Structural determinants of microtubule minus end preference in CAMSAP CKK domains.

CAMSAP/Patronins regulate microtubule minus-end dynamics. Their end specificity is mediated by their CKK domains, which we proposed recognise specific tubulin conformations found at minus ends. To critically test this idea, we compared the human CAMSAP1 CKK domain (HsCKK) with a CKK domain from Naegleria gruberi (NgCKK), which lacks minus-end specificity. Here we report near-atomic cryo-electron microscopy structures of HsCKK- and NgCKK-microtubule complexes, which show that these CKK domains share the same protein fold, bind at the intradimer interprotofilament tubulin junction, but exhibit different footprints on microtubules. NMR experiments show that both HsCKK and NgCKK are remarkably rigid. However, whereas NgCKK binding does not alter the microtubule architecture, HsCKK remodels its microtubule interaction site and changes the underlying polymer structure because the tubulin lattice conformation is not optimal for its binding. Thus, in contrast to many MAPs, the HsCKK domain can differentiate subtly specific tubulin conformations to enable microtubule minus-end recognition.

Centriole assembly at a glance.

The centriole organelle consists of microtubules (MTs) that exhibit a striking 9-fold radial symmetry. Centrioles play fundamental roles across eukaryotes, notably in cell signaling, motility and division. In this Cell Science at a Glance article and accompanying poster, we cover the cellular life cycle of this organelle - from assembly to disappearance - focusing on human centrioles. The journey begins at the end of mitosis when centriole pairs disengage and the newly formed centrioles mature to begin a new duplication cycle. Selection of a single site of procentriole emergence through focusing of polo-like kinase 4 (PLK4) and the resulting assembly of spindle assembly abnormal protein 6 (SAS-6) into a cartwheel element are evoked next. Subsequently, we cover the recruitment of peripheral components that include the pinhead structure, MTs and the MT-connecting A-C linker. The function of centrioles in recruiting pericentriolar material (PCM) and in forming the template of the axoneme are then introduced, followed by a mention of circumstances in which centrioles form de novo or are eliminated.

Ubiquitin-like Atg8 protein is expressed during autophagy and the encystation process in Naegleria gruberi.

Members of the Naegleria genus are free-living amoebae, and the only pathogenic specie described to date for humans is N. fowleri. However, as the complete genome of this specie has not been reported, non-pathogenic N. gruberi is employed to describe molecular pathways in N. fowleri. Regardless, certain mechanisms, such as autophagy, have not yet been characterized in N. gruberi. Autophagy is involved in different cellular processes in some protozoa, including the recycling of unnecessary organelles, development, and cell differentiation. In this work, we characterized autophagy in N. gruberi using the specific inducer rapamycin. The formation of autophagy vacuoles in treated trophozoites was observed by ultrastructural analysis, and real time quantitative PCR demonstrated overexpression of the atg8 gene. In addition, we detected an increase in the vacuolar acidification of treated amoebae using the LysoTracker. Finally, confocal microscopy was utilized to identify Atg8 protein signal in the cytoplasm of N. gruberi trophozoites induced with rapamycin and even in trophozoites induced to encyst. In conclusion, N. gruberi possesses an Atg8 protein homolog that is overexpressed during the autophagic mechanism induced by rapamycin and also during encystation of this free-living amoeba.

Corifungin, a new drug lead against Naegleria, identified from a high-throughput screen.

Primary amebic meningoencephalitis (PAM) is a rapidly fatal infection caused by the free-living ameba Naegleria fowleri. The drug of choice in treating PAM is the antifungal antibiotic amphotericin B, but its use is associated with severe adverse effects. Moreover, few patients treated with amphotericin B have survived PAM. Therefore, fast-acting and efficient drugs are urgently needed for the treatment of PAM. To facilitate drug screening for this pathogen, an automated, high-throughput screening methodology was developed and validated for the closely related species Naegleria gruberi. Five kinase inhibitors and an NF-kappaB inhibitor were hits identified in primary screens of three compound libraries. Most importantly for a preclinical drug discovery pipeline, we identified corifungin, a water-soluble polyene macrolide with a higher activity against Naegleria than that of amphotericin B. Transmission electron microscopy of N. fowleri trophozoites incubated with different concentrations of corifungin showed disruption of cytoplasmic and plasma membranes and alterations in mitochondria, followed by complete lysis of amebae. In vivo efficacy of corifungin in a mouse model of PAM was confirmed by an absence of detectable amebae in the brain and 100% survival of mice for 17 days postinfection for a single daily intraperitoneal dose of 9 mg/kg of body weight given for 10 days. The same dose of amphotericin B did not reduce ameba growth, and mouse survival was compromised. Based on these results, the U.S. FDA has approved orphan drug status for corifungin for the treatment of PAM.

The structure of the mitotic spindle and nucleolus during mitosis in the amebo-flagellate Naegleria.

Mitosis in the amebo-flagellate Naegleria pringsheimi is acentrosomal and closed (the nuclear membrane does not break down). The large central nucleolus, which occupies about 20% of the nuclear volume, persists throughout the cell cycle. At mitosis, the nucleolus divides and moves to the poles in association with the chromosomes. The structure of the mitotic spindle and its relationship to the nucleolus are unknown. To identify the origin and structure of the mitotic spindle, its relationship to the nucleolus and to further understand the influence of persistent nucleoli on cellular division in acentriolar organisms like Naegleria, three-dimensional reconstructions of the mitotic spindle and nucleolus were carried out using confocal microscopy. Monoclonal antibodies against three different nucleolar regions and α-tubulin were used to image the nucleolus and mitotic spindle. Microtubules were restricted to the nucleolus beginning with the earliest prophase spindle microtubules. Early spindle microtubules were seen as short rods on the surface of the nucleolus. Elongation of the spindle microtubules resulted in a rough cage of microtubules surrounding the nucleolus. At metaphase, the mitotic spindle formed a broad band completely embedded within the nucleolus. The nucleolus separated into two discreet masses connected by a dense band of microtubules as the spindle elongated. At telophase, the distal ends of the mitotic spindle were still completely embedded within the daughter nucleoli. Pixel by pixel comparison of tubulin and nucleolar protein fluorescence showed 70% or more of tubulin co-localized with nucleolar proteins by early prophase. These observations suggest a model in which specific nucleolar binding sites for microtubules allow mitotic spindle formation and attachment. The fact that a significant mass of nucleolar material precedes the chromosomes as the mitotic spindle elongates suggests that spindle elongation drives nucleolar division.

Ultrastructural study of the encystation and excystation processes in Naegleria sp.

An important aspect of the biology of Naegleria sp. is the differentiation processes that occur during encystation and excystation. We studied these using both fluorescence and transmission electron microscopy techniques. In the initial stages of encystation, the cisternae of the endoplasmic reticulum became densely filled with a fibrillar material. Vesicles with a similar content that appeared to be derived from the cisternae were also observed in close contact with the plasma membrane. As encystation progressed, the fibrillar material became localized on the surface of the amoeba. An irregular compaction was observed in some areas of the cyst wall, which contained thin extensions of the cyst wall fibrillar material. Completely formed cysts had two to three ostioles, each sealed by an operculum. The operculum contained two areas in which a differential compaction of the fibrillar structure was observed. When excystation was induced, small dense granules (DGs), which were in close contact with fibrillar material were observed in the cyst cytoplasm and in the peritrophic space. During excystation, the more compact component of the operculum moves to enable the pseudopod of the emerging trophozoite to penetrate the ostiole. Vacuoles containing a fibrillar material, probably derived from the cyst wall, were observed in the cytoplasm of the pseudopodia. Our results provide a platform for further studies using biochemical markers to investigate the origin of the cyst wall as well as the role of DGs during excystation in Naegleria.

Temperature-shock induction of multiple flagella induces additional synthesis of flagellum specific mRNAs and tubulin.

Four mRNAs (alpha- and beta-tubulin, flagellar calmodulin and Class-I), specifically expressed when Naegleria amebae differentiate into flagellates, were followed at 5-10 min intervals during the temperature-shock induction of multiple flagella in order to better understand how basal body and flagellum number are regulated. Surprisingly, tubulin synthesis continued during the 37 min temperature shock. An initial rapid decline in alpha- and beta-tubulin and flagellar calmodulin mRNAs was followed by a rapid re-accumulation of mRNAs before the temperature was lowered. mRNA levels continued to increase until they exceeded control levels by 4-21%. Temperature shock delayed flagella formation 37 min, produced twice as much tubulin protein synthesis and three fold more flagella. Labeling with an antibody against Naegleria centrin suggested that basal body formation was also delayed 30-40 min. An extended temperature shock demonstrated that lowering the temperature was not required for return of mRNAs to near control levels suggesting that induction of multiple flagella and the formation of flagella per se are affected in different ways. We suggest that temperature-shock induction of multiple flagella reflects increased mRNA accumulation combined with interference with the regulation of the recently reported microtubule-nucleating complex needed for basal body formation.

Cell surface differences of Naegleria fowleri and Naegleria lovaniensis exposed with surface markers.

Differences in the distribution of diverse cell surface coat markers were found between Naegleria fowleri and Naegleria lovaniensis. The presence of carbohydrate-containing components in the cell coat of the two species was detected by selective staining with ruthenium red and alcian blue. Using both markers, N. fowleri presented a thicker deposit than N. lovaniensis. The existence of exposed mannose or glucose residues was revealed by discriminatory agglutination with the plant lectin Concanavalin A. These sugar residues were also visualized at the cell surface of these parasites either by transmission electron microscopy or by fluorescein-tagged Concanavalin A. Using this lectin cap formation was induced only in N. fowleri. The anionic sites on the cell surface detected by means of cationized ferritin were more apparent in N. fowleri. Biotinylation assays confirmed that even though the two amoebae species have some analogous plasma membrane proteins, there is a clear difference in their composition.

Morphologic and molecular identification of Naegleria dunnebackei n. sp. isolated from a water sample.

Naegleria dunnebackei n. sp., a new species of the free-living amoeboflagellate Naegleria, is described in this report. The organism was isolated from a water sample taken from drinking troughs associated with cases of primary amoebic meningoencephalitis in cattle at a ranch in southern California. The isolate grew at, but not above 37 degrees C, and did not kill young mice upon intranasal inoculation suggesting that it was not pathogenic. The new species combines morphological features of non-pathogenic Naegleria gruberi and pathogenic Naegleria fowleri. The trophic amoeba resembled other members of the genus, with a prominent vesicular nucleus and mitochondria with discoidal cristae; a Golgi apparatus was not observed by electron microscopy. The cyst stage had pores in the wall typical of those seen in pathogenic N. fowleri. Upon suspension in distilled water, amoebae transformed into temporary, non-feeding flagellates, mostly with two anterior flagella but occasionally with four. The rationale for its description as a new species was based upon sequencing of the 5.8S rDNA and internal transcribed spacers of the amoeba, which is similar to but not identical to that of Naegleria gallica, differing from that organism's DNA by six base pairs. Virus-like elements were found in the cytoplasm of trophic amoebae, often in association with crystalloids, and may be the cause of lysis of amoebae in culture.

De novo formation of basal bodies in Naegleria gruberi: regulation by phosphorylation.

The de novo formation of basal bodies in Naegleria gruberi was preceded by the transient formation of a microtubule (MT)-nucleating complex containing gamma-tubulin, pericentrin, and myosin II complex (GPM complex). The MT-nucleating activity of GPM complexes was maximal just before the formation of visible basal bodies and then rapidly decreased. The regulation of MT-nucleating activity of GPM complexes was accomplished by a transient phosphorylation of the complex. Inhibition of dephosphorylation after the formation of basal bodies resulted in the formation of multiple flagella. 2D-gel electrophoresis and Western blotting showed a parallel relationship between the MT-nucleating activity of GPM complexes and the presence of hyperphosphorylated gamma-tubulin in the complexes. These data suggest that the nucleation of MTs by GPM complexes precedes the de novo formation of basal bodies and that the regulation of MT-nucleating activity of GPM complexes is essential to the regulation of basal body number.

The flagellar apparatus of heteroloboseans.

The flagellar apparatus of four heterolobosean species Percolomonas descissus, Percolomonas sulcatus, Tetramitus rostratus, and Naegleria gruberi were examined. P. descissus lives in oxygen-poor water. It is a quadriflagellated cell with a ventral groove. The two pairs of basal bodies are connected to an apical structure from which the peripheral dorso-lateral microtubules and a short striated rhizoplast originate. There is one major microtubular root, R1, which originates from the posterior basal body pair and splits into left and right portions that support the sides of the ventral groove. The anterior pair of basal bodies is associated with a root of four to five microtubules that runs to the left of the groove. This organisation is similar to that previously reported for Psalteriomonas, Lyromonas, and Percolomonas cosmopolitus. Percolomonas sulcatus has two parallel pairs of basal bodies, each of which is associated with a well-developed R1 root. These roots divide to give two distinct left portions and one merged right portion that support the margins of the slit-like ventral groove. Tetramitus rostratus has two pairs of basal bodies, several rhizoplast fibres, and two R1 roots. Each R1 root supports one wall of the ventral groove. Naegleria gruberi may have two pairs of basal bodies, each associated with a microtubular root and one long rhizoplast fibre. From available data, a 'double bikont'-like organisation of the heterolobosean flagellar apparatus is inferred, where both of the eldest basal bodies have largely 'mature' complements of microtubular roots. The cytoskeletal organisation of heteroloboseans is compared to those of (other) excavates. Our structural data and existing molecular phylogenies weaken the case that Percolomonas, Psalteriomonas, and Lyromonas are phylogenetically separable from other heteroloboseans, undermining many of the highest-level taxa proposed for these organisms, including Percolozoa, Striatorhiza, Percolomonada, Percolomonadea, and Lyromonadea.

Identity of Naegleria strains isolated from organs of freshwater fishes.

Eighteen Naegleria strains were isolated from organs of freshwater fishes belonging to 5 species. Morphometric study allowed the separation of the Naegleria strains from the non-vahlkampfiid amoeboflagellates, but was inadequate for species determination. Six strains, representatives of groups that had a slightly different cyst size, were selected and corresponding derived clones were subjected to sequence analysis and riboprinting restriction fragment length polymorphism (RFLP)-PCR analysis of the small subunit (SSU) rRNA genes. One strain isolated from the brain of a fish with systemic infection was characterised by an intronless 2 kb long SSU rRNA gene and was identified as N. australiensis. Another 5 strains had a 1.3 kb long group I intron in their SSU rRNA gene and, based on the SSU rRNA sequences and riboprints, RFLP-PCR patterns appeared in phylogenetic trees to be closely related to Naegleria clarki.

Distribution of pathogenic Naegleria spp in Thailand.

Research concerning the distribution, isolation, viability, ultrastructure, morphology and immunogenicity of Naegleria fowleri has been increasing in Thailand during 1988-2000. The distribution of the organism was carried out from 1985 to 1987 in Si Sa Ket and Ubon Rachathani Provinces, after the first fatal case was reported in Si Sa Ket. Since then in a 1998 survey of N. fowleri in stagnant water around industrial areas was carried out in Pathum Thani, Samut Prakan and Lopburi provinces. The results showed that 10% of pathogenic Naegleria belonged to species fowleri as characterized by morphology and the occurrence of pathogenesis in mice after nasal inoculation. In the same year, Nacapunchai et al (1999) determined the prevalence of amebae in aquatic habitat of human environments in five parts of Thailand during the summer. Fourteen percent of free living Naegleria spp were found in both soil and water resources. Recent studies of the ultrastructure, factors affecting the viability and SDS-PAGE electrophoretic patterns of 3 Thai strains of pathogenic Naegleria spp indicated their similarities in morphological characteristics of pathogenic reference control, Naegleria fowleri CDC VO 3081. Additional study using a genetic approach to species criteria using allozyme electrophoresis had been conducted.

mRNAs for microtubule proteins are specifically colocalized during the sequential formation of basal body, flagella, and cytoskeletal microtubules in the differentiation of Naegleria gruberi.

We have examined the distribution of four mRNAs-alpha-tubulin, beta-tubulin, flagellar calmodulin, and Class I mRNA-during differentiation of Naegleria gruberi amebas into flagellates by in situ hybridization. Three of the four mRNAs-alpha-tubulin, beta-tubulin, and Class I mRNA-began to be colocalized at the periphery of the cells as soon as transcription of the respective genes was activated and before any microtubular structures were observable. At 70 min after the initiation of differentiation, these mRNAs were relocalized to the base of the growing flagella, adjacent to the basal bodies and microtubule organizing center for the cytoskeletal microtubules. Within an additional 15 min, the mRNAs were translocated to the posterior of the flagellated cells, and by the end of differentiation (120 min), very low levels of the mRNAs were observed. Cytochalasin D inhibited stage-specific localization of the mRNAs, demonstrating that RNA localization was actin dependent. Since cytochalasin D also blocked differentiation, this raises the possibility that actin-dependent RNA movement is an essential process for differentiation.

Seasonal distribution of pathogenic free-living amebae in Oklahoma waters.

Pathogenic free-living amebae cause serious human disease, including infection of the eye and the central nervous system. The purpose of this study was to sample aquatic environments in the Tulsa, Oklahoma, area year-round for the presence of these disease-causing amebae. A total of 34 pathogenic isolates were obtained from 2,016 processed water and swab samples. Pathogenicity was determined by the ability of amebae to cause death in mice after intranasal inoculation. Pathogenic amebae were isolated during every month of the year and were identified as Naegleria australiensis (38%), Acanthamoeba species (35%), N. fowleri (18%), and leptomyxid amebae (9%). Pathogenic leptomyxids have not previously been reported from the environment. The greatest percentage of recovery of pathogens occurred during the spring and autumn. The prevalence of pathogenic free-living amebae in the sampled waters was 1 pathogen/3.4 l water.

Nucleolus-like morphology produced during the in vitro reassociation of nucleolar components.

Nucleoli, the sites of rRNA synthesis, rRNA processing, and the assembly of ribosomes, are dynamic organelles that, in most cells, disperse and reform during mitosis. The mechanisms that regulate nucleolar formation are unknown as is the relationship between nucleolar morphology and the pathway of ribosome biogenesis. In this report we describe the in vitro formation of nucleolus-like particles (NLPs) from soluble extracts of nucleoli. NLPs, which reached sizes comparable to nucleoli (1-3 microns), were found to contain 40% of the nucleolar DNA, RNA, and protein. The ultrastructure of NLPs resembled that of a number of in vivo structures including compact nucleoli, prenucleolar bodies, and pseudonucleoli. The particles were composed of two morphologically distinct regions. The core resembled the dense fibrillar component (DFC) of nucleoli while the cortex resembled the granular component (GC) of nucleoli. The cortex of NLPs contained numerous 15-20 nm osmophilic granules that resembled the preribosomes found in the GC of nucleoli. The distribution of nucleolar proteins in NLPs also resembled that in nucleoli. BN46/51, a component of the GC of nucleoli, was restricted to the GC-like cortex of NLPs. A mAb that bound to the DFC of nucleoli, bound only to the DFC-like core of NLPs while a second mAb that bound to both the DFC and GC of nucleoli, bound to both the core and cortex of NLPs. Thus solubilized components of nucleoli can reassociate in vitro to produce particles that resemble nucleoli in their size, ultrastructure, and protein distribution.

Flagella number among Naegleria flagellates.

Scanning electron microscopy was used to determine the number of flagella on the flagellates of Naegleria australiensis, N. fowleri, N. gruberi, and N. jadini. Although the majority of flagellates had 2 flagella, there was considerable variation among individual cells. The number of flagella per flagellate varied from 1-8, with 2.4 being the average number per cell. For the different species, the average number of flagella per cell ranged from 2.0 in N. jadini to 3.1 for N. australiensis. The greatest amount of variation occurred in N. australiensis, with only 43% of the cells having 2 flagella. By contrast, 92% of N. fowleri cells had 2 flagella. Naegleria jadini and N. gruberi were intermediate with 80% and 74% biflagellates, respectively.

Amoeboid locomotion of Naegleria gruberi: the effects of cytochalasin B on cell-substratum interactions and motile behavior.

The major manifestations of amoeboid locomotion in Naegleria-cytoplasmic streaming, pseudopod production, cell polarity and focal contact production-require that the actin-based cytoskeleton be extremely dynamic. Whether these features are causally linked is unclear. In an attempt to answer this question we have used the fungal product cytochalasin B (cyt B) to dissect the motility process. This drug can perturb the organisation of actin filaments both in vivo and in vitro. Essentially cyt B acts as a molecule which can cap the barbed ends of actin filaments. Not surprisingly, therefore cyt B has an effect on rates of actin polymerization and the dynamic state of actin in the cytoplasm. We have found that cyt B has a profound effect on focal contact production and breakdown. Within minutes of addition of cyt B focal contact production ceases, existing focal contacts are stabilised but cytoplasmic streaming and pseudopod production are not blocked. In conclusion it is now clear that the state of actin required for focal contact production is different from that required for pseudopod extension and cytoplasmic streaming.

Naegleria lovaniensis tarasca new subspecies, and the purepecha strain, a morphological variant of N. l. lovaniensis, isolated from natural thermal waters in Mexico.

Amoebae were isolated from a natural thermal water source in Michoacán, Mexico, in September 1986. Two 500-ml samples were taken from pools with water at 45 degrees C and 46 degrees C and concentrated at 2,000 g for 15 min. The sediment was seeded on nonnutritive agar plates and incubated at 42 degrees C. The isolates were axenized in bactocasitone-serum medium. The identification of the isolates was based on their morphology, total protein and isoenzyme patterns by agarose isoelectric focusing, serology, fine structure, agglutination with Concanavalin A, sensitivity to trimethoprim, capacity to kill mice, and their cytopathic effect in Vero cells. The results showed several morphophysiological, biochemical and serological differences between the isolates and the type strain Aq/9/1/45D of Naegleria lovaniensis. These remarkable differences provide sufficient evidence to consider one of the isolates a new subspecies, and the other one a morphological variant of N. l. lovaniensis, which can be differentiated from other Naegleriae by their morphology, biochemistry, serology and physiology. The authors propose the name tarasca for the subspecies and purepecha for the morphological variant.

The interaction of Naegleria fowleri amoebae with murine macrophage cell lines.

The present study was undertaken to determine whether murine macrophage cell lines exhibited in vitro amoebicidal activity comparable to that elicited by activated murine peritoneal macrophages. Peritoneal macrophages activated in vivo by bacillus Calmette-Guérin or Propionibacterium acnes demonstrated significant cytolysis of Naegleria fowleri amoebae. The macrophage cell line RAW264.7 also effected cytolysis of amoebae, but to a lesser extent than that elicited by activated peritoneal macrophages. However, the macrophage cell lines, J774A.1 and P388D1, did not exhibit amoebicidal activity. Macrophage conditioned medium prepared from RAW264.7 macrophages mediated cytolysis of L929 tumor cells but had no effect on N. fowleri amoebae. In addition, neither recombinant tumor necrosis factor nor recombinant interleukin-1 exhibited amoebicidal activity. Scanning electron microscopy of co-cultures revealed that N. fowleri bound to activated peritoneal macrophages and RAW264.7 macrophages. These results suggest that RAW264.7 macrophages treated in vitro with lipopolysaccharide are similar to macrophages activated in vivo in that they effect contact-dependent cytolysis of Naegleria fowleri amoebae. The RAW264.7 macrophages are unlike primary macrophage cultures in that they either do not release soluble amoebicidal factors into the conditioned medium or they release insufficient quantities.