Advertisement

Membrane-deforming Proteins Play Distinct Roles in Actin Pedestal Biogenesis by Enterohemorrhagic Escherichia coli*

Open AccessPublished:April 27, 2012DOI:https://doi.org/10.1074/jbc.M112.363473
      Many bacterial pathogens reorganize the host actin cytoskeleton during the course of infection, including enterohemorrhagic Escherichia coli (EHEC), which utilizes the effector protein EspFU to assemble actin filaments within plasma membrane protrusions called pedestals. EspFU activates N-WASP, a host actin nucleation-promoting factor that is normally auto-inhibited and found in a complex with the actin-binding protein WIP. Under native conditions, this N-WASP/WIP complex is activated by the small GTPase Cdc42 in concert with several different SH3 (Src-homology-3) domain-containing proteins. In the current study, we tested whether SH3 domains from the F-BAR (FCH-Bin-Amphiphysin-Rvs) subfamily of membrane-deforming proteins are involved in actin pedestal formation. We found that three F-BAR proteins: CIP4, FBP17, and TOCA1 (transducer of Cdc42-dependent actin assembly), play different roles during actin pedestal biogenesis. Whereas CIP4 and FBP17 inhibited actin pedestal assembly, TOCA1 stimulated this process. TOCA1 was recruited to pedestals by its SH3 domain, which bound directly to proline-rich sequences within EspFU. Moreover, EspFU and TOCA1 activated the N-WASP/WIP complex in an additive fashion in vitro, suggesting that TOCA1 can augment actin assembly within pedestals. These results reveal that EspFU acts as a scaffold to recruit multiple actin assembly factors whose functions are normally regulated by Cdc42.

      Introduction

      A variety of intracellular microbial pathogens trigger actin polymerization at their surface to drive directional motility within mammalian cells. For years, filamentous-actin (F-actin)
      The abbreviations used are: F-actin
      filamentous-actin
      EHEC
      enterohemorrhagic Escherichia coli
      I-BAR
      inverse-BAR
      GBD
      GTPase binding domain
      WCA
      WH2-connector-acidic
      TOCA
      transducer of Cdc42-dependent actin assembly
      F-BAR
      FCH-Bin-Amphiphysin-Rvs.
      “comet tail” formation by Listeria monocytogenes and Shigella flexneri have served as powerful model systems for studying the mechanisms by which cells control cytoplasmic actin assembly (
      • Stevens J.M.
      • Galyov E.E.
      • Stevens M.P.
      Actin-dependent movement of bacterial pathogens.
      ,
      • Haglund C.M.
      • Welch M.D.
      Pathogens and polymers: microbe-host interactions illuminate the cytoskeleton.
      ). More recently, a group of extracellular pathogens that adhere to the cell surface and reorganize the underlying cytoskeleton into dynamic F-actin “pedestals” have been used to help decipher how cells regulate actin assembly during plasma membrane remodeling (
      • Hayward R.D.
      • Leong J.M.
      • Koronakis V.
      • Campellone K.G.
      Exploiting pathogenic Escherichia coli to model transmembrane receptor signaling.
      ). These bacteria include enterohemorrhagic Escherichia coli (EHEC) (serotype O157), a major cause of bloody diarrhea and pediatric kidney failure (
      • Kaper J.B.
      • Nataro J.P.
      • Mobley H.L.
      Pathogenic Escherichia coli.
      ,
      • Spears K.J.
      • Roe A.J.
      • Gally D.L.
      A comparison of enteropathogenic and enterohaemorrhagic Escherichia coli pathogenesis.
      ).
      Actin pedestal biogenesis requires translocation of EHEC effector proteins into the host cell via a type III secretion system (
      • Dean P.
      • Maresca M.
      • Kenny B.
      EPEC's weapons of mass subversion.
      ,
      • Garmendia J.
      • Frankel G.
      • Crepin V.F.
      Enteropathogenic and enterohemorrhagic Escherichia coli infections: translocation, translocation, translocation.
      ,
      • Campellone K.G.
      Cytoskeleton-modulating effectors of enteropathogenic and enterohaemorrhagic Escherichia coli: Tir, EspFU, and actin pedestal assembly.
      ). Among the many effectors, only two are known to directly drive pedestal formation: Tir and EspFU (also known as TccP) (
      • Campellone K.G.
      • Cheng H.C.
      • Robbins D.
      • Siripala A.D.
      • McGhie E.J.
      • Hayward R.D.
      • Welch M.D.
      • Rosen M.K.
      • Koronakis V.
      • Leong J.M.
      Repetitive N-WASP-binding elements of the enterohemorrhagic Escherichia coli effector EspF(U) synergistically activate actin assembly.
      ). Tir acts as a plasma membrane receptor for intimin, an adhesin expressed on the bacterial surface, and intimin-Tir interactions result in clustering of the C-terminal cytoplasmic domain of Tir. A 12-residue peptide within this portion of Tir harbors its essential signaling function (
      • Allen-Vercoe E.
      • Waddell B.
      • Toh M.C.
      • DeVinney R.
      Amino acid residues within enterohemorrhagic Escherichia coli O157:H7 Tir involved in phosphorylation, α-actinin recruitment, and Nck-independent pedestal formation.
      ,
      • Brady M.J.
      • Campellone K.G.
      • Ghildiyal M.
      • Leong J.M.
      Enterohaemorrhagic and enteropathogenic Escherichia coli Tir proteins trigger a common Nck-independent actin assembly pathway.
      ,
      • Campellone K.G.
      • Brady M.J.
      • Alamares J.G.
      • Rowe D.C.
      • Skehan B.M.
      • Tipper D.J.
      • Leong J.M.
      Enterohaemorrhagic Escherichia coli Tir requires a C-terminal 12-residue peptide to initiate EspF-mediated actin assembly and harbours N-terminal sequences that influence pedestal length.
      ), and it binds to the I-BAR (inverse-BAR) domains of the membrane-deforming host cell proteins IRSp53 and IRTKS (
      • Vingadassalom D.
      • Kazlauskas A.
      • Skehan B.
      • Cheng H.C.
      • Magoun L.
      • Robbins D.
      • Rosen M.K.
      • Saksela K.
      • Leong J.M.
      Insulin receptor tyrosine kinase substrate links the E. coli O157:H7 actin assembly effectors Tir and EspF(U) during pedestal formation.
      ,
      • Weiss S.M.
      • Ladwein M.
      • Schmidt D.
      • Ehinger J.
      • Lommel S.
      • Städing K.
      • Beutling U.
      • Disanza A.
      • Frank R.
      • Jänsch L.
      • Scita G.
      • Gunzer F.
      • Rottner K.
      • Stradal T.E.
      IRSp53 links the enterohemorrhagic E. coli effectors Tir and EspFU for actin pedestal formation.
      ). SH3 domains from IRSp53 and IRTKS, in turn, recruit EspFU (
      • Vingadassalom D.
      • Kazlauskas A.
      • Skehan B.
      • Cheng H.C.
      • Magoun L.
      • Robbins D.
      • Rosen M.K.
      • Saksela K.
      • Leong J.M.
      Insulin receptor tyrosine kinase substrate links the E. coli O157:H7 actin assembly effectors Tir and EspF(U) during pedestal formation.
      ,
      • Weiss S.M.
      • Ladwein M.
      • Schmidt D.
      • Ehinger J.
      • Lommel S.
      • Städing K.
      • Beutling U.
      • Disanza A.
      • Frank R.
      • Jänsch L.
      • Scita G.
      • Gunzer F.
      • Rottner K.
      • Stradal T.E.
      IRSp53 links the enterohemorrhagic E. coli effectors Tir and EspFU for actin pedestal formation.
      ), which possesses multiple 47-residue proline-rich peptide repeats (EspFU(R1–6)) (
      • Campellone K.G.
      • Robbins D.
      • Leong J.M.
      EspFU is a translocated EHEC effector that interacts with Tir and N-WASP and promotes Nck-independent actin assembly.
      ,
      • Garmendia J.
      • Phillips A.D.
      • Carlier M.F.
      • Chong Y.
      • Schüller S.
      • Marches O.
      • Dahan S.
      • Oswald E.
      • Shaw R.K.
      • Knutton S.
      • Frankel G.
      TccP is an enterohaemorrhagic Escherichia coli O157:H7 type III effector protein that couples Tir to the actin-cytoskeleton.
      ). This repeat region is the most crucial bacterial component of the signaling machinery that triggers actin pedestal biogenesis, because a Tir-EspFU hybrid protein in which the C terminus of Tir is replaced with the EspFU repeats is fully capable of driving pedestal assembly (
      • Campellone K.G.
      • Cheng H.C.
      • Robbins D.
      • Siripala A.D.
      • McGhie E.J.
      • Hayward R.D.
      • Welch M.D.
      • Rosen M.K.
      • Koronakis V.
      • Leong J.M.
      Repetitive N-WASP-binding elements of the enterohemorrhagic Escherichia coli effector EspF(U) synergistically activate actin assembly.
      ).
      One major function of the EspFU repeat region is to recruit WASP and N-WASP (
      • Campellone K.G.
      • Robbins D.
      • Leong J.M.
      EspFU is a translocated EHEC effector that interacts with Tir and N-WASP and promotes Nck-independent actin assembly.
      ,
      • Garmendia J.
      • Phillips A.D.
      • Carlier M.F.
      • Chong Y.
      • Schüller S.
      • Marches O.
      • Dahan S.
      • Oswald E.
      • Shaw R.K.
      • Knutton S.
      • Frankel G.
      TccP is an enterohaemorrhagic Escherichia coli O157:H7 type III effector protein that couples Tir to the actin-cytoskeleton.
      ), a pair of actin nucleation-promoting factors. WASP, which is expressed in hematopoietic cells, and N-WASP, which is expressed ubiquitously, utilize C-terminal WH2-connector-acidic (WCA) domains to stimulate the Arp2/3 complex, the major nucleator of branched actin filament networks in mammalian cells (
      • Campellone K.G.
      • Welch M.D.
      A nucleator arms race: cellular control of actin assembly.
      ,
      • Rottner K.
      • Hänisch J.
      • Campellone K.G.
      WASH, WHAMM, and JMY: regulation of Arp2/3 complex and beyond.
      ). Normally, N-WASP adopts an auto-inhibited conformation in which this WCA region is prevented from activating Arp2/3 by an intramolecular interaction with a central GTPase-binding domain (GBD). The canonical mechanism of N-WASP activation involves binding of the small GTPase Cdc42 to a portion of the GBD that lies adjacent to the segment that contacts the WCA domain. This changes the GBD conformation, relieves auto-inhibition, and allows the WCA domain to stimulate the Arp2/3 complex (
      • Padrick S.B.
      • Rosen M.K.
      Physical mechanisms of signal integration by WASP family proteins.
      ).
      Although Cdc42 is sufficient to activate recombinant N-WASP in vitro, several N-WASP-interacting proteins, including WIP, are known to influence N-WASP regulation in the cytoplasm. For example, activation of the native N-WASP/WIP complex by Cdc42 requires a protein named TOCA1 (
      • Ho H.Y.
      • Rohatgi R.
      • Lebensohn A.M.
      • Le M.
      • Li J.
      • Gygi S.P.
      • Kirschner M.W.
      Toca-1 mediates Cdc42-dependent actin nucleation by activating the N-WASP-WIP complex.
      ). TOCA1 harbors an N-terminal F-BAR domain that tubulates membranes, a central homology region-1 (HR1) domain that binds Cdc42, and a C-terminal SH3 domain that can interact with N-WASP. Two TOCA1 homologs, FBP17 and CIP4, also contain this F-BAR-HR1-SH3 domain organization.
      Although TOCA1, FBP17, and CIP4 each participate in plasma membrane dynamics (
      • Itoh T.
      • Erdmann K.S.
      • Roux A.
      • Habermann B.
      • Werner H.
      • De Camilli P.
      Dynamin and the actin cytoskeleton cooperatively regulate plasma membrane invagination by BAR and F-BAR proteins.
      ,
      • Kamioka Y.
      • Fukuhara S.
      • Sawa H.
      • Nagashima K.
      • Masuda M.
      • Matsuda M.
      • Mochizuki N.
      A novel dynamin-associating molecule, formin-binding protein 17, induces tubular membrane invaginations and participates in endocytosis.
      ,
      • Tsujita K.
      • Suetsugu S.
      • Sasaki N.
      • Furutani M.
      • Oikawa T.
      • Takenawa T.
      Coordination between the actin cytoskeleton and membrane deformation by a novel membrane tubulation domain of PCH proteins is involved in endocytosis.
      ), their membrane tubulation activities differ significantly from one another (
      • Toguchi M.
      • Richnau N.
      • Ruusala A.
      • Aspenström P.
      Members of the CIP4 family of proteins participate in the regulation of platelet-derived growth factor receptor-beta-dependent actin reorganization and migration.
      ,
      • Bu W.
      • Lim K.B.
      • Yu Y.H.
      • Chou A.M.
      • Sudhaharan T.
      • Ahmed S.
      Cdc42 interaction with N-WASP and Toca-1 regulates membrane tubulation, vesicle formation, and vesicle motility: implications for endocytosis.
      ,
      • Kakimoto T.
      • Katoh H.
      • Negishi M.
      Regulation of neuronal morphology by Toca-1, an F-BAR/EFC protein that induces plasma membrane invagination.
      ), implying that they are regulated by divergent mechanisms or that they have distinct cellular roles. TOCA1 has been shown to also function with N-WASP during neuronal morphogenesis and in filopodial protrusions of the plasma membrane (
      • Kakimoto T.
      • Katoh H.
      • Negishi M.
      Regulation of neuronal morphology by Toca-1, an F-BAR/EFC protein that induces plasma membrane invagination.
      ,
      • Bu W.
      • Chou A.M.
      • Lim K.B.
      • Sudhaharan T.
      • Ahmed S.
      The Toca-1-N-WASP complex links filopodial formation to endocytosis.
      ,
      • Hu J.
      • Mukhopadhyay A.
      • Craig A.W.
      Transducer of Cdc42-dependent actin assembly promotes epidermal growth factor-induced cell motility and invasiveness.
      ). Moreover, TOCA1 appears to recruit the N-WASP/WIP complex to drive actin assembly at the tips of reconstituted filopodia-like structures in vitro (
      • Lee K.
      • Gallop J.L.
      • Rambani K.
      • Kirschner M.W.
      Self-assembly of filopodia-like structures on supported lipid bilayers.
      ). TOCA1 additionally contributes to the formation of actin tails that drive endosome motility (
      • Ho H.Y.
      • Rohatgi R.
      • Lebensohn A.M.
      • Le M.
      • Li J.
      • Gygi S.P.
      • Kirschner M.W.
      Toca-1 mediates Cdc42-dependent actin nucleation by activating the N-WASP-WIP complex.
      ,
      • Bu W.
      • Lim K.B.
      • Yu Y.H.
      • Chou A.M.
      • Sudhaharan T.
      • Ahmed S.
      Cdc42 interaction with N-WASP and Toca-1 regulates membrane tubulation, vesicle formation, and vesicle motility: implications for endocytosis.
      ), and can recruit N-WASP/WIP to liposomes in vitro, where actin assembly rates might be influenced by membrane curvature (
      • Takano K.
      • Toyooka K.
      • Suetsugu S.
      EFC/F-BAR proteins and the N-WASP-WIP complex induce membrane curvature-dependent actin polymerization.
      ). Similarly, TOCA1 has been implicated in actin tail assembly by Shigella. In the Shigella system, TOCA1 increases actin tail length, although the mechanisms by which it is recruited have not been defined (
      • Leung Y.
      • Ally S.
      • Goldberg M.B.
      Bacterial actin assembly requires toca-1 to relieve N-wasp autoinhibition.
      ).
      Like Cdc42, EspFU is able to activate WASP and N-WASP. However, in contrast to the aforementioned mechanism of WASP activation by Cdc42, EspFU directly competes with the WCA domain for binding to the auto-inhibitory segment of the GBD (
      • Cheng H.C.
      • Skehan B.M.
      • Campellone K.G.
      • Leong J.M.
      • Rosen M.K.
      Structural mechanism of WASP activation by the enterohaemorrhagic E. coli effector EspF(U).
      ,
      • Sallee N.A.
      • Rivera G.M.
      • Dueber J.E.
      • Vasilescu D.
      • Mullins R.D.
      • Mayer B.J.
      • Lim W.A.
      The pathogen protein EspF(U) hijacks actin polymerization using mimicry and multivalency.
      ). A short amphipathic helix near the N terminus of each 47-residue EspFU repeat binds to this region of the GBD with high affinity and therefore acts as a potent activator of WASP and N-WASP (
      • Cheng H.C.
      • Skehan B.M.
      • Campellone K.G.
      • Leong J.M.
      • Rosen M.K.
      Structural mechanism of WASP activation by the enterohaemorrhagic E. coli effector EspF(U).
      ,
      • Sallee N.A.
      • Rivera G.M.
      • Dueber J.E.
      • Vasilescu D.
      • Mullins R.D.
      • Mayer B.J.
      • Lim W.A.
      The pathogen protein EspF(U) hijacks actin polymerization using mimicry and multivalency.
      ), as well as the N-WASP/WIP complex (
      • Campellone K.G.
      • Cheng H.C.
      • Robbins D.
      • Siripala A.D.
      • McGhie E.J.
      • Hayward R.D.
      • Welch M.D.
      • Rosen M.K.
      • Koronakis V.
      • Leong J.M.
      Repetitive N-WASP-binding elements of the enterohemorrhagic Escherichia coli effector EspF(U) synergistically activate actin assembly.
      ). Importantly, actin assembly is further amplified by the multivalency of EspFU, because the repeats synergize in activating recombinant N-WASP derivatives and the N-WASP/WIP complex in vitro (
      • Campellone K.G.
      • Cheng H.C.
      • Robbins D.
      • Siripala A.D.
      • McGhie E.J.
      • Hayward R.D.
      • Welch M.D.
      • Rosen M.K.
      • Koronakis V.
      • Leong J.M.
      Repetitive N-WASP-binding elements of the enterohemorrhagic Escherichia coli effector EspF(U) synergistically activate actin assembly.
      ,
      • Sallee N.A.
      • Rivera G.M.
      • Dueber J.E.
      • Vasilescu D.
      • Mullins R.D.
      • Mayer B.J.
      • Lim W.A.
      The pathogen protein EspF(U) hijacks actin polymerization using mimicry and multivalency.
      ,
      • Padrick S.B.
      • Cheng H.C.
      • Ismail A.M.
      • Panchal S.C.
      • Doolittle L.K.
      • Kim S.
      • Skehan B.M.
      • Umetani J.
      • Brautigam C.A.
      • Leong J.M.
      • Rosen M.K.
      Hierarchical regulation of WASP/WAVE proteins.
      ).
      Interestingly, each N-WASP-binding helix within an EspFU repeat lies adjacent to a proline-rich sequence, suggesting that EspFU may interact with multiple SH3 domains that could potentially modulate N-WASP activity even further. In fact, these proline-rich motifs are involved in binding to the SH3 domain-containing I-BAR proteins IRSp53 and IRTKS (
      • Vingadassalom D.
      • Kazlauskas A.
      • Skehan B.
      • Cheng H.C.
      • Magoun L.
      • Robbins D.
      • Rosen M.K.
      • Saksela K.
      • Leong J.M.
      Insulin receptor tyrosine kinase substrate links the E. coli O157:H7 actin assembly effectors Tir and EspF(U) during pedestal formation.
      ,
      • Weiss S.M.
      • Ladwein M.
      • Schmidt D.
      • Ehinger J.
      • Lommel S.
      • Städing K.
      • Beutling U.
      • Disanza A.
      • Frank R.
      • Jänsch L.
      • Scita G.
      • Gunzer F.
      • Rottner K.
      • Stradal T.E.
      IRSp53 links the enterohemorrhagic E. coli effectors Tir and EspFU for actin pedestal formation.
      ). However, the ability of these and other BAR protein relatives to regulate N-WASP during pedestal biogenesis has not been explored. Given that the SH3 domain of the F-BAR protein TOCA1 is known to cooperate with Cdc42 to activate N-WASP in uninfected cells, we sought to test whether the TOCA1 family of proteins plays a role in EspFU-mediated actin assembly during pedestal formation. We found that TOCA1 stimulates actin pedestal assembly, whereas FBP17 and CIP4 inhibit this process. TOCA1 is recruited to sites of pedestal biogenesis in a manner dependent on its SH3 domain, which recognizes EspFU, and these two proteins activate the N-WASP/WIP complex in an additive manner. These results highlight an important role for TOCA1 in pedestal biogenesis and define a key scaffolding function for EspFU.

      DISCUSSION

      One of the best-characterized ways in which actin polymerization is regulated involves activation of the nucleation-promoting factor N-WASP by the small GTPase Cdc42 (
      • Padrick S.B.
      • Rosen M.K.
      Physical mechanisms of signal integration by WASP family proteins.
      ). More recently, a bacterial molecule, the EHEC O157 effector protein EspFU, has been estimated to activate N-WASP with a potency that is orders of magnitude higher than Cdc42 and other individual N-WASP-binding proteins (
      • Cheng H.C.
      • Skehan B.M.
      • Campellone K.G.
      • Leong J.M.
      • Rosen M.K.
      Structural mechanism of WASP activation by the enterohaemorrhagic E. coli effector EspF(U).
      ,
      • Sallee N.A.
      • Rivera G.M.
      • Dueber J.E.
      • Vasilescu D.
      • Mullins R.D.
      • Mayer B.J.
      • Lim W.A.
      The pathogen protein EspF(U) hijacks actin polymerization using mimicry and multivalency.
      ). EspFU is a relatively simple protein: a type III translocation sequence followed by a 47-residue peptide repeated between 2 and 8 times (
      • Garmendia J.
      • Ren Z.
      • Tennant S.
      • Midolli Viera M.A.
      • Chong Y.
      • Whale A.
      • Azzopardi K.
      • Dahan S.
      • Sircili M.P.
      • Franzolin M.R.
      • Trabulsi L.R.
      • Phillips A.
      • Gomes T.A.
      • Xu J.
      • Robins-Browne R.
      • Frankel G.
      Distribution of tccP in clinical enterohemorrhagic and enteropathogenic Escherichia coli isolates.
      ). Included within each repeat is an N-terminal amphipathic helix that binds to N-WASP (
      • Cheng H.C.
      • Skehan B.M.
      • Campellone K.G.
      • Leong J.M.
      • Rosen M.K.
      Structural mechanism of WASP activation by the enterohaemorrhagic E. coli effector EspF(U).
      ,
      • Sallee N.A.
      • Rivera G.M.
      • Dueber J.E.
      • Vasilescu D.
      • Mullins R.D.
      • Mayer B.J.
      • Lim W.A.
      The pathogen protein EspF(U) hijacks actin polymerization using mimicry and multivalency.
      ) and a C-terminal proline-rich sequence that is recognized by the SH3 domains of at least 2 proteins, IRSp53 and IRTKS (
      • Vingadassalom D.
      • Kazlauskas A.
      • Skehan B.
      • Cheng H.C.
      • Magoun L.
      • Robbins D.
      • Rosen M.K.
      • Saksela K.
      • Leong J.M.
      Insulin receptor tyrosine kinase substrate links the E. coli O157:H7 actin assembly effectors Tir and EspF(U) during pedestal formation.
      ,
      • Weiss S.M.
      • Ladwein M.
      • Schmidt D.
      • Ehinger J.
      • Lommel S.
      • Städing K.
      • Beutling U.
      • Disanza A.
      • Frank R.
      • Jänsch L.
      • Scita G.
      • Gunzer F.
      • Rottner K.
      • Stradal T.E.
      IRSp53 links the enterohemorrhagic E. coli effectors Tir and EspFU for actin pedestal formation.
      ). Because activation of the native N-WASP/WIP complex by Cdc42 normally requires the SH3 domain-containing F-BAR protein TOCA1 (
      • Ho H.Y.
      • Rohatgi R.
      • Lebensohn A.M.
      • Le M.
      • Li J.
      • Gygi S.P.
      • Kirschner M.W.
      Toca-1 mediates Cdc42-dependent actin nucleation by activating the N-WASP-WIP complex.
      ), we explored whether TOCA1 and other closely-related factors play a role in EspFU-mediated actin pedestal assembly.
      Through a combination of protein depletion and overexpression studies, we found that TOCA1 stimulates actin pedestal assembly, whereas surprisingly its homologs FBP17 and CIP4 inhibit this process. After 3-h infections, when pedestals have typically reached steady-state in quantity, length, and intensity, the frequency of pedestal formation decreased by approximately one-third when TOCA1 was depleted from cells, while the average intensity of F-actin staining within pedestals more than doubled when TOCA1 was overexpressed. In contrast, when either FBP17 or CIP4 were depleted, the efficiency of pedestal formation increased slightly, and the average intensity of F-actin staining more than doubled. Moreover, FBP17 overexpression caused a major decrease in the efficiency of actin pedestal formation. We do not yet understand the mechanistic basis for how FBP17 (and perhaps CIP4) normally inhibits pedestal assembly, but these results are consistent with previous observations suggesting that TOCA1, FBP17, and CIP4 have different abilities to promote plasma membrane remodeling (
      • Itoh T.
      • Erdmann K.S.
      • Roux A.
      • Habermann B.
      • Werner H.
      • De Camilli P.
      Dynamin and the actin cytoskeleton cooperatively regulate plasma membrane invagination by BAR and F-BAR proteins.
      ,
      • Toguchi M.
      • Richnau N.
      • Ruusala A.
      • Aspenström P.
      Members of the CIP4 family of proteins participate in the regulation of platelet-derived growth factor receptor-beta-dependent actin reorganization and migration.
      ,
      • Bu W.
      • Lim K.B.
      • Yu Y.H.
      • Chou A.M.
      • Sudhaharan T.
      • Ahmed S.
      Cdc42 interaction with N-WASP and Toca-1 regulates membrane tubulation, vesicle formation, and vesicle motility: implications for endocytosis.
      ). Given that TOCA1 was the only one of these factors to have a positive effect on pedestal assembly, we further explored its function during this process.
      GFP-TOCA1 localized to actin pedestals generated by EspFU, and the SH3 domain of TOCA1 was required for this recruitment. TOCA1 also bound to EspFU in an SH3 domain-dependent manner in multiple protein-protein interaction assays. A W518K substitution that disrupts SH3 function abrogated both EspFU binding in vitro and the dominant negative activity of this isolated domain in cells. Collectively, these results suggest that EspFU is capable of directly engaging TOCA1 via its SH3 domain during actin pedestal assembly.
      Importantly, although EspFU and Cdc42 each bind to both TOCA1 and N-WASP, they recognize different sequences in these proteins (Fig. 9, A and B). EspFU binds to the autoinhibitory portion of the N-WASP GBD, whereas Cdc42 binds to a nearby motif called the CRIB (
      • Cheng H.C.
      • Skehan B.M.
      • Campellone K.G.
      • Leong J.M.
      • Rosen M.K.
      Structural mechanism of WASP activation by the enterohaemorrhagic E. coli effector EspF(U).
      ,
      • Sallee N.A.
      • Rivera G.M.
      • Dueber J.E.
      • Vasilescu D.
      • Mullins R.D.
      • Mayer B.J.
      • Lim W.A.
      The pathogen protein EspF(U) hijacks actin polymerization using mimicry and multivalency.
      ). In addition, while EspFU interacts with the TOCA1 SH3 domain, Cdc42 recognizes its HR1 domain (
      • Ho H.Y.
      • Rohatgi R.
      • Lebensohn A.M.
      • Le M.
      • Li J.
      • Gygi S.P.
      • Kirschner M.W.
      Toca-1 mediates Cdc42-dependent actin nucleation by activating the N-WASP-WIP complex.
      ). Thus, EspFU has evolved strategies that are parallel to, but distinct from those used by Cdc42 to activate the N-WASP/WIP actin polymerization machinery.
      Figure thumbnail gr9
      FIGURE 9Model for EspFU-mediated recruitment of TOCA1 and enhancement of N-WASP activation. A, EspFU contains an N-terminal sequence (N) important for entry into mammalian cells and multiple 47-residue peptide repeats (R1-R6) that include an N-WASP-binding amphipathic helix and a proline-rich SH3-binding sequence. Interactions between an EspFU repeat and the N-WASP autoinhibitory (AI) motif within the GBD exposes the WCA region to promote Arp2/3 complex activation. The EspFU repeats also bind to the SH3 domain of TOCA1. In a simple model, one subunit of a TOCA1 homodimer may interact with EspFU, while the SH3 domain of the other subunit may enhance N-WASP activation by binding to the N-WASP proline-rich domain (PRD). Other N-WASP sequences include a WASP-homology-1 (WH1) domain, a basic (B) peptide, and a C-terminal WCA region. B, in contrast to EspFU, Cdc42 binds to the N-WASP Cdc42/Rac-interactive-binding (CRIB) motif within the GBD and the TOCA1 HR1 domain to promote actin assembly. The TOCA1 SH3 domain can facilitate N-WASP activation by binding to the PRD.
      Interestingly, pulldown assays, Far-Western blots, and peptide-binding ELISAs all indicated that multiple 47-residue repeats are necessary for the physical association of EspFU with TOCA1, implying that repeat cooperativity is required for recruitment of TOCA1 during pedestal biogenesis. Cooperativity among the EspFU repeats was previously shown to result in simultaneous engagement of multiple N-WASP molecules and a dramatic enhancement of subsequent Arp2/3 binding and activation (
      • Campellone K.G.
      • Cheng H.C.
      • Robbins D.
      • Siripala A.D.
      • McGhie E.J.
      • Hayward R.D.
      • Welch M.D.
      • Rosen M.K.
      • Koronakis V.
      • Leong J.M.
      Repetitive N-WASP-binding elements of the enterohemorrhagic Escherichia coli effector EspF(U) synergistically activate actin assembly.
      ,
      • Padrick S.B.
      • Cheng H.C.
      • Ismail A.M.
      • Panchal S.C.
      • Doolittle L.K.
      • Kim S.
      • Skehan B.M.
      • Umetani J.
      • Brautigam C.A.
      • Leong J.M.
      • Rosen M.K.
      Hierarchical regulation of WASP/WAVE proteins.
      ). Taken together, these findings suggest that, in addition to their synergistic effects on N-WASP activation, the tandem EspFU repeats may provide a scaffold for recruiting additional host proteins like TOCA1 that coalesce into a multisubunit complex capable of robust actin polymerization. Notably, the number of EspFU repeat units varies among pathogenic E. coli isolates, but all known EspFU proteins contain at least two repeats (
      • Garmendia J.
      • Ren Z.
      • Tennant S.
      • Midolli Viera M.A.
      • Chong Y.
      • Whale A.
      • Azzopardi K.
      • Dahan S.
      • Sircili M.P.
      • Franzolin M.R.
      • Trabulsi L.R.
      • Phillips A.
      • Gomes T.A.
      • Xu J.
      • Robins-Browne R.
      • Frankel G.
      Distribution of tccP in clinical enterohemorrhagic and enteropathogenic Escherichia coli isolates.
      ), implying that multivalent interactions, likely with multiple binding partners, are important for actin pedestal assembly in vivo.
      Consistent with previous observations indicating that N-WASP has higher levels of activity when it is engaged by multiple signaling molecules (
      • Padrick S.B.
      • Rosen M.K.
      Physical mechanisms of signal integration by WASP family proteins.
      ,
      • Ho H.Y.
      • Rohatgi R.
      • Lebensohn A.M.
      • Le M.
      • Li J.
      • Gygi S.P.
      • Kirschner M.W.
      Toca-1 mediates Cdc42-dependent actin nucleation by activating the N-WASP-WIP complex.
      ,
      • Rohatgi R.
      • Nollau P.
      • Ho H.Y.
      • Kirschner M.W.
      • Mayer B.J.
      Nck and phosphatidylinositol 4,5-bisphosphate synergistically activate actin polymerization through the N-WASP-Arp2/3 pathway.
      ,
      • Carlier M.F.
      • Nioche P.
      • Broutin-L'Hermite I.
      • Boujemaa R.
      • Le Clainche C.
      • Egile C.
      • Garbay C.
      • Ducruix A.
      • Sansonetti P.
      • Pantaloni D.
      GRB2 links signaling to actin assembly by enhancing interaction of neural Wiskott-Aldrich syndrome protein (N-WASp) with actin-related protein (ARP2/3) complex.
      ), pyrene-actin assembly assays demonstrated that EspFU and TOCA1 activate the N-WASP/WIP complex in an additive manner when present at low concentrations. However, EspFU and TOCA1 are each independently capable of potently stimulating assembly when used in higher quantities. Interestingly, our observation that TOCA1 alone activates the N-WASP/WIP complex in an SH3-dependent manner indicates that it does not always require coordination with other endogenous N-WASP activators such as Cdc42 to promote actin polymerization.
      The findings that the SH3 domain of TOCA1 can bind EspFU as well as activate N-WASP might be interpreted to mean that simultaneous interactions of TOCA1 with EspFU (to promote recruitment) and N-WASP (to promote activation) does not occur. However, F-BAR domains like the one present in TOCA1 dimerize, so native TOCA1 might harbor two (or more) SH3 domains that could promote coincident binding to EspFU and N-WASP (Fig. 9A). It is also possible that different cellular conditions dictate whether TOCA1 is bound to EspFU, N-WASP, both proteins, or neither protein within the pedestal. Finally, it is important to note that the proline-rich sequences within EspFU are additionally recognized by the SH3 domains of IRSp53 and IRTKS, I-BAR proteins that mediate the recruitment of EspFU to the EHEC transmembrane receptor, Tir (
      • Vingadassalom D.
      • Kazlauskas A.
      • Skehan B.
      • Cheng H.C.
      • Magoun L.
      • Robbins D.
      • Rosen M.K.
      • Saksela K.
      • Leong J.M.
      Insulin receptor tyrosine kinase substrate links the E. coli O157:H7 actin assembly effectors Tir and EspF(U) during pedestal formation.
      ,
      • Weiss S.M.
      • Ladwein M.
      • Schmidt D.
      • Ehinger J.
      • Lommel S.
      • Städing K.
      • Beutling U.
      • Disanza A.
      • Frank R.
      • Jänsch L.
      • Scita G.
      • Gunzer F.
      • Rottner K.
      • Stradal T.E.
      IRSp53 links the enterohemorrhagic E. coli effectors Tir and EspFU for actin pedestal formation.
      ). EspFU has also been reported to bind the SH3 domain of cortactin (
      • Cantarelli V.V.
      • Kodama T.
      • Nijstad N.
      • Abolghait S.K.
      • Nada S.
      • Okada M.
      • Iida T.
      • Honda T.
      Tyrosine phosphorylation controls cortactin binding to two enterohaemorrhagic Escherichia coli effectors: Tir and EspFu/TccP.
      ), another actin-associated factor. Thus, with numerous potential host cell targets, the mechanisms by which the EspFU repeat region organizes a complex signaling platform during actin pedestal formation are only beginning to be elucidated.
      Our preliminary investigations into how the SH3 domains from TOCA1 and FBP17, as well as IRSp53 and IRTKS, bind to EspFU and activate the N-WASP/WIP complex have begun to reveal some of the similarities and differences between the F-BAR and I-BAR subclasses of the BAR superfamily of membrane-deforming proteins. Interestingly, a monomeric form of the proline-rich EspFU peptide binds to the recombinant SH3 domains of IRTKS and (to an even greater extent) IRSp53, but not to TOCA1 or FBP17. Thus, there appears to be a fundamental difference in the way in which the SH3 domains from IRSp53/IRTKS and TOCA1 are recognized by EspFU. The SH3 domains from the I-BAR proteins also are slightly better than the SH3 domains from the F-BAR proteins at activating the N-WASP/WIP complex in vitro. However, it is not yet clear if such subtle differences are manifested in any significant effects on actin assembly in cells.
      Along with activating N-WASP/WIP, TOCA1 can also bind and deform membranes via its F-BAR domain, which recognizes charged phospholipids (
      • Itoh T.
      • Erdmann K.S.
      • Roux A.
      • Habermann B.
      • Werner H.
      • De Camilli P.
      Dynamin and the actin cytoskeleton cooperatively regulate plasma membrane invagination by BAR and F-BAR proteins.
      ,
      • Kamioka Y.
      • Fukuhara S.
      • Sawa H.
      • Nagashima K.
      • Masuda M.
      • Matsuda M.
      • Mochizuki N.
      A novel dynamin-associating molecule, formin-binding protein 17, induces tubular membrane invaginations and participates in endocytosis.
      ,
      • Tsujita K.
      • Suetsugu S.
      • Sasaki N.
      • Furutani M.
      • Oikawa T.
      • Takenawa T.
      Coordination between the actin cytoskeleton and membrane deformation by a novel membrane tubulation domain of PCH proteins is involved in endocytosis.
      ). In fact, membrane binding by TOCA1 may contribute to its ability to activate N-WASP (
      • Takano K.
      • Toyooka K.
      • Suetsugu S.
      EFC/F-BAR proteins and the N-WASP-WIP complex induce membrane curvature-dependent actin polymerization.
      ). Interestingly, the concave-shaped F-BAR dimer induces membrane invagination, in contrast to the convex membrane binding I-BAR domains of IRTKS and IRSp53 that induce protrusive structures (
      • Saarikangas J.
      • Zhao H.
      • Pykäläinen A.
      • Laurinmäki P.
      • Mattila P.K.
      • Kinnunen P.K.
      • Butcher S.J.
      • Lappalainen P.
      Molecular mechanisms of membrane deformation by I-BAR domain proteins.
      ). How these opposing activities might influence the morphology of the actin pedestal also remains to be determined.
      Bacterial comandeering of multiple host factors to promote actin-based motility has also been demonstrated for Shigella, which recruits both N-WASP and TOCA1 during actin tail assembly (
      • Leung Y.
      • Ally S.
      • Goldberg M.B.
      Bacterial actin assembly requires toca-1 to relieve N-wasp autoinhibition.
      ). In that experimental system, TOCA1 depletion reduces the average length of the actin tail, suggesting that TOCA1 facilitates tail elongation (
      • Leung Y.
      • Ally S.
      • Goldberg M.B.
      Bacterial actin assembly requires toca-1 to relieve N-wasp autoinhibition.
      ). However, the Shigella N-WASP activator, IcsA, does not bind TOCA1. Instead, TOCA1 recruitment requires a second unidentified Shigella effector protein that functions by an unknown mechanism. Thus, in contrast to a multifaceted strategy for N-WASP activation by Shigella, our results indicate that the mechanism of actin pedestal assembly driven by N-WASP and TOCA1 relies solely upon the versatile EHEC effector protein EspFU. Further characterizations of EspFU and its host protein targets will undoubtedly continue to shed light on how cells and pathogens control the actin assembly machinery.

      Acknowledgments

      We thank Henry Ho and Marc Kirschner for TOCA1 plasmids and Pietro DeCamilli for the GFP-FBP17 plasmid.

      References

        • Stevens J.M.
        • Galyov E.E.
        • Stevens M.P.
        Actin-dependent movement of bacterial pathogens.
        Nat. Rev. Microbiol. 2006; 4: 91-101
        • Haglund C.M.
        • Welch M.D.
        Pathogens and polymers: microbe-host interactions illuminate the cytoskeleton.
        J. Cell Biol. 2011; 195: 7-17
        • Hayward R.D.
        • Leong J.M.
        • Koronakis V.
        • Campellone K.G.
        Exploiting pathogenic Escherichia coli to model transmembrane receptor signaling.
        Nat. Rev. Microbiol. 2006; 4: 358-370
        • Kaper J.B.
        • Nataro J.P.
        • Mobley H.L.
        Pathogenic Escherichia coli.
        Nat. Rev. Microbiol. 2004; 2: 123-140
        • Spears K.J.
        • Roe A.J.
        • Gally D.L.
        A comparison of enteropathogenic and enterohaemorrhagic Escherichia coli pathogenesis.
        FEMS Microbiol. Lett. 2006; 255: 187-202
        • Dean P.
        • Maresca M.
        • Kenny B.
        EPEC's weapons of mass subversion.
        Curr. Opin. Microbiol. 2005; 8: 28-34
        • Garmendia J.
        • Frankel G.
        • Crepin V.F.
        Enteropathogenic and enterohemorrhagic Escherichia coli infections: translocation, translocation, translocation.
        Infect. Immun. 2005; 73: 2573-2585
        • Campellone K.G.
        Cytoskeleton-modulating effectors of enteropathogenic and enterohaemorrhagic Escherichia coli: Tir, EspFU, and actin pedestal assembly.
        FEBS J. 2010; 277: 2390-2402
        • Campellone K.G.
        • Cheng H.C.
        • Robbins D.
        • Siripala A.D.
        • McGhie E.J.
        • Hayward R.D.
        • Welch M.D.
        • Rosen M.K.
        • Koronakis V.
        • Leong J.M.
        Repetitive N-WASP-binding elements of the enterohemorrhagic Escherichia coli effector EspF(U) synergistically activate actin assembly.
        PLoS Pathog. 2008; 4: e1000191
        • Allen-Vercoe E.
        • Waddell B.
        • Toh M.C.
        • DeVinney R.
        Amino acid residues within enterohemorrhagic Escherichia coli O157:H7 Tir involved in phosphorylation, α-actinin recruitment, and Nck-independent pedestal formation.
        Infect Immun. 2006; 74: 6196-6205
        • Brady M.J.
        • Campellone K.G.
        • Ghildiyal M.
        • Leong J.M.
        Enterohaemorrhagic and enteropathogenic Escherichia coli Tir proteins trigger a common Nck-independent actin assembly pathway.
        Cell Microbiol. 2007; 9: 2242-2253
        • Campellone K.G.
        • Brady M.J.
        • Alamares J.G.
        • Rowe D.C.
        • Skehan B.M.
        • Tipper D.J.
        • Leong J.M.
        Enterohaemorrhagic Escherichia coli Tir requires a C-terminal 12-residue peptide to initiate EspF-mediated actin assembly and harbours N-terminal sequences that influence pedestal length.
        Cell Microbiol. 2006; 8: 1488-1503
        • Vingadassalom D.
        • Kazlauskas A.
        • Skehan B.
        • Cheng H.C.
        • Magoun L.
        • Robbins D.
        • Rosen M.K.
        • Saksela K.
        • Leong J.M.
        Insulin receptor tyrosine kinase substrate links the E. coli O157:H7 actin assembly effectors Tir and EspF(U) during pedestal formation.
        Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 6754-6759
        • Weiss S.M.
        • Ladwein M.
        • Schmidt D.
        • Ehinger J.
        • Lommel S.
        • Städing K.
        • Beutling U.
        • Disanza A.
        • Frank R.
        • Jänsch L.
        • Scita G.
        • Gunzer F.
        • Rottner K.
        • Stradal T.E.
        IRSp53 links the enterohemorrhagic E. coli effectors Tir and EspFU for actin pedestal formation.
        Cell Host Microbe. 2009; 5: 244-258
        • Campellone K.G.
        • Robbins D.
        • Leong J.M.
        EspFU is a translocated EHEC effector that interacts with Tir and N-WASP and promotes Nck-independent actin assembly.
        Dev. Cell. 2004; 7: 217-228
        • Garmendia J.
        • Phillips A.D.
        • Carlier M.F.
        • Chong Y.
        • Schüller S.
        • Marches O.
        • Dahan S.
        • Oswald E.
        • Shaw R.K.
        • Knutton S.
        • Frankel G.
        TccP is an enterohaemorrhagic Escherichia coli O157:H7 type III effector protein that couples Tir to the actin-cytoskeleton.
        Cell Microbiol. 2004; 6: 1167-1183
        • Campellone K.G.
        • Welch M.D.
        A nucleator arms race: cellular control of actin assembly.
        Nat. Rev. Mol. Cell Biol. 2010; 11: 237-251
        • Rottner K.
        • Hänisch J.
        • Campellone K.G.
        WASH, WHAMM, and JMY: regulation of Arp2/3 complex and beyond.
        Trends Cell Biol. 2010; 20: 650-661
        • Padrick S.B.
        • Rosen M.K.
        Physical mechanisms of signal integration by WASP family proteins.
        Annu. Rev. Biochem. 2010; 79: 707-735
        • Ho H.Y.
        • Rohatgi R.
        • Lebensohn A.M.
        • Le M.
        • Li J.
        • Gygi S.P.
        • Kirschner M.W.
        Toca-1 mediates Cdc42-dependent actin nucleation by activating the N-WASP-WIP complex.
        Cell. 2004; 118: 203-216
        • Itoh T.
        • Erdmann K.S.
        • Roux A.
        • Habermann B.
        • Werner H.
        • De Camilli P.
        Dynamin and the actin cytoskeleton cooperatively regulate plasma membrane invagination by BAR and F-BAR proteins.
        Dev. Cell. 2005; 9: 791-804
        • Kamioka Y.
        • Fukuhara S.
        • Sawa H.
        • Nagashima K.
        • Masuda M.
        • Matsuda M.
        • Mochizuki N.
        A novel dynamin-associating molecule, formin-binding protein 17, induces tubular membrane invaginations and participates in endocytosis.
        J. Biol. Chem. 2004; 279: 40091-40099
        • Tsujita K.
        • Suetsugu S.
        • Sasaki N.
        • Furutani M.
        • Oikawa T.
        • Takenawa T.
        Coordination between the actin cytoskeleton and membrane deformation by a novel membrane tubulation domain of PCH proteins is involved in endocytosis.
        J. Cell Biol. 2006; 172: 269-279
        • Toguchi M.
        • Richnau N.
        • Ruusala A.
        • Aspenström P.
        Members of the CIP4 family of proteins participate in the regulation of platelet-derived growth factor receptor-beta-dependent actin reorganization and migration.
        Biol. Cell. 2010; 102: 215-230
        • Bu W.
        • Lim K.B.
        • Yu Y.H.
        • Chou A.M.
        • Sudhaharan T.
        • Ahmed S.
        Cdc42 interaction with N-WASP and Toca-1 regulates membrane tubulation, vesicle formation, and vesicle motility: implications for endocytosis.
        PLoS One. 2010; 5: e12153
        • Kakimoto T.
        • Katoh H.
        • Negishi M.
        Regulation of neuronal morphology by Toca-1, an F-BAR/EFC protein that induces plasma membrane invagination.
        J. Biol. Chem. 2006; 281: 29042-29053
        • Bu W.
        • Chou A.M.
        • Lim K.B.
        • Sudhaharan T.
        • Ahmed S.
        The Toca-1-N-WASP complex links filopodial formation to endocytosis.
        J. Biol. Chem. 2009; 284: 11622-11636
        • Hu J.
        • Mukhopadhyay A.
        • Craig A.W.
        Transducer of Cdc42-dependent actin assembly promotes epidermal growth factor-induced cell motility and invasiveness.
        J. Biol. Chem. 2011; 286: 2261-2272
        • Lee K.
        • Gallop J.L.
        • Rambani K.
        • Kirschner M.W.
        Self-assembly of filopodia-like structures on supported lipid bilayers.
        Science. 2010; 329: 1341-1345
        • Takano K.
        • Toyooka K.
        • Suetsugu S.
        EFC/F-BAR proteins and the N-WASP-WIP complex induce membrane curvature-dependent actin polymerization.
        EMBO J. 2008; 27: 2817-2828
        • Leung Y.
        • Ally S.
        • Goldberg M.B.
        Bacterial actin assembly requires toca-1 to relieve N-wasp autoinhibition.
        Cell Host Microbe. 2008; 3: 39-47
        • Cheng H.C.
        • Skehan B.M.
        • Campellone K.G.
        • Leong J.M.
        • Rosen M.K.
        Structural mechanism of WASP activation by the enterohaemorrhagic E. coli effector EspF(U).
        Nature. 2008; 454: 1009-1013
        • Sallee N.A.
        • Rivera G.M.
        • Dueber J.E.
        • Vasilescu D.
        • Mullins R.D.
        • Mayer B.J.
        • Lim W.A.
        The pathogen protein EspF(U) hijacks actin polymerization using mimicry and multivalency.
        Nature. 2008; 454: 1005-1008
        • Padrick S.B.
        • Cheng H.C.
        • Ismail A.M.
        • Panchal S.C.
        • Doolittle L.K.
        • Kim S.
        • Skehan B.M.
        • Umetani J.
        • Brautigam C.A.
        • Leong J.M.
        • Rosen M.K.
        Hierarchical regulation of WASP/WAVE proteins.
        Mol. Cell. 2008; 32: 426-438
        • Campellone K.G.
        • Leong J.M.
        Nck-independent actin assembly is mediated by two phosphorylated tyrosines within enteropathogenic Escherichia coli Tir.
        Mol. Microbiol. 2005; 56: 416-432
        • Campellone K.G.
        • Webb N.J.
        • Znameroski E.A.
        • Welch M.D.
        WHAMM is an Arp2/3 complex activator that binds microtubules and functions in ER to Golgi transport.
        Cell. 2008; 134: 148-161
        • Rohatgi R.
        • Nollau P.
        • Ho H.Y.
        • Kirschner M.W.
        • Mayer B.J.
        Nck and phosphatidylinositol 4,5-bisphosphate synergistically activate actin polymerization through the N-WASP-Arp2/3 pathway.
        J. Biol. Chem. 2001; 276: 26448-26452
        • Vingadassalom D.
        • Campellone K.G.
        • Brady M.J.
        • Skehan B.
        • Battle S.E.
        • Robbins D.
        • Kapoor A.
        • Hecht G.
        • Snapper S.B.
        • Leong J.M.
        PLoS Pathog. 2010; 6: e1001056
        • Cameron L.A.
        • Footer M.J.
        • van Oudenaarden A.
        • Theriot J.A.
        Motility of ActA protein-coated microspheres driven by actin polymerization.
        Proc. Natl. Acad. Sci. U.S.A. 1999; 96: 4908-4913
        • Goldberg M.B.
        • Theriot J.A.
        Shigella flexneri surface protein IcsA is sufficient to direct actin-based motility.
        Proc. Natl. Acad. Sci. U.S.A. 1995; 92: 6572-6576
        • Loisel T.P.
        • Boujemaa R.
        • Pantaloni D.
        • Carlier M.F.
        Reconstitution of actin-based motility of Listeria and Shigella using pure proteins.
        Nature. 1999; 401: 613-616
        • Alto N.M.
        • Weflen A.W.
        • Rardin M.J.
        • Yarar D.
        • Lazar C.S.
        • Tonikian R.
        • Koller A.
        • Taylor S.S.
        • Boone C.
        • Sidhu S.S.
        • Schmid S.L.
        • Hecht G.A.
        • Dixon J.E.
        The type III effector EspF coordinates membrane trafficking by the spatiotemporal activation of two eukaryotic signaling pathways.
        J. Cell Biol. 2007; 178: 1265-1278
        • Aitio O.
        • Hellman M.
        • Kazlauskas A.
        • Vingadassalom D.F.
        • Leong J.M.
        • Saksela K.
        • Permi P.
        Recognition of tandem PXXP motifs as a unique Src homology 3-binding mode triggers pathogen-driven actin assembly.
        Proc. Natl. Acad. Sci. U.S.A. 2010; 107: 21743-21748
        • Carlier M.F.
        • Nioche P.
        • Broutin-L'Hermite I.
        • Boujemaa R.
        • Le Clainche C.
        • Egile C.
        • Garbay C.
        • Ducruix A.
        • Sansonetti P.
        • Pantaloni D.
        GRB2 links signaling to actin assembly by enhancing interaction of neural Wiskott-Aldrich syndrome protein (N-WASp) with actin-related protein (ARP2/3) complex.
        J. Biol. Chem. 2000; 275: 21946-21952
        • Garmendia J.
        • Ren Z.
        • Tennant S.
        • Midolli Viera M.A.
        • Chong Y.
        • Whale A.
        • Azzopardi K.
        • Dahan S.
        • Sircili M.P.
        • Franzolin M.R.
        • Trabulsi L.R.
        • Phillips A.
        • Gomes T.A.
        • Xu J.
        • Robins-Browne R.
        • Frankel G.
        Distribution of tccP in clinical enterohemorrhagic and enteropathogenic Escherichia coli isolates.
        J. Clin. Microbiol. 2005; 43: 5715-5720
        • Cantarelli V.V.
        • Kodama T.
        • Nijstad N.
        • Abolghait S.K.
        • Nada S.
        • Okada M.
        • Iida T.
        • Honda T.
        Tyrosine phosphorylation controls cortactin binding to two enterohaemorrhagic Escherichia coli effectors: Tir and EspFu/TccP.
        Cell Microbiol. 2007; 9: 1782-1795
        • Saarikangas J.
        • Zhao H.
        • Pykäläinen A.
        • Laurinmäki P.
        • Mattila P.K.
        • Kinnunen P.K.
        • Butcher S.J.
        • Lappalainen P.
        Molecular mechanisms of membrane deformation by I-BAR domain proteins.
        Curr. Biol. 2009; 19: 95-107