Hierarchical delivery of an essential host colonization factor in enteropathogenic Escherichia coli

Many significant bacterial pathogens use a type III secretion system to inject effector proteins into host cells to disrupt specific cellular functions, enabling disease progression. The injection of these effectors into host cells is often dependent on dedicated chaperones within the bacterial cell. In this report, we demonstrate that the enteropathogenic Escherichia coli (EPEC) chaperone CesT interacts with a variety of known and putative type III effector proteins. Using pull-down and secretion assays, a degenerate CesT binding domain was identified within multiple type III effectors. Domain exchange experiments between selected type III effector proteins revealed a modular nature for the CesT binding domain, as demonstrated by secretion, chaperone binding, and infection assays. The CesT-interacting type III effector Tir, which is crucial for in vivo intestinal colonization, had to be expressed and secreted for efficient secretion of other type III effectors. In contrast, the absence of other CesT-interacting type III effectors did not abrogate effector secretion, indicating an unexpected hierarchy with respect to Tir for type III effector delivery. Coordinating the expression of other type III effectors with cesT in the absence of tir partially restored total type III effector secretion, thereby implicating CesT in secretion events. Collectively, the results suggest a coordinated mechanism involving both Tir and CesT for type III effector injection into host cells.     

CesT is a bivalent enteropathogenic Escherichia coli chaperone required for translocation of both Tir and Map

Map is an enteropathogenic Escherichia coli (EPEC) protein that is translocated into eukaryotic cells by a type III secretion system. Although not required for the induction of attaching and effacing (A/E) lesion formation characteristic of EPEC infection, translocated Map is suggested to disrupt mitochondrial membrane potential, which may impact upon subsequent functions of the organelle such as control of cell death. Before secretion, many effector proteins are maintained in the bacterial cytosol by association with a specific chaperone. In EPEC, chaperones have been identified for the effector proteins translocated intimin receptor (Tir) and EspF, and for the translocator proteins EspB and EspD. In this study, we present evidence that the Tir-specific chaperone, CesT, also performs a chaperone function for Map. Using a combination of biochemical approaches, we demonstrate specific interaction between CesT and Map. Similar to other chaperone-effector pairings, binding is apparent at the amino-terminus of Map and is indicated to proceed by a similar mechanism to CesT:Tir interaction. Map secretion from a cesT mutant strain (SE884) is shown to be reduced and, importantly, its translocation from this strain after infection of HEp-2 cells is almost totally abrogated. Although other chaperones are reported to have a bivalent binding specificity, CesT is the first member of its family that chaperones more than one protein for translocation.     

The N-terminus of enteropathogenic Escherichia coli (EPEC) Tir mediates transport across bacterial and eukaryotic cell membranes

Enteropathogenic Escherichia coli (EPEC) uses a type III secretion system to translocate into host cells several effector molecules that are required for virulence. One of these, the translocated intimin receptor, Tir, inserts into the host cell cytoplasmic membrane, where it functions as the receptor for intimin, an outer membrane adhesin expressed by EPEC. A chaperone for Tir, called CesT, is required for stability of Tir in the EPEC cytoplasm. In this study, the cyaA gene reporter system was used to identify domains in Tir that mediate secretion into the culture supernatant and translocation into host cells. A Tir-CyaA fusion containing the first 15 N-terminal residues of Tir was secreted and translocated into HeLa cells by a deltatirdeltacesT mutant; however, maximal secretion and translocation was observed with the first 26 N-terminal residues of Tir. Fusions containing progressively larger N-terminal sequences of Tir were also efficiently secreted and translocated into HeLa cells by the deltatirdeltacesT strain. However, in a deltatir mutant that expresses CesT, Tir26-CyaA and an additional fusion containing the first 69 N-terminal residues of Tir were not secreted or translocated, but fusions containing larger N-terminal Tir sequences were secreted and translocated by the deltatir mutant. Wild-type EPEC secreted and translocated the Tir15-CyaA fusion, whereas longer fusions, such as Tir26-CyaA and Tir69-CyaA, were translocated to higher levels, similar to what was observed with the deltatirdeltacesT mutant. A Tir-CyaA fusion containing the CesT binding domain was translocated into HeLa cells more rapidly in the presence of CesT compared with translocation in the absence of CesT. Collectively, these results suggest that an N-terminal domain of 26 amino acids functions as a CesT-independent signal that is capable of delivering Tir into both the culture supernatant and the cytosol of host cells. Furthermore, in addition to its role in the stability of Tir, CesT may function in translocation by mediating rapid delivery of Tir into host cells.     

Esp-independent functional integration of the translocated intimin receptor (Tir) of enteropathogenic Escherichia coli (EPEC) into host cell membranes

The pathogenesis of enteropathogenic Escherichia coli (EPEC) is characterized by the type III secretion system-dependent exploitation of target cells that results in attaching and effacing (A/E) lesions, actin rearrangements and pedestal formation. This pathology is mediated by effector proteins which are translocated by the type III secretion system into the host cell such as the translocated intimin receptor (Tir) and several E. coli secreted proteins (Esp). Secretion of virulence proteins of EPEC is tightly regulated. In response to Ca(2+), Esp secretion is drastically reduced, whereas secretion of Tir is increased. Membrane insertion of Tir, secreted under low Ca(2+) conditions, is therefore independent of Esp. Furthermore, espB and espD mutant strains of EPEC, unable to form the translocation pore, still translocate Tir into host cells membranes. This autointegrated Tir is functional, as it is able to complement a tir mutant strain in recruiting actin to bacterial contact sites. The uptake of Tir into the host cell appears to depend on the C-terminal part of the protein, as deletion of this part of Tir prevents autointegration. Together, our results demonstrate that under conditions of limited Ca(2+) an alternative mechanism for Tir integration can trigger the induction of A/E lesions.     

CesT is a multi-effector chaperone and recruitment factor required for the efficient type III secretion of both LEE- and non-LEE-encoded effectors of enteropathogenic Escherichia coli

Enteropathogenic Escherichia coli (EPEC) is an intestinal attaching and effacing pathogen that utilizes a type III secretion system (T3SS) for the delivery of effectors into host cells. The chaperone CesT has been shown to bind and stabilize the type III translocated effectors Tir and Map in the bacterial cytoplasm prior to their delivery into host cells. In this study we demonstrate a role for CesT in effector recruitment to the membrane embedded T3SS. CesT-mediated effector recruitment was dependent on the presence of the T3SS membrane-associated ATPase EscN. EPEC DeltacesT carrying a C-terminal CesT variant, CesT(E142G), exhibited normal cytoplasmic Tir stability function, but was less efficient in secreting Tir, further implicating CesT in type III secretion. In vivo co-immunoprecipitation studies using CesT-FLAG containing EPEC lysates demonstrated that CesT interacts with Tir and EscN, consistent with the notion of CesT recruiting Tir to the T3SS. CesT was also shown to be required for the efficient secretion of several type III effectors encoded within and outside the locus of enterocyte effacement (LEE) in addition to Tir and Map. Furthermore, a CesT affinity column was shown to specifically retain multiple effector proteins from EPEC culture supernatants. These findings indicate that CesT is centrally involved in recruiting multiple type III effectors to the T3SS via EscN for efficient secretion, and functionally redefine the role of CesT in multiple type III effector interactions.     

Identification of CesT, a chaperone for the type III secretion of Tir in enteropathogenic Escherichia coli

The locus of enterocyte effacement of enteropathogenic Escherichia coli encodes a type III secretion system, an outer membrane protein adhesin (intimin, the product of eae ) and Tir, a translocated protein that becomes a host cell receptor for intimin. Many type III secreted proteins require chaperones, which function to stabilize proteins, prevent inappropriate protein-protein interactions and aid in secretion. An open reading frame located between tir and eae, previously named orfU, was predicted to encode a protein with partial similarity to the Yersinia SycH chaperone. We examined the potential of the orfU gene product to serve as a chaperone for Tir. The orfU gene encoded a 15 kDa cytoplasmic protein that specifically interacted with Tir as demonstrated by the yeast two-hybrid assay, column binding and coimmunoprecipitation experiments. An orfU mutant was defective in attaching-effacing lesion formation and Tir secretion, but was unaffected in expression of other virulence factors. OrfU appeared to stabilize Tir levels in the cytoplasm, but was not absolutely necessary for secretion of Tir. Based upon the physical similarities, phenotypic characteristics and the demonstrated interaction with Tir, orfU is redesignated as cesT for the chaperone for E. coli secretion of T ir.     

Enteropathogenic E. coli Tir binds Nck to initiate actin pedestal formation in host cells

Enteropathogenic Escherichia coli (EPEC) is a bacterial pathogen that causes infantile diarrhea worldwide. EPEC injects a bacterial protein, translocated intimin receptor (Tir), into the host-cell plasma membrane where it acts as a receptor for the bacterial outer membrane protein, intimin. The interaction of Tir and intimin triggers a marked rearrangement of the host actin cytoskeleton into pedestals beneath adherent bacteria. On delivery into host cells, EPEC Tir is phosphorylated on tyrosine 474 of the intracellular carboxy-terminal domain, an event that is required for pedestal formation. Despite its essential role, the function of Tir tyrosine phosphorylation has not yet been elucidated. Here we show that tyrosine 474 of Tir directly binds the host-cell adaptor protein Nck, and that Nck is required for the recruitment of both neural Wiskott-Aldrich-syndrome protein (N-WASP) and the actin-related protein (Arp)2/3 complex to the EPEC pedestal, directly linking Tir to the cytoskeleton. Cells with null alleles of both mammalian Nck genes are resistant to the effects of EPEC on the actin cytoskeleton. These results implicate Nck adaptors as host-cell determinants of EPEC virulence.     

Functional analysis of the enteropathogenic Escherichia coli type III secretion system chaperone CesT identifies domains that mediate substrate interactions

In many Gram-negative bacteria, a key indicator of pathogenic potential is the possession of a specialized type III secretion system, which is utilized to deliver virulence effector proteins directly into the host cell cytosol. Many of the proteins secreted from such systems require small cytosolic chaperones to maintain the secreted substrates in a secretion-competent state. One such protein, CesT, serves a chaperone function for the enteropathogenic Escherichia coli (EPEC) translocated intimin receptor (Tir) protein, which confers upon EPEC the ability to alter host cell morphology following intimate bacterial attachment. Using a combination of complementary biochemical approaches, functional domains of CesT that mediate intermolecular interactions, involved in both chaperone-chaperone and chaperone-substrate associations, were determined. The CesT N-terminal is implicated in chaperone dimerization, whereas the amphipathic alpha-helical region of the C-terminal, is intimately involved in substrate binding. By functional complementation of chaperone domains using the Salmonella SicA chaperone to generate chaperone chimeras, we show that CesT-Tir interaction proceeds by a mechanism potentially common to other type III secretion system chaperones.     

Identification of the intimin-binding domain of Tir of enteropathogenic Escherichia coli

Enteropathogenic Escherichia coli (EPEC) attaches intimately to mammalian cells via a bacterial outer membrane adhesion molecule, intimin, and its receptor in the host cell membrane, Tir. Tir is a bacterial protein translocated into the host cell membrane and tyrosine phosphorylated after insertion. Tir–intimin binding induces organized actin polymerization beneath the adherent bacteria, resulting in the formation of pedestal-like structures. A series of Tir deletion derivatives were constructed to analyse which Tir domains are involved in intimin binding. We have localized the intimin-binding domain (IBD) of Tir using a yeast two-hybrid system and a gel-overlay approach to a region of 109 amino acids that is predicted to be exposed on the surface of the plasma membrane. A truncated Tir protein lacking this domain was translocated to the host cell membrane and tyrosine phosphorylated, but failed to bind intimin or to induce either actin polymerization or Tir accumulation beneath the bacteria. These results indicate that only a small region of Tir is needed to bind intimin and support the predicted topology for Tir, with both N- and C-terminal regions in the mammalian cell cytosol. They also confirm that Tir–intimin interactions are needed for cytoskeletal organization. We have also identified N-terminal regions involved in Tir stability and Tir secretion to the media.     

Binding of intimin from enteropathogenic Escherichia coli to Tir and to host cells

Enteropathogenic Escherichia coli (EPEC) induce characteristic attaching and effacing (A/E) lesions on epithelial cells. This event is mediated, in part, by binding of the bacterial outer membrane protein, intimin, to a second EPEC protein, Tir (translocated intimin receptor), which is exported by the bacteria and integrated into the host cell plasma membrane. In this study, we have localized the intimin-binding domain of Tir to a central 107-amino-acid region, designated Tir-M. We provide evidence that both the amino- and carboxy-termini of Tir are located within the host cell. In addition, using immunogold labelling electron microscopy, we have confirmed that intimin can bind independently to host cells even in the absence of Tir. This Tir-independent interaction and the ability of EPEC to induce A/E lesions requires an intact lectin-like module residing at the carboxy-terminus of the intimin polypeptide. Using the yeast two-hybrid system and gel overlays, we show that intimin can bind both Tir and Tir-M even when the lectin-like domain is disrupted. These data provide strong evidence that intimin interacts not only with Tir but also in a lectin-like manner with a host cell intimin receptor.     

Tandem tyrosine phosphosites in the Enteropathogenic Escherichia coli chaperone CesT are required for differential type III effector translocation and virulence

Enteropathogenic Escherichia coli (EPEC) use a type 3 secretion system (T3SS) for injection of effectors into host cells and intestinal colonization. Here, we demonstrate that the multicargo chaperone CesT has two strictly conserved tyrosine phosphosites, Y152 and Y153 that regulate differential effector secretion in EPEC. Conservative substitution of both tyrosine residues to phenylalanine strongly attenuated EPEC type 3 effector injection into host cells, and limited Tir effector mediated intimate adherence during infection. EPEC expressing a CesT Y152F variant were deficient for NleA effector expression and exhibited significantly reduced translocation of NleA into host cells during infection. Other effectors were observed to be dependent on CesT Y152 for maximal translocation efficiency. Unexpectedly, EPEC expressing a CesT Y153F variant exhibited significantly enhanced effector translocation of many CesT‐interacting effectors, further implicating phosphosites Y152 and Y153 in CesT functionality. A mouse infection model of intestinal disease using Citrobacter rodentium revealed that CesT tyrosine substitution variants displayed delayed colonization and were more rapidly cleared from the intestine. These data demonstrate genetically separable functions for tandem tyrosine phosphosites within CesT. Therefore, CesT via its C‐terminal tyrosine phosphosites, has relevant roles beyond typical type III secretion chaperones that interact and stabilize effector proteins.     

Translocated intimin receptor and its chaperone interact with ATPase of the type III secretion apparatus of enteropathogenic Escherichia coli

Few interactions have been reported between effectors and components of the type III secretion apparatus, although many interactions have been demonstrated between type III effectors and their cognate chaperones. It is thought that chaperones may play a role in directing effectors to the type III secretion apparatus. The ATPase FliI in the flagellar assembly apparatus plays a pivotal role in interacting with other components of the apparatus and with substrates of the flagellar system. We performed experiments to determine if there were any interactions between the effector Tir and its chaperone CesT and the type III secretion apparatus of enteropathogenic Escherichia coli (EPEC). Specifically, based on analogies with the flagella system, we examined Tir-CesT interactions with the putative ATPase EscN. We showed by affinity chromatography that EscN and Tir bind CesT specifically. Tir is not necessary for CesT and EscN interactions, and EscN binds Tir specifically without its chaperone CesT. Moreover, Tir directly binds EscN, as shown via gel overlay and enzyme-linked immunosorbent assay, and coimmunoprecipitation experiments revealed that Tir interacts with EscN inside EPEC. These data provide evidence for direct interactions between a chaperone, effector, and type III component in the pathogenic type III secretion system and suggest a model for Tir translocation whereby its chaperone, CesT, brings Tir to the type III secretion apparatus by specifically interacting with the type III ATPase EscN.     

SepL, a protein required for enteropathogenic Escherichia coli type III translocation, interacts with secretion component SepD

Enteropathogenic Escherichia coli (EPEC), an important cause of infantile diarrhoea in the developing world, disrupts host cell microvilli, causes actin rearrangements and attaches intimately to the host cell surface. This characteristic phenotype, referred to as the attaching and effacing (A/E) effect, is encoded on a 36 kb pathogenicity island called the locus of enterocyte effacement (LEE). The LEE includes genes involved in type III secretion and translocation, the eae gene encoding an outer membrane adhesin known as intimin, the tir gene for the translocated intimin receptor, a regulator and various genes of unknown function. Among this last group is sepL. To determine the role of SepL in EPEC pathogenesis, we constructed and tested a non-polar sepL mutant. We found that this sepL mutant is deficient for A/E and that it secretes markedly reduced quantities of those proteins involved in translocation (EspA, EspB and EspD), but normal levels of those proteins presumed to be effectors (Tir, EspF and EspG). Despite normal levels of secretion, the mutant strain was unable to translocate EspF and Tir into host cells and formed no EspA filaments. Fractionation studies revealed that SepL is a soluble cytoplasmic protein. Yeast two-hybrid and affinity purification studies indicated that SepL interacts with the LEE-encoded protein SepD. In contrast to SepL, we found that SepD is required for type III secretion of both translocation and effector proteins. Together, these results demonstrate that SepL has a unique role in type III secretion as a functional component of the translocation system that interacts with an essential element of the secretion machinery.     

Tails of two Tirs: actin pedestal formation by enteropathogenic E. coli and enterohemorrhagic E. coli O157:H7

Enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli O157:H7 (EHEC) form characteristic lesions on infected mammalian cells called actin pedestals. Each of these two pathogens injects its own translocated intimin receptor (Tir) molecule into the plasma membranes of host cells. Interaction of translocated Tir with the bacterial outer membrane protein intimin is required to trigger the assembly of actin into focused pedestals beneath bound bacteria. Despite similarities between the Tir molecules and the host components that associate with pedestals, recent work indicates that EPEC and EHEC Tir are not functionally interchangeable. For EPEC, Tir-mediated binding of Nck, a host adaptor protein implicated in actin signaling, is both necessary and sufficient to initiate actin assembly. In contrast, for EHEC, pedestals are formed independently of Nck, and require translocation of bacterial factors in addition to Tir to trigger actin signaling.     

A tyrosine-phosphorylated 12-amino-acid sequence of enteropathogenic Escherichia coli Tir binds the host adaptor protein Nck and is required for Nck localization to actin pedestals

Enteropathogenic Escherichia coli (EPEC) and enterohaemorrhagic E. coli (EHEC) each promote the reorganization of actin into filamentous pedestal structures beneath attached bacteria during colonization of the intestinal epithelium. Central to this process is the translocation of the protein Tir (translocated intimin receptor) into the plasma membrane of host cells, where it interacts with the bacterial outer membrane protein intimin and triggers cellular signalling events that lead to actin rearrangement. Actin signalling by EPEC Tir requires a tyrosine residue, Y474, which is phosphorylated in the host cell. In contrast, EHEC Tir lacks this residue and generates pedestals independently of tyrosine phosphorylation. Consistent with this difference, recent work indicates that EHEC Tir cannot functionally replace EPEC Tir. To identify the role that tyrosine phosphorylation of EPEC Tir plays in actin signalling, we generated chimeric EHEC/EPEC Tir proteins and identified a 12-residue sequence of EPEC Tir containing Y474 that confers actin-signalling capabilities to EHEC Tir when the chimera is expressed in EPEC. Nck, a mammalian adaptor protein that has been implicated in the initiation of actin signalling, binds to this sequence in a Y474 phosphorylation-dependent manner and is recruited to the pedestals of EPEC, but not of EHEC.     

Binding of intimin with Tir on the bacterial surface is prerequisite for the barrier disruption induced by enteropathogenic Escherichia coli

Enteropathogenic Escherichia coli (EPEC) infects intestinal epithelial cells and perturbs the intestinal barrier that limits the paracellular movement of molecules. The disruption of the barrier is mediated by the effectors translocated into the host cells through the bacterial type III secretion system (TTSS). A previous report has described the importance of a bacterial outer membrane protein, intimin, in EPEC-mediated disruption of the barrier, and proposed that intimin, in concert with a host intimin receptor, controls the activity of the translocated barrier-disrupting effectors [P. Dean, B. Kenny, Intestinal barrier dysfunction by enteropathogenic Escherichia coli is mediated by two effector molecules and a bacterial surface protein, Mol. Microbiol. 54 (2004) 665-675]. In this study, we found that the importance of intimin is in its ability to bind a bacterial intimin receptor, Tir. Additionally, the impaired ability of an intimin-negative mutant was not restored by co-infection with intimin-expressing TTSS mutants. Collectively, the results in this study favor an alternative scenario explaining the importance of intimin, that the binding of intimin with Tir on the bacterial surface triggers or promotes the translocation of factors required for the efficient disruption of the barrier. Thus, the interaction of intimin with Tir may serve as a molecular switch that controls the delivery of virulence factors into the host cells.     

Enteropathogenic E. coli translocated intimin receptor, Tir, interacts directly with alpha-actinin

Enteropathogenic Escherichia coli (EPEC) triggers a dramatic rearrangement of the host epithelial cell actin cytoskeleton to form an attaching and effacing lesion, or pedestal. The pathogen remains attached extracellularly to the host cell through the pedestal for the duration of the infection. At the tip of the pedestal is a bacterial protein, Tir, which is secreted from the bacterium into the host cell plasma membrane, where it functions as the receptor for an EPEC outer membrane protein, intimin [1]. Delivery of Tir to the host cell results in its tyrosine phosphorylation, followed by Tir-intimin binding. Tir is believed to anchor EPEC firmly to the host cell, although its direct linkage to the cytoskeleton is unknown. Here, we show that Tir directly binds the cytoskeletal protein alpha-actinin. alpha-Actinin is recruited to the pedestal in a Tir-dependent manner and colocalizes with Tir in infected host cells. Binding is mediated through the amino terminus of Tir. Recruitment of alpha-actinin occurs independently of Tir tyrosine phosphorylation. Recruitment of actin, VASP, and N-WASP, however, is abolished in the absence of this tyrosine phosphorylation. These results suggest that Tir plays at least three roles in the host cell during infection: binding intimin on EPEC; mediating a stable anchor with alpha-actinin through its amino terminus in a phosphotyrosine-independent manner; and recruiting additional cytoskeletal proteins at the carboxyl terminus in a phosphotyrosine-dependent manner. These findings demonstrate the first known direct linkage between extracellular EPEC, through the transmembrane protein Tir, to the host cell actin cytoskeleton via alpha-actinin.     

Co-ordinate regulation of distinct host cell signalling pathways by multifunctional enteropathogenic Escherichia coli effector molecules

Enteropathogenic Escherichia coli (EPEC) is a major cause of paediatric diarrhoea and a model for the family of attaching and effacing (A/E) pathogens. A/E pathogens encode a type III secretion system to transfer effector proteins into host cells. The EPEC Tir effector protein acts as a receptor for the bacterial surface protein intimin and is involved in the formation of Cdc42-independent, actin-rich pedestal structures beneath the adhered bacteria. In this paper, we demonstrate that EPEC binding to HeLa cells also induces Tir-independent, cytoskeletal rearrangement evidenced by the early, transient formation of filopodia-like structures at sites of infection. Filopodia formation is dependent on expression of the EPEC Map effector molecule - a protein that targets mitochondria and induces their dysfunction. We show that Map-induced filopodia formation is independent of mitochondrial targeting and is abolished by cellular expression of the Cdc42 inhibitory WASP-CRIB domain, demonstrating that Map has at least two distinct functions in host cells. The transient nature of the filopodia is related to an ability of EPEC to downregulate Map-induced cell signalling that, like pedestal formation, was dependent on both Tir and intimin proteins. The ability of Tir to downregulate filopodia was impaired by disrupting a putative GTPase-activating protein (GAP) motif, suggesting that Tir may possess such a function, with its interaction with intimin triggering this activity. Furthermore, we also found that Map-induced cell signalling inhibits pedestal formation, revealing that the cellular effects of Tir and Map must be co-ordinately regulated during infection. Possible implications of the multifunctional nature of EPEC effector molecules in pathogenesis are discussed.     

Clustering of Nck by a 12-residue Tir phosphopeptide is sufficient to trigger localized actin assembly

Enteropathogenic Escherichia coli (EPEC) translocates effector proteins into mammalian cells to promote reorganization of the cytoskeleton into filamentous actin pedestals. One effector, Tir, is a transmembrane receptor for the bacterial surface adhesin intimin, and intimin binding by the extracellular domain of Tir is required for actin assembly. The cytoplasmic NH2 terminus of Tir interacts with focal adhesion proteins, and its tyrosine-phosphorylated COOH terminus binds Nck, a host adaptor protein critical for pedestal formation. To define the minimal requirements for EPEC-mediated actin assembly, Tir derivatives were expressed in mammalian cells in the absence of all other EPEC components. Replacement of the NH2 terminus of Tir with a viral membrane-targeting sequence promoted efficient surface expression of a COOH-terminal Tir fragment. Artificial clustering of this fusion protein revealed that the COOH terminus of Tir, by itself, is sufficient to initiate a complete signaling cascade leading to pedestal formation. Consistent with this finding, clustering of Nck by a 12-residue Tir phosphopeptide triggered actin tail formation in Xenopus egg extracts.