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Report

Teaser, summary, work performed and final results

Periodic Reporting for period 2 - SELFORGANICELL (Self-Organization of the Bacterial Cell)

Teaser

Summary

Even though bacteria have been studied for a long time, the mechanism of bacterial cell division is still not clear. With the rise of antibiotic resistance, however, a mechanistic understanding of how cells grow and divide is required to develop new drugs inhibiting bacterial cell division.

The aim of this project is to understand how bacteria divide, specifically the proteins required for cell division are able to self-organize into a complex machinery that splits the cell into two. For this aim, we are following an in vitro reconstitution approach, where we purify the components required for this process to rebuild complex cellular behavior from the bottom up.
This approach, combined with fluorescence microscopy and image analysis allows us to understand in detail the role of individual players for cell division. This project will help us understand how these proteins contribute to the spatiotemporal organization of the cell division machinery and its emerging.

We currently focus on three major questions:

1. How do the components of the cell division machinery modulate the architecture and dynamics of the Z-ring?
Cell division in bacteria depends on a cytoskeletal structure called the Z-ring, which is built by the prokaryotic tubulin FtsZ and FtsA. Bacteria contain several proteins that directly bind to FtsZ to stabilize the Z-ring and to increase the precision of division. Despite their important role, how they achieve this task is currently unclear, mainly because mechanistic studies are difficult to perform in the living cell. In this project, we are studying four previously described proteins from Escherichia coli known to bind directly to FtsZ. Even though all four of them were thought to have overlapping functions, they are structurally unrelated, raising the questions about their distinct roles for Z-ring assembly and cell division. Our in vitro assay allows studying the influence of these proteins on the Z-ring, their respective function for cell division outside the complexity of the living cell.

2. How does the Z-ring organize cell division machinery?
The Z-ring has long been known to play a central role for the assembly of the cell division machinery. Despite being highly dynamic, this cytoskeletal structure acts a scaffold that recruits proteins to the site of cell division. How this assembly process works on a molecular level however is not clear, mainly because a visualization of the underlying events in vivo at a sufficiently high spatiotemporal resolution has not yet been achieved. Accordingly, we currently do not know how proteins come together to build the cell division machinery. Using an in vitro reconstitution approach and high speed single molecule imaging, we were able to bypass these limitations. This has helped us to promote a new model for how the cell division machinery is assembled in the living cell.

3. How do enzymes build the cell wall?
The shape of bacteria is defined by the cell wall that represents a two-dimensional polymer surrounding the cell. The proteins required to build and remodel this structure are commonly the target of antibiotic drugs, as inhibiting them often leads to cell death.
Despite the pharmacological importance of the cell wall, how this structure is built from its starting material is currently unclear, mainly because we miss assays that would allow us to follow the underlying reactions in real time.
In this project, we are developing novel microscopy and spectroscopy assays that allow us to closely follow the biochemical reactions that give rise to the peptidoglycan layer surrounding the bacterial cell.

In sum, we believe that our aim of rebuilding the cell division machinery in vitro will not only help to understand this complex process better, but also to identify new targets and approaches for antibiotic therapies.

Work performed

Since the start of this project we made significant progress regarding all objectives of the grant proposal (â€œThe biochemical network underlying bacterial divisome assemblyâ€) and two research papers summarizing our results are under review or in revision.

First, we have developed an in vitro assay recapitulating cytokinesis signaling in E. coli, and now have a much better understanding for how how treadmilling filaments of FtsZ are able to spatiotemporally organize division proteins on the membrane. Using purified components combined with fluorescence microscopy and single-molecule imaging, we discovered that the cell division machinery self-organizes by a diffusion-and-capture mechanism. Strikingly, this mechanism gives rise to an apparent directional co-migration of cell division proteins: treadmilling filaments of FtsZ and FtsA drive the movement of the membrane proteins FtsN and FtsQ in a coordinated, synchronous fashion by the continuous hydrolysis of GTP. Our data suggests that FtsZ filaments act as a dynamic scaffold, which locally increases the concentration of cell division proteins. This establishes a confined zone of signaling activity on the membrane that initiates the assembly of the cell division machinery. FtsZ polymerization powers the movement of this signaling zone, providing directionality required for homogeneous cell wall synthesis at the cell division site.

Second, we have studied the influence of the FtsZ associated proteins ZapA and ZapB on the architecture and dynamics of FtsZ filaments. We have found that these proteins build a well-balanced system that is able to increase the spatial order of membrane-bound FtsZ filaments without slowing down the underlying polymerization dynamics. As FtsZ treadmilling was found to be essential for the homogeneous synthesis of septal peptidoglycan, our findings suggest a model where ZapA increases the spatial precision of cell division, without compromising cell division rate.

Finally, we focussed on the in vitro reconstitution of cell wall synthesis, where we were able to reconstitute full-length PBP1b into polymer supported lipid membranes preserving two dimensional diffusion of the protein and correct orientation between the enzyme and its substrate lipid II. We found that reconstituted PBP1b was active to synthesize peptidoglycan in this experimental assay, which was confirmed by a classical approach - an end-point HPLC assay with radioactively labelled lipid II. To detect peptidoglycan synthesis directly on supported lipid membrane, we developed an ensemble real-time FRET assay to monitor peptidoglycan synthesis. In this assay, lipid II labelled with two different fluorophores were mixed and polymerization and cross-linking of lipid II could be detected by an increase in FRET signal. In some cases, we could also detect clusters of lipid II, which were sensitive to amidase and cellosyl digestion, suggesting they represent polymerized and crosslinked glycan chains. Our current focus for this project is to develop novel methods to confirm the presence of a peptidoglycan layer on the membrane and to study the effect of PBP1b regulators, such as FtsN, PBP3 and LpoB as well as the formation of their complex on the fluid membrane.

Final results

In the course of this project, we have developed novel image analysis methods to analyse polymerization dynamics of filaments within dense cytoskeletal structures. Although we used this approach to quantitatively characterize growth and shrinkage of treadmilling FtsZ filaments in vitro, this approach is applicable to study the polymerization dynamics of other cytoskeletal systems as well. In contrast to previous methods, this method can be applied on any time-lapse movie of homogeneously labelled filaments as it does not require specific markers for polymer ends. It also allows to quantify both, growing and shrinking rates of dynamic cytoskeletal filaments. Furthermore, it can be easily extended with more complex analyses, for example to generate spatiotemporal maps of filament dynamics in living cells. In fact, combined with an appropriate detection procedure, the approach used here should also allow to visualize and track motion on every spatial and temporal scale, not only cytoskeletal structures in vivo and in vitro, but also crawling cells and migrating animals. To analyse multiple time-lapse movies of treadmilling filaments in an automated manner, we developed macros based on ImageJ plug-ins and Python. These are easy to use, require no programming knowledge and are available as supplementary material on Github: https://github.com/paulocaldas/Treadmilling-Speed-Analysis.