Active Membrane Viscoelasticity by the Bacterial FtsZ-Division Protein

Article pubs.acs.org/Langmuir

Active Membrane Viscoelasticity by the Bacterial FtsZ-Division Protein Iván López-Montero,† Pablo Mateos-Gil,‡ Michele Sferrazza,†,§ Pilar L. Navajas,∥ Germán Rivas,∥ Marisela Vélez,#,⊥ and Francisco Monroy*,† †

Departamento de Química Física I, Universidad Complutense de Madrid, 28040 Madrid, Spain Instituto Nicolás Cabrera, Facultad de Ciencias, Universidad Autónoma de Madrid, 28049 Cantoblanco, Spain § Département de Physique, Université Libre de Bruxelles, Boulevard du Triomphe, CP223 Bruxelles, Belgium ∥ Departamento de Ciencia de Proteínas, Centro de Investigaciones Biológicas, CIB-CSIC, 28040 Madrid, Spain ⊥ Instituto de Catálisis y Petroleoquímica, CSIC, c/Marie Curie 2, Cantoblanco, 28049 Madrid, Spain # Instituto Madrileño de Estudios Avanzados, IMDEA Nanociencia, 28049 Cantoblanco, Madrid, Spain ‡

S Supporting Information *

ABSTRACT: At the early stages of the division process in Escherichia coli, the protein FtsZ forms a septal ring at the midcell. This Z-ring causes membrane constriction during bacterial division. The Z-ring associates to the lipid membrane through several membrane proteins, ZipA among them. Here, a simplified FtsZ-ZipA model was reconstituted onto Langmuir monolayers based in E. coli polar lipid extract. Brewster angle and atomic force microscopy have revealed membrane FtsZ-polymerization upon GTP hydrolysis. The compression viscoelasticity of these monolayers has been also investigated. The presence of protein induced softening and fluidization with respect to the bare lipid membrane. An active mechanism, based on the internal forces stressed by FtsZ filaments and transduced to the lipid membrane by ZipA, was suggested to underlie the observed behavior.

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INTRODUCTION The influence of mechanical forces on individual cells and tissues is recognized as being essential for biological function.1−3 Protein-based machines have evolved to perform diverse mechanical functions engaging targeted forces.4 At the single cell level nonrandom forces are necessary for cytokinesis, to promote cell division, to initiate membrane protrusion, and to alter motility and affect metabolic reactions, among other important cell functions.5 A variety of proteins are involved in force generation, actin being the more prevalent in eukaryote cells.6 The ability of actin filaments exploiting polymerization reactions to drive active motion in cytoplasm structures is a fascinating process which has attracted much attention.7−16 Using methods of bulk rheology, the mechanical activity of actin fibers has been monitored as an effective fluidization of the solution due to the action of molecular motors.8−16 Very recently, Isanta et al.17 have reported the first experimental evidence for active fluidization in a membrane model system based on actin. Simple organisms are also provided with membrane machines that perform important biological functions as cell division.18 The cytokinetic apparatus of bacteria is mainly constituted by FtsZ, a 40 kDa globular GTPase able to polymerize in vitro as filaments. FtsZ forms filaments with a repeating head-to-tail arrangement.19 Upon binding one GTP molecule, two consecutive monomers are brought together into the polymerized state. However, different © 2012 American Chemical Society

than covalent polymers, FtsZ linkages can reversibly disengage by hydrolysis of the bound GTP into GDP. FtsZ filaments behave indeed as living polymers undergoing frequent reorganizations of their polymeric bonds under GTP hydrolysis.19 In vivo, FtsZ is recruited early in the membrane septal site where, together with some other proteins, it forms a contractile Z-ring that remains assembled along the whole division process.20−25 Although the precise mechanism of force generation has not yet been elucidated, the Z-ring is believed to be able to exploit the capacity of FtsZ polymers to function as molecular motors developing effective mechanical work under GTP consumption.26,27 Some data suggest a direct correlation of the constriction force with the spontaneous curvature of the FtsZ filaments,28,29 whereas others assume that radial forces directly stem from the lateral interactions that stabilize the Zring.30,31 Experiments on membrane reconstituted systems performed on supported32 and free-standing membranes27 evidenced a clear correlation between the polymerization activity and topological constraints (filament persistence, lateral interactions, substrate curvature, etc.), suggesting a tight interplay between the bulk filaments and the mechanical characteristics of the membrane. Recently, Osawa et al.26,27 have Received: December 1, 2011 Revised: February 6, 2012 Published: February 13, 2012 4744

dx.doi.org/10.1021/la204742b | Langmuir 2012, 28, 4744−4753

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Distilled water was from a Milli-Q source (Millipore, conductivity lower than 18MΩ cm; γ0 = 72.6 mN/m at 20 °C). Neither GTP nor GMPCPP have surfactant character, the surface tension of their aqueous solutions remaining equal to γ0 up to concentrations much higher than 1 mM. Protein Expression and Purification. FtsZ was purified as described in ref 43. The production and purification of sZipA was performed following the protocol described in ref 44. Both proteins were dissolved in buffer (50 mM Tris, 500 mM KCl, 5 mM MgCl2, 1 mM NiCl2, pH = 7.7) and FtsZ polymerized in GTP-containing buffer (1 mM GTP, 50 mM Tris, 500 mM KCl, 5 mM MgCl2, 1 mM NiCl2, pH 7.7). FtsZ-sZipA Complex Reconstituted onto E. coli Lipid Monolayers. The schematic construction of FtsZ−sZipA complexes on the lipid monolayers is shown in Figure 1. The whole system was reconstituted

recreated, in vitro, a functional constriction machine based on FtsZ filaments artificially anchored to phospholipid vesicles. In that work FtsZ was able to constrict locally the liposome membrane, demonstrating that FtsZ can generate a mechanical force working as a membrane motor. In that experiment an artificial membrane-targeted FtsZ construction was necessary to tether the FtsZ polymers to the lipid bilayer. This requirement highlights the fact that to exert the constriction force FtsZ must be linked to the membrane by other intermediate proteins.33 A minimal septal apparatus might be composed therefore by a ring-like structure based on FtsZ polymers attached to the inner side of the cytoplasmic membrane by anchoring proteins. In Escherichia coli cells, such a proto-ring is composed of FtsZ and the membrane-associated proteins ZipA and FtsA. ZipA is a transmembrane protein associated with FtsZ; in E. coli, the Cterminal end of FtsZ binds directly to the C-terminal of ZipA.34 Both, depletion or overproduction of ZipA leads to the inhibition of the division process.35 Consequently, even if FtsZ polymers are formed, adequate interaction with ZipA (and/or FtsA) is required for proper division of E. coli cells. Because ZipA is a transmembrane protein, it has been suggested that it functions as an anchor of FtsZ to the cytoplasmic side of the bacterial membrane.36 In the last years, the surface rheology of Langmuir films has become a powerful technique to determine the mechanical properties of biomembrane models.37−40 A variety of biophysical studies have taken advantage of rheological information to discuss the mechanical function of real membranes.3 Indeed, the rheological analysis is so useful in getting new physical insight that such methods are being currently used to reveal dynamical functional aspects of biological membranes in relation to their complex lipid composition.37,41 Here, we propose an experimental study of the mechanical response of an E. coli lipid membrane analogue under the action of associated FtsZ polymers. Our objective is to measure the deformation forces exerted by the FtsZ active polymers anchored to the membrane through sZipA. With this aim, we have reconstituted FtsZ-ZipA complexes onto Langmuir monolayers made of native E. coli lipids. In this construction, FtsZ was linked to the lipid membrane by a soluble ZipA mutant (sZipA), for which the native transmembrane fragment was substituted by a 6-fold histidin-tag able to specifically bind to NTA-lipids included in the membrane.42 We probed the rheological response of the membrane under reversible FtsZ polymerization induced by GTP. The evidence suggests the appearance of forced lateral mass transport along the lipid membrane, resulting in a strong surface softening and fluidization induced by FtsZ membrane motors.

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Figure 1. Schematic illustration of the FtsZ−sZipA reconstitution onto E. coli/DOGS-NTA (99:1 w/w) lipid monolayers. FtsZ filaments (in the presence of GTP) are anchored to the lipid monolayer through sZipA, which in turn was bound to NTA lipids. as follows: 75 mL of buffer containing 12 nM sZipA and 200 nM FtsZ were introduced into the Langmuir trough. The barriers were placed at the initial position delimiting a surface area of 100 cm2. Several microlitres (10−15 μL) of lipid stock solution were carefully spread dropwise up to a final lateral pressure of π = 30 mN/m. This pressure was chosen to correspond to equivalent lipid bilayer packing.45 In order to avoid surface-induced protein denaturalization, the free air/ water areas outside the barriers were also covered with a lipid monolayer. Proteins were incubated for 2 h, the time required for adequate interaction of sZipA with NTA lipids in the monolayer. Then, 1 mM (final concentration) of GTP dissolved in buffer was injected into the subphase and the system was allowed to equilibrate for 1 h. In order to ensure FtsZ polymerization during the whole experiment, GTP concentration was added in large excess compared to FtsZ. Because GTP is continuously hydrolyzed in the reversible polymerization/depolymerization process, sufficient excess GTP ensures both extensive and steady-state polymerization. A negative control of polymer activity was performed using GMPCPP (1 mM) instead of GTP (1 mM). GMPCPP is a nonhydrolyzable GTP-analogue causing irreversible GTP-binding, thus resulting in a nondepolymerizing, “frozen”, structure. Other controls lacking one or several components were performed following the same protocol described above. The temperature was maintained constant at 25.0 ± 0.1 °C. Infrared Reflection Absorption Spectroscopy (IRRAS). The FT-IRRAS spectra were recorded with a Nicolet spectrophotometer (750 FT-IR) equipped with an external reflection accessory allowing for grazing angle specular reflectance (Specac P/N 19650) in Langmuir monolayers. The spectral resolution was 8 cm−1. Optimal spectra were obtained at an incident angle of 80° relative to the surface normal. Prior to the measurement, dry nitrogen was flowed in the sample chamber for 3 h in order to reduce the ambient CO2 and H2O bands. Brewster Angle Microscopy. The BAM micrographs were taken with a Nanofilm EP3 imaging ellipsometer (Germany) with a polarized

MATERIALS AND METHODS

Lipids. Egg phosphatidylcholine (egg PC), E. coli polar lipid extract, and 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-[(N-(5-amino-1-carboxypentyl)iminodiacetic acid)succinyl] (ammonium salt) (DOGS-NTA) were supplied by Avanti Polar Lipids. E. coli PLE and DOGS-NTA were dissolved in chloroform (99:1 w:w) to achieve a final concentration of 1 mg/mL. The stock solution was stored at −20 °C. The E. coli PLE (PE 70%, PG 20%, CL 10%) was chosen to mimic the lipid component of the inner bacterial membrane. Chemicals. Langmuir monolayers were spread on a polymerization buffer subphase (50 mM Tris, 500 mM KCl, 5 mM MgCl2, 1 mM NiCl2, pH 7.7). Tris (hydroxymethyl) aminomethane hydrochloride buffer was from Fluka, KCl (>99.8%) was from Carlo Erba, and GTP, MgCl2 and NiCl2 (>99.8%) were from Sigma-Aldrich. GMPCPP (guanosine-5′-[(α, β)-methyleno]triphosphate, sodium salt) was from Jena Bioscience (Germany).The chloroform used to spread the lipid monolayers was ultrahigh HPLC purity (Chromasolv), supplied by Riedel-de Haën. 4745


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phase lag between the stress response and the deformation (ϕ = ϕπ − ϕu), the compression modulus ε and the viscosity κ were calculated as follows:

incident laser of λ = 532.0 nm. The monolayers were prepared in a Langmuir balance (302BAM, NIMA, U.K.) with a maximum area of 200 cm2. The surface pressure was simultaneously monitored. FtsZ-sZipA Complex Reconstituted onto Supported Lipid (E. coli) Bilayers. Liposomes were prepared from mixtures of lipids E. coli lipids with DOGS-NTA in molar ratio 10:1. Lipids were dissolved in chloroform/methanol solution (1:1 v/v), mixed in the desired amount, dried under a stream of nitrogen and resuspended at 4 mg/mL lipid concentration in Milli-Q water. The resulting multilamellar vesicles were extruded through a polycarbonate membrane with 200-nm nominal pore diameter (Avanti Mini extruder) resulting in large unilamellar vesicles (LUVs). Planar lipid bilayers on mica were formed by incubating 40 μL of 0.1 g/L LUVs solution in Tris-HCl 10 mM, 100 mM NaCl, 2 mM CaCl2 (pH 7.5) for 1 h resulting in the formation of a supported lipid bilayer as described in ref 46. The solution containing the nonfused vesicles was removed and replaced by working buffer (50 mM TrisHCl, 500 mM KCl, 5 mM MgCl2, pH 7.5). To form the ZipA-FtsZ bundles on the lipid bilayers, sZipA (3 μM) and FtsZ (12.5 μM) were incubated for 1 h in 50 mM TrisHCl, KCl 500 mM, MgCl2 5 mM, pH 7.5 buffer over the lipid bilayer previously fused on the mica surface. Excess protein was removed from solution and excess GTP (10 mM) was added to induce FtsZ polymerization before imaging. Partial film dewetting is induced during this washing step, resulting in incomplete film coverage over macroscopic scales, the images shown being representative of those regions where film wetting is prevalent. This protocol resulted in reproducible attachment of the ZipA−FtsZ complex to the lipid bilayer leaving the FtsZ protein responsive to the presence of GTP. Atomic Force Microscopy. AFM images were taken with a Nanotech Electrónica microscope (Spain), operated in the jump mode.47 The scanning piezo was calibrated using silicon calibrating gratings (NT-MDT, Russia). Silicon nitride tips (DI Instruments) with a force constant of 0.05 N/m were used. The images presented are statistically representative of different realizations in a given system. Compression Rheology.48−50 Experiments of surface rheology in the oscillatory compression mode were performed in a Langmuir balance (702BAM, NIMA, U.K.) equipped with two symmetrically moving barriers. The maximal surface area of 700 cm2 was reduced down to 200 cm2 by a volume reducer (Teflon) but maintaining a high compression ratio. Surface pressure was measured with a paper Wilhemy plate (Wathmann #1, 21 mm perimeter). The subphase temperature was controlled by recirculating water in a jacket from a thermostatic bath (Polyscience). The temperature was measured by a Pt-100 sensor. All experiments were performed at T = 25.0 ± 0.1 °C. The whole setup was placed in a transparent plexiglass case. The oscillatory movement of the barriers was computer controlled using the 554NIMA software. For an exhaustive revision of interfacial rheology, see refs 48 and 49. Briefly, the compression deformation was a sinusoidal of amplitude u0 and period 2π/ω, the surface area available in the monolayer varying as

u ⎡ ⎤ A(t ) = A 0 ⎢1 + 0 sin(ωt + ϕu)⎥ ⎣ ⎦ 2

ε = (σ0 /u 0) cos ϕ κ = (σ0 /ωu 0) sin ϕ

Relaxation Dynamics. These experiments were performed in a Langmuir trough (KSV, Finland; total area A0 = 245.8 cm2), the compression ratio being adjusted at u0 = 1%. At this strain the viscoelastic response was linear with a high signal-to-noise ratio. The stress relaxation is recorded as a function of time t after a sudden uniaxial inplane compression is performed with the barriers, i.e., σ(t) = Δπ(t) = π(t) − π(t → ∞). The relaxation profile was characterized by a generalized exponential function

σ(t ) = σ0 exp[− (t /τ)β ]

σ0 sin(ωt + ϕπ) 2

(4)

where σ0 is the amplitude of the relaxation, τ is the relaxation time, and β (≤1) is a stretching exponent accounting for deviations from single exponential behavior (β = 1). The viscoelastic moduli can be calculated as52 σ ε = 0 and κ = ετ u0 (5) Here, the compression elasticity ε represents the instantaneous elastic response of the film, the viscosity κ accounting for viscous friction.

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RESULTS Binding of sZipA to E. coli lipid monolayers. In the present construction, the soluble mutant sZipA is bound to NTA-functional lipids with high specificity and affinity,42 as evidenced by IRRAS spectroscopy. Figure 2 shows the IRRAS

Figure 2. IRRAS spectra of an E. coli/DOGS-NTA (99:1 mol; 1% mol) lipid monolayer (black line) and of an E. coli/DOGS-NTA (99:1 mol) lipid monolayer incubated with sZipA (red line) in a buffered subphase, both at lateral pressure of 30 mN/m. The lipid carbonyl band (1739 cm−1) was accompanied by the amide I and II protein (1635 and 1538 cm−1, respectively) bands when sZipA was present in the subphase. Amide bands become comparatively more intense at higher NTA content (10% mol; blue line), indicating specific binding with sZipA.

(1)

where A0 is the average area, u0 = (A − A0)/A0, is the relative deformation amplitude, ω is the frequency of the sinusoidal motion, and ϕu accounts for a possible initial phase. Two kinds of experiments were performed on the reconstituted system: First, the strain−stress curve was constructed from increasing the strain amplitude at a constant frequency (ω = 0.21 s−1). This allowed for an unambiguous determination of the linear regime. Then, the frequency was varied at a constant deformation amplitude in the linear regime (u0 = 1%). In the linear regime, the stress response π(t) followed a sinusoidal form with identical frequency than the imposed deformation48

π(t ) = π 0 +

(3) 51

spectrum of E. coli lipid monolayers containing sZipA (12nM) in the subphase. For clarity, only the relevant amide-protein region (1800−1500 cm−1) is plotted. The presence of lipids was identified from the stretching peak of the carbonyl groups (1739 cm−1; black line) linking the proximal ends of the hydrocarbon chains to the glycerol bridge. Other typical lipid band (ca. 1225 cm−1), corresponding to the PO stretching of the phosphate groups were also detected (data not shown). In the presence of NTA-functional lipid, two low intensity broad bands were also detected at 1635 and 1538 cm−1 (red line).

(2)

where π0 is the average pressure, σ0 the stress amplitude, and ϕπ a phase factor accounting for the delay imposed by viscous friction within the response. From the experimental values of the amplitudes (u0, σ0) and the 4746


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net positive charge, favoring Coulombic interactions with anionic lipids (PG and CL) in the ld phase where the NTAlinkers are homogenously dispersed. Therefore, when sZipA becomes bound to NTA sites in the ld phase, phase equilibrium is completely altered, the PE domains disappearing. Figure 3c shows as further incubation with monomeric FtsZ (200nM) caused membrane interaction mediated by sZipA (see Figure 3c). In this case, the monolayer became segregated again into coexistence domains. The domains are in this case similar in size (ca. 3 μm diameter) but darker than the former lipid domains (Figure 3a), suggesting a more disordered lipid packing caused by lipid association with the proteins. After GTP injection into the subphase (1 mM final), FtsZ polymerization proceeds, the domain distribution being strongly disturbed. Figure 3d shows the monolayer 1 h after GTP injection. A close inspection revealed the growth and coalescence of irregular protein patches from the pre-existing nonpolymerized FtsZ nuclei in Figure 3c. A similar behavior was previously reported by Lafontaine et al.55 for a different construction where FtsZ was weakly associated to the lipid monolayer through unspecific adsorption interactions, not involving the ZipA linker. These results constitute sufficient evidence of the presence of a ZipA-FtsZ protein layer interacting with the lipid membrane, a mutual interaction able to modify the phase behavior. Since, we were interested in how GTP-mediated polymerization affected the microscopic structure of the ZipA-FtsZ layer, a complementary AFM study was conducted. An extensive structural characterization in bilayer systems have been recently published in ref 46. Structural Characterization by AFM. The microscopic structure of ZipA-FtsZ polymers and its reorganization dynamics at the lipid interface were characterized by AFM. A construction of the whole sZipA/FtsZ(GTP) system was anchored to an E. coli lipid bilayer (containing NTA-linkers) supported on mica (see the Material and Methods section for details). Figure 4 shows a series of three representative images corresponding to the nonpolymerized systems (no GTP). Figure 4a shows film patches of E. coli (+NTA) lipids supported on mica. Expectedly, these very smooth, 5 nm height (h1), platforms corresponds to the bare lipid bilayer. Figure 4b shows similar patches obtained after incubation with sZipA. In this case, thicker layers with a higher roughness were observed, h2 ≈ 10 ± 1 nm. This thickness might be consistent with the dimensions of ZipA, DZipA ≈ h2 − h1 ≈ 5 ± 1 nm, which indeed correspond to the size of the stable globule of ZipA (ca. 4 nm)56 plus its unstructured domain close to the membrane (ca. 1 nm). There is no indeed evidence of stable structure in this domain, which comprises N = 156 aminoacids.36 If assumed to behave as a flexible polymer, its partial contribution to film thickness is expected to vary in the range from ca. 1 nm up to 6 nm. In the thinner limit, if spread flat over the lipid layer, the unstructured chain might occupy a ≈ 0.5−1 nm, the size of a monomer aminoacid. This flat conformation is compatible with the experimental observation (Figure 4b). However, if conformed as a bulk coil, its typical size could increase up to Rg ≈ aN1/2 ≈ 6 nm. This is consistent with AFM data in Figure 4c, which shows patches of the complete ZipA + FtsZ (no GTP) complex incubated on the lipid bilayer. In this case, the membrane film was found thicker and with a higher roughness that in the previous cases, h3 ≈ 20 ± 2 nm. FtsZ folds as a stable globule with a size DFtsZ ≈ 4 nm.57 Consequently, the thickness observed for the ZipA+FtsZ complex (Figure 4c) is compatible either with an oligomeric FtsZ stacking, hFtsZ = h3 − h2 = 10 nm ≈ 2−3 DFtsZ or,

These were assigned to amide I and amide II bands, respectively, which correspond to the peptide bonds of the protein. The intensities of these surface protein bands were found low, but increasing with NTA concentration, suggesting specific linking between NTA-lipids and sZipA. Expectedly, the amide protein fingerprints are not detected in the absence of NTA-linker, supporting the idea of specific NTA-mediated anchoring of sZipA to the membrane. FtsZ/sZipA interaction in E. coli Lipid Monolayers. Structural evidence for FtsZ polymerization mediated by sZipA interactions at the lipid surface was provided by BAM experiments performed at different conditions. Figure 3a shows a

Figure 3. BAM images of Langmuir monolayers made of (a) E. coli/ DOGS-NTA (99:1 mol), (b) E. coli/DOGS-NTA (99:1 mol) and sZipA, (c) E. coli/DOGS-NTA (99:1 mol), sZipA, and nonpolymerized FtsZ, and (d) E. coli/DOGS-NTA (99:1 mol), sZipA FtsZ in the presence of GTP. The scale bar represents 50 μm.

representative BAM image of an E. coli lipid monolayer at a surface pressure πbil = 30 mN/m, corresponding to the biologically relevant bilayer packing.45 Similar monolayer textures are observed when doped with the functional lipid (DOGSNTA, 1% molar). The dark continuous phase is a fluid phase mainly constituted by anionic lipids (PG and CL) in a expanded ld state. The denser phospholipid PE accumulates in the bright domains, which are more ordered and thicker than the continuous phase.53 These domains were found with a circular shape (ca. 3 μm diameter), suggesting an ordered molecular organization, like the lo phase. Such a phase behavior is compatible with previous results by fluorescence microscopy where the fluorescent probe was found excluded out from the ordered domains.54 Figure 3b shows how the presence of sZipA causes lipid domains to disappear. The fact that protein binding elicited domain disappearance suggests a strong interaction between the E. coli lipids and sZipA. Indeed, proximal to the transmembrane terminus, ZipA contains an unfolded domain rich in charged aminoacids (aa 29 to 85). This domain entails a 4747


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Figure 4. Topographic AFM images of (A) lipid bilayer patches (E. coli lipids + DOGS-NTA), characterized by a very smooth surface (bilayer thickness, h1 = 5.0 ± 0.2 nm). (B) E. coli (+NTA) lipid bilayer incubated with sZipA on top (total thickness, h2 = 10 ± 1 nm). (C) E. coli (+NTA) bilayer with sZipA/FtsZ complex in the absence of GTP. The thickness of these complex platforms was measured h3 = 20 ± 2 nm, a size compatible with the accumulated thickness of the bilayer (5 nm), sZipA (ca. 10 nm), and FtsZ (5 nm) (see text for a detailed discussion). Scale bar is 450 nm in all images.

alternatively, with ZipA extended in a bulky conformation, i.e., DZipA ≈ h3 − h1 − DFtsZ ≈ 11 ± 3 nm. Since no FtsZ oligomerization is expected in the absence of GTP, assuming conformational flexibility for ZipA, the thickness observed in Figure 4c is consistent with a stratified structure where the intermediate ZipA anchors occupy DZipA ≈ 10 nm, a size in agreement with previous TEM estimations assuming a semiflexible conformation.36 Therefore, assuming ZipA (DZipA ≈ Dfold + Dunstr ≈ 10 nm) sandwiched between the supporting lipid bilayer (Dlipid ≈ h1 ≈ 5 nm) and an outer FtsZ monolayer (DFtsZ ≈ 5 nm), one obtains h3 = h1 + DZipA + DFtsZ ≈ 20 nm, in agreement with experiments (Figure 4c). Figure 5 shows AFM data obtained in the presence of GTP. In this case, the system becomes extensively polymerized forming an unstructured mesh well attached to the supporting bilayer (Figure 5a). Then, the system organized as rigid filaments which, after two hours, evolved to a higher organization based on 2D bundles (Figure 5b). The individual filaments were found large, relatively thin (Figure 5b) and with a high conformational persistence,46 suggesting an internal structure as polymer chains made of linked FtsZ monomer units. In the high packing zones, the large filaments were observed to bundle into thicker structures, probably stabilized by cohesive lateral interactions (Figure 5b).46 In the sparser regions, individual FtsZ polymers associate end-to-end forming dynamic filament bundles of different curvature and length. Nonactive System: Monolayer Rigidity Remains High in the Absence of GTP. We measured the mechanical impact of nonpolymerized ZipA/FtsZ protein complexes on the compression response of the lipid monolayers where they are covalently attached. Figure 6 shows the results obtained in a first series of experiments where the two proteins are sequentially incubated in the subphase. For the bare lipid monolayer, at low frequencies, the dynamic compression modulus is compatible with the isothermal compressibility (K0) calculated from

Figure 5. Topographic AFM images of sZipA/FtsZ complex reconstituted on E. coli (+DOGS-NTA 1%mol) bilayer in presence of GTP. Images show the evolution of FtsZ network formation on E. coli bilayer upon addition of 1 mM GTP. (A) Initially no bundles are observed. (B) After 2 h a highly interconnected network is formed. Scale bar is 1 μm in all images.

the compression isotherm (see ref 54 for details); this is, ε(ω → 0) ≈ K0 (≈100 mN m−1, at the relevant packing state πbil = 30 mN/m). The dynamic modulus increased slightly but systematically with the deformation frequency, a relaxation effect related with the existence of diffusive transport between the domains and the continuous phase of the lipid layer.58,59 Surprisingly, although the presence of sZipA caused the lipid domains to disappear (Figure 3b), regarding the compression modulus, no appreciable change was observed with respect to the bare 4748


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by the presence of the flexible protein sZipA and only weakly by the presence of the monomer form of FtsZ. Membrane Softening by Living sZipA-FtsZ Polymers in the Presence of GTP. BAM experiments revealed that addition of GTP into the subphase caused FtsZ polymerization, inducing coalescence of the domains of Figure 3c into the larger protein/lipid regions (Figure 3d). We are now interested in the effect of reversible FtsZ polymerization on membrane mechanics. We preformed a lipid−protein monolayer by coincubating sZipA (12nM) and FtsZ (200nM) for 1 h under a lipid monolayer containing 1%NTA lipids. Then, GTP was injected at high excess (1 mM) into the subphase, a concentration sufficient to drive reversible polymerization for a long time ([GTP] ≫ [FtsZ]). The compression response of the monolayer to a periodic perturbation (1% amplitude, 30s period; ω = 0.21 s−1) was recorded during two hours after triggering the polymerization process with GTP. The transient compression modulus was evaluated for each cycle as

Figure 6. Frequency dependence of the dilational elasticity modulus as obtained from the oscillatory barrier experiments for monolayers made of lipid + sZipA (red circles) and lipid + sZipA + FtsZ in the lack of GTP (cyan circles). The frequency dependence of the compression elasticity modulus for the bare lipid monolayer is also represented (black circles). Typical experimental error, δε ≈ ± 5 mN/m.

ε(t ) = −A 0

bilayer when this protein was present (Figure 6). ZipA is a very disordered protein, thus its conformation can be regarded as a flexible coil (see AFM data), its impact in monolayer rigidity being negligible with respect to rigid lipids. The higher the frequency the more visible the differences are, a fact probably related to the absence of lipid domains induced by sZipA.59 Furthermore, when monomer FtsZ was also present, the dynamic compression modulus increased slightly with respect to the lipid layer at low frequencies (ε(ω → 0) ≈ 130 mN/m > K0). This weak stiffening could be related to the presence of FtsZ in the surface assembly, a rigid globule with a better defined folding than sZipA. In summary, the present results evidence that, in the absence of polymerization activity driven by GTP, the compression elasticity of the lipid monolayer is not affected

Δπ(t ) ΔA

(6)

where A0 is the average surface area. In the course of the experiment, progressive FtsZ polymerization into the filamentous structures of Figure 5a,b might confer strong changes on the mechanical response of the lipid monolayer, expectedly a membrane stiffening characterized by an increase of the elasticity modulus. However, just after GTP injection, a decrease of the stress response was observed. This is clearly visible in Figure 7, which shows the transient mechanical response of the system with high excess GTP (1 mM) against an oscillatory deformation exerted at constant amplitude (Figure 7a). The progressive response is shown in Figure 7b, which evidences a systematic decrease of stress as polymerization proceeds at progressively longer times. The transient values of ε(t) are plotted in Figure 7c. In excess GTP (1 mM), a two-step

Figure 7. (A) Typical time-trace of the strain function found for FtsZ living polymers reconstituted as active sZipA/FtsZ complex onto E. coli lipid monolayers (at ω0 = 0.21 s−1 and 1% strain amplitude). (B) Stress time-traces recorded at different times (see colored stars in (C) after GTP addition into the subphase. C) Softening decay of the compression modulus after GTP addition (+1 mM; blue symbols). After an initial diffusive period (ε ≈ −t1/2 at t < t0 ≈ 30 min; dashed line), the compression modulus decreases further until reaching a final steady-state value (εsoft ≈ 60 mN/m), approximately half than the initial value measured before GTP injection (ε0 ≈ 125 mN/m). A lower GTP content (+0.3 mM; red symbols) causes smaller softening, the initial rigid state being recovered after GTP exhaustion. As a negative control, permanent polymerization was induced by irreversible GTP-binding with the nonhydrolyzable analogue GMPCPP (green symbols). In this case, no softening is induced by permanent FtsZ filaments polymerized in a “frozen” passive state. 4749


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softening kinetics is clearly observed to progress over a time of 100 min. In this case, an initial decrease was detected along an induction period of 30−40 min after GTP injection. This is approximately the time necessary for domain coalescence and consequent protein accretion in the macroscopic scale (Figures 3c,d) and progressive filament growth at a molecular level (Figures 5a,b). During this initial step, the monolayer rigidity decreased by ca. 20% with respect to the initial nonpolymerized state (ε0 ≈ 125 mN/m). Then, a sudden drop of rigidity was observed down to a steady-state characterized by a low value of the compression modulus (εsoft ≈ 60 ± 10 mN/m). The monolayer remained soft during 1−2 h, the time probably necessary for total GTP exhaustion. At the steady-state, the mechanical response was observed to be unstable with large variations of the compression modulus (Figure 7c). The observed two-step kinetics is not only connected with the morphological heterogeneity of the monolayer (Figure 3c,d) and with the microscopic structure revealed by AFM (Figure 4b,c) but also probably to nonequilibrium effects associated to polymerization activity. Indeed, during the induction period, the incipiently formed FtsZ polymers had not organized yet into the mature bundled structures of Figure 5b. Membrane organization occurred during this slow limiting step following a diffusive transport mechanism, ε(t) ≈ −t1/2.60,61 Only when sufficient filamentous FtsZ became organized in the membrane region, sufficient FtsZ polymerization reactions could eventually give rise to a collective mechanical effect driven by active fluctuations between GTP- and GDP-bound states. Such dynamic process is sustained by GTP, consequently, the fewer GTP, the lesser softening effect should be observed during a shorter time. To verify this dynamic mechanism, a new experiment was performed at the same experimental conditions but with a lower GTP content (0.3 mM), practically equimolar ([GTP] ≈ [FtsZ] = 200 nM). In this case, a much weaker softening was observed, from ε0 ≈ 125 mN/m down to εsoft ≈ 110 mN/m (≈10% decrease with GTP 0.3 mM). Such a softening was observed maintained in steady-state during 1 h, then a progressive recovering to the initial elasticity value was observed. In this time, GTP probably became exhausted, so the monolayer returned to their initial nonpolymerized state. To discriminate the dynamic hydrolytic mechanism from a mere structural effect due to the conformational change occurred upon polymerization, a third experiment was designed. In this experiment, “frozen” polymerization was induced by irreversible GTP-binding using GMPCPP (1 mM), a nonhydrolyzable GTP analogue. In this case, no softening was observed, the elasticity modulus remaining in its initial value even though extensive FtsZ polymerization occurred. This negative control discards a structural scenario for membrane softening, becoming rather in support of the active mechanism. Figure 8 compares the frequency dependence of the elasticity modulus of the whole construction (lipids + sZipA + FtsZ) in the following cases: (1) no GTP, thus no FtsZ polymerization and FtsZ “living” polymerization under (2) excess GTP (1 mM), (3) low GTP (0.3 mM), and (4) excess GTP (1 mM) at nonanchoring conditions (no sZipA). Data were taken in steady-state (30−60 min after GTP incubation). Finally, 5) we consider FtsZ “frozen” polymerization under excess GMPCPP (1 mM). Although no clear changes are observed for the frequency dependences, a significant decrease of the elasticity modulus was observed in the presence of living polymers: the higher the GTP concentration, the larger the softening (Δε ≈ −15 mN/m with GTP 0.3 mM and Δε ≈ −60 mN/m

Figure 8. Frequency dependence of the dilational elasticity modulus found for the sZipA/FtsZ complex anchored to lipid monolayer. Every experiment was performed at steady-state. In the lack of GTP (black circles) the nonpolymerized system is very rigid (ε0 ≥ 125 mN/m). Under excess GTP (1 mM; blue circles), living FtsZ-polymers anchored to the lipid monolayer cause active membrane softening (εsoft ≈ 60 mN/m). Under anchoring conditions, weaker softening is observed at smaller GTP concentration (0.3 mM; red circles). Typical experimental uncertainty, δε ≈ ±5 mN/m. As a control of activity transduction, no softening is observed for nonanchored living polymers formed in the absence of sZipA (nonanchoring control; lipids + FtsZ/1 mM GTP, no sZipA; blue squares). Finally, no softening is observed under anchoring passive FtsZ-filaments produced in a “frozen” polymer state in excess GMPCPP (1 mM)(green circles).

with GTP 1 mM), thus supporting the dynamic mechanism. However, no softening is observed for living polymers (GTP 1 mM) in the absence of sZipA. In effect, if nonattached, living polymers cannot impart motion into the membrane, which remains in a “passive” rigid state. Furthermore, no softening was observed for a control system where frozen polymers were induced by the nonhydrolisable GTP-analogue. Therefore, the results in Figure 8 prove altogether that the softening effect is induced by living FtsZ polymers on the E. coli lipid membrane. This constitutes a strong piece of evidence on the existence of active motions caused by FtsZ when adequately tethered through the membrane anchor ZipA. Relaxation Behavior: Membrane Fluidization. A fundamental principle of complex fluids establishes that only active motions make an entangled polymer solution fluent at higher densities.62 Consequently, the fluidization effect observed in active matter can be only understood if slow thermal diffusion is superseded with directed motion driven by internal forces.63 To verify activity effects in the systems containing polymeric FtsZ, we have carried out a complementary study of the compression rheology based on the relaxation properties of the monolayers at different FtsZ concentration. A system is preformed by incubating sZipA (12nM) in the subphase under an E. coli lipid monolayer containing functional NTA-lipids. The subphase consists of polymerizing buffer (5 mM Mg2+) and different amounts of FtsZ ranging from 0 to 200 nM. The sZipA/FtsZ complex was incubated for 1 h in order to adequate NTA-binding and monolayer equilibration. Then, GTP (1 mM; final concentration) was injected in the subphase, the system being left to equilibrate again for 1 h (the time necessary for reaching the soften steady-state). When the surface pressure 4750


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remained constant, the monolayer is suddenly strained (less than 5s compression time) under a small lateral compression (−ΔA/A0 = 0.9%; a deformation regime where the mechanical response was observed linear). Figure 9a-b shows a typical

Figure 10. (A) Elasticity modulus (ε) and (B) effective compression viscosity (κeff) as a function of FtsZ concentration.

ε0 = 125 mN/m) down to a much smaller value at high FtsZ concentration (ε ≈ 60 mN/m at [FtsZ] = 200nM). The quantitative strength of this softening was found in agreement with data from the oscillatory experiments (Figure 9). Again, a structural mechanism is discarded in favor of a dynamic scenario driven by internal forces exerted by “active” filaments on the lipid membrane. Reduced compression rigidity implies a favorable reaction force upon filament deformation, probably driven by depolymerisation reactions. If direct forces exerted by active FtsZ filaments are present, they might confer a higher mobility to the membrane, thus decreasing viscosities might be also observed with increasing the FtsZ concentration. Figure 10b shows the compression viscosities, calculated from the relaxation experiments performed at different FtsZ concentration. In the absence of FtsZ polymers, the monolayer containing sZipA is characterized by a relatively high viscosity (κZipA = 10 N s/m) compared to the fluid character of the bare lipid monolayer (κlip = 0.1 N s/m).54 Addition of polymerizing FtsZ at low concentration ([FtsZ] < 25 nM) caused κ to increase near by a factor ten, an unequivocal sign of surface polymerization. However, a progressive decrease in viscosity was observed with increasing FtsZ concentration above 25 nM, a clear signature of the progressive action of directed forces exerted by an increasing number of living polymers. The induced fluidization is so significant that the compression viscosity decreases down to values close to those corresponding to the system devoid of FtsZ polymers.

Figure 9. (A) Step compression experiment performed for the reconstituted sZipA-FtsZ complex onto E. coli lipid monolayers in the presence of GTP. (B) Relaxation of the surface pressure after sudden compression. (C) Stress-relaxation curves of the reconstituted sZipAFtsZ complex on E. coli lipid monolayers at different FtsZ concentrations: 0 (black), 33 (navy), 67 (blue), and 100 nM (cyan). The relaxation times τ were obtained from fits to stretched exponentials (lines).

relaxation experiment. As a consequence of deformation, the monolayer developed an instantaneous surface stress Δπ0 which is partially released as the monolayer relaxed at times longer than a relaxation time τ. Once again stresses were fully relaxed, the monolayer raised a final pressure πf compatible with the equilibrium pressure of the compressed state (see the Material and Methods). The instantaneous elasticity ε = −A0(Δπ0/ΔA) and the compression viscosity κ = ετ might inform us, respectively about the elastic energy accumulated by the membrane and the energy dissipated by viscous friction after relaxation. In the absence of FtsZ (containing sZipA; but no FtsZ), a fast relaxation compatible with a single exponential decay was observed (Δπ(t) = Δπ0 exp(−t/τ); eq 4 with β = 1), as revealed form the linear profile of the experimental relaxation in the semilogarithmic representation (Figure 9a). This is the relaxation behavior previously observed in Langmuir films of entangled flexible chains,51,63 a system physically resembling ZipA. A strong retardation was observed when polymerized FtsZ was present, even at very low concentration. In those cases, the relaxation profile became visibly stretched at long times with respect to the single exponential form. The modified exponential function in eq 4, Δπ(t) = Δπ0 exp(−t/τ)β (with β ≤ 1) was found adequate to fit the experimental relaxation profiles. This non-Debye relaxation behavior is compatible with that found in percolated monolayers made of stiff polymer chains.64 By fitting the results to this equation, the relaxation parameters can be easily obtained as a function of the concentration of FtsZ present in the system (Figure 10). Figure 10a shows that the instantaneous compression modulus decreases monotonously with increasing FtsZ concentration. Higher contents in “passive” filaments are expected to induce higher stiffening in the membrane, however, we observed membrane softening, the compression modulus decreasing from a high value for the monolayer devoid of FtsZ (compatible with the bare lipid layer;

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DISCUSSION Previous surface rheology studies of E. coli lipid monolayers revealed a highly fluid and rigid monolayer with large mechanical stability under lateral compression.54,59,37 The compression (κE.coli = 0.1 N s/m) and shear (ηE.coli = 10−5 N s/m) viscosities were measured to be very low compared to those of typical fluid lipids, like POPC.37,58 However, the compression modulus (εE.coli = 125 mN/m) was similar to the values found in monolayer models of other fluid lipids.58 Here, to study the effect of the proteins on the mechanical properties of the E. coli membrane, we have reconstituted a protein subsystem (FtsZ/ ZipA) on the same lipid monolayer (E. coli lipids). Using a wellknown strategy for linking of His-tagged proteins to NTAcontaining lipid monolayers,42 we have anchored active structures based on FtsZ filaments by the intermediate of sZipA. The whole system exhibited evidence of molecular interactions 4751


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Figure 11. Network dynamics. Inside relatively low compacted regions (single arrow), individual filaments develop directed motion. Collective forces are stressed out from more compact areas (double arrow), causing motion on bundling nodes where several filaments are attached (circle). Scale bar in all images is 1 μm. The dynamic character of the network can be easily visualized in the movie S-Video-NetworkDynamics.avi in the Supporting Information.

between proteins and lipids, the artificial construction resembling the protein structure previously described in studies performed in vitro.32,46 The reconstituted FtsZ/ZipA complex had two major mechanical effects on the E. coli monolayer: (a) softening, evidenced as a decrease of the compression modulus, and (b) fluidization, evidenced as a decrease in viscosity. Although the fluidization observed here could be alternatively compatible with total FtsZ depolymerisation, the concomitant softening confirms an “active” character for the observed behavior; otherwise, the compression modulus might remain equal to the “non-polymerized” value (ε0 ≈ 125 mN/m; see Figure 7). A similar, but less pronounced, “active” softening/ fluidization was recently reported in lipid monolayers with fibrilar actin covalently attached through of an artificial membrane linker.17 Treadmilling motions driven by polymerization/depolymerisation reactions typical of actin under ATP hydrolysis were in that case suggested to underlie the observed fluidization behavior. In the present case, GTP-mediated softening/fluidization effects are only observed in the presence of the membrane linker NTA/sZipA assigning both, a motor function to living FtsZ polymers and a transducer role to the membrane tether sZipA. Consequently, ZipA can be considered not only a mediator of the interaction between FtsZ and the lipid surface but also as a force transducer between them. At a microscopic level, nondiffusive directed movements of FtsZ polymers have been observed in different systems.46,65 Precessive motion has been identified in individual FtsZ filaments with a curved configuration.65 In that work, individual FtsZ filaments were observed to behave as living circular polymers with the ability to become close/open depending on the polymerization/depolymerization state. The dynamic character of FtsZ filaments bounded to the membrane by the ZipA linker was demonstrated in a recent work considering a similar construction to that considered here (E. coli + NTA lipids/sZipA/FtsZ/GTP).46 From those results, the microscopic mechanism behind the macroscopic softening/fluidization can be envisaged. Figure 11 shows a sequence of AFM images taken for the whole construction in a low coverage area. Each frame corresponds to an image of the same surface area recorded at different consecutive times. Despite a high packing, imposing strong restrictions to motion, relatively large displacements/ reorganizations are observed in the lower covered regions (see Figure 11 and the movie S-Video-NetworkDynamics.avi in the Supporting Information). Qualitatively, the present results depicts a microscopic scenario as a very dense and highly connected assembly of living FtsZ filaments with the potential ability to reorganize by opening/recombining the polymer chain at single monomer-to-monomer binding sites. The existence of

membrane movements mediated by dynamic FtsZ filaments at the molecular level confirms a true “active” character for the softening process observed in the macroscale. The active and dynamic GTP-hydrolyzing system in Figure 11 could therefore be able to reorganize under external deformation. Therefore, we conceive polymerization−depolymerization reactions in the FtsZ assembly to cause the softening/fluidization effect observed in the lipid membrane where it is attached. The relaxation experiments provided further evidence about the “active” nature of the membrane softening. Indeed, the observed membrane softening (Figure 10a)/fluidization (Figure 10b) is progressively marked at higher FtsZ concentration, a behavior only compatible with the active mechanism, where the more dynamical FtsZ filaments produce the higher activity. This is however incompatible with a passive structural scenario, where FtsZ static filaments might give rise to effective stiffening but certainly to an increase in viscosity. The observed behavior resembles that of active bulk gels, e.g., actin solutions whose reduced viscoelasticity reflects the internal motions of individual filaments.5−17 In the present case, if an internal force exerted by living filaments is able to oversee random diffusion in the membrane assembly, mass transport will be meaningfully enhanced inside. Consequently, the surface structure is softened and fluidized, as experimentally observed. This effect assigns FtsZ filaments as true “molecular motors”, not only interacting with the lipid component of the membrane but also exerting direct forces on it. Therefore, a second conclusion can be stated about the role of FtsZ filaments as nanomechanical devices able to generate direct forces, which if adequately transmitted by anchoring transducers (ZipA), produce effective softening/ fluidization in the membrane.

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ASSOCIATED CONTENT

S Supporting Information *

Video showing the dynamic character of the network. This material is available free of charge via the Internet at http:// pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was partially financed under grants from Ministerio de Ciencia e Innovación (MCINN, Spain: FIS2009-14650-C02-01 4752


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to FM, BIO2008-04478-C03 to M.V. and G.R. and Consolider Ingenio en Nanociencia Molecular CSD2007-0010 to F.M. and M.V.); Comunidad de Madrid (CAM: S2005MAT-0283, S2009MAT-1507 to F.M. and M.V. and S2006-BIO-0260 to G.R.); European Commission (EC: DIVINOCELL FP7 HEALTH-F3-2009-223431 and FP/HEALTH-F3-2009223432 to G.R. and M.Ve.); and Human Frontier Science Program (HFSP-RGP0050/2010 to G.R.). I.L.-M. was supported by Juan de la Cierva program (MICINN). P.L.-N. and P.M. thank FPI-CAM for financial support. We thank the facility “CAI Infrarrojo-Raman-Correlador” of the Universidad Complutense for IRRAS and BAM time.

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Active Membrane Viscoelasticity by the Bacterial FtsZ-Division Protein

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