@misc{13116, abstract = {The emergence of large-scale order in self-organized systems relies on local interactions between individual components. During bacterial cell division, FtsZ -- a prokaryotic homologue of the eukaryotic protein tubulin -- polymerizes into treadmilling filaments that further organize into a cytoskeletal ring. In vitro, FtsZ filaments can form dynamic chiral assemblies. However, how the active and passive properties of individual filaments relate to these large-scale self-organized structures remains poorly understood. Here, we connect single filament properties with the mesoscopic scale by combining minimal active matter simulations and biochemical reconstitution experiments. We show that density and flexibility of active chiral filaments define their global order. At intermediate densities, curved, flexible filaments organize into chiral rings and polar bands. An effectively nematic organization dominates for high densities and for straight, mutant filaments with increased rigidity. Our predicted phase diagram captures these features quantitatively, demonstrating how the flexibility, density and chirality of active filaments affect their collective behaviour. Our findings shed light on the fundamental properties of active chiral matter and explain how treadmilling FtsZ filaments organize during bacterial cell division. }, author = {Dunajova, Zuzana and Prats Mateu, Batirtze and Radler, Philipp and Lim, Keesiang and Brandis, Dörte and Velicky, Philipp and Danzl, Johann G and Wong, Richard W. and Elgeti, Jens and Hannezo, Edouard B and Loose, Martin}, publisher = {Institute of Science and Technology Austria}, title = {{Chiral and nematic phases of flexible active filaments}}, doi = {10.15479/AT:ISTA:13116}, year = {2023}, } @article{13314, abstract = {The emergence of large-scale order in self-organized systems relies on local interactions between individual components. During bacterial cell division, FtsZ—a prokaryotic homologue of the eukaryotic protein tubulin—polymerizes into treadmilling filaments that further organize into a cytoskeletal ring. In vitro, FtsZ filaments can form dynamic chiral assemblies. However, how the active and passive properties of individual filaments relate to these large-scale self-organized structures remains poorly understood. Here we connect single-filament properties with the mesoscopic scale by combining minimal active matter simulations and biochemical reconstitution experiments. We show that the density and flexibility of active chiral filaments define their global order. At intermediate densities, curved, flexible filaments organize into chiral rings and polar bands. An effectively nematic organization dominates for high densities and for straight, mutant filaments with increased rigidity. Our predicted phase diagram quantitatively captures these features, demonstrating how the flexibility, density and chirality of the active filaments affect their collective behaviour. Our findings shed light on the fundamental properties of active chiral matter and explain how treadmilling FtsZ filaments organize during bacterial cell division.}, author = {Dunajova, Zuzana and Prats Mateu, Batirtze and Radler, Philipp and Lim, Keesiang and Brandis, Dörte and Velicky, Philipp and Danzl, Johann G and Wong, Richard W. and Elgeti, Jens and Hannezo, Edouard B and Loose, Martin}, issn = {1745-2481}, journal = {Nature Physics}, pages = {1916--1926}, publisher = {Springer Nature}, title = {{Chiral and nematic phases of flexible active filaments}}, doi = {10.1038/s41567-023-02218-w}, volume = {19}, year = {2023}, } @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}, } @misc{10934, abstract = {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. }, author = {Radler, Philipp}, keywords = {Bacterial cell division, in vitro reconstitution, FtsZ, FtsN, FtsA}, publisher = {Institute of Science and Technology Austria}, title = {{In vitro reconstitution of Escherichia coli divisome activation}}, doi = {10.15479/AT:ISTA:10934}, year = {2022}, } @article{11373, abstract = {The actin-homologue FtsA is essential for E. coli cell division, as it links FtsZ filaments in the Z-ring to transmembrane proteins. FtsA is thought to initiate cell constriction by switching from an inactive polymeric to an active monomeric conformation, which recruits downstream proteins and stabilizes the Z-ring. However, direct biochemical evidence for this mechanism is missing. Here, we use reconstitution experiments and quantitative fluorescence microscopy to study divisome activation in vitro. By comparing wild-type FtsA with FtsA R286W, we find that this hyperactive mutant outperforms FtsA WT in replicating FtsZ treadmilling dynamics, FtsZ filament stabilization and recruitment of FtsN. We could attribute these differences to a faster exchange and denser packing of FtsA R286W below FtsZ filaments. Using FRET microscopy, we also find that FtsN binding promotes FtsA self-interaction. We propose that in the active divisome FtsA and FtsN exist as a dynamic copolymer that follows treadmilling filaments of FtsZ.}, author = {Radler, Philipp and Baranova, Natalia S. and Dos Santos Caldas, Paulo R and Sommer, Christoph M and Lopez Pelegrin, Maria D and Michalik, David and Loose, Martin}, issn = {2041-1723}, journal = {Nature Communications}, keywords = {General Physics and Astronomy, General Biochemistry, Genetics and Molecular Biology, General Chemistry}, publisher = {Springer Nature}, title = {{In vitro reconstitution of Escherichia coli divisome activation}}, doi = {10.1038/s41467-022-30301-y}, volume = {13}, year = {2022}, } @article{9243, abstract = {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.}, author = {Hernández-Rocamora, Víctor M. and Baranova, Natalia S. and Peters, Katharina and Breukink, Eefjan and Loose, Martin and Vollmer, Waldemar}, issn = {2050-084X}, journal = {eLife}, publisher = {eLife Sciences Publications}, title = {{Real time monitoring of peptidoglycan synthesis by membrane-reconstituted penicillin binding proteins}}, doi = {10.7554/eLife.61525}, volume = {10}, year = {2021}, } @article{9414, abstract = {Microtubule plus-end depolymerization rate is a potentially important target of physiological regulation, but it has been challenging to measure, so its role in spatial organization is poorly understood. Here we apply a method for tracking plus ends based on time difference imaging to measure depolymerization rates in large interphase asters growing in Xenopus egg extract. We observed strong spatial regulation of depolymerization rates, which were higher in the aster interior compared with the periphery, and much less regulation of polymerization or catastrophe rates. We interpret these data in terms of a limiting component model, where aster growth results in lower levels of soluble tubulin and microtubule-associated proteins (MAPs) in the interior cytosol compared with that at the periphery. The steady-state polymer fraction of tubulin was ∼30%, so tubulin is not strongly depleted in the aster interior. We propose that the limiting component for microtubule assembly is a MAP that inhibits depolymerization, and that egg asters are tuned to low microtubule density.}, author = {Ishihara, Keisuke and Decker, Franziska and Dos Santos Caldas, Paulo R and Pelletier, James F. and Loose, Martin and Brugués, Jan and Mitchison, Timothy J.}, issn = {1939-4586}, journal = {Molecular Biology of the Cell}, number = {9}, pages = {869--879}, publisher = {American Society for Cell Biology}, title = {{Spatial variation of microtubule depolymerization in large asters}}, doi = {10.1091/MBC.E20-11-0723}, volume = {32}, year = {2021}, } @article{9907, abstract = {DivIVA is a protein initially identified as a spatial regulator of cell division in the model organism Bacillus subtilis, but its homologues are present in many other Gram-positive bacteria, including Clostridia species. Besides its role as topological regulator of the Min system during bacterial cell division, DivIVA is involved in chromosome segregation during sporulation, genetic competence, and cell wall synthesis. DivIVA localizes to regions of high membrane curvature, such as the cell poles and cell division site, where it recruits distinct binding partners. Previously, it was suggested that negative curvature sensing is the main mechanism by which DivIVA binds to these specific regions. Here, we show that Clostridioides difficile DivIVA binds preferably to membranes containing negatively charged phospholipids, especially cardiolipin. Strikingly, we observed that upon binding, DivIVA modifies the lipid distribution and induces changes to lipid bilayers containing cardiolipin. Our observations indicate that DivIVA might play a more complex and so far unknown active role during the formation of the cell division septal membrane. }, author = {Labajová, Naďa and Baranova, Natalia S. and Jurásek, Miroslav and Vácha, Robert and Loose, Martin and Barák, Imrich}, issn = {1422-0067}, journal = {International Journal of Molecular Sciences}, number = {15}, publisher = {MDPI}, title = {{Cardiolipin-containing lipid membranes attract the bacterial cell division protein diviva}}, doi = {10.3390/ijms22158350}, volume = {22}, year = {2021}, } @inbook{7572, abstract = {The polymerization–depolymerization dynamics of cytoskeletal proteins play essential roles in the self-organization of cytoskeletal structures, in eukaryotic as well as prokaryotic cells. While advances in fluorescence microscopy and in vitro reconstitution experiments have helped to study the dynamic properties of these complex systems, methods that allow to collect and analyze large quantitative datasets of the underlying polymer dynamics are still missing. Here, we present a novel image analysis workflow to study polymerization dynamics of active filaments in a nonbiased, highly automated manner. Using treadmilling filaments of the bacterial tubulin FtsZ as an example, we demonstrate that our method is able to specifically detect, track and analyze growth and shrinkage of polymers, even in dense networks of filaments. We believe that this automated method can facilitate the analysis of a large variety of dynamic cytoskeletal systems, using standard time-lapse movies obtained from experiments in vitro as well as in the living cell. Moreover, we provide scripts implementing this method as supplementary material.}, author = {Dos Santos Caldas, Paulo R and Radler, Philipp and Sommer, Christoph M and Loose, Martin}, booktitle = {Methods in Cell Biology}, editor = {Tran, Phong }, issn = {0091-679X}, pages = {145--161}, publisher = {Elsevier}, title = {{Computational analysis of filament polymerization dynamics in cytoskeletal networks}}, doi = {10.1016/bs.mcb.2020.01.006}, volume = {158}, year = {2020}, } @article{7387, abstract = {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.}, author = {Baranova, Natalia S. and Radler, Philipp and Hernández-Rocamora, Víctor M. and Alfonso, Carlos and Lopez Pelegrin, Maria D and Rivas, Germán and Vollmer, Waldemar and Loose, Martin}, issn = {2058-5276}, journal = {Nature Microbiology}, pages = {407--417}, publisher = {Springer Nature}, title = {{Diffusion and capture permits dynamic coupling between treadmilling FtsZ filaments and cell division proteins}}, doi = {10.1038/s41564-019-0657-5}, volume = {5}, year = {2020}, } @article{7197, abstract = {During bacterial cell division, the tubulin-homolog FtsZ forms a ring-like structure at the center of the cell. This Z-ring not only organizes the division machinery, but treadmilling of FtsZ filaments was also found to play a key role in distributing proteins at the division site. What regulates the architecture, dynamics and stability of the Z-ring is currently unknown, but FtsZ-associated proteins are known to play an important role. Here, using an in vitro reconstitution approach, we studied how the well-conserved protein ZapA affects FtsZ treadmilling and filament organization into large-scale patterns. Using high-resolution fluorescence microscopy and quantitative image analysis, we found that ZapA cooperatively increases the spatial order of the filament network, but binds only transiently to FtsZ filaments and has no effect on filament length and treadmilling velocity. Together, 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.}, author = {Dos Santos Caldas, Paulo R and Lopez Pelegrin, Maria D and Pearce, Daniel J. G. and Budanur, Nazmi B and Brugués, Jan and Loose, Martin}, issn = {2041-1723}, journal = {Nature Communications}, publisher = {Springer Nature}, title = {{Cooperative ordering of treadmilling filaments in cytoskeletal networks of FtsZ and its crosslinker ZapA}}, doi = {10.1038/s41467-019-13702-4}, volume = {10}, year = {2019}, }