FtsZ filament capping by MciZ, a developmentalregulator of bacterial divisionAlexandre W. Bisson-Filhoa, Karen F. Discolab,c,d, Patrícia Castellena,e, Valdir Blasiosa, Alexandre Martinsb,c,d,Maurício L. Sforçae, Wanius Garciaf, Ana Carolina M. Zerie, Harold P. Ericksong, Andréa Dessenb,c,d,e,and Frederico J. Gueiros-Filhoa,1

aDepartamento de Bioquímica, IQ, Universidade de São Paulo, 05508000, São Paulo, SP, Brazil; bUniversité Grenoble Alpes, cCentre National de la RechercheScientifique, and dCommissariat à l’Energie Atomique et aux Energies Alternatives, Institut de Biologie Structurale, F-38044 Grenoble, France;eBrazilian Biosciences National Laboratory, LNBio, Centro Nacional de Pesquisa em Energia e Materiais (CNPEM), 13083-970, Campinas, SP, Brazil; fCentro deCiências Naturais e Humanas, Universidade Federal do ABC, 09210580, Santo André, SP, Brazil; and gDepartment of Cell Biology, Duke UniversityMedical Center, Durham, NC 27710-3709

Edited by Edward D. Korn, National Heart, Lung and Blood Institute, Bethesda, MD, and approved March 6, 2015 (received for review July 28, 2014)

Cytoskeletal structures are dynamically remodeled with the aid ofregulatory proteins. FtsZ (filamentation temperature-sensitive Z)is the bacterial homolog of tubulin that polymerizes into ringslocalized to cell-division sites, and the constriction of these ringsdrives cytokinesis. Here we investigate the mechanism by whichthe Bacillus subtilis cell-division inhibitor, MciZ (mother cell inhibitorof FtsZ), blocks assembly of FtsZ. The X-ray crystal structure revealsthat MciZ binds to the C-terminal polymerization interface of FtsZ, theequivalent of the minus end of tubulin. Using in vivo and in vitroassays and microscopy, we show that MciZ, at substoichiometric lev-els to FtsZ, causes shortening of protofilaments and blocks the assem-bly of higher-order FtsZ structures. The findings demonstrate anunanticipated capping-based regulatory mechanism for FtsZ.

FtsZ | cell division | cytokinesis | filament capping | bacterial cytoskeleton

The discovery that bacteria have actin-, tubulin-, and inter-mediate filament-like proteins demonstrated that the cyto-

skeleton is an ancient invention, predating the divergencebetween prokaryotes and eukaryotes (1). The GTPase FtsZ(filamentation temperature-sensitive Z) was the first prokaryoticprotein to be recognized as a cytoskeletal element (2, 3). FtsZ isa tubulin-like protein, which is widely conserved in bacteria andthe main component of the bacterial cytokinesis machine, or“divisome.” FtsZ self-assembles into single-stranded protofila-ments and these associate further inside cells to form a super-structure known as the Z ring (4, 5). FtsZ alone can generatea constriction force to initiate division (6). The Z ring also pro-vides a scaffold onto which several other components of thedivisome—mostly cell wall synthesizing enzymes—are recruitedand oriented so as to build the division septum, a cross-wallseparating a progenitor cell into two isogenic daughter cells (7).FtsZ and tubulin share several essential properties: their as-

sembly is cooperative, stimulated by GTP, and leads to GTPhydrolysis; they form dynamic polymers whose turnover is de-pendent on nucleotide hydrolysis (8); they use essentially thesame bond for polymer formation (9); and recent evidenceindicates that they undergo similar allosteric transitions uponpolymerization (10, 11). Not surprisingly, however, the func-tional specialization of these proteins led to some significantdifferences between them, the most prominent being that FtsZexists as single protofilaments, whereas tubulin always adopts amultifilament tubular structure. This difference in their higher-order structure implies that the reactions that lead to cooperativityand subunit turnover are likely different. It has also representeda significant technical challenge for the study of FtsZ. Because FtsZfilaments are smaller than the resolution of optical microscopy, sofar it has been impossible to determine essential properties associ-ated with its dynamic behavior.Similarly to actin filaments and microtubules, the assembly of

FtsZ protofilaments into a Z ring is regulated by a number ofproteins that bind directly to FtsZ and modulate its polymeri-

zation (4). Among these proteins, negative modulators haveattracted the most attention because of their crucial role in de-termining when and where a Z ring should form. The strikinglyprecise positioning of the division site in rod-shaped bacteria,such as Escherichia coli and Bacillus subtilis, is because of thecombined action of Min and nucleoid occlusion systems, twonegative modulators that work together to ensure that the Z ringwill be formed only at midcell. The Min system, whose corecomponent is the polarly localized MinCD complex, prevents theZ ring from forming at the cell poles, reducing the chances ofminicell formation (12, 13). The nucleoid occlusion system, inturn, is based on DNA binding proteins (Noc in B. subtilis andSlmA in E. coli) that recognize specific sequence signatures andinhibit Z ring formation around the bacterial chromosomes(14–16). The combination of Min and nucleoid occlusion in-hibition prevent division from happening at the cell poles andover the chromosomes, leaving only the central region of the cellfree for Z-ring formation. Min/nucleoid occlusion also provide ameans to regulate the cell-cycle timing of Z-ring formation be-cause the creation of an inhibitor-free zone at midcell depends onproper DNA replication and segregation. Another well-studied FtsZmodulator is the checkpoint protein SulA (suppressor of Lon A),which makes bacterial cytokinesis responsive to environmental

Significance

Division of bacteria is executed by a contractile ring whosecytoskeletal framework is FtsZ (filamentation temperature-sensitive Z), a protein evolutionarily related to eukaryotic tubulin.The FtsZ ring is made of filaments of head-to-tail FtsZ subunitsbut its architecture and the rules governing its assembly are stillpoorly known. Here we show that MciZ, an inhibitor of FtsZ ringformation, functions by capping the minus end of FtsZ filaments.Capping by MciZ makes FtsZ filaments shorter than normal, likelyby blocking filament annealing; this represents fundamental in-formation to understand how FtsZ filaments grow and shrink,and attain their normal size. The powerful inhibition of Z-ringassembly by MciZ also suggests that an FtsZ ring cannot formfrom filaments smaller than a certain size.

Author contributions: A.W.B.-F., A.C.M.Z., H.P.E., A.D., and F.J.G.-F. designed research;A.W.B.-F., K.F.D., P.C., V.B., A.M., M.L.S., and W.G. performed research; A.W.B.-F., K.F.D.,P.C., V.B., M.L.S., W.G., A.C.M.Z., H.P.E., A.D., and F.J.G.-F. analyzed data; and A.W.B.-F.,K.F.D., P.C., H.P.E., A.D., and F.J.G.-F. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Data deposition: The atomic coordinates and structure factors have been deposited in theProtein Data Bank, www.pdb.org [PDB ID codes 4U39 (FtsZ:MciZ complex), and 2MRW(free MciZ)].1To whom correspondence should be addressed. Email: fgueiros@iq.usp.br.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1414242112/-/DCSupplemental.

E2130–E2138 | PNAS | Published online April 6, 2015 www.pnas.org/cgi/doi/10.1073/pnas.1414242112

Dow

nloa

ded

by g

uest

on

May

17,

202

0

stresses. SulA is expressed in response to DNA damage as part ofthe SOS system in E. coli and halts Z-ring formation and celldivision until DNA damage is repaired by the cell (17, 18). Morerecently, other negative modulators of FtsZ have been reportedwhose function is to control cytokinesis in response to the nutri-tional [UgtP (19), KidO (20), and OpgH (21)] and developmental[MciZ (mother cell inhibitor of FtsZ) (22) and Maf (23)] state ofthe cell.Eukaryotic cytoskeletal modulators use a variety of strategies

to achieve their effect, including nucleation, monomer seques-tration, filament capping, and severing (24, 25). The conservationof the structure and principles that govern cytoskeletal filamentformation suggest that the general mechanisms operating ineukaryotes should also be present in prokaryotes. However, littleis known about the molecular details of how modulators affectFtsZ assembly. SulA is one of the few inhibitors whose mecha-nism has been studied in detail. The crystal structure of SulA incomplex with FtsZ showed that it binds to the C-terminal poly-merization interface of FtsZ (26). In addition, in vitro experi-ments demonstrated that SulA inhibits FtsZ polymerization bya simple sequestration mechanism (27, 28).Here we focused on MciZ (mother cell inhibitor of FtsZ),

a poorly understood 40-aa peptide discovered in a yeast two-hybrid screen for FtsZ binding partners (22). MciZ is an intriguingexample of a developmentally regulated division inhibitor. It is

expressed during sporulation in B. subtilis and blocks Z-ringformation after cells commit to the terminally differentiatedspore fate. Although it has been shown that MciZ directly in-hibits FtsZ polymerization in vitro (22, 29), the nature of itsinteraction with FtsZ and its inhibition mechanism are still un-known. We determined the crystal structure of MciZ in complexwith FtsZ from B. subtilis and, by using fluorescence and electronmicroscopy as well as biochemical experiments, showed thatMciZ binds to the C-terminal subdomain of FtsZ and acts as acapper of the minus end of FtsZ filaments. Minus-end capping isan unusual way to inhibit filament formation and indicates thatannealing plays an important role in FtsZ filament dynamics.

ResultsCrystal Structure of the FtsZ:MciZ Complex. To investigate themechanism of MciZ inhibition, we first determined the crystalstructure of the FtsZ:MciZ complex to a resolution of 3.2 Å. Wecoexpressed his-tagged MciZ (His6-MciZ) and a truncated ver-sion of FtsZ that included only the globular portion of the pro-tein (FtsZ12-315) in E. coli and purified the complex by metalaffinity and gel-filtration chromatographies. As expected fromthe high affinity of MciZ for FtsZ (Kd of 150 ± 10 nM, measuredby tryptophan fluorescence) (Fig. S1A), the complex was verystable; it could be produced with high purity in milligramquantities and it crystallized under standard conditions. Crystals

Fig. 1. MciZ binds to the minus end of FtsZ and displaces the T7 loop. (A) Cartoon representation of the FtsZ:MciZ complex structure (PDB ID code 4U39). TheN-terminal domain of FtsZ (residues 13–178) is represented in blue, the C-terminal domain (residues 209–314) in green, the H7 α-helix (residues 179–202) inyellow, and MciZ (residues 2–37) in red. Cocrystallized phosphates are represented as orange sticks. (B) Superposition of 20 representative low-energystructures of MciZ (red) solved by 1H NMR (PDB ID code 2MRW). Only a segment of the α-helix H1 appears to get structured in absence of FtsZ. (C) Alignmentbetween the FtsZ:MciZ complex (FtsZ in green/blue/yellow and MciZ in red) and the FtsZ monomer of B. subtilis (gray, PDB ID code 2VAM) showing the overlapbetween MciZ and the T7 loop of the monomer structure, and the absence of the T7 loop in the FtsZ:MciZ structure (blue dotted line). (D) Superposition ofMciZ (red, surface rendering) onto the FtsZ dimer structure of S. aureus (PDB 3VOA). The B. subtilis FtsZ:MciZ complex structure was aligned with the uppersubunit of the dimer (gray FtsZ). Note the steric clash between MciZ and the bottom subunit of the dimer (orange FtsZ).

Bisson-Filho et al. PNAS | Published online April 6, 2015 | E2131

BIOCH

EMISTR

YPN

ASPL

US

Dow

nloa

ded

by g

uest

on

May

17,

202

0

belonged to space group P6522 and displayed nine FtsZ:MciZcomplexes in the asymmetric unit (only one will be describedfor clarity, FtsZ:A and MciZ:J). In FtsZ, residues 203–208 and219–220 could not be modeled in the map potentially because ofinherent flexibility. Our FtsZ structure has no guanine nucleo-tide bound. MciZ (residues 2–37) folds into a β-hairpin followedby a loop, an α-helix, and a helical turn. It binds to the C-terminalsubdomain of FtsZ by aligning with FtsZ’s C-terminal β-sheetthrough hydrogen bonds established between β9 of FtsZ and β2of MciZ, thus generating a six-stranded β-sheet (Fig. 1A). Thismode of protein–protein interaction, generally termed β-strandaddition, is found in diverse protein complexes (30).Binding of MciZ to FtsZ leads to significant restructuring of

MciZ’s N-terminal region, as seen by comparing its structure inthe crystallized complex with the structure of the free peptide insolution, determined by 1H NMR (Fig. 1B). Free MciZ displaysthe same segments of α-helix as the complexed peptide (aminoacids 16–27 and 32–35) but its N terminus is unstructured (Fig. 1B).This N terminus thus becomes structured into two β-segments uponinteraction with β9 of FtsZ, as noted above. The C terminus ofMciZ is unstructured in both the free and bound forms.Binding of MciZ buries a solvent-accessible area on FtsZ of

188 Å2. In addition to the backbone hydrogen bonds involved inthe β-sheet extension, hydrophobic interactions between helicesH1 of MciZ and H10 of FtsZ, and between H1/β2 of MciZ withH10/β9 of FtsZ could stabilize the interaction (Fig. S1B). Recog-nition between FtsZ and MciZ is further strengthened by a saltbridge between the carboxylate of Asp280 of FtsZ and the gua-nidinium group of Arg20 of MciZ (Fig. S1B).To further analyze the FtsZ:MciZ interaction, we introduced

site-directed modifications into FtsZ. FtsZ carrying an Asp toArg mutation at position 280 (Asp280Arg) could not stably bindMciZ (Fig. S1C), probably because of disruption of the aforemen-tioned salt bridge with Arg20. The reciprocal mutation in MciZ, inwhich we substituted Arg20 to an aspartate (Arg20Asp), also dis-rupted the FtsZ:MciZ interaction (Fig. S1C). Thus, the Asp280-Arg20 salt bridge is critical for complex stability.

MciZ Inhibits FtsZ Polymerization by Steric Hindrance. The bindingsite of MciZ is at one of the polymerization surfaces of FtsZ,which suggests that the inhibitory effect of MciZ is simply causedby steric hindrance. Indeed, modeling of MciZ onto the structureof the Staphylococcus aureus FtsZ filament (PDB ID code 3VOA)(10) showed that the presence of MciZ would completely preventthe association of FtsZ monomers (Fig. 1D). Alignment of ourstructure with five other B. subtilis FtsZ structures (PDB IDcodes 2VAM, 2VXY, 2RHL, 2RHO, and 2RHH) showed thatbinding of MciZ causes no major conformational changes (rmsdof 0.610 Å) (Fig. S1D), other than the displacement the T7 loopfrom its usual position. In fact, we cannot observe the densitycorresponding to the T7 loop (residues 203–208) in our structure(Fig. 1C), suggesting that it becomes more flexible or unstructuredupon binding of MciZ. The T7 loop is critical for FtsZ function,contributing contacts to filament formation and the highly con-served 208NLDFAD213 for GTP hydrolysis (3, 31). Despite thisfact, T7 loop displacement is unlikely to be the direct cause of theinhibition of FtsZ polymerization by MciZ; it could be simply aconsequence of steric hindrance by the latter molecule.

MciZ Does Not Compete with GTP Binding. Ray et al. (29) recentlysuggested that MciZ competes with GTP for binding to FtsZ.Because GTP binds to the N-terminal subdomain of FtsZ; this isat odds with the binding site for MciZ identified in our crystalstructure. We revisited the possibility of competition betweenMciZ and GTP by comparing the binding of GTP to free FtsZ andto the FtsZ:MciZ complex using the fluorescent analog mant-GTP. This analysis showed that mant-GTP bound equally well tofree and MciZ-bound FtsZ (Kd of 1.7 ± 0.2 μM and 1.4 ± 0.3 μM,

respectively) (Fig. S1E). Thus, we cannot detect the competi-tion between MciZ and GTP reported by Ray et al., a findingthat is consistent with our structural data that indicates thatMciZ and GTP bind at opposite surfaces of the FtsZ molecule.

Substoichimetric MciZ Shortens FtsZ Protofilaments. There are avariety of mechanisms by which modulatory proteins can inhibitcytoskeletal filament formation. Binding of MciZ to the poly-merization interface of FtsZ is compatible with MciZ acting byeither monomer sequestration or filament capping. Monomersequesterers inhibit assembly by reducing the effective concen-tration of monomer and, consequently, shifting the equilibriumtoward depolymerization. A sequestered subunit is incapable ofbinding at either end. Thus, sequesterers tend to be effectiveonly when present at close to stoichiometric concentrations rel-ative to the filament-forming protein (28). In contrast, cappersblock the ability of a subunit to bind at one end but leave theother end available to bind to a filament and block further as-sembly. Cappers thus tend to exhibit effects even at substoi-chiometric concentrations (32, 33). To study the mechanism ofMciZ inhibition, we monitored the effect of various concen-trations of MciZ on the polymerization of FtsZ by light scattering.This showed that as little as a 1:10 ratio of MciZ to FtsZ causeda marked inhibition (about 50%) of the light-scattering signal (Fig.2A). In contrast, treatment of FtsZ with the MciZ-R20D mutantdid not affect the levels of light scattering, suggesting that theMciZ effect is specific and dependent on the interaction with FtsZ(Fig. S2A). Because the light-scattering signal is disproportionatelysensitive to polymer size, the 50% inhibition of light scattering by1:10 MciZ does not necessarily mean that there was a reduction byhalf in the polymer mass (or polymer bonds). More likely, thissubstoichiometric inhibition of the light-scattering signal reflectsa decrease in length or width (bundling) of the polymers.To investigate further this substoichiometric poisoning, we

carried out electron microscopy (EM) of negatively stained FtsZpolymers formed in the presence of MciZ. Polymerization of3 μM FtsZ in HMK buffer produced filaments with a length of200 ± 75 nm (Fig. 2C). In contrast, reactions with 3 μM FtsZ and0.3 μM MciZ, the same 1:10 ratio that inhibited light scatteringby 50%, showed a reduction in protofilament length to 120 ±45 nm (Fig. 2D). This reduction in length caused by 0.3 μMMciZcannot be explained by a sequestration mechanism, in which theeffect of MciZ would be to reduce the concentration of activeFtsZ monomers from 3.0 to 2.7 μM. Some filaments withoutMciZ seem to have higher contrast than those with MciZ, sug-gesting that they may be small bundles. Thus, the inhibition oflight scattering by MciZ may also involve a reduction of bun-dling, probably as a result of shortening of the protofilaments.Nevertheless, to confirm that the primary effect of MciZ is toreduce the length of protofilaments rather than inhibiting bun-dling, we tested the effect of higher concentrations of MciZ (0.6and 1.0 μM) on the polymerization of 3 μM of FtsZ (Fig. S2B).The results showed shorter protofilaments as MciZ concentrationwas increased, and no visible structures could be identified at 1.0μM of MciZ. Because the critical concentration (Cc) of FtsZ inthe conditions of our experiments is between 1 and 1.5 μM (seeFig. 5 A and B), it is conceivable that the inhibitory effect of 1.0μM MciZ added to 3 μM FtsZ could be explained by MciZ se-questering FtsZ to a concentration of active monomers too closeto the Cc to allow for assembly. To rule out this possibility, we alsodid EM experiments with FtsZ-L69W, a mutant protein that hasa significantly lower Cc than the wild-type protein (0.5 μM). MciZshortened FtsZ-L69W filaments as well as affecting wild-type FtsZ(Fig. S2C), suggesting that it is not acting by sequestration.

A FtsZ–MciZ Fusion Protein Inhibits Assembly Similarly to MciZ. Thesubstoichiometric effect of MciZ suggests that it is acting as acapper. However, to decisively rule out sequestration of monomers

E2132 | www.pnas.org/cgi/doi/10.1073/pnas.1414242112 Bisson-Filho et al.

Dow

nloa

ded

by g

uest

on

May

17,

202

0

as the mechanism of MciZ inhibition, we tested the effect ofadding MciZ in the form of an FtsZ:MciZ complex on the as-sembly of FtsZ. If MciZ works as a sequesterer, adding MciZthat is already complexed with FtsZ should have no effect onFtsZ assembly. We tested both the noncovalent FtsZ:MciZcomplex produced by copurification, as well as a covalent com-plex, in which we fused MciZ to the C-terminal end of FtsZ(FtsZ–MciZ). The C-terminal end of FtsZ is a mostly unstructured,flexible chain with a contour length of 23 nm (34, 35), more thanenough to allow MciZ to bind to the same FtsZ molecule that it iscovalently connected to. Strikingly, both the FtsZ:MciZ complex(Fig. S3A) and the FtsZ–MciZ fusion were highly effective atinhibiting FtsZ assembly (Fig. 2B). Plotting the data from allexperiments together (free MciZ, FtsZ:MciZ complex, and FtsZ–MciZ fusion) indicates that the complexes are as capable as freeMciZ to promote inhibition (Fig. S3B). Similarly, EM determi-nation of filament lengths demonstrated that the FtsZ–MciZ fu-sion shortened FtsZ filaments somewhat more than MciZ alone(100 ± 35 nm vs. 120 ± 45 nm) (Fig. 2E). These results demon-strate that MciZ does not act as a simple monomer sequesterer andsuggest that MciZ-bound FtsZ monomers can interact with FtsZfilaments to block their growth. In the simplest model, the N-ter-minal polymerization face of MciZ-poisoned FtsZ should be held ina polymerization-competent conformation that can bind to and capthe minus end of protofilaments.

Substoichiometric MciZ Inhibits Z-Ring Formation in Vivo. MciZ is avery potent inhibitor of cell division in vivo, being capable of

causing a complete block of Z-ring formation and robust fila-mentation when induced with as little as 0.1% xylose from asingle integrated chromosomal copy (Fig. 3 A and B). This findingsuggested that MciZ also works substoichiometrically in vivo. Toascertain that theory, we carried out quantitative Western blotsof cells expressing GFP-MciZ, using anti-GFP serum and puri-fied GFP as a standard (Fig. 3C). This process revealed thatinduction with 0.1% xylose leads to the accumulation of 350 ± 150molecules of GFP-MciZ per cell (Fig. S3D). We also quantified thelevels of endogenous FtsZ in the same extract (Fig. S3 C and E),and found 4,200 molecules per cell, similarly to published data thatindicates that B. subtilis contains at least 5,000 molecules of FtsZper cell when grown in rich medium (36, 37). This result suggeststhat a ratio of MciZ to FtsZ of less than 1:10 is enough to com-pletely block Z-ring formation and cell division in vivo.

MciZ Binds to FtsZ Filaments in Vitro and to FtsZ Polymers in Vivo. Acapping protein should be able bind to the polymeric form of itstarget. This ability of MciZ is implied by the results above, but wealso aimed to demonstrate the binding of MciZ to FtsZ filamentsdirectly, both in vitro and in vivo. To show binding of MciZ toFtsZ filaments in vitro, we carried out sedimentation experi-ments using rhodamine-labeled MciZ and FITC-labeled FtsZ(Fig. 4 A and B and Fig. S4). Reactions with 10 μM FtsZ and noMciZ resulted in ∼50% of FtsZ in the pellet. The addition of250 nM MciZ produced a clear reduction in pelleted FtsZ to25%. Importantly, MciZ efficiently cosedimented with the FtsZpolymers in these reactions, being found almost equally partitioned

Fig. 2. MciZ inhibits FtsZ polymerization substoichiometrically in vitro. All assembly experiments were done in HMK buffer (50mMHepes, 5 mMMgAc, 100mMKAc,pH 7.7). (A and B) Effect of various concentrations (0, 0.2, 0.5, 1, and 2.5 μM) of MciZ (A) or the FtsZ-MciZ fusion (B) on the polymerization of FtsZ (5 μM) reported byright-angle light scattering. (C–E) Effect of MciZ or FtsZ-MciZ on FtsZ filament length visualized by negative-staining EM. For this, 3 μM FtsZ was polymerized without(C) and with 0.3 μM MciZ (D) or 0.3 μM FtsZ-MciZ (E). The filament length distribution in each situation was measured and is plotted (Right). (Scale bar, 100 nm.)

Bisson-Filho et al. PNAS | Published online April 6, 2015 | E2133

BIOCH

EMISTR

YPN

ASPL

US

Dow

nloa

ded

by g

uest

on

May

17,

202

0

between supernatant and pellet fractions. As a control for thespecificity of the cosedimentation, we also tested the MciZ-R20D mutant and found that it neither inhibited FtsZ sedi-mentation nor cosedimented with FtsZ. Even though sedimen-tation experiments are not fully quantitative (they underestimatepolymer because individual protofilaments do not pellet well),the distribution of MciZ between pellet and supernatant frac-tions suggests it binds preferentially to FtsZ filaments.To demonstrate binding of MciZ to FtsZ polymers in vivo, we

imaged GFP-MciZ and attempted to detect decoration of Zrings with GFP-MciZ. Induction of even low levels of GFP-MciZin a wild-type strain led to very fast disassembly of Z rings and

only rarely could we observe fluorescent Z rings. To improve ourobservation window, we repeated the experiment in a strainbearing a mutation in FtsZ (T45M) that makes it partially re-sistant to MciZ. This strain also expressed a low level of FtsZ-mCherry, to allow for colocalization of MciZ and FtsZ. In-duction of GFP-MciZ for short times (15 min) allowed us todetect fluorescent Z rings in a high proportion of the cells (Fig. 4C).The FtsZ-mCherry rings in Fig. 4C are dimmer than typical Z rings(compare Fig. 4C, Upper and Lower) and tended to localize at di-vision sites that already started to constrict. This result was alsoobserved for the GFP-MciZ rings in the same cells and suggests thatthe inhibitory effect of MciZ is probably stronger at newer Z ringsthan in mature rings, in which FtsZ is associated with other divi-some proteins that could stabilize it. Despite the atypical appear-ance, 100% of the GFP-MciZ rings colocalized with FtsZ-mCherryrings (Fig. 4C, Upper). As a control for the specificity of the GFP-MciZ localization, we also imaged a strain expressing GFP-MciZ-R20D, which exhibited no inhibitory effect on Z-ring formation andwas found to be completely dispersed in the cytosol, despite thepresence of robust FtsZ-mCherry rings in this strain (Fig. 4C,Lower). These results indicate that MciZ associates with FtsZpolymers in vivo. Because detection of GFP-MciZ rings requiresthat more than a few GFP-MciZ molecules decorate each Z ring,these results are further support for the idea that the Z ring isformed by multiple short protofilaments with exposed minus ends.

MciZ Does Not Promote Extensive Depolymerization of FtsZ Filaments.Substoichiometric effects have been reported before for cappersof actin filaments and microtubules (32, 38). These cappersusually bind to the growing (plus) end of filaments and restrictexchange of subunits to the minus end. Because the minus endhas a higher Cc than the plus end, blocking the plus end leads toextensive depolymerization of filaments, until the free monomerconcentration becomes equal to the minus-end Cc (39, 40). Suchdepolymerization induced by eukaryotic capping proteins is de-pendent on, and a hallmark of, treadmilling (41). To deter-mine if the shortening of FtsZ protofilaments by MciZ involved

Fig. 3. MciZ blocks cell division substoichiometrically in vivo. (A and B)B. subtilis cells without (A) and with (B) GFP-MciZ expression induced by 0.1%xylose for 2 h. The cells were stained with FM 5-95 membrane dye to revealsepta. (Scale bar, 3 μm.) (C) Quantification of GFP-MciZ from cell extracts. GFP-MciZ was induced with 0.1% xylose for 2 h. GFP-MciZ was detected by im-munoblotting using anti-GFP antibody and quantification was carried out bycomparing the intensities of the bands with that of purified GFP standards.

Fig. 4. MciZ binds to FtsZ filaments. (A and B) Cosedimentation of FtsZ polymers and MciZ. FtsZ was added to the reaction to a final concentration of 10 μM (9 μMof unlabeledwild type FtsZ and 1 μMof FtsZ-S152C labeledwith FITC). MciZ-W36C andMciZ-R20D+W36Cwere labeled with TMR and added to a final concentrationof 250 nM (when indicated). Fluorescent measurements from supernatant (light gray) and pellet (dark gray) fractions were obtained by excitation at 495 nm (FITC)and 550 nm (TMR). NF, normalized fluorescence; P, pellet; S, supernatant. (C) Colocalization of Ftsz-mCherry and GFP-MciZ in B. subtilis live cells. (Upper) Cellsexpressing FtsZ-mCherry (Left) and GFP-MciZ (Right). (Lower) The mislocalization of GFP-MciZ(R20D) mutant compared with the wild-type MciZ. (Scale bar, 2 μm.)

E2134 | www.pnas.org/cgi/doi/10.1073/pnas.1414242112 Bisson-Filho et al.

Dow

nloa

ded

by g

uest

on

May

17,

202

0

substoichiometric depolymerization of FtsZ, we used a fluores-cence-quenching assay capable of accurately quantifying polymeri-zation (42). For this assay we constructed the double-mutantFtsZ(S152C/S223W) and labeled the Cys with BODIPY. As withthe equivalent construct for E. coli FtsZ, the Trp quenching of theBODIPY fluorescence is decreased by the conformational changeaccompanying assembly, and the fluorescence increase reportsthe total protofilament polymer. We used the BODIPY-labeledFtsZ mixed with a ninefold excess of wild-type FtsZ to ensurethat the assembly is dominated by the wild-type FtsZ and avoidthe bundling observed previously for fully labeled FtsZ (42).Fig. S5 shows that assembly kinetics reached the overshoot

peak in 12 s, a short time consistent with protofilament assemblyas opposed to slower bundle formation (40 s to overshoot peakin Fig. 2 A and B) (42). Fig. 5A plots the plateau assembly fora range of FtsZ up to 9 μM, and for MciZ added at 0, 1, 2, and3 μM. Similarly to the previously reported inhibition of FtsZ bySulA (27, 28), MciZ caused an increase in the apparent criticalconcentration, Ccapp, of FtsZ from 1.5 to 2.4, 3.1, and 4.2 μM,respectively. The increase in Ccapp was almost equal to theamount of added MciZ, consistent with MciZ binding FtsZ withhigher affinity than the ∼1.5 μM Cc. This finding means thateach MciZ molecule is roughly preventing the formation of onlyone FtsZ–FtsZ bond and indicates that capping of the minus enddoes not induce extensive depolymerization of the plus end.

MciZ Increases the Dynamics of FtsZ Filaments. We measured theGTPase activity of FtsZ in the presence of MciZ to assess whethercapping by MciZ affected the dynamics of FtsZ filaments. Reac-tions were set up with a similar range of FtsZ concentrations andwith MciZ concentrations of 0, 2, 4, and 6 μM. As expected, MciZdecreased the absolute GTPase activity of FtsZ and increased theapparent Cc in proportion to its concentration (Fig. 5B), corrob-orating the BODIPY results. Strikingly, however, the specific ac-tivity (the slope of the line in GTP per minute per FtsZ) was

increased from 2.8 in the absence of MciZ, to 5.4 at all concen-trations of MciZ. The converse experiment, in which we assayed afixed FtsZ concentration (10 μM) in the presence of varying MciZconcentrations, also revealed a twofold increase in the GTPaseactivity of FtsZ in the presence of low MciZ concentrations (Fig.S6). However, at a ratio of MciZ to FtsZ higher than 1:10, theGTPase activity started to decrease (Fig. S6), probably because athigh MciZ the reduction in the amount of polymerized FtsZ beginsto offset the stimulatory effect of MciZ.The increase in GTPase specific activity indicates that poly-

mers assembled in the presence of MciZ are more dynamic. Thefaster dynamics could be because of MciZ increasing the rate ofGTP hydrolysis, or the rate of subunit exchange, as both limit theoverall GTP turnover by FtsZ polymers (43, 44). To further in-vestigate the effect of MciZ on polymer dynamics, we appliedagain the BODIPY fluorescence-quenching assay, but this timeto follow the disassembly kinetics of FtsZ filaments after dilutionto below the Cc. Disassembly curves were fit to a single expo-nential with decay time τ. The disassembly of FtsZ polymers inHMK buffer (Fig. 5C) was much faster in reactions with 1:10MciZ (τ = 3 ± 2 s), compared with those with FtsZ alone (τ =10 ± 2 s). A similar effect was observed in reactions in whichGTP hydrolysis was blocked by the addition of EDTA (Fig. 5D).Disassembly in EDTA was slower than with GTP hydrolysis, asreported previously (43). However, polymers poisoned with MciZagain exhibited faster disassembly than FtsZ alone (τ = 9 ± 2 and20 ± 3 s, respectively). Because the effect of MciZ is independentof GTP hydrolysis, we conclude that MciZ increases the turnoverof FtsZ subunits in filaments, as we explain in the discussion.

DiscussionWe applied a combination of structural, biochemical, and in vivoapproaches to elucidate the mechanism of MciZ, a develop-mentally regulated inhibitor of FtsZ polymerization of B. subtilis.Clarifying the molecular effects of the growing roster of FtsZ

Fig. 5. MciZ enhances the turnover of FtsZ filaments. (A) FtsZ polymerization reported by fluorescence quenching with BODIPY-conjugated FtsZ-S152C+S223W. Assembly curves with different FtsZ and MciZ concentrations were run to steady state and the ΔFluorescence was plotted. MciZ concentrations areindicated above the lines. (B) Effect of MciZ on the GTPase activity of FtsZ. (C and D) Kinetics of disassembly of FtsZ protofilaments reported by BODIPY-quench system: (C) in 5 mM Mg2+ (HMK buffer, which supports GTPase on), (D) in 1 mM EDTA (MEK buffer, GTPase blocked). Five micromolars of FtsZ waspreassembled in the absence (gray) or presence of 0.5 μM of MciZ (red) and the rate of disassembly was measured after dilution in the appropriate buffer.

Bisson-Filho et al. PNAS | Published online April 6, 2015 | E2135

BIOCH

EMISTR

YPN

ASPL

US

Dow

nloa

ded

by g

uest

on

May

17,

202

0

modulators will be key for a better understanding of Z-ringformation. Our data show that MciZ is a capping protein, andthat the inhibition of FtsZ assembly at low MciZ stoichiometriesis primarily a result of capping. At high stoichiometries, MciZ iscapable of both capping and sequestration of FtsZ. Thus, theidentification of the first FtsZ capping protein indicates that thepalette of biochemical activities available for the regulation ofthe bacterial cytoskeleton could be as diverse as the one knownto operate on actin and tubulin.We also exploited MciZ as a probe to try to answer some

longstanding questions about FtsZ. The small size of FtsZ poly-mers has posed a formidable challenge to the understandingof its dynamic behavior. Simple questions, such as whether thepolymerization of filaments is asymmetric, or if FtsZ undergoestreadmilling or dynamic instability, have remained unansweredlargely because individual FtsZ filaments are shorter than theresolution of the light microscope. By carefully analyzing theeffect of MciZ in bulk measurements of FtsZ polymerization, weprovide evidence suggesting that fragmentation and annealingare important determinants of FtsZ filament size and that, simi-larly to tubulin, the plus end is more dynamic than the minus endof FtsZ filaments. We anticipate that the use of MciZ as a reagentin further studies, together with the application of sophisticatedimaging methods to visualize filaments, will soon allow the con-struction of a realistic model of FtsZ polymerization dynamics.

MciZ Binds to the Minus End of FtsZ and Functions as a Filament-Capping Protein. Our crystal structure of the FtsZ:MciZ com-plex showed that MciZ binds to the C-terminal polymerizationinterface of FtsZ—the minus end of the molecule—and impedesfurther polymerization by steric hindrance. Because binding ofMciZ to the minus end does not affect the plus end of FtsZ(Fig. 1 and Fig. S1D), MciZ should be able to function as a capperand our data show that this is indeed the case.The evidence that MciZ is a capper is manifold. First, it binds

to filaments in addition to FtsZ monomers (Fig. 4). Second, itacts at substoichiometric concentrations. At 1:10 MciZ:FtsZ, thelight-scattering signal was substantially reduced, and EM showeda reduction in the length of filaments (Fig. 2). These effects wereapproximately the same when MciZ was added alone, in a 1:1complex with FtsZ, and when MciZ was fused to the C terminusof FtsZ (Fig. 2 and Fig. S3). The similar effects of free andprebound MciZ can only be explained if the FtsZ:MciZ complexcaps the minus end of FtsZ filaments. Third, MciZ increases thedynamics of FtsZ polymers (Fig. 5 B–D). Above the Cc, FtsZfilaments are thought to be in a steady-state on-off reaction withthe pool of subunits. When diluted below the Cc, the on-reactionis eliminated, leaving only the off-reaction. If disassembly occursprimarily from the filament ends, its rate will be proportional tofilament length. If the filaments are half as long, and thereforepresenting twice as many ends, they should take half as long todisassemble. Because the 1:10 MciZ:FtsZ filaments disassembledtwo- to three-times faster than FtsZ without MciZ, this suggeststhat they are two- to three-times shorter. The twofold increase inGTPase promoted by MciZ is also consistent with shorter fila-ments and increased number of ends. Both results are in goodagreement with the EM data, and represent independent dem-onstrations of filament shortening by MciZ.SulA, the only other FtsZ inhibitor whose mechanism is well

established, also binds to the minus end of FtsZ (26). Interest-ingly, however, SulA functions as a monomer sequesterer insteadof a capper (27, 28). Because SulA does not directly occlude theplus end of FtsZ, a monomer bound to SulA could still poly-merize using its free plus end, but this does not seem to occur.The reason for the difference in the mechanisms of MciZ andSulA may reside in the details of how these inhibitors interactwith the minus end of FtsZ, or may be related to the differentaffinities that these inhibitors display for FtsZ. SulA displays 10-fold

lower affinity for FtsZ than MciZ and may not be able to competeeffectively with monomeric FtsZ for binding to filament ends.

How Does MciZ Shorten FtsZ Filaments?Minus-end cappers (γ-tubulin,Tropomodulin, Arp2/3) generally exert a stabilizing or nucleat-ing role for actin and microtubules (45–47). Because the minusend is usually the site of subunit loss (40, 48), capping of theminus end prevents disassembly and stabilizes filaments. In con-trast to the eukaryotic paradigm, MciZ is a minus-end capperthat shortens FtsZ filaments and destabilizes the Z ring. To ex-plain this apparent paradox we recall that in addition to affectingthe exchange of subunits at filament ends, cappers will also affectannealing, the reaction by which two filaments become joined bytheir ends. Capping of either end of a filament will block annealingand, depending on how frequent annealing is, it could havea marked effect on filament sizes. Thus, to explain the effect ofMciZ on FtsZ filaments we propose a model in which filamentlength at steady state will depend on the gain and loss of subunits atfilament ends and on the balance between filament fragmentationand annealing (Fig. 6). Because capping of the minus end does notinduce extensive depolymerization from the plus end (Fig. 5A), wepostulate that blocking annealing may be an important pathway bywhich MciZ reduces filament size. Fragmentation and annealing ofFtsZ filaments have been observed for filaments adsorbed tomica or supported lipid bilayers (49–51), and also in solution(43), although the rates of these reactions are yet to be preciselydetermined. Annealing may be particularly important in vivo,when the filaments are concentrated on the membrane at thecenter of the cell (5, 52). Thus, blocking annealing in vivo wouldbe a powerful way to block the overall assembly of the Z ring.Our observation that a minus-end capper destabilizes FtsZ fila-

ments is also supported by recent work by Arumugam et al. (51),who produced a truncated FtsZ termed “NZ” that included only theN-terminal subdomain, and found that it also worked as a capperand caused the disassembly of FtsZ bundles on a supported lipidbilayer. This finding indicates that FtsZ filaments are susceptible tocapping when assembled in higher-order structures tethered to themembrane, a more physiological situation than our in vitro system,

Fig. 6. Mechanism of FtsZ polymerization inhibition by MciZ. FtsZ filamentlength is determined by the rates of subunit gain and loss at filament ends,and the balance between filament fragmentation (kfrag) and annealing(kanneal). Capping of filament minus ends by MciZ (red circles) will inhibitsubunit addition at these ends, but in principle, growth at the free plus endscould compensate that. In contrast, inhibition of annealing by MciZ turnsfragmentation into an irreversible reaction, leading to shortening of FtsZfilaments. At close to stoichiometric concentrations, MciZ should also pro-mote subunit sequestration in addition to capping.

E2136 | www.pnas.org/cgi/doi/10.1073/pnas.1414242112 Bisson-Filho et al.

Dow

nloa

ded

by g

uest

on

May

17,

202

0

and supports the conclusion that the powerful inhibition of Z-ringformation by MciZ in vivo is a consequence of capping.

MciZ Acts as a Sequesterer at High Concentrations. Cytoskeletal mod-ulatory proteins rarely have a single effect on their targets and it isnot unexpected that a capper may also exhibit sequestration effects.For example, tropomodulin, which is a minus-end capper like MciZ,has been shown to bind to actin monomers and promote seques-tration at high concentrations (53). MciZ behaves as a sequestererat high concentrations, as demonstrated by the increase in Ccapp thatis approximately equal to the concentration of addedMciZ (Fig. 5 Aand B). SulA produces an increase in Ccapp somewhat lower than itsconcentration, because its 0.7 μM Kd is comparable to the Kd forFtsZ filament formation [which is equal to the Cc (28)]. MciZ bindswith a much higher affinity, Kd ∼0.1 μM, measured from trp fluo-rescence (Fig. S1A), so the sequestration is approximately equal tothe concentration of MciZ. Sequestering activity may be explainedas follows. The FtsZ:MciZ complex is sterically blocked at the minusend, making it impossible to bind to the plus end of a filament. Thecomplex can bind to the minus end of a filament but at high MciZconcentration all filament minus ends should already be capped.Thus, the bound subunit is effectively sequestered.

The Polarity of FtsZ Filaments. Some of our results are consistentwith FtsZ exhibiting the same kinetic polarity as tubulin. A sig-nificant observation is that the capped filaments apparently do notdisassemble at the plus end (Fig. 5A), indicating that the Cc of theplus end is similar or lower than the Cc of the minus end. In ad-dition, because the rate of polymerization in the presence of MciZis not markedly changed (Fig. S5), this suggests that the plus enddominates the kinetics of assembly. A more dynamic plus endmeans that FtsZ filaments should be inherently capable of tread-milling and, in fact, treadmilling has recently been reported forFtsZ filaments tethered to planar lipid monolayers by FtsA (54).However, our conclusion that assembly of FtsZ is maintainedprimarily at the plus end is the opposite of a previous treadmillinginterpretation. Redick et al. made an effort to create FtsZ cappersby making debilitating point mutants of the top and bottominterfaces of E. coli FtsZ (55). Surprisingly, top-cap mutants, witha debilitating mutation on the upper GTP binding surface werecompletely inactive. Bottom-cap mutants showed dominant-nega-tive effects in vivo, and blocked GTPase activity when mixed withwild-type FtsZ in vitro; however, these were extremely weak, re-quiring approximately 10-times excess of the cap mutant relative towild-type. The differential effect of the top and bottom caps sug-gested treadmilling, but with reversed polarity relative to tubulin(subunits added primarily to the minus end, and dissociated pri-marily at the plus end). It is possible that the differential effect oftop- and bottom-cap mutants is simply a result of a poorer ability ofthe top-mutant subunits to associate with filaments. Nevertheless,we will need more information, preferably the direct observation offilaments, before we can resolve the issue of FtsZ polarity.

A Threshold Size for FtsZ Filaments in Vivo? The substoichiometricinhibition and the high affinity for FtsZ means that MciZ is themost powerful inhibitor of Z-ring formation described to date. This

finding makes sense in light of MciZ’s function. In contrast withother negative modulators, such as SulA and MinC, whose activityneeds to be restricted in space/time or reversed, MciZ acts ona terminally differentiating cell that is going to die. Thus, MciZeffects do not need to be reversed. The substoichiometric effect ofMciZ in vivo also suggests that FtsZ filaments have a minimum size,below which a Z ring does not form. Assuming similar effects invitro and in vivo, our data suggest that halving filament lengthsuffices to block Z-ring formation in vivo. Why do shorter filamentsnot work? Shorter filaments seem less capable of interacting withFtsA and getting recruited to the membrane (54). In addition, theymay have a lower tendency to make the lateral interactions necessaryto form a coherent ring. Further investigation of this hypothesis mayproduce new insights on the structure and assembly of the Z ring.

Materials and MethodsGeneral Methods. All B. subtilis strains were derived from the wild-type PY79strain. Final data collection, structure solution, and refinement statistics arein Tables S1 and S2. Oligonucleotides are listed in Table S3 and plasmids inTable S4. Purification of FtsZ and the FtsZ:MciZ complex is described in SIMaterials and Methods. Immunoblots were performed using anti-GFP andanti-FtsZ antisera. The detailed protocol and procedure to estimate cellularprotein concentration can be found in SI Materials and Methods.

Structure Determination. The crystal structure of the FtsZ:MciZ complex wassolved to 3.2 Å using molecular replacement techniques, and the solutionstructure of free MciZ was solved by 2D 1H-NMR. Both methodologies aredescribed in SI Materials and Methods.

Biochemical Experiments. The affinity of MciZ for FtsZ was measured by tryp-tophan fluorescence. The affinity of the FtsZ:MciZ complex for GTP was mea-suredusing the fluorescent analogmant-GTP. FtsZ polymerizationwasmeasuredby right angle light scattering or by a BODIPY-based fluorescence quenchingsystem. The GTPase activity of FtsZ was determined using the malachite greenmethod. Detailed protocols for each procedure are in SI Materials andMethods.

Microscopy. FtsZ protofilaments were imaged by negative stain EM. Cellfilamentation and GFP-MciZ localization were determined by live-cell fluo-rescence microscopy. Detailed protocols for microscopy procedures are in SIMaterials and Methods.

ACKNOWLEDGMENTS. We thank Ethan Garner, Jessica Polka, Masaki Osawa,and José Manuel Andreu for discussions; the Brazilian Biosciences NationalLaboratory for access to their facilities; J. Marquez and the High-ThroughputCrystallization Laboratory team (Grenoble Partnership for Structural Biology)for access to and help with high-throughput crystallization; and the EuropeanSynchrotron Radiation Facility for access to beamlines. This work used the plat-forms of the Grenoble Instruct Center (UMS 3518 Centre National de laRecherche Scientifique-Commissariat à l’Energie Atomique et aux EnergiesAlternatives-Université Joseph Fourier-European Molecular Biology Laboratory)with support from the French Infrastructure for Structural Biology (ANR-10-INSB-05-02) and the Genoble Alliance for Integrated Structural Cell Biology(ANR-10-LABX-49-01) within the Grenoble Partnership for Structural Biology.This work was supported by Grants 10/51866-0 (Smolbnet 2.0) from Fundaçãode Amparo à Pesquisa do Estado de São Paulo (FAPESP) and 1169/2013 fromCoordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) (toF.J.G.-F.); National Institute of Health Grant GM66014 (to H.P.E.); a doctoralfellowship from FAPESP and a doctoral sandwich fellowship from the Sciencewithout Borders program (to A.W.B.-F.); a doctoral fellowship from FAPESP(to V.B.); a postdoctoral fellowship from FAPESP (to P.C.); and a PQ-2 fellowshipfrom Conselho Nacional de Desenvolvimento Científico e Tecnológico (to F.J.G.-F.).

1. Theriot JA (2013) Why are bacteria different from eukaryotes? BMC Biol 11:119.2. Bi EF, Lutkenhaus J (1991) FtsZ ring structure associated with division in Escherichia

coli. Nature 354(6349):161–164.3. Nogales E, Downing KH, Amos LA, Löwe J (1998) Tubulin and FtsZ form a distinct

family of GTPases. Nat Struct Biol 5(6):451–458.4. Adams DW, Errington J (2009) Bacterial cell division: Assembly, maintenance and

disassembly of the Z ring. Nat Rev Microbiol 7(9):642–653.5. Erickson HP, Anderson DE, Osawa M (2010) FtsZ in bacterial cytokinesis: Cytoskeleton

and force generator all in one. Microbiol Mol Biol Rev 74(4):504–528.6. Osawa M, Anderson DE, Erickson HP (2008) Reconstitution of contractile FtsZ rings in

liposomes. Science 320(5877):792–794.7. De Boer PA (2010) Advances in understanding E. coli cell fission. Curr Opin Microbiol

13(6):730–737.

8. Mukherjee A, Lutkenhaus J (1998) Dynamic assembly of FtsZ regulated by GTP hy-drolysis. EMBO J 17(2):462–469.

9. Aylett CHS, Löwe J, Amos LA (2011) New insights into the mechanisms of cytomotiveactin and tubulin filaments. Int Rev Cell Mol Biol 292(40):1–71.

10. Matsui T, et al. (2012) Structural reorganization of the bacterial cell-division proteinFtsZ from Staphylococcus aureus. Acta Crystallogr D Biol Crystallogr 68(Pt 9):1175–1188.

11. Ravelli RBG, et al. (2004) Insight into tubulin regulation from a complex with col-chicine and a stathmin-like domain. Nature 428(6979):198–202.

12. Rothfield L, Taghbalout A, Shih YL (2005) Spatial control of bacterial division-siteplacement. Nat Rev Microbiol 3(12):959–968.

13. Lutkenhaus J (2007) Assembly dynamics of the bacterial MinCDE system and spatialregulation of the Z ring. Annu Rev Biochem 76:539–562.

Bisson-Filho et al. PNAS | Published online April 6, 2015 | E2137

BIOCH

EMISTR

YPN

ASPL

US

Dow

nloa

ded

by g

uest

on

May

17,

202

0

14. Wu LJ, et al. (2009) Noc protein binds to specific DNA sequences to coordinate celldivision with chromosome segregation. EMBO J 28(13):1940–1952.

15. Cho H, McManus HR, Dove SL, Bernhardt TG (2011) Nucleoid occlusion factor SlmA isa DNA-activated FtsZ polymerization antagonist. Proc Natl Acad Sci USA 108(9):3773–3778.

16. Tonthat NK, et al. (2011) Molecular mechanism by which the nucleoid occlusionfactor, SlmA, keeps cytokinesis in check. EMBO J 30(1):154–164.

17. Huisman O, D’Ari R, Gottesman S (1984) Cell-division control in Escherichia coli:Specific induction of the SOS function SfiA protein is sufficient to block septation.Proc Natl Acad Sci USA 81(14):4490–4494.

18. Bi E, Lutkenhaus J (1993) Cell division inhibitors SulA and MinCD prevent formation ofthe FtsZ ring. J Bacteriol 175(4):1118–1125.

19. Weart RB, et al. (2007) A metabolic sensor governing cell size in bacteria. Cell 130(2):335–347.

20. Radhakrishnan SK, Pritchard S, Viollier PH (2010) Coupling prokaryotic cell fate anddivision control with a bifunctional and oscillating oxidoreductase homolog. Dev Cell18(1):90–101.

21. Hill NS, Buske PJ, Shi Y, Levin PA (2013) A moonlighting enzyme links Escherichia colicell size with central metabolism. PLoS Genet 9(7):e1003663.

22. Handler AA, Lim JE, Losick R (2008) Peptide inhibitor of cytokinesis during sporulationin Bacillus subtilis. Mol Microbiol 68(3):588–599.

23. Briley K, Jr, Prepiak P, Dias MJ, Hahn J, Dubnau D (2011) Maf acts downstream ofComGA to arrest cell division in competent cells of B. subtilis. Mol Microbiol 81(1):23–39.

24. Pollard TD, Cooper JA (1986) Actin and actin-binding proteins. A critical evaluation ofmechanisms and functions. Annu Rev Biochem 55:987–1035.

25. Winder SJ, Ayscough KR (2005) Actin-binding proteins. J Cell Sci 118(Pt 4):651–654.26. Cordell SC, Robinson EJ, Lowe J (2003) Crystal structure of the SOS cell division in-

hibitor SulA and in complex with FtsZ. Proc Natl Acad Sci USA 100(13):7889–7894.27. Dajkovic A, Mukherjee A, Lutkenhaus J (2008) Investigation of regulation of FtsZ

assembly by SulA and development of a model for FtsZ polymerization. J Bacteriol190(7):2513–2526.

28. Chen Y, Milam SL, Erickson HP (2012) SulA inhibits assembly of FtsZ by a simple se-questration mechanism. Biochemistry 51(14):3100–3109.

29. Ray S, Kumar A, Panda D (2013) GTP regulates the interaction between MciZ and FtsZ:A possible role of MciZ in bacterial cell division. Biochemistry 52(2):392–401.

30. Remaut H, Waksman G (2006) Protein–protein interaction through beta-strand ad-dition. Trends Biochem Sci 31(8):436–444.

31. Oliva MA, Cordell SC, Löwe J (2004) Structural insights into FtsZ protofilament for-mation. Nat Struct Mol Biol 11(12):1243–1250.

32. MacLean-Fletcher S, Pollard TD (1980) Mechanism of action of cytochalasin B on actin.Cell 20(2):329–341.

33. Kilimann MW, Isenberg G (1982) Actin filament capping protein from bovine brain.EMBO J 1(7):889–894.

34. Buske PJ, Levin PA (2013) A flexible C-terminal linker is required for proper FtsZ as-sembly in vitro and cytokinetic ring formation in vivo. Mol Microbiol 89(2):249–263.

35. Gardner KAJA, Moore DA, Erickson HP (2013) The C-terminal linker of Escherichia coliFtsZ functions as an intrinsically disordered peptide. Mol Microbiol 89(2):264–275.

36. Feucht A, Lucet I, Yudkin MD, Errington J (2001) Cytological and biochemical char-acterization of the FtsA cell division protein of Bacillus subtilis. Mol Microbiol 40(1):115–125.

37. Haeusser DP, Schwartz RL, Smith AM, Oates ME, Levin PA (2004) EzrA prevents ab-errant cell division by modulating assembly of the cytoskeletal protein FtsZ. MolMicrobiol 52(3):801–814.

38. Pecqueur L, et al. (2012) A designed ankyrin repeat protein selected to bind to tubulincaps the microtubule plus end. Proc Natl Acad Sci USA 109(30):12011–12016.

39. Wegner A, Isenberg G (1983) 12-fold difference between the critical monomer con-centrations of the two ends of actin filaments in physiological salt conditions. ProcNatl Acad Sci USA 80(16):4922–4925.

40. Pollard TD, Mooseker MS (1981) Direct measurement of actin polymerization rateconstants by electron microscopy of actin filaments nucleated by isolated microvilluscores. J Cell Biol 88(3):654–659.

41. Neuhaus JM, Wanger M, Keiser T, Wegner A (1983) Treadmilling of actin. J Muscle ResCell Motil 4(5):507–527.

42. Chen Y, Erickson HP (2011) Conformational changes of FtsZ reported by tryptophanmutants. Biochemistry 50(21):4675–4684.

43. Chen Y, Erickson HP (2009) FtsZ filament dynamics at steady state: Subunit exchangewith and without nucleotide hydrolysis. Biochemistry 48(28):6664–6673.

44. Romberg L, Mitchison TJ (2004) Rate-limiting guanosine 5′-triphosphate hydrolysisduring nucleotide turnover by FtsZ, a prokaryotic tubulin homologue involved inbacterial cell division. Biochemistry 43(1):282–288.

45. Li Q, Joshi HC (1995) gamma-tubulin is a minus end-specific microtubule bindingprotein. J Cell Biol 131(1):207–214.

46. Fischer RS, Fowler VM (2003) Tropomodulins: Life at the slow end. Trends Cell Biol13(11):593–601.

47. Mullins RD, Heuser JA, Pollard TD (1998) The interaction of Arp2/3 complex with actin:Nucleation, high affinity pointed end capping, and formation of branching networksof filaments. Proc Natl Acad Sci USA 95(11):6181–6186.

48. Margolis RL, Wilson L (1978) Opposite end assembly and disassembly of microtubulesat steady state in vitro. Cell 13(1):1–8.

49. Mingorance J, et al. (2005) Visualization of single Escherichia coli FtsZ filament dy-namics with atomic force microscopy. J Biol Chem 280(21):20909–20914.

50. Mateos-Gil P, et al. (2012) Depolymerization dynamics of individual filaments ofbacterial cytoskeletal protein FtsZ. Proc Natl Acad Sci USA 109(21):8133–8138.

51. Arumugam S, Petra�sek Z, Schwille P (2014) MinCDE exploits the dynamic nature ofFtsZ filaments for its spatial regulation. Proc Natl Acad Sci USA 111(13):E1192–E1200.

52. Surovtsev IV, Morgan JJ, Lindahl PA (2008) Kinetic modeling of the assembly, dynamicsteady state, and contraction of the FtsZ ring in prokaryotic cytokinesis. PLOS ComputBiol 4(7):e1000102.

53. Fischer RS, et al. (2006) Tropomodulin 3 binds to actin monomers. J Biol Chem 281(47):36454–36465.

54. Loose M, Mitchison TJ (2014) The bacterial cell division proteins FtsA and FtsZ self-organize into dynamic cytoskeletal patterns. Nat Cell Biol 16(1):38–46.

55. Redick SD, Stricker J, Briscoe G, Erickson HP (2005) Mutants of FtsZ targeting theprotofilament interface: Effects on cell division and GTPase activity. J Bacteriol 187(8):2727–2736.

E2138 | www.pnas.org/cgi/doi/10.1073/pnas.1414242112 Bisson-Filho et al.

Dow

nloa

ded

by g

uest

on

May

17,

202

0