <a href=\"https://doi.org/10.1523/JNEUROSCI.1514-22.2023\">https://doi.org/10.1523/JNEUROSCI.1514-22.2023</a>.","ista":"Eguchi K, Le Monnier E, Shigemoto R. 2023. Nanoscale phosphoinositide distribution on cell membranes of mouse cerebellar neurons. The Journal of Neuroscience. 43(23), 4197–4216.","mla":"Eguchi, Kohgaku, et al. “Nanoscale Phosphoinositide Distribution on Cell Membranes of Mouse Cerebellar Neurons.” <i>The Journal of Neuroscience</i>, vol. 43, no. 23, Society for Neuroscience, 2023, pp. 4197–216, doi:<a href=\"https://doi.org/10.1523/JNEUROSCI.1514-22.2023\">10.1523/JNEUROSCI.1514-22.2023</a>.","apa":"Eguchi, K., Le Monnier, E., &#38; Shigemoto, R. (2023). Nanoscale phosphoinositide distribution on cell membranes of mouse cerebellar neurons. <i>The Journal of Neuroscience</i>. Society for Neuroscience. <a href=\"https://doi.org/10.1523/JNEUROSCI.1514-22.2023\">https://doi.org/10.1523/JNEUROSCI.1514-22.2023</a>"}},{"date_updated":"2023-08-03T06:08:01Z","_id":"10889","type":"journal_article","doi":"10.1093/jmicro/dfab048","article_processing_charge":"No","publisher":"Oxford Academic","main_file_link":[{"url":"https://doi.org/10.1093/jmicro/dfab048","open_access":"1"}],"quality_controlled":"1","page":"i72-i80","year":"2022","isi":1,"external_id":{"pmid":["35275179"],"isi":["000768384100011"]},"pmid":1,"ec_funded":1,"date_published":"2022-03-01T00:00:00Z","acknowledgement":"European Research Council Advanced Grant (694539 to R.S.).","project":[{"_id":"25CA28EA-B435-11E9-9278-68D0E5697425","call_identifier":"H2020","name":"In situ analysis of single channel subunit composition in neurons: physiological implication in synaptic plasticity and behaviour","grant_number":"694539"}],"publication":"Microscopy","status":"public","volume":71,"article_type":"original","date_created":"2022-03-20T23:01:39Z","author":[{"full_name":"Shigemoto, Ryuichi","id":"499F3ABC-F248-11E8-B48F-1D18A9856A87","last_name":"Shigemoto","first_name":"Ryuichi","orcid":"0000-0001-8761-9444"}],"day":"01","scopus_import":"1","title":"Electron microscopic visualization of single molecules by tag-mediated metal particle labeling","oa_version":"Published Version","publication_status":"published","publication_identifier":{"eissn":["2050-5701"],"issn":["2050-5698"]},"abstract":[{"lang":"eng","text":"Genetically encoded tags have introduced extensive lines of application from purification of tagged proteins to their visualization at the single molecular, cellular, histological and whole-body levels. Combined with other rapidly developing technologies such as clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system, proteomics, super-resolution microscopy and proximity labeling, a large variety of genetically encoded tags have been developed in the last two decades. In this review, I focus on the current status of tag development for electron microscopic (EM) visualization of proteins with metal particle labeling. Compared with conventional immunoelectron microscopy using gold particles, tag-mediated metal particle labeling has several advantages that could potentially improve the sensitivity, spatial and temporal resolution, and applicability to a wide range of proteins of interest (POIs). It may enable researchers to detect single molecules in situ, allowing the quantitative measurement of absolute numbers and exact localization patterns of POI in the ultrastructural context. Thus, genetically encoded tags for EM could revolutionize the field as green fluorescence protein did for light microscopy, although we still have many challenges to overcome before reaching this goal."}],"intvolume":"        71","department":[{"_id":"RySh"}],"month":"03","issue":"Supplement_1","citation":{"ama":"Shigemoto R. Electron microscopic visualization of single molecules by tag-mediated metal particle labeling. <i>Microscopy</i>. 2022;71(Supplement_1):i72-i80. doi:<a href=\"https://doi.org/10.1093/jmicro/dfab048\">10.1093/jmicro/dfab048</a>","short":"R. Shigemoto, Microscopy 71 (2022) i72–i80.","ieee":"R. Shigemoto, “Electron microscopic visualization of single molecules by tag-mediated metal particle labeling,” <i>Microscopy</i>, vol. 71, no. Supplement_1. Oxford Academic, pp. i72–i80, 2022.","ista":"Shigemoto R. 2022. Electron microscopic visualization of single molecules by tag-mediated metal particle labeling. Microscopy. 71(Supplement_1), i72–i80.","chicago":"Shigemoto, Ryuichi. “Electron Microscopic Visualization of Single Molecules by Tag-Mediated Metal Particle Labeling.” <i>Microscopy</i>. Oxford Academic, 2022. <a href=\"https://doi.org/10.1093/jmicro/dfab048\">https://doi.org/10.1093/jmicro/dfab048</a>.","mla":"Shigemoto, Ryuichi. “Electron Microscopic Visualization of Single Molecules by Tag-Mediated Metal Particle Labeling.” <i>Microscopy</i>, vol. 71, no. Supplement_1, Oxford Academic, 2022, pp. i72–80, doi:<a href=\"https://doi.org/10.1093/jmicro/dfab048\">10.1093/jmicro/dfab048</a>.","apa":"Shigemoto, R. (2022). Electron microscopic visualization of single molecules by tag-mediated metal particle labeling. <i>Microscopy</i>. Oxford Academic. <a href=\"https://doi.org/10.1093/jmicro/dfab048\">https://doi.org/10.1093/jmicro/dfab048</a>"},"user_id":"4359f0d1-fa6c-11eb-b949-802e58b17ae8","oa":1,"language":[{"iso":"eng"}]},{"month":"02","article_number":"846615","file":[{"checksum":"51ec9b90e7da919e22c01a15489eaacd","relation":"main_file","access_level":"open_access","content_type":"application/pdf","success":1,"file_name":"2022_FrontiersNeuroanatomy_Eguchi.pdf","file_id":"10911","date_updated":"2022-03-21T09:41:19Z","creator":"dernst","file_size":2416395,"date_created":"2022-03-21T09:41:19Z"}],"department":[{"_id":"RySh"}],"language":[{"iso":"eng"}],"oa":1,"user_id":"4359f0d1-fa6c-11eb-b949-802e58b17ae8","citation":{"chicago":"Eguchi, Kohgaku, Jacqueline-Claire Montanaro-Punzengruber, Elodie Le Monnier, and Ryuichi Shigemoto. “The Number and Distinct Clustering Patterns of Voltage-Gated Calcium Channels in Nerve Terminals.” <i>Frontiers in Neuroanatomy</i>. Frontiers, 2022. <a href=\"https://doi.org/10.3389/fnana.2022.846615\">https://doi.org/10.3389/fnana.2022.846615</a>.","ista":"Eguchi K, Montanaro-Punzengruber J-C, Le Monnier E, Shigemoto R. 2022. The number and distinct clustering patterns of voltage-gated Calcium channels in nerve terminals. Frontiers in Neuroanatomy. 16, 846615.","mla":"Eguchi, Kohgaku, et al. “The Number and Distinct Clustering Patterns of Voltage-Gated Calcium Channels in Nerve Terminals.” <i>Frontiers in Neuroanatomy</i>, vol. 16, 846615, Frontiers, 2022, doi:<a href=\"https://doi.org/10.3389/fnana.2022.846615\">10.3389/fnana.2022.846615</a>.","apa":"Eguchi, K., Montanaro-Punzengruber, J.-C., Le Monnier, E., &#38; Shigemoto, R. (2022). The number and distinct clustering patterns of voltage-gated Calcium channels in nerve terminals. <i>Frontiers in Neuroanatomy</i>. Frontiers. <a href=\"https://doi.org/10.3389/fnana.2022.846615\">https://doi.org/10.3389/fnana.2022.846615</a>","ama":"Eguchi K, Montanaro-Punzengruber J-C, Le Monnier E, Shigemoto R. The number and distinct clustering patterns of voltage-gated Calcium channels in nerve terminals. <i>Frontiers in Neuroanatomy</i>. 2022;16. doi:<a href=\"https://doi.org/10.3389/fnana.2022.846615\">10.3389/fnana.2022.846615</a>","short":"K. Eguchi, J.-C. Montanaro-Punzengruber, E. Le Monnier, R. Shigemoto, Frontiers in Neuroanatomy 16 (2022).","ieee":"K. Eguchi, J.-C. Montanaro-Punzengruber, E. Le Monnier, and R. Shigemoto, “The number and distinct clustering patterns of voltage-gated Calcium channels in nerve terminals,” <i>Frontiers in Neuroanatomy</i>, vol. 16. Frontiers, 2022."},"oa_version":"Published Version","title":"The number and distinct clustering patterns of voltage-gated Calcium channels in nerve terminals","author":[{"last_name":"Eguchi","id":"2B7846DC-F248-11E8-B48F-1D18A9856A87","full_name":"Eguchi, Kohgaku","orcid":"0000-0002-6170-2546","first_name":"Kohgaku"},{"first_name":"Jacqueline-Claire","id":"3786AB44-F248-11E8-B48F-1D18A9856A87","full_name":"Montanaro-Punzengruber, Jacqueline-Claire","last_name":"Montanaro-Punzengruber"},{"full_name":"Le Monnier, Elodie","id":"3B59276A-F248-11E8-B48F-1D18A9856A87","last_name":"Le Monnier","first_name":"Elodie"},{"id":"499F3ABC-F248-11E8-B48F-1D18A9856A87","full_name":"Shigemoto, Ryuichi","last_name":"Shigemoto","orcid":"0000-0001-8761-9444","first_name":"Ryuichi"}],"scopus_import":"1","day":"24","article_type":"original","date_created":"2022-03-20T23:01:39Z","volume":16,"intvolume":"        16","tmp":{"name":"Creative Commons Attribution 4.0 International Public License (CC-BY 4.0)","legal_code_url":"https://creativecommons.org/licenses/by/4.0/legalcode","image":"/images/cc_by.png","short":"CC BY (4.0)"},"abstract":[{"text":"Upon the arrival of action potentials at nerve terminals, neurotransmitters are released from synaptic vesicles (SVs) by exocytosis. CaV2.1, 2.2, and 2.3 are the major subunits of the voltage-gated calcium channel (VGCC) responsible for increasing intraterminal calcium levels and triggering SV exocytosis in the central nervous system (CNS) synapses. The two-dimensional analysis of CaV2 distributions using sodium dodecyl sulfate (SDS)-digested freeze-fracture replica labeling (SDS-FRL) has revealed their numbers, densities, and nanoscale clustering patterns in individual presynaptic active zones. The variation in these properties affects the coupling of VGCCs with calcium sensors on SVs, synaptic efficacy, and temporal precision of transmission. In this study, we summarize how the morphological parameters of CaV2 distribution obtained using SDS-FRL differ depending on the different types of synapses and could correspond to functional properties in synaptic transmission.","lang":"eng"}],"has_accepted_license":"1","publication_identifier":{"eissn":["16625129"]},"publication_status":"published","file_date_updated":"2022-03-21T09:41:19Z","external_id":{"isi":["000766662700001"],"pmid":["35280978"]},"year":"2022","isi":1,"project":[{"_id":"25CA28EA-B435-11E9-9278-68D0E5697425","grant_number":"694539","name":"In situ analysis of single channel subunit composition in neurons: physiological implication in synaptic plasticity and behaviour","call_identifier":"H2020"},{"_id":"05970B30-7A3F-11EA-A408-12923DDC885E","grant_number":"I04638","name":"LGI1 antibody-induced pathophysiology in synapses"}],"publication":"Frontiers in Neuroanatomy","status":"public","acknowledgement":"This work was supported by the European Research Council advanced grant No. 694539 and the joint German-Austrian DFG and FWF project SYNABS (FWF: I-4638-B) to RS.\r\nThe authors thank Walter Kaufmann for his critical comments on the manuscript.","date_published":"2022-02-24T00:00:00Z","pmid":1,"ec_funded":1,"publisher":"Frontiers","doi":"10.3389/fnana.2022.846615","article_processing_charge":"No","type":"journal_article","date_updated":"2024-10-29T07:57:26Z","_id":"10890","ddc":["570"],"quality_controlled":"1"},{"file":[{"date_created":"2020-10-19T13:31:28Z","file_size":4915964,"creator":"dernst","date_updated":"2020-10-19T13:31:28Z","file_id":"8678","success":1,"file_name":"2021_CurrentBiology_Fredes.pdf","access_level":"open_access","content_type":"application/pdf","relation":"main_file","checksum":"b7b9c8bc84a08befce365c675229a7d1"}],"department":[{"_id":"MaJö"},{"_id":"RySh"}],"month":"01","user_id":"4359f0d1-fa6c-11eb-b949-802e58b17ae8","citation":{"mla":"Fredes Tolorza, Felipe A., et al. “Ventro-Dorsal Hippocampal Pathway Gates Novelty-Induced Contextual Memory Formation.” <i>Current Biology</i>, vol. 31, no. 1, Elsevier, 2021, p. P25–38.E5, doi:<a href=\"https://doi.org/10.1016/j.cub.2020.09.074\">10.1016/j.cub.2020.09.074</a>.","apa":"Fredes Tolorza, F. A., Silva Sifuentes, M. A., Koppensteiner, P., Kobayashi, K., Jösch, M. A., &#38; Shigemoto, R. (2021). Ventro-dorsal hippocampal pathway gates novelty-induced contextual memory formation. <i>Current Biology</i>. Elsevier. <a href=\"https://doi.org/10.1016/j.cub.2020.09.074\">https://doi.org/10.1016/j.cub.2020.09.074</a>","chicago":"Fredes Tolorza, Felipe A, Maria A Silva Sifuentes, Peter Koppensteiner, Kenta Kobayashi, Maximilian A Jösch, and Ryuichi Shigemoto. “Ventro-Dorsal Hippocampal Pathway Gates Novelty-Induced Contextual Memory Formation.” <i>Current Biology</i>. Elsevier, 2021. <a href=\"https://doi.org/10.1016/j.cub.2020.09.074\">https://doi.org/10.1016/j.cub.2020.09.074</a>.","ista":"Fredes Tolorza FA, Silva Sifuentes MA, Koppensteiner P, Kobayashi K, Jösch MA, Shigemoto R. 2021. Ventro-dorsal hippocampal pathway gates novelty-induced contextual memory formation. Current Biology. 31(1), P25–38.E5.","short":"F.A. Fredes Tolorza, M.A. Silva Sifuentes, P. Koppensteiner, K. Kobayashi, M.A. Jösch, R. Shigemoto, Current Biology 31 (2021) P25–38.E5.","ieee":"F. A. Fredes Tolorza, M. A. Silva Sifuentes, P. Koppensteiner, K. Kobayashi, M. A. Jösch, and R. Shigemoto, “Ventro-dorsal hippocampal pathway gates novelty-induced contextual memory formation,” <i>Current Biology</i>, vol. 31, no. 1. Elsevier, p. P25–38.E5, 2021.","ama":"Fredes Tolorza FA, Silva Sifuentes MA, Koppensteiner P, Kobayashi K, Jösch MA, Shigemoto R. Ventro-dorsal hippocampal pathway gates novelty-induced contextual memory formation. <i>Current Biology</i>. 2021;31(1):P25-38.E5. doi:<a href=\"https://doi.org/10.1016/j.cub.2020.09.074\">10.1016/j.cub.2020.09.074</a>"},"issue":"1","language":[{"iso":"eng"}],"oa":1,"date_created":"2020-02-28T10:56:18Z","article_type":"original","volume":31,"title":"Ventro-dorsal hippocampal pathway gates novelty-induced contextual memory formation","oa_version":"Published Version","day":"11","author":[{"first_name":"Felipe A","last_name":"Fredes Tolorza","id":"384825DA-F248-11E8-B48F-1D18A9856A87","full_name":"Fredes Tolorza, Felipe A"},{"first_name":"Maria A","id":"371B3D6E-F248-11E8-B48F-1D18A9856A87","full_name":"Silva Sifuentes, Maria A","last_name":"Silva Sifuentes"},{"first_name":"Peter","last_name":"Koppensteiner","id":"3B8B25A8-F248-11E8-B48F-1D18A9856A87","full_name":"Koppensteiner, Peter"},{"last_name":"Kobayashi","full_name":"Kobayashi, Kenta","first_name":"Kenta"},{"orcid":"0000-0002-3937-1330","first_name":"Maximilian A","last_name":"Jösch","id":"2BD278E6-F248-11E8-B48F-1D18A9856A87","full_name":"Jösch, Maximilian A"},{"full_name":"Shigemoto, Ryuichi","id":"499F3ABC-F248-11E8-B48F-1D18A9856A87","last_name":"Shigemoto","orcid":"0000-0001-8761-9444","first_name":"Ryuichi"}],"file_date_updated":"2020-10-19T13:31:28Z","publication_status":"published","tmp":{"image":"/images/cc_by_nc_nd.png","name":"Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0)","legal_code_url":"https://creativecommons.org/licenses/by-nc-nd/4.0/legalcode","short":"CC BY-NC-ND (4.0)"},"abstract":[{"text":"Novelty facilitates formation of memories. The detection of novelty and storage of contextual memories are both mediated by the hippocampus, yet the mechanisms that link these two functions remain to be defined. Dentate granule cells (GCs) of the dorsal hippocampus fire upon novelty exposure forming engrams of contextual memory. However, their key excitatory inputs from the entorhinal cortex are not responsive to novelty and are insufficient to make dorsal GCs fire reliably. Here we uncover a powerful glutamatergic pathway to dorsal GCs from ventral hippocampal mossy cells (MCs) that relays novelty, and is necessary and sufficient for driving dorsal GCs activation. Furthermore, manipulation of ventral MCs activity bidirectionally regulates novelty-induced contextual memory acquisition. Our results show that ventral MCs activity controls memory formation through an intra-hippocampal interaction mechanism gated by novelty.","lang":"eng"}],"intvolume":"        31","has_accepted_license":"1","related_material":{"link":[{"url":"https://ist.ac.at/en/news/remembering-novelty/","description":"News on IST Homepage","relation":"press_release"}]},"external_id":{"isi":["000614361000020"]},"isi":1,"year":"2021","acknowledgement":"We thank Peter Jonas and Peter Somogyi for critically reading the manuscript, Satoshi Kida for helpful discussion, Taijia Makinen for providing the Prox1-creERT2 mouse line, and Hiromu Yawo for the VAMP2-Venus construct. We also thank Vivek Jayaraman, Ph.D.; Rex A. Kerr, Ph.D.; Douglas S. Kim, Ph.D.; Loren L. Looger, Ph.D.; and Karel Svoboda, Ph.D. from the GENIE Project, Janelia Farm Research Campus, Howard Hughes Medical Institute for the viral constructs used for GCaMP6s expression. We also thank Jacqueline Montanaro, Vanessa Zheden, David Kleindienst, and Laura Burnett for technical assistance, as well as Robert Beattie for imaging assistance. This work was supported by a European Research Council Advanced Grant 694539 to R.S.","date_published":"2021-01-11T00:00:00Z","ec_funded":1,"status":"public","publication":"Current Biology","project":[{"call_identifier":"H2020","name":"In situ analysis of single channel subunit composition in neurons: physiological implication in synaptic plasticity and behaviour","grant_number":"694539","_id":"25CA28EA-B435-11E9-9278-68D0E5697425"}],"type":"journal_article","_id":"7551","date_updated":"2023-08-04T10:47:11Z","publisher":"Elsevier","article_processing_charge":"No","doi":"10.1016/j.cub.2020.09.074","quality_controlled":"1","ddc":["570"],"page":"P25-38.E5"},{"file_date_updated":"2021-04-19T10:10:56Z","publication_identifier":{"eissn":["1091-6490"]},"publication_status":"published","has_accepted_license":"1","acknowledged_ssus":[{"_id":"EM-Fac"}],"tmp":{"name":"Creative Commons Attribution 4.0 International Public License (CC-BY 4.0)","legal_code_url":"https://creativecommons.org/licenses/by/4.0/legalcode","image":"/images/cc_by.png","short":"CC BY (4.0)"},"abstract":[{"lang":"eng","text":"In nerve cells the genes encoding for α2δ subunits of voltage-gated calcium channels have been linked to synaptic functions and neurological disease. Here we show that α2δ subunits are essential for the formation and organization of glutamatergic synapses. Using a cellular α2δ subunit triple-knockout/knockdown model, we demonstrate a failure in presynaptic differentiation evidenced by defective presynaptic calcium channel clustering and calcium influx, smaller presynaptic active zones, and a strongly reduced accumulation of presynaptic vesicle-associated proteins (synapsin and vGLUT). The presynaptic defect is associated with the downscaling of postsynaptic AMPA receptors and the postsynaptic density. The role of α2δ isoforms as synaptic organizers is highly redundant, as each individual α2δ isoform can rescue presynaptic calcium channel trafficking and expression of synaptic proteins. Moreover, α2δ-2 and α2δ-3 with mutated metal ion-dependent adhesion sites can fully rescue presynaptic synapsin expression but only partially calcium channel trafficking, suggesting that the regulatory role of α2δ subunits is independent from its role as a calcium channel subunit. Our findings influence the current view on excitatory synapse formation. First, our study suggests that postsynaptic differentiation is secondary to presynaptic differentiation. Second, the dependence of presynaptic differentiation on α2δ implicates α2δ subunits as potential nucleation points for the organization of synapses. Finally, our results suggest that α2δ subunits act as transsynaptic organizers of glutamatergic synapses, thereby aligning the synaptic active zone with the postsynaptic density."}],"intvolume":"       118","volume":118,"date_created":"2021-04-18T22:01:40Z","article_type":"original","day":"06","scopus_import":"1","author":[{"first_name":"Clemens L.","last_name":"Schöpf","full_name":"Schöpf, Clemens L."},{"full_name":"Ablinger, Cornelia","last_name":"Ablinger","first_name":"Cornelia"},{"full_name":"Geisler, Stefanie M.","last_name":"Geisler","first_name":"Stefanie M."},{"first_name":"Ruslan I.","last_name":"Stanika","full_name":"Stanika, Ruslan I."},{"last_name":"Campiglio","full_name":"Campiglio, Marta","first_name":"Marta"},{"first_name":"Walter","orcid":"0000-0001-9735-5315","last_name":"Kaufmann","id":"3F99E422-F248-11E8-B48F-1D18A9856A87","full_name":"Kaufmann, Walter"},{"last_name":"Nimmervoll","full_name":"Nimmervoll, Benedikt","first_name":"Benedikt"},{"full_name":"Schlick, Bettina","last_name":"Schlick","first_name":"Bettina"},{"last_name":"Brockhaus","full_name":"Brockhaus, Johannes","first_name":"Johannes"},{"full_name":"Missler, Markus","last_name":"Missler","first_name":"Markus"},{"orcid":"0000-0001-8761-9444","first_name":"Ryuichi","full_name":"Shigemoto, Ryuichi","id":"499F3ABC-F248-11E8-B48F-1D18A9856A87","last_name":"Shigemoto"},{"first_name":"Gerald J.","full_name":"Obermair, Gerald J.","last_name":"Obermair"}],"title":"Presynaptic α2δ subunits are key organizers of glutamatergic synapses","oa_version":"Published Version","citation":{"ama":"Schöpf CL, Ablinger C, Geisler SM, et al. Presynaptic α2δ subunits are key organizers of glutamatergic synapses. <i>PNAS</i>. 2021;118(14). doi:<a href=\"https://doi.org/10.1073/pnas.1920827118\">10.1073/pnas.1920827118</a>","ieee":"C. L. Schöpf <i>et al.</i>, “Presynaptic α2δ subunits are key organizers of glutamatergic synapses,” <i>PNAS</i>, vol. 118, no. 14. National Academy of Sciences, 2021.","short":"C.L. Schöpf, C. Ablinger, S.M. Geisler, R.I. Stanika, M. Campiglio, W. Kaufmann, B. Nimmervoll, B. Schlick, J. Brockhaus, M. Missler, R. Shigemoto, G.J. Obermair, PNAS 118 (2021).","chicago":"Schöpf, Clemens L., Cornelia Ablinger, Stefanie M. Geisler, Ruslan I. Stanika, Marta Campiglio, Walter Kaufmann, Benedikt Nimmervoll, et al. “Presynaptic Α2δ Subunits Are Key Organizers of Glutamatergic Synapses.” <i>PNAS</i>. National Academy of Sciences, 2021. <a href=\"https://doi.org/10.1073/pnas.1920827118\">https://doi.org/10.1073/pnas.1920827118</a>.","ista":"Schöpf CL, Ablinger C, Geisler SM, Stanika RI, Campiglio M, Kaufmann W, Nimmervoll B, Schlick B, Brockhaus J, Missler M, Shigemoto R, Obermair GJ. 2021. Presynaptic α2δ subunits are key organizers of glutamatergic synapses. PNAS. 118(14).","apa":"Schöpf, C. L., Ablinger, C., Geisler, S. M., Stanika, R. I., Campiglio, M., Kaufmann, W., … Obermair, G. J. (2021). Presynaptic α2δ subunits are key organizers of glutamatergic synapses. <i>PNAS</i>. National Academy of Sciences. <a href=\"https://doi.org/10.1073/pnas.1920827118\">https://doi.org/10.1073/pnas.1920827118</a>","mla":"Schöpf, Clemens L., et al. “Presynaptic Α2δ Subunits Are Key Organizers of Glutamatergic Synapses.” <i>PNAS</i>, vol. 118, no. 14, National Academy of Sciences, 2021, doi:<a href=\"https://doi.org/10.1073/pnas.1920827118\">10.1073/pnas.1920827118</a>."},"issue":"14","user_id":"4359f0d1-fa6c-11eb-b949-802e58b17ae8","oa":1,"language":[{"iso":"eng"}],"department":[{"_id":"EM-Fac"},{"_id":"RySh"}],"file":[{"file_size":2603911,"date_created":"2021-04-19T10:10:56Z","date_updated":"2021-04-19T10:10:56Z","creator":"dernst","file_id":"9340","file_name":"2021_PNAS_Schoepf.pdf","success":1,"content_type":"application/pdf","access_level":"open_access","relation":"main_file","checksum":"dd014f68ae9d7d8d8fc4139a24e04506"}],"month":"04","quality_controlled":"1","ddc":["570"],"_id":"9330","date_updated":"2023-08-08T13:08:47Z","type":"journal_article","article_processing_charge":"No","doi":"10.1073/pnas.1920827118","publisher":"National Academy of Sciences","ec_funded":1,"date_published":"2021-04-06T00:00:00Z","acknowledgement":"We thank Arnold Schwartz for providing α2δ-1 knockout mice; Ariane Benedetti, Sabine Baumgartner, Sandra Demetz, and Irene Mahlknecht for technical support; Nadine Ortner and Andreas Lieb for electrophysiological experiments; the team of the Electron Microscopy Facility at the Institute of Science and Technology Austria for technical support related to ultrastructural analysis; Hermann Dietrich and Anja Beierfuß and her team for animal care; Jutta Engel and Jörg Striessnig for critical discussions; and Bruno Benedetti and Bernhard Flucher for critical discussions and reading the manuscript. This study was supported by Austrian Science Fund Grants P24079, F44060, F44150, and DOC30-B30 (to G.J.O.) and T855 (to M.C.), European Research Council Grant AdG 694539 (to R.S.), Deutsche Forschungsgemeinschaft\r\nGrant SFB1348-TP A03 (to M.M.), and Interdisziplinäre Zentrum für Klinische Forschung Münster Grant Mi3/004/19 (to M.M.). This work is part of the PhD theses of C.L.S., S.M.G., and C.A.","publication":"PNAS","status":"public","project":[{"call_identifier":"H2020","grant_number":"694539","name":"In situ analysis of single channel subunit composition in neurons: physiological implication in synaptic plasticity and behaviour","_id":"25CA28EA-B435-11E9-9278-68D0E5697425"}],"year":"2021","isi":1,"external_id":{"isi":["000637398300002"]}},{"publication_identifier":{"eissn":["2050-084X"]},"publication_status":"published","file_date_updated":"2021-05-31T09:43:09Z","has_accepted_license":"1","tmp":{"name":"Creative Commons Attribution 4.0 International Public License (CC-BY 4.0)","legal_code_url":"https://creativecommons.org/licenses/by/4.0/legalcode","image":"/images/cc_by.png","short":"CC BY (4.0)"},"abstract":[{"lang":"eng","text":"The synaptic connection from medial habenula (MHb) to interpeduncular nucleus (IPN) is critical for emotion-related behaviors and uniquely expresses R-type Ca2+ channels (Cav2.3) and auxiliary GABAB receptor (GBR) subunits, the K+-channel tetramerization domain-containing proteins (KCTDs). Activation of GBRs facilitates or inhibits transmitter release from MHb terminals depending on the IPN subnucleus, but the role of KCTDs is unknown. We therefore examined the localization and function of Cav2.3, GBRs, and KCTDs in this pathway in mice. We show in heterologous cells that KCTD8 and KCTD12b directly bind to Cav2.3 and that KCTD8 potentiates Cav2.3 currents in the absence of GBRs. In the rostral IPN, KCTD8, KCTD12b, and Cav2.3 co-localize at the presynaptic active zone. Genetic deletion indicated a bidirectional modulation of Cav2.3-mediated release by these KCTDs with a compensatory increase of KCTD8 in the active zone in KCTD12b-deficient mice. The interaction of Cav2.3 with KCTDs therefore scales synaptic strength independent of GBR activation."}],"intvolume":"        10","volume":10,"article_type":"original","date_created":"2021-05-30T22:01:23Z","author":[{"first_name":"Pradeep","orcid":"0000-0003-0863-4481","full_name":"Bhandari, Pradeep","id":"45EDD1BC-F248-11E8-B48F-1D18A9856A87","last_name":"Bhandari"},{"orcid":"0000-0001-7577-1676","first_name":"David H","last_name":"Vandael","id":"3AE48E0A-F248-11E8-B48F-1D18A9856A87","full_name":"Vandael, David H"},{"last_name":"Fernández-Fernández","full_name":"Fernández-Fernández, Diego","first_name":"Diego"},{"first_name":"Thorsten","full_name":"Fritzius, Thorsten","last_name":"Fritzius"},{"first_name":"David","last_name":"Kleindienst","id":"42E121A4-F248-11E8-B48F-1D18A9856A87","full_name":"Kleindienst, David"},{"id":"4659D740-F248-11E8-B48F-1D18A9856A87","full_name":"Önal, Hüseyin C","last_name":"Önal","orcid":"0000-0002-2771-2011","first_name":"Hüseyin C"},{"id":"3786AB44-F248-11E8-B48F-1D18A9856A87","full_name":"Montanaro-Punzengruber, Jacqueline-Claire","last_name":"Montanaro-Punzengruber","first_name":"Jacqueline-Claire"},{"full_name":"Gassmann, Martin","last_name":"Gassmann","first_name":"Martin"},{"first_name":"Peter M","orcid":"0000-0001-5001-4804","full_name":"Jonas, Peter M","id":"353C1B58-F248-11E8-B48F-1D18A9856A87","last_name":"Jonas"},{"full_name":"Kulik, Akos","last_name":"Kulik","first_name":"Akos"},{"first_name":"Bernhard","full_name":"Bettler, Bernhard","last_name":"Bettler"},{"full_name":"Shigemoto, Ryuichi","id":"499F3ABC-F248-11E8-B48F-1D18A9856A87","last_name":"Shigemoto","orcid":"0000-0001-8761-9444","first_name":"Ryuichi"},{"last_name":"Koppensteiner","id":"3B8B25A8-F248-11E8-B48F-1D18A9856A87","full_name":"Koppensteiner, Peter","first_name":"Peter","orcid":"0000-0002-3509-1948"}],"day":"29","scopus_import":"1","title":"GABAB receptor auxiliary subunits modulate Cav2.3-mediated release from medial habenula terminals","oa_version":"Published Version","citation":{"ieee":"P. Bhandari <i>et al.</i>, “GABAB receptor auxiliary subunits modulate Cav2.3-mediated release from medial habenula terminals,” <i>eLife</i>, vol. 10. eLife Sciences Publications, 2021.","short":"P. Bhandari, D.H. Vandael, D. Fernández-Fernández, T. Fritzius, D. Kleindienst, H.C. Önal, J.-C. Montanaro-Punzengruber, M. Gassmann, P.M. Jonas, A. Kulik, B. Bettler, R. Shigemoto, P. Koppensteiner, ELife 10 (2021).","ama":"Bhandari P, Vandael DH, Fernández-Fernández D, et al. GABAB receptor auxiliary subunits modulate Cav2.3-mediated release from medial habenula terminals. <i>eLife</i>. 2021;10. doi:<a href=\"https://doi.org/10.7554/ELIFE.68274\">10.7554/ELIFE.68274</a>","apa":"Bhandari, P., Vandael, D. H., Fernández-Fernández, D., Fritzius, T., Kleindienst, D., Önal, H. C., … Koppensteiner, P. (2021). GABAB receptor auxiliary subunits modulate Cav2.3-mediated release from medial habenula terminals. <i>ELife</i>. eLife Sciences Publications. <a href=\"https://doi.org/10.7554/ELIFE.68274\">https://doi.org/10.7554/ELIFE.68274</a>","mla":"Bhandari, Pradeep, et al. “GABAB Receptor Auxiliary Subunits Modulate Cav2.3-Mediated Release from Medial Habenula Terminals.” <i>ELife</i>, vol. 10, e68274, eLife Sciences Publications, 2021, doi:<a href=\"https://doi.org/10.7554/ELIFE.68274\">10.7554/ELIFE.68274</a>.","ista":"Bhandari P, Vandael DH, Fernández-Fernández D, Fritzius T, Kleindienst D, Önal HC, Montanaro-Punzengruber J-C, Gassmann M, Jonas PM, Kulik A, Bettler B, Shigemoto R, Koppensteiner P. 2021. GABAB receptor auxiliary subunits modulate Cav2.3-mediated release from medial habenula terminals. eLife. 10, e68274.","chicago":"Bhandari, Pradeep, David H Vandael, Diego Fernández-Fernández, Thorsten Fritzius, David Kleindienst, Hüseyin C Önal, Jacqueline-Claire Montanaro-Punzengruber, et al. “GABAB Receptor Auxiliary Subunits Modulate Cav2.3-Mediated Release from Medial Habenula Terminals.” <i>ELife</i>. eLife Sciences Publications, 2021. <a href=\"https://doi.org/10.7554/ELIFE.68274\">https://doi.org/10.7554/ELIFE.68274</a>."},"user_id":"4359f0d1-fa6c-11eb-b949-802e58b17ae8","oa":1,"language":[{"iso":"eng"}],"department":[{"_id":"RySh"},{"_id":"PeJo"}],"file":[{"file_size":8174719,"date_created":"2021-05-31T09:43:09Z","date_updated":"2021-05-31T09:43:09Z","creator":"cziletti","file_id":"9440","success":1,"file_name":"2021_eLife_Bhandari.pdf","access_level":"open_access","content_type":"application/pdf","relation":"main_file","checksum":"6ebcb79999f889766f7cd79ee134ad28"}],"article_number":"e68274","month":"04","quality_controlled":"1","ddc":["570"],"date_updated":"2024-03-25T23:30:16Z","_id":"9437","type":"journal_article","doi":"10.7554/ELIFE.68274","article_processing_charge":"No","publisher":"eLife Sciences Publications","ec_funded":1,"date_published":"2021-04-29T00:00:00Z","acknowledgement":"We are grateful to Akari Hagiwara and Toshihisa Ohtsuka for CAST antibody, and Masahiko Watanabe for neurexin antibody. We thank David Adams for kindly providing the stable Cav2.3 cell line. Cav2.3 KO mice were kindly provided by Tsutomu Tanabe. This project has received funding from the European Research Council (ERC) and European Commission (EC), under the European Union’s Horizon 2020 research and innovation programme (ERC grant agreement no. 694539 to Ryuichi Shigemoto, no. 692692 to Peter Jonas, and the Marie Skłodowska-Curie grant agreement no. 665385 to Cihan Önal), the Swiss National Science Foundation Grant 31003A-172881 to Bernhard Bettler and Deutsche Forschungsgemeinschaft (For 2143) and BIOSS-2 to Akos Kulik.","project":[{"call_identifier":"H2020","grant_number":"694539","name":"In situ analysis of single channel subunit composition in neurons: physiological implication in synaptic plasticity and behaviour","_id":"25CA28EA-B435-11E9-9278-68D0E5697425"},{"_id":"25B7EB9E-B435-11E9-9278-68D0E5697425","name":"Biophysics and circuit function of a giant cortical glumatergic synapse","grant_number":"692692","call_identifier":"H2020"},{"_id":"2564DBCA-B435-11E9-9278-68D0E5697425","grant_number":"665385","name":"International IST Doctoral Program","call_identifier":"H2020"}],"status":"public","publication":"eLife","year":"2021","isi":1,"external_id":{"isi":["000651761700001"]},"related_material":{"record":[{"relation":"dissertation_contains","status":"public","id":"9562"}],"link":[{"url":"https://doi.org/10.1101/2020.04.16.045112","relation":"earlier_version"}]}},{"date_created":"2021-07-11T22:01:16Z","article_type":"original","volume":183,"oa_version":"Published Version","title":"The role of hippocampal mossy cells in novelty detection","day":"30","scopus_import":"1","author":[{"first_name":"Felipe","full_name":"Fredes, Felipe","last_name":"Fredes"},{"last_name":"Shigemoto","full_name":"Shigemoto, Ryuichi","id":"499F3ABC-F248-11E8-B48F-1D18A9856A87","orcid":"0000-0001-8761-9444","first_name":"Ryuichi"}],"file_date_updated":"2021-07-19T13:46:06Z","publication_status":"published","publication_identifier":{"issn":["10747427"],"eissn":["10959564"]},"intvolume":"       183","tmp":{"image":"/images/cc_by_nc_nd.png","name":"Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0)","legal_code_url":"https://creativecommons.org/licenses/by-nc-nd/4.0/legalcode","short":"CC BY-NC-ND (4.0)"},"abstract":[{"lang":"eng","text":"At the encounter with a novel environment, contextual memory formation is greatly enhanced, accompanied with increased arousal and active exploration. Although this phenomenon has been widely observed in animal and human daily life, how the novelty in the environment is detected and contributes to contextual memory formation has lately started to be unveiled. The hippocampus has been studied for many decades for its largely known roles in encoding spatial memory, and a growing body of evidence indicates a differential involvement of dorsal and ventral hippocampal divisions in novelty detection. In this brief review article, we discuss the recent findings of the role of mossy cells in the ventral hippocampal moiety in novelty detection and put them in perspective with other novelty-related pathways in the hippocampus. We propose a mechanism for novelty-driven memory acquisition in the dentate gyrus by the direct projection of ventral mossy cells to dorsal dentate granule cells. By this projection, the ventral hippocampus sends novelty signals to the dorsal hippocampus, opening a gate for memory encoding in dentate granule cells based on information coming from the entorhinal cortex. We conclude that, contrary to the presently accepted functional independence, the dorsal and ventral hippocampi cooperate to link the novelty and contextual information, and this dorso-ventral interaction is crucial for the novelty-dependent memory formation."}],"has_accepted_license":"1","file":[{"date_created":"2021-07-19T13:46:06Z","file_size":1994793,"date_updated":"2021-07-19T13:46:06Z","creator":"cziletti","file_id":"9694","success":1,"file_name":"2021_NeurobLearnMemory_Fredes.pdf","access_level":"open_access","content_type":"application/pdf","relation":"main_file","checksum":"8e8298a9e8c7df146ad23f32c2a63929"}],"article_number":"107486","department":[{"_id":"RySh"}],"month":"06","user_id":"4359f0d1-fa6c-11eb-b949-802e58b17ae8","citation":{"ieee":"F. Fredes and R. Shigemoto, “The role of hippocampal mossy cells in novelty detection,” <i>Neurobiology of Learning and Memory</i>, vol. 183. Elsevier, 2021.","short":"F. Fredes, R. Shigemoto, Neurobiology of Learning and Memory 183 (2021).","ama":"Fredes F, Shigemoto R. The role of hippocampal mossy cells in novelty detection. <i>Neurobiology of Learning and Memory</i>. 2021;183. doi:<a href=\"https://doi.org/10.1016/j.nlm.2021.107486\">10.1016/j.nlm.2021.107486</a>","apa":"Fredes, F., &#38; Shigemoto, R. (2021). The role of hippocampal mossy cells in novelty detection. <i>Neurobiology of Learning and Memory</i>. Elsevier. <a href=\"https://doi.org/10.1016/j.nlm.2021.107486\">https://doi.org/10.1016/j.nlm.2021.107486</a>","mla":"Fredes, Felipe, and Ryuichi Shigemoto. “The Role of Hippocampal Mossy Cells in Novelty Detection.” <i>Neurobiology of Learning and Memory</i>, vol. 183, 107486, Elsevier, 2021, doi:<a href=\"https://doi.org/10.1016/j.nlm.2021.107486\">10.1016/j.nlm.2021.107486</a>.","chicago":"Fredes, Felipe, and Ryuichi Shigemoto. “The Role of Hippocampal Mossy Cells in Novelty Detection.” <i>Neurobiology of Learning and Memory</i>. Elsevier, 2021. <a href=\"https://doi.org/10.1016/j.nlm.2021.107486\">https://doi.org/10.1016/j.nlm.2021.107486</a>.","ista":"Fredes F, Shigemoto R. 2021. The role of hippocampal mossy cells in novelty detection. Neurobiology of Learning and Memory. 183, 107486."},"language":[{"iso":"eng"}],"oa":1,"type":"journal_article","_id":"9641","date_updated":"2023-08-10T14:10:37Z","publisher":"Elsevier","article_processing_charge":"No","doi":"10.1016/j.nlm.2021.107486","quality_controlled":"1","ddc":["610"],"external_id":{"isi":["000677694900004"],"pmid":["34214666"]},"isi":1,"year":"2021","acknowledgement":"This work was supported by a European Research Council Advanced Grant 694539 to Ryuichi Shigemoto.","date_published":"2021-06-30T00:00:00Z","ec_funded":1,"pmid":1,"publication":"Neurobiology of Learning and Memory","status":"public","project":[{"name":"In situ analysis of single channel subunit composition in neurons: physiological implication in synaptic plasticity and behaviour","grant_number":"694539","call_identifier":"H2020","_id":"25CA28EA-B435-11E9-9278-68D0E5697425"}]},{"citation":{"ieee":"W. Kaufmann, D. Kleindienst, H. Harada, and R. Shigemoto, “High-Resolution localization and quantitation of membrane proteins by SDS-digested freeze-fracture replica labeling (SDS-FRL),” in <i> Receptor and Ion Channel Detection in the Brain</i>, vol. 169, New York: Humana, 2021, pp. 267–283.","short":"W. Kaufmann, D. Kleindienst, H. Harada, R. Shigemoto, in:,  Receptor and Ion Channel Detection in the Brain, Humana, New York, 2021, pp. 267–283.","ama":"Kaufmann W, Kleindienst D, Harada H, Shigemoto R. High-Resolution localization and quantitation of membrane proteins by SDS-digested freeze-fracture replica labeling (SDS-FRL). In: <i> Receptor and Ion Channel Detection in the Brain</i>. Vol 169. Neuromethods. New York: Humana; 2021:267-283. doi:<a href=\"https://doi.org/10.1007/978-1-0716-1522-5_19\">10.1007/978-1-0716-1522-5_19</a>","apa":"Kaufmann, W., Kleindienst, D., Harada, H., &#38; Shigemoto, R. (2021). High-Resolution localization and quantitation of membrane proteins by SDS-digested freeze-fracture replica labeling (SDS-FRL). In <i> Receptor and Ion Channel Detection in the Brain</i> (Vol. 169, pp. 267–283). New York: Humana. <a href=\"https://doi.org/10.1007/978-1-0716-1522-5_19\">https://doi.org/10.1007/978-1-0716-1522-5_19</a>","mla":"Kaufmann, Walter, et al. “High-Resolution Localization and Quantitation of Membrane Proteins by SDS-Digested Freeze-Fracture Replica Labeling (SDS-FRL).” <i> Receptor and Ion Channel Detection in the Brain</i>, vol. 169, Humana, 2021, pp. 267–83, doi:<a href=\"https://doi.org/10.1007/978-1-0716-1522-5_19\">10.1007/978-1-0716-1522-5_19</a>.","chicago":"Kaufmann, Walter, David Kleindienst, Harumi Harada, and Ryuichi Shigemoto. “High-Resolution Localization and Quantitation of Membrane Proteins by SDS-Digested Freeze-Fracture Replica Labeling (SDS-FRL).” In <i> Receptor and Ion Channel Detection in the Brain</i>, 169:267–83. Neuromethods. New York: Humana, 2021. <a href=\"https://doi.org/10.1007/978-1-0716-1522-5_19\">https://doi.org/10.1007/978-1-0716-1522-5_19</a>.","ista":"Kaufmann W, Kleindienst D, Harada H, Shigemoto R. 2021.High-Resolution localization and quantitation of membrane proteins by SDS-digested freeze-fracture replica labeling (SDS-FRL). In:  Receptor and Ion Channel Detection in the Brain. Neuromethods, vol. 169, 267–283."},"place":"New York","user_id":"D865714E-FA4E-11E9-B85B-F5C5E5697425","language":[{"iso":"eng"}],"department":[{"_id":"RySh"},{"_id":"EM-Fac"}],"month":"07","publication_status":"published","publication_identifier":{"eisbn":["9781071615225"],"isbn":["9781071615218"]},"has_accepted_license":"1","intvolume":"       169","abstract":[{"text":"High-resolution visualization and quantification of membrane proteins contribute to the understanding of their functions and the roles they play in physiological and pathological conditions. Sodium dodecyl sulfate-digested freeze-fracture replica labeling (SDS-FRL) is a powerful electron microscopy method to study quantitatively the two-dimensional distribution of transmembrane proteins and their tightly associated proteins. During treatment with SDS, intracellular organelles and proteins not anchored to the replica are dissolved, whereas integral membrane proteins captured and stabilized by carbon/platinum deposition remain on the replica. Their intra- and extracellular domains become exposed on the surface of the replica, facilitating the accessibility of antibodies and, therefore, providing higher labeling efficiency than those obtained with other immunoelectron microscopy techniques. In this chapter, we describe the protocols of SDS-FRL adapted for mammalian brain samples, and optimization of the SDS treatment to increase the labeling efficiency for quantification of Cav2.1, the alpha subunit of P/Q-type voltage-dependent calcium channels utilizing deep learning algorithms.","lang":"eng"}],"volume":169,"date_created":"2021-07-30T09:34:56Z","author":[{"orcid":"0000-0001-9735-5315","first_name":"Walter","last_name":"Kaufmann","full_name":"Kaufmann, Walter","id":"3F99E422-F248-11E8-B48F-1D18A9856A87"},{"last_name":"Kleindienst","id":"42E121A4-F248-11E8-B48F-1D18A9856A87","full_name":"Kleindienst, David","first_name":"David"},{"last_name":"Harada","full_name":"Harada, Harumi","id":"2E55CDF2-F248-11E8-B48F-1D18A9856A87","orcid":"0000-0001-7429-7896","first_name":"Harumi"},{"full_name":"Shigemoto, Ryuichi","id":"499F3ABC-F248-11E8-B48F-1D18A9856A87","last_name":"Shigemoto","first_name":"Ryuichi","orcid":"0000-0001-8761-9444"}],"day":"27","title":"High-Resolution localization and quantitation of membrane proteins by SDS-digested freeze-fracture replica labeling (SDS-FRL)","oa_version":"None","ec_funded":1,"date_published":"2021-07-27T00:00:00Z","acknowledgement":"This work was supported by the European Union (European Research Council Advanced grant no. 694539 and Human Brain Project Ref. 720270 to R. S.) and the Austrian Academy of Sciences (DOC fellowship to D.K.).","project":[{"_id":"25CA28EA-B435-11E9-9278-68D0E5697425","name":"In situ analysis of single channel subunit composition in neurons: physiological implication in synaptic plasticity and behaviour","grant_number":"694539","call_identifier":"H2020"},{"call_identifier":"H2020","grant_number":"720270","name":"Human Brain Project Specific Grant Agreement 1 (HBP SGA 1)","_id":"25CBA828-B435-11E9-9278-68D0E5697425"}],"publication":" Receptor and Ion Channel Detection in the Brain","status":"public","keyword":["Freeze-fracture replica: Deep learning","Immunogold labeling","Integral membrane protein","Electron microscopy"],"year":"2021","related_material":{"record":[{"id":"9562","relation":"dissertation_contains","status":"public"}]},"quality_controlled":"1","page":"267-283","ddc":["573"],"date_updated":"2024-03-25T23:30:16Z","_id":"9756","type":"book_chapter","series_title":"Neuromethods","doi":"10.1007/978-1-0716-1522-5_19","article_processing_charge":"No","alternative_title":["Neuromethods"],"publisher":"Humana"},{"day":"14","scopus_import":"1","author":[{"first_name":"David","id":"42E121A4-F248-11E8-B48F-1D18A9856A87","full_name":"Kleindienst, David","last_name":"Kleindienst"},{"last_name":"Montanaro-Punzengruber","id":"3786AB44-F248-11E8-B48F-1D18A9856A87","full_name":"Montanaro-Punzengruber, Jacqueline-Claire","first_name":"Jacqueline-Claire"},{"orcid":"0000-0003-0863-4481","first_name":"Pradeep","full_name":"Bhandari, Pradeep","id":"45EDD1BC-F248-11E8-B48F-1D18A9856A87","last_name":"Bhandari"},{"id":"44B7CA5A-F248-11E8-B48F-1D18A9856A87","full_name":"Case, Matthew J","last_name":"Case","first_name":"Matthew J"},{"full_name":"Fukazawa, Yugo","last_name":"Fukazawa","first_name":"Yugo"},{"orcid":"0000-0001-8761-9444","first_name":"Ryuichi","last_name":"Shigemoto","id":"499F3ABC-F248-11E8-B48F-1D18A9856A87","full_name":"Shigemoto, Ryuichi"}],"oa_version":"Published Version","title":"Deep learning-assisted high-throughput analysis of freeze-fracture replica images applied to glutamate receptors and calcium channels at hippocampal synapses","volume":21,"date_created":"2020-09-20T22:01:35Z","article_type":"original","has_accepted_license":"1","tmp":{"name":"Creative Commons Attribution 4.0 International Public License (CC-BY 4.0)","legal_code_url":"https://creativecommons.org/licenses/by/4.0/legalcode","image":"/images/cc_by.png","short":"CC BY (4.0)"},"abstract":[{"text":"The molecular anatomy of synapses defines their characteristics in transmission and plasticity. Precise measurements of the number and distribution of synaptic proteins are important for our understanding of synapse heterogeneity within and between brain regions. Freeze–fracture replica immunogold electron microscopy enables us to analyze them quantitatively on a two-dimensional membrane surface. Here, we introduce Darea software, which utilizes deep learning for analysis of replica images and demonstrate its usefulness for quick measurements of the pre- and postsynaptic areas, density and distribution of gold particles at synapses in a reproducible manner. We used Darea for comparing glutamate receptor and calcium channel distributions between hippocampal CA3-CA1 spine synapses on apical and basal dendrites, which differ in signaling pathways involved in synaptic plasticity. We found that apical synapses express a higher density of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors and a stronger increase of AMPA receptors with synaptic size, while basal synapses show a larger increase in N-methyl-D-aspartate (NMDA) receptors with size. Interestingly, AMPA and NMDA receptors are segregated within postsynaptic sites and negatively correlated in density among both apical and basal synapses. In the presynaptic sites, Cav2.1 voltage-gated calcium channels show similar densities in apical and basal synapses with distributions consistent with an exclusion zone model of calcium channel-release site topography.","lang":"eng"}],"intvolume":"        21","file_date_updated":"2020-09-21T14:08:58Z","publication_identifier":{"issn":["16616596"],"eissn":["14220067"]},"publication_status":"published","month":"09","department":[{"_id":"RySh"}],"file":[{"success":1,"file_name":"2020_JournMolecSciences_Kleindienst.pdf","access_level":"open_access","content_type":"application/pdf","relation":"main_file","checksum":"2e4f62f3cfe945b7391fc3070e5a289f","date_created":"2020-09-21T14:08:58Z","file_size":5748456,"date_updated":"2020-09-21T14:08:58Z","creator":"dernst","file_id":"8551"}],"article_number":"6737","oa":1,"language":[{"iso":"eng"}],"citation":{"ama":"Kleindienst D, Montanaro-Punzengruber J-C, Bhandari P, Case MJ, Fukazawa Y, Shigemoto R. Deep learning-assisted high-throughput analysis of freeze-fracture replica images applied to glutamate receptors and calcium channels at hippocampal synapses. <i>International Journal of Molecular Sciences</i>. 2020;21(18). doi:<a href=\"https://doi.org/10.3390/ijms21186737\">10.3390/ijms21186737</a>","short":"D. Kleindienst, J.-C. Montanaro-Punzengruber, P. Bhandari, M.J. Case, Y. Fukazawa, R. Shigemoto, International Journal of Molecular Sciences 21 (2020).","ieee":"D. Kleindienst, J.-C. Montanaro-Punzengruber, P. Bhandari, M. J. Case, Y. Fukazawa, and R. Shigemoto, “Deep learning-assisted high-throughput analysis of freeze-fracture replica images applied to glutamate receptors and calcium channels at hippocampal synapses,” <i>International Journal of Molecular Sciences</i>, vol. 21, no. 18. MDPI, 2020.","chicago":"Kleindienst, David, Jacqueline-Claire Montanaro-Punzengruber, Pradeep Bhandari, Matthew J Case, Yugo Fukazawa, and Ryuichi Shigemoto. “Deep Learning-Assisted High-Throughput Analysis of Freeze-Fracture Replica Images Applied to Glutamate Receptors and Calcium Channels at Hippocampal Synapses.” <i>International Journal of Molecular Sciences</i>. MDPI, 2020. <a href=\"https://doi.org/10.3390/ijms21186737\">https://doi.org/10.3390/ijms21186737</a>.","ista":"Kleindienst D, Montanaro-Punzengruber J-C, Bhandari P, Case MJ, Fukazawa Y, Shigemoto R. 2020. Deep learning-assisted high-throughput analysis of freeze-fracture replica images applied to glutamate receptors and calcium channels at hippocampal synapses. International Journal of Molecular Sciences. 21(18), 6737.","mla":"Kleindienst, David, et al. “Deep Learning-Assisted High-Throughput Analysis of Freeze-Fracture Replica Images Applied to Glutamate Receptors and Calcium Channels at Hippocampal Synapses.” <i>International Journal of Molecular Sciences</i>, vol. 21, no. 18, 6737, MDPI, 2020, doi:<a href=\"https://doi.org/10.3390/ijms21186737\">10.3390/ijms21186737</a>.","apa":"Kleindienst, D., Montanaro-Punzengruber, J.-C., Bhandari, P., Case, M. J., Fukazawa, Y., &#38; Shigemoto, R. (2020). Deep learning-assisted high-throughput analysis of freeze-fracture replica images applied to glutamate receptors and calcium channels at hippocampal synapses. <i>International Journal of Molecular Sciences</i>. MDPI. <a href=\"https://doi.org/10.3390/ijms21186737\">https://doi.org/10.3390/ijms21186737</a>"},"issue":"18","user_id":"4359f0d1-fa6c-11eb-b949-802e58b17ae8","article_processing_charge":"No","doi":"10.3390/ijms21186737","publisher":"MDPI","_id":"8532","date_updated":"2024-03-25T23:30:16Z","type":"journal_article","ddc":["570"],"quality_controlled":"1","isi":1,"year":"2020","related_material":{"record":[{"relation":"dissertation_contains","status":"public","id":"9562"}]},"external_id":{"isi":["000579945300001"]},"status":"public","publication":"International Journal of Molecular Sciences","project":[{"call_identifier":"H2020","name":"In situ analysis of single channel subunit composition in neurons: physiological implication in synaptic plasticity and behaviour","grant_number":"694539","_id":"25CA28EA-B435-11E9-9278-68D0E5697425"},{"_id":"25D32BC0-B435-11E9-9278-68D0E5697425","name":"Mechanism of formation and maintenance of input side-dependent asymmetry in the hippocampus"},{"grant_number":"785907","name":"Human Brain Project Specific Grant Agreement 2 (HBP SGA 2)","call_identifier":"H2020","_id":"26436750-B435-11E9-9278-68D0E5697425"}],"ec_funded":1,"date_published":"2020-09-14T00:00:00Z","acknowledgement":"This research was funded by Austrian Academy of Sciences, DOC fellowship to D.K., European Research\r\nCouncil Advanced Grant 694539 and European Union Human Brain Project (HBP) SGA2 785907 to R.S.\r\nWe acknowledge Elena Hollergschwandtner for technical support."},{"file":[{"date_updated":"2020-07-14T12:48:01Z","creator":"dernst","file_size":9227283,"date_created":"2020-04-20T10:59:49Z","file_id":"7668","content_type":"application/pdf","access_level":"open_access","file_name":"2020_FrontiersCellularNeurosc_Eguchi.pdf","checksum":"1c145123c6f8dc3e2e4bd5a66a1ad60e","relation":"main_file"}],"article_number":"63","department":[{"_id":"JoDa"},{"_id":"RySh"}],"month":"03","user_id":"4359f0d1-fa6c-11eb-b949-802e58b17ae8","citation":{"short":"K. Eguchi, P. Velicky, E. Saeckl, M. Itakura, Y. Fukazawa, J.G. Danzl, R. Shigemoto, Frontiers in Cellular Neuroscience 14 (2020).","ieee":"K. Eguchi <i>et al.</i>, “Advantages of acute brain slices prepared at physiological temperature in the characterization of synaptic functions,” <i>Frontiers in Cellular Neuroscience</i>, vol. 14. Frontiers Media, 2020.","ama":"Eguchi K, Velicky P, Saeckl E, et al. Advantages of acute brain slices prepared at physiological temperature in the characterization of synaptic functions. <i>Frontiers in Cellular Neuroscience</i>. 2020;14. doi:<a href=\"https://doi.org/10.3389/fncel.2020.00063\">10.3389/fncel.2020.00063</a>","mla":"Eguchi, Kohgaku, et al. “Advantages of Acute Brain Slices Prepared at Physiological Temperature in the Characterization of Synaptic Functions.” <i>Frontiers in Cellular Neuroscience</i>, vol. 14, 63, Frontiers Media, 2020, doi:<a href=\"https://doi.org/10.3389/fncel.2020.00063\">10.3389/fncel.2020.00063</a>.","apa":"Eguchi, K., Velicky, P., Saeckl, E., Itakura, M., Fukazawa, Y., Danzl, J. G., &#38; Shigemoto, R. (2020). Advantages of acute brain slices prepared at physiological temperature in the characterization of synaptic functions. <i>Frontiers in Cellular Neuroscience</i>. Frontiers Media. <a href=\"https://doi.org/10.3389/fncel.2020.00063\">https://doi.org/10.3389/fncel.2020.00063</a>","ista":"Eguchi K, Velicky P, Saeckl E, Itakura M, Fukazawa Y, Danzl JG, Shigemoto R. 2020. Advantages of acute brain slices prepared at physiological temperature in the characterization of synaptic functions. Frontiers in Cellular Neuroscience. 14, 63.","chicago":"Eguchi, Kohgaku, Philipp Velicky, Elena Saeckl, Makoto Itakura, Yugo Fukazawa, Johann G Danzl, and Ryuichi Shigemoto. “Advantages of Acute Brain Slices Prepared at Physiological Temperature in the Characterization of Synaptic Functions.” <i>Frontiers in Cellular Neuroscience</i>. Frontiers Media, 2020. <a href=\"https://doi.org/10.3389/fncel.2020.00063\">https://doi.org/10.3389/fncel.2020.00063</a>."},"language":[{"iso":"eng"}],"oa":1,"date_created":"2020-04-19T22:00:55Z","article_type":"original","volume":14,"oa_version":"Published Version","title":"Advantages of acute brain slices prepared at physiological temperature in the characterization of synaptic functions","day":"19","scopus_import":"1","author":[{"last_name":"Eguchi","full_name":"Eguchi, Kohgaku","id":"2B7846DC-F248-11E8-B48F-1D18A9856A87","first_name":"Kohgaku","orcid":"0000-0002-6170-2546"},{"last_name":"Velicky","id":"39BDC62C-F248-11E8-B48F-1D18A9856A87","full_name":"Velicky, Philipp","orcid":"0000-0002-2340-7431","first_name":"Philipp"},{"id":"3C054040-F248-11E8-B48F-1D18A9856A87","full_name":"Hollergschwandtner, Elena","last_name":"Hollergschwandtner","first_name":"Elena"},{"full_name":"Itakura, Makoto","last_name":"Itakura","first_name":"Makoto"},{"first_name":"Yugo","last_name":"Fukazawa","full_name":"Fukazawa, Yugo"},{"first_name":"Johann G","orcid":"0000-0001-8559-3973","last_name":"Danzl","id":"42EFD3B6-F248-11E8-B48F-1D18A9856A87","full_name":"Danzl, Johann G"},{"first_name":"Ryuichi","orcid":"0000-0001-8761-9444","last_name":"Shigemoto","id":"499F3ABC-F248-11E8-B48F-1D18A9856A87","full_name":"Shigemoto, Ryuichi"}],"file_date_updated":"2020-07-14T12:48:01Z","publication_status":"published","publication_identifier":{"issn":["16625102"]},"abstract":[{"lang":"eng","text":"Acute brain slice preparation is a powerful experimental model for investigating the characteristics of synaptic function in the brain. Although brain tissue is usually cut at ice-cold temperature (CT) to facilitate slicing and avoid neuronal damage, exposure to CT causes molecular and architectural changes of synapses. To address these issues, we investigated ultrastructural and electrophysiological features of synapses in mouse acute cerebellar slices prepared at ice-cold and physiological temperature (PT). In the slices prepared at CT, we found significant spine loss and reconstruction, synaptic vesicle rearrangement and decrease in synaptic proteins, all of which were not detected in slices prepared at PT. Consistent with these structural findings, slices prepared at PT showed higher release probability. Furthermore, preparation at PT allows electrophysiological recording immediately after slicing resulting in higher detectability of long-term depression (LTD) after motor learning compared with that at CT. These results indicate substantial advantages of the slice preparation at PT for investigating synaptic functions in different physiological conditions."}],"tmp":{"name":"Creative Commons Attribution 4.0 International Public License (CC-BY 4.0)","legal_code_url":"https://creativecommons.org/licenses/by/4.0/legalcode","image":"/images/cc_by.png","short":"CC BY (4.0)"},"intvolume":"        14","has_accepted_license":"1","external_id":{"isi":["000525582200001"]},"isi":1,"year":"2020","date_published":"2020-03-19T00:00:00Z","ec_funded":1,"publication":"Frontiers in Cellular Neuroscience","status":"public","project":[{"_id":"2659CC84-B435-11E9-9278-68D0E5697425","name":"Ultrastructural analysis of phosphoinositides in nerve terminals: distribution, dynamics and physiological roles in synaptic transmission","grant_number":"793482","call_identifier":"H2020"},{"_id":"25CA28EA-B435-11E9-9278-68D0E5697425","call_identifier":"H2020","name":"In situ analysis of single channel subunit composition in neurons: physiological implication in synaptic plasticity and behaviour","grant_number":"694539"},{"_id":"265CB4D0-B435-11E9-9278-68D0E5697425","grant_number":"I03600","name":"Optical control of synaptic function via adhesion molecules","call_identifier":"FWF"},{"name":"IST Austria Open Access Fund","_id":"B67AFEDC-15C9-11EA-A837-991A96BB2854"}],"type":"journal_article","_id":"7665","date_updated":"2023-08-21T06:12:48Z","publisher":"Frontiers Media","article_processing_charge":"Yes (via OA deal)","doi":"10.3389/fncel.2020.00063","quality_controlled":"1","ddc":["570"]},{"page":"995-1000","ddc":["570"],"quality_controlled":"1","article_processing_charge":"No","doi":"10.1246/bcsj.20190034","publisher":"Bulletin of the Chemical Society of Japan","_id":"6659","date_updated":"2021-01-12T08:08:26Z","type":"journal_article","publication":"Bulletin of the Chemical Society of Japan","status":"public","project":[{"_id":"25CA28EA-B435-11E9-9278-68D0E5697425","call_identifier":"H2020","name":"In situ analysis of single channel subunit composition in neurons: physiological implication in synaptic plasticity and behaviour","grant_number":"694539"}],"ec_funded":1,"date_published":"2019-05-15T00:00:00Z","acknowledgement":"his work was supported by the Grant-in-Aid for Scientific Research B (JSPS KAKENHI grant no. JP17H03090 to A. O.); the Scientific Research on Innovative Areas “Chemistry for Multimolecular Crowding Biosystems” (JSPS KAKENHI grant no. JP17H06349 to A. O.); and the European Union (European Research Council Advanced grant no. 694539 and Human Brain Project Ref. 720270 to R. S.). A. O. acknowledges the financial support of the Takeda Science Foundation.","year":"2019","has_accepted_license":"1","intvolume":"        92","abstract":[{"lang":"eng","text":"Chemical labeling of proteins with synthetic molecular probes offers the possibility to probe the functions of proteins of interest in living cells. However, the methods for covalently labeling targeted proteins using complementary peptide tag-probe pairs are still limited, irrespective of the versatility of such pairs in biological research. Herein, we report the new CysHis tag-Ni(II) probe pair for the specific covalent labeling of proteins. A broad-range evaluation of the reactivity profiles of the probe and the CysHis peptide tag afforded a tag-probe pair with an optimized and high labeling selectivity and reactivity. In particular, the labeling specificity of this pair was notably improved compared to the previously reported one. This pair was successfully utilized for the fluorescence imaging of membrane proteins on the surfaces of living cells, demonstrating its potential utility in biological research."}],"file_date_updated":"2020-10-02T08:49:58Z","publication_status":"published","publication_identifier":{"issn":["00092673"]},"day":"15","scopus_import":"1","author":[{"last_name":"Zenmyo","full_name":"Zenmyo, Naoki","first_name":"Naoki"},{"first_name":"Hiroki","last_name":"Tokumaru","full_name":"Tokumaru, Hiroki"},{"first_name":"Shohei","last_name":"Uchinomiya","full_name":"Uchinomiya, Shohei"},{"first_name":"Hirokazu","full_name":"Fuchida, Hirokazu","last_name":"Fuchida"},{"id":"4427179E-F248-11E8-B48F-1D18A9856A87","full_name":"Tabata, Shigekazu","last_name":"Tabata","first_name":"Shigekazu"},{"first_name":"Itaru","full_name":"Hamachi, Itaru","last_name":"Hamachi"},{"last_name":"Shigemoto","full_name":"Shigemoto, Ryuichi","id":"499F3ABC-F248-11E8-B48F-1D18A9856A87","orcid":"0000-0001-8761-9444","first_name":"Ryuichi"},{"full_name":"Ojida, Akio","last_name":"Ojida","first_name":"Akio"}],"oa_version":"Published Version","title":"Optimized reaction pair of the CysHis tag and Ni(II)-NTA probe for highly selective chemical labeling of membrane proteins","volume":92,"date_created":"2019-07-21T21:59:16Z","article_type":"original","oa":1,"language":[{"iso":"eng"}],"citation":{"ista":"Zenmyo N, Tokumaru H, Uchinomiya S, Fuchida H, Tabata S, Hamachi I, Shigemoto R, Ojida A. 2019. Optimized reaction pair of the CysHis tag and Ni(II)-NTA probe for highly selective chemical labeling of membrane proteins. Bulletin of the Chemical Society of Japan. 92(5), 995–1000.","chicago":"Zenmyo, Naoki, Hiroki Tokumaru, Shohei Uchinomiya, Hirokazu Fuchida, Shigekazu Tabata, Itaru Hamachi, Ryuichi Shigemoto, and Akio Ojida. “Optimized Reaction Pair of the CysHis Tag and Ni(II)-NTA Probe for Highly Selective Chemical Labeling of Membrane Proteins.” <i>Bulletin of the Chemical Society of Japan</i>. Bulletin of the Chemical Society of Japan, 2019. <a href=\"https://doi.org/10.1246/bcsj.20190034\">https://doi.org/10.1246/bcsj.20190034</a>.","mla":"Zenmyo, Naoki, et al. “Optimized Reaction Pair of the CysHis Tag and Ni(II)-NTA Probe for Highly Selective Chemical Labeling of Membrane Proteins.” <i>Bulletin of the Chemical Society of Japan</i>, vol. 92, no. 5, Bulletin of the Chemical Society of Japan, 2019, pp. 995–1000, doi:<a href=\"https://doi.org/10.1246/bcsj.20190034\">10.1246/bcsj.20190034</a>.","apa":"Zenmyo, N., Tokumaru, H., Uchinomiya, S., Fuchida, H., Tabata, S., Hamachi, I., … Ojida, A. (2019). Optimized reaction pair of the CysHis tag and Ni(II)-NTA probe for highly selective chemical labeling of membrane proteins. <i>Bulletin of the Chemical Society of Japan</i>. Bulletin of the Chemical Society of Japan. <a href=\"https://doi.org/10.1246/bcsj.20190034\">https://doi.org/10.1246/bcsj.20190034</a>","ama":"Zenmyo N, Tokumaru H, Uchinomiya S, et al. Optimized reaction pair of the CysHis tag and Ni(II)-NTA probe for highly selective chemical labeling of membrane proteins. <i>Bulletin of the Chemical Society of Japan</i>. 2019;92(5):995-1000. doi:<a href=\"https://doi.org/10.1246/bcsj.20190034\">10.1246/bcsj.20190034</a>","short":"N. Zenmyo, H. Tokumaru, S. Uchinomiya, H. Fuchida, S. Tabata, I. Hamachi, R. Shigemoto, A. Ojida, Bulletin of the Chemical Society of Japan 92 (2019) 995–1000.","ieee":"N. Zenmyo <i>et al.</i>, “Optimized reaction pair of the CysHis tag and Ni(II)-NTA probe for highly selective chemical labeling of membrane proteins,” <i>Bulletin of the Chemical Society of Japan</i>, vol. 92, no. 5. Bulletin of the Chemical Society of Japan, pp. 995–1000, 2019."},"issue":"5","user_id":"2DF688A6-F248-11E8-B48F-1D18A9856A87","month":"05","department":[{"_id":"RySh"}],"file":[{"checksum":"186de511d6e0ca93f5d981e2443eb8cd","relation":"main_file","access_level":"open_access","content_type":"application/pdf","success":1,"file_name":"2019_BCSJ_Zenmyo.pdf","file_id":"8594","creator":"dernst","date_updated":"2020-10-02T08:49:58Z","file_size":2464903,"date_created":"2020-10-02T08:49:58Z"}]},{"page":"256-268","ddc":["570"],"quality_controlled":"1","article_processing_charge":"No","doi":"10.1016/j.isci.2019.11.025","publisher":"Elsevier","_id":"7391","date_updated":"2024-03-25T23:30:07Z","type":"journal_article","status":"public","publication":"iScience","project":[{"_id":"25CA28EA-B435-11E9-9278-68D0E5697425","call_identifier":"H2020","grant_number":"694539","name":"In situ analysis of single channel subunit composition in neurons: physiological implication in synaptic plasticity and behaviour"},{"grant_number":"720270","name":"Human Brain Project Specific Grant Agreement 1 (HBP SGA 1)","call_identifier":"H2020","_id":"25CBA828-B435-11E9-9278-68D0E5697425"}],"ec_funded":1,"pmid":1,"date_published":"2019-12-20T00:00:00Z","year":"2019","related_material":{"record":[{"relation":"dissertation_contains","status":"public","id":"11393"}]},"external_id":{"pmid":["31786521"],"isi":[":000504652000020"]},"has_accepted_license":"1","abstract":[{"lang":"eng","text":"Electron microscopy (EM) is a technology that enables visualization of single proteins at a nanometer resolution. However, current protein analysis by EM mainly relies on immunolabeling with gold-particle-conjugated antibodies, which is compromised by large size of antibody, precluding precise detection of protein location in biological samples. Here, we develop a specific chemical labeling method for EM detection of proteins at single-molecular level. Rational design of α-helical peptide tag and probe structure provided a complementary reaction pair that enabled specific cysteine conjugation of the tag. The developed chemical labeling with gold-nanoparticle-conjugated probe showed significantly higher labeling efficiency and detectability of high-density clusters of tag-fused G protein-coupled receptors in freeze-fracture replicas compared with immunogold labeling. Furthermore, in ultrathin sections, the spatial resolution of the chemical labeling was significantly higher than that of antibody-mediated labeling. These results demonstrate substantial advantages of the chemical labeling approach for single protein visualization by EM."}],"tmp":{"name":"Creative Commons Attribution 4.0 International Public License (CC-BY 4.0)","legal_code_url":"https://creativecommons.org/licenses/by/4.0/legalcode","image":"/images/cc_by.png","short":"CC BY (4.0)"},"intvolume":"        22","file_date_updated":"2020-07-14T12:47:57Z","publication_status":"published","publication_identifier":{"issn":["2589-0042"]},"day":"20","scopus_import":"1","author":[{"last_name":"Tabata","full_name":"Tabata, Shigekazu","id":"4427179E-F248-11E8-B48F-1D18A9856A87","first_name":"Shigekazu"},{"id":"4BE3BC94-F248-11E8-B48F-1D18A9856A87","full_name":"Jevtic, Marijo","last_name":"Jevtic","first_name":"Marijo"},{"first_name":"Nobutaka","full_name":"Kurashige, Nobutaka","last_name":"Kurashige"},{"last_name":"Fuchida","full_name":"Fuchida, Hirokazu","first_name":"Hirokazu"},{"first_name":"Munetsugu","full_name":"Kido, Munetsugu","last_name":"Kido"},{"first_name":"Kazushi","last_name":"Tani","full_name":"Tani, Kazushi"},{"first_name":"Naoki","full_name":"Zenmyo, Naoki","last_name":"Zenmyo"},{"first_name":"Shohei","last_name":"Uchinomiya","full_name":"Uchinomiya, Shohei"},{"last_name":"Harada","id":"2E55CDF2-F248-11E8-B48F-1D18A9856A87","full_name":"Harada, Harumi","orcid":"0000-0001-7429-7896","first_name":"Harumi"},{"full_name":"Itakura, Makoto","last_name":"Itakura","first_name":"Makoto"},{"last_name":"Hamachi","full_name":"Hamachi, Itaru","first_name":"Itaru"},{"orcid":"0000-0001-8761-9444","first_name":"Ryuichi","id":"499F3ABC-F248-11E8-B48F-1D18A9856A87","full_name":"Shigemoto, Ryuichi","last_name":"Shigemoto"},{"first_name":"Akio","full_name":"Ojida, Akio","last_name":"Ojida"}],"oa_version":"Published Version","title":"Electron microscopic detection of single membrane proteins by a specific chemical labeling","volume":22,"date_created":"2020-01-29T15:56:56Z","article_type":"original","oa":1,"language":[{"iso":"eng"}],"citation":{"ista":"Tabata S, Jevtic M, Kurashige N, Fuchida H, Kido M, Tani K, Zenmyo N, Uchinomiya S, Harada H, Itakura M, Hamachi I, Shigemoto R, Ojida A. 2019. Electron microscopic detection of single membrane proteins by a specific chemical labeling. iScience. 22(12), 256–268.","chicago":"Tabata, Shigekazu, Marijo Jevtic, Nobutaka Kurashige, Hirokazu Fuchida, Munetsugu Kido, Kazushi Tani, Naoki Zenmyo, et al. “Electron Microscopic Detection of Single Membrane Proteins by a Specific Chemical Labeling.” <i>IScience</i>. Elsevier, 2019. <a href=\"https://doi.org/10.1016/j.isci.2019.11.025\">https://doi.org/10.1016/j.isci.2019.11.025</a>.","mla":"Tabata, Shigekazu, et al. “Electron Microscopic Detection of Single Membrane Proteins by a Specific Chemical Labeling.” <i>IScience</i>, vol. 22, no. 12, Elsevier, 2019, pp. 256–68, doi:<a href=\"https://doi.org/10.1016/j.isci.2019.11.025\">10.1016/j.isci.2019.11.025</a>.","apa":"Tabata, S., Jevtic, M., Kurashige, N., Fuchida, H., Kido, M., Tani, K., … Ojida, A. (2019). Electron microscopic detection of single membrane proteins by a specific chemical labeling. <i>IScience</i>. Elsevier. <a href=\"https://doi.org/10.1016/j.isci.2019.11.025\">https://doi.org/10.1016/j.isci.2019.11.025</a>","ama":"Tabata S, Jevtic M, Kurashige N, et al. Electron microscopic detection of single membrane proteins by a specific chemical labeling. <i>iScience</i>. 2019;22(12):256-268. doi:<a href=\"https://doi.org/10.1016/j.isci.2019.11.025\">10.1016/j.isci.2019.11.025</a>","short":"S. Tabata, M. Jevtic, N. Kurashige, H. Fuchida, M. Kido, K. Tani, N. Zenmyo, S. Uchinomiya, H. Harada, M. Itakura, I. Hamachi, R. Shigemoto, A. Ojida, IScience 22 (2019) 256–268.","ieee":"S. Tabata <i>et al.</i>, “Electron microscopic detection of single membrane proteins by a specific chemical labeling,” <i>iScience</i>, vol. 22, no. 12. Elsevier, pp. 256–268, 2019."},"issue":"12","user_id":"c635000d-4b10-11ee-a964-aac5a93f6ac1","month":"12","department":[{"_id":"RySh"}],"file":[{"access_level":"open_access","content_type":"application/pdf","file_name":"2019_iScience_Tabata.pdf","checksum":"f3e90056a49f09b205b1c4f8c739ffd1","relation":"main_file","date_updated":"2020-07-14T12:47:57Z","creator":"dernst","date_created":"2020-02-04T10:48:36Z","file_size":7197776,"file_id":"7448"}]}]