FtsZ: interactions, anchoring and torsion
FtsZ is a protein we can find in prokaryotes. It is present in bacteria, archaea and even chloroplasts and some mitochondria (both believed to have a prokaryotic origin through symbiotic events in the early days of life). FtsZ is an old relative of animal tubulin, although it is more similar to actine in function—as far as we know.
When prokaryotic cells need to divide they gather a complex of proteins that form a constriction ring in the middle of the cell. The first protein identified in this system was FtsZ, which was given the name Filamenting temperature-sensitive mutant Z, because mutations that affected this protein impaired the division process and produced very long cells.
We know that FtsZ needs other proteins, FtsA or ZipA, to bind to the cellular membrane, and suspect that the Z-ring is responsible for producing the forces needed to split the cell in two. However, the process is yet very far from being understood, and we lack a satisfactory model to describe the division event.
As one can imagine, the complete picture is much more complicated than the simple description I have hinted above , even if we do not consider all the other systems and reactions that are taking place simultaneously in vivo. For this reason, we need simplified approaches to help us unravel the cell-division mystery.
We are working in close collaboration with the group of Marisela Vélez, who provide atomic force microscopy (AFM) images of FtsZ filaments anchored on flat surfaces. The advantage of this approach is that it greatly simplifies the experimental conditions, so that we can better understand FtsZ properties and then extrapolate that knowledge to more complicated situations.
However, AFM images proved too difficult to interpret by direct observation, specially considering the highly dynamic nature of the filaments, which continuously break and rearrange. Our goal is to develop simple models containing only the truly essential interactions between FtsZ monomers which can explain the AFM observations. The idea is that, if you are able to reproduce the results with only a few parameters, then the other interactions and details are not as essential at that level; this is very helpful information for biologists. We test our models in Montecarlo simulations.
The basic version of the model considers a longitudinal bond between monomers of similar orientation on the surface. This bond can have some curvature (with an energy cost depending on the direction of curvature). There is also a weaker isotropic (unspecific) interaction between monomers which promotes bundling. The monomers live on an hexagonal lattice finer than the proteins so that it allows for small steps in movement that will not necessarily break protein bonds .
In this image we see how experiment (a) and simulation compares. Snapshot (b) is missing the weaker isotropic interaction and also allows the filaments to overlap each other: the results are a clear disaster. In snapshot (c) we have excluded area, that is, filaments cannot cross over each other. The results are better, but we see how the weaker unspecific interaction is also needed to explain the aggregation of the filaments in more complex structures (d). In (e) we are missing curvature. In this way the model was used to identify the most basic properties of FtsZ filaments: longitudinal bond, isotropic weak interaction and curvature.
Strong Lateral Bond
My first research objective was to understand new aggregates that were being observed. The group of Marisela Vélez had begun working with FtsZ mutants that can directly bond to a lipid bilayer without need of FtsA or ZipA. These mutants offer some control over the orientation of the monomer with respect to the surface. The new experiments produced qualitatively new structures that could not be reproduced with the model above.
We considered a new interaction at specific angles between two monomers, not as strong as the longitudinal bond, not as weak as the isotropic interaction. This new interaction, called Strong Lateral Bond (SLB) can assume different variations in order to account for the different experimental conditions .
In this image we can see two different experimental set-ups, (a) and (c), and the new aggregates that are formed. In particular (c) is rather different from previous aggregates. Beside them we can see the results of the simulations including the new interaction, SLB, with the specific configuration shown in the inset. In this project we also studied the impact of the initial conditions of the system on the final aggregates, which can also affect the shape of the aggregates.
The SLB also explained some new details found in straight aggregates (found under other experimental conditions). For example, we see how the SLB favours covering the short ends of straight aggregates, something which
will not happen in the older version of the model.
But there were more problems with the FtsZ model. Some experiments showed filaments with a peculiar curvature distribution that could not be understood with models that assumed a simple preferential curvature. This is why we started thinking about torsion:
When an specific anchoring angle is favoured, all the monomers want to have the same angle with respect to the surface. If the filaments have a twist, this torsion will want the angle with respect to the surface to change from one monomer to another. Thus a competence arises and several compromises can be found—you can either sacrifice torsion and have well-anchored filaments (with a nice natural curvature) or you can prefer to sacrifice a bit of anchoring and have filaments with torsion, which will look straightish from a distance (like a stretched spring). This was also suspected by other groups analysing other conflicting data .
Molecular Dynamics studies by the group of Paulino Gómez-Puertas showed an FtsZ helix. From these simulations we extracted the bending and torsion angles of the bonds, as well as their variations. We used that data to build a model for the bond which could include the new torsion angle, and we studied how the bonds of the helical filament would behave if they were forced to remain on a surface (as in the experiments).
The new model predicted different results depending on the anchoring conditions: for a strong anchoring with the bending plane parallel to the surface we find filaments with high curvature. If the anchoring angle is perpendicular (so that filaments try to curve outwards from the surface) we find straight filaments and, in the case of a weaker anchoring, we see how torsion produces seemingly straight filaments. If we analyse the flexibility of the filaments by measuring the direction of each monomer, loosely-anchored filaments would appear to be more flexible, which could explain some previous observations regarding the flexibility of FtsZ filaments on different substrates.
According to this model filaments will produce some stress on the surface depending on the preferred anchoring angle, and this could contribute to the generation of force in the division of the cell .
After the previous exploration of the model in ideal filaments, we implemented the new bond model into the fine-grained model for Montecarlo simulations. We see how FtsZ filament-rings increase their curvature as the anchoring angle changes, up to the point when open filaments with different degrees of curvature are found. This more complete model also produces coexistence between long and short filaments, which is always observed in AFM images but was elusive in the results from the simpler model.
Under certain experimental conditions the different lipids used for the membrane segregate and form domains. This results in an unexpected polymorphism of the protein aggregates which cannot be explained by the previous model.
Segregation of lipids sometimes happen in vivo, and this domains and rafts have received a lot of attention recently, as they have been linked to
many biological processes.
Previous attempts to describe lipid domains have included proteins as a rigid scaffold to guide lipid segregation . In our case the proteins are clearly playing a more dynamical role. For this reason we modified our fine-grained lattice model so that it could include two different lipid species (an anchoring lipid and a neutral lipid). Each cell of the lattice will now have a lipid density accounting for the mixture of anchoring and neutral lipids at that particular point. Lipids tend to segregate to minimize the energy of the system, while entropy tends to mix them. Temperature will decide which factor is more important.
We also included an interaction between monomers and lipids, representing the fact that monomers need one of the two lipid species to correctly anchor to the substrate.
Under this conditions we could see how lipids affect protein dynamics and vice versa. With a temperature closer to the critical temperature proteins will lead this interaction, whereas lipids lead with lower temperatures. Our results suggest that this interaction can stabilize certain lipid structures as hinted by the experimental results. We also learnt that lipid segregation can promote protein aggregation through a local increase in protein concentration. This could be of importance in vivo, where certain lipids are known to gather in the division region of the cell during
the division process.
From our research we can conclude that the details in anchoring of FtsZ have a huge impact on the resulting aggregates and their dynamics. Anchoring orientation and the intensity of this anchoring are usually taken for granted in division models, but our results show that it is an essential element in the system, specially if one tries to explain the constriction force as a direct result of the mechanical properties of the filaments.
On top of that, anchoring is a plausible mechanism for constriction-force regulation. Filaments attached with a natural bending orientation on the plane surface will not produce constriction forces, as will do filaments attached in a perpendicular (off-plane) direction.
Torsion and anchoring have antagonistic roles, since well-anchored filaments cannot have torsion. This results in richer aggregates. Subtle variations in the anchoring conditions can thus drive qualitative changes in the system. This may be exploited to regulate the division process.
Other features we have studied (SLB, lipid segregation) would seem to have an auxiliary role in the FtsZ dynamics, perhaps triggering the transition between different aggregation states or localizing and stabilizing the Z-ring, collaborating in this way with the regulation of the division process.
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