---
OA_place: publisher
_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.
corr_author: '1'
date_created: 2020-09-08T08:53:53Z
date_published: 2020-09-08T00:00:00Z
date_updated: 2026-04-08T07:24:56Z
day: '08'
ddc:
- '570'
degree_awarded: PhD
department:
- _id: MaLo
doi: 10.15479/AT:ISTA:8341
file:
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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
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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: ba8df636-2132-11f1-aed0-ed93e2281fdd
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 of the United States of America. 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 of the United States of America.
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 of the United States
of America. 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 of the United States of America, vol.
117, no. 12. 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 of the United States of America. 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 of the
United States of America, vol. 117, no. 12, 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 of the United States of America 117 (2020)
6504–6549.
date_created: 2020-03-12T05:32:26Z
date_published: 2020-03-24T00:00:00Z
date_updated: 2026-04-08T07:24:55Z
day: '24'
department:
- _id: MaLo
- _id: CaBe
doi: 10.1073/pnas.1921027117
external_id:
isi:
- '000521821800040'
pmid:
- '32161136'
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
pmid: 1
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 of the United States
of America
publication_identifier:
eissn:
- 1091-6490
issn:
- 0027-8424
publication_status: published
publisher: 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: 2DF688A6-F248-11E8-B48F-1D18A9856A87
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: 2025-09-18T10:41:35Z
day: '01'
ddc:
- '570'
department:
- _id: MaLo
doi: 10.1016/j.ymthe.2016.10.005
external_id:
isi:
- '000391901600013'
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'
isi: 1
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: 317138e5-6ab7-11ef-aa6d-ffef3953e345
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'
...