Isolation of Naegleria fowleri from a domestic water tank associated with a fatal encephalitis in a 4 month-old Venezuelan child.

This study describes the association of household water system contamination with the pathogenic Free-Living Amoeba (FLA) Naegleria fowleri and a case of fatal Primary Amoebic Meningoencephalitis (PAM) in a child from the state of Monagas in Venezuela. Amoebae were initially identified by microscopy from a sample of cerebrospinal fluid (CSF) from the child. Direct DNA extraction and specific PCR/sequencing for N. fowleri was also carried out from the same CSF sample. In order to determine a possible environmental source of infection, water samples from the water tank of the child's home and also water bodies recently visited by the child and his family, were examined for the presence of N. fowleri by culture and PCR/sequencing. The results obtained from the collected water samples revealed that only the water tank of the house was positive for N. fowleri. PCR/sequencing showed that the strains isolated from the patient and the water tanks were 100 % identical. Therefore, the house water tank was confirmed as the source of infection in this case, possibly as a result of the occasional immersion of the child´s head under the water while bathing. This case highlights a novel source of thermally polluted water and another threat of N. fowleri infection.

Binding of gelsolin domain 2 to actin. An actin interface distinct from that of gelsolin domain 1 and from ADF/cofilin.

It is generally assumed that of the six domains that comprise gelsolin, domain 2 is primarily responsible for the initial contact with the actin filament that will ultimately result in the filament being severed. Other actin-binding regions within domains 1 and 4 are involved in gelsolin's severing and subsequent capping activity. The overall fold of all gelsolin repeated domains are similar to the actin depolymerizing factor (ADF)/cofilin family of actin-binding proteins and it has been proposed that there is a similarity in the actin-binding interface. Gelsolin domains 1 and 4 bind G-actin in a similar manner and compete with each other, whereas domain 2 binds F-actin at physiological salt concentrations, and does not compete with domain 1. Here we investigate the domain 2 : actin interface and compare this to our recent studies of the cofilin : actin interface. We conclude that important differences exist between the interfaces of actin with gelsolin domains 1 and 2, and with ADF/cofilin. We present a model for F-actin binding of domain 2 with respect to the F-actin severing and capping activity of the whole gelsolin molecule.

The second ADF/cofilin actin-binding site exists in F-actin, the cofilin-G-actin complex, but not in G-actin.

ADF/cofilins are actin binding proteins that bind actin close to both the N- and C-termini (site 1), and we have found a second cofilin binding site (site 2) centered around helix 112-125 [Renoult, C., Ternent, D., Maciver, S.K., Fattoum, A., Astier, C., Benyamin, Y. & Roustan, C. (1999) J. Biol. Chem. 274, 28893-28899]. We proposed a model in which ADF/cofilin intercalated between subdomains 1 and 2 of two longitudinally associated actin monomers within the actin:cofilin cofilament, explaining the change in twist that ADF/cofilins induce in the filament [McGough, A. Pope, B., Chiu, W. & Weeds, A. (1998) J. Cell Biol. 138, 771-781]. Here, we have determined the fuller extent of the cofilin footprint on site 1 of actin. Site 1 is primarily the G-actin binding site. Experiments with both peptide mimetics and fluorescently labeled cofilin suggest that site 2 only becomes available for cofilin binding within the filament, possibly due to motion between subdomains 1 and 2 within an actin monomer. We have detected motion between subdomains 1 and 2 of G-actin by FRET induced by cofilin, to reveal the second cofilin-binding site. This motion may also explain how cofilins inhibit the nucleotide exchange of actin, and why the actin:cofilin complex is polymerizable without dissociation.

Interaction of elongation factor 1alpha from Zea mays (ZmEF-1alpha) with F-actin and interplay with the maize actin severing protein, ZmADF3.

EF-1alpha is an abundant eukaryotic protein whose principle function appears to be to bind aminoacyl-tRNA to the ribosome. However, it is also known that EF-1alpha from other sources binds both microtubules and microfilaments. We report the expression of Zea mays EF-1alpha (ZmEF-1alpha) in bacteria and that this protein has similar actin-binding properties as other EF-1alpha members. ZmEF-1alpha bundles actin filaments at low pH (6.5) and inhibits the addition of monomer at both filament ends, possibly as a consequence. ZmEF-1alpha binds actin filaments at all pH values tested (pH 6.0-8.0), indicating that one actin binding site is not pH sensitive. One of the actin-binding sites was determined to reside within domain I (1-223) of ZmEF-1alpha, but this domain did not affect the kinetics of polymerisation. We show that the bundling activity of ZmEF-1alpha is modulated by ZmADF3 a (a Zea mays ADF/cofilin), an actin filament severing protein, in vitro. Bundling of actin filaments caused by ZmEF-1alpha was enhanced in the presence of ZmADF3. The pH-dependent activities of both proteins in vitro suggests that they may work together to respond to temporal and spatial intracellular pH changes to regulate the pattern of the growth of plant cells.

Interaction of pollen-specific actin-depolymerizing factor with actin.

We have examined the interaction of recombinant lily pollen ADF, LlADF1, with actin and found that whilst it bound both G- and F-actin, it had a much smaller effect on the polymerization and depolymerization rate constants than the maize vegetative ADF, ZmADF3. An antiserum specific to pollen ADF, antipADF, was raised and used to localize pollen ADF in daffodil--a plant in which massive reorganizations of the actin cytoskeleton have been seen to occur as pollen enters and exits dormancy. We show, for the first time, an ADF decorating F-actin in cells that did not result from artificial increase in ADF concentration. In dehydrated pollen this ADF : actin array is replaced by actin : ADF rodlets and aggregates of actin, which presumably act as a storage form of actin during dormancy. In germinated pollen ADF has no specific localization, except when an adhesion is made at the tip where actin and ADF now co-localize. These activities of pollen ADF are discussed with reference to the activities of ZmADF3 and other members of the ADF/cofilin group of proteins.

Rapid formation and high diffusibility of actin-cofilin cofilaments at low pH.

Cofilin is a small actin-binding protein that is known to bind both F-actin and G-actin, severing the former. The interaction of cofilin with actin is pH-sensitive, F-actin being preferentially bound at low pH and G-actin at higher pH, within the physiological range. Diffusion coefficients of F-actin with cofilin were measured by the fluorescence recovery after photobleaching (FRAP) technique. This has the potential for simultaneous and direct measurement of average polymer length via the average diffusion coefficient of the polymers (DLM) as well as the fraction of polymerized actin, fLM, present in solution. In the range of cofilin-actin ratios up to 1 : 1 and at both pH 6.5 and pH 8.0, the diffusion coefficients of the polymers increased with the amount of cofilin present in the complex, in a co-operative manner to a plateau. We interpret this as indicating co-operative binding/severing and that filaments less than a certain length cannot be severed further. Under the conditions used here, filaments were found to be more motile at pH 6.5 than at pH 8.0. At pH 8.0, some actin is expected to be sequestered as ADP-actin-cofilin complexes, with the remaining actin being present as long slowly diffusing filaments. At pH 6.5, however, cofilin binds to F-actin to form short rapidly diffusing cofilaments. These filaments form very rapidly from cofilin-actin monomeric complexes, possibly indicating that this complex is able to polymerize without dissociation. These findings may be relevant to the nuclear import of actin-cofilin complexes.

Domain 2 of gelsolin binds directly to tropomyosin.

Gelsolin is an actin filament severing protein composed of six similar structured domains that differ with respect to actin, calcium and polyphospho-inositide binding. Previous work has established that gelsolin binds tropomyosin [Koepf, E.K. and Burtnick, L.D. (1992) FEBS Lett. 309, 56-58]. We have produced various specific gelsolin domains in Escherichia coli in order to establish which of the six domains binds tropomyosin. Gelsolin domains 1-3 (G1-3), G1-2 and G2 all bind tropomyosin in a pH and calcium insensitive manner whereas binding of G4-6 to tropomyosin was barely detectable under the conditions tested. We conclude that gelsolin binds tropomyosin via domain 2 (G2).

The identification of a second cofilin binding site on actin suggests a novel, intercalated arrangement of F-actin binding.

The cofilins are members of a protein family that binds monomeric and filamentous actin, severs actin filaments, and increases monomer off-rate from the pointed end. Here, we characterize the cofilin-actin interface. We confirm earlier work suggesting the importance of the lower region of subdomain 1 encompassing the N and C termini (site 1) in cofilin binding. In addition, we report the discovery of a new cofilin binding site (site 2) from residues 112-125 that form a helix toward the upper, rear surface of subdomain 1 in the standard actin orientation (Kabsch, W., Mannherz, H. G., Suck, D., Pai, E. F., and Holmes, K. C. (1990) Nature 347, 37-44). We propose that cofilin binds "behind" one monomer and "in front" of the other longitudinally associated monomer, accounting for the fact that cofilin alters the twist in the actin (McGough, A., Pope, B., Chiu, W., and Weeds, A. (1997) J. Cell Biol. 138, 771-781). The characterization of the cofilin-actin interface will facilitate an understanding of how cofilin severs and depolymerizes filaments and may shed light on the mechanism of the gelsolin family because they share a similar fold with the cofilins (Hatanaka, H., Ogura, K., Moriyama, K., Ichikawa, S., Yahara, I., and Inagiki, F. (1996) Cell 85, 1047-1055).

The effect of two actin depolymerizing factors (ADF/cofilins) on actin filament turnover: pH sensitivity of F-actin binding by human ADF, but not of Acanthamoeba actophorin.

Actin depolymerizing factor (ADF) from vertebrates and actophorin from Acanthamoeba castellanii are members of a protein family that bind monomeric and polymeric actin and have been shown by microscopy to sever filaments. Here, we compare the properties of recombinant human ADF and actophorin using rabbit muscle actin. ADF binds tenfold more strongly than actophorin to monomeric actin (G-actin)-ATP, and both bind co-operatively to F-actin. ADF decorates filaments below pH 7.3 and induces substantial depolymerization at higher pH values [Hawkins, M., Pope, B., Maciver, S. K. & Weeds, A. G. (1993) Human actin depolymerizing factor mediates a pH-sensitive destruction of actin filaments, Biochemistry 32, 9985-9993], but, at all pH values tested, actophorin binds to filaments in a similar manner to ADF at pH 6.5. Both proteins increase the depolymerization rate at the pointed ends of gelsolin-capped filaments, but the effect of ADF is more marked at pH 8.0. Both proteins accelerate the nucleating activity when mixed with filamentous actin (F-actin), but not with gelsolin-capped filaments, and they rapidly decrease the lengths of filaments as evidenced by electron microscopy. Both of these effects are best explained by a weak severing activity. Our results are discussed in relation to earlier models and to the structural changes observed when ADF binds F-actin [McGough, A., Pope, B., Chiu, W. & Weeds, A. (1997) Cofilin changes the twist of F-actin: implications for actin filament dynamics and cellular function, J. Cell Biol. 138, 771-781]. We also discuss the relevance of these observations to their possible roles in facilitating actin turnover in cells, thereby regulating filament dynamics in cell motility.

Pollen specific expression of maize genes encoding actin depolymerizing factor-like proteins.

In pollen development, a dramatic reorganization of the actin cytoskeleton takes place during the passage of the pollen grain into dormancy and on activation of pollen tube growth. A role for actin-binding proteins is implicated and we report here the identification of a small gene family in maize that encodes actin depolymerizing factor (ADF)-like proteins. The ADF group of proteins are believed to control actin polymerization and depolymerization in response to both intracellular and extracellular signals. Two of the maize genes ZmABP1 and ZmABP2 are expressed specifically in pollen and germinating pollen suggesting that the protein products may be involved in pollen actin reorganization. A third gene, ZmABP3, encodes a protein only 56% and 58% identical to ZmABP1 and ZmABP2, respectively, and its expression is suppressed in pollen and germinated pollen. The fundamental biochemical characteristics of the ZmABP proteins has been elucidated using bacterially expressed ZmABP3 protein. This has the ability to bind monomeric actin (G-actin) and filamentous actin (F-actin). Moreover, it decreases the viscosity of polymerized actin solutions consistent with an ability to depolymerize filaments. These biochemical characteristics, taken together with the sequence comparisons, support the inclusion of the ZmABP proteins in the ADF group.

Myosin II function in non-muscle cells.

Amongst the remarkable variety of motility that cells display, cytokinesis (cell division) is particularly striking. Dramatic changes in cell shape occur before, during and after cytokinesis. Myosin II is implicated in the 'rounding up' of cells prior to cytokinesis, and is essential in the formation of the contractile cleavage furrow during cytokinesis. Now it appears that myosin II plays a role in all stages of cytokinesis, as a recent report suggests that myosin II drives post-mitotic cell spreading. A similar type of motile mechanism operating in cell spreading may occur in other cell types in other situations.

Two actin binding proteins, actin depolymerizing factor and cofilin, are associated with Hirano bodies.

Hirano bodies are intracellular, paracrystalline, rod-like structures which contain actin, tropomyosin, vinculin, alpha-actinin, amyloid beta-protein precursor and several microtubule associated proteins (MAPs). These bodies are observed more frequently in the elderly and in a number of neurodegenerative diseases including Alzheimer's disease. Many of the proteins known to be associated with Hirano bodies are actin binding proteins. We present immunohistological evidence that actin depolymerizing factor (ADF) and cofilin, two closely related proteins that bind and sever actin filaments, are also components of Hirano bodies. However, we could detect no difference in the levels of expression of either ADF or cofilin in the hippocampal tissue from normal individuals and Alzheimer's disease patients.

An actin-related protein from Dictyostelium discoideum is developmentally regulated and associated with mitochondria.

An actin-related protein (ACLA) has been identified in the cellular slime mould Dictyostelium discoideum. The complete cDNA sequence indicates that within the actin superfamily it belongs to the ARP3 family of actin-related proteins together with Arp66B from Drosophila melanogaster, Actin2 from Bos taurus, act2 from Schizosaccharomyces pombe and possibly act2 from Caenorhabditis elegans. The ACLA mRNA is regulated during development, showing a maximum between 2 and 4 h after starvation. The protein has been expressed in E. coli and antibodies raised against it. Immunofluorescence microscopy shows that ACLA protein co-localises with mitochondria; the protein copurifies with Dictyostelium mitochondria.

Utrophin actin binding domain: analysis of actin binding and cellular targeting.

Utrophin, or dystrophin-related protein, is an autosomal homologue of dystrophin. The protein is apparently ubiquitously expressed and in muscle tissues the expression is developmentally regulated. Since utrophin has a similar domain structure to dystrophin it has been suggested that it could substitute for dystrophin in dystrophic muscle. Like dystrophin, utrophin has been shown to be associated with a membrane-bound glycoprotein complex. Here we demonstrate that expressed regions of the predicted actin binding domain in the NH2 terminus of utrophin are able to bind to F-actin in vitro, but do not interact with G-actin. The utrophin actin binding domain was also able to associate with actin-containing structures, stress fibres and focal contacts, when microinjected into chick embryo fibroblasts. The expressed NH2-terminal 261 amino acid domain of utrophin has an affinity for skeletal F-action (Kd 19 +/- 2.8 microM), midway between that of the corresponding domains of alpha-actinin (Kd 4 microM) and dystrophin (Kd 44 microM). Moreover, this utrophin domain binds to non-muscle actin with a approximately 4-fold higher affinity than to skeletal muscle actin. These data (together with those of Matsumura et al. (1992) Nature, 360, 588-591) demonstrate for the first time that utrophin is capable of performing a functionally equivalent role to that of dystrophin. The NH2 terminus of utrophin binds to actin and the COOH terminus binds to the membrane associated glycoprotein complex, thus in non-muscle and developing muscle utrophin performs the same predicted 'spacer' or 'shock absorber' role as dystrophin in mature muscle tissues. These data suggest that utrophin could replace dystrophin functionally in dystrophic muscle.

Actophorin preferentially binds monomeric ADP-actin over ATP-bound actin: consequences for cell locomotion.

Actophorin from Acanthamoeba castellanii severs actin filaments and sequesters actin monomers. Here we report that actophorin binds ADP-bound monomers with higher affinity than ATP-bound monomers. Actophorin is therefore much less efficient at severing actin filaments in the presence of ADP compared to ATP, particularly taking account of the higher critical concentration in ADP. Monomer binding is also reduced in the presence of 25 mM inorganic phosphate (which is assumed to form ADP.Pi-actin). These findings are discussed in the light of observations on the nucleotide specificity of other monomer binding proteins and related to the role of actin in lamellar protrusion and cell locomotion.

Human actin depolymerizing factor mediates a pH-sensitive destruction of actin filaments.

ADF (actin depolymerizing factor) is an M(r) 19,000 actin-binding protein present in many vertebrate tissues and particularly abundant in neuronal cells. We have cloned human ADF and here show it to be identical in sequence to porcine destrin. Human ADF expressed in Escherichia coli behaves like native ADF from porcine brain. It binds to G-actin at pH 8 with a 1:1 stoichiometry and Kd approximately 0.2 microM, thereby sequestering monomers and preventing polymerization. It does not cosediment with F-actin at this pH, but severs actin filaments in a calcium-insensitive manner. The severing activity is only about 0.1% efficient. By contrast, at pH values below 7, ADF binds to actin filaments in a highly cooperative manner and at a 1:1 ratio to filament subunits. When the pH is raised to 8.0, the decorated filaments are rapidly severed and depolymerized.

Primary structure of and studies on Acanthamoeba actophorin.

We determined the amino acid sequence of the actin monomer binding/actin filament severing protein actophorin from Acanthamoeba castellanii by automated Edman degradation of peptide fragments and by sequencing of full-length cDNA. Actophorin consists of 138 amino acids (calculated molecular weight of 15,543) and shares a high degree of sequence similarity to other low molecular weight actin monomer sequestering proteins, especially vertebrate cofilin, vertebrate actin depolymerizing factor/destrin, and echinoderm depactin. Actophorin is smaller and does not contain a nuclear localization sequence like the related vertebrate proteins. Southern blot analysis indicates that actophorin is a single-copy gene; however, Northern blots show two distinct mRNA species of 1 and 0.9 kb in size. Homogeneous recombinant actophorin purified from Escherichia coli is indistinguishable from the native protein in its physical properties and in biochemical assays of its interaction with actin, but is less reactive with three monoclonal antibodies raised against the native protein. The NH2 terminus of native actophrin is blocked, while the initiating methionine residue is removed from recombinant actophorin. This difference has no measurable effect on activity. By fluorescent antibody staining of Acanthamoeba, actophorin colocalizes with actin filaments in the cortical cytoplasm, especially at the leading edge of the cell. Additionally, actophorin binds phosphatidylinositol 4',5'-bisphosphate. The recombinant actophorin forms X-ray diffraction quality crystals of superior quality in poly(ethylene glycol)/2-propanol and, like the native crystal form, belongs to space group P2(1)2(1)2(1).

Characterization of actin filament severing by actophorin from Acanthamoeba castellanii.

Actophorin is an abundant 15-kD actinbinding protein from Acanthamoeba that is thought to form a nonpolymerizable complex with actin monomers and also to reduce the viscosity of polymerized actin by severing filaments (Cooper et al., 1986. J. Biol. Chem. 261:477-485). Homologous proteins have been identified in sea urchin, chicken, and mammalian tissues. Chemical crosslinking produces a 1:1 covalent complex of actin and actophorin. Actophorin and profilin compete for crosslinking to actin monomers. The influence of actophorin on the steady-state actin polymer concentration gave a Kd of 0.2 microM for the complex of actophorin with actin monomers. Several new lines of evidence, including assays for actin filament ends by elongation rate and depolymerization rate, show that actophorin severs actin filaments both at steady state and during spontaneous polymerization. This is confirmed by direct observation in the light microscope and by showing that the effects of actophorin on the low shear viscosity of polymerized actin cannot be explained by monomer sequestration. The severing activity of actophorin is strongly inhibited by stoichiometric concentrations of phalloidin or millimolar concentrations of inorganic phosphate.