RESUMEN
The Arp2/3 complex is an actin nucleator with well-characterized activities in cell morphogenesis and movement, but its roles in nuclear processes are relatively understudied. We investigated how the Arp2/3 complex affects genomic integrity and cell cycle progression using mouse fibroblasts containing an inducible knockout (iKO) of the ArpC2 subunit. We show that permanent Arp2/3 complex ablation results in DNA damage, the formation of cytosolic micronuclei, and cellular senescence. Micronuclei arise in ArpC2 iKO cells due to chromatin segregation defects during mitosis and premature mitotic exits. Such phenotypes are explained by the presence of damaged DNA fragments that fail to attach to the mitotic spindle, abnormalities in actin assembly during metaphase, and asymmetric microtubule architecture during anaphase. In the nuclei of Arp2/3-depleted cells, the tumor suppressor p53 is activated and the cell cycle inhibitor Cdkn1a/p21 mediates a G1 arrest. In the cytosol, micronuclei are recognized by the DNA sensor cGAS, which is important for stimulating a STING- and IRF3-associated interferon response. These studies establish functional requirements for the mammalian Arp2/3 complex in mitotic spindle organization and genome stability. They also expand our understanding of the mechanisms leading to senescence and suggest that cytoskeletal dysfunction is an underlying factor in biological aging.
Asunto(s)
Complejo 2-3 Proteico Relacionado con la Actina , Actinas , Animales , Ratones , Complejo 2-3 Proteico Relacionado con la Actina/genética , Complejo 2-3 Proteico Relacionado con la Actina/metabolismo , Actinas/metabolismo , Senescencia Celular/genética , ADN/metabolismo , Inestabilidad Genómica/genética , Mitosis/genéticaRESUMEN
The actin cytoskeleton is a well-known player in most vital cellular processes, but comparably little is understood about how the actin assembly machinery impacts programmed cell death pathways. In the current study, we explored roles for the human Wiskott-Aldrich Syndrome Protein (WASP) family of actin nucleation factors in DNA damage-induced apoptosis. Inactivation of each WASP-family gene revealed that two of them, JMY and WHAMM, are necessary for rapid apoptotic responses. JMY and WHAMM participate in a p53-dependent cell death pathway by enhancing mitochondrial permeabilization, initiator caspase cleavage, and executioner caspase activation. JMY-mediated apoptosis requires actin nucleation via the Arp2/3 complex, and actin filaments are assembled in cytoplasmic territories containing clusters of cytochrome c and active caspase-3. The loss of JMY additionally results in significant changes in gene expression, including upregulation of the WHAMM-interacting G-protein RhoD. Depletion or deletion of RHOD increases cell death, suggesting that RhoD normally contributes to cell survival. These results give rise to a model in which JMY and WHAMM promote intrinsic cell death responses that can be opposed by RhoD.
Asunto(s)
Proteínas de la Membrana/genética , Proteínas Asociadas a Microtúbulos/genética , Proteínas Nucleares/genética , Transactivadores/genética , Proteína p53 Supresora de Tumor/genética , Síndrome de Wiskott-Aldrich/genética , Proteínas de Unión al GTP rho/genética , Citoesqueleto de Actina/genética , Proteína 2 Relacionada con la Actina/genética , Complejo 2-3 Proteico Relacionado con la Actina/genética , Proteína 3 Relacionada con la Actina/genética , Apoptosis/genética , Citocromos c/genética , Daño del ADN/genética , Humanos , Mitocondrias/genética , Mitocondrias/metabolismo , ARN Interferente Pequeño/genética , Proteína del Síndrome de Wiskott-Aldrich/genéticaRESUMEN
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.
Asunto(s)
Complejo 2-3 Proteico Relacionado con la Actina/metabolismo , Retículo Endoplásmico/metabolismo , Aparato de Golgi/metabolismo , Microtúbulos/metabolismo , Actinas/metabolismo , Animales , Células COS , Chlorocebus aethiops , Humanos , Ratones , Células 3T3 NIH , SpodopteraRESUMEN
Cell motility is governed by cooperation between the Arp2/3 complex and nucleation-promoting factors from the Wiskott-Aldrich Syndrome Protein (WASP) family, which together assemble actin filament networks to drive membrane protrusion. Here we identify WHIMP (WAVE Homology In Membrane Protrusions) as a new member of the WASP family. The Whimp gene is encoded on the X chromosome of a subset of mammals, including mice. Murine WHIMP promotes Arp2/3-dependent actin assembly, but is less potent than other nucleation factors. Nevertheless, WHIMP-mediated Arp2/3 activation enhances both plasma membrane ruffling and wound healing migration, whereas WHIMP depletion impairs protrusion and slows motility. WHIMP expression also increases Src-family kinase activity, and WHIMP-induced ruffles contain the additional nucleation-promoting factors WAVE1, WAVE2, and N-WASP, but not JMY or WASH. Perturbing the function of Src-family kinases, WAVE proteins, or Arp2/3 complex inhibits WHIMP-driven ruffling. These results suggest that WHIMP-associated actin assembly plays a direct role in membrane protrusion, but also results in feedback control of tyrosine kinase signaling to modulate the activation of multiple WASP-family members.
Asunto(s)
Citoesqueleto de Actina/metabolismo , Movimiento Celular/fisiología , Extensiones de la Superficie Celular/fisiología , Proteína del Síndrome de Wiskott-Aldrich/metabolismo , Familia-src Quinasas/metabolismo , Complejo 2-3 Proteico Relacionado con la Actina/metabolismo , Actinas/metabolismo , Animales , Línea Celular , Endocitosis/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Células 3T3 NIH , Dominios Proteicos , Transducción de Señal , Familia de Proteínas del Síndrome de Wiskott-Aldrich/metabolismoRESUMEN
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.
Asunto(s)
Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Complejo 2-3 Proteico Relacionado con la Actina/metabolismo , Animales , Humanos , Proteínas Nucleares/fisiología , Transactivadores/fisiología , Familia de Proteínas del Síndrome de Wiskott-Aldrich/fisiologíaRESUMEN
Enteropathogenic and enterohemorrhagic E. coli (EPEC and EHEC) are closely related extracellular pathogens that reorganize host cell actin into "pedestals" beneath the tightly adherent bacteria. This pedestal-forming activity is both a critical step in pathogenesis, and it makes EPEC and EHEC useful models for studying the actin rearrangements that underlie membrane protrusions. To generate pedestals, EPEC relies on the tyrosine phosphorylated bacterial effector protein Tir to bind host adaptor proteins that recruit N-WASP, a nucleation-promoting factor that activates the Arp2/3 complex to drive actin polymerization. In contrast, EHEC depends on the effector EspFU to multimerize N-WASP and promote Arp2/3 activation. Although these core pathways of pedestal assembly are well-characterized, the contributions of additional actin nucleation factors are unknown. We investigated potential cooperation between the Arp2/3 complex and other classes of nucleators using chemical inhibitors, siRNAs, and knockout cell lines. We found that inhibition of formins impairs actin pedestal assembly, motility, and cellular colonization for bacteria using the EPEC, but not the EHEC, pathway of actin polymerization. We also identified mDia1 as the formin contributing to EPEC pedestal assembly, as its expression level positively correlates with the efficiency of pedestal formation, and it localizes to the base of pedestals both during their initiation and once they have reached steady state. Collectively, our data suggest that mDia1 enhances EPEC pedestal biogenesis and maintenance by generating seed filaments to be used by the N-WASP-Arp2/3-dependent actin nucleation machinery and by sustaining Src-mediated phosphorylation of Tir.
Asunto(s)
Complejo 2-3 Proteico Relacionado con la Actina/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Escherichia coli Enteropatógena/metabolismo , Infecciones por Escherichia coli/metabolismo , Interacciones Huésped-Patógeno/fisiología , Citoesqueleto de Actina , Células CACO-2 , Escherichia coli Enteropatógena/patogenicidad , Proteínas de Escherichia coli/metabolismo , Forminas , Células HeLa , Humanos , Proteína Neuronal del Síndrome de Wiskott-Aldrich/metabolismoRESUMEN
Enteropathogenic and enterohemorrhagic Escherichia coli (EPEC and EHEC) are closely-related pathogens that attach tightly to intestinal epithelial cells, efface microvilli, and promote cytoskeletal rearrangements into protrusions called actin pedestals. To trigger pedestal formation, EPEC employs the tyrosine phosphorylated transmembrane receptor Tir, while EHEC relies on the multivalent scaffolding protein EspFU. The ability to generate these structures correlates with bacterial colonization in several animal models, but the precise function of pedestals in infection remains unclear. To address this uncertainty, we characterized the colonization properties of EPEC and EHEC during infection of polarized epithelial cells. We found that EPEC and EHEC both formed distinct bacterial communities, or "macrocolonies," that encompassed multiple host cells. Tir and EspFU, as well as the host Arp2/3 complex, were all critical for the expansion of macrocolonies over time. Unexpectedly, EspFU accelerated the formation of larger macrocolonies compared to EPEC Tir, as EspFU-mediated actin assembly drove faster bacterial motility to cell junctions, where bacteria formed a secondary pedestal on a neighboring cell and divided, allowing one of the daughters to disengage and infect the second cell. Collectively, these data reveal that EspFU enhances epithelial colonization by increasing actin-based motility and promoting an efficient method of cell-to-cell transmission.
Asunto(s)
Escherichia coli Enterohemorrágica/patogenicidad , Infecciones por Escherichia coli/microbiología , Actinas/metabolismo , Células CACO-2 , Proteínas Portadoras/metabolismo , Quimiotaxis/fisiología , Citoesqueleto/metabolismo , Escherichia coli Enterohemorrágica/metabolismo , Infecciones por Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Células HeLa , Humanos , Péptidos y Proteínas de Señalización Intracelular , Microscopía Electrónica de Transmisión , Microscopía FluorescenteRESUMEN
During infection, enterohaemorrhagic Escherichia coli (EHEC) takes over the actin cytoskeleton of eukaryotic cells by injecting the EspF(U) protein into the host cytoplasm. EspF(U) controls actin by activating members of the Wiskott-Aldrich syndrome protein (WASP) family. Here we show that EspF(U) 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-EspF(U) complex, EspF(U) forms an amphipathic helix that binds the GBD, mimicking interactions of the VCA domain in autoinhibited WASP. Thus, EspF(U) 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 EspF(U) and Cdc42/SH2, plus high-affinity EspF(U) binding, enable EHEC to hijack the eukaryotic cytoskeletal machinery effectively.
Asunto(s)
Proteínas Portadoras/metabolismo , Escherichia coli Enterohemorrágica/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteína del Síndrome de Wiskott-Aldrich/metabolismo , Actinas/metabolismo , Secuencia de Aminoácidos , Animales , Proteínas Portadoras/química , Células Cultivadas , Escherichia coli Enterohemorrágica/química , Escherichia coli Enterohemorrágica/genética , Proteínas de Escherichia coli/química , Fibroblastos/citología , Péptidos y Proteínas de Señalización Intracelular , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , Proteína del Síndrome de Wiskott-Aldrich/química , Proteína Neuronal del Síndrome de Wiskott-Aldrich/química , Proteína Neuronal del Síndrome de Wiskott-Aldrich/metabolismoRESUMEN
Autophagy is an intracellular degradation process that maintains homeostasis, responds to stress, and plays key roles in the prevention of aging and disease. Autophagosome biogenesis, vesicle rocketing, and autolysosome tubulation are controlled by multiple actin nucleation factors, but the impact of actin assembly on completion of the autophagic pathway is not well understood. Here we studied autophagosome and lysosome remodeling in fibroblasts harboring an inducible knockout (iKO) of the Arp2/3 complex, an essential actin nucleator. Arp2/3 complex ablation resulted in increased basal levels of autophagy receptors and lipidated membrane proteins from the LC3 and GABARAP families. Under both steady-state and starvation conditions, Arp2/3 iKO cells accumulated abnormally high numbers of autolysosomes, suggesting a defect in autophagic flux. The inability of Arp2/3 complex-deficient cells to complete autolysosome degradation and turnover is explained by the presence of damaged, leaky lysosomes. In cells treated with an acute lysosomal membrane-damaging agent, the Arp2/3-activating protein WHAMM is recruited to lysosomes, where Arp2/3 complex-dependent actin assembly is crucial for restoring intact lysosomal structure. These results establish the Arp2/3 complex as a central player late in the canonical autophagy pathway and reveal a new role for the actin nucleation machinery in maintaining lysosomal integrity.
RESUMEN
The actin cytoskeleton is essential for many functions of eukaryotic cells, but the factors that nucleate actin assembly are not well understood at the organismal level or in the context of disease. To explore the function of the actin nucleation factor WHAMM in mice, we examined how Whamm inactivation impacts kidney physiology and cellular proteostasis. We show that male WHAMM knockout mice excrete elevated levels of albumin, glucose, phosphate, and amino acids, and display structural abnormalities of the kidney proximal tubule, suggesting that WHAMM activity is important for nutrient reabsorption. In kidney tissue, the loss of WHAMM results in the accumulation of the lipidated autophagosomal membrane protein LC3, indicating an alteration in autophagy. In mouse fibroblasts and human proximal tubule cells, WHAMM and its binding partner the Arp2/3 complex control autophagic membrane closure and cargo receptor recruitment. These results reveal a role for WHAMM-mediated actin assembly in maintaining kidney function and promoting proper autophagosome membrane remodeling.
Asunto(s)
Actinas , Autofagosomas , Autofagia , Riñón , Ratones Noqueados , Animales , Ratones , Actinas/metabolismo , Autofagia/fisiología , Humanos , Autofagosomas/metabolismo , Riñón/metabolismo , Masculino , Túbulos Renales Proximales/metabolismo , Citoesqueleto de Actina/metabolismo , Complejo 2-3 Proteico Relacionado con la Actina/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Polimerizacion , Fibroblastos/metabolismoRESUMEN
The actin cytoskeleton is essential for many functions of eukaryotic cells, but the factors that nucleate actin assembly are not well understood at the organismal level or in the context of disease. To explore the function of the actin nucleation factor WHAMM in mice, we examined how Whamm inactivation impacts kidney physiology and cellular proteostasis. We show that male WHAMM knockout mice excrete elevated levels of albumin, glucose, phosphate, and amino acids, and display abnormalities of the kidney proximal tubule, suggesting that WHAMM activity is important for nutrient reabsorption. In kidney tissue, the loss of WHAMM results in the accumulation of the lipidated autophagosomal membrane protein LC3, indicating an alteration in autophagy. In mouse fibroblasts and human proximal tubule cells, WHAMM and its binding partner the Arp2/3 complex control autophagic membrane closure and cargo receptor recruitment. These results reveal a role for WHAMM-mediated actin assembly in maintaining kidney function and promoting proper autophagosome membrane remodeling.
RESUMEN
Many bacterial pathogens reorganize the host actin cytoskeleton during the course of infection, including enterohemorrhagic Escherichia coli (EHEC), which utilizes the effector protein EspF(U) to assemble actin filaments within plasma membrane protrusions called pedestals. EspF(U) 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 EspF(U). Moreover, EspF(U) 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 EspF(U) acts as a scaffold to recruit multiple actin assembly factors whose functions are normally regulated by Cdc42.
Asunto(s)
Proteínas Portadoras/metabolismo , Proteínas del Citoesqueleto/metabolismo , Escherichia coli Enterohemorrágica/metabolismo , Infecciones por Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteína Neuronal del Síndrome de Wiskott-Aldrich/metabolismo , Proteína de Unión al GTP cdc42/metabolismo , Animales , Células COS , Proteínas Portadoras/genética , Chlorocebus aethiops , Proteínas del Citoesqueleto/genética , Escherichia coli Enterohemorrágica/genética , Infecciones por Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Unión a Ácidos Grasos , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Antígenos de Histocompatibilidad Menor , Proteína Neuronal del Síndrome de Wiskott-Aldrich/genética , Proteína de Unión al GTP cdc42/genética , Dominios Homologos srcRESUMEN
The actin cytoskeleton is a ubiquitous participant in cellular functions that maintain viability, but how it controls programmed cell death is not well understood. Here we show that in response to DNA damage, human cells form a juxtanuclear F-actin-rich territory that coordinates the organized progression of apoptosome assembly to caspase activation. This cytoskeletal compartment is created by the actin nucleation factors JMY, WHAMM, and the Arp2/3 complex, and it excludes proteins that inhibit JMY and WHAMM activity. Within the territory, mitochondria undergo outer membrane permeabilization and JMY localization overlaps with punctate structures containing the core apoptosome components cytochrome c and Apaf-1. The F-actin-rich area also encompasses initiator caspase-9 and clusters of a cleaved form of executioner caspase-3 but restricts accessibility of the caspase inhibitor XIAP. The clustering and potency of caspase-3 activation are positively regulated by the amount of actin polymerized by JMY and WHAMM. These results indicate that JMY-mediated actin reorganization functions in apoptotic signaling by coupling the biogenesis of apoptosomes to the localized processing of caspases.
Asunto(s)
Actinas , Apoptosomas , Humanos , Actinas/metabolismo , Caspasa 3 , Apoptosomas/metabolismo , Apoptosis/fisiología , Caspasas/metabolismo , Citoesqueleto de Actina/metabolismo , Daño del ADN , Proteínas de la Membrana/metabolismo , Proteínas Asociadas a MicrotúbulosRESUMEN
The actin cytoskeleton impacts practically every function of a eukaryotic cell. Historically, the best-characterized cytoskeletal activities are in cell morphogenesis, motility, and division. The structural and dynamic properties of the actin cytoskeleton are also crucial for establishing, maintaining, and changing the organization of membrane-bound organelles and other intracellular structures. Such activities are important in nearly all animal cells and tissues, although distinct anatomical regions and physiological systems rely on different regulatory factors. Recent work indicates that the Arp2/3 complex, a broadly expressed actin nucleator, drives actin assembly during several intracellular stress response pathways. These newly described Arp2/3-mediated cytoskeletal rearrangements are coordinated by members of the Wiskott-Aldrich Syndrome Protein (WASP) family of actin nucleation-promoting factors. Thus, the Arp2/3 complex and WASP-family proteins are emerging as crucial players in cytoplasmic and nuclear activities including autophagy, apoptosis, chromatin dynamics, and DNA repair. Characterizations of the functions of the actin assembly machinery in such stress response mechanisms are advancing our understanding of both normal and pathogenic processes, and hold great promise for providing insights into organismal development and interventions for disease.
Asunto(s)
Actinas , Familia de Proteínas del Síndrome de Wiskott-Aldrich , Animales , Familia de Proteínas del Síndrome de Wiskott-Aldrich/metabolismo , Actinas/metabolismo , Complejo 2-3 Proteico Relacionado con la Actina/metabolismo , Citoesqueleto de Actina/metabolismo , Citoesqueleto/metabolismo , Proteína del Síndrome de Wiskott-Aldrich/genética , Proteína del Síndrome de Wiskott-Aldrich/metabolismo , Proteína 3 Relacionada con la Actina/metabolismoRESUMEN
Upon infection of mammalian cells, enterohemorrhagic E. coli (EHEC) O157:H7 utilizes a type III secretion system to translocate the effectors Tir and EspF(U) (aka TccP) that trigger the formation of F-actin-rich 'pedestals' beneath bound bacteria. EspF(U) is localized to the plasma membrane by Tir and binds the nucleation-promoting factor N-WASP, which in turn activates the Arp2/3 actin assembly complex. Although N-WASP has been shown to be required for EHEC pedestal formation, the precise steps in the process that it influences have not been determined. We found that N-WASP and actin assembly promote EHEC-mediated translocation of Tir and EspF(U) into mammalian host cells. When we utilized the related pathogen enteropathogenic E. coli to enhance type III translocation of EHEC Tir and EspF(U), we found surprisingly that actin pedestals were generated on N-WASP-deficient cells. Similar to pedestal formation on wild type cells, Tir and EspF(U) were the only bacterial effectors required for pedestal formation, and the EspF(U) sequences required to interact with N-WASP were found to also be essential to stimulate this alternate actin assembly pathway. In the absence of N-WASP, the Arp2/3 complex was both recruited to sites of bacterial attachment and required for actin assembly. Our results indicate that actin assembly facilitates type III translocation, and reveal that EspF(U), presumably by recruiting an alternate host factor that can signal to the Arp2/3 complex, exhibits remarkable versatility in its strategies for stimulating actin polymerization.
Asunto(s)
Actinas/metabolismo , Escherichia coli Enterohemorrágica/metabolismo , Infecciones por Escherichia coli/metabolismo , Proteína Neuronal del Síndrome de Wiskott-Aldrich/metabolismo , Animales , Proteínas Portadoras/metabolismo , Escherichia coli Enterohemorrágica/patogenicidad , Proteínas de Escherichia coli/metabolismo , Técnicas de Inactivación de Genes , Humanos , Immunoblotting , Péptidos y Proteínas de Señalización Intracelular , Ratones , Microscopía Fluorescente , Transporte de Proteínas/fisiología , Receptores de Superficie Celular/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/fisiologíaRESUMEN
The bacteriophage Lambda (λ) "Red" recombination system has enabled the development of efficient methods for engineering bacterial chromosomes. This system has been particularly important to the field of bacterial pathogenesis, where it has advanced the study of virulence factors from Shiga toxin-producing and enteropathogenic Escherichia coli (STEC and EPEC). Transient plasmid-driven expression of Lambda Red allows homologous recombination between PCR-derived linear DNA substrates and target loci in the STEC/EPEC chromosomes. Red-associated techniques can be used to create individual gene knockouts, generate deletions of large pathogenicity islands, and make markerless allelic exchanges. This chapter describes specific strategies and procedures for performing Lambda Red-mediated genome engineering in STEC.
Asunto(s)
Bacteriófago lambda/metabolismo , Escherichia coli Enteropatógena/metabolismo , Recombinación Genética , Escherichia coli Shiga-Toxigénica/metabolismo , Proteínas Virales/metabolismo , Bacteriófago lambda/genética , Escherichia coli Enteropatógena/genética , Infecciones por Escherichia coli/genética , Infecciones por Escherichia coli/metabolismo , Escherichia coli Shiga-Toxigénica/genética , Proteínas Virales/genéticaRESUMEN
Enterohemorrhagic Escherichia coli (EHEC) generate F-actin-rich adhesion pedestals by delivering effector proteins into mammalian cells. These effectors include the translocated receptor Tir, along with EspF(U), a protein that associates indirectly with Tir and contains multiple peptide repeats that stimulate actin polymerization. In vitro, the EspF(U) repeat region is capable of binding and activating recombinant derivatives of N-WASP, a host actin nucleation-promoting factor. In spite of the identification of these important bacterial and host factors, the underlying mechanisms of how EHEC so potently exploits the native actin assembly machinery have not been clearly defined. Here we show that Tir and EspF(U) are sufficient for actin pedestal formation in cultured cells. Experimental clustering of Tir-EspF(U) fusion proteins indicates that the central role of the cytoplasmic portion of Tir is to promote clustering of the repeat region of EspF(U). Whereas clustering of a single EspF(U) repeat is sufficient to bind N-WASP and generate pedestals on cultured cells, multi-repeat EspF(U) derivatives promote actin assembly more efficiently. Moreover, the EspF(U) repeats activate a protein complex containing N-WASP and the actin-binding protein WIP in a synergistic fashion in vitro, further suggesting that the repeats cooperate to stimulate actin polymerization in vivo. One explanation for repeat synergy is that simultaneous engagement of multiple N-WASP molecules can enhance its ability to interact with the actin nucleating Arp2/3 complex. These findings define the minimal set of bacterial effectors required for pedestal formation and the elements within those effectors that contribute to actin assembly via N-WASP-Arp2/3-mediated signaling pathways.
Asunto(s)
Citoesqueleto de Actina/metabolismo , Proteínas Portadoras/química , Proteínas Portadoras/metabolismo , Escherichia coli Enterohemorrágica/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Secuencias Repetitivas de Aminoácido , Proteína Neuronal del Síndrome de Wiskott-Aldrich/metabolismo , Complejo 2-3 Proteico Relacionado con la Actina/metabolismo , Secuencia de Aminoácidos , Animales , Encéfalo/metabolismo , Proteínas Portadoras/genética , Proteínas del Citoesqueleto/genética , Proteínas del Citoesqueleto/metabolismo , ADN Bacteriano/genética , ADN Bacteriano/metabolismo , Escherichia coli Enterohemorrágica/genética , Proteínas de Escherichia coli/genética , GTP Fosfohidrolasas/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Transducción de Señal/genética , PorcinosRESUMEN
Filopodia are actin-rich protrusions important for sensing and responding to the extracellular environment, but the repertoire of factors required for filopodia formation is only partially understood. Jarsch et al. (2020. J. Cell. Biol. https://doi.org/10.1083/jcb.201909178) combine an in vitro system of filopodia biogenesis with a phage display screen to show that SNX9 drives filopodial assembly.
Asunto(s)
Actinas , SeudópodosRESUMEN
Several microbial pathogens including enteropathogenic E. coli (EPEC) exploit mammalian tyrosine-kinase signaling cascades to recruit Nck adaptor proteins and activate N-WASP-Arp2/3-mediated actin assembly. To promote localized actin "pedestal formation," EPEC translocates the bacterial effector protein Tir into the plasma membrane, where it is tyrosine-phosphorylated and binds Nck. Enterohemorrhagic E. coli (EHEC) also generates Tir-dependent pedestals, but in the absence of phosphotyrosines and Nck recruitment. To identify additional EHEC effectors that stimulate phosphotyrosine-independent actin assembly, we systematically generated EHEC mutants containing specific deletions in putative pathogenicity-islands. Among 0.33 Mb of deleted sequences, only one ORF was critical for pedestal formation. It lies within prophage-U, and encodes a protein similar to the known effector EspF. This proline-rich protein, EspFU, is the only EHEC effector of actin assembly absent from EPEC. Whereas EHEC Tir cannot efficiently recruit N-WASP or trigger actin polymerization, EspFU associates with Tir, binds N-WASP, and potently stimulates Nck-independent actin assembly.
Asunto(s)
Proteínas Portadoras/fisiología , Escherichia coli O157/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/fisiología , Proteínas del Tejido Nervioso/metabolismo , Proteínas Oncogénicas/metabolismo , Receptores de Superficie Celular/metabolismo , Actinas/química , Actinas/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Western Blotting , Proteínas Portadoras/metabolismo , Núcleo Celular/metabolismo , Escherichia coli/metabolismo , Eliminación de Gen , Prueba de Complementación Genética , Islas Genómicas , Células HeLa , Humanos , Immunoblotting , Péptidos y Proteínas de Señalización Intracelular , Microscopía Fluorescente , Mutación , Fosfotirosina/química , Plásmidos/metabolismo , Pruebas de Precipitina , Prolina/química , Unión Proteica , Estructura Terciaria de Proteína , Transporte de Proteínas , Fracciones Subcelulares , Técnicas del Sistema de Dos Híbridos , Proteína Neuronal del Síndrome de Wiskott-AldrichRESUMEN
Many microbial pathogens manipulate the actin cytoskeleton of eukaryotic target cells to promote their internalization, intracellular motility and dissemination. Enteropathogenic and enterohaemorrhagic Escherichia coli, which both cause severe diarrhoeal disease, can adhere to mammalian intestinal cells and induce reorganization of the actin cytoskeleton into 'pedestal-like' pseudopods beneath the extracellular bacteria. As pedestal assembly is triggered by E. coli virulence factors that mimic several host cell-signalling components, such as transmembrane receptors, their cognate ligands and cytoplasmic adaptor proteins, it can serve as a powerful model system to study eukaryotic transmembrane signalling. Here, we consider the impact of recent data on our understanding of both E. coli pathogenesis and cell biology, and the rich prospects for exploiting these bacterial factors as versatile tools to probe cellular signalling pathways.