@phdthesis{20741,
  abstract     = {Life on Earth emerged when biomacromolecules were membrane-enclosed in a confined space where many essential chemical reactions were more likely to happen and thereby accelerate evolution. These kinds of membranes separated internal reactions from the outside chaos while staying flexible so that those primordial cells can move, adopt their shape and, most importantly, propagate. Such membrane plasticity still remains a defining feature of all modern cell types. This remarkable ability to change their shape is most prominently observed during their propagation (i.e., cell division). Throughout division, a cell undergoes drastic change in its shape, usually at the middle of the cell, pulling the two opposite membrane sides inward, closer to each other, and, finally, culminating in pinching off to separate the cell into two daughter cells. To achieve this, a cell needs to employ a protein machinery, usually termed divisome, that can coordinate all necessary intracellular processes with membrane remodelling and synthesis of other extracellular structures that decorate a cell. The focus of this dissertation is a membrane-remodelling FtsZ system that is present across all domains of life. FtsZ forms filaments that further self-organize into ring-like structures at the cell septum and together with other division proteins perform cell envelope synthesis and constriction. However, there are still knowledge gaps in our mechanistic understanding of division in both archaea and bacteria. My work presented in this dissertation centres around a simple yet not well understood question: How is the divisome positioned correctly at the mid-cell? To achieve the proper positioning, the divisome needs to (i) be recruited to the mid-cell and (ii) localized orthogonally to the long cell axis. I tackle these processes in two different systems by applying an in vitro biochemical bottom-up reconstitution approach. I use purified components of Haloferax volcanii and Escherichia coli divisome to explore how divisome is recruited to the mid-cell in archaea and how the Z-ring positions orthogonally to the long cell axis in bacteria, respectively. 

Firstly, I collaborate with archaeal cell and structural biologists to explore the assembly of early division proteins in two FtsZ-containing archaeon H. volcanii, a standard model system for understudied archaeal organisms. I particularly address the hierarchy of interactions that allow a tripartite complex formation (SepF-CdpB1-CdpB2) and how the hierarchy of interactions ultimately leads to the recruitment of FtsZ filaments to the septum. This part of work has been published in (Nußbaum et al., 2024). In collaboration with evolutionary biologists, I shed light on ancient features that archaeal divisome has retained to this day and also speculate on a property that it might have lost during the course of evolution. 

Next, I switch my attention to E. coli divisome. Particularly, I address the FtsZ’s intrinsic biophysical property that drives the Z-ring diameter, and thereby the perpendicular orientation of the Z-ring to the long cell axis based on suggested membrane curvature sensing mechanism (Vanhille-Campos et al., 2024). This property allows formation of different Z-ring diameters that match the variety of cell diameters present in prokaryotes. The results showcase that the distribution of charged amino acids in the intrinsically disordered linker at the C-terminus (CTL) of FtsZ is the major determining factor of Z-ring diameter with inter-CTL interactions as an underlying mechanism. 

Finally, I thoroughly explain the methodology I used to address the abovementioned projects, and I finish with a discussion on how early archaeal divisome assembly and curvature sensing mechanism in bacteria, at first sight unrelated topics, are interconnected and important groundwork for both fundamental and translational research. },
  author       = {Kojic, Marko},
  isbn         = {978-3-99078-073-2},
  issn         = {2663-337X},
  publisher    = {Institute of Science and Technology Austria},
  title        = {{Towards understanding the assembly mechanisms of the Z-ring in Archaea and Bacteria}},
  doi          = {10.15479/AT-ISTA-20741},
  year         = {2025},
}

@phdthesis{14280,
  abstract     = {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. 
A 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?
We 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. 
Following 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. 
We 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.},
  author       = {Radler, Philipp},
  isbn         = {978-3-99078-033-6},
  issn         = {2663-337X},
  keywords     = {Cell Division, Reconstitution, FtsZ, FtsA, Divisome, E.coli},
  pages        = {156},
  publisher    = {Institute of Science and Technology Austria},
  title        = {{Spatiotemporal signaling during assembly of the bacterial divisome}},
  doi          = {10.15479/at:ista:14280},
  year         = {2023},
}

@phdthesis{14510,
  abstract     = {Clathrin-mediated endocytosis (CME) is vital for the regulation of plant growth and
development by controlling plasma membrane protein composition and cargo uptake. CME
relies on the precise recruitment control of protein regulators for vesicle maturation and
release. During the early stages of endocytosis, an area of flat membrane is remodelled by
proteins to create a spherical vesicle against intracellular forces. After the Clathrin-coated
vesicle (CCV) is fully formed, scission machinery releases it from the plasma membrane,
and cargo proceeds for recycling or degradation through early endosomes / Trans Golgi
network. Protein machineries that mediate membrane bending and vesicle release in plants
are unknown. However, studies show, that plant endocytosis is actin independent, thus
indicating that plants utilize a unique mechanism to mediate membrane bending against highturgor pressure compared to other model systems. First, by using biochemical and advanced
live microscopy approaches we investigate the TPLATE complex, a plant-specific
endocytosis protein complex. We found that TPLATE is peripherally associated with
clathrin-coated vesicles and localises at the rim of endocytosis events. Next, our study of
plant Dynamin-related protein 1C (DRP1C), which was hypothesised previously to play a
role in vesicle release, shows the recruitment of the protein already at the early stages of
endocytosis. Moreover, DRP1C assembles into organised ring-like structures and is able to
induce membrane deformation and tubulation, suggesting its role also in membrane bending
during early CME. Based on the data from mammalian and yeast systems, plant DynaminRelated Proteins 2 and SH3P2 protein are strong candidates to be part of the plant vesicle
scission machinery; however, their precise role in plant CME has not been yet elucidated.
Here, we characterised DRP2s and SH3P2 roles in CME by combining high-resolution
imaging of endocytic events in vivo and protein characterisation. Although DRP2s and
SH3P2 arrive together during late CME and physically interact, genetic analysis using
∆sh3p1,2,3 mutant and complementation with non-DRP2-interacting SH3P2 variants suggest
that SH3P2 does not directly recruit DRP2s to the site of endocytosis. Summarising our
research, these observations provide new important insights into the mechanism of plant
CME and show that, despite plants posses many homologues of mammalian and yeast CME
components, they do not necessarily act in the same manner. },
  author       = {Gnyliukh, Nataliia},
  isbn         = {978-3-99078-037-4},
  issn         = {2663-337X},
  keywords     = {Clathrin-Mediated Endocytosis, vesicle scission, Dynamin-Related Protein 2, SH3P2, TPLATE complex, Total internal reflection fluorescence microscopy, Arabidopsis thaliana},
  pages        = {180},
  publisher    = {Institute of Science and Technology Austria},
  title        = {{Mechanism of clathrin-coated vesicle  formation during endocytosis in plants}},
  doi          = {10.15479/at:ista:14510},
  year         = {2023},
}

@phdthesis{8341,
  abstract     = {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
of 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
in 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
point 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
findings 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.},
  author       = {Bezeljak, Urban},
  issn         = {2663-337X},
  pages        = {215},
  publisher    = {Institute of Science and Technology Austria},
  title        = {{In vitro reconstitution of a Rab activation switch}},
  doi          = {10.15479/AT:ISTA:8341},
  year         = {2020},
}

@phdthesis{8358,
  abstract     = {During bacterial cell division, the tubulin-homolog FtsZ forms a ring-like structure at the center of the cell. This so-called Z-ring acts as a scaffold recruiting several division-related proteins to mid-cell and plays a key role in distributing proteins at the division site, a feature driven by the treadmilling motion of FtsZ filaments around the septum. What regulates the architecture, dynamics and stability of the Z-ring is still poorly understood, but FtsZ-associated proteins (Zaps) are known to play an important role. 
Advances in fluorescence microscopy and in vitro reconstitution experiments have helped to shed light into some of the dynamic properties of these complex systems, but methods that allow to collect and analyze large quantitative data sets of the underlying polymer dynamics are still missing.
Here, using an in vitro reconstitution approach, we studied how different Zaps affect FtsZ filament dynamics and organization into large-scale patterns, giving special emphasis to the role of the well-conserved protein ZapA. For this purpose, we use high-resolution fluorescence microscopy combined with novel image analysis workfows to study pattern organization and polymerization dynamics of active filaments. We quantified the influence of Zaps on FtsZ on three diferent spatial scales: the large-scale organization of the membrane-bound filament network, the underlying
polymerization dynamics and the behavior of single molecules.
We found that ZapA cooperatively increases the spatial order of the filament network, binds only transiently to FtsZ filaments and has no effect on filament length and treadmilling velocity. Our data provides a model for how FtsZ-associated proteins can increase the precision and stability of the bacterial cell division machinery in a
switch-like manner, without compromising filament dynamics. Furthermore, we believe that our automated quantitative methods can be used to analyze a large variety of dynamic cytoskeletal systems, using standard time-lapse
movies of homogeneously labeled proteins obtained from experiments in vitro or even inside the living cell.
},
  author       = {Dos Santos Caldas, Paulo R},
  isbn         = {978-3-99078-009-1},
  issn         = {2663-337X},
  pages        = {135},
  publisher    = {Institute of Science and Technology Austria},
  title        = {{Organization and dynamics of treadmilling filaments in cytoskeletal networks of FtsZ and its crosslinkers}},
  doi          = {10.15479/AT:ISTA:8358},
  year         = {2020},
}