FtsZ filament capping by MciZ, a developmental PNAS PLUS regulator of bacterial division

Alexandre 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 Recherche Scientifique, 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 de Ciências Naturais e Humanas, Universidade Federal do ABC, 09210580, Santo André, SP, Brazil; and gDepartment of Cell , Duke University Medical 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 of zation (4). Among these , negative modulators have regulatory proteins. FtsZ (filamentation temperature-sensitive Z) attracted the most attention because of their crucial role in de- is the bacterial homolog of that polymerizes into rings termining when and where a Z ring should form. The strikingly localized to cell-division sites, and the constriction of these rings precise positioning of the division site in rod-shaped , drives . Here we investigate the mechanism by which such as and , is because of the the Bacillus subtilis cell-division inhibitor, MciZ (mother cell inhibitor combined action of Min and occlusion systems, two of FtsZ), blocks assembly of FtsZ. The X-ray crystal structure reveals negative modulators that work together to ensure that the Z ring that MciZ binds to the C-terminal polymerization interface of FtsZ, the will be formed only at midcell. The Min system, whose core equivalent of the minus end of tubulin. Using in vivo and in vitro component is the polarly localized MinCD complex, prevents the assays and microscopy, we show that MciZ, at substoichiometric lev- Z ring from forming at the cell poles, reducing the chances of els to FtsZ, causes shortening of protofilaments and blocks the assem- minicell formation (12, 13). The nucleoid occlusion system, in

bly of higher-order FtsZ structures. The findings demonstrate an turn, is based on DNA binding proteins (Noc in B. subtilis and BIOCHEMISTRY unanticipated capping-based regulatory mechanism for FtsZ. SlmA in E. coli) that recognize specific sequence signatures and inhibit Z ring formation around the bacterial chromosomes FtsZ | | cytokinesis | filament capping | bacterial (14–16). The combination of Min and nucleoid occlusion in- hibition prevent division from happening at the cell poles and he discovery that bacteria have -, tubulin-, and inter- over the chromosomes, leaving only the central region of the cell Tmediate filament-like proteins demonstrated that the cyto- free for Z-ring formation. Min/nucleoid occlusion also provide a skeleton is an ancient invention, predating the divergence means to regulate the cell-cycle timing of Z-ring formation be- between and (1). The GTPase FtsZ cause the creation of an inhibitor-free zone at midcell depends on (filamentation temperature-sensitive Z) was the first prokaryotic proper DNA replication and segregation. Another well-studied FtsZ to be recognized as a cytoskeletal element (2, 3). FtsZ is modulator is the checkpoint protein SulA (suppressor of Lon A), a tubulin-like protein, which is widely conserved in bacteria and which makes bacterial cytokinesis responsive to environmental the main component of the bacterial cytokinesis machine, or “.” FtsZ self-assembles into single-stranded protofila- ments and these associate further inside cells to form a super- Significance structure known as the Z ring (4, 5). FtsZ alone can generate a constriction force to initiate division (6). The Z ring also pro- Division of bacteria is executed by a contractile ring whose vides a scaffold onto which several other components of the cytoskeletal framework is FtsZ (filamentation temperature- divisome—mostly synthesizing enzymes—are recruited sensitive Z), a protein evolutionarily related to eukaryotic tubulin. and oriented so as to build the division septum, a cross-wall The FtsZ ring is made of filaments of head-to-tail FtsZ subunits separating a progenitor cell into two isogenic daughter cells (7). but its architecture and the rules governing its assembly are still FtsZ and tubulin share several essential properties: their as- poorly known. Here we show that MciZ, an inhibitor of FtsZ ring sembly is cooperative, stimulated by GTP, and leads to GTP formation, functions by capping the minus end of FtsZ filaments. hydrolysis; they form dynamic polymers whose turnover is de- Capping by MciZ makes FtsZ filaments shorter than normal, likely pendent on nucleotide hydrolysis (8); they use essentially the by blocking filament annealing; this represents fundamental in- same bond for polymer formation (9); and recent evidence formation to understand how FtsZ filaments grow and shrink, indicates that they undergo similar allosteric transitions upon and attain their normal size. The powerful inhibition of Z-ring polymerization (10, 11). Not surprisingly, however, the func- assembly by MciZ also suggests that an FtsZ ring cannot form tional specialization of these proteins led to some significant from filaments smaller than a certain size. differences between them, the most prominent being that FtsZ exists as single protofilaments, whereas tubulin always adopts a 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., multifilament tubular structure. This difference in their higher- 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., order structure implies that the reactions that lead to cooperativity K.F.D., P.C., H.P.E., A.D., and F.J.G.-F. wrote the paper. and subunit turnover are likely different. It has also represented The authors declare no conflict of interest. a significant technical challenge for the study of FtsZ. Because FtsZ This article is a PNAS Direct Submission. filamentsaresmallerthantheresolution of optical microscopy, so Data deposition: The atomic coordinates and structure factors have been deposited in the far it has been impossible to determine essential properties associ- , www.pdb.org [PDB ID codes 4U39 (FtsZ:MciZ complex), and 2MRW ated with its dynamic behavior. (free MciZ)]. Similarly to actin filaments and , the assembly of 1To whom correspondence should be addressed. Email: [email protected]. FtsZ protofilaments into a Z ring is regulated by a number of This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. proteins that bind directly to FtsZ and modulate its polymeri- 1073/pnas.1414242112/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1414242112 PNAS Early Edition | 1of9 Downloaded by guest on October 2, 2021 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). The N-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) in yellow, and MciZ (residues 2–37) in red. Cocrystallized phosphates are represented as orange sticks. (B) Superposition of 20 representative low-energy structures 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) Alignment between 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 overlap between 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 of MciZ (red, surface rendering) onto the FtsZ dimer structure of S. aureus (PDB 3VOA). The B. subtilis FtsZ:MciZ complex structure was aligned with the upper subunit of the dimer (gray FtsZ). Note the steric clash between MciZ and the bottom subunit of the dimer (orange FtsZ).

stresses. SulA is expressed in response to DNA damage as part of expressed during sporulation in B. subtilis and blocks Z-ring the SOS system in E. coli and halts Z-ring formation and cell formation after cells commit to the terminally differentiated division until DNA damage is repaired by the cell (17, 18). More spore fate. Although it has been shown that MciZ directly in- recently, other negative modulators of FtsZ have been reported hibits FtsZ polymerization in vitro (22, 29), the nature of its whose function is to control cytokinesis in response to the nutri- interaction with FtsZ and its inhibition mechanism are still un- tional [UgtP (19), KidO (20), and OpgH (21)] and developmental known. We determined the crystal structure of MciZ in complex [MciZ (mother cell inhibitor of FtsZ) (22) and Maf (23)] state of with FtsZ from B. subtilis and, by using fluorescence and electron the cell. microscopy as well as biochemical experiments, showed that Eukaryotic cytoskeletal modulators use a variety of strategies MciZ binds to the C-terminal subdomain of FtsZ and acts as a to achieve their effect, including nucleation, monomer seques- capper of the minus end of FtsZ filaments. Minus-end capping is tration, filament capping, and severing (24, 25). The conservation an unusual way to inhibit filament formation and indicates that of the structure and principles that govern cytoskeletal filament annealing plays an important role in FtsZ filament dynamics. formation suggest that the general mechanisms operating in eukaryotes should also be present in prokaryotes. However, little Results is known about the molecular details of how modulators affect Crystal Structure of the FtsZ:MciZ Complex. To investigate the FtsZ assembly. SulA is one of the few inhibitors whose mecha- mechanism of MciZ inhibition, we first determined the crystal nism has been studied in detail. The crystal structure of SulA in structure of the FtsZ:MciZ complex to a resolution of 3.2 Å. We complex with FtsZ showed that it binds to the C-terminal poly- coexpressed his-tagged MciZ (His6-MciZ) and a truncated ver- merization interface of FtsZ (26). In addition, in vitro experi- sion of FtsZ that included only the globular portion of the pro- ments demonstrated that SulA inhibits FtsZ polymerization by tein (FtsZ12-315)inE. coli and purified the complex by metal a simple sequestration mechanism (27, 28). affinity and gel-filtration chromatographies. As expected from Here we focused on MciZ (mother cell inhibitor of FtsZ), the high affinity of MciZ for FtsZ (Kd of 150 ± 10 nM, measured a poorly understood 40-aa peptide discovered in a yeast two- by tryptophan fluorescence) (Fig. S1A), the complex was very hybrid screen for FtsZ binding partners (22). MciZ is an intriguing stable; it could be produced with high purity in milligram example of a developmentally regulated division inhibitor. It is quantities and it crystallized under standard conditions. Crystals

2of9 | www.pnas.org/cgi/doi/10.1073/pnas.1414242112 Bisson-Filho et al. Downloaded by guest on October 2, 2021 PNAS PLUS belonged to space group P6522 and displayed nine FtsZ:MciZ respectively) (Fig. S1E). Thus, we cannot detect the competi- complexes in the asymmetric unit (only one will be described tion between MciZ and GTP reported by Ray et al., a finding for clarity, FtsZ:A and MciZ:J). In FtsZ, residues 203–208 and that is consistent with our structural data that indicates that 219–220 could not be modeled in the map potentially because of MciZ and GTP bind at opposite surfaces of the FtsZ molecule. inherent flexibility. Our FtsZ structure has no guanine nucleo- tide bound. MciZ (residues 2–37) folds into a β-hairpin followed Substoichimetric MciZ Shortens FtsZ Protofilaments. There are a by a loop, an α-helix, and a helical turn. It binds to the C-terminal variety of mechanisms by which modulatory proteins can inhibit subdomain of FtsZ by aligning with FtsZ’s C-terminal β-sheet cytoskeletal filament formation. Binding of MciZ to the poly- through hydrogen bonds established between β9 of FtsZ and β2 merization interface of FtsZ is compatible with MciZ acting by of MciZ, thus generating a six-stranded β-sheet (Fig. 1A). This either monomer sequestration or filament capping. Monomer mode of protein–protein interaction, generally termed β-strand sequesterers inhibit assembly by reducing the effective concen- addition, is found in diverse protein complexes (30). tration of monomer and, consequently, shifting the equilibrium Binding of MciZ to FtsZ leads to significant restructuring of toward depolymerization. A sequestered subunit is incapable of MciZ’s N-terminal region, as seen by comparing its structure in binding at either end. Thus, sequesterers tend to be effective the crystallized complex with the structure of the free peptide in only when present at close to stoichiometric concentrations rel- solution, determined by 1H NMR (Fig. 1B). Free MciZ displays ative to the filament-forming protein (28). In contrast, cappers the same segments of α-helix as the complexed peptide (amino block the ability of a subunit to bind at one end but leave the acids 16–27 and 32–35) but its N terminus is unstructured (Fig. 1B). other end available to bind to a filament and block further as- This N terminus thus becomes structured into two β-segments upon sembly. Cappers thus tend to exhibit effects even at substoi- interaction with β9 of FtsZ, as noted above. The C terminus of chiometric concentrations (32, 33). To study the mechanism of MciZ is unstructured in both the free and bound forms. MciZ inhibition, we monitored the effect of various concen- Binding of MciZ buries a solvent-accessible area on FtsZ of trations of MciZ on the polymerization of FtsZ by light scattering. 188 Å2. In addition to the backbone hydrogen bonds involved in This showed that as little as a 1:10 ratio of MciZ to FtsZ caused the β-sheet extension, hydrophobic interactions between helices a marked inhibition (about 50%) of the light-scattering signal (Fig. H1 of MciZ and H10 of FtsZ, and between H1/β2 of MciZ with 2A). In contrast, treatment of FtsZ with the MciZ-R20D mutant H10/β9 of FtsZ could stabilize the interaction (Fig. S1B). Recog- did not affect the levels of light scattering, suggesting that the nition between FtsZ and MciZ is further strengthened by a salt MciZ effect is specific and dependent on the interaction with FtsZ

bridge between the carboxylate of Asp280 of FtsZ and the gua- (Fig. S2A). Because the light-scattering signal is disproportionately BIOCHEMISTRY nidinium group of Arg20 of MciZ (Fig. S1B). sensitive to polymer size, the 50% inhibition of light scattering by To further analyze the FtsZ:MciZ interaction, we introduced 1:10 MciZ does not necessarily mean that there was a reduction by site-directed modifications into FtsZ. FtsZ carrying an Asp to half in the polymer mass (or polymer bonds). More likely, this Arg mutation at position 280 (Asp280Arg) could not stably bind substoichiometric inhibition of the light-scattering signal reflects MciZ (Fig. S1C), probably because of disruption of the aforemen- a decrease in length or width (bundling) of the polymers. tioned salt bridge with Arg20. The reciprocal mutation in MciZ, in To investigate further this substoichiometric poisoning, we whichwesubstitutedArg20toanaspartate(Arg20Asp),alsodis- carried out electron microscopy (EM) of negatively stained FtsZ rupted the FtsZ:MciZ interaction (Fig. S1C). Thus, the Asp280- polymers formed in the presence of MciZ. Polymerization of Arg20 salt bridge is critical for complex stability. 3 μM FtsZ in HMK buffer produced filaments with a length of 200 ± 75 nm (Fig. 2C). In contrast, reactions with 3 μM FtsZ and MciZ Inhibits FtsZ Polymerization by Steric Hindrance. The binding 0.3 μM MciZ, the same 1:10 ratio that inhibited light scattering site of MciZ is at one of the polymerization surfaces of FtsZ, by 50%, showed a reduction in protofilament length to 120 ± which suggests that the inhibitory effect of MciZ is simply caused 45 nm (Fig. 2D). This reduction in length caused by 0.3 μM MciZ by steric hindrance. Indeed, modeling of MciZ onto the structure cannot be explained by a sequestration mechanism, in which the of the Staphylococcus aureus FtsZ filament (PDB ID code 3VOA) effect of MciZ would be to reduce the concentration of active (10) showed that the presence of MciZ would completely prevent FtsZ monomers from 3.0 to 2.7 μM. Some filaments without the association of FtsZ monomers (Fig. 1D). Alignment of our MciZ seem to have higher contrast than those with MciZ, sug- structure with five other B. subtilis FtsZ structures (PDB ID gesting that they may be small bundles. Thus, the inhibition of codes 2VAM, 2VXY, 2RHL, 2RHO, and 2RHH) showed that light scattering by MciZ may also involve a reduction of bun- binding of MciZ causes no major conformational changes (rmsd dling, probably as a result of shortening of the protofilaments. of 0.610 Å) (Fig. S1D), other than the displacement the T7 loop Nevertheless, to confirm that the primary effect of MciZ is to from its usual position. In fact, we cannot observe the density reduce the length of protofilaments rather than inhibiting bun- corresponding to the T7 loop (residues 203–208) in our structure dling, we tested the effect of higher concentrations of MciZ (0.6 (Fig. 1C), suggesting that it becomes more flexible or unstructured and 1.0 μM) on the polymerization of 3 μM of FtsZ (Fig. S2B). upon binding of MciZ. The T7 loop is critical for FtsZ function, The results showed shorter protofilaments as MciZ concentration contributing contacts to filament formation and the highly con- was increased, and no visible structures could be identified at 1.0 served 208NLDFAD213 for GTP hydrolysis (3, 31). Despite this μM of MciZ. Because the critical concentration (Cc) of FtsZ in fact, T7 loop displacement is unlikely to be the direct cause of the the conditions of our experiments is between 1 and 1.5 μM (see inhibition of FtsZ polymerization by MciZ; it could be simply a Fig. 5 A and B), it is conceivable that the inhibitory effect of 1.0 consequence of steric hindrance by the latter molecule. μM MciZ added to 3 μM FtsZ could be explained by MciZ se- questering FtsZ to a concentration of active monomers too close MciZ Does Not Compete with GTP Binding. Ray et al. (29) recently to the Cc to allow for assembly. To rule out this possibility, we also suggested that MciZ competes with GTP for binding to FtsZ. did EM experiments with FtsZ-L69W, a mutant protein that has Because GTP binds to the N-terminal subdomain of FtsZ; this is a significantly lower Cc than the wild-type protein (0.5 μM). MciZ at odds with the binding site for MciZ identified in our crystal shortened FtsZ-L69W filaments as well as affecting wild-type FtsZ structure. We revisited the possibility of competition between (Fig. S2C), suggesting that it is not acting by sequestration. MciZ and GTP by comparing the binding of GTP to free FtsZ and to the FtsZ:MciZ complex using the fluorescent analog mant- A FtsZ–MciZ Fusion Protein Inhibits Assembly Similarly to MciZ. The GTP. This analysis showed that mant-GTP bound equally well to substoichiometric effect of MciZ suggests that it is acting as a free and MciZ-bound FtsZ (Kd of 1.7 ± 0.2 μMand1.4± 0.3 μM, capper. However, to decisively rule out sequestration of monomers

Bisson-Filho et al. PNAS Early Edition | 3of9 Downloaded by guest on October 2, 2021 Fig. 2. MciZ inhibits FtsZ polymerization substoichiometrically in vitro. All assembly experiments were done in HMK buffer (50 mM Hepes, 5 mM MgAc, 100 mM KAc, 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 by right-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)andwith0.3μMMciZ(D)or0.3μM FtsZ-MciZ (E). The filament length distribution in each situation was measured and is plotted (Right). (Scale bar, 100 nm.)

as the mechanism of MciZ inhibition, we tested the effect of causing a complete block of Z-ring formation and robust fila- adding MciZ in the form of an FtsZ:MciZ complex on the as- mentation when induced with as little as 0.1% xylose from a sembly of FtsZ. If MciZ works as a sequesterer, adding MciZ single integrated chromosomal copy (Fig. 3 A and B). This finding that is already complexed with FtsZ should have no effect on suggested that MciZ also works substoichiometrically in vivo. To FtsZ assembly. We tested both the noncovalent FtsZ:MciZ ascertain that theory, we carried out quantitative Western blots complex produced by copurification, as well as a covalent com- of cells expressing GFP-MciZ, using anti-GFP serum and puri- plex, in which we fused MciZ to the C-terminal end of FtsZ fied GFP as a standard (Fig. 3C). This process revealed that (FtsZ–MciZ). The C-terminal end of FtsZ is a mostly unstructured, induction with 0.1% xylose leads to the accumulation of 350 ± 150 flexible chain with a contour length of 23 nm (34, 35), more than molecules of GFP-MciZ per cell (Fig. S3D). We also quantified the enough to allow MciZ to bind to the same FtsZ molecule that it is levels of endogenous FtsZ in the same extract (Fig. S3 C and E), covalently connected to. Strikingly, both the FtsZ:MciZ complex and found 4,200 molecules per cell, similarly to published data that (Fig. S3A) and the FtsZ–MciZ fusion were highly effective at indicates that B. subtilis contains at least 5,000 molecules of FtsZ inhibiting FtsZ assembly (Fig. 2B). Plotting the data from all per cell when grown in rich medium (36, 37). This result suggests experiments together (free MciZ, FtsZ:MciZ complex, and FtsZ– that a ratio of MciZ to FtsZ of less than 1:10 is enough to com- MciZ fusion) indicates that the complexes are as capable as free pletely block Z-ring formation and cell division in vivo. MciZ to promote inhibition (Fig. S3B). Similarly, EM determi- nation of filament lengths demonstrated that the FtsZ–MciZ fu- MciZ Binds to FtsZ Filaments in Vitro and to FtsZ Polymers in Vivo. A sion shortened FtsZ filaments somewhat more than MciZ alone capping protein should be able bind to the polymeric form of its (100 ± 35 nm vs. 120 ± 45 nm) (Fig. 2E). These results demon- target. This ability of MciZ is implied by the results above, but we strate that MciZ does not act as a simple monomer sequesterer and also aimed to demonstrate the binding of MciZ to FtsZ filaments suggest that MciZ-bound FtsZ monomers can interact with FtsZ directly, both in vitro and in vivo. To show binding of MciZ to filaments to block their growth. In the simplest model, the N-ter- FtsZ filaments in vitro, we carried out sedimentation experi- minal polymerization face of MciZ-poisoned FtsZ should be held in ments using rhodamine-labeled MciZ and FITC-labeled FtsZ a polymerization-competent conformation that can bind to and cap (Fig. 4 A and B and Fig. S4). Reactions with 10 μM FtsZ and no the minus end of protofilaments. MciZ resulted in ∼50% of FtsZ in the pellet. The addition of 250 nM MciZ produced a clear reduction in pelleted FtsZ to Substoichiometric MciZ Inhibits Z-Ring Formation in Vivo. MciZ is a 25%. Importantly, MciZ efficiently cosedimented with the FtsZ very potent inhibitor of cell division in vivo, being capable of polymers in these reactions, being found almost equally partitioned

4of9 | www.pnas.org/cgi/doi/10.1073/pnas.1414242112 Bisson-Filho et al. Downloaded by guest on October 2, 2021 only rarely could we observe fluorescent Z rings. To improve our PNAS PLUS observation window, we repeated the experiment in a strain bearing 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 to detect 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 also observed for the GFP-MciZ rings in the same cells and suggests that theinhibitoryeffectofMciZisprobablystrongeratnewerZrings than 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-mCherry rings (Fig. 4C, Upper). As a control for the specificity of the GFP- MciZ localization, we also imaged a strain expressing GFP-MciZ- Fig. 3. MciZ blocks cell division substoichiometrically in vivo. (A and B) R20D, which exhibited no inhibitory effect on Z-ring formation and B. subtilis cells without (A)andwith(B) GFP-MciZ expression induced by 0.1% was found to be completely dispersed in the cytosol, despite the xylose for 2 h. The cells were stained with FM 5-95 membrane dye to reveal C septa. (Scale bar, 3 μm.) (C) Quantification of GFP-MciZ from cell extracts. GFP- presence of robust FtsZ-mCherry rings in this strain (Fig. 4 , MciZ was induced with 0.1% xylose for 2 h. GFP-MciZ was detected by im- Lower). These results indicate that MciZ associates with FtsZ munoblotting using anti-GFP antibody and quantification was carried out by polymers in vivo. Because detection of GFP-MciZ rings requires comparing the intensities of the bands with that of purified GFP standards. that more than a few GFP-MciZ molecules decorate each Z ring, these results are further support for the idea that the Z ring is formed by multiple short protofilaments with exposed minus ends. between supernatant and pellet fractions. As a control for the specificity of the cosedimentation, we also tested the MciZ- MciZ Does Not Promote Extensive Depolymerization of FtsZ Filaments. BIOCHEMISTRY R20D mutant and found that it neither inhibited FtsZ sedi- Substoichiometric effects have been reported before for cappers mentation nor cosedimented with FtsZ. Even though sedimen- of actin filaments and microtubules (32, 38). These cappers tation experiments are not fully quantitative (they underestimate usually bind to the growing (plus) end of filaments and restrict polymer because individual protofilaments do not pellet well), exchange of subunits to the minus end. Because the minus end the distribution of MciZ between pellet and supernatant frac- has a higher Cc than the plus end, blocking the plus end leads to tions suggests it binds preferentially to FtsZ filaments. extensive depolymerization of filaments, until the free monomer To demonstrate binding of MciZ to FtsZ polymers in vivo, we concentration becomes equal to the minus-end Cc (39, 40). Such imaged GFP-MciZ and attempted to detect decoration of Z depolymerization induced by eukaryotic capping proteins is de- rings with GFP-MciZ. Induction of even low levels of GFP-MciZ pendent on, and a hallmark of, (41). To deter- in a wild-type strain led to very fast disassembly of Z rings and mine if the shortening of FtsZ protofilaments by MciZ involved

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(9μM of unlabeled wild type FtsZ and 1 μM of FtsZ-S152C labeled with FITC). MciZ-W36C and MciZ-R20D+W36C were labeled with TMR and added to a final concentration of 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)Cells expressing FtsZ-mCherry (Left)andGFP-MciZ(Right). (Lower) The mislocalization of GFP-MciZ(R20D) mutant compared with the wild-type MciZ. (Scale bar, 2 μm.)

Bisson-Filho et al. PNAS Early Edition | 5of9 Downloaded by guest on October 2, 2021 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 are indicated 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 was preassembled 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.

substoichiometric depolymerization of FtsZ, we used a fluores- increased from 2.8 in the absence of MciZ, to 5.4 at all concen- cence-quenching assay capable of accurately quantifying polymeri- trations of MciZ. The converse experiment, in which we assayed a zation (42). For this assay we constructed the double-mutant fixed FtsZ concentration (10 μM)inthepresenceofvaryingMciZ FtsZ(S152C/S223W) and labeled the Cys with BODIPY. As with concentrations, also revealed a twofold increase in the GTPase the equivalent construct for E. coli FtsZ, the Trp quenching of the activity of FtsZ in the presence of low MciZ concentrations (Fig. BODIPY fluorescence is decreased by the conformational change S6).However,ataratioofMciZtoFtsZhigherthan1:10,the accompanying assembly, and the fluorescence increase reports GTPase activity started to decrease (Fig. S6), probably because at the total protofilament polymer. We used the BODIPY-labeled high MciZ the reduction in the amount of polymerized FtsZ begins FtsZ mixed with a ninefold excess of wild-type FtsZ to ensure to offset the stimulatory effect of MciZ. that the assembly is dominated by the wild-type FtsZ and avoid The increase in GTPase specific activity indicates that poly- the bundling observed previously for fully labeled FtsZ (42). mers assembled in the presence of MciZ are more dynamic. The Fig. S5 shows that assembly kinetics reached the overshoot faster dynamics could be because of MciZ increasing the rate of peak in 12 s, a short time consistent with protofilament assembly GTP hydrolysis, or the rate of subunit exchange, as both limit the as opposed to slower bundle formation (40 s to overshoot peak overall GTP turnover by FtsZ polymers (43, 44). To further in- in Fig. 2 A and B) (42). Fig. 5A plots the plateau assembly for vestigate the effect of MciZ on polymer dynamics, we applied a range of FtsZ up to 9 μM, and for MciZ added at 0, 1, 2, and again the BODIPY fluorescence-quenching assay, but this time 3 μM. Similarly to the previously reported inhibition of FtsZ by to follow the disassembly kinetics of FtsZ filaments after dilution SulA (27, 28), MciZ caused an increase in the apparent critical to below the Cc. Disassembly curves were fit to a single expo- τ concentration, Ccapp, of FtsZ from 1.5 to 2.4, 3.1, and 4.2 μM, nential with decay time . The disassembly of FtsZ polymers in C respectively. The increase in Ccapp was almost equal to the HMK buffer (Fig. 5 ) was much faster in reactions with 1:10 amount of added MciZ, consistent with MciZ binding FtsZ with MciZ (τ = 3 ± 2 s), compared with those with FtsZ alone (τ = higher affinity than the ∼1.5 μM Cc. This finding means that 10 ± 2 s). A similar effect was observed in reactions in which each MciZ molecule is roughly preventing the formation of only GTP hydrolysis was blocked by the addition of EDTA (Fig. 5D). one FtsZ–FtsZ bond and indicates that capping of the minus end Disassembly in EDTA was slower than with GTP hydrolysis, as does not induce extensive depolymerization of the plus end. reported previously (43). However, polymers poisoned with MciZ again exhibited faster disassembly than FtsZ alone (τ = 9 ± 2and MciZ Increases the Dynamics of FtsZ Filaments. We measured the 20 ± 3 s, respectively). Because the effect of MciZ is independent GTPase activity of FtsZ in the presence of MciZ to assess whether of GTP hydrolysis, we conclude that MciZ increases the turnover capping by MciZ affected the dynamics of FtsZ filaments. Reac- of FtsZ subunits in filaments, as we explain in the discussion. tions were set up with a similar range of FtsZ concentrations and with MciZ concentrations of 0, 2, 4, and 6 μM. As expected, MciZ Discussion decreased the absolute GTPase activity of FtsZ and increased the We applied a combination of structural, biochemical, and in vivo apparent Cc in proportion to its concentration (Fig. 5B), corrob- approaches to elucidate the mechanism of MciZ, a develop- orating the BODIPY results. Strikingly, however, the specific ac- mentally regulated inhibitor of FtsZ polymerization of B. subtilis. tivity (the slope of the line in GTP per minute per FtsZ) was Clarifying the molecular effects of the growing roster of FtsZ

6of9 | www.pnas.org/cgi/doi/10.1073/pnas.1414242112 Bisson-Filho et al. Downloaded by guest on October 2, 2021 modulators will be key for a better understanding of Z-ring lower affinity for FtsZ than MciZ and may not be able to compete PNAS PLUS formation. Our data show that MciZ is a capping protein, and effectively with monomeric FtsZ for binding to filament ends. that the inhibition of FtsZ assembly at low MciZ stoichiometries is primarily a result of capping. At high stoichiometries, MciZ is How Does MciZ Shorten FtsZ Filaments? Minus-end cappers (γ-tubulin, capable of both capping and sequestration of FtsZ. Thus, the , Arp2/3) generally exert a stabilizing or nucleat- identification of the first FtsZ capping protein indicates that the ing role for actin and microtubules (45–47). Because the minus palette of biochemical activities available for the regulation of end is usually the site of subunit loss (40, 48), capping of the the bacterial cytoskeleton could be as diverse as the one known minus end prevents disassembly and stabilizes filaments. In con- to operate on actin and tubulin. trast to the eukaryotic paradigm, MciZ is a minus-end capper We also exploited MciZ as a probe to try to answer some that shortens FtsZ filaments and destabilizes the Z ring. To ex- longstanding questions about FtsZ. The small size of FtsZ poly- plain this apparent paradox we recall that in addition to affecting mers has posed a formidable challenge to the understanding the exchange of subunits at filament ends, cappers will also affect of its dynamic behavior. Simple questions, such as whether the annealing, the reaction by which two filaments become joined by polymerization of filaments is asymmetric, or if FtsZ undergoes their ends. Capping of either end of a filament will block annealing treadmilling or dynamic instability, have remained unanswered and, depending on how frequent annealing is, it could have largely because individual FtsZ filaments are shorter than the a marked effect on filament sizes. Thus, to explain the effect of resolution of the light microscope. By carefully analyzing the MciZ on FtsZ filaments we propose a model in which filament effect of MciZ in bulk measurements of FtsZ polymerization, we length at steady state will depend on the gain and loss of subunits at provide evidence suggesting that fragmentation and annealing filament ends and on the balance between filament fragmentation are important determinants of FtsZ filament size and that, simi- and annealing (Fig. 6). Because capping of the minus end does not larly to tubulin, the plus end is more dynamic than the minus end induce extensive depolymerization from the plus end (Fig. 5A), we of FtsZ filaments. We anticipate that the use of MciZ as a reagent postulate that blocking annealing may be an important pathway by in further studies, together with the application of sophisticated which MciZ reduces filament size. Fragmentation and annealing of imaging methods to visualize filaments, will soon allow the con- FtsZ filaments have been observed for filaments adsorbed to struction of a realistic model of FtsZ polymerization dynamics. mica or supported lipid bilayers (49–51), and also in solution (43), although the rates of these reactions are yet to be precisely MciZ Binds to the Minus End of FtsZ and Functions as a Filament- determined. Annealing may be particularly important in vivo,

Capping Protein. Our crystal structure of the FtsZ:MciZ com- when the filaments are concentrated on the membrane at the BIOCHEMISTRY plex showed that MciZ binds to the C-terminal polymerization center of the cell (5, 52). Thus, blocking annealing in vivo would interface of FtsZ—the minus end of the molecule—and impedes be a powerful way to block the overall assembly of the Z ring. further polymerization by steric hindrance. Because binding of Our observation that a minus-end capper destabilizes FtsZ fila- MciZ to the minus end does not affect the plus end of FtsZ ments is also supported by recent work by Arumugam et al. (51), (Fig. 1 and Fig. S1D), MciZ should be able to function as a capper who produced a truncated FtsZ termed “NZ” that included only the and our data show that this is indeed the case. N-terminal subdomain, and found that it also worked as a capper The evidence that MciZ is a capper is manifold. First, it binds and caused the disassembly of FtsZ bundles on a supported lipid to filaments in addition to FtsZ monomers (Fig. 4). Second, it bilayer. This finding indicates that FtsZ filaments are susceptible to acts at substoichiometric concentrations. At 1:10 MciZ:FtsZ, the capping when assembled in higher-order structures tethered to the light-scattering signal was substantially reduced, and EM showed membrane, a more physiological situation than our in vitro system, a reduction in the length of filaments (Fig. 2). These effects were approximately the same when MciZ was added alone, in a 1:1 complex with FtsZ, and when MciZ was fused to the C terminus of FtsZ (Fig. 2 and Fig. S3). The similar effects of free and prebound MciZ can only be explained if the FtsZ:MciZ complex caps the minus end of FtsZ filaments. Third, MciZ increases the dynamics of FtsZ polymers (Fig. 5 B–D). Above the Cc, FtsZ filaments are thought to be in a steady-state on-off reaction with the pool of subunits. When diluted below the Cc, the on-reaction is eliminated, leaving only the off-reaction. If disassembly occurs primarily from the filament ends, its rate will be proportional to filament length. If the filaments are half as long, and therefore presenting twice as many ends, they should take half as long to disassemble. Because the 1:10 MciZ:FtsZ filaments disassembled two- to three-times faster than FtsZ without MciZ, this suggests that they are two- to three-times shorter. The twofold increase in GTPase promoted by MciZ is also consistent with shorter fila- ments and increased number of ends. Both results are in good agreement 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 instead Fig. 6. Mechanism of FtsZ polymerization inhibition by MciZ. FtsZ filament of a capper (27, 28). Because SulA does not directly occlude the length is determined by the rates of subunit gain and loss at filament ends, plus end of FtsZ, a monomer bound to SulA could still poly- and the balance between filament fragmentation (kfrag) and annealing (kanneal). Capping of filament minus ends by MciZ (red circles) will inhibit merize using its free plus end, but this does not seem to occur. subunit addition at these ends, but in principle, growth at the free plus ends The reason for the difference in the mechanisms of MciZ and could compensate that. In contrast, inhibition of annealing by MciZ turns SulA may reside in the details of how these inhibitors interact fragmentation into an irreversible reaction, leading to shortening of FtsZ with the minus end of FtsZ, or may be related to the different filaments. At close to stoichiometric concentrations, MciZ should also pro- affinities that these inhibitors display for FtsZ. SulA displays 10-fold mote subunit sequestration in addition to capping.

Bisson-Filho et al. PNAS Early Edition | 7of9 Downloaded by guest on October 2, 2021 and supports the conclusion that the powerful inhibition of Z-ring finding makes sense in light of MciZ’s function. In contrast with formation by MciZ in vivo is a consequence of capping. other negative modulators, such as SulA and MinC, whose activity needs to be restricted in space/time or reversed, MciZ acts on MciZ Acts as a Sequesterer at High Concentrations. Cytoskeletal mod- a terminally differentiating cell that is going to die. Thus, MciZ ulatory proteins rarely have a single effect on their targets and it is effects do not need to be reversed. The substoichiometric effect of not unexpected that a capper may also exhibit sequestration effects. MciZ in vivo also suggests that FtsZ filaments have a minimum size, For example, tropomodulin, which is a minus-end capper like MciZ, below which a Z ring does not form. Assuming similar effects in has been shown to bind to actin monomers and promote seques- vitro and in vivo, our data suggest that halving filament length tration at high concentrations (53). MciZ behaves as a sequesterer suffices to block Z-ring formation in vivo. Why do shorter filaments at high concentrations, as demonstrated by the increase in Ccapp that not work? Shorter filaments seem less capable of interacting with A is approximately equal to the concentration of added MciZ (Fig. 5 FtsA and getting recruited to the membrane (54). In addition, they and B). SulA produces an increase in Ccapp somewhat lower than its μ K K may have a lower tendency to make the lateral interactions necessary concentration, because its 0.7 M d is comparable to the d for to form a coherent ring. Further investigation of this hypothesis may FtsZ filament formation [which is equal to the Cc (28)]. MciZ binds produce new insights on the structure and assembly of the Z ring. with a much higher affinity, Kd ∼0.1 μM, measured from trp fluo- rescence (Fig. S1A), so the sequestration is approximately equal to Materials and Methods the concentration of MciZ. Sequestering activity may be explained General Methods. All B. subtilis strains were derived from the wild-type PY79 as follows. The FtsZ:MciZ complex is sterically blocked at the minus strain. Final data collection, structure solution, and refinement statistics are end, making it impossible to bind to the plus end of a filament. The in Tables S1 and S2. Oligonucleotides are listed in Table S3 and plasmids in complexcanbindtotheminusendofafilamentbutathighMciZ Table S4. Purification of FtsZ and the FtsZ:MciZ complex is described in SI concentration all filament minus ends should already be capped. Materials and Methods. Immunoblots were performed using anti-GFP and Thus, the bound subunit is effectively sequestered. anti-FtsZ antisera. The detailed protocol and procedure to estimate cellular protein concentration can be found in SI Materials and Methods. The Polarity of FtsZ Filaments. Some of our results are consistent with FtsZ exhibiting the same kinetic polarity as tubulin. A sig- Structure Determination. The crystal structure of the FtsZ:MciZ complex was nificant observation is that the capped filaments apparently do not solved to 3.2 Å using molecular replacement techniques, and the solution 1 disassemble at the plus end (Fig. 5A), indicating that the Cc of the structure of free MciZ was solved by 2D H-NMR. Both methodologies are plus end is similar or lower than the Cc of the minus end. In ad- described in SI Materials and Methods. dition, because the rate of polymerization in the presence of MciZ is not markedly changed (Fig. S5), this suggests that the plus end 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- dominates the kinetics of assembly. A more dynamic plus end sured using the fluorescent analog mant-GTP. FtsZ polymerization was measured means that FtsZ filaments should be inherently capable of tread- by right angle light scattering or by a BODIPY-based fluorescence quenching milling and, in fact, treadmilling has recently been reported for system. The GTPase activity of FtsZ was determined using the malachite green FtsZ filaments tethered to planar lipid monolayers by FtsA (54). method. Detailed protocols for each procedure are in SI Materials and Methods. However, our conclusion that assembly of FtsZ is maintained primarily at the plus end is the opposite of a previous treadmilling Microscopy. FtsZ protofilaments were imaged by negative stain EM. Cell interpretation. Redick et al. made an effort to create FtsZ cappers filamentation and GFP-MciZ localization were determined by live-cell fluo- by making debilitating point mutants of the top and bottom rescence microscopy. Detailed protocols for microscopy procedures are in SI interfaces of E. coli FtsZ (55). Surprisingly, top-cap mutants, with Materials and Methods. a debilitating mutation on the upper GTP binding surface were completely inactive. Bottom-cap mutants showed dominant-nega- ACKNOWLEDGMENTS. We thank Ethan Garner, Jessica Polka, Masaki Osawa, and José Manuel Andreu for discussions; the Brazilian Biosciences National tive effects in vivo, and blocked GTPase activity when mixed with Laboratory for access to their facilities; J. Marquez and the High-Throughput wild-type FtsZ in vitro; however, these were extremely weak, re- Crystallization Laboratory team (Grenoble Partnership for Structural Biology) quiring approximately 10-times excess of the cap mutant relative to for access to and help with high-throughput crystallization; and the European wild-type. The differential effect of the top and bottom caps sug- Synchrotron Radiation Facility for access to beamlines. This work used the plat- forms of the Grenoble Instruct Center (UMS 3518 Centre National de la gested treadmilling, but with reversed polarity relative to tubulin Recherche Scientifique-Commissariat à l’Energie Atomique et aux Energies (subunits added primarily to the minus end, and dissociated pri- Alternatives-Université Joseph Fourier-European Molecular Biology Laboratory) marily at the plus end). It is possible that the differential effect of with support from the French Infrastructure for Structural Biology (ANR-10- top- and bottom-cap mutants is simply a result of a poorer ability of INSB-05-02) and the Genoble Alliance for Integrated Structural Cell Biology (ANR-10-LABX-49-01) within the Grenoble Partnership for Structural Biology. the top-mutant subunits to associate with filaments. Nevertheless, This work was supported by Grants 10/51866-0 (Smolbnet 2.0) from Fundação we will need more information, preferably the direct observation of de Amparo à Pesquisa do Estado de São Paulo (FAPESP) and 1169/2013 from filaments, before we can resolve the issue of FtsZ polarity. Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) (to F.J.G.-F.); National Institute of Health Grant GM66014 (to H.P.E.); a doctoral A Threshold Size for FtsZ Filaments in Vivo? fellowship from FAPESP and a doctoral sandwich fellowship from the Science The substoichiometric without Borders program (to A.W.B.-F.); a doctoral fellowship from FAPESP inhibition and the high affinity for FtsZ means that MciZ is the (to V.B.); a postdoctoral fellowship from FAPESP (to P.C.); and a PQ-2 fellowship most powerful inhibitor of Z-ring formation described to date. This from Conselho Nacional de Desenvolvimento Científico e Tecnológico (to F.J.G.-F.).

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