Division accuracy in a stochastic model of Min oscillations in Escherichia coli.
ABSTRACT: Accurate cell division in Escherichia coli requires the Min proteins MinC, MinD, and MinE as well as the presence of nucleoids. MinD and MinE exhibit spatial oscillations, moving from pole to pole of the bacterium, resulting in an average MinD concentration that is low at the center of the cell and high at the poles. This concentration minimum is thought to signal the site of cell division. Deterministic models of the Min oscillations reproduce many observed features of the system, including the concentration minimum of MinD. However, there are only a few thousand Min proteins in a bacterium, so stochastic effects are likely to play an important role. Here, we show that Monte Carlo simulations with a large number of proteins agree well with the results from a deterministic treatment of the equations. The location of minimum local MinD concentration is too variable to account for cell division accuracy in wild-type, but is consistent with the accuracy of cell division in cells without nucleoids. This finding confirms the need to include additional mechanisms, such as reciprocal interactions with the cell division ring or positioning of the nucleoids, to explain wild-type accuracy.
Project description:The E. coli?Min system forms a cell-pole-to-cell-pole oscillator that positions the divisome at mid-cell. The MinD ATPase binds the membrane and recruits the cell division inhibitor MinC. MinE interacts with and releases MinD (and MinC) from the membrane. The chase of MinD by MinE creates the in vivo oscillator that maintains a low level of the division inhibitor at mid-cell. In vitro reconstitution and visualization of Min proteins on a supported lipid bilayer has provided significant advances in understanding Min patterns in vivo. Here we studied the effects of flow, lipid composition, and salt concentration on Min patterning. Flow and no-flow conditions both supported Min protein patterns with somewhat different characteristics. Without flow, MinD and MinE formed spiraling waves. MinD and, to a greater extent MinE, have stronger affinities for anionic phospholipid. MinD-independent binding of MinE to anionic lipid resulted in slower and narrower waves. MinE binding to the bilayer was also more susceptible to changes in ionic strength than MinD. We find that modulating protein diffusion with flow, or membrane binding affinities with changes in lipid composition or salt concentration, can differentially affect the retention time of MinD and MinE, leading to spatiotemporal changes in Min patterning.
Project description:The Min protein system creates a dynamic spatial pattern in Escherichia coli cells where the proteins MinD and MinE oscillate from pole to pole. MinD positions MinC, an inhibitor of FtsZ ring formation, contributing to the mid-cell localization of cell division. In this paper, Fourier analysis is used to decompose experimental and model MinD spatial distributions into time-dependent harmonic components. In both experiment and model, the second harmonic component is responsible for producing a mid-cell minimum in MinD concentration. The features of this harmonic are robust in both experiment and model. Fourier analysis reveals a close correspondence between the time-dependent behaviour of the harmonic components in the experimental data and model. Given this, each molecular species in the model was analysed individually. This analysis revealed that membrane-bound MinD dimer shows the mid-cell minimum with the highest contrast when averaged over time, carrying the strongest signal for positioning the cell division ring. This concurs with previous data showing that the MinD dimer binds to MinC inhibiting FtsZ ring formation. These results show that non-linear interactions of Min proteins are essential for producing the mid-cell positioning signal via the generation of second-order harmonic components in the time-dependent spatial protein distribution.
Project description:Proper cell division requires an accurate definition of the division plane. In bacteria, this plane is determined by a polymeric ring of the FtsZ protein. The site of Z ring assembly in turn is controlled by the Min system, which suppresses FtsZ polymerization at noncentral membrane sites. The Min proteins in Escherichia coli undergo a highly dynamic localization cycle, during which they oscillate between the membrane of both cell halves. By using computer simulations we show that Min protein dynamics can be described accurately by using the following assumptions: (i) the MinD ATPase self-assembles on the membrane and recruits both MinC, an inhibitor of Z ring formation, and MinE, a protein required for MinC/MinD oscillation, (ii) a local accumulation of MinE is generated by a pattern formation reaction that is based on local self-enhancement and a long range antagonistic effect, and (iii) it displaces MinD from the membrane causing its own local destabilization and shift toward higher MinD concentrations. This local destabilization results in a wave of high MinE concentration traveling from the cell center to a pole, where it disappears. MinD reassembles on the membrane of the other cell half and attracts a new accumulation of MinE, causing a wave-like disassembly of MinD again. The result is a pole-to-pole oscillation of MinC/D. On time average, MinC concentration is highest at the poles, forcing FtsZ assembly to the center. The mechanism is self-organizing and does not require any other hypothetical topological determinant.
Project description:The Min proteins (MinC, MinD, and MinE) form a pole-to-pole oscillator that controls the spatial assembly of the division machinery in Escherichia coli cells. Previous studies identified that interactions of MinD with phospholipids positioned the Min machinery at the membrane. We extend these studies by measuring the affinity, kinetics, and ATPase activity of E. coli MinD, MinE, and MinDE binding to supported lipid bilayers containing varying compositions of anionic phospholipids. Using quartz crystal microbalance measurements, we found that the binding affinity (K(d)) for the interaction of recombinant E. coli MinD and MinE with lipid bilayers increased with increasing concentration of the anionic phospholipids phosphatidylglycerol and cardiolipin. The K(d) for MinD (1.8 ?M) in the presence of ATP was smaller than for MinE (12.1 ?M) binding to membranes consisting of 95:5 phosphatidylcholine/cardiolipin. The simultaneous binding of MinD and MinE to membranes revealed that increasing the concentration of anionic phospholipid stimulates the initial rate of adsorption (k(on)). The ATPase activity of MinD decreased in the presence of anionic phospholipids. These results indicate that anionic lipids, which are concentrated at the poles, increase the retention of MinD and MinE and explain its dwell time at this region of bacterial cells. These studies provide insight into interactions between MinD and MinE and between these proteins and membranes that are relevant to understanding the process of bacterial cell division, in which the interaction of proteins and membranes is essential.
Project description:The Escherichia coli Min system plays an important role in the proper placement of the septum ring at mid-cell during cell division. MinE forms a pole-to-pole spatial oscillator with the membrane-bound ATPase MinD, resulting in MinD concentration being the lowest at mid-cell. MinC, the direct inhibitor of the septum initiator protein FtsZ, forms a complex with MinD at the membrane, mirroring its polar gradients. Therefore, MinC-mediated FtsZ inhibition occurs away from mid-cell. Min oscillations are often studied in living cells by time-lapse microscopy using fluorescently labelled Min proteins. Here, we show that, despite permitting oscillations to occur in a range of protein concentrations, the enhanced yellow fluorescent protein (eYFP) C-terminally fused to MinE impairs its function. Combining in vivo, in vitro and in silico approaches, we demonstrate that eYFP compromises the ability of MinE to displace MinC from MinD, to stimulate MinD ATPase activity and to directly bind to the membrane. Moreover, we reveal that MinE-eYFP is prone to aggregation. In silico analyses predict that other fluorescent proteins are also likely to compromise several functionalities of MinE, suggesting that the results presented here are not specific to eYFP.
Project description:Oscillations of the Min protein system are involved in the correct midcell placement of the divisome during Escherichia coli cell division. Based on molecular interactions of the Min system, we formulated a mathematical model that reproduces Min patterning during cell growth and division. Specifically, the increase in the residence time of MinD attached to the membrane as its own concentration increases, is accounted for by dimerisation of membrane-bound MinD and its interaction with MinE. Simulation of this system generates unparalleled correlation between the waveshape of experimental and theoretical MinD distributions, suggesting that the dominant interactions of the physical system have been successfully incorporated into the model. For cells where MinD is fully-labelled with GFP, the model reproduces the stationary localization of MinD-GFP for short cells, followed by oscillations from pole to pole in larger cells, and the transition to the symmetric distribution during cell filamentation. Cells containing a secondary, GFP-labelled MinD display a contrasting pattern. The model is able to account for these differences, including temporary midcell localization just prior to division, by increasing the rate constant controlling MinD ATPase and heterotetramer dissociation. For both experimental conditions, the model can explain how cell division results in an equal distribution of MinD and MinE in the two daughter cells, and accounts for the temperature dependence of the period of Min oscillations. Thus, we show that while other interactions may be present, they are not needed to reproduce the main characteristics of the Min system in vivo.
Project description:The pole-to-pole oscillation of the Min proteins in Escherichia coli results in the inhibition of aberrant polar division, thus facilitating placement of the division septum at the midcell. MinE of the Min system forms a ring-like structure that plays a critical role in triggering the oscillation cycle. However, the mechanism underlying the formation of the MinE ring remains unclear. This study demonstrates that MinE self-assembles into fibrillar structures on the supported lipid bilayer. The MinD-interacting domain of MinE shows amyloidogenic properties, providing a possible mechanism for self-assembly of MinE. Supporting the idea, mutations in residues Ile-24 and Ile-25 of the MinD-interacting domain affect fibril formation, membrane binding ability of MinE and MinD, and subcellular localization of three Min proteins. Additional mutations in residues Ile-72 and Ile-74 suggest a role of the C-terminal domain of MinE in regulating the folding propensity of the MinD-interacting domain for different molecular interactions. The study suggests a self-assembly mechanism that may underlie the ring-like structure formed by MinE-GFP observed in vivo.
Project description:The bacterial cell division regulators MinD and MinE together with the division inhibitor MinC localize to the membrane in concentrated zones undergoing coordinated pole-to-pole oscillation to help ensure that the cytokinetic division septum forms only at the mid-cell position. This dynamic localization is driven by MinD-catalyzed ATP hydrolysis, stimulated by interactions with MinE's anti-MinCD domain. This domain is buried in the 6-?-stranded MinE "closed" structure, but is liberated for interactions with MinD, giving rise to a 4-?-stranded "open" structure through an unknown mechanism. Here we show that MinE-membrane interactions induce a structural change into a state resembling the open conformation. However, MinE mutants lacking the MinE membrane-targeting sequence stimulated higher ATP hydrolysis rates than the full-length protein, indicating that binding to MinD is sufficient to trigger this conformational transition in MinE. In contrast, conformational change between the open and closed states did not affect stimulation of ATP hydrolysis rates in the absence of membrane binding, although the MinD-binding residue Ile-25 is critical for this conformational transition. We therefore propose an updated model where MinE is brought to the membrane through interactions with MinD. After stimulation of ATP hydrolysis, MinE remains bound to the membrane in a state that does not catalyze additional rounds of ATP hydrolysis. Although the molecular basis for this inhibited state is unknown, previous observations of higher-order MinE self-association may explain this inhibition. Overall, our findings have general implications for Min protein oscillation cycles, including those that regulate cell division in bacterial pathogens.
Project description:Protein patterning is vital for many fundamental cellular processes. This raises two intriguing questions: Can such intrinsically complex processes be reduced to certain core principles and, if so, what roles do the molecular details play in individual systems? A prototypical example for protein patterning is the bacterial Min system, in which self-organized pole-to-pole oscillations of MinCDE proteins guide the cell division machinery to midcell. These oscillations are based on cycling of the ATPase MinD and its activating protein MinE between the membrane and the cytoplasm. Recent biochemical evidence suggests that MinE undergoes a reversible, MinD-dependent conformational switch from a latent to a reactive state. However, the functional relevance of this switch for the Min network and pattern formation remains unclear. By combining mathematical modeling and in vitro reconstitution of mutant proteins, we dissect the two aspects of MinE's switch, persistent membrane binding and a change in MinE's affinity for MinD. Our study shows that the MinD-dependent change in MinE's binding affinity for MinD is essential for patterns to emerge over a broad and physiological range of protein concentrations. Mechanistically, our results suggest that conformational switching of an ATPase-activating protein can lead to the spatial separation of its distinct functional states and thereby confer robustness on an intracellular protein network with vital roles in bacterial cell division.
Project description:MinD is a cell division ATPase in Escherichia coli that oscillates from pole to pole and regulates the spatial position of the cell division machinery. Together with MinC and MinE, the Min system restricts assembly of the FtsZ-ring to midcell, oscillating between the opposite ends of the cell and preventing FtsZ-ring misassembly at the poles. Here, we show that the ATP-dependent bacterial proteasome complex ClpXP degrades MinD in reconstituted degradation reactions in vitro and in vivo through direct recognition of the MinD N-terminal region. MinD degradation is enhanced during stationary phase, suggesting that ClpXP regulates levels of MinD in cells that are not actively dividing. ClpXP is a major regulator of growth phase-dependent proteins, and these results suggest that MinD levels are also controlled during stationary phase. In vitro, MinC and MinD are known to coassemble into linear polymers; therefore, we monitored copolymers assembled in vitro after incubation with ClpXP and observed that ClpXP promotes rapid MinCD copolymer destabilization and direct MinD degradation by ClpXP. The N terminus of MinD, including residue Arg 3, which is near the ATP-binding site in sequence, is critical for degradation by ClpXP. Together, these results demonstrate that ClpXP degradation modifies conformational assemblies of MinD in vitro and depresses Min function in vivo during periods of reduced proliferation.