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.     

Membrane-associated components of the bacterial flagellar apparatus

At the position of insertion of the flagellum into the Gram-negative bacterial cell envelope, a specialized membrane differentiation has been observed by electron microscopy. This structure, termed concentric membrane rings, is harboured on the under-side of the outer membrane of Spirillum serpens, and forms a plate-like array of up to seven rings (diameter 90 nm) and an interior supporting collar. The concentric membrane rings are sensitive to proteolytic digestion, but are lysozyme and phospholipase resistant. The structures are disrupted by ionic detergents, yet resistant to the action of non-ionic detergents. A model integrating the basal organelle of the bacterial flagellum and the outer membrane of the cell wall is presented.     

The flagellar apparatus of spermatozoa in fish. Ultrastructure and evolution

Chondrichthyes possess an evolved type of spermatozoa. Their flagellar apparatus is characterized by the presence of flagellar roots which form the axis of the midpiece, and the existence of one or two lateral elements associated with the axoneme. Osteichthyes, mainly teleosteans, show a great diversity of spermatic forms. The primitive spermatozoon with a 9 + 2 pattern flagellum is common. The primitive spermatozoon has evolved along different lines. The spermatic diversity which results from this is mainly evident in the structure of the flagellar apparatus. In the animal kingdom the primitive spermatozoon with a 9 + 2 pattern flagellum, present in primitive metazoa, is retained in phyla where external fertilization is maintained. The main evolutionary tendencies--elongation, aflagellarity or biflagellarity--are generally connected with the acquisition of internal fertilization. These evolutionary tendencies are found in teleosteans. It is not possible to link aflagellarity or biflagellarity of the gamete in certain fishes to this method of fertilization. Only the elongation of the spermatozoon is connected, in certain cases, with internal fertilization, but this cannot be taken as general.     

The flagellar apparatus and cytoskeleton of the dinoflagellates

Summary Modern microscopical approaches have allowed more accurate investigations of the three-dimensional nature of the dinoflagellate flagellar apparatus (FA) and several other cytoskeletal protein complexes. Our presentation overviews the nature of the dinoflagellate FA and cytoskeleton in a number of taxa and compares them with those of other protists. As with other protists, the FA of the dinoflagellates can be characterized by the presence of fibrous and microtubular components. Our studies and others indicate that the dinoflagellate FA can be expected to possess a striated fibrous root on the basal body of the transverse flagellum and a multimembered microtubular root on the basal body of the longitudinal flagellum. Two other features that appear widespread in the group are the transverse striated root associated microtubule (tsrm) and the transverse microtubular root (tmr). The tsrm extends at least half the length of the transverse striated root while the tmr extends from the transverse basal body toward the exit aperture of the transverse flagellum. In most cases, the tmr gives rise to several cytoplasmic microtubules at a right angle. The apparent conserved nature of these roots leads us to the conclusion that the dinoflagellate FA can be compared to the FA of the cryptomonads, chrysophytes, and the ciliates for phylogenetic purposes. Of these groups, the chrysophytes possess an FA with the most structures in common with the dinoflagellates. Our immunomicroscopical investigations of the microtubular, actin and centrin components of the dinoflagellate cytoskeleton point to the comparative usefulness of these cytological features.Abbreviations aptb apical transverse microtubular band - FA flagellar apparatus - Imr longitudinal microtubular root - mls multilayered structure - tmr transverse microtubular root - tmre transverse microtubular root extension - tsr transverse striated fibrous root - tsrm transverse striated root associated microtubule     

Membrane associated components of the bacterial flagellar apparatus

At the position of insertion of the flagellum into the Gram-negative bacterial cell envelope, a specialized membrane differentiation has been observed by electron microscopy. This structure, termed concentric membrane rings, is harboured on the under-side of the outer membrane of Spirillum serpens, and forms a plate-like array of up to seven rings (diameter 90 nm) and an interior supporting collar. The concentric membrane rings are sensitive to proteolytic digestion, but are lysozyme and phospholipase resistant. The structures are disrupted by ionic detergents, yet resistant to the action of non-ionic detergents. A model integrating the basal organelle of the bacterial flagellum and the outer membrane of the cell wall is presented.     

Ultrastructure of the flagellar apparatus of Oxyrrhis marina

Summary— Oxyrrhis marina, like all dinoflagellates, possesses one transverse and one longitudinal flagellum, which show structural differences. The transverse flagellum contains a small fibre, 20 nm in diameter, associated with doublet no.7, whereas the longitudinal flagellum is substantially by a large (200–300 nm) hollows structure closely resembling the paraflagellar rod described by several authors in kinetoplastidae and in euglenoids. This structure is made up of a hemicylindrical network of filaments which are often linked on one side to the outer doublet no. 4, and on the other side to a dense plate. Another thinner filamentous network closes this hemicyclinder. In cross-section, the wall of this structure is made up of 8 filaments 2–4 nm in diameter that show a thicker periodic structure. In longitudinal section the same filaments appear arranged in periodic rhombus meshes or a helicoidal pattern, depending on the orientation of the section relative to the axoneme.     

The flagellar apparatus of the glaucophyte Cyanophora cuspidata

Glaucophytes are a kingdom‐scale lineage of unicellular algae with uniquely underived plastids. The genus Cyanophora is of particular interest because it is the only glaucophyte that is a flagellate throughout its life cycle, making its morphology more directly comparable than other glaucophytes to other eukaryote flagellates. The ultrastructure of Cyanophora has already been studied, primarily in the 1960s and 1970s. However, the usefulness of that work has been undermined by its own limitations, subsequent misinterpretations, and a recent taxonomic revision of the genus. For example, Cyanophora's microtubular roots have been widely reported as cruciate, with rotationally symmetrical wide and thin roots, although the first ultrastructural work described it as having three wide and one narrow root. We examine Cyanophora cuspidata using scanning and transmission electron microscopy, and construct a model of its cytoskeleton using serial‐section TEM. We confirm the earlier model, with asymmetric roots. We describe previously unknown and unsuspected features of its microtubular roots, including (i) a rearrangement of individual microtubules within the posterior right root, (ii) a splitting of the posterior left root into two subroots, and (iii) the convergence and termination of the narrow roots against wider ones in both the anterior and posterior subsystems of the flagellar apparatus. We also describe a large complement of nonmicrotubular components of the cytoskeleton, including a substantial connective between the posterior right root and the anterior basal body. Our work should serve as the starting point for a re‐examination of both internal glaucophyte diversity and morphological evolution in eukaryotes.     

Regulation of flagellar dynein by the axonemal central apparatus

Numerous studies indicate that the central apparatus, radial spokes, and dynein regulatory complex form a signaling pathway that regulates dynein activity in eukaryotic flagella. This regulation involves the action of several kinases and phosphatases anchored to the axoneme. To further investigate the role of the central apparatus in this signaling pathway, we have taken advantage of a microtubule-sliding assay to assess dynein activity in central apparatus defective mutants of Chlamydomonas. Axonemes isolated from both pf18 and pf15 (lacking the entire central apparatus) and from pf16 (lacking the C1 central microtubule) have reduced microtubule-sliding velocity compared with wild-type axonemes. Based on functional analyses of axonemes isolated from radial spokeless mutants, we hypothesized that inhibitors of casein kinase 1 (CK1) and cAMP dependent protein kinase (PKA) would rescue dynein activity and increase microtubule-sliding velocity in central pairless mutants. Treatment of axonemes isolated from both pf18 and pf16 with DRB, a CK1 inhibitor, but not with PKI, a PKA inhibitor, restored dynein activity to wild-type levels. The DRB-induced increase in dynein-driven microtubule sliding was inhibited if axonemes were first incubated with the phosphatase inhibitor, microcystin. Inhibiting CK1 in pf15 axonemes, which lack the central pair as well as PP2A [Yang et al., 2000: J. Cell Sci. 113:91-102], did not increase microtubule-sliding velocity. These data are consistent with a model in which the central apparatus, and specifically the C1 microtubule, regulate dynein through interactions with the radial spokes that ultimately alter the activity of CK1 and PP2A. These data are also consistent with localization of axonemal CK1 and PP2A near the dynein arms.     

Ultrastructure of the flagellar apparatus inPleurochrysis (classPrymnesiophyceae)

Summary The ultrastructure of the flagellar apparatus of aPleurochrysis, a coccolithophorid was studied in detail. Three major fibrous connecting bands and several accessory fibrous bands link the basal bodies, haptonema and microtubular flagellar roots. The asymmetrical flagellar root system is composed of three different microtubular roots (referred to here as roots 1,2, and 3) and a fibrous root. Root 1, associated with one of the basal bodies, is of the compound type, constructed of two sets of microtubules,viz. a broad sheet consisting of up to twenty closely aligned microtubules, and a secondary bundle made up of 100–200 microtubules which arises at right angles to the former. A thin electron-dense plate occurs on the surface of the microtubular sheet opposite the secondary bundle. The fibrous root arises from the same basal body and passes along the plasmalemma together with the microtubular sheet of root 1. Root 2 is also of the compound type and arises from one of the major connecting bands (called a distal band) as a four-stranded microtubular root and extends in the opposite direction to the haptonema. From this stranded root a secondary bundle of microtubules arises at approximately right angle. Root 3 is a more simple type, composed of at least six microtubules which are associated with the basal body. The flagellar transition region was found to be unusual for the classPrymnesiophyceae. The phylogenetic significance of the flagellar apparatus in thePrymnesiophyceae is discussed.     

Ultrastructure of the flagellar apparatus inPyramimonas octopus (Prasinophyceae)

Summary While green algae usually lack one of the outer dynein arms in the axoneme, flagella of the octoflagellated prasinophytePyramimonas octopus possess dynein arms on all peripheral doublets. The outer dynein arm on doublet no. 1 is modified, and additional structures are associated with doublets no. 2 and 6. The flagellar scales are asymmetrically arranged. Thus the two rows of thick flagellar hairscales are displaced towards doublet no. 6,i.e., in the direction of the effective stroke of each flagellum. The underlayer of small scales includes two nearly opposite double rows scales, arranged in the longitudinal direction of the flagellum. The hairscales emerge from these rows. The double rows are separated on one side by 9, on the other by 11 rows of helically arranged scales. The central pair of microtubules twists, but the axoneme itself (represented by the 9 peripheral doublets), does not seem to rotate. The flagella are arranged in two groups, showing modified 180° rotational symmetry. The effective strokes of the two central flagella are exactly opposite, while the other flagella beat in six intermediate directions.     

Absolute orientation of the flagellar apparatus of Hibberdia magna comb. nov. (Chrysophyceae)

The first flagellum of Hibberdia magna comb. nov. bears mastigonemes that have both short and long lateral filaments attached to the tubular shaft. The second flagellum is very short (ca. 850 nm) and is directed posteriorly approximately 160° from the first flagellum. Three microtubular flagellar roots (R₁, R₂ and R₄) and a rhizoplast (= striated root) are present. The R₁ root consists of four microtubules that arise near the right surface of the first flagellum basal body; the R₁ root extends to the dorsal side of the cell and then curves back along the left side of the cell. Cytoskeletal microtubules are nucleated from the R₁ root including one loose cluster of cytoskeletal microtubules that extends down the left side of the cell adjacent to the contractile vacuole. The R₂ root is a single microtubule that arises along the left surface of the first flagellum basal body and extends to the left side of the cell. The R₄ root consists of three microtubules that arise along the left side of the second flagellum basal body. A helical band wraps around two microtubules at the proximal end of the R₄ root. Two of the three R₄ root microtubules extend along the left side of the second flagellum, curve around to the right side of that flagellum and terminate. No R₃ root was found. The orientation of the basal bodies of Hibberdia gen. nov. is similar to that of the Xanthophyceae and Oomycetes. There are apparent homologies in the R₁, R₂ and R₄ roots of Hibberdia and these and other protists, but only Hibberdia lacks a R₃ root. Three long flagella are present in preprophase but later one is endocytosized and the axoneme extends to the posterior of the cell. During metaphase the nuclear envelope is more or less intact except at the poles; the flagellar apparatuses are at the poles and the spindle microtubules originate near the basal bodies. Two stages are known in the life history: 1) a capsoidlike state with non-swimming flagellate cells inside a colonial gel, and 2) a free-swimming single-celled monad state. Vegetative cell division occurs in both stages. The flagellar apparatus, the cell division process and the life history combined with the previously described unique light-harvesting antheraxanthin make H. magna distinct from other algae. A new genus, Hibberdia gen. nov., a new family, Hibberdiaceae fam. nov. and a new order, Hibberdiales, ord. nov. are described.     

Genes for the hook-basal body proteins of the flagellar apparatus in Escherichia coli.

Of the more than 30 genes required for flagellar function, 6 are located between pyrC and ptsG on the Escherichia coli genetic man. This cluster of genes is called flagellar region I. Four-point transductional crosses were used to establish the position and order of the region I flagellar genes with respect to the outside markers ptsG and pyrC. Bacteriophage lambda-E. coli hybrids that contained most of the genes necessary for flagellar formation were constructed. The properties of specific hybrids that carried the region I fla genes were examined by genetic complementation and by measuring the capacity of the hybrids to direct the synthesis of specific polypeptides. The results of these tests with lambda hybrids and with a series of deletion mutations derived from the lambda hybrids demonstrated the existence of at least six flagellar-specific cistrons. These directed the synthesis of polypeptides with the following apparent molecular weights: flaV, 11,000; flaK, 42,000; flaL, 30,000 and 27,000; flaM, 38,000; flS, 60,000; and flaT, 35,000. Plasmid ColE1-E. coli hybrids with region I flagellar genes were also used to program the synthesis of polypeptides in minicell-producing strains. The polypeptides synthesized in these experiments were identical to polypeptides of the hook-basal body structure and helped to confirm the assignment of genes to specific polypeptides. The synthesis of all of these polypeptides was regulated by the same mechanism that regulates the synthesis of other flagellar-related structural components.     

Flagellar apparatus duplication and partition, flagellar transformation during division in Entosiphon sulcatum.

Electron microscopic examination of serial sections of developmental stages of the flagellar apparatus during the cell cycle indicates that the basal bodies replicate in a semi-conservative manner and that there is a flagellar transformation over two cell cycles in euglenoids as in other algal flagellate groups. Two new pairs of basal bodies are formed, each pair comprising one parental and one newly developed basal body. There is a transformation of the parental dorsal flagellum containing a thin paraxonemal rod into a ventral flagellum bearing a large paraxonemal rod. Observation of the roots associated with the basal bodies shows that the dorsal root transforms into an intermediate root over two cell cycles following the transformation of the dorsal basal body/flagellum to a ventral one. Also the two ventral roots are newly formed in relation to the formation of two new phagotrophic apparatuses during the division. After the breakage of the connection between the parental basal bodies the two new pairs move apart and are guided/drawn by transverse microfibrillar bundles which connect them to opposite sides of the pellicle. The axis of the separation/migration of the pairs of basal bodies is parallel to the axis of elongation of the dividing nucleus.     

Centrin association with the flagellar apparatus in spores ofPhytophthora cinnamomi

Summary Antibodies raised against the calcium-binding protein centrin, were used to identify and localise centrin containing structures in the flagellar apparatus of zoospores and cysts of the oomycetePhytophthora cinnamomi. Immunoblotting of extracts from zoospores indicates that theP. cinnamomi centrin homologue is a 20 kDa protein. Immunofluorescence microscopy with anti-centrin antibodies reveals labelling in the flagella, the basal body connector and co-localisation along the microtubular R1 root (formerly called AR3) that runs from the right side of the basal body of the anterior flagellum into the anterior of the zoospore close to the ventral surface. The centrin (R1^cen) and tubulin components of the R1 root split into four loops on the right hand side of the ventral groove and rejoin along the left hand side of the groove. The R1 root continues down the left hand side of the zoospore past the basal bodies and parallel to the R4 root. We propose that at least inP. cinnamomi there is no R2 root. Immunogold labelling confirms that centrin is a component of the basal body connector complex. When the zoospores become spherical during encystment, the R1^cen pivots by approximately 90 ° with respect to the nucleus.     

Ultrastructure of the flagellar apparatus of the green algaTetraselmis subcordiformis

Summary The ultrastructure of the flagellar apparatus of the marine quadriflagellate green algaTetraselmis subcordiformis is described in detail. Special consideration is given to the functional significance of the contractile rhizoplast and also to a complex structure which anchors the flagellar apparatus to the cell membrane and theca. The flagellar apparatus lies at the base of a deep apical depression. Four basal bodies lie in a zigzag row with their long axes nearly parallel. Outer adjacent pairs of basal bodies are structurally linked by a Z-shaped, ribbon-like structure. A striated fiber (transfiber) connects each outer basal body with the inner basal body of the opposite, mirror image pair. A complex system of four laminated oval discs (rhizanchora), microtubule rootlets and fibrous material anchor the flagellar apparatus and rhizoplasts to the plasma membrane and theca. A 4-2-4-2 arrangement of microtubule rootlets is present. Rhizoplasts, which are contractile organelles, branch into five distinct arms and associate with the near outer basal body and each of the four rhizanchora. Rhizoplast contraction is thought to be linked to flagellar activity and may act to alter the direction of motion of the cell.     

Ultrastructure of the flagellar apparatus in the green flagellateSpermatozopsis similis

The ultrastructure of the flagellar apparatus of the naked, biflagellate green algaSpermatozopsis similis Preisig & Melkonian has been studied in detail using an absolute configuration analysis. The two basal bodies are displaced by 350 nm in the 1/7 o'clock direction and do not overlap proximally. They are interconnected by a principal distal connecting fibre consisting of a bundle of 5–8 nm filaments and possibly two proximal striated connecting fibres. The flagellar root system is cruciate (5-2-5-2 or 4-2-4-2 system) and contains a prominent continuous system I fibre overlying the two opposite two-stranded roots. A system II fibre is absent. Pronounced structural differences have been observed in the flagellar apparatus ultrastructure at two types of flagella orientation: During backward swimming basal bodies are parallel, the distal connecting fibre is extremely contracted; during forward swimming basal bodies assume various angles (from 20° to 180°) and the connecting fibre is about five times longer compared to the contracted state. The function of the connecting fibre as a contractile organelle and the mechanism of its contraction are discussed. On the basis of the flagellar apparatus ultrastructure,Spermatozopsis similis is related toChlamydomonas-type green algae.     

An ultrastructural comparison of the mitotic apparatus,feeding apparatus,flagellar apparatus and cytoskeleton in euglenoids and kinetoplastids

Summary The euglenoids and kinetoplastids form a diverse assemblage of organisms which show no obvious phylogenetic relationship with other flagellates. An ultrastructural examination and comparison of the flagellar apparatus, the feeding apparatus, and mitotic nucleus indicate a number of shared morphological features which support a common ancestry for the two groups. Of particular interest is the euglenoid,Petalomonas cantuscygni, which shares many of the ultrastructural features common to both groups. Based on the data presented, we hypothesize that a euglenoid with features similar to those now present inP. cantuscygni was ancestral to both the euglenoid and kinetoplastid lines.Abbrevation MTR complex of reinforcing microtubules