RESUMEN
Capping the barbed ends of actin filaments is a critical step for regulating actin-based motility in nonmuscle cells. The in vivo function of CapG, a calcium-sensitive barbed end capping protein and member of the gelsolin/villin family, has been assessed using a null Capg allele engineered into mice. Both CapG-null mice and CapG/gelsolin double-null mice appear normal and have no gross functional abnormalities. However, the loss of CapG in bone marrow macrophages profoundly inhibits macrophage colony stimulating factor-stimulated ruffling; reintroduction of CapG protein by microinjection fully restores this function. CapG-null macrophages also demonstrate approximately 50% impairment of immunoglobulin G, and complement-opsonized phagocytosis and lanthanum-induced vesicle rocketing. These motile functions are not impaired in gelsolin-null macrophages and no additive effects are observed in CapG/gelsolin double-null macrophages, establishing that CapG function is distinct from, and does not overlap with, gelsolin in macrophages. Our observations indicate that CapG is required for receptor-mediated ruffling, and that it is a major functional component of macrophage phagocytosis. These primary effects on macrophage motile function suggest that CapG may be a useful target for the regulation of macrophage-mediated inflammatory responses.
Asunto(s)
Actinas/metabolismo , Movimiento Celular/fisiología , Gelsolina/genética , Macrófagos/fisiología , Proteínas de Microfilamentos/genética , Proteínas Nucleares/genética , Animales , Células de la Médula Ósea/citología , Células de la Médula Ósea/fisiología , Fibroblastos/citología , Fibroblastos/fisiología , Membranas Intracelulares/fisiología , Macrófagos/citología , Ratones , Ratones Noqueados , Mutagénesis Sitio-Dirigida , Neutrófilos/citología , Neutrófilos/fisiología , Fagocitosis/fisiologíaRESUMEN
Mounting evidence supports the role of truncated vinculin in the intracellular actin-based motility of Shigella flexneri. Vinculin's role was recently questioned by Goldberg [1997: Cell Motil Cytoskeleton 37:44-53] who observed Shigella motility in mouse embryonal carcinoma 5.51 cells, a genetically modified cell line that reputedly lacked vinculin. That challenge implicitly relied on the assumption that 5.51 cells had no detectable vinculin polypeptide and lacked full-length vinculin mRNA. Despite the appearance of being an unambiguous test of vinculin's role in Shigella motility, 5.51 cells were shown to contain adequate amounts of truncated vinculin (as well as the corresponding mRNA transcript) to support bacterial locomotion. We also examined Shigella locomotion in gamma229 cells, a related embryonal carcinoma cell line containing approximately one-half the vinculin content found in 5.51 cells. We observed that there was a commensurate twofold decrease in the Shigella motility rate, as compared to 5.51 cells; this finding raises the possibility that vinculin can become a rate-limiting factor under some circumstances. Immunofluorescence microscopy using vin 11-5 monoclonal antibody directed against the vinculin head domain showed intense staining of Shigella rocket tails in both gamma229 and 5.51 cells. Our findings clearly demonstrate that motility in 5.51 cells cannot be regarded as a valid criterion for evaluating the role of truncated vinculin in Shigella motility.
Asunto(s)
Actinas/metabolismo , Shigella flexneri/fisiología , Vinculina/fisiología , Animales , Western Blotting , Movimiento Celular , Sistema Libre de Células , Ratones , Microscopía Fluorescente , Unión Proteica , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Shigella flexneri/metabolismo , Factores de Tiempo , Células Tumorales Cultivadas , Vinculina/farmacologíaRESUMEN
The mechanism of profilin-promoted actin polymerization has been systematically reinvestigated. Rates of barbed-end elongation onto Spectrin.4.1. Actin seeds were measured by right angle light scattering to avoid confounding effects of pyrenyl-actin, and KINSIM was used to analyze elongation progress curves. Without thymosin-beta4, both actin and Profilin. Actin (P.A) are competent in barbed-end polymerization, and kinetic simulations yielded the same bimolecular rate constant ( approximately 10 x 10(6) M(-1) s(-1)) for actin monomer or Profilin. Actin. When measured in the absence of profilin, actin assembly curves over a 0.7-4 microM thymosin-beta4 concentration range fit a simple monomer sequestering model (1 microM K(D) for Thymosin-beta4. Actin). The corresponding constant for thymosin-beta4.pyrenyl-Actin, however, was significantly higher ( approximately 9-10 microM), suggesting that the fluorophore markedly weakens binding to thymosin-beta4. With solutions of actin (2 microM) and thymosin-beta4 (2 or 4 microM), the barbed-end assembly rate rose with increasing profilin concentration (0.7-2 microM). Actin assembly in presence of thymosin-beta4 and profilin fit a simple thermodynamic energy cycle, thereby disproving an earlier claim (D. Pantaloni and M.-F. Carlier (1993) Cell 75, 1007-1014) that profilin promotes nonequilibrium filament assembly by accelerating hydrolysis of filament-bound ATP. Our findings indicate that profilin serves as a polymerization catalyst that captures actin monomers from Thymosin-beta4. Actin and ushers actin as a Profilin. Actin complex onto growing barbed filament ends.
Asunto(s)
Actinas/química , Proteínas Contráctiles , Proteínas de Microfilamentos/farmacología , Animales , Humanos , Luz , Polímeros/química , Profilinas , Conejos , Dispersión de Radiación , Termodinámica , Timosina/farmacologíaRESUMEN
Actin-Based Motility motifs [ABM-1 sequence = (D/E)FPPPPX(D/E), where X = P or T, and ABM-2 sequence = XPPPPP, where X denotes G, A, L, P, and S] facilitate assembly of an activated motility complex. Potent inhibition of intracellular motility of pathogens by ABM-1 and ABM-2 peptide analogues has served as a criterion for investigating actin-based motility. To assess the specificity of ABM-1 peptide inhibitors, we microinjected proline-rich peptides into Listeria-infected PtK2 host cells. Use of a combinatorial ABM-1 peptide library (empirical formula = D1E2F2P4T1) demonstrated that high-potency inhibition requires a precise sequence, and not merely a particular amino acid composition. Calculated concentrations of specific sequences in this library indicate that the entire (D/E)FPPPPX(D/E) motif is needed to achieve high-affinity inhibition in living cells. The failure of the well known proline-rich SH3 binding antagonists VSL-12 or APP-12 to inhibit Listeria motility also indicates that SH3 interactions are unlikely to control actin-based motility directly.
Asunto(s)
Actinas/fisiología , Proteínas Bacterianas/fisiología , Listeria monocytogenes/citología , Listeria monocytogenes/fisiología , Adhesión Bacteriana , Péptidos/fisiologíaRESUMEN
Vaccinia uses actin-based motility for virion movement in host cells, but the specific protein components have yet to be defined. A cardinal feature of Listeria and Shigella actin-based motility is the involvement of vasodilator-stimulated phosphoprotein (VASP). This essential adapter recognizes and binds to actin-based motility 1 (ABM-1) consensus sequences [(D/E)FPPPPX(D/E), X = P or T] contained in Listeria ActA and in the p90 host-cell vinculin fragment generated by Shigella infection. VASP, in turn, provides the ABM-2 sequences [XPPPPP, X = G, P, L, S, A] for binding profilin, an actin-regulatory protein that stimulates actin filament assembly. Immunolocalization using rabbit anti-VASP antibody revealed that VASP concentrates behind motile virions in HeLa cells. Profilin was also present in these actin-rich rocket tails, and microinjection of 10 microM (intracellular) ABM-2 peptide (GPPPPP)3 blocked vaccinia actin-based motility. Vinculin did not colocalize with VASP on motile virions and remained in focal adhesion contacts; however, another ABM-1-containing host protein, zyxin, was concentrated at the rear of motile virions. We also examined time-dependent changes in the location of these cytoskeletal proteins during vaccinia infection. VASP and zyxin were redistributed dramatically several hours before the formation of actin rocket tails, concentrating in the viral factories of the perinuclear cytoplasm. Our findings underscore the universal involvement of ABM-1 and ABM-2 docking sites in actin-based motility of Listeria, Shigella, and now vaccinia.
Asunto(s)
Actinas/fisiología , Virus Vaccinia/fisiología , Animales , Transporte Biológico Activo , Citoesqueleto/virología , Células HeLa , Humanos , Fragmentos de Péptidos , Conejos , Replicación ViralRESUMEN
The actin-based motility of Listeria monocytogenes requires the addition of actin monomers to the barbed or plus ends of actin filaments. Immunofluorescence micrographs have demonstrated that gelsolin, a protein that both caps barbed ends and severs actin filaments, is concentrated directly behind motile bacteria at the junction between the actin filament rocket tail and the bacterium. In contrast, CapG, a protein that strictly caps actin filaments, fails to localize near intracellular Listeria. To explore the effect of increasing concentrations of gelsolin on bacterial motility, NIH 3T3 fibroblasts stably transfected with gelsolin cDNA were infected with Listeria. The C5 cell line containing 2.25 times control levels of gelsolin supported significantly higher velocities of bacterial movement than did control fibroblasts (mean +/- standard error of the mean, 0.09 +/- 0.003 micro(m)/s [n = 176] versus 0.05 +/- 0.003 micro(m)/s [n = 65]). The rate of disassembly of the Listeria-induced actin filament rocket tail was found to be independent of gelsolin content. Therefore, if increases in gelsolin content result in increases in Listeria-induced rocket tail assembly rates, a positive correlation between gelsolin content and tail length would be expected. BODIPY-phalloidin staining of four different stably transfected NIH 3T3 fibroblast cell lines confirmed this expectation (r = 0.92). Rocket tails were significantly longer in cells with a high gelsolin content. Microinjection of gelsolin 1/2 (consisting of the amino-terminal half of native gelsolin) also increased bacterial velocity by more than 2.2 times. Microinjection of CapG had no effect on bacterial movement. Cultured skin fibroblasts derived from gelsolin-null mice were capable of supporting intracellular Listeria motility at velocities comparable to those supported by wild-type skin fibroblasts. These experiments demonstrated that the surface of Listeria contains a polymerization zone that can block the barbed-end-capping activity of both gelsolin and CapG. The ability of Listeria to uncap actin filaments combined with the severing activity of gelsolin can accelerate actin-based motility. However, gelsolin is not absolutely required for the actin-based intracellular movement of Listeria because its function can be replaced by other actin regulatory proteins in gelsolin-null cells, demonstrating the functional redundancy of the actin system.
Asunto(s)
Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Gelsolina/metabolismo , Listeria monocytogenes/metabolismo , Proteínas de Microfilamentos/metabolismo , Proteínas Nucleares/metabolismo , Células 3T3 , Animales , Anticuerpos/inmunología , Técnica del Anticuerpo Fluorescente Indirecta , Gelsolina/genética , Gelsolina/inmunología , Humanos , Ratones , Proteínas de Microfilamentos/inmunología , Microinyecciones , Proteínas Nucleares/inmunología , Conejos , TransfecciónAsunto(s)
Actinas/fisiología , Listeria/fisiología , Listeria/patogenicidad , Shigella/fisiología , Shigella/patogenicidad , Secuencia de Aminoácidos , Animales , Sitios de Unión/genética , Moléculas de Adhesión Celular/fisiología , Disentería Bacilar/etiología , Humanos , Listeria/crecimiento & desarrollo , Listeriosis/etiología , Proteínas de Microfilamentos , Movimiento/fisiología , Oligopéptidos/química , Oligopéptidos/farmacología , Fagocitosis , Fosfoproteínas/fisiología , Shigella/crecimiento & desarrolloRESUMEN
To generate the forces needed for motility, the plasma membranes of nonmuscle cells adopt an activated state that dynamically reorganizes the actin cytoskeleton. By usurping components from focal contacts and the actin cytoskeleton, the intracellular pathogens Shigella flexneri and Listeria monocytogenes use molecular mimicry to create their own actin-based motors. We raised an antibody (designated FS-1) against the FEFPPPPTDE sequence of Listeria ActA, and this antibody: (a) localized at the trailing end of motile intracellular Shigella, (b) inhibited intracellular locomotion upon microinjection of Shigella-infected cells, and (c) cross-reacted with the proteolytically derived 90-kD human vinculin head fragment that contains the Vinc-1 oligoproline sequence, PDFPPPPPDL. Antibody FS-1 reacted only weakly with full-length vinculin, suggesting that the Vinc-1 sequence in full-length vinculin may be masked by its tail region and that this sequence is unmasked by proteolysis. Immunofluoresence staining with a monoclonal antibody against the head region of vinculin (Vin 11-5) localized to the back of motile bacteria (an identical staining pattern observed with the anti-ActA FS-1 antibody), indicating that motile bacteria attract a form of vinculin containing an unmasked Vinc-1 oligoproline sequence. Microinjection of submicromolar concentrations of a synthetic Vinc-1 peptide arrested Shigella intracellular motility, underscoring the functional importance of this sequence. Western blots revealed that Shigella infection induces vinculin proteolysis in PtK2 cells and generates p90 head fragment over the same 1-3 h time frame when intracellular bacteria move within the host cell cytoplasm. We also discovered that microinjected p90, but not full-length vinculin, accelerates rates of pathogen motility by a factor of 3 +/- 0.4 in Shigella-infected PtK2 cells. These experiments suggest that vinculin p90 is a rate-limiting component in actin-based Shigella motility, and that supplementing cells with p90 stimulates rocket tail growth. Earlier findings demonstrated that vinculin p90 binds to IcsA (Suzuki, T.A., S. Saga, and C. Sasakawa. 1996. J. Biol. Chem. 271:21878-21885) and to vasodilator-stimulated phosphoprotein (VASP) (Brindle, N.P.J., M. R. Hold, J.E. Davies, C.J. Price, and D.R. Critchley. 1996. Biochem. J. 318:753-757). We now offer a working model in which proteolysis unmasks vinculin's ActA-like oligoproline sequence. Unmasking of this site serves as a molecular switch that initiates assembly of an actin-based motility complex containing VASP and profilin.
Asunto(s)
Proteínas Bacterianas/metabolismo , Movimiento Celular/fisiología , Disentería Bacilar/microbiología , Proteínas de la Membrana/metabolismo , Shigella flexneri/citología , Vinculina/metabolismo , Actinas/fisiología , Animales , Especificidad de Anticuerpos , Proteínas Bacterianas/química , Proteínas Bacterianas/inmunología , Plaquetas/química , Moléculas de Adhesión Celular/metabolismo , Células Cultivadas , Reacciones Cruzadas , Disentería Bacilar/metabolismo , Técnica del Anticuerpo Fluorescente , Humanos , Riñón/citología , Macropodidae , Proteínas de la Membrana/química , Proteínas de la Membrana/inmunología , Proteínas de Microfilamentos/metabolismo , Microinyecciones , Fragmentos de Péptidos/inmunología , Fragmentos de Péptidos/farmacología , Fosfoproteínas/metabolismo , Prolina/metabolismo , Shigella flexneri/química , Vinculina/química , Vinculina/farmacologíaRESUMEN
Intracellular actin-based motility of Listeria monocytogenes requires protein-protein interactions involving two different proline-rich sequences: first, the tightly bound bacterial surface protein ActA uses its multiple oligoproline registers [consensus sequence = FE(D)FPPPPTD(E)E(D)] to tether vasodilator-stimulated phosphoprotein (VASP) to the bacterial surface; and second, VASP then deploys its own multiple GPPPPP (or GP5) registers to localize the actin-regulatory protein profilin to promote actin polymerization. We now report that fluorescence titration showed that GP5GP5GP5 peptide binds to profilin (KD of 84 microM), and the peptide weakly inhibits exchange of actin-bound nucleotide in the absence or presence of profilin. Microinjection of synthetic GPPPPP triplet into Listeria-infected PtK2 cells promptly arrested motility at an intracellular concentration of 10 microM. This inhibition was completely neutralized when equimolar concentrations of profilin and GP5GP5GP5 were simultaneously microinjected. Fluorescence studies with [His-133-Ser]-profilin, a site-directed mutant previously shown to be defective in binding poly-l-proline [Bjorkegren, C., Rozycki, M., Schutt, C. E., Lindberg, U., & Karlsson, R. (1993) FEBS Lett. 333, 123-126], exhibits little or no evidence of saturable GP5GP5GP5 binding. When an equimolar concentration of this [His-133-Ser]-profilin mutant was co-injected with GP5GP5GP5, the peptide's inhibitory action remained completely unaffected, indicating that GP5GP5GP5 binding to wild-type profilin represents a key step in actin-based pathogen motility. We also present a model that shows how the focal binding of VASP with its GPPPPP registers can greatly increase the local concentration of profilin and/or profilin-actin-ATP complex at the bacteria/rocket-tail interface.
Asunto(s)
Actinas/metabolismo , Moléculas de Adhesión Celular/química , Moléculas de Adhesión Celular/metabolismo , Listeria monocytogenes/fisiología , Proteínas de Microfilamentos/metabolismo , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Actinas/farmacología , Adenosina Difosfato/metabolismo , Adenosina Trifosfato/metabolismo , Secuencia de Aminoácidos , Proteínas Contráctiles , Humanos , Proteínas de Microfilamentos/química , Movimiento/efectos de los fármacos , Fragmentos de Péptidos/farmacología , Cloruro de Potasio/farmacología , Profilinas , Proteínas Recombinantes/metabolismo , Espectrometría de FluorescenciaRESUMEN
Actin-based motility involves a cascade of binding interactions designed to assemble actin regulatory proteins into functional locomotory units. Listeria ActA surface protein contains a series of nearly identical EFPPPPTDE-type oligoproline sequences for binding vasodilator-stimulated phosphoprotein (VASP). The latter is a tetrameric protein with numerous GPP-PPP docking sites for profilin, a 15 kDa regulatory protein that promotes actin filament assembly. Analysis of known actin regulatory proteins led to the identification of distinct Actin-Based Motility homology sequences ABM-1; (D/E)FPPPPX(D/E); and ABM-2, XPPPPP (where X denotes G, A, L, and S).
Asunto(s)
Actinas/química , Proteínas Bacterianas/química , Moléculas de Adhesión Celular/química , Proteínas Contráctiles , Proteínas de la Membrana/química , Proteínas de Microfilamentos/química , Fosfoproteínas/química , Secuencia de Aminoácidos , Animales , Sitios de Unión , Secuencia de Consenso , Humanos , Datos de Secuencia Molecular , Profilinas , Prolina/química , Unión ProteicaRESUMEN
The gram negative rod Shigella flexneri uses it surface protein IcsA to induce host cell actin assembly and to achieve intracellular motility. Yet, the IcsA protein lacks the oligoproline sequences found in ActA, the surface protein required for locomotion of the gram positive rod Listeria monocytogenes. Microinjection of a peptide matching the second ActA oligoproline repeat (FEFPPPPTDE) stops Listeria locomotion (Southwick, F.S., and D.L. Purich. 1994a. Proc. Natl. Acad. Sci. USA. 91:5168-5172), and submicromolar concentrations (intracellular concentration 80-800 nM) similarly arrest Shigella rocket-tail assembly and intracellular motility. Coinjection of a binary solution containing profilin and the ActA analogue increased the observed rates of intracellular motility by a factor of three (mean velocity 0.90 +/- 0.07 mu m/s, SD n=16 before injection vs 0.3 +/- 0.1 mu m/s, n=33 postinjection, intracellular concentration = 80 nM profilin plus 80 nM ActA analogue). Recent evidence suggests the ActA analogue may act by displacing the profilin-binding protein VASP (Pistor, S.C., T. Chakaborty, V. Walter, and J. Wehland. 1995. Curr. Biol. 5:517-525). At considerably higher intracellular concentrations (10 muM), the VASP oligoproline sequence (GPPPPP)3 thought to represent the profilin-binding site (Reinhard, M., K. Giehl, K. Abel, C. Haffner, T. Jarchau, V. Hoppe, B.M. Jockusch, and U. Walter. 1995. EMBO (Eur. Mol. Biol. Organ.) J. 14:1583-1589) also inhibited Shigella movement. A binary mixture of the VASP analogue and profilin (each 10 muM intracellular concentration) led to a doubling of Shigella intracellular migration velocity (0.09 +/- 0.06 mu m/s, n = 25 preinjection vs 0.18 +/- 0.10 mu m/s, n = 61 postinjection). Thus, the two structurally divergent bacteria, Listeria and Shigella, have adopted convergent mechanisms involving profilin recognition of VASP oligoproline sequences and VASP recognition of oligoproline sequences in ActA or an ActA-like host protein to induce host cell actin assembly and to provide the force for intracellular locomotion and cell-cell spread.
Asunto(s)
Actinas/metabolismo , Proteínas Contráctiles , Proteínas de Microfilamentos/fisiología , Oligopéptidos/farmacología , Péptidos/farmacología , Shigella flexneri/fisiología , Actinina/análisis , Actinas/análisis , Secuencia de Aminoácidos , Animales , Proteínas Bacterianas/química , Moléculas de Adhesión Celular/química , Línea Celular , Epitelio/microbiología , Listeria monocytogenes/química , Listeria monocytogenes/citología , Macropodidae , Proteínas de la Membrana/química , Proteínas de Microfilamentos/farmacología , Microinyecciones , Datos de Secuencia Molecular , Movimiento , Oligopéptidos/síntesis química , Péptidos/síntesis química , Fosfoproteínas/química , Profilinas , Shigella flexneri/química , Shigella flexneri/citologíaAsunto(s)
Listeria monocytogenes/fisiología , Listeriosis/etiología , Actinas/biosíntesis , Antibacterianos/uso terapéutico , Enfermedades Fetales/microbiología , Humanos , Listeria monocytogenes/crecimiento & desarrollo , Listeria monocytogenes/patogenicidad , Listeriosis/tratamiento farmacológico , Listeriosis/epidemiología , Meningitis por Listeria/microbiología , Meningoencefalitis/microbiología , Movimiento/fisiologíaRESUMEN
In human polymorphonuclear leukocytes (PMN), changes in the actin architecture are critical for the shape changes required for chemotaxis and phagocytosis. Barbed-end capping proteins are likely to regulate actin assembly in PMN. The previously identified barbed-end blocking proteins in PMN, gelsolin and CapG, require Ca(2+) to initiate capping of actin filaments. Because chemoattractants can stimulate PMN actin assembly by a calcium-independent signal transduction pathway, we sought to purify a calcium-independent barbed-end capping activity from PMN cytoplasmic extracts. A Ca(2+) -insensitive actin polymerization inhibitory activity was partially purified from human PMN [Southwick & Stossel (1981) J. Biol. Chem 256, 3030]. Using five column chromatography steps, we purified the protein to homogeneity as assessed by silver staining. Purification was associated with an increase in specific activity of greater than 40 X. Western blot analysis identified the protein as the nonmuscle isoform of the heterodimeric capping protein capZ. Human PMN capZ has an apparent disassociation constant of 3 nM for capping in the presence or absence of micromolar Ca(2+), as assessed by both pyrenylactin elongation and depolymerization assays. Similar to the activity reported for the actin polymerization inhibitor, activity of PMN capZ was inhibited by increasing the KC1 concentration from 0.1 M to 0.6 M. The capping function was also inhibited by phosphatidylinositol 4,5-bisphosphate (PIP(2)) micelles, with half-maximal inhibition occurring at 5.5 micrograms mL(-1). PMN capZ did not nucleate actin assembly, sequester actin monomers, or sever actin filaments. Quantitative Western blot analysis revealed that capZ levels corresponded to 0.7-1.0% of the total human PMN cytoplasmic protein. Given its abundance and high affinity for barbed filament ends, capZ is likely to play an important role in the calcium-independent regulation of actin filament assembly associated with PMN chemotaxis.
Asunto(s)
Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Quimiotaxis de Leucocito , Proteínas del Citoesqueleto , Proteínas de Microfilamentos/aislamiento & purificación , Proteínas de Microfilamentos/metabolismo , Proteínas Musculares/aislamiento & purificación , Neuropéptidos , Neutrófilos/química , Factores Despolimerizantes de la Actina , Proteína CapZ , Movimiento Celular , Destrina , Gelsolina/metabolismo , Humanos , Proteínas de la Membrana/metabolismo , Peso Molecular , Proteínas Musculares/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Unión Proteica , Espectrina/metabolismoAsunto(s)
Infecciones Bacterianas , Trombosis de los Senos Intracraneales/complicaciones , Senos Craneales/anatomía & histología , Diagnóstico Diferencial , Humanos , Trombosis de los Senos Intracraneales/diagnóstico , Trombosis de los Senos Intracraneales/tratamiento farmacológico , Resultado del TratamientoRESUMEN
Infection of host cells by Listeria monocytogenes results in the recruitment of cytoplasmic actin into a tail-like appendage that projects from one end of the bacterium. Each filamentous actin tail progressively lengthens, providing the force which drives the bacterium in a forward direction through the cytoplasm and later results in Listeria cell-to-cell spread. Host cell actin monomers are incorporated into the filamentous actin tail at a discrete site, the bacterial-actin tail interface. We have studied the consequences of microinjecting three different actin monomer-binding proteins on the actin tail assembly and Listeria intracellular movement. Introduction of high concentrations of profilin (estimated injected intracellular concentration 11-22 microM) into infected PtK2 cells causes a marked slowing of actin tail elongation and bacterial migration. Lower intracellular concentrations of two other injected higher affinity monomer-sequestering proteins, Vitamin D-binding protein (DBP; 1-2 microM) and DNase I (6-7 microM) completely block bacterial-induced actin assembly and bacterial migration. The onset of inhibition by each protein is gradual (10-20 min) indicating that the mechanisms by which these proteins interfere with Listeria-induced actin assembly are likely to be complex. To exclude the possibility that Listeria recruits preformed actin filaments to generate the tails and that these monomer-binding proteins act by depolymerizing such performed actin filaments, living infected cells have been injected with fluorescently labeled phalloidin (3 microM). Although the stress fibers are labeled, no fluorescent phalloidin is found in the tails of the moving bacteria. These results demonstrate that Listeria-induced actin assembly in PtK2 cells is the result of assembly of actin monomers into new filaments and that Listeria's ability to recruit polymerization competent monomeric actin is very sensitive to the introduction of exogenous actin monomer-binding proteins.
Asunto(s)
Actinas/metabolismo , Movimiento Celular/efectos de los fármacos , Proteínas Contráctiles , Citoplasma/microbiología , Desoxirribonucleasa I/farmacología , Listeria monocytogenes/fisiología , Proteínas de Microfilamentos/farmacología , Proteína de Unión a Vitamina D/farmacología , Actinina/metabolismo , Animales , Biopolímeros , Bovinos , Células Cultivadas , Pollos , Listeria monocytogenes/efectos de los fármacos , Listeria monocytogenes/ultraestructura , Macropodidae , Microinyecciones , Microscopía Fluorescente , Faloidina/farmacología , ProfilinasRESUMEN
Nonmuscle cell motility requires marked changes in the consistency and shape of the peripheral cytoplasm. These changes are regulated by a gel-sol transformation of the actin filament network, and actin filament-severing proteins are responsible for network solation. Macrophage Cap G, unlike all other proteins in the gelsolin family, caps but does not sever actin filaments. Two amino acid stretches in Cap G diverge markedly from the severing proteins: 84LNTLLGE and 124AFHKTS. Discrete mutations in Cap G have been generated to determine if these amino acid sequences are critical for actin filament severing. Conversion of 84LNTLLGE to the gelsolin actin-binding helix sequence (84LDDYLGG) renders Cap G capable of severing actin filaments (half-maximal severing, 1-2 microM). Adding a second set of mutations, converting 124AFHKTS to 124GFKHV, enhances severing by 10-fold (half-maximal severing, 0.1-0.2 microM). These experiments support a critical role for these two regions in actin filament severing and showcase the power of gain-of-function mutations in clarifying structure-function relationships.
Asunto(s)
Actinas/metabolismo , Macrófagos/metabolismo , Proteínas de Microfilamentos/metabolismo , Proteínas Nucleares/metabolismo , Secuencia de Aminoácidos , Humanos , Proteínas de Microfilamentos/genética , Datos de Secuencia Molecular , Mutación , Proteínas Nucleares/genética , Homología de Secuencia de Aminoácido , Relación Estructura-ActividadRESUMEN
Mosquito oostatic factor, a naturally occurring decapeptide (YDPAPPPPPP), strikingly resembles the primary structure of oligoproline-rich regions within the protein ActA, a bacterial surface protein required for Listeria motility in host cells. When microinjected into Listeria-infected PtK2 cells, the insect oostatic factor rapidly blocks Listeria-induced actin rocket tail assembly as well as intracellular locomotion of this pathogen. At intracellular concentrations of about 90 nM, transient inhibition of rocket tail formation and bacterial locomotion occurs, followed by full recovery of tail length and motility. However, at 0.9 microM oostatic factor, both processes are permanently arrested. Introduction of oostatic factor by microinjection also causes PtK2 peripheral membrane retraction in both Listeria-infected and uninfected cells. Epifluorescence microscopy with bodipy-phallacidin reveals that cells microinjected with the insect factor lose all actin stress fibers and accumulate F-actin in regions of membrane retraction. When the insect peptide is combined with profilin as an equimolar binary solution (1 microM [final concentration] each), intracellular addition fails to inhibit Listeria rocket-tail formation, fails to block intracellular bacterial movement, and no longer causes marked membrane retraction. The ability of profilin to neutralize the inhibitory action of oostatic factor is consistent with complex formation, and this finding suggests that profilin may interact directly with ActA peptide as well as a host cell peripheral membrane component to promote actin filament assembly by locally generating ATP-actin. Dispersal of profilin from such sites by oligoproline-rich peptide inhibitors suggests that profilin is directly involved in intracellular pathogen locomotion and reorganization of actin cytoskeleton of the host cell peripheral membrane.
Asunto(s)
Proteínas Bacterianas/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Proteínas Contráctiles , Listeria monocytogenes/efectos de los fármacos , Proteínas de la Membrana/efectos de los fármacos , Proteínas de Microfilamentos/farmacología , Oligopéptidos/farmacología , Secuencia de Aminoácidos , Animales , Proteínas Bacterianas/química , Células Cultivadas , Relación Dosis-Respuesta a Droga , Interacciones Farmacológicas , Proteínas de la Membrana/química , Microinyecciones , Microscopía por Video , Modelos Biológicos , Datos de Secuencia Molecular , ProfilinasRESUMEN
The bacterial pathogen Listeria monocytogenes displays the remarkable ability to reorganize the actin cytoskeleton within host cells as a means for promoting cell-to-cell transfer of the pathogen, in a manner that evades humoral immunity. In a series of events commencing with the biosynthesis of the bacterial surface protein ActA, host cell actin and many actin-associated proteins self-assemble to form rocket-tail structures that continually grow at sites proximal to the bacterium and depolymerize distally. Widespread interest in the underlying molecular mechanism of Listeria locomotion stems from the likelihood that the dynamic remodeling of the host cell actin cytoskeleton at the cell's leading edge involves mechanistically analogous interactions. Recent advances in our understanding of these fundamental cytoskeletal rearrangements have been achieved through a clearer recognition of the central role of oligo-proline sequence repeats present in ActA, and these findings provide a basis for inferring the role of analogous host cell proteins in the force-producing and position-securing steps in pseudopod and lamellipod formation at the peripheral membrane.