ISTA Thesis

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

In vitro reconstitution of Escherichia coli divisome activation

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

A dynamic duo: Understanding the roles of FtsZ and FtsA for Escherichia coli cell division through in vitro approaches

Bacteria divide by binary fission. The protein machine responsible for this process is the divisome, a transient assembly of more than 30 proteins in and on the surface of the cytoplasmic membrane. Together, they constrict the cell envelope and remodel the peptidoglycan layer to eventually split the cell into two. For Escherichia coli, most molecular players involved in this process have probably been identified, but obtaining the quantitative information needed for a mechanistic understanding can often not be achieved from experiments in vivo alone. Since the discovery of the Z-ring more than 30 years ago, in vitro reconstitution experiments have been crucial to shed light on molecular processes normally hidden in the complex environment of the living cell. In this review, we summarize how rebuilding the divisome from purified components – or at least parts of it - have been instrumental to obtain the detailed mechanistic understanding of the bacterial cell division machinery that we have today

A novel non-invasive, non-conductive method for measuring respiration

Accompanying datasets to the article "A novel non-invasive, non-conductive method for measuring Respiration" published in the Journal of Sensors and Sensor Systems by the same authors as this data sets

In vitro reconstitution of Escherichia coli divisome activation (Nature Communications, (2022), 13, 1, (2635), 10.1038/s41467-022-30301-y)

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Chiral and nematic phases of flexible active filaments

The emergence of large-scale order in self-organized systems relies on local interactions between individual components. During bacterial cell division, the tubulin-homolog FtsZ polymerizes into treadmilling filaments that further assemble into a cytoskeletal ring. Although minimal in vitro assays have shown the striking self-organization capacity of FtsZ filaments, such as dynamic chiral assemblies, how these large-scale structures emerge and relate to individual filament properties remains poorly understood. To understand this quantitatively, we combined minimal chiral active matter simulations with biochemical reconstitution experiments. Using STED and TIRF microscopy as well as high-speed AFM, we imaged the behavior of FtsZ filaments on different spatial scales. Simulations and experiments revealed that filament density and flexibility define the local and global order of the system: At intermediate densities, flexible filaments organize into chiral rings and polar bands, while an effectively nematic organization dominates for high filament densities and for mutant filaments with increased rigidity. Our predicted phase diagram captured these features quantitatively, demonstrating how filament flexibility, density and chirality cooperate with activity to give rise to a large repertoire of collective behaviors. These properties are likely important for the dynamic organization of soft chiral matter, including that of treadmilling FtsZ filaments during bacterial cell division

Chiral and nematic phases of flexible active filaments

The emergence of large-scale order in self-organized systems relies on local interactions between individual components. During bacterial cell division, the tubulin-homolog FtsZ polymerizes into treadmilling filaments that further assemble into a cytoskeletal ring. Although minimal in vitro assays have shown the striking self-organization capacity of FtsZ filaments, such as dynamic chiral assemblies, how these large-scale structures emerge and relate to individual filament properties remains poorly understood. To understand this quantitatively, we combined minimal chiral active matter simulations with biochemical reconstitution experiments. Using STED and TIRF microscopy as well as high-speed AFM, we imaged the behavior of FtsZ filaments on different spatial scales. Simulations and experiments revealed that filament density and flexibility define the local and global order of the system: At intermediate densities, flexible filaments organize into chiral rings and polar bands, while an effectively nematic organization dominates for high filament densities and for mutant filaments with increased rigidity. Our predicted phase diagram captured these features quantitatively, demonstrating how filament flexibility, density and chirality cooperate with activity to give rise to a large repertoire of collective behaviors. These properties are likely important for the dynamic organization of soft chiral matter, including that of treadmilling FtsZ filaments during bacterial cell division

Diffusion and capture permits dynamic coupling between treadmilling FtsZ filaments and cell division proteins

60 p.-11 fig.-3 tab.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.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.Peer reviewe

In vitro reconstitution of Escherichia coli divisome activation

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