Kenneth Geno Campellone

A nucleator arms race: cellular control of actin assembly

For over a decade, the actin-related protein 2/3 (ARP2/3) complex, a handful of nucleation-promoting factors and formins were the only molecules known to directly nucleate actin filament formation de novo. However, the past several years have seen a surge in the discovery of mammalian proteins with roles in actin nucleation and dynamics. Newly recognized nucleation-promoting factors, such as WASP and SCAR homologue (WASH), WASP homologue associated with actin, membranes and microtubules (WHAMM), and junction-mediating regulatory protein (JMY), stimulate ARP2/3 activity at distinct cellular locations. Formin nucleators with additional biochemical and cellular activities have also been uncovered. Finally, the Spire, cordon-bleu and leiomodin nucleators have revealed new ways of overcoming the kinetic barriers to actin polymerization.

Lambda Red-mediated recombinogenic engineering of enterohemorrhagic and enteropathogenic E. coli

BackgroundThe λ Red recombineering technology has been used extensively in Escherichia coli and Salmonella typhimurium for easy PCR-mediated generation of deletion mutants, but less so in pathogenic species of E. coli such as EHEC and EPEC. Our early experiments with the use of λ Red in EHEC and EPEC have led to sporadic results, leading to the present study to identify factors that might improve the efficiency of Red recombineering in these pathogenic strains of E. coli.ResultsIn this report, we have identified conditions that optimize the use of λ Red for recombineering in EHEC and EPEC. Using plasmids that contain a Ptac-red-gam operon and a temperature-sensitive origin of replication, we have generated multiple mutations (both marked and unmarked) in known virulence genes. In addition, we have easily deleted five O157-specific islands (O-islands) of EHEC suspected of containing virulence factors. We have examined the use of both PCR-generated substrates (40 bp of flanking homology) and plasmid-derived substrates (~1 kb of flanking homology); both work well and each have their own advantages. The establishment of the hyper-rec phenotype requires only a 20 minute IPTG induction period of red and gam. This recombinogenic window is important as constitutive expression of red and gam induces a 10-fold increase in spontaneous resistance to rifampicin. Other factors such as the orientation of the drug marker in recombination substrates and heat shock effects also play roles in the success of Red-mediated recombination in EHEC and EPEC.ConclusionsThe λ Red recombineering technology has been optimized for use in pathogenic species of E. coli, namely EHEC and EPEC. As demonstration of this technology, five O-islands of EHEC were easily and precisely deleted from the chromosome by electroporation with PCR-generated substrates containing drug markers flanked with 40 bp of target DNA. These results should encourage the use of λ Red recombineering in these and other strains of pathogenic bacteria for faster identification of virulence factors and the speedy generation of bacterial mutants for vaccine development.

PCR-mediated gene replacement in Escherichia coli.

The hyper-recombinogenic properties of an E. coli strain in which the recBCD genes have been replaced by lambda red recombination functions were exploited in the development of a general PCR-mediated gene replacement scheme for Escherichia coli. Linear DNA substrates generated by recombinant PCR are introduced by electroporation into strains containing the recBCDDelta::red substitution. This technique allows for gene replacement in E. coli without prior cloning of the gene of interest. In addition, the counter-selectable marker sacB has been used to construct unmarked precise gene deletions without the need to form sacB-containing plasmid integrates. In other experiments, electroporation of recBCDDelta::red strains with high concentrations of linear DNA fragments (derived from plasmid digests) gave linear transformation rates approaching 1% of the survivors of electroporation. The placement of lambda red and gam at a locus in the chromosome other than recBCD (galK) resulted in a strain that was as hyper-rec as one containing the lambda red for recBCD substitution. The gene replacement technique described here has been used for the construction of deletion-substitution alleles of lacZ and sulA, as well as six genes important for general homologous recombination in E. coli. Three of these replacements were performed without prior cloning of the genes.

WHAMM Is an Arp2/3 Complex Activator That Binds Microtubules and Functions in ER to Golgi Transport

The Arp2/3 complex is an actin nucleator that plays a critical role in many cellular processes. Its activities are regulated by nucleation-promoting factors (NPFs) that function primarily during plasma membrane dynamics. Here we identify a mammalian NPF called WHAMM (WASP homolog associated with actin, membranes, and microtubules) that localizes to the cis-Golgi apparatus and tubulo-vesicular membrane transport intermediates. The modular organization of WHAMM includes an N-terminal domain that mediates Golgi membrane association, a coiled-coil region that binds microtubules, and a WCA segment that stimulates Arp2/3-mediated actin polymerization. Overexpression and depletion studies indicate that WHAMM is important for maintaining Golgi structure and facilitating anterograde membrane transport. The ability of WHAMM to interact with microtubules plays a role in membrane tubulation, while its capacity to induce actin assembly promotes tubule elongation. Thus, WHAMM is an important regulator of membrane dynamics functioning at the interface of the microtubule and actin cytoskeletons.

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.

WASH, WHAMM and JMY: regulation of Arp2/3 complex and beyond

Arp2/3 complex mediates the nucleation of actin filaments in multiple subcellular processes, and is activated by nucleation-promoting factors (NPFs) from the Wiskott-Aldrich Syndrome family. In exciting new developments, this family has grown by three members: WASH, WHAMM and JMY, which extend the repertoire of dynamic membrane structures that are remodeled following Arp2/3 activation in vivo. These novel NPFs share an actin- and Arp2/3-interacting WCA module, and combine Arp2/3 activation with additional biochemical functions, including capping protein inhibition, microtubule engagement or Arp2/3-independent actin nucleation, none of which had been previously associated with canonical WCA-harboring proteins. Uncovering the physiological relevance of these unique activities will require concerted efforts from multiple disciplines, and is sure to impact our understanding of how the cytoskeleton controls so many dynamic subcellular events.

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.

Nck‐independent actin assembly is mediated by two phosphorylated tyrosines within enteropathogenic Escherichia coli Tir

Enteropathogenic Escherichia coli (EPEC) stimulates tyrosine‐kinase signalling cascades to trigger localized actin assembly within mammalian cells. During actin ‘pedestal’ formation, the EPEC effector protein Tir is translocated into the plasma membrane, becomes phosphorylated on tyrosine‐474 (Y474) and promotes recruitment of the mammalian adaptor protein Nck to efficiently activate N‐WASP‐Arp2/3‐mediated actin polymerization. Tir also triggers localized actin assembly in the absence of Nck, but the Tir sequences involved in this signalling cascade have not been defined. To identify and characterize the phosphotyrosines that contribute to Nck‐independent pedestal formation, we investigated the regulation of Tir tyrosine phosphorylation and found that phosphorylation is stimulated by Tir clustering. In addition to Y474, residue Y454 is also phosphorylated, although at lower efficiency. These tyrosines differentially contribute to actin polymerization in a fashion reminiscent of actin ‘tail’ formation mediated by the vaccinia virus envelope protein A36R, which utilizes two similarly spaced phosphotyrosines to recruit the adaptors Nck and Grb2, respectively, in order to stimulate N‐WASP. Neither phosphorylated Y454 nor Y474 directly bind Grb2, but Tir derivatives harbouring these residues ultimately recruit N‐WASP and Arp2/3 independently of Nck, suggesting that EPEC exploits additional phosphotyrosine‐binding adaptors capable of initiating actin assembly.

Structural mechanism of WASP activation by the enterohaemorrhagic E. coli effector EspF(U).

During infection, enterohaemorrhagic Escherichia coli (EHEC) takes over the actin cytoskeleton of eukaryotic cells by injecting the EspFU protein into the host cytoplasm. EspFU controls actin by activating members of the Wiskott–Aldrich syndrome protein (WASP) family. Here we show that EspFU binds to the autoinhibitory GTPase binding domain (GBD) in WASP proteins and displaces it from the activity-bearing VCA domain (for verprolin homology, central hydrophobic and acidic regions). This interaction potently activates WASP and neural (N)-WASP in vitro and induces localized actin assembly in cells. In the solution structure of the GBD–EspFU complex, EspFU forms an amphipathic helix that binds the GBD, mimicking interactions of the VCA domain in autoinhibited WASP. Thus, EspFU activates WASP by competing directly for the VCA binding site on the GBD. This mechanism is distinct from that used by the eukaryotic activators Cdc42 and SH2 domains, which globally destabilize the GBD fold to release the VCA. Such diversity of mechanism in WASP proteins is distinct from other multimodular systems, and may result from the intrinsically unstructured nature of the isolated GBD and VCA elements. The structural incompatibility of the GBD complexes with EspFU and Cdc42/SH2, plus high-affinity EspFU binding, enable EHEC to hijack the eukaryotic cytoskeletal machinery effectively.