[{"year":"2020","publisher":"eLife Sciences Publications","publication_status":"published","author":[{"full_name":"Fribourgh, Jennifer L","last_name":"Fribourgh","first_name":"Jennifer L"},{"first_name":"Ashutosh","last_name":"Srivastava","full_name":"Srivastava, Ashutosh"},{"full_name":"Sandate, Colby R","last_name":"Sandate","first_name":"Colby R"},{"full_name":"Michael, Alicia Kathleen","last_name":"Michael","first_name":"Alicia Kathleen","id":"6437c950-2a03-11ee-914d-d6476dd7b75c"},{"first_name":"Peter L","last_name":"Hsu","full_name":"Hsu, Peter L"},{"last_name":"Rakers","first_name":"Christin","full_name":"Rakers, Christin"},{"full_name":"Nguyen, Leslee T","last_name":"Nguyen","first_name":"Leslee T"},{"last_name":"Torgrimson","first_name":"Megan R","full_name":"Torgrimson, Megan R"},{"full_name":"Parico, Gian Carlo G","last_name":"Parico","first_name":"Gian Carlo G"},{"full_name":"Tripathi, Sarvind","last_name":"Tripathi","first_name":"Sarvind"},{"full_name":"Zheng, Ning","first_name":"Ning","last_name":"Zheng"},{"last_name":"Lander","first_name":"Gabriel C","full_name":"Lander, Gabriel C"},{"full_name":"Hirota, Tsuyoshi","first_name":"Tsuyoshi","last_name":"Hirota"},{"first_name":"Florence","last_name":"Tama","full_name":"Tama, Florence"},{"full_name":"Partch, Carrie L","last_name":"Partch","first_name":"Carrie L"}],"volume":9,"date_created":"2024-03-21T07:55:12Z","date_updated":"2024-03-25T12:25:02Z","article_number":"55275","extern":"1","main_file_link":[{"open_access":"1","url":"https://doi.org/10.7554/eLife.55275"}],"oa":1,"quality_controlled":"1","doi":"10.7554/elife.55275","language":[{"iso":"eng"}],"publication_identifier":{"issn":["2050-084X"]},"month":"02","_id":"15153","user_id":"2DF688A6-F248-11E8-B48F-1D18A9856A87","intvolume":" 9","status":"public","title":"Dynamics at the serine loop underlie differential affinity of cryptochromes for CLOCK:BMAL1 to control circadian timing","oa_version":"Published Version","type":"journal_article","abstract":[{"text":"Mammalian circadian rhythms are generated by a transcription-based feedback loop in which CLOCK:BMAL1 drives transcription of its repressors (PER1/2, CRY1/2), which ultimately interact with CLOCK:BMAL1 to close the feedback loop with ~24 hr periodicity. Here we pinpoint a key difference between CRY1 and CRY2 that underlies their differential strengths as transcriptional repressors. Both cryptochromes bind the BMAL1 transactivation domain similarly to sequester it from coactivators and repress CLOCK:BMAL1 activity. However, we find that CRY1 is recruited with much higher affinity to the PAS domain core of CLOCK:BMAL1, allowing it to serve as a stronger repressor that lengthens circadian period. We discovered a dynamic serine-rich loop adjacent to the secondary pocket in the photolyase homology region (PHR) domain that regulates differential binding of cryptochromes to the PAS domain core of CLOCK:BMAL1. Notably, binding of the co-repressor PER2 remodels the serine loop of CRY2, making it more CRY1-like and enhancing its affinity for CLOCK:BMAL1.","lang":"eng"}],"citation":{"chicago":"Fribourgh, Jennifer L, Ashutosh Srivastava, Colby R Sandate, Alicia K. Michael, Peter L Hsu, Christin Rakers, Leslee T Nguyen, et al. “Dynamics at the Serine Loop Underlie Differential Affinity of Cryptochromes for CLOCK:BMAL1 to Control Circadian Timing.” ELife. eLife Sciences Publications, 2020. https://doi.org/10.7554/elife.55275.","mla":"Fribourgh, Jennifer L., et al. “Dynamics at the Serine Loop Underlie Differential Affinity of Cryptochromes for CLOCK:BMAL1 to Control Circadian Timing.” ELife, vol. 9, 55275, eLife Sciences Publications, 2020, doi:10.7554/elife.55275.","short":"J.L. Fribourgh, A. Srivastava, C.R. Sandate, A.K. Michael, P.L. Hsu, C. Rakers, L.T. Nguyen, M.R. Torgrimson, G.C.G. Parico, S. Tripathi, N. Zheng, G.C. Lander, T. Hirota, F. Tama, C.L. Partch, ELife 9 (2020).","ista":"Fribourgh JL, Srivastava A, Sandate CR, Michael AK, Hsu PL, Rakers C, Nguyen LT, Torgrimson MR, Parico GCG, Tripathi S, Zheng N, Lander GC, Hirota T, Tama F, Partch CL. 2020. Dynamics at the serine loop underlie differential affinity of cryptochromes for CLOCK:BMAL1 to control circadian timing. eLife. 9, 55275.","ieee":"J. L. Fribourgh et al., “Dynamics at the serine loop underlie differential affinity of cryptochromes for CLOCK:BMAL1 to control circadian timing,” eLife, vol. 9. eLife Sciences Publications, 2020.","apa":"Fribourgh, J. L., Srivastava, A., Sandate, C. R., Michael, A. K., Hsu, P. L., Rakers, C., … Partch, C. L. (2020). Dynamics at the serine loop underlie differential affinity of cryptochromes for CLOCK:BMAL1 to control circadian timing. ELife. eLife Sciences Publications. https://doi.org/10.7554/elife.55275","ama":"Fribourgh JL, Srivastava A, Sandate CR, et al. Dynamics at the serine loop underlie differential affinity of cryptochromes for CLOCK:BMAL1 to control circadian timing. eLife. 2020;9. doi:10.7554/elife.55275"},"publication":"eLife","article_type":"original","date_published":"2020-02-26T00:00:00Z","scopus_import":"1","keyword":["General Immunology and Microbiology","General Biochemistry","Genetics and Molecular Biology","General Medicine","General Neuroscience"],"article_processing_charge":"No","day":"26"},{"file_date_updated":"2021-03-01T23:30:04Z","date_updated":"2023-09-07T13:20:03Z","date_created":"2020-02-26T10:56:37Z","author":[{"full_name":"Bhandari, Pradeep","first_name":"Pradeep","last_name":"Bhandari","id":"45EDD1BC-F248-11E8-B48F-1D18A9856A87","orcid":"0000-0003-0863-4481"}],"department":[{"_id":"RySh"}],"publisher":"Institute of Science and Technology Austria","publication_status":"published","year":"2020","publication_identifier":{"issn":["2663-337X"]},"month":"02","language":[{"iso":"eng"}],"acknowledged_ssus":[{"_id":"EM-Fac"}],"supervisor":[{"last_name":"Shigemoto","first_name":"Ryuichi","orcid":"0000-0001-8761-9444","id":"499F3ABC-F248-11E8-B48F-1D18A9856A87","full_name":"Shigemoto, Ryuichi"}],"degree_awarded":"PhD","doi":"10.15479/AT:ISTA:7525","oa":1,"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"}],"alternative_title":["ISTA Thesis"],"type":"dissertation","oa_version":"Published Version","file":[{"relation":"main_file","embargo":"2021-02-28","file_id":"7538","title":"Localization and functional role of Cav2.3 in the medial habenula to interpeduncular nucleus pathway","checksum":"4589234fdb12b4ad72273b311723a7b4","date_updated":"2021-03-01T23:30:04Z","date_created":"2020-02-28T08:37:53Z","access_level":"open_access","file_name":"Pradeep Bhandari Thesis.pdf","content_type":"application/pdf","file_size":9646346,"creator":"pbhandari"},{"file_id":"7539","title":"Localization and functional role of Cav2.3 in the medial habenula to interpeduncular nucleus pathway","relation":"source_file","checksum":"aa79490553ca0a5c9b6fbcd152e93928","date_created":"2020-02-28T08:47:14Z","date_updated":"2021-03-01T23:30:04Z","access_level":"closed","file_name":"Pradeep Bhandari Thesis.docx","embargo_to":"open_access","creator":"pbhandari","file_size":35252164,"content_type":"application/vnd.openxmlformats-officedocument.wordprocessingml.document"}],"status":"public","ddc":["570"],"title":"Localization and functional role of Cav2.3 in the medial habenula to interpeduncular nucleus pathway","user_id":"c635000d-4b10-11ee-a964-aac5a93f6ac1","_id":"7525","article_processing_charge":"No","has_accepted_license":"1","day":"28","keyword":["Cav2.3","medial habenula (MHb)","interpeduncular nucleus (IPN)"],"date_published":"2020-02-28T00:00:00Z","page":"79","citation":{"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). Localization and functional role of Cav2.3 in the medial habenula to interpeduncular nucleus pathway. Institute of Science and Technology Austria. https://doi.org/10.15479/AT:ISTA:7525","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.","ama":"Bhandari P. Localization and functional role of Cav2.3 in the medial habenula to interpeduncular nucleus pathway. 2020. doi:10.15479/AT:ISTA:7525","chicago":"Bhandari, Pradeep. “Localization and Functional Role of Cav2.3 in the Medial Habenula to Interpeduncular Nucleus Pathway.” Institute of Science and Technology Austria, 2020. https://doi.org/10.15479/AT:ISTA:7525.","mla":"Bhandari, Pradeep. Localization and Functional Role of Cav2.3 in the Medial Habenula to Interpeduncular Nucleus Pathway. Institute of Science and Technology Austria, 2020, doi:10.15479/AT:ISTA:7525.","short":"P. Bhandari, Localization and Functional Role of Cav2.3 in the Medial Habenula to Interpeduncular Nucleus Pathway, Institute of Science and Technology Austria, 2020."}},{"ddc":["570"],"status":"public","title":"3D printed cell culture grid holders for improved cellular specimen preparation in cryo-electron microscopy","intvolume":" 212","user_id":"4359f0d1-fa6c-11eb-b949-802e58b17ae8","_id":"8586","file":[{"access_level":"open_access","file_name":"2020_JourStrucBiology_Faessler.pdf","creator":"dernst","content_type":"application/pdf","file_size":7076870,"file_id":"8937","relation":"main_file","success":1,"checksum":"c48cbf594e84fc2f91966ffaafc0918c","date_updated":"2020-12-10T14:01:10Z","date_created":"2020-12-10T14:01:10Z"}],"oa_version":"Published Version","type":"journal_article","abstract":[{"lang":"eng","text":"Cryo-electron microscopy (cryo-EM) of cellular specimens provides insights into biological processes and structures within a native context. However, a major challenge still lies in the efficient and reproducible preparation of adherent cells for subsequent cryo-EM analysis. This is due to the sensitivity of many cellular specimens to the varying seeding and culturing conditions required for EM experiments, the often limited amount of cellular material and also the fragility of EM grids and their substrate. Here, we present low-cost and reusable 3D printed grid holders, designed to improve specimen preparation when culturing challenging cellular samples directly on grids. The described grid holders increase cell culture reproducibility and throughput, and reduce the resources required for cell culturing. We show that grid holders can be integrated into various cryo-EM workflows, including micro-patterning approaches to control cell seeding on grids, and for generating samples for cryo-focused ion beam milling and cryo-electron tomography experiments. Their adaptable design allows for the generation of specialized grid holders customized to a large variety of applications."}],"issue":"3","article_type":"original","publication":"Journal of Structural Biology","citation":{"chicago":"Fäßler, Florian, Bettina Zens, Robert Hauschild, and Florian KM Schur. “3D Printed Cell Culture Grid Holders for Improved Cellular Specimen Preparation in Cryo-Electron Microscopy.” Journal of Structural Biology. Elsevier, 2020. https://doi.org/10.1016/j.jsb.2020.107633.","mla":"Fäßler, Florian, et al. “3D Printed Cell Culture Grid Holders for Improved Cellular Specimen Preparation in Cryo-Electron Microscopy.” Journal of Structural Biology, vol. 212, no. 3, 107633, Elsevier, 2020, doi:10.1016/j.jsb.2020.107633.","short":"F. Fäßler, B. Zens, R. Hauschild, F.K. Schur, Journal of Structural Biology 212 (2020).","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.","apa":"Fäßler, F., Zens, B., Hauschild, R., & Schur, F. K. (2020). 3D printed cell culture grid holders for improved cellular specimen preparation in cryo-electron microscopy. Journal of Structural Biology. Elsevier. https://doi.org/10.1016/j.jsb.2020.107633","ieee":"F. Fäßler, B. Zens, R. Hauschild, and F. K. Schur, “3D printed cell culture grid holders for improved cellular specimen preparation in cryo-electron microscopy,” Journal of Structural Biology, vol. 212, no. 3. Elsevier, 2020.","ama":"Fäßler F, Zens B, Hauschild R, Schur FK. 3D printed cell culture grid holders for improved cellular specimen preparation in cryo-electron microscopy. Journal of Structural Biology. 2020;212(3). doi:10.1016/j.jsb.2020.107633"},"date_published":"2020-12-01T00:00:00Z","keyword":["electron microscopy","cryo-EM","EM sample preparation","3D printing","cell culture"],"scopus_import":"1","day":"01","has_accepted_license":"1","article_processing_charge":"Yes (via OA deal)","publication_status":"published","department":[{"_id":"FlSc"}],"publisher":"Elsevier","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.","year":"2020","date_updated":"2024-03-28T23:30:05Z","date_created":"2020-09-29T13:24:06Z","volume":212,"author":[{"first_name":"Florian","last_name":"Fäßler","id":"404F5528-F248-11E8-B48F-1D18A9856A87","orcid":"0000-0001-7149-769X","full_name":"Fäßler, Florian"},{"full_name":"Zens, Bettina","orcid":"0000-0002-9561-1239","id":"45FD126C-F248-11E8-B48F-1D18A9856A87","last_name":"Zens","first_name":"Bettina"},{"full_name":"Hauschild, Robert","last_name":"Hauschild","first_name":"Robert","orcid":"0000-0001-9843-3522","id":"4E01D6B4-F248-11E8-B48F-1D18A9856A87"},{"orcid":"0000-0003-4790-8078","id":"48AD8942-F248-11E8-B48F-1D18A9856A87","last_name":"Schur","first_name":"Florian KM","full_name":"Schur, Florian KM"}],"related_material":{"record":[{"relation":"used_in_publication","status":"public","id":"14592"},{"id":"12491","relation":"dissertation_contains","status":"public"}]},"article_number":"107633","license":"https://creativecommons.org/licenses/by/4.0/","file_date_updated":"2020-12-10T14:01:10Z","quality_controlled":"1","isi":1,"project":[{"name":"Structure and isoform diversity of the Arp2/3 complex","grant_number":"P33367","_id":"9B954C5C-BA93-11EA-9121-9846C619BF3A"},{"name":"NÖ-Fonds Preis für die Jungforscherin des Jahres am IST Austria","_id":"059B463C-7A3F-11EA-A408-12923DDC885E"}],"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","short":"CC BY (4.0)","image":"/images/cc_by.png"},"external_id":{"isi":["000600997800008"]},"oa":1,"acknowledged_ssus":[{"_id":"ScienComp"},{"_id":"LifeSc"},{"_id":"Bio"},{"_id":"EM-Fac"}],"language":[{"iso":"eng"}],"doi":"10.1016/j.jsb.2020.107633","month":"12","publication_identifier":{"issn":["1047-8477"]}},{"month":"10","publication_identifier":{"issn":["2663-337X"],"isbn":["978-3-99078-011-4"]},"oa":1,"acknowledged_ssus":[{"_id":"LifeSc"},{"_id":"M-Shop"}],"supervisor":[{"full_name":"Tkačik, Gašper","id":"3D494DCA-F248-11E8-B48F-1D18A9856A87","orcid":"0000-0002-6699-1455","first_name":"Gašper","last_name":"Tkačik"},{"full_name":"Bollenbach, Mark Tobias","last_name":"Bollenbach","first_name":"Mark Tobias","orcid":"0000-0003-4398-476X","id":"3E6DB97A-F248-11E8-B48F-1D18A9856A87"}],"degree_awarded":"PhD","language":[{"iso":"eng"}],"doi":"10.15479/AT:ISTA:8657","file_date_updated":"2021-10-07T22:30:03Z","publication_status":"published","publisher":"Institute of Science and Technology Austria","department":[{"_id":"GaTk"}],"acknowledgement":"I thank Life Science Facilities for their continuous support with providing top-notch laboratory materials, keeping the devices humming, and coordinating the repairs and building of custom-designed laboratory equipment with the MIBA Machine shop.","year":"2020","date_updated":"2023-09-07T13:20:48Z","date_created":"2020-10-13T16:46:14Z","author":[{"full_name":"Kavcic, Bor","last_name":"Kavcic","first_name":"Bor","orcid":"0000-0001-6041-254X","id":"350F91D2-F248-11E8-B48F-1D18A9856A87"}],"related_material":{"record":[{"relation":"part_of_dissertation","status":"public","id":"7673"},{"id":"8250","relation":"part_of_dissertation","status":"public"}]},"day":"14","article_processing_charge":"No","has_accepted_license":"1","page":"271","citation":{"apa":"Kavcic, B. (2020). Perturbations of protein synthesis: from antibiotics to genetics and physiology. Institute of Science and Technology Austria. https://doi.org/10.15479/AT:ISTA:8657","ieee":"B. Kavcic, “Perturbations of protein synthesis: from antibiotics to genetics and physiology,” Institute of Science and Technology Austria, 2020.","ista":"Kavcic B. 2020. Perturbations of protein synthesis: from antibiotics to genetics and physiology. Institute of Science and Technology Austria.","ama":"Kavcic B. Perturbations of protein synthesis: from antibiotics to genetics and physiology. 2020. doi:10.15479/AT:ISTA:8657","chicago":"Kavcic, Bor. “Perturbations of Protein Synthesis: From Antibiotics to Genetics and Physiology.” Institute of Science and Technology Austria, 2020. https://doi.org/10.15479/AT:ISTA:8657.","short":"B. Kavcic, Perturbations of Protein Synthesis: From Antibiotics to Genetics and Physiology, Institute of Science and Technology Austria, 2020.","mla":"Kavcic, Bor. Perturbations of Protein Synthesis: From Antibiotics to Genetics and Physiology. Institute of Science and Technology Austria, 2020, doi:10.15479/AT:ISTA:8657."},"date_published":"2020-10-14T00:00:00Z","alternative_title":["ISTA Thesis"],"type":"dissertation","abstract":[{"lang":"eng","text":"Synthesis of proteins – translation – is a fundamental process of life. Quantitative studies anchor translation into the context of bacterial physiology and reveal several mathematical relationships, called “growth laws,” which capture physiological feedbacks between protein synthesis and cell growth. Growth laws describe the dependency of the ribosome abundance as a function of growth rate, which can change depending on the growth conditions. Perturbations of translation reveal that bacteria employ a compensatory strategy in which the reduced translation capability results in increased expression of the translation machinery.\r\nPerturbations of translation are achieved in various ways; clinically interesting is the application of translation-targeting antibiotics – translation inhibitors. The antibiotic effects on bacterial physiology are often poorly understood. Bacterial responses to two or more simultaneously applied antibiotics are even more puzzling. The combined antibiotic effect determines the type of drug interaction, which ranges from synergy (the effect is stronger than expected) to antagonism (the effect is weaker) and suppression (one of the drugs loses its potency).\r\nIn the first part of this work, we systematically measure the pairwise interaction network for translation inhibitors that interfere with different steps in translation. We find that the interactions are surprisingly diverse and tend to be more antagonistic. To explore the underlying mechanisms, we begin with a minimal biophysical model of combined antibiotic action. We base this model on the kinetics of antibiotic uptake and binding together with the physiological response described by the growth laws. The biophysical model explains some drug interactions, but not all; it specifically fails to predict suppression.\r\nIn the second part of this work, we hypothesize that elusive suppressive drug interactions result from the interplay between ribosomes halted in different stages of translation. To elucidate this putative mechanism of drug interactions between translation inhibitors, we generate translation bottlenecks genetically using in- ducible control of translation factors that regulate well-defined translation cycle steps. These perturbations accurately mimic antibiotic action and drug interactions, supporting that the interplay of different translation bottlenecks partially causes these interactions.\r\nWe extend this approach by varying two translation bottlenecks simultaneously. This approach reveals the suppression of translocation inhibition by inhibited translation. We rationalize this effect by modeling dense traffic of ribosomes that move on transcripts in a translation factor-mediated manner. This model predicts a dissolution of traffic jams caused by inhibited translocation when the density of ribosome traffic is reduced by lowered initiation. We base this model on the growth laws and quantitative relationships between different translation and growth parameters.\r\nIn the final part of this work, we describe a set of tools aimed at quantification of physiological and translation parameters. We further develop a simple model that directly connects the abundance of a translation factor with the growth rate, which allows us to extract physiological parameters describing initiation. We demonstrate the development of tools for measuring translation rate.\r\nThis thesis showcases how a combination of high-throughput growth rate mea- surements, genetics, and modeling can reveal mechanisms of drug interactions. Furthermore, by a gradual transition from combinations of antibiotics to precise genetic interventions, we demonstrated the equivalency between genetic and chemi- cal perturbations of translation. These findings tile the path for quantitative studies of antibiotic combinations and illustrate future approaches towards the quantitative description of translation."}],"status":"public","ddc":["571","530","570"],"title":"Perturbations of protein synthesis: from antibiotics to genetics and physiology","user_id":"c635000d-4b10-11ee-a964-aac5a93f6ac1","_id":"8657","file":[{"creator":"bkavcic","content_type":"application/pdf","file_size":52636162,"access_level":"open_access","file_name":"kavcicB_thesis202009.pdf","checksum":"d708ecd62b6fcc3bc1feb483b8dbe9eb","date_created":"2020-10-15T06:41:20Z","date_updated":"2021-10-07T22:30:03Z","file_id":"8663","embargo":"2021-10-06","relation":"main_file"},{"checksum":"bb35f2352a04db19164da609f00501f3","date_updated":"2021-10-07T22:30:03Z","date_created":"2020-10-15T06:41:53Z","file_id":"8664","relation":"source_file","creator":"bkavcic","content_type":"application/zip","file_size":321681247,"access_level":"closed","file_name":"2020b.zip","embargo_to":"open_access"}],"oa_version":"Published Version"},{"month":"03","publication_identifier":{"issn":["0896-6273"]},"language":[{"iso":"eng"}],"doi":"10.1016/j.neuron.2019.12.022","isi":1,"quality_controlled":"1","project":[{"_id":"25B7EB9E-B435-11E9-9278-68D0E5697425","grant_number":"692692","name":"Biophysics and circuit function of a giant cortical glumatergic synapse","call_identifier":"H2020"},{"_id":"25BAF7B2-B435-11E9-9278-68D0E5697425","grant_number":"708497","call_identifier":"H2020","name":"Presynaptic calcium channels distribution and impact on coupling at the hippocampal mossy fiber synapse"},{"grant_number":"Z00312","_id":"25C5A090-B435-11E9-9278-68D0E5697425","name":"The Wittgenstein Prize","call_identifier":"FWF"},{"_id":"25C3DBB6-B435-11E9-9278-68D0E5697425","grant_number":"W01205","name":"Zellkommunikation in Gesundheit und Krankheit","call_identifier":"FWF"}],"tmp":{"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)","image":"/images/cc_by_nc_nd.png"},"external_id":{"pmid":["31928842"],"isi":["000520854700008"]},"oa":1,"license":"https://creativecommons.org/licenses/by-nc-nd/4.0/","file_date_updated":"2020-11-20T08:58:53Z","ec_funded":1,"date_updated":"2024-03-28T23:30:07Z","date_created":"2020-02-10T15:59:45Z","volume":105,"author":[{"full_name":"Borges Merjane, Carolina","last_name":"Borges Merjane","first_name":"Carolina","orcid":"0000-0003-0005-401X","id":"4305C450-F248-11E8-B48F-1D18A9856A87"},{"full_name":"Kim, Olena","first_name":"Olena","last_name":"Kim","id":"3F8ABDDA-F248-11E8-B48F-1D18A9856A87"},{"id":"353C1B58-F248-11E8-B48F-1D18A9856A87","orcid":"0000-0001-5001-4804","first_name":"Peter M","last_name":"Jonas","full_name":"Jonas, Peter M"}],"related_material":{"link":[{"relation":"press_release","description":"News on IST Homepage","url":"https://ist.ac.at/en/news/flash-and-freeze-reveals-dynamics-of-nerve-connections/"}],"record":[{"status":"public","relation":"dissertation_contains","id":"11196"}]},"publication_status":"published","publisher":"Elsevier","department":[{"_id":"PeJo"}],"year":"2020","acknowledgement":"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. 692692 and Marie Sklodowska-Curie 708497) and from Fonds zur Förderung der Wissenschaftlichen Forschung (Z 312-B27 Wittgenstein award and DK W1205-B09). We thank Johann Danzl and Ryuichi Shigemoto for critically reading the manuscript; Walter Kaufmann, Daniel Gutl, and Vanessa Zheden for extensive EM training, advice, and experimental assistance; Benjamin Suter for substantial help with light stimulation, ImageJ plugins for analysis, and manuscript editing; Florian Marr and Christina Altmutter for technical support; Eleftheria Kralli-Beller for manuscript editing; Julia König and Paul Wurzinger (Leica Microsystems) for helpful technical discussions; and Taija Makinen for providing the Prox1-CreERT2 mouse line.","pmid":1,"day":"18","article_processing_charge":"No","has_accepted_license":"1","scopus_import":"1","date_published":"2020-03-18T00:00:00Z","article_type":"original","page":"992-1006","publication":"Neuron","citation":{"short":"C. Borges Merjane, O. Kim, P.M. Jonas, Neuron 105 (2020) 992–1006.","mla":"Borges Merjane, Carolina, et al. “Functional Electron Microscopy (‘Flash and Freeze’) of Identified Cortical Synapses in Acute Brain Slices.” Neuron, vol. 105, Elsevier, 2020, pp. 992–1006, doi:10.1016/j.neuron.2019.12.022.","chicago":"Borges Merjane, Carolina, Olena Kim, and Peter M Jonas. “Functional Electron Microscopy (‘Flash and Freeze’) of Identified Cortical Synapses in Acute Brain Slices.” Neuron. Elsevier, 2020. https://doi.org/10.1016/j.neuron.2019.12.022.","ama":"Borges Merjane C, Kim O, Jonas PM. Functional electron microscopy (“Flash and Freeze”) of identified cortical synapses in acute brain slices. Neuron. 2020;105:992-1006. doi:10.1016/j.neuron.2019.12.022","apa":"Borges Merjane, C., Kim, O., & Jonas, P. M. (2020). Functional electron microscopy (“Flash and Freeze”) of identified cortical synapses in acute brain slices. Neuron. Elsevier. https://doi.org/10.1016/j.neuron.2019.12.022","ieee":"C. Borges Merjane, O. Kim, and P. M. Jonas, “Functional electron microscopy (‘Flash and Freeze’) of identified cortical synapses in acute brain slices,” Neuron, vol. 105. Elsevier, pp. 992–1006, 2020.","ista":"Borges Merjane C, Kim O, Jonas PM. 2020. Functional electron microscopy (“Flash and Freeze”) of identified cortical synapses in acute brain slices. Neuron. 105, 992–1006."},"abstract":[{"lang":"eng","text":"How structural and functional properties of synapses relate to each other is a fundamental question in neuroscience. Electrophysiology has elucidated mechanisms of synaptic transmission, and electron microscopy (EM) has provided insight into morphological properties of synapses. Here we describe an enhanced method for functional EM (“flash and freeze”), combining optogenetic stimulation with high-pressure freezing. We demonstrate that the improved method can be applied to intact networks in acute brain slices and organotypic slice cultures from mice. As a proof of concept, we probed vesicle pool changes during synaptic transmission at the hippocampal mossy fiber-CA3 pyramidal neuron synapse. Our findings show overlap of the docked vesicle pool and the functionally defined readily releasable pool and provide evidence of fast endocytosis at this synapse. Functional EM with acute slices and slice cultures has the potential to reveal the structural and functional mechanisms of transmission in intact, genetically perturbed, and disease-affected synapses."}],"type":"journal_article","file":[{"file_id":"8778","relation":"main_file","date_updated":"2020-11-20T08:58:53Z","date_created":"2020-11-20T08:58:53Z","success":1,"checksum":"3582664addf26859e86ac5bec3e01416","file_name":"2020_Neuron_BorgesMerjane.pdf","access_level":"open_access","creator":"dernst","content_type":"application/pdf","file_size":9712957}],"oa_version":"Published Version","title":"Functional electron microscopy (“Flash and Freeze”) of identified cortical synapses in acute brain slices","ddc":["570"],"status":"public","intvolume":" 105","_id":"7473","user_id":"4359f0d1-fa6c-11eb-b949-802e58b17ae8"},{"language":[{"iso":"eng"}],"doi":"10.1038/s41467-020-17734-z","project":[{"call_identifier":"FWF","name":"Revealing the mechanisms underlying drug interactions","grant_number":"P27201-B22","_id":"25E9AF9E-B435-11E9-9278-68D0E5697425"},{"_id":"254E9036-B435-11E9-9278-68D0E5697425","grant_number":"P28844-B27","name":"Biophysics of information processing in gene regulation","call_identifier":"FWF"}],"isi":1,"quality_controlled":"1","oa":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","short":"CC BY (4.0)","image":"/images/cc_by.png"},"external_id":{"isi":["000562769300008"]},"publication_identifier":{"issn":["2041-1723"]},"month":"08","volume":11,"date_updated":"2024-03-28T23:30:08Z","date_created":"2020-08-12T09:13:50Z","related_material":{"record":[{"id":"8657","relation":"dissertation_contains","status":"public"}]},"author":[{"full_name":"Kavcic, Bor","id":"350F91D2-F248-11E8-B48F-1D18A9856A87","orcid":"0000-0001-6041-254X","first_name":"Bor","last_name":"Kavcic"},{"full_name":"Tkačik, Gašper","id":"3D494DCA-F248-11E8-B48F-1D18A9856A87","orcid":"0000-0002-6699-1455","first_name":"Gašper","last_name":"Tkačik"},{"full_name":"Bollenbach, Tobias","first_name":"Tobias","last_name":"Bollenbach","id":"3E6DB97A-F248-11E8-B48F-1D18A9856A87","orcid":"0000-0003-4398-476X"}],"publisher":"Springer Nature","department":[{"_id":"GaTk"}],"publication_status":"published","year":"2020","acknowledgement":"We thank M. Hennessey-Wesen, I. Tomanek, K. Jain, A. Staron, K. Tomasek, M. Scott,\r\nK.C. Huang, and Z. Gitai for reading the manuscript and constructive comments. B.K. is\r\nindebted to C. Guet for additional guidance and generous support, which rendered this\r\nwork possible. B.K. thanks all members of Guet group for many helpful discussions and\r\nsharing of resources. B.K. additionally acknowledges the tremendous support from A.\r\nAngermayr and K. Mitosch with experimental work. We further thank E. Brown for\r\nhelpful comments regarding lamotrigine, and A. Buskirk for valuable suggestions\r\nregarding the ribosome footprint size. This work was supported in part by Austrian\r\nScience Fund (FWF) standalone grants P 27201-B22 (to T.B.) and P 28844 (to G.T.),\r\nHFSP program Grant RGP0042/2013 (to T.B.), German Research Foundation (DFG)\r\nstandalone grant BO 3502/2-1 (to T.B.), and German Research Foundation (DFG)\r\nCollaborative Research Centre (SFB) 1310 (to T.B.). Open access funding provided by\r\nProjekt DEAL.","file_date_updated":"2020-08-17T07:36:57Z","article_number":"4013","date_published":"2020-08-11T00:00:00Z","article_type":"original","citation":{"chicago":"Kavcic, Bor, Gašper Tkačik, and Mark Tobias Bollenbach. “Mechanisms of Drug Interactions between Translation-Inhibiting Antibiotics.” Nature Communications. Springer Nature, 2020. https://doi.org/10.1038/s41467-020-17734-z.","short":"B. Kavcic, G. Tkačik, M.T. Bollenbach, Nature Communications 11 (2020).","mla":"Kavcic, Bor, et al. “Mechanisms of Drug Interactions between Translation-Inhibiting Antibiotics.” Nature Communications, vol. 11, 4013, Springer Nature, 2020, doi:10.1038/s41467-020-17734-z.","apa":"Kavcic, B., Tkačik, G., & Bollenbach, M. T. (2020). Mechanisms of drug interactions between translation-inhibiting antibiotics. Nature Communications. Springer Nature. https://doi.org/10.1038/s41467-020-17734-z","ieee":"B. Kavcic, G. Tkačik, and M. T. Bollenbach, “Mechanisms of drug interactions between translation-inhibiting antibiotics,” Nature Communications, vol. 11. Springer Nature, 2020.","ista":"Kavcic B, Tkačik G, Bollenbach MT. 2020. Mechanisms of drug interactions between translation-inhibiting antibiotics. Nature Communications. 11, 4013.","ama":"Kavcic B, Tkačik G, Bollenbach MT. Mechanisms of drug interactions between translation-inhibiting antibiotics. Nature Communications. 2020;11. doi:10.1038/s41467-020-17734-z"},"publication":"Nature Communications","has_accepted_license":"1","article_processing_charge":"No","day":"11","file":[{"checksum":"986bebb308850a55850028d3d2b5b664","success":1,"date_created":"2020-08-17T07:36:57Z","date_updated":"2020-08-17T07:36:57Z","relation":"main_file","file_id":"8275","content_type":"application/pdf","file_size":1965672,"creator":"dernst","access_level":"open_access","file_name":"2020_NatureComm_Kavcic.pdf"}],"oa_version":"Published Version","intvolume":" 11","ddc":["570"],"title":"Mechanisms of drug interactions between translation-inhibiting antibiotics","status":"public","_id":"8250","user_id":"4359f0d1-fa6c-11eb-b949-802e58b17ae8","abstract":[{"text":"Antibiotics that interfere with translation, when combined, interact in diverse and difficult-to-predict ways. Here, we explain these interactions by “translation bottlenecks”: points in the translation cycle where antibiotics block ribosomal progression. To elucidate the underlying mechanisms of drug interactions between translation inhibitors, we generate translation bottlenecks genetically using inducible control of translation factors that regulate well-defined translation cycle steps. These perturbations accurately mimic antibiotic action and drug interactions, supporting that the interplay of different translation bottlenecks causes these interactions. We further show that growth laws, combined with drug uptake and binding kinetics, enable the direct prediction of a large fraction of observed interactions, yet fail to predict suppression. However, varying two translation bottlenecks simultaneously supports that dense traffic of ribosomes and competition for translation factors account for the previously unexplained suppression. These results highlight the importance of “continuous epistasis” in bacterial physiology.","lang":"eng"}],"type":"journal_article"},{"date_updated":"2024-03-28T23:30:08Z","date_created":"2020-04-22T08:27:56Z","oa_version":"Preprint","author":[{"last_name":"Kavcic","first_name":"Bor","orcid":"0000-0001-6041-254X","id":"350F91D2-F248-11E8-B48F-1D18A9856A87","full_name":"Kavcic, Bor"},{"full_name":"Tkačik, Gašper","first_name":"Gašper","last_name":"Tkačik","id":"3D494DCA-F248-11E8-B48F-1D18A9856A87","orcid":"0000-0002-6699-1455"},{"orcid":"0000-0003-4398-476X","id":"3E6DB97A-F248-11E8-B48F-1D18A9856A87","last_name":"Bollenbach","first_name":"Tobias","full_name":"Bollenbach, Tobias"}],"related_material":{"record":[{"id":"8997","status":"public","relation":"later_version"},{"relation":"dissertation_contains","status":"public","id":"8657"}]},"publication_status":"published","status":"public","title":"A minimal biophysical model of combined antibiotic action","department":[{"_id":"GaTk"}],"publisher":"Cold Spring Harbor Laboratory","_id":"7673","year":"2020","user_id":"2DF688A6-F248-11E8-B48F-1D18A9856A87","abstract":[{"text":"Combining drugs can improve the efficacy of treatments. However, predicting the effect of drug combinations is still challenging. The combined potency of drugs determines the drug interaction, which is classified as synergistic, additive, antagonistic, or suppressive. While probabilistic, non-mechanistic models exist, there is currently no biophysical model that can predict antibiotic interactions. Here, we present a physiologically relevant model of the combined action of antibiotics that inhibit protein synthesis by targeting the ribosome. This model captures the kinetics of antibiotic binding and transport, and uses bacterial growth laws to predict growth in the presence of antibiotic combinations. We find that this biophysical model can produce all drug interaction types except suppression. We show analytically that antibiotics which cannot bind to the ribosome simultaneously generally act as substitutes for one another, leading to additive drug interactions. Previously proposed null expectations for higher-order drug interactions follow as a limiting case of our model. We further extend the model to include the effects of direct physical or allosteric interactions between individual drugs on the ribosome. Notably, such direct interactions profoundly change the combined drug effect, depending on the kinetic parameters of the drugs used. The model makes additional predictions for the effects of resistance genes on drug interactions and for interactions between ribosome-targeting antibiotics and antibiotics with other targets. These findings enhance our understanding of the interplay between drug action and cell physiology and are a key step toward a general framework for predicting drug interactions.","lang":"eng"}],"type":"preprint","language":[{"iso":"eng"}],"doi":"10.1101/2020.04.18.047886","date_published":"2020-04-18T00:00:00Z","project":[{"call_identifier":"FWF","name":"Revealing the mechanisms underlying drug interactions","_id":"25E9AF9E-B435-11E9-9278-68D0E5697425","grant_number":"P27201-B22"},{"name":"Biophysics of information processing in gene regulation","call_identifier":"FWF","grant_number":"P28844-B27","_id":"254E9036-B435-11E9-9278-68D0E5697425"}],"publication":"bioRxiv","main_file_link":[{"open_access":"1","url":"https://doi.org/10.1101/2020.04.18.047886 "}],"oa":1,"citation":{"ama":"Kavcic B, Tkačik G, Bollenbach MT. A minimal biophysical model of combined antibiotic action. bioRxiv. 2020. doi:10.1101/2020.04.18.047886","ieee":"B. Kavcic, G. Tkačik, and M. T. Bollenbach, “A minimal biophysical model of combined antibiotic action,” bioRxiv. Cold Spring Harbor Laboratory, 2020.","apa":"Kavcic, B., Tkačik, G., & Bollenbach, M. T. (2020). A minimal biophysical model of combined antibiotic action. bioRxiv. Cold Spring Harbor Laboratory. https://doi.org/10.1101/2020.04.18.047886","ista":"Kavcic B, Tkačik G, Bollenbach MT. 2020. A minimal biophysical model of combined antibiotic action. bioRxiv, 10.1101/2020.04.18.047886.","short":"B. Kavcic, G. Tkačik, M.T. Bollenbach, BioRxiv (2020).","mla":"Kavcic, Bor, et al. “A Minimal Biophysical Model of Combined Antibiotic Action.” BioRxiv, Cold Spring Harbor Laboratory, 2020, doi:10.1101/2020.04.18.047886.","chicago":"Kavcic, Bor, Gašper Tkačik, and Mark Tobias Bollenbach. “A Minimal Biophysical Model of Combined Antibiotic Action.” BioRxiv. Cold Spring Harbor Laboratory, 2020. https://doi.org/10.1101/2020.04.18.047886."},"month":"04","day":"18","article_processing_charge":"No"},{"year":"2020","pmid":1,"publication_status":"published","department":[{"_id":"JiFr"},{"_id":"EvBe"}],"publisher":"Proceedings of the National Academy of Sciences","author":[{"full_name":"Hörmayer, Lukas","orcid":"0000-0001-8295-2926","id":"2EEE7A2A-F248-11E8-B48F-1D18A9856A87","last_name":"Hörmayer","first_name":"Lukas"},{"full_name":"Montesinos López, Juan C","first_name":"Juan C","last_name":"Montesinos López","id":"310A8E3E-F248-11E8-B48F-1D18A9856A87","orcid":"0000-0001-9179-6099"},{"first_name":"Petra","last_name":"Marhavá","id":"44E59624-F248-11E8-B48F-1D18A9856A87","full_name":"Marhavá, Petra"},{"last_name":"Benková","first_name":"Eva","orcid":"0000-0002-8510-9739","id":"38F4F166-F248-11E8-B48F-1D18A9856A87","full_name":"Benková, Eva"},{"full_name":"Yoshida, Saiko","last_name":"Yoshida","first_name":"Saiko","id":"2E46069C-F248-11E8-B48F-1D18A9856A87"},{"orcid":"0000-0002-8302-7596","id":"4159519E-F248-11E8-B48F-1D18A9856A87","last_name":"Friml","first_name":"Jiří","full_name":"Friml, Jiří"}],"related_material":{"record":[{"id":"9992","status":"public","relation":"dissertation_contains"}],"link":[{"relation":"press_release","description":"News on IST Homepage","url":"https://ist.ac.at/en/news/how-wounded-plants-coordinate-their-healing/"}]},"date_updated":"2024-03-28T23:30:10Z","date_created":"2020-06-22T13:33:52Z","volume":117,"article_number":"202003346","file_date_updated":"2020-07-14T12:48:07Z","ec_funded":1,"external_id":{"isi":["000565729700033"],"pmid":["32541049"]},"tmp":{"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)","image":"/images/cc_by_nc_nd.png"},"oa":1,"isi":1,"quality_controlled":"1","project":[{"grant_number":"742985","_id":"261099A6-B435-11E9-9278-68D0E5697425","name":"Tracing Evolution of Auxin Transport and Polarity in Plants","call_identifier":"H2020"},{"name":"RNA-directed DNA methylation in plant development","call_identifier":"FWF","_id":"262EF96E-B435-11E9-9278-68D0E5697425","grant_number":"P29988"}],"doi":"10.1073/pnas.2003346117","acknowledged_ssus":[{"_id":"Bio"},{"_id":"LifeSc"}],"language":[{"iso":"eng"}],"month":"06","publication_identifier":{"issn":["0027-8424"],"eissn":["1091-6490"]},"user_id":"c635000d-4b10-11ee-a964-aac5a93f6ac1","_id":"8002","status":"public","title":"Wounding-induced changes in cellular pressure and localized auxin signalling spatially coordinate restorative divisions in roots","ddc":["580"],"intvolume":" 117","oa_version":"None","file":[{"access_level":"open_access","file_name":"2020_PNAS_Hoermayer.pdf","creator":"dernst","content_type":"application/pdf","file_size":2407102,"file_id":"8009","relation":"main_file","checksum":"908b09437680181de9990915f2113aca","date_created":"2020-06-23T11:30:53Z","date_updated":"2020-07-14T12:48:07Z"}],"type":"journal_article","abstract":[{"text":"Wound healing in plant tissues, consisting of rigid cell wall-encapsulated cells, represents a considerable challenge and occurs through largely unknown mechanisms distinct from those in animals. Owing to their inability to migrate, plant cells rely on targeted cell division and expansion to regenerate wounds. Strict coordination of these wound-induced responses is essential to ensure efficient, spatially restricted wound healing. Single-cell tracking by live imaging allowed us to gain mechanistic insight into the wound perception and coordination of wound responses after laser-based wounding in Arabidopsis root. We revealed a crucial contribution of the collapse of damaged cells in wound perception and detected an auxin increase specific to cells immediately adjacent to the wound. This localized auxin increase balances wound-induced cell expansion and restorative division rates in a dose-dependent manner, leading to tumorous overproliferation when the canonical TIR1 auxin signaling is disrupted. Auxin and wound-induced turgor pressure changes together also spatially define the activation of key components of regeneration, such as the transcription regulator ERF115. Our observations suggest that the wound signaling involves the sensing of collapse of damaged cells and a local auxin signaling activation to coordinate the downstream transcriptional responses in the immediate wound vicinity.","lang":"eng"}],"issue":"26","publication":"Proceedings of the National Academy of Sciences","citation":{"chicago":"Hörmayer, Lukas, Juan C Montesinos López, Petra Marhavá, Eva Benková, Saiko Yoshida, and Jiří Friml. “Wounding-Induced Changes in Cellular Pressure and Localized Auxin Signalling Spatially Coordinate Restorative Divisions in Roots.” Proceedings of the National Academy of Sciences. Proceedings of the National Academy of Sciences, 2020. https://doi.org/10.1073/pnas.2003346117.","short":"L. Hörmayer, J.C. Montesinos López, P. Marhavá, E. Benková, S. Yoshida, J. Friml, Proceedings of the National Academy of Sciences 117 (2020).","mla":"Hörmayer, Lukas, et al. “Wounding-Induced Changes in Cellular Pressure and Localized Auxin Signalling Spatially Coordinate Restorative Divisions in Roots.” Proceedings of the National Academy of Sciences, vol. 117, no. 26, 202003346, Proceedings of the National Academy of Sciences, 2020, doi:10.1073/pnas.2003346117.","apa":"Hörmayer, L., Montesinos López, J. C., Marhavá, P., Benková, E., Yoshida, S., & Friml, J. (2020). Wounding-induced changes in cellular pressure and localized auxin signalling spatially coordinate restorative divisions in roots. Proceedings of the National Academy of Sciences. Proceedings of the National Academy of Sciences. https://doi.org/10.1073/pnas.2003346117","ieee":"L. Hörmayer, J. C. Montesinos López, P. Marhavá, E. Benková, S. Yoshida, and J. Friml, “Wounding-induced changes in cellular pressure and localized auxin signalling spatially coordinate restorative divisions in roots,” Proceedings of the National Academy of Sciences, vol. 117, no. 26. Proceedings of the National Academy of Sciences, 2020.","ista":"Hörmayer L, Montesinos López JC, Marhavá P, Benková E, Yoshida S, Friml J. 2020. Wounding-induced changes in cellular pressure and localized auxin signalling spatially coordinate restorative divisions in roots. Proceedings of the National Academy of Sciences. 117(26), 202003346.","ama":"Hörmayer L, Montesinos López JC, Marhavá P, Benková E, Yoshida S, Friml J. Wounding-induced changes in cellular pressure and localized auxin signalling spatially coordinate restorative divisions in roots. Proceedings of the National Academy of Sciences. 2020;117(26). doi:10.1073/pnas.2003346117"},"article_type":"original","date_published":"2020-06-30T00:00:00Z","scopus_import":"1","day":"30","has_accepted_license":"1","article_processing_charge":"No"},{"month":"04","publication_identifier":{"eissn":["2663-337X"]},"doi":"10.15479/AT:ISTA:7680","supervisor":[{"full_name":"Janovjak, Harald L","id":"33BA6C30-F248-11E8-B48F-1D18A9856A87","orcid":"0000-0002-8023-9315","first_name":"Harald L","last_name":"Janovjak"}],"degree_awarded":"PhD","language":[{"iso":"eng"}],"oa":1,"file_date_updated":"2021-10-31T23:30:05Z","author":[{"full_name":"Kainrath, Stephanie","last_name":"Kainrath","first_name":"Stephanie","id":"32CFBA64-F248-11E8-B48F-1D18A9856A87"}],"related_material":{"record":[{"id":"1028","status":"public","relation":"dissertation_contains"}]},"date_created":"2020-04-24T16:00:51Z","date_updated":"2023-09-22T09:20:10Z","year":"2020","publication_status":"published","publisher":"Institute of Science and Technology Austria","department":[{"_id":"CaGu"}],"day":"24","has_accepted_license":"1","article_processing_charge":"No","date_published":"2020-04-24T00:00:00Z","citation":{"chicago":"Kainrath, Stephanie. “Synthetic Tools for Optogenetic and Chemogenetic Inhibition of Cellular Signals.” Institute of Science and Technology Austria, 2020. https://doi.org/10.15479/AT:ISTA:7680.","short":"S. Kainrath, Synthetic Tools for Optogenetic and Chemogenetic Inhibition of Cellular Signals, Institute of Science and Technology Austria, 2020.","mla":"Kainrath, Stephanie. Synthetic Tools for Optogenetic and Chemogenetic Inhibition of Cellular Signals. Institute of Science and Technology Austria, 2020, doi:10.15479/AT:ISTA:7680.","apa":"Kainrath, S. (2020). Synthetic tools for optogenetic and chemogenetic inhibition of cellular signals. Institute of Science and Technology Austria. https://doi.org/10.15479/AT:ISTA:7680","ieee":"S. Kainrath, “Synthetic tools for optogenetic and chemogenetic inhibition of cellular signals,” Institute of Science and Technology Austria, 2020.","ista":"Kainrath S. 2020. Synthetic tools for optogenetic and chemogenetic inhibition of cellular signals. Institute of Science and Technology Austria.","ama":"Kainrath S. Synthetic tools for optogenetic and chemogenetic inhibition of cellular signals. 2020. doi:10.15479/AT:ISTA:7680"},"page":"98","abstract":[{"text":"Proteins and their complex dynamic interactions regulate cellular mechanisms from sensing and transducing extracellular signals, to mediating genetic responses, and sustaining or changing cell morphology. To manipulate these protein-protein interactions (PPIs) that govern the behavior and fate of cells, synthetically constructed, genetically encoded tools provide the means to precisely target proteins of interest (POIs), and control their subcellular localization and activity in vitro and in vivo. Ideal synthetic tools react to an orthogonal cue, i.e. a trigger that does not activate any other endogenous process, thereby allowing manipulation of the POI alone.\r\nIn optogenetics, naturally occurring photosensory domain from plants, algae and bacteria are re-purposed and genetically fused to POIs. Illumination with light of a specific wavelength triggers a conformational change that can mediate PPIs, such as dimerization or oligomerization. By using light as a trigger, these tools can be activated with high spatial and temporal precision, on subcellular and millisecond scales. Chemogenetic tools consist of protein domains that recognize and bind small molecules. By genetic fusion to POIs, these domains can mediate PPIs upon addition of their specific ligands, which are often synthetically designed to provide highly specific interactions and exhibit good bioavailability.\r\nMost optogenetic tools to mediate PPIs are based on well-studied photoreceptors responding to red, blue or near-UV light, leaving a striking gap in the green band of the visible light spectrum. Among both optogenetic and chemogenetic tools, there is an abundance of methods to induce PPIs, but tools to disrupt them require UV illumination, rely on covalent linkage and subsequent enzymatic cleavage or initially result in protein clustering of unknown stoichiometry.\r\nThis work describes how the recently structurally and photochemically characterized green-light responsive cobalamin-binding domains (CBDs) from bacterial transcription factors were re-purposed to function as a green-light responsive optogenetic tool. In contrast to previously engineered optogenetic tools, CBDs do not induce PPI, but rather confer a PPI already upon expression, which can be rapidly disrupted by illumination. This was employed to mimic inhibition of constitutive activity of a growth factor receptor, and successfully implement for cell signalling in mammalian cells and in vivo to rescue development in zebrafish. This work further describes the development and application of a chemically induced de-dimerizer (CDD) based on a recently identified and structurally described bacterial oxyreductase. CDD forms a dimer upon expression in absence of its cofactor, the flavin derivative F420. Safety and of domain expression and ligand exposure are demonstrated in vitro and in vivo in zebrafish. The system is further applied to inhibit cell signalling output from a chimeric receptor upon F420 treatment.\r\nCBDs and CDD expand the repertoire of synthetic tools by providing novel mechanisms of mediating PPIs, and by recognizing previously not utilized cues. In the future, they can readily be combined with existing synthetic tools to functionally manipulate PPIs in vitro and in vivo.","lang":"eng"}],"type":"dissertation","alternative_title":["ISTA Thesis"],"file":[{"file_name":"Thesis_without-signatures_PDFA.pdf","access_level":"open_access","creator":"stgingl","file_size":3268017,"content_type":"application/pdf","embargo":"2021-10-30","file_id":"7692","relation":"main_file","date_updated":"2021-10-31T23:30:05Z","date_created":"2020-04-28T11:19:21Z","checksum":"fb9a4468eb27be92690728e35c823796"},{"access_level":"closed","file_name":"Thesis_without signatures.docx","embargo_to":"open_access","creator":"stgingl","file_size":5167703,"content_type":"application/octet-stream","file_id":"7693","relation":"source_file","checksum":"f6c80ca97104a631a328cb79a2c53493","date_updated":"2021-10-31T23:30:05Z","date_created":"2020-04-28T11:19:24Z"}],"oa_version":"None","_id":"7680","user_id":"c635000d-4b10-11ee-a964-aac5a93f6ac1","ddc":["570"],"title":"Synthetic tools for optogenetic and chemogenetic inhibition of cellular signals","status":"public"},{"abstract":[{"text":"The development of the human brain occurs through a tightly regulated series of dynamic and adaptive processes during prenatal and postnatal life. A disruption of this strictly orchestrated series of events can lead to a number of neurodevelopmental conditions, including Autism Spectrum Disorders (ASDs). ASDs are a very common, etiologically and phenotypically heterogeneous group of disorders sharing the core symptoms of social interaction and communication deficits and restrictive and repetitive interests and behaviors. They are estimated to affect one in 59 individuals in the U.S. and, over the last three decades, mutations in more than a hundred genetic loci have been convincingly linked to ASD pathogenesis. Yet, for the vast majority of these ASD-risk genes their role during brain development and precise molecular function still remain elusive.\r\nDe novo loss of function mutations in the ubiquitin ligase-encoding gene Cullin 3 (CUL3) lead to ASD. In the study described here, we used Cul3 mouse models to evaluate the consequences of Cul3 mutations in vivo. Our results show that Cul3 heterozygous knockout mice exhibit deficits in motor coordination as well as ASD-relevant social and cognitive impairments. Cul3+/-, Cul3+/fl Emx1-Cre and Cul3fl/fl Emx1-Cre mutant brains display cortical lamination abnormalities due to defective migration of post-mitotic excitatory neurons, as well as reduced numbers of excitatory and inhibitory neurons. In line with the observed abnormal cortical organization, Cul3 heterozygous deletion is associated with decreased spontaneous excitatory and inhibitory activity in the cortex. At the molecular level we show that Cul3 regulates cytoskeletal and adhesion protein abundance in the mouse embryonic cortex. Abnormal regulation of cytoskeletal proteins in Cul3 mutant neural cells results in atypical organization of the actin mesh at the cell leading edge. Of note, heterozygous deletion of Cul3 in adult mice does not induce the majority of the behavioral defects observed in constitutive Cul3 haploinsufficient animals, pointing to a critical time-window for Cul3 deficiency.\r\nIn conclusion, our data indicate that Cul3 plays a critical role in the regulation of cytoskeletal proteins and neuronal migration. ASD-associated defects and behavioral abnormalities are primarily due to dosage sensitive Cul3 functions at early brain developmental stages.","lang":"eng"}],"type":"dissertation","alternative_title":["ISTA Thesis"],"oa_version":"Published Version","file":[{"file_id":"8621","embargo":"2021-10-15","relation":"main_file","checksum":"7ee83e42de3e5ce2fedb44dff472f75f","date_updated":"2021-10-16T22:30:04Z","date_created":"2020-10-07T14:41:49Z","access_level":"open_access","file_name":"Jasmin_Morandell_Thesis-2020_final.pdf","creator":"jmorande","content_type":"application/pdf","file_size":16155786},{"date_created":"2020-10-07T14:45:07Z","date_updated":"2021-10-16T22:30:04Z","checksum":"5e0464af453734210ce7aab7b4a92e3a","file_id":"8622","relation":"source_file","creator":"jmorande","file_size":24344152,"content_type":"application/x-zip-compressed","file_name":"Jasmin_Morandell_Thesis-2020_final.zip","embargo_to":"open_access","access_level":"closed"}],"_id":"8620","user_id":"c635000d-4b10-11ee-a964-aac5a93f6ac1","title":"Illuminating the role of Cul3 in autism spectrum disorder pathogenesis","ddc":["610"],"status":"public","has_accepted_license":"1","article_processing_charge":"No","day":"12","date_published":"2020-10-12T00:00:00Z","citation":{"short":"J. Morandell, Illuminating the Role of Cul3 in Autism Spectrum Disorder Pathogenesis, Institute of Science and Technology Austria, 2020.","mla":"Morandell, Jasmin. Illuminating the Role of Cul3 in Autism Spectrum Disorder Pathogenesis. Institute of Science and Technology Austria, 2020, doi:10.15479/AT:ISTA:8620.","chicago":"Morandell, Jasmin. “Illuminating the Role of Cul3 in Autism Spectrum Disorder Pathogenesis.” Institute of Science and Technology Austria, 2020. https://doi.org/10.15479/AT:ISTA:8620.","ama":"Morandell J. Illuminating the role of Cul3 in autism spectrum disorder pathogenesis. 2020. doi:10.15479/AT:ISTA:8620","apa":"Morandell, J. (2020). Illuminating the role of Cul3 in autism spectrum disorder pathogenesis. Institute of Science and Technology Austria. https://doi.org/10.15479/AT:ISTA:8620","ieee":"J. Morandell, “Illuminating the role of Cul3 in autism spectrum disorder pathogenesis,” Institute of Science and Technology Austria, 2020.","ista":"Morandell J. 2020. Illuminating the role of Cul3 in autism spectrum disorder pathogenesis. Institute of Science and Technology Austria."},"page":"138","file_date_updated":"2021-10-16T22:30:04Z","related_material":{"record":[{"status":"public","relation":"part_of_dissertation","id":"7800"},{"relation":"part_of_dissertation","status":"public","id":"8131"}]},"author":[{"full_name":"Morandell, Jasmin","id":"4739D480-F248-11E8-B48F-1D18A9856A87","first_name":"Jasmin","last_name":"Morandell"}],"date_created":"2020-10-07T14:53:13Z","date_updated":"2023-09-07T13:22:14Z","year":"2020","acknowledgement":"I would like to especially thank Armel Nicolas from the Proteomics and Christoph Sommer from the Bioimaging Facilities for the data analysis, and to thank the team of the Preclinical Facility, especially Sabina Deixler, Angela Schlerka, Anita Lepold, Mihalea Mihai and Michael Schun for taking care of the mouse line maintenance and their great support.","department":[{"_id":"GaNo"}],"publisher":"Institute of Science and Technology Austria","publication_status":"published","publication_identifier":{"issn":["2663-337X"]},"month":"10","doi":"10.15479/AT:ISTA:8620","language":[{"iso":"eng"}],"degree_awarded":"PhD","acknowledged_ssus":[{"_id":"Bio"},{"_id":"PreCl"}],"supervisor":[{"full_name":"Novarino, Gaia","orcid":"0000-0002-7673-7178","id":"3E57A680-F248-11E8-B48F-1D18A9856A87","last_name":"Novarino","first_name":"Gaia"}],"oa":1,"project":[{"grant_number":"W1232-B24","_id":"2548AE96-B435-11E9-9278-68D0E5697425","call_identifier":"FWF","name":"Molecular Drug Targets"},{"grant_number":"F07807","_id":"05A0D778-7A3F-11EA-A408-12923DDC885E","name":"Neural stem cells in autism and epilepsy"}]}]