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FEBS Letters 581 (2007) 853–857

Capping of filaments by vinculin activated by the Shigella IpaA carboxyl-terminal domainq

Nalini Ramaraoa,1, Christophe Le Claincheb, Tina Izardc, Raphae¨lle Bourdet-Sicarda,2, Elisabeth Agerona, Philippe J. Sansonettia, Marie-France Carlierb, Guy Tran Van Nhieua,* a Unite´ de Pathoge´nie Microbienne Mole´culaire, Inserm U786, Institut Pasteur, 75724 Paris Cedex 15, France b Dynamique du Cytosquelette et Motilite´ Cellulaire, Laboratoire d’Enzymologie et Biochimie Structurale, CNRS, 91198 Gif-sur-Yvette, France c Department of Hematology-Oncology, St. Jude Children’s Research Hospital, Memphis, TN 38105, USA

Received 26 October 2006; revised 15 January 2007; accepted 22 January 2007

Available online 2 February 2007

Edited by Veli-Pekka Lehto

lular adhesion and motility, as well as in embryonic develop- Abstract Shigella, the causative agent of bacillary dysentery, invades epithelial cells. Upon bacterial–cell contact, the type ment and tumorigenesis [5–7]. Vinculin is comprised of five a- III bacterial effector IpaA binds to the cytoskeletal vin- helical bundle domains that form a large globular head, and culin to promote actin reorganization required for efficient bac- a five-helical bundle tail (Vt) domain, which is connected to terial uptake. We show that the last 74 C-terminal residues of the head via a -rich hinge region [8]. The isolated head IpaA (A559) bind to human vinculin (HV) and promotes its asso- domain of vinculin binds to and a-, while the iso- ciation with actin filaments. Polymerisation experiments demon- lated tail domain binds F-actin [9,10]. However, in its native strated that A559 was sufficient to induce HV-dependent partial state an intramolecular interaction between the N-terminal se- capping of the barbed ends of actin filaments. These results sug- ven-helical bundle domain (Vh1) of vinculin with its Vt tail do- gest that IpaA regulates actin polymerisation/depolymerisation main clamps vinculin in a closed conformation, and masks its at sites of Shigella invasion by modulating the barbed end cap- binding sites to some of its ligands such as F-actin and ping activity of vinculin. 2007 Federation of European Biochemical Societies. Published [11]. The binding of the vinculin-binding sites (VBSs) present in by Elsevier B.V. All rights reserved. the central rod domains of talin or a-actinin with the Vh1 domain induces conformational changes that displace the Keywords: IpaA; Vinculin; Actin; Capping; Shigella head–tail interaction and are alone sufficient to trigger vinculin binding to F-actin, a hallmark of vinculin activation [11–13]. Here we report that the 74-residues C-terminal domain of IpaA activates vinculin binding to actin and mediates vinculin- dependent capping of the barbed end of actin filaments. 1. Introduction

Shigella, a gram-negative bacillus known as the major etio- 2. Materials and methods logical agent of dysentery in developing countries, mediates its entry into epithelial cells by a trigger-like process involving 2.1. Antibodies a type III secretion apparatus and injection of several bacterial The rabbit anti-IpaA antiserum was previously described [3]. The type III effectors [1,2]. Bacterial entry into host cells requires mouse anti-vinculin monoclonal antibody Vin11.5, and the rabbit anti-actin were from Sigma Corp (St. Louis, MO). The anti-rabbit the dynamic polymerisation and depolymerisation of actin, IgG HRP antibody was from Amersham Corp. which serves to mould the contact site to allow efficient entry of the pathogen [2]. The type III effector IpaA binds to vincu- 2.2. Bacterial strains and plasmid constructs lin, and the IpaA–vinculin complex induces actin depolymer- S. flexneri strains are derivatives of the wild-type strain M90T [14]. isation, which is required for effective bacterial entry [3,4]. Strain SF635, in which the ipaA, B, C and D genes have been deleted Vinculin, a highly conserved cytoskeletal component of focal (Dipa) was used for purification of IpaA [15]. Bacteria were grown in adhesions, plays critical roles in various processes such as cel- tryptic casein soy broth (TCBS) at 37 C. To obtain the IpaA deriva- tives AD1, AD2 and AD3, internal deletions of the ipaA gene in the plasmid pBad18::ipaA [16] were performed between residues 69 (Bsu36I) to 139 (PshA1)(AD1), 139 (PshA1) to 420 (StuI)(AD2) and q 419 (StuI), as well as a C-terminal deletion from residues 552–633 While this paper was submitted, a work by Demali et al. reported (SnaBI)(AD3). The Dipa strain was transformed with plasmid pBa- that the first 500 amino-terminal residues of IpaA could induce d18::AD1(Dipa-AD1), pBad18::AD2(Dipa-AD2) or pBad18::AD3 vinculin-independent actin depolymerisation following cell transfec- (Dipa-AD3) or with pBad18::ipaA (Dipa-IpaA). A559 is a fusion pro- tion [24]. tein containing GST fused to the C-terminal residues 513–633, or 560–633 of IpaA, respectively, expressed from a derivative of pGEX- *Corresponding author. Fax: +33 1 45 68 89 53. 4T2 in E. coli. E-mail address: [email protected] (G. Tran Van Nhieu).

1Present address: Unite´ de Ge´ne´tique Microbienne et Environnement, 2.3. Purification of and overlay assays INRA La Minie`re, 78285 Guyancourt, France. IpaA was purified from cultures of a Dipa/pBad: IpaA strain as 2Present address: Danone Vitapole, Nutrivaleur, 91767 Palaiseau previously described [4]. GST-N and A559 fusions were purified using Cedex, France. glutathione beads, according to the manufacturer’s instructions.

0014-5793/$32.00 2007 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2007.01.057 854 N. Ramarao et al. / FEBS Letters 581 (2007) 853–857

Human recombinant vinculin was purified from E. coli as described [8]. Vinculin overlay assays were performed as previously described [3,17].

2.4. Actin sedimentation assays Recombinant vinculin (1.8 lM) and IpaA or the A559 derivative (200, 400 or 800 nM), were added to 3 lM G-actin () dur- ing the polymerisation reaction in F-buffer (2 mM Tris pH 7.5, 0.2 mM CaCl2, 0.2 mM ATP, 100 mM KCl and 2 mM MgCl2) for 1 h at room temperature (RT). Proteins were centrifuged at 70000 · g for 30 min in a TL-100 ultracentrifuge (Beckman). Pellet and supernatant fractions were analysed by SDS–PAGE and Coomassie staining.

2.5. Polymerisation assays using pyrenyl-actin fluorescence Actin was purified from rabbit muscle and labelled on cysteine 374 by pyrenyl iodoacetamide [18,19]. For measurements of the initial rate of barbed end growth, samples of 2 lM actin (10% pyrenyl-labelled) containing 0.2 nM -actin seeds were supplemented with vincu- lin (0–20 lM) and/or A559 (0–15 lM). Polymerisation was started by addition of 100 mM KCl, 0.2 mM EGTA and 1 mM MgCl2 to the solution in G-buffer. For measurements of dilution-induced depoly- merisation at barbed ends, 25 lM actin (75% pyrenyl labelled) was polymerised by addition of 1 mM MgCl2, 0.2 mM EGTA and 100 mM KCl for 20 min at RT. F-actin was then diluted to 67 nM in F buffer and supplemented with vinculin (0–2 lM) and/or A559 (0–2 lM). The initial rate of decrease in fluorescence of pyrenyl-F-actin was measured. Changes in fluorescence were recorded using a Safas flx spectrofluorometer. The excitation and emission monochromators wavelengths were set at 366 nm and 407 nm, respectively.

3. Results

3.1. The C-terminus of IpaA is sufficient to reveal the latent F-actin binding potential of vinculin To identify the domain of IpaA that mediates its interactions with vinculin overlay assays were performed. We generated IpaA derivatives containing internal deletions between residues 69–139 (AD1) and 139–420 (AD2), carboxyl-terminal deletions of residues 419–633 (AD3) and 559–633 (ADCT), as well as a GST-fusion protein containing IpaA C-terminal residues Fig. 1. The 74 C-terminal residues of IpaA mediate binding to vinculin. (A) The structure of IpaA derivatives containing either 560–633 (A559) (Fig. 1A). These IpaA derivatives were ex- internal deletions between residues 69–139 (AD1), 139–420 (AD2), pressed and migrated at their expected sizes in SDS–PAGE 419–633 (AD3), or 559–633 (ADCT), or GST fused to the C-terminal (Fig. 1B). Full length IpaA, the internal IpaA deletions AD1 residues 514–633 (A513) or 560–633 (A559) of IpaA is shown. (B) and AD2, as well as the C-terminal fusion A559, all bound Supernatants from Dipa-IpaA, AD1, AD2, AD3, ADCT Shigella to full-length human vinculin (HV, Fig. 1C). However, the strains, and purified GST and A559, were analysed by SDS–PAGE and anti IpaA-immunoblotting. A559 was detected by staining with AD3 and ADCT mutant IpaA proteins failed to bind to HV Coomassie blue. (C) Binding of the IpaA derivatives to vinculin was (Fig. 1C). tested in overlay assays. To address the role of the IpaA vinculin-binding domain in regulating vinculin–F-actin interactions, actin sedimentation assays were performed with the A559 and HV proteins. As ex- ments was measured by monitoring changes in pyrenyl-labeled pected, when HV (1.8 lM) and F-actin (3 lM) were incubated actin fluorescence. Effects of barbed end elongation were stud- together the majority of the actin pool was recovered in its ied using spectrin-actin seeds, which are short actin filaments polymerised form in the pellet fraction whereas HV was mostly capped at their pointed ends. When A559 was added at con- found in the soluble fraction (Fig. 2A). Addition of A559, centrations ranging from 0 to 875 nm to HV at a concentration however, led to a significant shift of HV into the pellet fraction of 2 lM, partial inhibition of actin polymerisation was ob- with polymerised actin (Fig. 2A). Quantification by scanning served, plateauing at 40% inhibition at saturating amounts of densitometry indicated that the percentage of the total pool A559 (>800 nM, Fig. 3A). A similar limited 40% inhibition of HV associated with F-actin shifted from 13% in the absence was observed at higher concentrations of HV (8 lM) in the of A559 to 86% in the presence of 2 lM A559 (Fig. 2B). presence of 10 lM A559, demonstrating that the partial inhibi- Therefore, the last 74 C-terminal residues of IpaA are suffi- tion of barbed ends did not result from partial saturation of cient to induce the association of vinculin with actin filaments. barbed ends (not shown). When the reverse experiments were performed in which increasing concentrations of HV were 3.2. A559–HV induces vinculin-dependent partial barbed-end added to 0.9 lM of A559, a partial inhibition of actin polymer- capping of actin filaments isation was again observed, with 40% inhibition occurring at To analyse the effects of the A559–vinculin interaction on concentrations of HV greater than 1.8 lM(Fig. 3B, inset). actin dynamics, the rate of barbed end growth of actin fila- In contrast, neither HV alone nor A559 alone affected the ini- N. Ramarao et al. / FEBS Letters 581 (2007) 853–857 855

tial rate of actin polymerisation at concentrations of up to 15 lM indicating that inhibition was mediated by the A559– HV complex (Fig. 3C). Inhibition mediated by A559–HV was specific for barbed ends, since no detectable effects on pointed end growth could be observed when -actin seeds were used (Fig. 3D). A559–HV did not affect actin polymerisation in steady state experiments in the absence or presence of gelsolin, indicating that the A559–HV complex does not prevent actin polymerisa- tion by sequestering actin monomers (Figs. 4A and B). Initial rates of filaments elongation grown from spectrin-actin seeds were plotted as a function of actin concentration [20].As shown in Fig. 4C, addition of A559–HV did not affect the crit- ical concentration at barbed ends, since the abscissa intercept of the plots indicated a value indistinguishable from actin alone, corresponding to the expected value of 0.1 lM. The rate of association of monomer to barbed ends was, however, sig- nificantly affected by saturating concentrations of A559 (0.9 lM) and HV (1.8 lM), with the association constant k+ Fig. 2. A559 induces vinculin co-sedimentation with F-actin. (A) Actin shifting from 9.2 lMÀ1 sÀ1 for actin alone to 6.5 lMÀ1 sÀ1 in (3 lM), was allowed to polymerise in F-buffer in the presence of 2 lM the presence of A559–HV (Fig. 4C). vinculin (0), or 2 lM vinculin and increasing concentrations of A559. Proteins were separated by ultracentrifugation. Pellets (P) and super- Collectively, these results demonstrate that A559 induces natants (S) were analysed by SDS–PAGE and Coomassie blue dye vinculin-dependent leaky capping of actin filaments at their staining. The apparent migration of proteins is indicated by an arrow. barbed end, thus modulating the rate of association of mono- The concentration of A559 is indicated in lM above the lanes. (B) The mers at this end. intensity of the band corresponding to HV in S or P fractions was determined by scanning densitometry and expressed as the percentage of the total (P + S).

Fig. 3. A559 mediates vinculin-dependent capping of actin filaments at barbed ends. For polymerisation studies at barbed ends kinetics of 2 lM pyrenyl-labelled (10%) actin assembly was monitored in the presence of spectrin-actin seeds. (A) Kinetics of barbed ends elongation in the presence of increasing concentrations of A559 (values are indicated in nM) and 1.8 lM H. (B) Plots of the initial elongation rates (Vo) as a function of increasing concentrations of A559 (0–8 lM) in the presence of 0.9 lM HV (solid circles) or 8 lM HV (empty squares); inset: increasing concentrations of HV (0–2 lM) added to 0.9 lM A559. (C) Plots of the initial elongation rates (V0) in the presence of increasing concentrations of HV alone or A559 alone (0–20 lM). (D) For polymerisation at pointed ends, kinetics of pyrenyl-labelled actin assembly was monitored in the presence of gelsolin seeds with buffer alone (red), 2 lM HV (green), or 2 lM HV and 1 lM A559 (orange). 856 N. Ramarao et al. / FEBS Letters 581 (2007) 853–857

Fig. 4. Steady state actin polymerization indicates an interference of HV–A559 on actin monomer association with the filament’s barbed ends. Actin (2 lM) (A), or actin in the presence of gelsolin (B) was allowed to polymerise for 16 h with HV (0–2 lM) (solid diamonds), A559 (0–1 lM) (empty circles), or 2 lM HV and A559 (0–1 lM) (solid squares). (C), The initial rate of actin polymerisation was plotted as a function of actin concentration (Cc plot) in the presence of 0.9 lM A559 and 1.8 lM vinculin.

4. Discussion growth of protrusions at sites of bacteria–cell contact, to avoid repulsion of the bacterium during the initial phase of bacterial Remodeling of the cellular actin filament network is regu- invasion. At later stages of infection, the capping activity of lated by actin binding proteins that either nucleate actin poly- the IpaA–vinculin complex could combine with the effect of merisation or destabilise F-actin to promote rapid spatial sequestering or depolymerising proteins to account for actin control of filament assembly and disassembly, respectively depolymerisation during the completion of the entry process [20,21]. Here we show that a C-terminal domain of Shigella’s [4]. As opposed to full length IpaA, however, A559 did not in- IpaA protein (A559) binds to vinculin and induces its associa- duce actin depolymerisation in vitro in the presence of vincu- tion with F-actin in stoichiometric amounts in the micromolar lin, or after cell microinjection (data not shown) or range. This suggests that at these concentrations, the A559– transfection [24]. This suggests that depolymerisation involved vinculin complex essentially binds to the sides of actin fila- IpaA domains other than the last 74 carboxyterminal residues. ments. Steady state polymerisation experiments, however, indi- Unfortunately, the fact that full length IpaA was soluble only cate that binding of the A559–vinculin complex to the sides of in the presence of detergents that interfered with the measure actin filaments does not impact the dynamics of actin polymer- of pyrenyl–actin assembly, did not allow us to test this hypoth- ization. Instead, we show that the effects of A559–HV complex esis (our unpublished data). Interestingly, the crystal structure on actin dynamics is linked to partial capping of the barbed of the Vh1 domain of vinculin in complex with IpaA’s binding ends of actin filaments. ‘‘Leaky cappers’’ provide a means to region, has established that vinculin undergoes structural control actin dynamics in various manners. For example, changes that are similar to those observed upon activation mDia1 formin acts as a leaky capper that slows down associa- with a talin-VBS [25]. Therefore, the leaky barbed end capping tion and dissociation of actin at barbed ends [22]. Yet, in the activity of vinculin may also reflect a function of vinculin that presence of profilin, mDia1 increases the rate of association is manifest when it is bound to its native ligands in focal adhe- of profilin–actin at barbed ends and induces processive poly- sions. merisation [23]. Along these lines, IpaA-mediated vinculin cap- ping of actin filaments may exert distinct effects during the Acknowledgements: We are pleased to acknowledge Philippe Bois for process of bacterial entry. For example, IpaA-mediated vincu- lengthy and stimulating discussions, as well as members of the PMM lin-dependent capping of actin filaments may inhibit the laboratory. We thank Dominique Didry for technical help. N.R. was N. Ramarao et al. / FEBS Letters 581 (2007) 853–857 857 supported by an EMBO fellowship. P.J.S. is a Howard Hughes Med- [13] Bois, P.R., O’Hara, B.P., Nietlispach, D., Kirkpatrick, J. and ical Institute Scholar. This work was supported in part by grants Izard, T. (2006) The vinculin binding sites of talin and alpha- AI067949 and GM7 1596 from the National Institute of Health to T.I. actinin are sufficient to activate vinculin. J. Biol. Chem. 281, 7228–7236. [14] Sansonetti, P.J., Kopecko, D.J. and Formal, S.B. 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