Periodic Reporting for period 4 - SELFORGANICELL (Self-Organization of the Bacterial Cell)
Okres sprawozdawczy: 2020-10-01 do 2021-03-31
Bacteria, like eukaryotic cells, have precisely regulated intracellular anatomies. Due to their small size, however, obtaining quantitative information about the underlying protein dynamics has been extremely difficult to obtain from experiments in living cells. A thorough understanding of the bacterial cell however is required for the development of new classes of antibiotic drugs, which is of critical importance given rapidly evolving antibiotic resistances.
For example, bacterial cell division is initiated and controlled by a cytoskeletal structure called the Z-ring, which is organized by treadmilling filaments of the tubulin homolog FtsZ. This Z-ring not only recruits other proteins to the division site, it is also thought to distribute cell wall synthesis around the diameter of the cell. Despite being essential for the life of bacteria and its important role as a pharmacological target, we still do not know how the bacterial cell division machinery works.
In this proposal, we used a “bottom-up” synthetic biology approach that combines protein biochemistry with high resolution microscopy and quantitative image analysis. Importantly, our in vitro reconstitution experiments aim to replicate physiological conditions, while still allowing for a high degree of experimental control. Using this approach, we were able to provide important new answers to three outstanding questions: 1. How do FtsZ associated proteins modulate the organization of FtsZ filaments? 2. How do treadmilling FtsZ filaments organize cell division proteins in space and time? 3. How do peptidoglycan synthases build the cell wall?
In summary, our work was able to provide a quantitative characterization of the emergent properties of the biochemical systems that control bacterial cell division. We thereby could provide mechanistic information otherwise hidden in the complexity of the living cell.
For example, bacterial cell division is initiated and controlled by a cytoskeletal structure called the Z-ring, which is organized by treadmilling filaments of the tubulin homolog FtsZ. This Z-ring not only recruits other proteins to the division site, it is also thought to distribute cell wall synthesis around the diameter of the cell. Despite being essential for the life of bacteria and its important role as a pharmacological target, we still do not know how the bacterial cell division machinery works.
In this proposal, we used a “bottom-up” synthetic biology approach that combines protein biochemistry with high resolution microscopy and quantitative image analysis. Importantly, our in vitro reconstitution experiments aim to replicate physiological conditions, while still allowing for a high degree of experimental control. Using this approach, we were able to provide important new answers to three outstanding questions: 1. How do FtsZ associated proteins modulate the organization of FtsZ filaments? 2. How do treadmilling FtsZ filaments organize cell division proteins in space and time? 3. How do peptidoglycan synthases build the cell wall?
In summary, our work was able to provide a quantitative characterization of the emergent properties of the biochemical systems that control bacterial cell division. We thereby could provide mechanistic information otherwise hidden in the complexity of the living cell.
Using an in vitro reconstitution approach my research group studied the mechanism of bacterial cell division. In the course of this project, we were able to provide answers on three important questions:
1. How do FtsZ associated proteins modulate the organization of FtsZ filaments? The Z-ring has long been known to act as a dynamic scaffold for the assembly of the bacterial cell division machinery. What governs the architecture, dynamics and stability of this cytoskeletal structure was unclear, but FtsZ-associated proteins (Zaps) had been suggested to play an important role. We used an in vitro reconstitution approach to study the influence of the well-conserved protein ZapA on the self-organization of FtsZ and FtsA on lipid membranes. Combining high-resolution fluorescence microscopy with novel quantitative image analysis (Caldas et al., 2020), we found that ZapA aligns FtsZ filaments and stabilizes filament bundles in a highly cooperative manner. Despite its strong influence on their large-scale organization, we found that ZapA binds only transiently to FtsZ filaments and has no effect on their treadmilling velocity. Our results led to a new model for how FtsZ-associated proteins can increase the precision and stability of bacterial cell division. Furthermore, we propose that the ultrasensitive response of FtsZ filaments to ZapA is important for the directional maturation of the cell division machinery
Relevant publications:
• Caldas, P., Radler, P., Sommer, C. & Loose, M. in Methods in Cell Biology (Academic Press, 2020).
• Caldas, P. et al. Cooperative ordering of treadmilling filaments in cytoskeletal networks of FtsZ and its crosslinker ZapA. Nat Commun 10, 5744 (2019).
2. How do treadmilling FtsZ filaments organize cell division proteins in space and time? The Z-ring not only defines the site of cell division, treadmilling of FtsZ filaments in this structure was also found to actively distribute cell wall synthesis around the cell thereby promoting the integrity of the new formed cell. To understand how FtsZ filament dynamics are coupled to cell wall synthesis, we reconstituted part of the bacterial cell division machinery using its purified components FtsZ, FtsA and truncated transmembrane proteins essential for cell division (Baranova et al., 2020). We found that the membrane-bound cytosolic peptides of FtsN and FtsQ co-migrated with treadmilling FtsZ-FtsA filaments. Remarkably, despite their directed motion on the ensemble level, individual peptides showed random diffusion and transient confinement. Our findings reveal that divisome proteins dynamically follow treadmilling FtsZ filaments by a diffusion-and-capture mechanism, which can help to establish a moving signaling zone at the division site in vivo. This work represents an important experimental demonstration for how transient interactions can lead to the self-organization of complex biological structures.
Relevant publications:
• Baranova, N. et al. Diffusion and capture permits dynamic coupling between treadmilling FtsZ filaments and cell division proteins. Nat Microbiol 16, 1–11 (2020).
• Baranova, N. & Loose, M. Single-molecule measurements to study polymerization dynamics of FtsZ-FtsA copolymers. Methods Cell Biol 137, 355–370 (2017).
3. How do peptidoglycan synthases build the cell wall? 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 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. We developed a novel FRET-based assay to follow in real time both reactions of class A PBPs reconstituted in liposomes or supported lipid bilayers and we demonstrate 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 allows 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 (Hernández-Rocamora, Baranova et al., eLife, 2020).
Relevant publications:
• Hernandez-Rocamora, V. M.*, Baranova, N.* et al. Real-time monitoring of peptidoglycan synthesis by membrane-reconstituted penicillin-binding proteins. eLife 10, (2021).
In sum, by rebuilding parts of the cell division machinery in vitro we were able to understand this complex process better, and identified new targets and approaches for antibiotic therapies.
1. How do FtsZ associated proteins modulate the organization of FtsZ filaments? The Z-ring has long been known to act as a dynamic scaffold for the assembly of the bacterial cell division machinery. What governs the architecture, dynamics and stability of this cytoskeletal structure was unclear, but FtsZ-associated proteins (Zaps) had been suggested to play an important role. We used an in vitro reconstitution approach to study the influence of the well-conserved protein ZapA on the self-organization of FtsZ and FtsA on lipid membranes. Combining high-resolution fluorescence microscopy with novel quantitative image analysis (Caldas et al., 2020), we found that ZapA aligns FtsZ filaments and stabilizes filament bundles in a highly cooperative manner. Despite its strong influence on their large-scale organization, we found that ZapA binds only transiently to FtsZ filaments and has no effect on their treadmilling velocity. Our results led to a new model for how FtsZ-associated proteins can increase the precision and stability of bacterial cell division. Furthermore, we propose that the ultrasensitive response of FtsZ filaments to ZapA is important for the directional maturation of the cell division machinery
Relevant publications:
• Caldas, P., Radler, P., Sommer, C. & Loose, M. in Methods in Cell Biology (Academic Press, 2020).
• Caldas, P. et al. Cooperative ordering of treadmilling filaments in cytoskeletal networks of FtsZ and its crosslinker ZapA. Nat Commun 10, 5744 (2019).
2. How do treadmilling FtsZ filaments organize cell division proteins in space and time? The Z-ring not only defines the site of cell division, treadmilling of FtsZ filaments in this structure was also found to actively distribute cell wall synthesis around the cell thereby promoting the integrity of the new formed cell. To understand how FtsZ filament dynamics are coupled to cell wall synthesis, we reconstituted part of the bacterial cell division machinery using its purified components FtsZ, FtsA and truncated transmembrane proteins essential for cell division (Baranova et al., 2020). We found that the membrane-bound cytosolic peptides of FtsN and FtsQ co-migrated with treadmilling FtsZ-FtsA filaments. Remarkably, despite their directed motion on the ensemble level, individual peptides showed random diffusion and transient confinement. Our findings reveal that divisome proteins dynamically follow treadmilling FtsZ filaments by a diffusion-and-capture mechanism, which can help to establish a moving signaling zone at the division site in vivo. This work represents an important experimental demonstration for how transient interactions can lead to the self-organization of complex biological structures.
Relevant publications:
• Baranova, N. et al. Diffusion and capture permits dynamic coupling between treadmilling FtsZ filaments and cell division proteins. Nat Microbiol 16, 1–11 (2020).
• Baranova, N. & Loose, M. Single-molecule measurements to study polymerization dynamics of FtsZ-FtsA copolymers. Methods Cell Biol 137, 355–370 (2017).
3. How do peptidoglycan synthases build the cell wall? 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 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. We developed a novel FRET-based assay to follow in real time both reactions of class A PBPs reconstituted in liposomes or supported lipid bilayers and we demonstrate 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 allows 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 (Hernández-Rocamora, Baranova et al., eLife, 2020).
Relevant publications:
• Hernandez-Rocamora, V. M.*, Baranova, N.* et al. Real-time monitoring of peptidoglycan synthesis by membrane-reconstituted penicillin-binding proteins. eLife 10, (2021).
In sum, by rebuilding parts of the cell division machinery in vitro we were able to understand this complex process better, and identified new targets and approaches for antibiotic therapies.
The Z-ring is formed from transient assemblies of treadmilling filaments that align on the membrane surface into a dynamic, coherent cytoskeletal structure19,46. The nature of these interactions, however, are so far not known. Previous approaches to visualize the dynamic lateral interactions between treadmilling filaments were limited by imaging techniques available. Fluorescence microscopy techniques such as TIRF and SPIM allow for fast imaging of protein dynamics, but their spatial resolution is not sufficient to resolve the behavior of individual filaments within dense cytoskeletal networks. Electron microscopy and standard atomic force microscopy experiments were able to shed light onto the organization of filaments at much higher resolution, but fast dynamic information is lost from these snapshots.
In first pilot experiments, we succeeded to use super-resolution STED (stimulated emission depletion) microscopy and use high-speed atomic force microscopy (HS-AFM) to visualize how treadmilling filaments laterally interact at high spatiotemporal resolution. This information will help us understand how dynamic, transient interactions contribute to the organization of membrane-bound FtsZ filaments from an isotropic, random distribution into a well-ordered, large structure. Following a quantitative characterization, we will develop a minimal model of active-matter to understand the emergence of order. In a collaboration with the group of Edouard Hannezo (IST Austria), we will develop Brownian dynamics simulations to recapitulate the observed behavior. We will establish a theoretical phase diagram, which can be quantitatively tested. Together, these experiments will not only offer us unprecedented insight into the dynamics of FtsZ protofilament interactions. Eventually, they will also help us understand the biophysical principles for how local behavior of nanometer-sized filaments results in the self-organization of a cytoskeletal structure on a much larger scale.
In first pilot experiments, we succeeded to use super-resolution STED (stimulated emission depletion) microscopy and use high-speed atomic force microscopy (HS-AFM) to visualize how treadmilling filaments laterally interact at high spatiotemporal resolution. This information will help us understand how dynamic, transient interactions contribute to the organization of membrane-bound FtsZ filaments from an isotropic, random distribution into a well-ordered, large structure. Following a quantitative characterization, we will develop a minimal model of active-matter to understand the emergence of order. In a collaboration with the group of Edouard Hannezo (IST Austria), we will develop Brownian dynamics simulations to recapitulate the observed behavior. We will establish a theoretical phase diagram, which can be quantitatively tested. Together, these experiments will not only offer us unprecedented insight into the dynamics of FtsZ protofilament interactions. Eventually, they will also help us understand the biophysical principles for how local behavior of nanometer-sized filaments results in the self-organization of a cytoskeletal structure on a much larger scale.