Dynamic localization cycle of the cell division regulator MinE in Escherichia coli

The MinC protein directs placement of the division septum to the middle of Escherichia coli cells by blocking assembly of the division apparatus at other sites. MinD and MinE regulate MinC activity by modulating its cellular location in a unique fashion. MinD recruits MinC to the membrane, and MinE induces MinC/MinD to oscillate rapidly between the membrane of opposite cell halves. Using fixed cells, we previously found that a MinE-green fluorescent protein fusion accumulated in an annular structure at or near the midcell, as well as along the membrane on only one side of the ring. Here we show that in living cells, MinE undergoes a rapid localization cycle that appears coupled to MinD oscillation. The results show that MinE is not a fixed marker for septal ring assembly. Rather, they support a model in which MinE stimulates the removal of MinD from the membrane in a wave-like fashion. These waves run from a midcell position towards the poles in an alternating sequence such that the time-averaged concentration of division inhibitor is lowest at midcell.     

Direct MinE–membrane interaction contributes to the proper localization of MinDE in E. coli

Dynamic oscillation of the Min system in Escherichia coli determines the placement of the division plane at the midcell. In addition to stimulating MinD ATPase activity, we report here that MinE can directly interact with the membrane and this interaction contributes to the proper MinDE localization and dynamics. The N‐terminal domain of MinE is involved in direct contact between MinE and the membranes that may subsequently be stabilized by the C‐terminal domain of MinE. In an in vitro system, MinE caused liposome deformation into membrane tubules, a property similar to that previously reported for MinD. We isolated a mutant MinE containing residue substitutions in R10, K11 and K12 that was fully capable of stimulating MinD ATPase activity, but was deficient in membrane binding. Importantly, this mutant was unable to support normal MinDE localization and oscillation, suggesting that direct MinE interaction with the membrane is critical for the dynamic behavior of the Min system.     

ATP-dependent interactions between Escherichia coli Min proteins and the phospholipid membrane in vitro

Proper placement of the division apparatus in Escherichia coli requires pole-to-pole oscillation of the MinC division inhibitor. MinC dynamics involves a membrane association-dissociation cycle that is driven by the activities of the MinD ATPase and the MinE topological specificity factor, which themselves undergo coupled oscillatory localization cycles. To understand the biochemical mechanisms underlying Min protein dynamics, we studied the interactions of purified Min proteins with phospholipid vesicles and the role of ATP in these interactions. We show that (i) the ATP-bound form of MinD (MinD.ATP) readily associates with phospholipid vesicles in the presence of Mg(2+), whereas the ADP-bound form (MinD.ADP) does not; (ii) MinD.ATP binds membrane in a self-enhancing fashion; (iii) both MinC and MinE can be recruited to MinD.ATP-decorated vesicles; (iv) MinE stimulates dissociation of MinD.ATP from the membrane in a process requiring hydrolysis of the nucleotide; and (v) MinE stimulates dissociation of MinC from MinD.ATP-membrane complexes, even when ATP hydrolysis is blocked. The results support and extend recent work by Z. Hu et al. (Z. Hu, E. P. Gogol, and J. Lutkenhaus, Proc. Natl. Acad. Sci. USA 99:6761-6766, 2002) and support models of protein oscillation wherein MinE induces Min protein dynamics by stimulating the conversion of the membrane-bound form of MinD (MinD.ATP) to the cytoplasmic form (MinD.ADP). The results also indicate that MinE-stimulated dissociation of MinC from the MinC-MinD.ATP-membrane complex can, and may, occur prior to hydrolysis of the nucleotide.     

Mobility of Min-proteins in Escherichia coli measured by fluorescence correlation spectroscopy

In the bacterium Escherichia coli, selection of the division site involves pole-to-pole oscillations of the proteins MinD and MinE. Different oscillation mechanisms based on cooperative effects between Min-proteins and on the exchange of Min-proteins between the cytoplasm and the cytoplasmic membrane have been proposed. The parameters characterizing the dynamics of the Min-proteins in vivo are not known. It has therefore been difficult to compare the models quantitatively with experiments. Here, we present in vivo measurements of the mobility of MinD and MinE using fluorescence correlation spectroscopy. Two distinct timescales are clearly visible in the correlation curves. While the faster timescale can be attributed to cytoplasmic diffusion, the slower timescale could result from diffusion of membrane-bound proteins or from protein exchange between the cytoplasm and the membrane. We determine the diffusion constant of cytoplasmic MinD to be approximately 16 microm(2) s(-1), while for MinE we find about 10 microm(2) s(-1), independently of the processes responsible for the slower time-scale. The implications of the measured values for the oscillation mechanism are discussed.     

Differences in MinC/MinD sensitivity between polar and internal Z rings in Escherichia coli

In Escherichia coli the Z ring has the potential to assemble anywhere along the cell length but is restricted to midcell by the action of negative regulatory systems, including Min. In the current model for the Min system, the MinC/MinD division inhibitory complex is evenly distributed on the membrane and can disrupt Z rings anywhere in the cell; however, MinE spatially regulates MinC/MinD by restricting it to the cell poles, thus allowing Z ring formation at midcell. This model assumes that Z rings formed at different cellular locations have equal sensitivity to MinC/MinD in the absence of MinE. However, here we report evidence that differences in MinC/MinD sensitivity between polar and nonpolar Z rings exists even when there is no MinE. MinC/MinD at proper levels is able to block minicell production in Δmin strains without increasing the cell length, indicating that polar Z rings are preferentially blocked. In the FtsZ-I374V strain (which is resistant to MinC(C)/MinD), wild-type morphology can be easily achieved with MinC/MinD in the absence of MinE. We also show that MinC/MinD at proper levels can rescue the lethal phenotype of a min slmA double deletion mutant, which we think is due to the elimination of polar Z rings (or FtsZ structures), which frees up FtsZ molecules for assembly of Z rings at internal sites to rescue division and growth. Taken together, these data indicate that polar Z rings are more susceptible to MinC/MinD than internal Z rings, even when MinE is absent.     

Role of MinD-membrane association in Min protein interactions

Division site placement in Escherichia coli involves interactions of the MinD protein with MinC and MinE and with other MinD molecules to form membrane-associated polymeric structures. In this work, as part of a study of these interactions, we established that heterologous membrane-associated proteins such as MinD can be targeted to the yeast nuclear membrane, dependent only on the presence of a membrane-binding domain and a nuclear targeting sequence. Targeting to the nuclear membrane was equally effective using the intrinsic MinD membrane-targeting domain or the completely unrelated membrane-targeting domain of cytochrome b(5). The chimeric proteins differing in their membrane-targeting sequences were then used to establish the roles of membrane association and specificity of the membrane anchor in MinD interactions, using the yeast two-hybrid system. The chimeric proteins were also used to show that the membrane association of MinD and MinE in E. coli cells had no specificity for the membrane anchor, whereas formation of MinDE polar zones and MinE rings required the presence of the native MinD membrane-targeting sequence.     

The N terminus of MinD contains determinants which affect its dynamic localization and enzymatic activity

MinD is involved in regulating the proper placement of the cytokinetic machinery in some bacteria, including Neisseria gonorrhoeae and Escherichia coli. Stimulation of the ATPase activity of MinD by MinE has been proposed to induce dynamic, pole-to-pole oscillations of MinD in E. coli. Here, we investigated the effects of deleting or mutating conserved residues within the N terminus of N. gonorrhoeae MinD (MinD(Ng)) on protein dynamism, localization, and interactions with MinD(Ng) and with MinE(Ng). Deletions or mutations were generated in the first five residues of MinD(Ng), and mutant proteins were evaluated by several functional assays. Truncation or mutation of N-terminal residues disrupted MinD(Ng) interactions with itself and with MinE. Although the majority of green fluorescent protein (GFP)-MinD(Ng) mutants could still oscillate from pole to pole in E. coli, the GFP-MinD(Ng) oscillation cycles were significantly faster and were accompanied by increased cytoplasmic localization. Interestingly, in vitro ATPase assays indicated that MinD(Ng) proteins lacking the first three residues or with an I5E substitution possessed higher MinE(Ng)-independent ATPase activities than the wild-type protein. These results indicate that determinants found within the extreme N terminus of MinD(Ng) are implicated in regulating the enzymatic activity and dynamic localization of the protein.     

Analysis of MinD mutations reveals residues required for MinE stimulation of the MinD ATPase and residues required for MinC interaction

The MinD ATPase is critical to the oscillation of the Min proteins, which limits formation of the Z ring to midcell. In the presence of ATP, MinD binds to the membrane and recruits MinC, forming a complex that can destabilize the cytokinetic Z ring. MinE, which is also recruited to the membrane by MinD, displaces MinC and stimulates the MinD ATPase, resulting in the oscillation of the Min proteins. In this study we have investigated the role of lysine 11, present in the deviant Walker A motif of MinD, and the three residues in helix 7 (E146, S148, and D152) that interact electrostatically with lysine 11. Lysine 11 is required for interaction of MinD with the membrane, MinC, MinE, and itself. In contrast, the three residues in helix 7 that interact with lysine 11 are not required for binding to the membrane or activation of MinC. They are also not required for MinE binding; however, they are required for MinE to stimulate the MinD ATPase. Interestingly, the D152A mutant self-interacts, binds to the membrane, and recruits MinC and MinE in the presence of ADP as well as ATP. This mutant provides evidence that dimerization of MinD is sufficient for MinD to bind the membrane and recruit its partners.     

Positioning of the MinE binding site on the MinD surface suggests a plausible mechanism for activation of the Escherichia coli MinD ATPase during division site selection

Division site selection in Escherichia coli requires that the MinD protein interact with itself and with MinC and MinE. MinD is a member of the NifH-ArsA-Par-MinD subgroup of ATPases. The MinE-MinD interaction results in activation of MinD ATPase activity in the presence of membrane vesicles. The sites within MinD responsible for its interaction with MinC and MinE were studied by site-directed mutagenesis and yeast two-hybrid analysis, guided by the known three-dimensional structure of MinD proteins. This provided evidence that MinC and MinE bind to overlapping sites on the MinD surface. The results also suggested that MinE and the invariant Lys11 residue in the ATPase P-loop of MinD compete for binding to a common site within the MinD structure, thereby providing a plausible structural basis for the ability of MinE to activate the ATPase activity of MinD.     

Expression of Escherichia coli Min system in Bacillus subtilis and its effect on cell division

In both rod-shaped Bacillus subtilis and Escherichia coli cells, Min proteins are involved in the regulation of division septa formation. In E. coli , dynamic oscillation of MinCD inhibitory complex and MinE, a topological specificity protein, prevents improper polar septation. However, in B. subtilis no MinE is present and no oscillation of Min proteins can be observed. The function of MinE is substituted by that of an unrelated DivIVA protein, which targets MinCD to division sites and retains them at the cell poles. We inspected cell division when the E. coli Min system was introduced into B. subtilis cells. Expression of these heterologous Min proteins resulted in cell elongation. We demonstrate here that E. coli MinD can partially substitute for the function of its B. subtilis protein counterpart. Moreover, E. coli MinD was observed to have similar helical localization as B. subtilis MinD.     

Determination of the structure of the MinD-ATP complex reveals the orientation of MinD on the membrane and the relative location of the binding sites for MinE and MinC

The three Min proteins spatially regulate Z ring positioning in Escherichia coli and are dynamically associated with the membrane. MinD binds to vesicles in the presence of ATP and can recruit MinC or MinE. Biochemical and genetic evidence indicate the binding sites for these two proteins on MinD overlap. Here we solved the structure of a hydrolytic-deficient mutant of MinD truncated for the C-terminal amphipathic helix involved in binding to the membrane. The structure solved in the presence of ATP is a dimer and reveals the face of MinD abutting the membrane. Using a combination of random and extensive site-directed mutagenesis additional residues important for MinE and MinC binding were identified. The location of these residues on the MinD structure confirms that the binding sites overlap and reveals that the binding sites are at the dimer interface and exposed to the cytosol. The location of the binding sites at the dimer interface offers a simple explanation for the ATP dependence of MinC and MinE binding to MinD.     

Recruitment of MinC,an inhibitor of Z-ring formation,to the membrane in Escherichia coli: role of MinD and MinE

In Escherichia coli, the min system prevents division away from midcell through topological regulation of MinC, an inhibitor of Z-ring formation. The topological regulation involves oscillation of MinC between the poles of the cell under the direction of the MinDE oscillator. Since the mechanism of MinC involvement in the oscillation is unknown, we investigated the interaction of MinC with the other Min proteins. We observed that MinD dimerized in the presence of ATP and interacted with MinC. In the presence of a phospholipid bilayer, MinD bound to the bilayer and recruited MinC in an ATP-dependent manner. Addition of MinE to the MinCD-bilayer complex resulted in release of both MinC and MinD. The release of MinC did not require ATP hydrolysis, indicating that MinE could displace MinC from the MinD-bilayer complex. In contrast, MinC was unable to displace MinE bound to the MinD-bilayer complex. These results suggest that MinE induces a conformational change in MinD bound to the bilayer that results in the release of MinC. Also, it is argued that binding of MinD to the membrane activates MinC.     

Molecular Interactions of the Min Protein System Reproduce Spatiotemporal Patterning in Growing and Dividing Escherichia coli Cells

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.     

The switch I and II regions of MinD are required for binding and activating MinC

MinD and MinC cooperate to form an efficient inhibitor of Z-ring formation that is spatially regulated by MinE. MinD activates MinC by recruiting it to the membrane and targeting it to a septal component. To better understand this activation, we have isolated loss-of-function mutations in minD and carried out site-directed mutagenesis. Many of these mutations block MinC-MinD interaction; however, they also prevent MinD self-interaction and membrane binding, suggesting that they affect nucleotide interaction or protein folding. Two mutations in the switch I region (MinD box) and one mutation in the switch II region had little affect on most MinD functions, such as MinD self-interaction, membrane binding, and MinE stimulation; however, they did eliminate MinD-MinC interaction. Two additional mutations in the switch II region did not affect MinC binding. Further study revealed that one of these allowed the MinCD complex to target to the septum but was still deficient in blocking division. These results indicate that the switch I and II regions of MinD are required for interaction with MinC but not MinE and that the switch II region has a role in activating MinC.     

The Min oscillator uses MinD-dependent conformational changes in MinE to spatially regulate cytokinesis

In E. coli, MinD recruits MinE to the membrane, leading to a coupled oscillation required for spatial regulation of the cytokinetic Z ring. How these proteins interact, however, is not clear because the MinD-binding regions of MinE are sequestered within a six-stranded β sheet and masked by N-terminal helices. minE mutations that restore interaction between some MinD and MinE mutants were isolated. These mutations alter the MinE structure leading to release of the MinD-binding regions and the N-terminal helices that bind the membrane. Crystallization of MinD-MinE complexes revealed a four-stranded β sheet MinE dimer with the released β strands (MinD-binding regions) converted to α helices bound to MinD dimers. These results identify the MinD-dependent conformational changes in MinE that convert it from a latent to an active form and lead to a model of how MinE persists at the MinD-membrane surface.     

MinD and MinE Interact with Anionic Phospholipids and Regulate Division Plane Formation in Escherichia coli

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 μm) in the presence of ATP was smaller than for MinE (12.1 μm) 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.     

Membrane redistribution of the Escherichia coli MinD protein induced by MinE

Escherichia coli cells contain potential division sites at midcell and adjacent to the cell poles. Selection of the correct division site at midcell is controlled by three proteins: MinC, MinD, and MinE. It has previously been shown (D. Raskin and P. de Boer, Cell 91:685-694, 1997) that MinE-Gfp localizes to the midcell site in an MinD-dependent manner. We use here Gfp-MinD to show that MinD associates with the membrane around the entire periphery of the cell in the absence of the other Min proteins and that MinE is capable of altering the membrane distribution pattern of Gfp-MinD. Studies with the isolated N-terminal and C-terminal MinE domains indicated different roles for the two MinE domains in the redistribution of membrane-associated MinD.     

The plastid division protein AtMinD1 is a Ca2+-ATPase stimulated by AtMinE1

Bacteria and plastids divide symmetrically through binary fission by accurately placing the division site at midpoint, a process initiated by FtsZ polymerization, which forms a Z-ring. In Escherichia coli precise Z-ring placement at midcell depends on controlled oscillatory behavior of MinD and MinE: In the presence of ATP MinD interacts with the FtsZ inhibitor MinC and migrates to the membrane where the MinD-MinC complex recruits MinE, followed by MinD-mediated ATP hydrolysis and membrane release. Although correct Z-ring placement during Arabidopsis plastid division depends on the precise localization of the bacterial homologs AtMinD1 and AtMinE1, the underlying mechanism of this process remains unknown. Here we have shown that AtMinD1 is a Ca2+-dependent ATPase and through mutation analysis demonstrated the physiological importance of this activity where loss of ATP hydrolysis results in protein mislocalization within plastids. The observed mislocalization is not due to disrupted AtMinD1 dimerization, however; the active site AtMinD1(K72A) mutant is unable to interact with the topological specificity factor AtMinE1. We have shown that AtMinE1, but not E. coli MinE, stimulates AtMinD1-mediated ATP hydrolysis, but in contrast to prokaryotes stimulation occurs in the absence of membrane lipids. Although AtMinD1 appears highly evolutionarily conserved, we found that important biochemical and cell biological properties have diverged. We propose that correct intraplastidic AtMinD1 localization is dependent on AtMinE1-stimulated, Ca2+-dependent AtMinD1 ATP hydrolysis, ultimately ensuring precise Z-ring placement and symmetric plastid division.     

MinDE-Dependent Pole-to-Pole Oscillation of Division Inhibitor MinC in Escherichia coli

By inhibiting FtsZ ring formation near the cell ends, the MinC protein plays a critical role in proper positioning of the division apparatus in Escherichia coli. MinC activity requires that of MinD, and the MinE peptide provides topological specificity by suppressing MinC-MinD-mediated division inhibition specifically at the middle of the cell. We recently presented evidence that MinE not only accumulates in an FtsZ-independent ring structure at the cell's middle but also imposes a unique dynamic localization pattern upon MinD in which the latter accumulates alternately in either one of the cell halves in what appears to be a rapidly oscillating membrane association-dissociation cycle. Here we show that functional green fluorescent protein-MinC displays a very similar oscillatory behavior which is dependent on both MinD and MinE and independent of FtsZ. The results support a model in which MinD recruits MinC to its site of action and in which FtsZ ring assembly at each of the cell ends is blocked in an intermittent and alternate fashion.     

Membrane Binding of MinE Allows for a Comprehensive Description of Min-Protein Pattern Formation

The rod-shaped bacterium Escherichia coli selects the cell center as site of division with the help of the proteins MinC, MinD, and MinE. This protein system collectively oscillates between the two cell poles by alternately binding to the membrane in one of the two cell halves. This dynamic behavior, which emerges from the interaction of the ATPase MinD and its activator MinE on the cell membrane, has become a paradigm for protein self-organization. Recently, it has been found that not only the binding of MinD to the membrane, but also interactions of MinE with the membrane contribute to Min-protein self-organization. Here, we show that by accounting for this finding in a computational model, we can comprehensively describe all observed Min-protein patterns in vivo and in vitro. Furthermore, by varying the system''s geometry, our computations predict patterns that have not yet been reported. We confirm these predictions experimentally.