[{"project":[{"name":"Self-Organization of the Bacterial Cell","call_identifier":"H2020","grant_number":"679239","_id":"2595697A-B435-11E9-9278-68D0E5697425"},{"grant_number":"P34607","_id":"fc38323b-9c52-11eb-aca3-ff8afb4a011d","name":"In vitro reconstitution of bacterial cell division"},{"name":"Synthesis of bacterial cell wall","_id":"2596EAB6-B435-11E9-9278-68D0E5697425","grant_number":"ALTF 2015-1163"},{"name":"Reconstitution of bacterial cell wall synthesis","_id":"259B655A-B435-11E9-9278-68D0E5697425","grant_number":"LT000824/2016"}],"has_accepted_license":"1","date_published":"2023-09-25T00:00:00Z","oa_version":"Published Version","year":"2023","date_updated":"2026-04-07T14:06:05Z","user_id":"ba8df636-2132-11f1-aed0-ed93e2281fdd","page":"156","status":"public","tmp":{"short":"CC BY (4.0)","legal_code_url":"https://creativecommons.org/licenses/by/4.0/legalcode","image":"/images/cc_by.png","name":"Creative Commons Attribution 4.0 International Public License (CC-BY 4.0)"},"type":"dissertation","day":"25","publisher":"Institute of Science and Technology Austria","ddc":["572"],"doi":"10.15479/at:ista:14280","OA_place":"publisher","alternative_title":["ISTA Thesis"],"_id":"14280","title":"Spatiotemporal signaling during assembly of the bacterial divisome","month":"09","article_processing_charge":"No","citation":{"ista":"Radler P. 2023. Spatiotemporal signaling during assembly of the bacterial divisome. Institute of Science and Technology Austria.","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>.","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>","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>","ieee":"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>.","short":"P. Radler, Spatiotemporal Signaling during Assembly of the Bacterial Divisome, Institute of Science and Technology Austria, 2023."},"related_material":{"record":[{"id":"10934","relation":"research_data","status":"public"},{"id":"11373","relation":"part_of_dissertation","status":"public"},{"status":"public","relation":"part_of_dissertation","id":"7387"}]},"file_date_updated":"2024-10-05T22:30:03Z","author":[{"full_name":"Radler, Philipp","last_name":"Radler","id":"40136C2A-F248-11E8-B48F-1D18A9856A87","first_name":"Philipp","orcid":"0000-0001-9198-2182 "}],"keyword":["Cell Division","Reconstitution","FtsZ","FtsA","Divisome","E.coli"],"ec_funded":1,"file":[{"embargo_to":"open_access","access_level":"closed","relation":"source_file","date_updated":"2024-10-05T22:30:03Z","checksum":"87eef11fbc5c7df0826f12a3a629b444","content_type":"application/vnd.openxmlformats-officedocument.wordprocessingml.document","file_size":114932847,"creator":"pradler","file_name":"PhD Thesis_Philipp Radler_20231004.docx","file_id":"14390","date_created":"2023-10-04T10:11:53Z"},{"creator":"pradler","file_name":"PhD Thesis_Philipp Radler_20231004.pdf","file_id":"14391","date_created":"2023-10-04T10:11:21Z","file_size":37838778,"content_type":"application/pdf","checksum":"3253e099b7126469d941fd9419d68b4f","date_updated":"2024-10-05T22:30:03Z","embargo":"2024-10-04","access_level":"open_access","relation":"main_file"}],"publication_identifier":{"isbn":["978-3-99078-033-6"],"issn":["2663-337X"]},"oa":1,"degree_awarded":"PhD","publication_status":"published","department":[{"_id":"GradSch"},{"_id":"MaLo"}],"date_created":"2023-09-06T10:58:25Z","corr_author":"1","language":[{"iso":"eng"}],"license":"https://creativecommons.org/licenses/by/4.0/","abstract":[{"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.","lang":"eng"}],"supervisor":[{"last_name":"Loose","full_name":"Loose, Martin","first_name":"Martin","id":"462D4284-F248-11E8-B48F-1D18A9856A87","orcid":"0000-0001-7309-9724"}],"acknowledged_ssus":[{"_id":"Bio"},{"_id":"LifeSc"}]},{"tmp":{"short":"CC BY (4.0)","legal_code_url":"https://creativecommons.org/licenses/by/4.0/legalcode","image":"/images/cc_by.png","name":"Creative Commons Attribution 4.0 International Public License (CC-BY 4.0)"},"status":"public","publisher":"eLife Sciences Publications","day":"24","type":"journal_article","oa_version":"Published Version","year":"2021","date_updated":"2024-10-22T10:04:21Z","date_published":"2021-02-24T00:00:00Z","has_accepted_license":"1","project":[{"grant_number":"679239","_id":"2595697A-B435-11E9-9278-68D0E5697425","call_identifier":"H2020","name":"Self-Organization of the Bacterial Cell"},{"name":"Synthesis of bacterial cell wall","grant_number":"ALTF 2015-1163","_id":"2596EAB6-B435-11E9-9278-68D0E5697425"},{"_id":"259B655A-B435-11E9-9278-68D0E5697425","grant_number":"LT000824/2016","name":"Reconstitution of bacterial cell wall synthesis"}],"user_id":"4359f0d1-fa6c-11eb-b949-802e58b17ae8","title":"Real time monitoring of peptidoglycan synthesis by membrane-reconstituted penicillin binding proteins","month":"02","_id":"9243","citation":{"chicago":"Hernández-Rocamora, Víctor M., Natalia S. Baranova, Katharina Peters, Eefjan Breukink, Martin Loose, and Waldemar Vollmer. “Real Time Monitoring of Peptidoglycan Synthesis by Membrane-Reconstituted Penicillin Binding Proteins.” <i>ELife</i>. eLife Sciences Publications, 2021. <a href=\"https://doi.org/10.7554/eLife.61525\">https://doi.org/10.7554/eLife.61525</a>.","ista":"Hernández-Rocamora VM, Baranova NS, Peters K, Breukink E, Loose M, Vollmer W. 2021. Real time monitoring of peptidoglycan synthesis by membrane-reconstituted penicillin binding proteins. eLife. 10, 1–32.","ama":"Hernández-Rocamora VM, Baranova NS, Peters K, Breukink E, Loose M, Vollmer W. Real time monitoring of peptidoglycan synthesis by membrane-reconstituted penicillin binding proteins. <i>eLife</i>. 2021;10. doi:<a href=\"https://doi.org/10.7554/eLife.61525\">10.7554/eLife.61525</a>","apa":"Hernández-Rocamora, V. M., Baranova, N. S., Peters, K., Breukink, E., Loose, M., &#38; Vollmer, W. (2021). Real time monitoring of peptidoglycan synthesis by membrane-reconstituted penicillin binding proteins. <i>ELife</i>. eLife Sciences Publications. <a href=\"https://doi.org/10.7554/eLife.61525\">https://doi.org/10.7554/eLife.61525</a>","ieee":"V. M. Hernández-Rocamora, N. S. Baranova, K. Peters, E. Breukink, M. Loose, and W. Vollmer, “Real time monitoring of peptidoglycan synthesis by membrane-reconstituted penicillin binding proteins,” <i>eLife</i>, vol. 10. eLife Sciences Publications, 2021.","short":"V.M. Hernández-Rocamora, N.S. Baranova, K. Peters, E. Breukink, M. Loose, W. Vollmer, ELife 10 (2021).","mla":"Hernández-Rocamora, Víctor M., et al. “Real Time Monitoring of Peptidoglycan Synthesis by Membrane-Reconstituted Penicillin Binding Proteins.” <i>ELife</i>, vol. 10, 1–32, eLife Sciences Publications, 2021, doi:<a href=\"https://doi.org/10.7554/eLife.61525\">10.7554/eLife.61525</a>."},"article_processing_charge":"No","ddc":["570"],"doi":"10.7554/eLife.61525","article_type":"original","oa":1,"publication_identifier":{"eissn":["2050-084X"]},"file":[{"relation":"main_file","access_level":"open_access","success":1,"date_updated":"2021-03-22T07:36:08Z","content_type":"application/pdf","checksum":"79897a09bfecd9914d39c4aea2841855","file_size":2314698,"file_id":"9268","date_created":"2021-03-22T07:36:08Z","file_name":"2021_eLife_HernandezRocamora.pdf","creator":"dernst"}],"acknowledgement":"We thank Alexander Egan (Newcastle University) for purified proteins LpoB(sol) and LpoPPa(sol), Federico Corona (Newcastle University) for purified MepM, and Oliver Birkholz and Jacob Piehler (Department of Biology and Center of Cellular Nanoanalytics, University of Osnabru¨ ck) for their help with PBP1B reconstitution into polymer-SLBs and initial guidance on single particle tracking. We also acknowledge Christian P Richter and Changjiang You (Department of Biology and Center of Cellular Nanoanalytics, University of Osnabru¨ ck) for providing SLIMfast software and tris-DODA-NTA reagent, respectively. This work was funded by the BBSRC grant BB/R017409/1 (to WV), the European Research Council through grant ERC-2015-StG-679239 (to ML), and long-term fellowships HFSP LT 000824/2016-L4 and EMBO ALTF 1163–2015 (to NB). ","ec_funded":1,"intvolume":"        10","isi":1,"file_date_updated":"2021-03-22T07:36:08Z","publication":"eLife","scopus_import":"1","author":[{"first_name":"Víctor M.","full_name":"Hernández-Rocamora, Víctor M.","last_name":"Hernández-Rocamora"},{"orcid":"0000-0002-3086-9124","id":"38661662-F248-11E8-B48F-1D18A9856A87","first_name":"Natalia S.","full_name":"Baranova, Natalia S.","last_name":"Baranova"},{"first_name":"Katharina","last_name":"Peters","full_name":"Peters, Katharina"},{"last_name":"Breukink","full_name":"Breukink, Eefjan","first_name":"Eefjan"},{"orcid":"0000-0001-7309-9724","last_name":"Loose","full_name":"Loose, Martin","first_name":"Martin","id":"462D4284-F248-11E8-B48F-1D18A9856A87"},{"full_name":"Vollmer, Waldemar","last_name":"Vollmer","first_name":"Waldemar"}],"quality_controlled":"1","volume":10,"abstract":[{"lang":"eng","text":"Peptidoglycan is an essential component of the bacterial cell envelope that surrounds the cytoplasmic membrane to protect the cell from osmotic lysis. Important antibiotics such as β-lactams and glycopeptides target peptidoglycan biosynthesis. Class A penicillin-binding proteins (PBPs) are bifunctional membrane-bound peptidoglycan synthases that polymerize glycan chains and connect adjacent stem peptides by transpeptidation. How these enzymes work in their physiological membrane environment is poorly understood. Here, we developed a novel Förster resonance energy transfer-based assay to follow in real time both reactions of class A PBPs reconstituted in liposomes or supported lipid bilayers and applied this assay with PBP1B homologues from Escherichia coli, Pseudomonas aeruginosa, and Acinetobacter baumannii in the presence or absence of their cognate lipoprotein activator. Our assay will allow unravelling the mechanisms of peptidoglycan synthesis in a lipid-bilayer environment and can be further developed to be used for high-throughput screening for new antimicrobials."}],"article_number":"1-32","publication_status":"published","language":[{"iso":"eng"}],"external_id":{"isi":["000627596400001"]},"department":[{"_id":"MaLo"}],"date_created":"2021-03-14T23:01:33Z"},{"type":"journal_article","day":"20","publisher":"Springer Nature","status":"public","user_id":"4359f0d1-fa6c-11eb-b949-802e58b17ae8","page":"407-417","project":[{"_id":"2595697A-B435-11E9-9278-68D0E5697425","grant_number":"679239","name":"Self-Organization of the Bacterial Cell","call_identifier":"H2020"},{"name":"Reconstitution of bacterial cell wall synthesis","grant_number":"LT000824/2016","_id":"259B655A-B435-11E9-9278-68D0E5697425"},{"_id":"2596EAB6-B435-11E9-9278-68D0E5697425","grant_number":"ALTF 2015-1163","name":"Synthesis of bacterial cell wall"}],"date_published":"2020-01-20T00:00:00Z","oa_version":"Submitted Version","year":"2020","date_updated":"2026-04-28T22:30:27Z","article_processing_charge":"No","citation":{"apa":"Baranova, N. S., Radler, P., Hernández-Rocamora, V. M., Alfonso, C., Lopez Pelegrin, M. D., Rivas, G., … Loose, M. (2020). Diffusion and capture permits dynamic coupling between treadmilling FtsZ filaments and cell division proteins. <i>Nature Microbiology</i>. Springer Nature. <a href=\"https://doi.org/10.1038/s41564-019-0657-5\">https://doi.org/10.1038/s41564-019-0657-5</a>","ama":"Baranova NS, Radler P, Hernández-Rocamora VM, et al. Diffusion and capture permits dynamic coupling between treadmilling FtsZ filaments and cell division proteins. <i>Nature Microbiology</i>. 2020;5:407-417. doi:<a href=\"https://doi.org/10.1038/s41564-019-0657-5\">10.1038/s41564-019-0657-5</a>","ista":"Baranova NS, Radler P, Hernández-Rocamora VM, Alfonso C, Lopez Pelegrin MD, Rivas G, Vollmer W, Loose M. 2020. Diffusion and capture permits dynamic coupling between treadmilling FtsZ filaments and cell division proteins. Nature Microbiology. 5, 407–417.","chicago":"Baranova, Natalia S., Philipp Radler, Víctor M. Hernández-Rocamora, Carlos Alfonso, Maria D Lopez Pelegrin, Germán Rivas, Waldemar Vollmer, and Martin Loose. “Diffusion and Capture Permits Dynamic Coupling between Treadmilling FtsZ Filaments and Cell Division Proteins.” <i>Nature Microbiology</i>. Springer Nature, 2020. <a href=\"https://doi.org/10.1038/s41564-019-0657-5\">https://doi.org/10.1038/s41564-019-0657-5</a>.","mla":"Baranova, Natalia S., et al. “Diffusion and Capture Permits Dynamic Coupling between Treadmilling FtsZ Filaments and Cell Division Proteins.” <i>Nature Microbiology</i>, vol. 5, Springer Nature, 2020, pp. 407–17, doi:<a href=\"https://doi.org/10.1038/s41564-019-0657-5\">10.1038/s41564-019-0657-5</a>.","short":"N.S. Baranova, P. Radler, V.M. Hernández-Rocamora, C. Alfonso, M.D. Lopez Pelegrin, G. Rivas, W. Vollmer, M. Loose, Nature Microbiology 5 (2020) 407–417.","ieee":"N. S. Baranova <i>et al.</i>, “Diffusion and capture permits dynamic coupling between treadmilling FtsZ filaments and cell division proteins,” <i>Nature Microbiology</i>, vol. 5. Springer Nature, pp. 407–417, 2020."},"related_material":{"link":[{"relation":"press_release","url":"https://ist.ac.at/en/news/little-cell-big-cover-story/","description":"News on IST Homepage"}],"record":[{"id":"14280","relation":"dissertation_contains","status":"public"}]},"_id":"7387","title":"Diffusion and capture permits dynamic coupling between treadmilling FtsZ filaments and cell division proteins","month":"01","doi":"10.1038/s41564-019-0657-5","article_type":"letter_note","main_file_link":[{"open_access":"1","url":"http://europepmc.org/article/PMC/7048620"}],"acknowledgement":"We acknowledge members of the Loose laboratory at IST Austria for helpful discussions—in particular, P. Caldas for help with the treadmilling analysis, M. Jimenez, A. Raso and N. Ropero for providing Alexa Fluor 488- and Alexa Fluor 647-labelled FtsA for the MST and analytical ultracentrifugation experiments. We thank C. You for providing the DODA-tris-NTA phospholipids, as well as J. Piehler and C. Richter (Department of Biology, University of Osnabruck, Germany) for the SLIMfast single-molecule tracking software and help with the confinement analysis. We thank J. Errington and H. Murray (both at Newcastle University, UK) for critical reading of the manuscript, and J. Brugués (MPI-CBG and MPI-PKS, Dresden, Germany) for help with the MATLAB programming and reading of the manuscript. This work was supported by the European Research Council through grant ERC-2015-StG-679239 to M.L. and grants HFSP LT 000824/2016-L4 and EMBO ALTF 1163-2015 to N.B., a grant from the Ministry of Economy and Competitiveness of the Spanish Government (BFU2016-75471-C2-1-P) to C.A. and G.R., and a Wellcome Trust Senior Investigator award (101824/Z/13/Z) and a grant from the BBSRC (BB/R017409/1) to W.V.","pmid":1,"oa":1,"publication_identifier":{"issn":["2058-5276"]},"publication":"Nature Microbiology","scopus_import":"1","author":[{"first_name":"Natalia S.","id":"38661662-F248-11E8-B48F-1D18A9856A87","last_name":"Baranova","full_name":"Baranova, Natalia S.","orcid":"0000-0002-3086-9124"},{"first_name":"Philipp","id":"40136C2A-F248-11E8-B48F-1D18A9856A87","full_name":"Radler, Philipp","last_name":"Radler","orcid":"0000-0001-9198-2182 "},{"first_name":"Víctor M.","last_name":"Hernández-Rocamora","full_name":"Hernández-Rocamora, Víctor M."},{"first_name":"Carlos","full_name":"Alfonso, Carlos","last_name":"Alfonso"},{"first_name":"Maria D","id":"319AA9CE-F248-11E8-B48F-1D18A9856A87","last_name":"Lopez Pelegrin","full_name":"Lopez Pelegrin, Maria D"},{"first_name":"Germán","last_name":"Rivas","full_name":"Rivas, Germán"},{"first_name":"Waldemar","last_name":"Vollmer","full_name":"Vollmer, Waldemar"},{"orcid":"0000-0001-7309-9724","full_name":"Loose, Martin","last_name":"Loose","id":"462D4284-F248-11E8-B48F-1D18A9856A87","first_name":"Martin"}],"ec_funded":1,"isi":1,"intvolume":"         5","abstract":[{"lang":"eng","text":"Most bacteria accomplish cell division with the help of a dynamic protein complex called the divisome, which spans the cell envelope in the plane of division. Assembly and activation of this machinery are coordinated by the tubulin-related GTPase FtsZ, which was found to form treadmilling filaments on supported bilayers in vitro1, as well as in live cells, in which filaments circle around the cell division site2,3. Treadmilling of FtsZ is thought to actively move proteins around the division septum, thereby distributing peptidoglycan synthesis and coordinating the inward growth of the septum to form the new poles of the daughter cells4. However, the molecular mechanisms underlying this function are largely unknown. Here, to study how FtsZ polymerization dynamics are coupled to downstream proteins, we reconstituted part of the bacterial cell division machinery using its purified components FtsZ, FtsA and truncated transmembrane proteins essential for cell division. We found that the membrane-bound cytosolic peptides of FtsN and FtsQ co-migrated with treadmilling FtsZ–FtsA filaments, but despite their directed collective behaviour, individual peptides showed random motion and transient confinement. Our work suggests that divisome proteins follow treadmilling FtsZ filaments by a diffusion-and-capture mechanism, which can give rise to a moving zone of signalling activity at the division site."}],"volume":5,"quality_controlled":"1","department":[{"_id":"MaLo"}],"external_id":{"pmid":["31959972"],"isi":["000508584700007"]},"date_created":"2020-01-28T16:14:41Z","corr_author":"1","language":[{"iso":"eng"}],"publication_status":"published"}]