Cytochalasin D inhibits actin polymerization and induces depolymerization of actin filaments formed during platelet shape change.
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Actins↗
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'Superpolyamines'. Macrocyclic polyamines induce highly efficient actin polymerization.
Macrocyclic polyammonium ions induce much more efficient polymerization of actin than the natural polyamines, behaving thus as 'superpolyamines'. The processes observed display large increases in rate, marked structural dependence, macrocyclic and ditopic binding effects and positive cooperativity.
Actins↗
Neomycin induces stimulatory and inhibitory effects on leukotriene generation, guanine triphosphatase activity, and actin polymerization within human neutrophils.
The effects of neomycin on human neutrophils (PMN) were studied with respect to the generation of leukotrienes, the involvement of guanine triphosphate binding proteins (G proteins) and the polymerization of actin. Incubation of neutrophils with neomycin induced the generation of low amounts of leukotrienes. Co-incubation of neutrophils with neomycin and the direct G-protein activator sodium fluoride (NaF) resulted in an enhanced leukotriene formation at 0.5 mM neomycin and an inhibition at a concentration of 10 mM. Simultaneous incubation with neomycin and formyl-methionyl-leucyl-phenylalanine (FLMP) did not affect the FMLP-induced leukotriene formation. However, pretreatment of neutrophils with 10 mM neomycin followed by the addition of NaF or FMLP resulted in an enhanced generation of leukotrienes. Crude membrane fractions of PMN incubated with neomycin at different concentrations showed an enhanced (0.5 mM) as well as a reduced (10 mM) guanine triphosphatase activity. Furthermore, incubation of neutrophils with neomycin above a concentration of 0.5 mM led to the depolymerization of actin. The presented results show inhibitory and stimulatory effects of neomycin on various cell functions, which may reflect differences in transmembrane signalling.
Actins↗
Release of oxygen metabolites from chemoattractant-stimulated neutrophils is inhibited by resting platelets: role of extracellular adenosine and actin polymerization.
The effect of human platelets on chemoattractant-induced generation of oxygen metabolites in neutrophils was investigated, using luminol-enhanced chemiluminescence (CL). Resting platelets inhibited the extracellular, but not the intracellular, production of oxygen radicals in formyl-methionyl-leucyl-phenylalanine (fMet-Leu-Phe)-stimulated neutrophils. Maximal effect was obtained at the physiological neutrophil/platelet ratio of 1/50. Similar results were acquired by adding supernatants of platelets, indicating a role for a soluble factor. Removal of extracellular adenosine by adenosine deaminase (ADA), or blocking of adenosine-receptors by theophylline, antagonized the inhibitory effects of platelets (or the equivalent supernatant) on the neutrophil respiratory burst. In contrast, accumulation of adenosine by apyrase enhanced the inhibition. Exogenous adenosine mimicked the effects of platelets on the fMet-Leu-Phe-induced respiratory burst. To further assess the role of platelet-derived adenosine, the platelets were fixed with paraformaldehyde. We found that fixed platelets, as well as their supernatant, inhibited the fMet-Leu-Phe-induced CL-response to the same extent as viable cells. These effects were also reversed by ADA and theophylline, respectively. A prior removal of adenosine in the platelet suspension by ADA, followed by treatment with erythro-9-(2-hydroxy-3-nonyl)-adenine (EHNA) to inactivate ADA, did not reverse the inhibitory action of platelets on the fMet-Leu-Phe-induced CL-response in neutrophils. However, if adenosine receptors of neutrophil at the same time were blocked with theophyline, the inhibition was significantly reduced. Platelets markedly increased the generation of adenosine in a neutrophil suspension. The effect was antagonized by S-(4-Nitrobenzyl)-6-thioguanosine (NBTG), but unaffected by alpha, beta-methyl-eneadenosine5'diphosphate (AMP-CP), indicating that the platelet-dependent accumulation of adenosine is due to an increased release of endogenous adenosine from neutrophils and not to a degradation of extracellular AMP. In correlation, NBTG, but not AMP-CP, reversed the platelet-mediated inhibition of the fMet-Leu-Phe-induced CL-response in neutrophils. Consequently, these data suggest that a platelet-derived factor increases the release of endogenously formed adenosine from neutrophils, terminating the production of oxygen radicals. The inhibition of oxidase activity was also associated with a platelet-induced polymerization of actin in the margin of the neutrophils. Treatment of neutrophils with cytochalasin B reversed the effects of platelets, both on F-actin content and CL-response. In summary, resting platelets limit the release of oxygen radicals from chemoattractant-stimulated neutrophils, thus preventing excessive damage to host tissues in the vascular space. This effect is suggested to be associated with an increase generation of neutrophil-derived adenosine enhancing an autoregulatory inhibitory pathway, and a peripheral accumulation of actin filaments forming a barrier for extracellular release of reactive oxygen radicals.
Actin Cytoskeleton↗
Role of actin polymerization and adhesion to extracellular matrix in Rac- and Rho-induced cytoskeletal reorganization.
Most animal cells use a combination of actin-myosin-based contraction and actin polymerization- based protrusion to control their shape and motility. The small GTPase Rho triggers the formation of contractile stress fibers and focal adhesion complexes (Ridley, A.J., and A. Hall. 1992. Cell. 70:389-399) while a close relative, Rac, induces lamellipodial protrusions and focal complexes in the lamellipodium (Nobes, C.D., and A. Hall. 1995. Cell. 81:53-62; Ridley, A.J., H.F. Paterson, C.L. Johnston, D. Diekmann, and A. Hall. 1992. Cell. 70:401-410); the Rho family of small GTPases may thus play an important role in regulating cell movement. Here we explore the roles of actin polymerization and extracellular matrix in Rho- and Rac-stimulated cytoskeletal changes. To examine the underlying mechanisms through which these GTPases control F-actin assembly, fluorescently labeled monomeric actin, Cy3-actin, was introduced into serum-starved Swiss 3T3 fibroblasts. Incorporation of Cy3- actin into lamellipodial protrusions is concomitant with F-actin assembly after activation of Rac, but Cy3-actin is not incorporated into stress fibers formed immediately after Rho activation. We conclude that Rac induces rapid actin polymerization in ruffles near the plasma membrane, whereas Rho induces stress fiber assembly primarily by the bundling of actin filaments. Activation of Rho or Rac also leads to the formation of integrin adhesion complexes. Integrin clustering is not required for the Rho-induced assembly of actin-myosin filament bundles, or for vinculin association with actin bundles, but is required for stress fiber formation. Integrin-dependent focal complex assembly is not required for the Rac-induced formation of lamellipodia or membrane ruffles. It appears, therefore, that the assembly of large integrin complexes is not required for most of the actin reorganization or cell morphology changes induced by Rac or Rho activation in Swiss 3T3 fibroblasts.
3T3 Cells↗
Heat shock protein 27 is a substrate of cGMP-dependent protein kinase in intact human platelets: phosphorylation-induced actin polymerization caused by HSP27 mutants.
Phosphorylation of heat shock protein 27 (Hsp27) in human platelets by mitogen-activated protein kinase-activated protein kinase (MAPKAP) 2 is associated with signaling events involved in platelet aggregation and regulation of microfilament organization. We now show that Hsp27 is also phosphorylated by cGMP-dependent protein kinase (cGK), a signaling system important for the inhibition of platelet aggregation. Stimulation of washed platelets with 8-para-chlorophenylthio-cGMP, a cGK specific activator, resulted in a time-dependent phosphorylation of Hsp27. This is supported by the ability of cGK to phosphorylate Hsp27 in vitro to an extent comparable with the cGK-mediated phosphorylation of its established substrate vasodilator-stimulated phosphoprotein. Studies with Hsp27 mutants identified threonine 143 as a yet uncharacterized phosphorylation site in Hsp27 specifically targeted by cGK. To test the hypothesis that cGK could inhibit platelet aggregation by phosphorylating Hsp27 and interfering with the MAPKAP kinase phosphorylation of Hsp27, the known MAPKAP kinase 2-phosphorylation sites (Ser15, Ser78, and Ser82) as well as Thr143 were replaced by negatively charged amino acids, which are considered to mimic phosphate groups, and tested in actin polymerization experiments. Mimicry at the MAPKAP kinase 2 phosphorylation sites led to mutants with a stimulating effect on actin polymerization. Mutation of the cGK-specific site Thr143 alone had no effect on actin polymerization, but in the MAPKAP kinase 2 phosphorylation-mimicking mutant, this mutation reduced the stimulation of actin polymerization significantly. These data suggest that phosphorylation of Hsp27 and Hsp27-dependent regulation of actin microfilaments contribute to the inhibitory effects of cGK on platelet function.
Actins↗
Massive actin polymerization induced by phosphatidylinositol-4-phosphate 5-kinase in vivo.
The Rho family GTP-binding proteins have been known to mediate extracellular signals to the actin cytoskeleton. Although several Rho interacting proteins have been found, downstream signals have yet to be determined. Many actin-binding proteins are known to be regulated by phosphatidylinositol 4,5-bisphosphate in vitro. Rho has been shown to enhance the activity of phosphatidylinositol-4-phosphate 5-kinase (PI4P5K), the phosphatidylinositol 4,5-bisphosphate synthesizing enzyme. Recently we isolated several isoforms of type I PI4P5K. Here we report that PI4P5K Ialpha induces massive actin polymerization resembling "pine needles" in COS-7 cells in vivo. When truncated from the C terminus to amino acid 308 of PI4P5K Ialpha, both kinase activity and actin polymerizing activity were lost. Although the dominant negative form of Rho, RhoN19, alone decreased actin fibers, those induced by PI4P5K were not affected by the coexpression of RhoN19. These results suggest that PI4P5K is located downstream from Rho and mediates signals for actin polymerization through its phosphatidylinositol-4-phosphate 5-kinase activity.
Actins↗
Binding of IgG containing immune complexes to human neutrophil Fc gamma RII and Fc gamma RIII induces actin polymerization by a pertussis toxin-insensitive transduction pathway.
The ability of a phagocytic stimulus, rabbit IgG anti-BSA/BSA immune complexes, to increase the F-actin content of human polymorphonuclear leukocytes was quantitated by flow cytometry following staining with nitrobenzoxadiazole-phallacidin. A significant rise in F-actin assembly was induced by addition of 5 micrograms/ml immune complex. Concentrations of immune complex of more than 200 micrograms/ml caused a maximal (approximately twofold) increase in F-actin content. After a delay of 5 s, the F-actin levels rose and reached maximum levels by 60 s after adding immune complexes. The twofold elevation in F-actin persisted for up to 60 min. Both anti-Fc gamma RII and anti-Fc gamma RIII mAb blocked immune complex stimulated actin polymerization. Exposure to pertussis toxin failed to affect the rate or extent of immune complex-induced actin polymerization. Cells incubated with immune complexes and then lysed with Triton had an increased number of sites able to nucleate actin polymerization. These findings suggest that immune complex binding to both polymorphonuclear leukocytes Fc gamma RII and Fc gamma RIII is required for actin filament assembly and that the induction of assembly occurs via transduction pathways that differ from those used by chemoattractants. As with adhesion this phagocytic stimulus induces actin assembly by a pertussis toxin insensitive pathway and produces a rise in actin filament content that persists for prolonged periods of time.
Actins↗
What initiates actin polymerization?
For those working on the actin cytoskeleton, a major theme of the 39th annual meeting of the American Society for Cell Biology [http://www.faseb.org/ascb/meetings/am99/+ ++main99mtg.htm] (Washington DC, December 11-15, 1999) was the elucidation of how actin polymerization is initiated. The emphasis was on the regulation and localization of the Arp2/3 complex, which over the last two years has been shown to stimulate actin nucleation, and on the identification of additional proteins that interact with actin and Arp2/3 in a variety of organisms.
Actins↗
Tip-localized actin polymerization and remodeling, reflected by the localization of ADF, profilin and villin, are fundamental for gravity-sensing and polar growth in characean rhizoids.
Polar organization and gravity-oriented, polarized growth of characean rhizoids are dependent on the actin cytoskeleton. In this report, we demonstrate that the prominent center of the Spitzenkörper serves as the apical actin polymerization site in the extending tip. After cytochalasin D-induced disruption of the actin cytoskeleton, the regeneration of actin microfilaments (MFs) starts with the reappearance of a flat, brightly fluorescing actin array in the outermost tip. The actin array rounds up, produces actin MFs that radiate in all directions and is then relocated into its original central position in the center of the Spitzenkörper. The emerging actin MFs rearrange and cross-link to form the delicate, subapical meshwork, which then controls the statolith positioning, re-establishes the tip-high calcium gradient and mediates the reorganization of the Spitzenkörper with its central ER aggregate and the accumulation of secretory vesicles. Tip growth and gravitropic sensing, which includes control of statolith positioning and gravity-induced sedimentation, are not resumed until the original polar actin organization is completely restored. Immunolocalization of the actin-binding proteins, actin-depolymerizing factor (ADF) and profilin, which both accumulate in the center of the Spitzenkörper, indicates high actin turnover and gives additional support for the actin-polymerizing function of this central, apical area. Association of villin immunofluorescence with two populations of thick undulating actin cables with uniform polarity underlying rotational cytoplasmic streaming in the basal region suggests that villin is the major actin-bundling protein in rhizoids. Our results provide evidence that the precise coordination of apical actin polymerization and dynamic remodeling of actin MFs by actin-binding proteins play a fundamental role in cell polarization, gravity sensing and gravity-oriented polarized growth of characean rhizoids.
Actin Depolymerizing Factors↗
Mechanical stretch modulates the promoter activity of the profibrotic factor CCN2 through increased actin polymerization and NF-kappaB activation.
The connective tissue growth factor known as CCN2 is an inducible, profibrotic molecule that becomes aberrantly expressed in mechanical overload-bearing tissues. In this study, we found that CCN2 gene expression is rapidly induced in cyclically stretched bladder smooth muscle cells (SMCs) in vitro and in the detrusor muscle of a mechanically overloaded bladder in a rat model of experimental urethral obstruction. The activity of CCN2 promoter constructs, transiently transfected into cultured SMCs, was increased (up to 6-fold) by continuous cyclic stretching. Molecular analyses of the CCN2 promoter by serial construct deletions, cis-element mutagenesis, and electrophoretic mobility shift assays revealed that a highly conserved NF-kappaB binding site located within the CCN2 proximal promoter region is responsible for the activation of the promoter by stretch. Chromatin immunoprecipitation assays showed that NF-kappaB binds to the endogenous CCN2 promoter in both stretched cells and mechanically overloaded bladder tissues. Furthermore, stretch-dependent CCN2 promoter activity was significantly reduced upon inhibition of either phosphatidylinositol 3-kinase, p38 stress-activated kinase, or RhoA GTPase and was completely abolished upon inhibition of actin polymerization. Concordantly, actin polymerization was increased in either mechanically stretched cells or overloaded bladder tissues. Incubation of cultured SMCs with a cell-penetrating peptide containing the N-terminal sequence, Ac-EEED, of smooth muscle alpha-actin, altered both actin cytoskeleton organization and stretch-mediated nuclear relocation of NF-kappaB, and subsequently, it reduced CCN2 promoter activity. Thus, mechanical stretch-induced changes in actin dynamics mediate NF-kappaB activation and induce CCN2 gene expression, which probably initiates the fibrotic reactions observed in mechanical overload-associated pathologies.
Amino Acid Sequence↗
Saccharomyces cerevisiae Bzz1p is implicated with type I myosins in actin patch polarization and is able to recruit actin-polymerizing machinery in vitro.
In Saccharomyces cerevisiae, the WASP (Wiskott-Aldrich syndrome protein) homologue Las17p (also called Bee1p) is an important component of cortical actin patches. Las17p is part of a high-molecular-weight protein complex that regulates Arp2/3 complex-dependent actin polymerization at the cell cortex and that includes the type I myosins Myo3p and Myo5p and verprolin (Vrp1p). To identify other factors implicated with this complex in actin regulation, we isolated proteins that bind to Las17p by two-hybrid screening and affinity chromatography. Here, we report the characterization of Lsb7/Bzz1p (for Las seventeen binding protein 7), an Src homology 3 (SH3) domain protein that interacts directly with Las17p via a polyproline-SH3 interaction. Bzz1p coimmunoprecipitates in a complex with Las17p, Vrp1p, Myo3/5p, Bbc1p, Hsp70p, and actin. It colocalizes with cortical actin patches and with Las17p. This localization is dependent on Las17p, but not on F-actin. Bzz1p interacts physically and genetically with type I myosins. While deletion of BZZ1 shows no obvious phenotype, simultaneous deletion of the BZZ1, MYO3, and MYO5 genes is lethal. Overexpression of Bzz1p inhibits cell growth, and a bzz1Delta myo5Delta double mutant is unable to restore actin polarity after NaCl stress. Finally, Bzz1p in vitro is able to recruit a functional actin polymerization machinery through its SH3 domains. Its interactions with Las17p, Vrp1p, and the type I myosins are essential for this process. This suggests that Bzz1p could be implicated in the regulation of actin polymerization.
Actins↗
Inhibition of rat platelet aggregation by mycalolide-B, a novel inhibitor of actin polymerization with a different mechanism of action from cytochalasin-D.
In vitro effects of mycalolide-B (MB), isolated from marine sponge, were investigated with regard to the activation of rat platelets. Collagen-induced platelet aggregation in platelet-rich plasma (PRP) was slightly but significantly potentiated by lower concentrations of MB (0.3 and 1 microM) but was inhibited by higher concentrations (3 and 10 microM). ADP-induced platelet aggregation in PRP was also significantly prevented by MB (1-10 microM). Potentiation of ADP-induced aggregation by MB (0.3 microM) was hardly observed. G-actin contents, determined by DNase I inhibition assay, were increased in resting washed platelets incubated with MB (3 microM). In contrast, cytochalasin-D (CD) at 3 microM slightly reduced G-actin contents in resting platelets. After platelet aggregation with collagen (3 microg/ml) or ADP (10 microM), G-actin contents in platelets were reduced, indicating de novo actin polymerization. MB (3 microM) and CD (3 microM) abolished both ADP (10 microM)- and collagen (3 microg/ml)-induced platelet aggregation and actin polymerization in washed platelets. MB (1-10 microM) had no effects on intracellular Ca2+ concentrations in ADP (10 microM)-stimulated platelets. [125I]-fibrinogen binding to activated platelets with ADP (10 microM)(was inhibited by MB (0.3-3 microM) in a concentration-dependent manner. Thrombin-induced platelet-fibrin clot retraction was inhibited by MB (1 and 10 microM). These results suggest that MB inhibits platelet activation by interfering with actin polymerization through a different mechanism of action from CD. MB may be a useful tool for studying the role of actin polymerization in various cells.
Actins↗
Stimulation of neutrophil actin polymerization and degranulation by opsonized and unopsonized Candida albicans hyphae and zymosan.
We previously showed that unopsonized Candida albicans hyphae stimulated a delayed rise in the putative neutrophil second messengers Ca2+ and inositol 1,4,5-trisphosphate and subsequent O2- release, as compared with opsonized hyphae or zymosan. Therefore, cytoskeletal and degranulation temporal responses to these stimuli were examined. Unopsonized zymosan elicited no neutrophil responses under the experimental condition used. Neutrophil actin polymerization (quantitated by fluorescent measurements of NBD phallacidin) was rapid after stimulation by opsonized hyphae or zymosan (peaking at 1 and 2 min, respectively). This corresponded to observed changes in microscopic actin polymerization, measured with rhodamine phalloidin, which progressed from initially diffuse to collarlike to cylinderlike staining patterns surrounding the hyphae. Compared with opsonized hyphae, unopsonized hyphae resulted in a delayed appearance of the last two visible patterns (P less than 0.05) and in quantitative actin polymerization despite similarly rapid initial contact and spreading over the hyphae by neutrophils. Unlike other neutrophil responses, degranulation did not follow the delayed patterns of responses to stimulation with unopsonized hyphae. In the absence of the release of the cytoplasmic marker lactate dehydrogenase, the release of beta-glucuronidase, an azurophil granule marker, gradually and progressively rose in response to all of the stimuli but unopsonized zymosan. The low but significant levels observed were within a range consistent with published results for degranulation responses to particulate stimuli without cytochalasin B. A quantitative immunoassay of lactoferrin, a specific granule marker, detected no release into supernatants, and immunofluorescent staining indicated concomitant depletion of lactoferrin from neutrophil granules and binding to hyphal and neutrophil surfaces after stimulation by unopsonized hyphae. Thus, the delayed actin polymerization response to unopsonized hyphae occurred subsequent to neutrophil attachment and spreading and resembled the temporal sequence of other neutrophil responses linked to the respiratory burst. In contrast, the degranulation responses to all stimuli appeared to begin and progress gradually after observed attachment and spreading of the neutrophil over hyphal surfaces without a clear temporal relationship to rises in cytoplasmic Ca2+ or F-actin. In addition, the avid binding of released lactoferrin to cell surfaces eliminates its value as a quantitative marker of enzyme release but raises the possibility that it might participate in fungicidal activity.(ABSTRACT TRUNCATED AT 400 WORDS)
Actin Cytoskeleton↗
Cytochalasins inhibit nuclei-induced actin polymerization by blocking filament elongation.
Polylysine was found to induce polymerization of muscle actin in a low ionic strength buffer containing 0.4 mM MgCl2. The rate of induced polymerization was dependent on the amount and on the molecular size of the polylysine added. A similar effect was obtained by adding actin nuclei (containing about 2-4 actin subunits) cross-linked by p-N,N'-phenylenebismaleimide to G-actin under the same conditions, suggesting that the effect of polylysine is due to promotion of the formation of actin nuclei. Polymerization induced by polylysine and by cross-linked actin nuclei was inhibited by low concentrations (10(-8)-10(-6)M) of cytochalasins. Binding experiments showed that actin filaments, but not actin monomers, contained high-affinity binding sites for [3H]cytochalasin B (one site per 600 actin monomers). The relative affinity of several cytochalasins for these sites (determined by competitive displacement of [3H]dihydrocytochalasin B) was: cytochalasin D greater than cytochalasin E approximately equal to dihydrocytochalasin B. The results of this study suggest that cytochalasins inhibit nuclei-induced actin polymerization by binding to highly specific sites at the point of monomer addition, i.e., the elongation site, in actin nuclei and filaments.
Actins↗
Cyclic nucleotides modulate store-mediated calcium entry through the activation of protein-tyrosine phosphatases and altered actin polymerization in human platelets.
Agonists elevate the cytosolic calcium concentration in human platelets via a receptor-operated mechanism, involving both Ca(2+) release from intracellular stores and subsequent Ca(2+) entry, which can be inhibited by platelet inhibitors, such as prostaglandin E(1) and nitroprusside which elevate cAMP and cGMP, respectively. In the present study we investigated the mechanisms by which cAMP and cGMP modulate store-mediated Ca(2+) entry. Both prostaglandin E(1) and sodium nitroprusside inhibited thapsigargin-evoked store-mediated Ca(2+) entry and actin polymerization. However, addition of these agents after induction of store-mediated Ca(2+) entry did not affect either Ca(2+) entry or actin polymerization. Furthermore, prostaglandin E(1) and sodium nitroprusside dramatically inhibited the tyrosine phosphorylation induced by depletion of the internal Ca(2+) stores or agonist stimulation without affecting the activation of Ras or the Ras-activated phosphatidylinositol 3-kinase or extracellular signal-related kinase (ERK) pathways. Inhibition of cyclic nucleotide-dependent protein kinases prevented inhibition of agonist-evoked Ca(2+) release but it did not have any effect on the inhibition of Ca(2+) entry or actin polymerization. Phenylarsine oxide and vanadate, inhibitors of protein-tyrosine phosphatases prevented the inhibitory effects of the cGMP and cAMP elevating agents on Ca(2+) entry and actin polymerization. These results suggest that Ca(2+) entry in human platelets is directly down-regulated by cGMP and cAMP by a mechanism involving the inhibition of cytoskeletal reorganization via the activation of protein tyrosine phosphatases.
Actins↗
TccP2 of O157:H7 and non-O157 enterohemorrhagic Escherichia coli (EHEC): challenging the dogma of EHEC-induced actin polymerization.
Enterohemorrhagic Escherichia coli (EHEC) O157:H7 and enteropathogenic E. coli (EPEC) trigger actin polymerization at the site of bacterial adhesion by inducing different signaling pathways. Actin assembly by EPEC requires tyrosine phosphorylation of Tir, which subsequently binds the host adaptor protein Nck. In contrast, Tir(EHEC O157) is not tyrosine phosphorylated and instead of Nck utilizes the bacterially encoded Tir-cytoskeleton coupling protein (TccP)/EspF(U), which mimics the function of Nck. tccP is carried on prophage CP-933U/Sp14 (TccP). Typical isolates of EHEC O157:H7 harbor a pseudo-tccP gene that is carried on prophage CP-933 M/Sp4 (tccP2). Here we report that atypical, beta-glucuronidase-positive and sorbitol-fermenting, strains of EHEC O157 harbor intact tccP and tccP2 genes, both of which are secreted by the LEE-encoded type III secretion system. Non-O157 EHEC strains, including O26, O103, O111, and O145, are typically tccP negative and translocate a Tir protein that encompasses an Nck binding site. Unexpectedly, we found that most clinical non-O157 EHEC isolates carry a functional tccP2 gene that encodes a secreted protein that can complement an EHEC O157:H7 DeltatccP mutant. Using discriminatory, allele-specific PCR, we have demonstrated that over 90% of tccP2-positive non-O157 EHEC strains contain a Tir protein that can be tyrosine phosphorylated. These results suggest that the TccP pathway can be used by both O157 and non-O157 EHEC and that non-O157 EHEC can also trigger actin polymerization via the Nck pathway.
Actins↗
Signals for actin polymerization in neutrophils.
The role of the G proteins and of changes in the cytoplasmic levels of calcium and hydrogen ions in the initiation of actin polymerization in the neutrophils are discussed. The presently available data allow us to draw the following conclusions: 1) G proteins play a mediatory role in the transmission of the signal(s) generated upon receptor occupancy that leads to the observed cytoskeletal changes. 2) Increases in cytoplasmic free calcium appear to be neither necessary nor sufficient for the initiation of actin polymerization. 3) The activation of the Na+/H+ antiport is unrelated to the initiation of actin polymerization. 4) A possible relationship holds between the chemotactic factor elicited cytoplasmic acidification and the initiation of the reorganization of the cytoskeletal structures of these cells. However, further studies are necessary in order to further examine this hypothesis. Several avenues of research still need to be explored in order to derive a more comprehensive understanding of the mechanisms regulating the dynamic nature of the cytoskeleton of the neutrophils. These include, among others, potential roles of the hydrolysis of the polyphosphoinositides, and of the surface glycoproteins in this process.
Actins↗