[{"keyword":["Cell Division","Reconstitution","FtsZ","FtsA","Divisome","E.coli"],"language":[{"iso":"eng"}],"has_accepted_license":"1","month":"09","project":[{"call_identifier":"H2020","_id":"2595697A-B435-11E9-9278-68D0E5697425","name":"Self-Organization of the Bacterial Cell","grant_number":"679239"},{"_id":"fc38323b-9c52-11eb-aca3-ff8afb4a011d","name":"Understanding bacterial cell division by in vitro\r\nreconstitution","grant_number":"P34607"},{"_id":"2596EAB6-B435-11E9-9278-68D0E5697425","grant_number":"ALTF 2015-1163","name":"Synthesis of bacterial cell wall"},{"name":"Reconstitution of bacterial cell wall sythesis","grant_number":"LT000824/2016","_id":"259B655A-B435-11E9-9278-68D0E5697425"}],"acknowledged_ssus":[{"_id":"Bio"},{"_id":"LifeSc"}],"oa_version":"Published Version","related_material":{"record":[{"status":"public","relation":"part_of_dissertation","id":"11373"},{"id":"7387","relation":"part_of_dissertation","status":"public"},{"status":"public","relation":"research_data","id":"10934"}]},"user_id":"8b945eb4-e2f2-11eb-945a-df72226e66a9","status":"public","file":[{"date_updated":"2023-10-04T10:28:35Z","content_type":"application/vnd.openxmlformats-officedocument.wordprocessingml.document","file_name":"PhD Thesis_Philipp Radler_20231004.docx","date_created":"2023-10-04T10:11:53Z","checksum":"87eef11fbc5c7df0826f12a3a629b444","file_size":114932847,"file_id":"14390","creator":"pradler","relation":"source_file","access_level":"closed"},{"file_id":"14391","creator":"pradler","access_level":"closed","relation":"main_file","date_updated":"2023-10-04T10:28:35Z","file_name":"PhD Thesis_Philipp Radler_20231004.pdf","content_type":"application/pdf","date_created":"2023-10-04T10:11:21Z","embargo":"2024-10-04","embargo_to":"open_access","checksum":"3253e099b7126469d941fd9419d68b4f","file_size":37838778}],"type":"dissertation","date_published":"2023-09-25T00:00:00Z","tmp":{"legal_code_url":"https://creativecommons.org/licenses/by/4.0/legalcode","short":"CC BY (4.0)","image":"/images/cc_by.png","name":"Creative Commons Attribution 4.0 International Public License (CC-BY 4.0)"},"supervisor":[{"id":"462D4284-F248-11E8-B48F-1D18A9856A87","last_name":"Loose","first_name":"Martin","full_name":"Loose, Martin","orcid":"0000-0001-7309-9724"}],"publication_identifier":{"isbn":["978-3-99078-033-6"],"issn":["2663-337X"]},"file_date_updated":"2023-10-04T10:28:35Z","ec_funded":1,"page":"156","publisher":"Institute of Science and Technology Austria","author":[{"orcid":"0000-0001-9198-2182 ","full_name":"Radler, Philipp","first_name":"Philipp","last_name":"Radler","id":"40136C2A-F248-11E8-B48F-1D18A9856A87"}],"_id":"14280","title":"Spatiotemporal signaling during assembly of the bacterial divisome","alternative_title":["ISTA Thesis"],"department":[{"_id":"GradSch"},{"_id":"MaLo"}],"article_processing_charge":"No","date_created":"2023-09-06T10:58:25Z","publication_status":"published","ddc":["572"],"citation":{"ista":"Radler P. 2023. Spatiotemporal signaling during assembly of the bacterial divisome. Institute of Science and Technology Austria.","short":"P. Radler, Spatiotemporal Signaling during Assembly of the Bacterial Divisome, Institute of Science and Technology Austria, 2023.","mla":"Radler, Philipp. <i>Spatiotemporal Signaling during Assembly of the Bacterial Divisome</i>. Institute of Science and Technology Austria, 2023, doi:<a href=\"https://doi.org/10.15479/at:ista:14280\">10.15479/at:ista:14280</a>.","chicago":"Radler, Philipp. “Spatiotemporal Signaling during Assembly of the Bacterial Divisome.” Institute of Science and Technology Austria, 2023. <a href=\"https://doi.org/10.15479/at:ista:14280\">https://doi.org/10.15479/at:ista:14280</a>.","ieee":"P. Radler, “Spatiotemporal signaling during assembly of the bacterial divisome,” Institute of Science and Technology Austria, 2023.","ama":"Radler P. Spatiotemporal signaling during assembly of the bacterial divisome. 2023. doi:<a href=\"https://doi.org/10.15479/at:ista:14280\">10.15479/at:ista:14280</a>","apa":"Radler, P. (2023). <i>Spatiotemporal signaling during assembly of the bacterial divisome</i>. Institute of Science and Technology Austria. <a href=\"https://doi.org/10.15479/at:ista:14280\">https://doi.org/10.15479/at:ista:14280</a>"},"year":"2023","date_updated":"2024-02-21T12:35:18Z","abstract":[{"lang":"eng","text":"Cell division in Escherichia coli is performed by the divisome, a multi-protein complex composed of more than 30 proteins. The divisome spans from the cytoplasm through the inner membrane to the cell wall and the outer membrane. Divisome assembly is initiated by a cytoskeletal structure, the so-called Z-ring, which localizes at the center of the E. coli cell and determines the position of the future cell septum. The Z-ring is composed of the highly conserved bacterial tubulin homologue FtsZ, which forms treadmilling filaments. These filaments are recruited to the inner membrane by FtsA, a highly conserved bacterial actin homologue. FtsA interacts with other proteins in the periplasm and thus connects the cytoplasmic and periplasmic components of the divisome. \r\nA previous model postulated that FtsA regulates maturation of the divisome by switching from an oligomeric, inactive state to a monomeric and active state. This model was based mostly on in vivo studies, as a biochemical characterization of FtsA has been hampered by difficulties in purifying the protein. Here, we studied FtsA using an in vitro reconstitution approach and aimed to answer two questions: (i) How are dynamics from cytoplasmic, treadmilling FtsZ filaments coupled to proteins acting in the periplasmic space and (ii) How does FtsA regulate the maturation of the divisome?\r\nWe found that the cytoplasmic peptides of the transmembrane proteins FtsN and FtsQ interact directly with FtsA and can follow the spatiotemporal signal of FtsA/Z filaments. When we investigated the underlying mechanism by imaging single molecules of FtsNcyto, we found the peptide to interact transiently with FtsA. An in depth analysis of the single molecule trajectories helped to postulate a model where PG synthases follow the dynamics of FtsZ by a diffusion and capture mechanism. \r\nFollowing up on these findings we were interested in how the self-interaction of FtsA changes when it encounters FtsNcyto and if we can confirm the proposed oligomer-monomer switch. For this, we compared the behavior of the previously identified, hyperactive mutant FtsA R286W with wildtype FtsA. The mutant outperforms WT in mirroring and transmitting the spatiotemporal signal of treadmilling FtsZ filaments. Surprisingly however, we found that this was not due to a difference in the self-interaction strength of the two variants, but a difference in their membrane residence time. Furthermore, in contrast to our expectations, upon binding of FtsNcyto the measured self-interaction of FtsA actually increased. \r\nWe propose that FtsNcyto induces a rearrangement of the oligomeric architecture of FtsA. In further consequence this change leads to more persistent FtsZ filaments which results in a defined signalling zone, allowing formation of the mature divisome. The observed difference between FtsA WT and R286W is due to the vastly different membrane turnover of the proteins. R286W cycles 5-10x faster compared to WT which allows to sample FtsZ filaments at faster frequencies. These findings can explain the observed differences in toxicity for overexpression of FtsA WT and R286W and help to understand how FtsA regulates divisome maturation."}],"day":"25","doi":"10.15479/at:ista:14280","degree_awarded":"PhD"},{"user_id":"2DF688A6-F248-11E8-B48F-1D18A9856A87","status":"public","related_material":{"record":[{"status":"public","relation":"used_in_publication","id":"11373"},{"relation":"used_in_publication","id":"14280","status":"public"}],"link":[{"url":"https://doi.org/10.5281/zenodo.6400639","description":"A custom written code (FRAPdiff) to quantify the Off binding rate and Diffusion coefficient of membrane bound proteins. 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helpful discussions—in particular L. Lindorfer for his assistance with cloning and purifications. We thank J. Löwe and T. Nierhaus (MRC-LMB Cambridge, UK) for sharing unpublished work and helpful discussions, as well as D. Vavylonis and D. Rutkowski (Lehigh University, Bethlehem, PA, USA) as well as S. Martin (University of Lausanne, Switzerland) for sharing their code for FRAP analysis. We are also thankful for the support by the Scientific Service Units (SSU) of IST Austria through resources provided by the Imaging and Optics Facility (IOF) and the Lab Support Facility (LSF). This work was supported by the European Research Council through grant ERC 2015-StG-679239 and by the Austrian Science Fund (FWF) StandAlone P34607 to M.L. and HFSP LT 000824/2016-L4 to N.B. For the purpose of open access, we have applied a CC BY public copyright licence to any Author Accepted Manuscript version arising from this submission.","date_updated":"2024-02-21T12:35:18Z","year":"2022","citation":{"ieee":"P. Radler, “In vitro reconstitution of Escherichia coli divisome activation.” Institute of Science and Technology Austria, 2022.","chicago":"Radler, Philipp. “In Vitro Reconstitution of Escherichia Coli Divisome Activation.” Institute of Science and Technology Austria, 2022. <a href=\"https://doi.org/10.15479/AT:ISTA:10934\">https://doi.org/10.15479/AT:ISTA:10934</a>.","ama":"Radler P. In vitro reconstitution of Escherichia coli divisome activation. 2022. doi:<a href=\"https://doi.org/10.15479/AT:ISTA:10934\">10.15479/AT:ISTA:10934</a>","apa":"Radler, P. (2022). In vitro reconstitution of Escherichia coli divisome activation. Institute of Science and Technology Austria. <a href=\"https://doi.org/10.15479/AT:ISTA:10934\">https://doi.org/10.15479/AT:ISTA:10934</a>","ista":"Radler P. 2022. In vitro reconstitution of Escherichia coli divisome activation, Institute of Science and Technology Austria, <a href=\"https://doi.org/10.15479/AT:ISTA:10934\">10.15479/AT:ISTA:10934</a>.","short":"P. Radler, (2022).","mla":"Radler, Philipp. <i>In Vitro Reconstitution of Escherichia Coli Divisome Activation</i>. Institute of Science and Technology Austria, 2022, doi:<a href=\"https://doi.org/10.15479/AT:ISTA:10934\">10.15479/AT:ISTA:10934</a>."},"abstract":[{"lang":"eng","text":"FtsA is crucial for assembly of the E. coli divisome, as it dynamically links cytoplasmic FtsZ filaments with transmembrane cell division proteins. FtsA allegedly initiates cell division by switching from an inactive polymeric to an active monomeric confirmation, which recruits downstream proteins and stabilizes FtsZ filaments. Here, we use biochemical reconstitution experiments combined with quantitative fluorescence microscopy to study divisome activation in vitro. We compare wildtype-FtsA with FtsA-R286W, a constantly active gain-of-function mutant and find that R286W outperforms the wildtype protein in replicating FtsZ treadmilling dynamics, stabilizing FtsZ filaments and recruiting FtsN. We attribute these differences to a faster membrane exchange of FtsA-R286W and its higher packing density below FtsZ filaments.  Using FRET microscopy, we find that FtsN binding does not compete with, but promotes FtsA self-interaction. Our findings suggest a model where FtsA always forms dynamic polymers on the membrane, which re-organize during assembly and activation of the divisome. "}],"doi":"10.15479/AT:ISTA:10934","day":"05","file_date_updated":"2022-04-22T10:15:19Z","ec_funded":1,"publisher":"Institute of Science and Technology Austria","author":[{"orcid":" 0000-0001-9198-2182 ","full_name":"Radler, Philipp","first_name":"Philipp","last_name":"Radler","id":"40136C2A-F248-11E8-B48F-1D18A9856A87"}],"_id":"10934","title":"In vitro reconstitution of Escherichia coli divisome activation","date_created":"2022-03-31T11:32:32Z","article_processing_charge":"No","department":[{"_id":"GradSch"},{"_id":"MaLo"}]}]