--- _id: '8341' abstract: - lang: eng text: "One of the most striking hallmarks of the eukaryotic cell is the presence of intracellular vesicles and organelles. Each of these membrane-enclosed compartments has a distinct composition of lipids and proteins, which is essential for accurate membrane traffic and homeostasis. Interestingly, their biochemical identities are achieved with the help\r\nof small GTPases of the Rab family, which cycle between GDP- and GTP-bound forms on the selected membrane surface. While this activity switch is well understood for an individual protein, how Rab GTPases collectively transition between states to generate decisive signal propagation in space and time is unclear. In my PhD thesis, I present\r\nin vitro reconstitution experiments with theoretical modeling to systematically study a minimal Rab5 activation network from bottom-up. We find that positive feedback based on known molecular interactions gives rise to bistable GTPase activity switching on system’s scale. Furthermore, we determine that collective transition near the critical\r\npoint is intrinsically stochastic and provide evidence that the inactive Rab5 abundance on the membrane can shape the network response. Finally, we demonstrate that collective switching can spread on the lipid bilayer as a traveling activation wave, representing a possible emergent activity pattern in endosomal maturation. Together, our\r\nfindings reveal new insights into the self-organization properties of signaling networks away from chemical equilibrium. Our work highlights the importance of systematic characterization of biochemical systems in well-defined physiological conditions. This way, we were able to answer long-standing open questions in the field and close the gap between regulatory processes on a molecular scale and emergent responses on system’s level." acknowledged_ssus: - _id: Bio - _id: LifeSc - _id: NanoFab acknowledgement: My thanks goes to the Loose lab members, BioImaging, Life Science and Nanofabrication Facilities and the wonderful international community at IST for sharing this experience with me. alternative_title: - ISTA Thesis article_processing_charge: No author: - first_name: Urban full_name: Bezeljak, Urban id: 2A58201A-F248-11E8-B48F-1D18A9856A87 last_name: Bezeljak orcid: 0000-0003-1365-5631 citation: ama: Bezeljak U. In vitro reconstitution of a Rab activation switch. 2020. doi:10.15479/AT:ISTA:8341 apa: Bezeljak, U. (2020). In vitro reconstitution of a Rab activation switch. Institute of Science and Technology Austria. https://doi.org/10.15479/AT:ISTA:8341 chicago: Bezeljak, Urban. “In Vitro Reconstitution of a Rab Activation Switch.” Institute of Science and Technology Austria, 2020. https://doi.org/10.15479/AT:ISTA:8341. ieee: U. Bezeljak, “In vitro reconstitution of a Rab activation switch,” Institute of Science and Technology Austria, 2020. ista: Bezeljak U. 2020. In vitro reconstitution of a Rab activation switch. Institute of Science and Technology Austria. mla: Bezeljak, Urban. In Vitro Reconstitution of a Rab Activation Switch. Institute of Science and Technology Austria, 2020, doi:10.15479/AT:ISTA:8341. short: U. Bezeljak, In Vitro Reconstitution of a Rab Activation Switch, Institute of Science and Technology Austria, 2020. date_created: 2020-09-08T08:53:53Z date_published: 2020-09-08T00:00:00Z date_updated: 2023-09-07T13:17:06Z day: '08' ddc: - '570' degree_awarded: PhD department: - _id: MaLo doi: 10.15479/AT:ISTA:8341 file: - access_level: closed checksum: 70871b335a595252a66c6bbf0824fb02 content_type: application/x-zip-compressed creator: dernst date_created: 2020-09-08T09:00:29Z date_updated: 2021-09-16T12:49:12Z file_id: '8342' file_name: 2020_Urban_Bezeljak_Thesis_TeX.zip file_size: 65246782 relation: source_file - access_level: open_access checksum: 59a62275088b00b7241e6ff4136434c7 content_type: application/pdf creator: dernst date_created: 2020-09-08T09:00:27Z date_updated: 2021-09-16T12:49:12Z file_id: '8343' file_name: 2020_Urban_Bezeljak_Thesis.pdf file_size: 31259058 relation: main_file file_date_updated: 2021-09-16T12:49:12Z has_accepted_license: '1' language: - iso: eng license: https://creativecommons.org/licenses/by-nc-sa/4.0/ month: '09' oa: 1 oa_version: Published Version page: '215' publication_identifier: issn: - 2663-337X publication_status: published publisher: Institute of Science and Technology Austria related_material: record: - id: '7580' relation: part_of_dissertation status: public status: public supervisor: - first_name: Martin full_name: Loose, Martin id: 462D4284-F248-11E8-B48F-1D18A9856A87 last_name: Loose orcid: 0000-0001-7309-9724 title: In vitro reconstitution of a Rab activation switch tmp: image: /images/cc_by_nc_sa.png legal_code_url: https://creativecommons.org/licenses/by-nc-sa/4.0/legalcode name: Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International (CC BY-NC-SA 4.0) short: CC BY-NC-SA (4.0) type: dissertation user_id: c635000d-4b10-11ee-a964-aac5a93f6ac1 year: '2020' ... --- _id: '7580' abstract: - lang: eng text: The eukaryotic endomembrane system is controlled by small GTPases of the Rab family, which are activated at defined times and locations in a switch-like manner. While this switch is well understood for an individual protein, how regulatory networks produce intracellular activity patterns is currently not known. Here, we combine in vitro reconstitution experiments with computational modeling to study a minimal Rab5 activation network. We find that the molecular interactions in this system give rise to a positive feedback and bistable collective switching of Rab5. Furthermore, we find that switching near the critical point is intrinsically stochastic and provide evidence that controlling the inactive population of Rab5 on the membrane can shape the network response. Notably, we demonstrate that collective switching can spread on the membrane surface as a traveling wave of Rab5 activation. Together, our findings reveal how biochemical signaling networks control vesicle trafficking pathways and how their nonequilibrium properties define the spatiotemporal organization of the cell. acknowledged_ssus: - _id: Bio - _id: LifeSc article_processing_charge: No article_type: original author: - first_name: Urban full_name: Bezeljak, Urban id: 2A58201A-F248-11E8-B48F-1D18A9856A87 last_name: Bezeljak orcid: 0000-0003-1365-5631 - first_name: Hrushikesh full_name: Loya, Hrushikesh last_name: Loya - first_name: Beata M full_name: Kaczmarek, Beata M id: 36FA4AFA-F248-11E8-B48F-1D18A9856A87 last_name: Kaczmarek - first_name: Timothy E. full_name: Saunders, Timothy E. last_name: Saunders - first_name: Martin full_name: Loose, Martin id: 462D4284-F248-11E8-B48F-1D18A9856A87 last_name: Loose orcid: 0000-0001-7309-9724 citation: ama: Bezeljak U, Loya H, Kaczmarek BM, Saunders TE, Loose M. Stochastic activation and bistability in a Rab GTPase regulatory network. Proceedings of the National Academy of Sciences. 2020;117(12):6504-6549. doi:10.1073/pnas.1921027117 apa: Bezeljak, U., Loya, H., Kaczmarek, B. M., Saunders, T. E., & Loose, M. (2020). Stochastic activation and bistability in a Rab GTPase regulatory network. Proceedings of the National Academy of Sciences. Proceedings of the National Academy of Sciences. https://doi.org/10.1073/pnas.1921027117 chicago: Bezeljak, Urban, Hrushikesh Loya, Beata M Kaczmarek, Timothy E. Saunders, and Martin Loose. “Stochastic Activation and Bistability in a Rab GTPase Regulatory Network.” Proceedings of the National Academy of Sciences. Proceedings of the National Academy of Sciences, 2020. https://doi.org/10.1073/pnas.1921027117. ieee: U. Bezeljak, H. Loya, B. M. Kaczmarek, T. E. Saunders, and M. Loose, “Stochastic activation and bistability in a Rab GTPase regulatory network,” Proceedings of the National Academy of Sciences, vol. 117, no. 12. Proceedings of the National Academy of Sciences, pp. 6504–6549, 2020. ista: Bezeljak U, Loya H, Kaczmarek BM, Saunders TE, Loose M. 2020. Stochastic activation and bistability in a Rab GTPase regulatory network. Proceedings of the National Academy of Sciences. 117(12), 6504–6549. mla: Bezeljak, Urban, et al. “Stochastic Activation and Bistability in a Rab GTPase Regulatory Network.” Proceedings of the National Academy of Sciences, vol. 117, no. 12, Proceedings of the National Academy of Sciences, 2020, pp. 6504–49, doi:10.1073/pnas.1921027117. short: U. Bezeljak, H. Loya, B.M. Kaczmarek, T.E. Saunders, M. Loose, Proceedings of the National Academy of Sciences 117 (2020) 6504–6549. date_created: 2020-03-12T05:32:26Z date_published: 2020-03-24T00:00:00Z date_updated: 2023-09-07T13:17:06Z day: '24' department: - _id: MaLo - _id: CaBe doi: 10.1073/pnas.1921027117 external_id: isi: - '000521821800040' intvolume: ' 117' isi: 1 issue: '12' language: - iso: eng main_file_link: - open_access: '1' url: https://doi.org/10.1101/776567 month: '03' oa: 1 oa_version: Preprint page: 6504-6549 project: - _id: 2599F062-B435-11E9-9278-68D0E5697425 grant_number: RGY0083/2016 name: Reconstitution of cell polarity and axis determination in a cell-free system publication: Proceedings of the National Academy of Sciences publication_identifier: eissn: - 1091-6490 issn: - 0027-8424 publication_status: published publisher: Proceedings of the National Academy of Sciences quality_controlled: '1' related_material: link: - description: News on IST Homepage relation: press_release url: https://ist.ac.at/en/news/proteins-as-molecular-switches/ record: - id: '8341' relation: dissertation_contains status: public scopus_import: '1' status: public title: Stochastic activation and bistability in a Rab GTPase regulatory network type: journal_article user_id: c635000d-4b10-11ee-a964-aac5a93f6ac1 volume: 117 year: '2020' ... --- _id: '7360' abstract: - lang: eng text: Inflammation, which is a highly regulated host response against danger signals, may be harmful if it is excessive and deregulated. Ideally, anti-inflammatory therapy should autonomously commence as soon as possible after the onset of inflammation, should be controllable by a physician, and should not systemically block beneficial immune response in the long term. We describe a genetically encoded anti-inflammatory mammalian cell device based on a modular engineered genetic circuit comprising a sensor, an amplifier, a “thresholder” to restrict activation of a positive-feedback loop, a combination of advanced clinically used biopharmaceutical proteins, and orthogonal regulatory elements that linked modules into the functional device. This genetic circuit was autonomously activated by inflammatory signals, including endogenous cecal ligation and puncture (CLP)-induced inflammation in mice and serum from a systemic juvenile idiopathic arthritis (sIJA) patient, and could be reset externally by a chemical signal. The microencapsulated anti-inflammatory device significantly reduced the pathology in dextran sodium sulfate (DSS)-induced acute murine colitis, demonstrating a synthetic immunological approach for autonomous anti-inflammatory therapy. article_processing_charge: No article_type: original author: - first_name: Anže full_name: Smole, Anže last_name: Smole - first_name: Duško full_name: Lainšček, Duško last_name: Lainšček - first_name: Urban full_name: Bezeljak, Urban id: 2A58201A-F248-11E8-B48F-1D18A9856A87 last_name: Bezeljak orcid: 0000-0003-1365-5631 - first_name: Simon full_name: Horvat, Simon last_name: Horvat - first_name: Roman full_name: Jerala, Roman last_name: Jerala citation: ama: Smole A, Lainšček D, Bezeljak U, Horvat S, Jerala R. A synthetic mammalian therapeutic gene circuit for sensing and suppressing inflammation. Molecular Therapy. 2017;25(1):102-119. doi:10.1016/j.ymthe.2016.10.005 apa: Smole, A., Lainšček, D., Bezeljak, U., Horvat, S., & Jerala, R. (2017). A synthetic mammalian therapeutic gene circuit for sensing and suppressing inflammation. Molecular Therapy. Elsevier. https://doi.org/10.1016/j.ymthe.2016.10.005 chicago: Smole, Anže, Duško Lainšček, Urban Bezeljak, Simon Horvat, and Roman Jerala. “A Synthetic Mammalian Therapeutic Gene Circuit for Sensing and Suppressing Inflammation.” Molecular Therapy. Elsevier, 2017. https://doi.org/10.1016/j.ymthe.2016.10.005. ieee: A. Smole, D. Lainšček, U. Bezeljak, S. Horvat, and R. Jerala, “A synthetic mammalian therapeutic gene circuit for sensing and suppressing inflammation,” Molecular Therapy, vol. 25, no. 1. Elsevier, pp. 102–119, 2017. ista: Smole A, Lainšček D, Bezeljak U, Horvat S, Jerala R. 2017. A synthetic mammalian therapeutic gene circuit for sensing and suppressing inflammation. Molecular Therapy. 25(1), 102–119. mla: Smole, Anže, et al. “A Synthetic Mammalian Therapeutic Gene Circuit for Sensing and Suppressing Inflammation.” Molecular Therapy, vol. 25, no. 1, Elsevier, 2017, pp. 102–19, doi:10.1016/j.ymthe.2016.10.005. short: A. Smole, D. Lainšček, U. Bezeljak, S. Horvat, R. Jerala, Molecular Therapy 25 (2017) 102–119. date_created: 2020-01-25T15:55:39Z date_published: 2017-01-01T00:00:00Z date_updated: 2021-01-12T08:13:14Z day: '01' ddc: - '570' department: - _id: MaLo doi: 10.1016/j.ymthe.2016.10.005 external_id: pmid: - '28129106' file: - access_level: open_access checksum: ea8b1b28606dd1edab7379ba4fa3641f content_type: application/pdf creator: dernst date_created: 2020-03-03T10:55:13Z date_updated: 2020-07-14T12:47:56Z file_id: '7561' file_name: 2017_MolecularTherapy_Smole.pdf file_size: 3404806 relation: main_file file_date_updated: 2020-07-14T12:47:56Z has_accepted_license: '1' intvolume: ' 25' issue: '1' language: - iso: eng license: https://creativecommons.org/licenses/by-nc-nd/4.0/ month: '01' oa: 1 oa_version: Published Version page: 102-119 pmid: 1 publication: Molecular Therapy publication_identifier: issn: - 1525-0016 publication_status: published publisher: Elsevier quality_controlled: '1' status: public title: A synthetic mammalian therapeutic gene circuit for sensing and suppressing inflammation tmp: image: /images/cc_by_nc_nd.png legal_code_url: https://creativecommons.org/licenses/by-nc-nd/4.0/legalcode name: Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0) short: CC BY-NC-ND (4.0) type: journal_article user_id: 2DF688A6-F248-11E8-B48F-1D18A9856A87 volume: 25 year: '2017' ... --- _id: '7361' abstract: - lang: eng text: Bistable switches are fundamental regulatory elements of complex systems, ranging from electronics to living cells. Designed genetic toggle switches have been constructed from pairs of natural transcriptional repressors wired to inhibit one another. The complexity of the engineered regulatory circuits can be increased using orthogonal transcriptional regulators based on designed DNA-binding domains. However, a mutual repressor-based toggle switch comprising DNA-binding domains of transcription-activator-like effectors (TALEs) did not support bistability in mammalian cells. Here, the challenge of engineering a bistable switch based on monomeric DNA-binding domains is solved via the introduction of a positive feedback loop composed of activators based on the same TALE domains as their opposing repressors and competition for the same DNA operator site. This design introduces nonlinearity and results in epigenetic bistability. This principle could be used to employ other monomeric DNA-binding domains such as CRISPR for applications ranging from reprogramming cells to building digital biological memory. article_number: '5007' article_processing_charge: No article_type: original author: - first_name: Tina full_name: Lebar, Tina last_name: Lebar - first_name: Urban full_name: Bezeljak, Urban id: 2A58201A-F248-11E8-B48F-1D18A9856A87 last_name: Bezeljak orcid: 0000-0003-1365-5631 - first_name: Anja full_name: Golob, Anja last_name: Golob - first_name: Miha full_name: Jerala, Miha last_name: Jerala - first_name: Lucija full_name: Kadunc, Lucija last_name: Kadunc - first_name: Boštjan full_name: Pirš, Boštjan last_name: Pirš - first_name: Martin full_name: Stražar, Martin last_name: Stražar - first_name: Dušan full_name: Vučko, Dušan last_name: Vučko - first_name: Uroš full_name: Zupančič, Uroš last_name: Zupančič - first_name: Mojca full_name: Benčina, Mojca last_name: Benčina - first_name: Vida full_name: Forstnerič, Vida last_name: Forstnerič - first_name: Rok full_name: Gaber, Rok last_name: Gaber - first_name: Jan full_name: Lonzarić, Jan last_name: Lonzarić - first_name: Andreja full_name: Majerle, Andreja last_name: Majerle - first_name: Alja full_name: Oblak, Alja last_name: Oblak - first_name: Anže full_name: Smole, Anže last_name: Smole - first_name: Roman full_name: Jerala, Roman last_name: Jerala citation: ama: Lebar T, Bezeljak U, Golob A, et al. A bistable genetic switch based on designable DNA-binding domains. Nature Communications. 2014;5(1). doi:10.1038/ncomms6007 apa: Lebar, T., Bezeljak, U., Golob, A., Jerala, M., Kadunc, L., Pirš, B., … Jerala, R. (2014). A bistable genetic switch based on designable DNA-binding domains. Nature Communications. Springer Nature. https://doi.org/10.1038/ncomms6007 chicago: Lebar, Tina, Urban Bezeljak, Anja Golob, Miha Jerala, Lucija Kadunc, Boštjan Pirš, Martin Stražar, et al. “A Bistable Genetic Switch Based on Designable DNA-Binding Domains.” Nature Communications. Springer Nature, 2014. https://doi.org/10.1038/ncomms6007. ieee: T. Lebar et al., “A bistable genetic switch based on designable DNA-binding domains,” Nature Communications, vol. 5, no. 1. Springer Nature, 2014. ista: Lebar T, Bezeljak U, Golob A, Jerala M, Kadunc L, Pirš B, Stražar M, Vučko D, Zupančič U, Benčina M, Forstnerič V, Gaber R, Lonzarić J, Majerle A, Oblak A, Smole A, Jerala R. 2014. A bistable genetic switch based on designable DNA-binding domains. Nature Communications. 5(1), 5007. mla: Lebar, Tina, et al. “A Bistable Genetic Switch Based on Designable DNA-Binding Domains.” Nature Communications, vol. 5, no. 1, 5007, Springer Nature, 2014, doi:10.1038/ncomms6007. short: T. Lebar, U. Bezeljak, A. Golob, M. Jerala, L. Kadunc, B. Pirš, M. Stražar, D. Vučko, U. Zupančič, M. Benčina, V. Forstnerič, R. Gaber, J. Lonzarić, A. Majerle, A. Oblak, A. Smole, R. Jerala, Nature Communications 5 (2014). date_created: 2020-01-25T15:57:17Z date_published: 2014-09-29T00:00:00Z date_updated: 2021-01-12T08:13:15Z day: '29' doi: 10.1038/ncomms6007 extern: '1' external_id: pmid: - '25264186' intvolume: ' 5' issue: '1' language: - iso: eng month: '09' oa_version: None pmid: 1 publication: Nature Communications publication_identifier: issn: - 2041-1723 publication_status: published publisher: Springer Nature quality_controlled: '1' status: public title: A bistable genetic switch based on designable DNA-binding domains type: journal_article user_id: 2DF688A6-F248-11E8-B48F-1D18A9856A87 volume: 5 year: '2014' ...