Free kinetosomes in Austrialian flagellates. I. Types and spatial arrangement

An electron microscope study was made of Deltotrichonympha and Koruga, two closely-related hypermastigote flagellates that live in the hindgut of the Australian termite, Mastotermes darwiniensis These symbiotic protozoans have a typical flagellated rostrum and long body flagella. Their "giant centrioles" (centriolar apparatus) are large, fibrillar, and granular bodies which do not resemble typical centrioles in structure. The unique feature of interphase cells is the presence of more than half a million free kinetosomes in the anterior cytoplasm. Two classes of free kinetosomes, differing in length and spatial arrangement, were found. 500,000–750,000 short free kinetosomes are concentrated in a dense column which extends from the centriolar apparatus in the rostrum to the anterior side of the nucleus Most of the short free kinetosomes in the column are arranged end-to-end in chains of varying lengths. Within a kinetosomal chain, all of the individual kinetosomes face in the same direction with respect to their cartwheel ends In most flagellates, the short free kinetosomes are 0 07–0.13 µ long, and are remarkably similar in length within any cell Occasionally, cells with uniformly "longer" short free kinetosomes are found. 70,000–120,000 long free kinetosomes are scattered singly throughout the cytoplasm between the column of short free kinetosomes and the cell surface These long free kinetosomes are 0 4–0 7 µ long, similar in length to the kinetosomes of the body flagella, and are oriented parallel to the anterior-posterior axis of the cell. The significance of this remarkable accumulation of free kinetosomes is discussed.     

The Fine Structure of the Centriolar Apparatus and Associated Structures in the Flagellates Deltotrichonympha and Koruga. II. Division

SYNOPSIS. At division of Deltotrichonympha operculata and Koruga bonita from the Australian termite, Mastotermes darwiniensis , the 2 centriolar bodies separate, each becoming a mitotic center. Spindle microtubules develop from the lower end of each centriolar body and radiate towards the elongating nucleus. A new rostrum is formed in association with each centriolar body. Thus, centriolar bodies which lack the structure of typical centrioles can nevertheless function as division centers during mitosis.     

Ultrastructure and ATPase activity of the centriolar body at the mitotic stage in neoblasts of the planarianDugesia sp.

The fine structure and ATPase activity of the mitotic spindle in neoblasts of planaria were examined. In neoblasts, the cells have a large nucleus and nucleolus. Mitochondria are aggregated around the nucleus with chromatoid bodies adjacent. The cytoplasm contains little endoplasmic reticulum (ER) and few Golgi bodies but many free ribosomes, forming polysomes, can be seen throughout the cytoplasmic and spindle ground areas. In addition, centriolar bodies, atypical centrioles, can also be recognized in the cytoplasm. Cells in the G₂ stage contain a pair of electron-dense bodies, both consisting of fibrogranules but differing from each other in fine structure and, in the mitotic stage, only one fibrogranular body can be recognized at each pole. ATPase activity was detected in the centriolar bodies in the G₂ and mitotic stages and in the ground area of the cytoplasm and spindle apparatus filled by free ribosomes. The activity associated with the microtubules differed with the developmental stage.     

Ciliogenesis and centriole formation in the mouse embryonic nervous system. An ultrastructural analysis

Serial ultrathin sections were used to study the formation of the primary cilium and the centriolar apparatus, basal body, and centriole in the neuroepithelial primordial cell of the embryonic nervous system in the mouse. At the end of mitosis, the centrioles seem to migrate toward the ventricular process of the neuroepithelial cell, near the ventricular surface. One of these centrioles, the nearest to the ventricular surface, begins to mature to form a basal body, since its tip is capped by a vesicle probably originating in the cytoplasm. This vesicle fuses with the plasmalemma and the cilium growth by the centrifugal extension of the 9 sets of microtubule doublets. These 9 sets invade the thick base of the cilium which is initially capped by a ball-shaped tip with the appearance of a mushroom cilium. The secondary extension of 7, then 5, and finally 2 sets of microtubule doublets contribute to form the tip of the mature cilium, which is associated with a mature centriolar apparatus formed by a basal body and a centriole. Centriologenesis occurs before mitosis and is concomitant with the progressive resorption of the cilium. The daughter centriole, or procentriole, begins to take form near the tips of fibrils that extend perpendicularly and at a short distance from the wall of the parent centriole. Osmiophilic material accumulates around these fibrils, and gives rise to the microtubules of the mature daughter centriole. These centrioles formed by a centriolar process are further engaged in mitosis, after the total resorption of the cilium. This pattern of development suggests that in the primordial cells of the embryonic nervous system, centriologenesis and ciliogenesis are 2 independent phenomena.     

DEVELOPMENT OF THE FLAGELLAR APPARATUS OF NAEGLERIA

Flagellates of Naegleria gruberi have an interconnected flagellar apparatus consisting of nucleus, rhizoplast and accessory filaments, basal bodies, and flagella. The structures of these components have been found to be similar to those in other flagellates. The development of methods for obtaining the relatively synchronous transformation of populations of Naegleria amebae into flagellates has permitted a study of the development of the flagellar apparatus. No indications of rhizoplast, basal body, or flagellum structures could be detected in amebae. A basal body appears and assumes a position at the cell surface with its filaments perpendicular to the cell membrane. Axoneme filaments extend from the basal body filaments into a progressive evagination of the cell membrane which becomes the flagellum sheath. Continued elongation of the axoneme filaments leads to differentiation of a fully formed flagellum with a typical "9 + 2" organization, within 10 min after the appearance of basal bodies.     

THE FINE STRUCTURE OF MITOSIS IN RAT THYMIC LYMPHOCYTES

The fine structure of rat thymic lymphocytes from early prophase to late telophase of mitosis is described, using material fixed at pH 7.3 either in 1 per cent OsO4 or in glutaraldehyde followed by 2 per cent OsO4. The structure of the centriolar complex of interphase thymocytes is analyzed and compared with that of centrioles during division. The appearance of daughter centrioles is the earliest clearly recognizable sign of prophase. Daughter centrioles probably retain a secondary relation to the primary centriole, while the latter appears to be related, both genetically and spatially, to the spindle apparatus. The nuclear envelope persists in recognizable form to help reconstitute the envelopes of the daughter nuclei. Ribosome bodies (dense aggregates of ribosomes) accumulate, beginning at late prophase, and are retained by the daughter cells. Cytokinesis proceeds by formation of a ribosome-free plate at the equator with a central plate of vesicles which may coalesce to form the new plasma membrane of the daughter cells. Stages in the formation of the midbody are illustrated.     

Ultrastructure of the flagellate Cruzella marina (Kinetoplastidea)

The ultrastructure of a marine, free-living heterotrophic kinetoplastid Cruzella marina was investigated with special attention being paid to the mitochondrion and flagellar organization. The flagellates have a polykinetoplastidal mitochondrion. Two flagella emerge from the pocket; one of these turns anteriorly being forward-directed, while the other is posteriorly directed to be adjacent to the ventral cell surface. The transition zone of both the flagella includes central filaments. The cytostome opens on the tip of the rostrum. The cytostome leads to the channel of cytopharynx, which penetrates the rostrum and proceeds into the flagellate body cytoplasm. The comparison of the relevant morphological and molecular data suggest that C. marina may arise early in the Kinetoplastidea lineage, before divergence of the majority taxa of the kinetoplastid flagellates.     

Ultrastructure of Lamblia duodenalis. I. Body Surface, Sucking Disc and Median Bodies

SYNOPSIS. The body surface, sucking disc and median bodies of Lamblia duodenalis have been studied on ultrathin sections in the electron microscope. The body is covered by a pellicle, displaying a striated structure in the area of the sucking disc. The striation is due to 150 Å thick dense ridges which are spaced in distances of 200–400 Å. The ridges are formed by the internal pellicular membrane and have a triangular cross section with a very dense apex. They are arranged concentrically and run parallel to the surface of the sucking disc lobes. Anteriad to the nuclei in the median line a space is free of ridges. The margin of the sucker is elevated above the body forming a sharp crest of the ridged pellicle.
This crest is the inner wall of a marginal groove delimiting the sucker from the body. The outer margin is circumscribed by a fold in the body tapering to the posterior end. A ventral groove containing the two ventral flagella lies in the median line. The movement of the ventral flagella pushes the medium through the marginal and ventral grooves thus producing vacuum in the sucker area.
The median bodies are composed of numerous 150 Å thick tubular fibrils. They differ in their ultrastructure from the parabasal apparatus in other flagellates and have nothing in common with the kinetoplasts. Their functional significance awaits elucidation.     

Localization of Tubulin and a Centrin-Homologue in Vegetative Cells and Developing Gametangia of Ectocarpus siliculosus (Dillw.) Lyngb. (Phaeophyceae,Ectocarpales)

The distribution of tubulin and centrin in vegetative cells and during gametogenesis of Ectocarpus siliculosus was studied by immunofluorescence. In interphase cells bundles of microtubules are focused on the centriolar region near the nuclear surface. Some of the bundles ensheath the nucleus while others traverse the cytoplasm in various directions, sometimes reaching the cell cortex. Evaluation of serial optical sections by confocal laser scanning microscopy (CLSM) revealed that the perinuclear and “cytoplasmic” microtubule bundles presumably constitute a single complex. In interphase cells centrin is localized as a single bright spot in the centriolar region. In dividing cells duplication and separation of the microtubular complex and the centrin spot takes place. In post-mitotic cells with two nuclei, the centrioles are located at opposite cell poles, short microtubule bundles emanate from them and partially encompass the nucleus. During gametogenesis a gradual transformation of the vegetative cytoskeleton to the gametic flagellar apparatus occurs.     

Light and electron microscopical studies onHafniomonas gen. nov. (Chlorophyceae,Volvocales), a genus resemblingPyramimonas (Prasinophyceae)

Based on light and electron microscopical studies ofPyramimonas reticulata the genusPyramimonas is shown to contain a number of unrelated flagellates.P. reticulata andP. montana are transferred to the new genusHafniomonas, cells of which differ fromPyramimonas in shape, in the absence of scales and hairs on the body and flagellar surfaces, in details of the chloroplast, the position of the nucleus, the Golgi apparatus, the internal structure of the flagellar apparatus, and in cell division. The prasinophytePyramimonas contains a characteristic association of a large microbody and a rhizoplast, situated on the nuclear surface. A similar association is being found in an increasing number of prasinophycean flagellates, but is absent inHafniomonas, which is considered related to chlorophycean rather than prasinophycean flagellates. The phylogenetic position ofHafniomonas is discussed, based in particular on details of the unique flagellar apparatus.     

The oral apparatus of Tetrahymena pyriformis, strain WH-6. IV. Observations on the organization of microtubules and filaments in the isolated oral apparatus and the differential effect of potassium chloride on the stability of oral apparatus microtubules.

This report is an ultrastructural analysis of the organization of the isolated oral apparatus of Tetrahymena pyriformis, strain WH-6, syngen 1. Attention has been focused on the organization of microtubules and filaments in oral apparatus membranelles. Oral apparatus membranellar basal bodies were characterized with respect to structural differentiations at the distal and proximal ends. The distal region of membranellar basal bodies contains the basal plate, accessory microtubules and filaments. The proximal end contains a dense material from which emanate accessory microtubules and filaments. There are at least two possibly three different arrangements of accessory structures at the proximal end of membranellar basal bodies. All membranellar basal bodies appear to have a dense material at the proximal end from which filaments emanate. Some of these basal bodies have accessory microtubules and filaments emanating from this dense material. A possible third arrangement is represented by basal bodies which have lateral projections, from the proximal end, of accessory microtubules and filaments which constitute cross or peripheral connectives. There are at least three examples of direct associations between oral apparatus microtubules and filaments: (1) filaments which form links between basal body triplet microtubules, (2) filaments which link the material of the basal plate to internal basal body microtubules, (3) filaments which link together microtubule bundles from membranellar connectives. KCl extraction of the isolated oral apparatus resulted in the selective solubilization of oral apparatus basal bodies, remnants of ciliary axonemes and fused basal plates. Based on their response to KCl extraction two distinct sets of morphologically similar micro tubules can be identified: (a) microtubules which constitute the internal structure of basal bodies and ciliary axonemes, (b) microtubules which constitute the fiber connectives between basal bodies.     

Cell cycle-dependent, in vitro assembly of microtubules onto pericentriolar material of HeLa cells

A centriolar complex comprising a pair of centrioles and a cloud of pericentriolar materials is located at the point of covergence of the microtubules of the mitotic apparatus. The in vitro assembly of microtubules was observed onto these complexes in the 1,400 g supernatant fraction of colcemid-blocked, mitotic HeLa cells lysed into solutions containing tubulin and Triton X-100. Dark-field microscopy provided a convenient means by which this process could be visualized directly. When this 1,400 g supernate was incubated at 30 degrees C and centrifuged into a discontinuous sucrose gradient, a band containing centriolar complexes and assembled microtubles was obtained at 50-60% sucrose interface. Ultrastructual analysis indicated that the majority of the microtubules assembled predominantly from the pericentriolar material but also onto the centrioles. When cells were synchronized by a double thymide block, the assembly of microtubules onto centriolar complexes was observed only in lysates of mitotic cells; no assembly was seen in lysed material of interphase cells. Microtubule assembly occured onto centriolar complexes in solutions of either 100,000 g brain supernate, 2 X cycled tubulin, or purified tubulin dimers. This study demonstrates that the pericentriolar material becomes competent as a microtubule-organizing center (MTOC) at the time of mitosis. With use of the techniques described, a method for the isolation of centriolar complexes may be developed.     

Transformation of the flagella and associated flagellar components during cell division in the coccolithophoridPleurochrysis carterae

Summary Immunofluorescence microscopy, conventional and high voltage transmission electron microscopy were used to describe changes in the flagellar apparatus during cell division in the motile, coccolithbearing cells ofPleurochrysis carterae (Braarud and Fagerlund) Christensen. New basal bodies appear alongside the parental basal bodies before mitosis and at prophase the large microtubular (crystalline) roots disassemble as their component microtubules migrate to the future spindle poles. By prometaphase the crystalline roots have disappeared; the flagellar axonemes shorten and the two pairs of basal bodies (each consisting of one parental and one daughter basal body) separate so that each pair is distal to a spindle pole. By late prometaphase the pairs of basal bodies bear diminutive flagellar roots for the future daughter cells. The long flagellum of each daughter cell is derived from the parental basal bodies; thus, the basal body that produces a short flagellum in the parent produces a long flagellum in the daughter cell. We conclude that each basal body in these cells is inherently identical but that a first generation basal body generates a short flagellum and in succeeding generations it produces a long flagellum. At metaphase a fibrous band connecting the basal bodies appears and the roots and basal bodies reorient to their interphase configuration. By telophase the crystalline roots have begun to reform and the rootlet microtubules have assumed their interphase appearance by early cytokinesis.Abbreviations CR1, CR2 crystalline roots 1 and 2 - CT cytoplasmic tongue microtubules - DIC differential interference contrast light microscopy - H haptonema - HVEM high voltage transmission electron microscopy - IMF immunofluorescence microscopy - L left flagellum/basal body - M metaphase plate - MT microtubule - N nucleus - R right flagellum/basal body - R1, R2, R3 roots 1, 2, and 3 - TEM transmission electron microscopy     

Evidence for a direct role of nascent basal bodies during spindle pole initiation in the green alga Spermatozopsis similis.

Basal body replication in the naked biflagellate green alga Spermatozopsis similis was analyzed using standard electron microscopy and immunogold localization of centrin, an ubiquitous centrosomal protein, and p210, a recently characterized basal apparatus component of S. similis. Fibrous disks representing probasal bodies appear at the proximal end of parental basal bodies at the end of interphase and development proceeds via a ring of nine singlet microtubules. Nascent basal bodies dock early to the plasma membrane but p210, usually present in basal body-membrane-linkers of S. similis, was already present on the cytosolic basal body precursors. In addition to the distal connecting fiber and the nuclear basal body connectors (NBBC) of the parental basal bodies, centrin was present on the fibrous probasal bodies, in a linker between probasal bodies and the basal apparatus, in the connecting fiber between nascent basal bodies and their corresponding parent, and, finally, a fiber linking the nascent basal bodies to the nucleus. This NBBC probably is present only in mitotic cells. During elongation a cartwheel of up to seven layers is formed, protruding from the proximal end of nascent basal bodies. Microtubules develop on the cartwheel indicating that it temporarily functions as a microtubule organizing center (MTOC). These microtubules and probably the cartwheels, touch the nuclear envelope at both sides of a nuclear projection. We propose that spindle assembly is initiated at these attachment sites. During metaphase, the spindle poles were close to thylakoid-free lobes of the chloroplast, and the basal bodies were not in the spindle axis. The role of nascent basal bodies during the initial steps of spindle assembly is discussed.     

The centriolar rim. The structure that maintains the configuration of centrioles and basal bodies in the absence of their microtubules

Preparations of centrioles from bovine spleen were incubated in solutions of NaCl, MgCl2, HCl, NaOH, EDTA and heparin. Their effects on the centrioles were studied by electron microscopy of ultrathin sections. It was found that the microtubules of centriolar cylinders gradually disintegrate at a higher than physiological ionic strength and at a pH value lower than 3.5 and higher than 8.5. After microtubule extraction, a closely apposed rim or sheath of dense centriolar matrix remains which has the same dimensions of length and width as the original centriole. Some other centriolar structures, including the pericentriolar satellites and certain structures in the cylinders (hub) are also preserved. The basal bodies of fish spermatozoa revealed similar structures, including the centriolar rim and hub, after microtubule extraction. Thus, the microtubule triplets are not involved in maintaining the structure of the centriolar cylinder; this role is rather carried out by amorphous material--the matrix, surrounding the microtubules.     

Studies on the rumen flagellate Sphaeromonas communis.

The rumen flagellate Sphaeromonas communis showed a significant increase in population density 1 to 2 h after the host sheep commenced feeding, followed by a reduction in numbers to the pre-feeding level after a further 2 to 3 h. The life-history of the organism was shown to consist of a motile flagellate which germinated to produce a vegetative stage comprising a limited rhizoidal system on which up to three reproductive bodies were borne together with (in vitro) other spherical bodies of unknown function; in vivo, the reproductive bodies were stimulated to liberate flagellates by a component of the diet of the host. The vegetative stage strongly resembled that of certain species of aquatic phycomycete fungi, and the flagellates may therefore by zoospores. Flagellates liberated in vivo lost their motility within 2 to 3 h and developed into the reproductive vegetative phase, producing a rapid decrease in numbers of flagellates. Conditions of maximum flagellate production (pH 6.5, 39 degrees C, presence of CO2, absnece of oxygen) approximated to those found in the rumen. The organism was cultured in vitro in an undefined medium in the absnece of bacteria and other flagellates.     

Effects of brefeldin A on the Golgi apparatus, the nuclear envelope, and the endoplasmic reticulum in a green alga,Scenedesmus acutus

Summary The effects of brefeldin A (BFA) on the structure of the Golgi apparatus, the nuclear envelope, and the endoplasmic reticulum (ER), and on the thiamine pyrophosphatase (TPPase) activity in these organelles were examined in a green alga,Scenedesmus acutus, to obtain evidence for the existence of a retrograde transport from the Golgi apparatus to the ER via the nuclear envelope. InScenedesmus, Golgi bodies are situated close to the nuclear envelope throughout the cell cycle and receive the transition vesicles not directly from the ER, but from the nuclear envelope. BFA induced the disassembly of Golgi bodies and an increase in the ER cisternae at the trans-side of decomposed Golgi bodies in interphase cells and multinuclear cells before septum formation. The accumulated ER cisternae connected to the nuclear envelope at one part. TPPase activity was detected in all cisternae of Golgi bodies, but not in the nuclear envelope or the ER in nontreated cells. On the contrary, in BFA-treated cells, TPPase activity was detected in the nuclear envelope and the ER in addition to the decomposed Golgi bodies. When septum-forming cells were treated with BFA, the disassembly of Golgi bodies was less than that in interphase cells, and TPPase activity was detected in the Golgi cisternae but not in the nuclear envelope or the ER. These results suggest mat BFA blocks the anterograde transport from the nuclear envelope to the Golgi bodies but does not block the retrograde transport from the Golgi bodies to the nuclear envelope in interphase and multinuclear cells.Abbreviations BFA brefeldin A - ER endoplasmic reticulum - TPPase thiamine pyrophosphatase     

Feeding spectra and pseudoconoid structure in predatory alveolate flagellates

It is revealed experimentally that predatory alveolate flagellates Colpodella angusta Simpson et Patterson, C. edax Simpson et Patterson, and Voromonas pontica (Mylnikov, 2000) Cavalier-Smith, 2004 eat its prey by sucking out the insides through the front of its own body (rostrum). Feeding spectra of these predators include bodonids, percolomonads and colorless chrysomonads, while colorless euglenoids, cryptomonads, ciliates and naked amoebas are unsuitable food for them. Predators’ rostrum contains a microtubular structure (pseudoconoid) complemented by microtubular bands, micronemas or rhoptries. It is shown that the pseudoconoid begins near the kinetosomes of flagella and passes along the flagellate pocket into the rostrum. The pseudoconoid forms an unlocked cone in Colpodella angusta and Voromonas pontica, while, in Colpodella edax, it coils significantly without forming a cone. The similarity between the pseudoconoid and associated structures of predatory flagellates with analogous structures in other colpodellids, percinzoids, dinoflagellates and sporozoans is discussed.     

Centriolar changes induced by vinblastine sulphate in the seminiferous epithelium of the mouse

The effects of a single dose of vinblastine sulphate on the ultrastructure of the centrioles and the microtubular system has been studied in mitotic spermatogonia and in spermatocytes at meiotic division. With this dose the alkaloid induces a decrease in the number of cytoplasmic microtubules, inhibits centriolar migration and produces characteristic changes in the morphology of the centrioles and kinetochores. Centriolar changes consist of the appearance of dense bodies attached to the outer surface of the centriolar wall, the outgrowth of the microtubules found in the centriolar wall and a less regular array of these microtubules as compared with normal centrioles. A delay in the appearance of these effects was observed in the meiotic spermatocytes as compared with spermatogonia in the same seminiferous tubules. These effects on the morphology of the centriole are discussed in relation with current hypothesis on the relationships between centrioles and microtubules.