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PubMedDate not supplied

Observations on a trypanosomatid flagellate in a flea, Peromyscopsylla silvatica spectabilis.

The method of attachment of a trypanosomatid parasite to the cuticular epithelium of the hindgut of a flea Peromyscopsylla silvatica spectabilis by a desmosome-like structure is described; the flagella are modified and have enlarged intraflagellar areas which occupy the whole surface area of the gut, including clefts and folds of the epithelium. Unusual features of the parasite itself include a very much reduced flagellar pocket and pellicular microtubules which are made up of doublets and triplets. The problems of differentiation and characterisation of monogenetic insect flagellates are discussed in the light of these findings.

Animals↗
PubMed1976

Surface membrane carbohydrate alterations of a flagellated protozoan mediated by bacterial endosymbiotes.

Crithidia oncopelti, a parasitic trypanosomatid protozoan of insects, normally contains intracellular symbiotic bacteria. As shown earlier, the protozoa can be rid of their endosymbiotes by chloramphenicol, producing a symbiote-free cell line. Here surface-membrane carbohydrate ligands of the symbiote-containing and symbiote-free strains were compared by lectin-mediated agglutination, lectin-ultrastructure localization. [3H] lectin-binding, and fluorescent lectin staining. Symbiote-free organisms consistently had 3-fold higher agglutination titers than symbiote-containing cells with concanavalin A. Conversely, symbiote-containing flagellates had 2- to 3-fold greater agglutination titers with a fucose-binding lectin than symbiote-free organisms. Ultrastructure results showed that more of concanavalin A-horseradish peroxidase-diaminobenzidine reaction product was present at the surface of symbiote-free than on symbiote-containing cells. Treatment with [3H]concanavalin A revealed that surface membrane sites available per cell for [3H]lectin-binding ranged from 6.2 to 7.4 x 10(4) and from 24 to 27 x 10(4) for symbiote-containing and symbiote-free organisms, respectively, i.e., the mean binding level of the latter for the lectin was 3.5 times greater than that of the former. Moreover, symbiote-free cells fluoresced more than symbiote-containing organisms after staining with fluorescein-labeled concanavalin A. Apparently, the prokaryotic endosymbiotes somehow alter the quantity of saccharide ligands in the C. oncopelti surface membrane.

Animals↗
PubMed1984

Isolation of flagellated membrane vesicles from Caulobacter crescentus cells: evidence for functional differentiation of polar membrane domains.

An immunoaffinity chromatography procedure is described for the separation of membrane vesicles from Caulobacter crescentus cells into flagellated (polar) vesicles and nonflagellated (nonpolar) vesicles. Analysis by two-dimensional gel electrophoresis shows that a number of proteins are associated primarily with either the polar or the nonpolar fraction, and this result suggests that the envelope of these cells is organized into at least two relatively stable domains. Radioimmunoassay also shows that the membrane pool of flagellin, which is known to behave as a precursor in the assembly of the flagellar filament, may be localized exclusively in the polar membrane domain. Thus, the results provide biochemical evidence for the structural and functional differentiation of the C. crescentus cell envelope. These findings are consistent with a model we proposed previously to explain the targeting of surface structures to the new cell pole of C. crescentus. The immunoadsorption approach described here should be useful in the further investigation of this problem, as well as in the fractionation of membrane domains with characteristic surface antigens in other systems.

Bacteria↗
PubMed2003

Mitochondrial and nuclear localization of topoisomerase II in the flagellate Bodo saltans (Kinetoplastida), a species with non-catenated kinetoplast DNA.

We have studied topoisomerase II (topo II) in the cells of Bodo saltans, a free-living bodonid (Kinetoplastida). Phylogenetic analysis based on the sequence of the entire topo II gene, which is a single-copy gene, confirmed that B. saltans is a predecessor of parasitic trypanosomatids. Antibodies generated against either an overexpressed unique C-terminal region of topo II or a synthetic oligopeptide derived from the same region did not cross-react with cell lysates of related trypanosomatids, while they recognized a single specific band in the B. saltans lysate. Immunolocalization experiments using both antibodies showed that topo II is evenly dispersed throughout the kinetoplast. This is in striking difference from the localization of topo II in other flagellates, where it occurs in two antipodal centers flanking the kinetoplast disk. Moreover, the same topo II has a distinct localization in multiple loci at the periphery of the nucleus of B. saltans. With a minicircle probe derived from the conserved region we have shown that all relaxed non-catenated minicircles are confined to the globular kinetoplast DNA bundle. Therefore, in the mitochondrion of this primitive eukaryote topo II does not catenate relaxed DNA circles into a network in vivo, while a decatenating activity is present in partially purified cell lysates.

Amino Acid Sequence↗
PubMed2001

Calcium is involved in the gravitactic orientation in colorless flagellates.

The colorless flagellate Astasia longa shows a pronounced negative gravitaxis. The calcium fluorescence indicator Calcium Crimson was used to detect changes of the intracellular calcium concentration during gravitactical orientation. Astasia shows an increase of the fluorescence after a lag phase of about 10 s, a maximum after about 30 s and a decrease to the basic level within 60 s during gravitactic reorientation. The observed change in fluorescence corresponds to an almost doubling of the initial free calcium concentration. The influence of inhibitors, known to impair gravitaxis, on the calcium concentration of Astasia longa was tested. Addition of caffeine, an inhibitor of phosphodiesterase, increases, while addition of gadolinium, an inhibitor of mechanosensitive ion channels decreases the fluorescence signal. While gravitactic stimulation of caffeine-treated cells resulted in a kinetics of fluorescence intensity changes comparable to control cells the addition of gadolinium inhibited any calcium concentration change. Dynamic fluorescence imaging was used during a sounding rocket experiment (MAXUS 3 campaign). Different accelerations interrupted by microgravity intervals were applied to Astasia cells. The cells show an increase in the calcium signal upon acceleration and a decrease during the microgravity state. The results strongly reemphasize the working model of gravitaxis which is based on the activation of mechano-sensitive ion channels as one of the primary events in signal perception.

Acceleration↗
PubMed2002

Physiological parameters of gravitaxis in the flagellate Euglena gracilis obtained during a parabolic flight campaign.

The unicellular freshwater flagellate Euglena gracilis and its close relative Astasia longa show a pronounced negative gravitaxis. Previous experiments revealed that gravitaxis is most likely mediated by an active physiological mechanism in which changes of the internal calcium concentration and the membrane potential play an important role. In a recent parabolic flight experiment on board an aircraft (ESA 29th parabolic flight campaign), changes of graviorientation, membrane potential and the cytosolic calcium concentration upon changes of the acceleration (between 1 x g(n), 1.8 x g(n), microgravity) were monitored by image analysis and photometric methods using Oxonol VI (membrane potential) and Calcium Crimson (cytosolic calcium concentration). The parabolic flight maneuvers performed by the aircraft resulted in transient phases of 1.8 x g(n) (about 20 s), microgravity (about 22 s) followed by 1.8 x g(n) (about 20 s). A transient increase in the intracellular calcium concentration was detected from lower to higher accelerations (1 x g(n) to 1.8 x g(n) or microgravity to 1.8 x g(n)). Oxonol VI-labeled cells showed a signal, which indicates a depolarization during the transition from 1 x g(n) to 1.8 x g(n), a weak repolarization in microgravity followed by a rapid repolarization in the subsequent 1 x g(n) phase. The results show good coincidence with observations of recent terrestrial and space experiments.

Acceleration↗
PubMed2000

Ultrastructure and 18S rDNA sequence analysis of Wobblia lunata gen. et sp. nov., a new heterotrophic flagellate (Stramenopiles, Incertae sedis).

A new heterotrophic flagellate Wobblia lunata gen. et sp. nov. is described. This organism usually attaches to the substratum showing a wobbling motion, and sometimes glides on the substratum or swims freely in the medium. W. lunata has various features characteristic of the stramenopiles. These include a hairy flagellum with tripartite tubular hairs, a mitochondrion with tubular cristae, arrangement of flagellar apparatus components and a double helix in the flagellar transition zone. W. lunata shares a double helix with heterotrophic stramenopiles, including Developayella elegans, oomycetes, hyphochytrids, opalinids and proteromonads, and could be placed in the phylum Bigyra Cavalier-Smith. However, from 18S rDNA tree analysis, these organisms form two distantly-related clades in the stramenopiles, and Wobblia appears at the base of the stramenopiles. Evaluation of morphological features and comparison of 18S rDNA sequences indicate that W. lunata is a member of the stramenopiles, but it is distinct from any other stramenopiles so far described. Its phylogenetic position within the stramenopiles is uncertain and therefore W. lunata is described as a stramenopile incertae sedis.

Animals↗
PubMed2003

Phylogenetic diversity and whole-cell hybridization of oxymonad flagellates from the hindgut of the wood-feeding lower termite Reticulitermes flavipes.

SSU rRNA genes of oxymonad protists from the hindgut of the wood-feeding termite Reticulitermes flavipes were PCR-amplified using a newly designed oxymonad-specific forward primer and a newly designed reverse primer specific for termite gut flagellates. After cloning, the clone library was sorted into four groups by RFLP analysis and nearly full-length SSU rRNA gene sequences were obtained for representative clones from each group. Phylogenetic analysis revealed that sequences of all four groups formed a monophyletic cluster with the only other existing SSU rRNA gene sequence of oxymonads. Using whole-cell hybridization with clone-specific fluorescently labeled probes, each of the four clone groups could be assigned to a specific morphotype, which were identified as Dinenympha gracilis, Dinenympha fimbriata, and so-far undescribed species of Pyrsonympha and Dinenympha. Our results demonstrate that the morphological variety of oxymonads is not caused by the presence of different developmental stages of the same organism, but that the various morphotypes represent different species.

Animals↗
PubMed2001

Overexpresssion of a Legionella pneumophila homologue of the E. coli regulator csrA affects cell size, flagellation, and pigmentation.

Legionella pneumophila is an inhabitant of the aquatic environment and the causative agent of a bacterial pneumonia. We identified the presence of an L. pneumophila homologue of csrA of E. coli and rsmA of Erwinia carotovora, genes which regulate gene expression by destabilising mRNA and which have been shown to relate to environmental fitness and pathogenicity. The Legionella csrA was able to complement a csrA-negative mutant of E. coli. Overproduction of csrA in L. pneumophila lead to a reduction of flagellation and pigmentation and an increase in bacterial cell size. csrA overproduction was associated with a reduction of fliA and flaA transcripts. This suggests that similar to E. coli and Erwinia, L. pneumophila csrA is a regulator of gene expression and may contribute to the capability of the pathogen to rapidly adapt to changing environments.

Amino Acid Sequence↗
PubMed2000

Complete gene map of the plastid genome of the nonphotosynthetic euglenoid flagellate Astasia longa.

Astasia longa is a colourless heterotrophic flagellate closely related to the photoautotrophic Euglena gracilis. A circular 73 kb plastid DNA (ptDNA) has been isolated from A. longa that is about half the size of the chloroplast DNA of E. gracilis (143 kb). We have determined the complete sequence of the ptDNA of A. longa and established a complete gene map. All chloroplast genes for photosynthesis-related proteins are completely absent from the A. longa plastid DNA except for rbcL, the gene for the ribulose-1,5-bisphosphate carboxylase large subunit. Identified genes encode components of the plastid transcriptional and translational machinery: genes for three subunits of a chloroplast RNA polymerase, 20 chloroplast ribosomal protein genes, a gene for a plastid elongation factor Tu, 27 plastidic tRNA genes and three tandemly arranged repeats of 16S, 23S and 5S rDNA. Transcripts of a number of genes were detected by Northern hybridisation. The ribulose-1,5-bisphosphate carboxylase large subunit protein has been identified by immunoblotting.

Animals↗
PubMed1986

New poly(A)+RNAs appear coordinately during the differentiation of Naegleria gruberi amebae into flagellates.

We have examined the nature of the requirement for RNA synthesis during the differentiation of Naegleria gruberi amebae into flagellates (Fulton, C., and C. Walsh, 1980, J. Cell Biol., 85:346-360) by looking for poly(A)+RNAs that are specific to differentiating cells. A cDNA library prepared from poly(A)+RNA extracted from cells 40 min after initiation of the differentiation (40-min RNA), the time when formation of flagella becomes insensitive to inhibitors of RNA synthesis, was cloned into pBR322. Recombinant clones were screened for sequences that were complementary to 40-min RNA but not to RNA from amebae (0-min RNA). Ten of these differentiation-specific (DS) plasmids were identified. The DS plasmids were found to represent at least four different poly(A)+RNAs based on cross-hybridization, restriction mapping, and Northern blot analysis. Dot blot analysis was used to quantify changes in DS RNA concentration. The four DS RNAs appeared coordinately during the differentiation. They were first detectable at 10-15 min after initiation, reached a peak at 70 min as flagella formed, and then declined to low levels by 120 min when flagella reached full length. The concentration of the DS RNAs was found to be at least 20-fold higher in cells at 70 min than in amebae. The changes in DS RNA concentration closely parallel changes in tubulin mRNA as measured by in vitro translation (Lai, E.Y., C. Walsh, D. Wardell, and C. Fulton, 1979, Cell, 17:867-878).

Amoeba↗
PubMed1987

mRNAs for alpha- and beta-tubulin and flagellar calmodulin are among those coordinately regulated when Naegleria gruberi amebae differentiate into flagellates.

Three of four mRNAs that are specific to the differentiation of Naegleria gruberi amebae into flagellates (Mar, J., J. H. Lee, D. Shea, and C. J. Walsh, 1986, J. Cell Biol., 102:353-361) have been identified as coding for flagellar proteins. The products of these mRNAs, which are coordinately regulated during the differentiation, were identified by in vitro translation of hybrid-selected RNA followed by two-dimensional gel electrophoresis and antibody binding. Six cross-hybridizing clones complementary to a 1.7-kb RNA (class II) all selected mRNA that was translated into two alpha-tubulins. The principal in vitro product, alpha-1, comigrated with a cytoplasmic alpha-tubulin, while the minor product with a more acidic pI, alpha-2, comigrated with flagellar alpha-tubulin. While Naegleria flagellar alpha-tubulin was found to be acetylated based on its reaction with a monoclonal antibody specific to this form, we suggest that alpha-2 is not likely to arise due to acetylation in vitro but probably represents the product of a second alpha-tubulin gene. The class III clone, also complementary to a 1.7-kb RNA, selected beta-tubulin mRNA. In the course of this work it was found, using monoclonal antibodies to the alpha- and beta-subunits of tubulin, that Naegleria alpha-tubulin migrated faster than beta-tubulin on SDS-PAGE. The class IV clone, which hybridizes with a 0.5-kb RNA, selected an mRNA that was translated into a heat stable calcium-binding protein, flagellar calmodulin.

Animals↗
PubMed1965

THE FINE STRUCTURE AND FUNCTION OF THE CONTRACTILE AXOSTYLES OF CERTAIN FLAGELLATES.

The axostyles of the flagellates Oxymonas, Saccinobaculus, and Notila are large ribbon-shaped structures which undulate actively in the cytoplasm. The form of their movements is described and illustrated. Axostyles consist of regular arrays of longitudinal fibres, the number of which varies between 100 and 5000 in different species. The fibres are about 240 A in diameter, apparently hollow, regularly cross-banded with a periodicity of about 150 A, and connected by delicate cross-links, also at regular intervals of about 150 A. They resemble very closely the central fibres of cilia and flagella. No other structural components are present, except at the anterior end, where the fibres are attached to one or more basal bodies, and at the posterior tip, where they are anchored to the plasma membrane. The relevance of the findings to an understanding of the mechanism of ciliary and flagellar movements is discussed.

Adenosine Triphosphatases↗
PubMed1959

Cytoplasmic membranes and the nuclear membrane in the flagellate Trichonympha.

The structure of the nuclear and cytoplasmic membranes of Trichonympha, a complex flagellate, has been studied in the electron microscope. The nuclear membrane consists of two 70 A membranes, penetrated by numerous pores. Small (100 A) granules occur on the outer surface, around the rims of the pores. Granule-bearing membranes, only 30 to 40 A thick, form long, ribbon-shaped sacs, with 100 A granules on their outer surface. They apparently form close to the nucleus, from which they probably derive their granules. Smooth membranes occur in the parabasal bodies, which consist of stacks of 70 A membranes, joined at their edges in pairs to form flattened sacs. These can inflate and form cytoplasmic vesicles. A protein fibre is applied laterally to the pile of sacs. New sacs, replacing those lost by inflation, appear to form by a process involving the granular membranes, and there may be a transformation of one into the other. Starving eliminates granular membranes and results in a failure in the formation of new parabasal sacs. Refeeding reverses these effects. A parabasal body is a steady-state system, in which the rates of loss and gain of sacs are normally approximately equal. Parabasal bodies resemble the Golgi apparatus.

Animals↗
PubMed1960

On flagellar structure in certain flagellates.

This paper describes the structure of the flagella, basal bodies, and some of the associated fibre systems in three genera of complex flagellates, Trichonympha, Pseudotrichonympha, and Holomastigotoides. Three groups of longitudinal fibres occur in a flagellum: two central and nine outer fibres such as have been repeatedly described in other material, and an additional set of nine smaller secondary fibres not previously identified as such. Each central fibre shows a helical substructure; the pair of them are enveloped in a common sheath. Each outer fibre is a doublet with one subfibre bearing projections-called arms-that extend toward the adjacent outer fibre. The basal body is formed by a cylinder of nine triplet outer fibres. Two subfibres of each triplet continue into the flagellum and constitute the doublets. The third subfibre terminates at the transition of basal body to flagellum, possibly giving rise to the nine radial transitional fibres that seem to attach the end of the basal body to the surface of the organism. The central and secondary flagellar fibres are not present in the lumen of the basal body, but other complex structures occur there. The form of these intraluminal structures differs from genus to genus. The flagellar unit is highly asymmetrical. All the flagella examined have possessed the same one of the two possible enantiomorphic forms. At least two systems of fibres are associated with the basal bodies of all three genera.

Animals↗
PubMed1978

Laser microbeam study of a rotary motor in termite flagellates. Evidence that the axostyle complex generates torque.

A rotary motor in a termite flagellate continually turns the anterior part of the cell (head) in a clockwise direction. Previous descriptive observations implicated the noncontractile axostyle, which runs through the cell like a drive shaft, in the motile mechanism. This study demonstrates directly that the axostyle complex generates torque, and describes serval of its dynamic properties. By laser microbeam irradiation, the axostyle is broken into an anterior segment attached to the cell's head, and a posterior segment which projects caudally as a thin spike, or axostylar projection. Before lasing, both head and axostylar projection rotate at the same speed. After breaking the axostyle, the rotation velocity of the head decreases, depending on the length of the anterior segment. Head speed is not a linear function of axostyle length, however. In contrast, the rotation velocity of the axostylar projection always increases about 1.5 times after lasing, regardless of the length of the posterior segment. Turning the head is thus a load on the axostylar rotary motor, but the speed of the posterior segment represents the free-running motor. A third, middle segment of the axostyle, not connected to the head or axostylar projection, can also rotate independently. No ultrastructural differences were found along the length of the axostyle complex, except at the very anterior end; lenth-velocity data suggest that this region may not be able to generate torque. An electric model of the axostylar rotary motor is presented to help understand the length-velocity data.

Animals↗
PubMed1979

Membrane movements and fluidity during rotational motility of a termite flagellate. A freeze-fracture study.

Freeze-fracture electron microscopy was used to examine the structure of a region of plasma membrane that undergoes continual, unidirectional shear. Membrane shear arises from the continual clockwise rotation of one part (head) of a termite flagellate relative to the rest of the cell. Freeze-fracture replicas show that the lipid bilayer is continuous across the shear zone. Thus, the relative movements of adjacent membrane regions are visible evidence of membrane fluidity. The distribution and density of intramembrane particles within the membrane of the shear zone is not different from that in other regions of the cell membrane. Also, an additional membrane shear zone arises when body membrane becomes closely applied to the rotating axostyle as cells change shape in vitro. This suggests that the entire membrane is potentially as fluid as the membrane between head and body but that this fluidity is only expressed at certain locations for geometrical and/or mechanical reasons. Membrane movements may be explained solely by cell shape and proximity to rotating structures, although specific membrane-cytoskeletal connections cannot be ruled out. The membrane of this cell may thus be viewed as a fluid which adheres to the underlying cytoplasm/cytoskeleton and passively follows its movements.

Animals↗
PubMed1982

ATP reactivation of the rotary axostyle in termite flagellates: effects of dynein ATPase inhibitors.

The anterior end or head of a devescovinid flagellate from termites continually rotates in a clockwise direction relative to the rest of the cell. Previous laser microbeam experiments showed that rotational motility is caused by a noncontractile axostyle complex which runs from the head through the cell body and generates torque along its length. We report here success in obtaining glycerinated cell models of the rotary axostyle which, upon addition of ATP, undergo reactivation and exhibit rotational movements similar to those observed in vivo. Reactivation of rotational motility and flagellar beating of the models requires ATP or ADP and is competitively inhibited by nonhydrolyzable ATP analogs (AMP-PNP and ATP-gamma-S). N-ethylmaleimide, p-hydroxymercuribenzoate, and mersalyl acid also blocked reactivation of both the rotary axostyle and flagella. Vanadate and erythro-9-[3-(2-hydroxynonyl)]-adenine (EHNA) selectively inhibited flagellar reactivation without effecting rotational motility. These results, together with previous ultrastructural findings, suggest that the rotary axostyle does not operate by a dynein-based mechanism but may be driven by an actomyosin system with a circular arrangement of interacting elements.

Adenine↗