Cytogeometrical determination of ciliary pattern formation in the hypotrich ciliate Stylonychia mytilus: II. Stability and field regulation

The first regeneration sequence after folding of right fragments of Stylonychia mytilus results in formation of mirror-imaged incomplete doublets illustrating independent determination of polar and lateral axes. Analysis of the second morphogenetic sequence illustrates that the independent determination of polar and lateral axes is stable through a subsequent cellular reorganization and confirms that cytogeometry participates in determination of ciliary pattern. The morphogenetic inversion of cirral row primordia in this type of fragment is reflected in the structure of the individual cirri. These data not only extend and confirm our earlier study on this type of fragment, but also are consistent with the conclusion derived from data on a different type of mirror-imaged doublet, that global patterning and assembly of ciliature are independently determined.     

Reproducing Singlets with an Inverted Oral Apparatus in Glaucoma scintillans (Ciliophora,Hymenostomatida)1

A fragment with only an abnormal oral apparatus (OA) was obtained by operation from a doublet of Glaucoma scintillans possessing one normal and one abnormal OA. This singlet could reproduce and produced a cell line. Singlets frequently possessed an inverted OA, whose antero-posterior axis was rotated 180°. This inversion of the OA has been perpetuated through a considerable number of generations. Oral replacement commonly occurred in singlets with an abnormal OA regardless of growth phases of a culture. The position of the contractile vacuole pore, the direction of curvature of ciliary rows surrounding the OA, and the organization of postoral ciliary rows were mirror-images of those of a normal singlet.     

Ciliary sieving and active ciliary response in capture of particles by suspension-feeding brachiopod larvae

Larvae of a brachiopod, Glottidia pyramidata, used at least two ciliary mechanisms to capture algal cells upstream from the lateral band of cilia that produces a feeding/swimming current. (1) Filtration: the larvae retained algal cells on the upstream (frontal) side of a sieve composed of a row of stationary laterofrontal cilia. Movement of the laterofrontal cilia could not be observed during capture or rejection of particles, but the laterofrontal cilia can bend toward the beating lateral cilia, a possible mechanism for releasing rejected particles from the ciliary sieve. (2) Localized changes of ciliary beat: the larvae may also concentrate particles by a local change in beat of lateral cilia in response to particles. The evidence is that the beat of lateral cilia changed coincident with captures of algal cells and that captured particles moved on paths consistent with a current redirected toward the frontal side of the tentacle by an induced local reversal of the lateral cilia. The change of beat of lateral cilia could have been an arrest rather than a reversal of ciliary beat, however. The similar ciliary bands in adult and larval lophophorates (brachiopods, phoronids, and bryozoans) suggest that these animals share a range of ciliary behaviours. The divergent accounts of ciliary feeding of lophophorates could be mostly the result of different authors observing different aspects of ciliary feeding.     

Biogenesis of the ciliary roots: participation of the dictyosomes of Golgi's complex.

In this study, the hypothesis of a possible biogenesis of the ciliary roots is suggested, after observing the cilia neurons under the electron microscope, which were found as an exception in the periaqueductal nucleus of the mesencephalon in the domestic cat, conserving the potential to differentiate the cilia, basal bodies and ciliary roots. The dictyosomes of Golgi's complex and Golgi's vesicles participated in this biogenesis. Vesicles of approximately 71.6 nm in diameter had become separated from the periphery of the flattened discoid cisterns of the dictyosome and were aligned normally, in tangential contact with each other, forming rows of vesicles or 'ringed chains', whose points of contact formed the beginning of the 'periodic striation' of a thin ciliary root. Later, the lateral walls of the vesicles and the molecules of the intracisternal proteins gave rise to the interperiodic microfilaments, when the carrier proteins were transformed into structural proteins of the ciliary roots. The parallel apposition of several ringed chains or thin ciliary roots, with their rings joined at the same level (or transversal striations), gave rise to thicker striated roots. This hypothesis of an ultrastructural biogenesis of the striated ciliary roots involves the following six stages: stage I = separation of Golgi's vesicles from the periphery of the flattened disk of dictyosomes near the basal body, with a diameter of over 71.6 nm; stage II = reinforcement of the membrane of the vesicles at the two opposite polar ends of its larger diameter; stage III = alignment of vesicles to form ringed chains, due to the tangential contact between their reinforced points; initiation of the 71.6-nm striation period, roots ringed linearly; stage IV = formation of joining microfilaments between periods (69.2 nm) with the lateral walls of the vesicles and the molecules of the proteins in their content; stage V = lengthening of the thin ciliary roots due to the coupling of new Golgi's vesicles at their ends so that their length increases as a result of the addition of terminal vesicles; stage VI = increase in thickness of the thin ciliary roots, due to the parallel apposition of several ringed chains or thin ringed ciliary roots, at the point where their transversal striation points coincide.     

Ciliary beating in three dimensions: Steps of a quantitative description

We document a novel approach for quantitative assessment of ciliary activity, exemplified in rapid three-dimensional cyclic motion of the frontal cirri of Stylonychia. Cells held under voltage-clamp control are stimulated by step pulses to elicit reproducible hyperpolarization- or depolarization-induced ciliary motor responses. High-speed video recording at 200 fields per second is used for imaging ciliary organelles of the same cell in two perspectives: the axial view and, following cell rotation by 90 degrees, the lateral view. From video sequences of typically 1 s, the contours of the cirral images are determined and digitized. Computer programs are established to (1) reduce an observed image to a "ciliary axis", (2) sort series of axes by template to generate an averaged ciliary cycle in 2D-projection, and (3) to associate the generalized axial and lateral 2D-images for generation of a sequence of three-dimensional images, which quantitatively represent the cycle in space and time. The method allows us to produce predetermined perspectives of images selected from the ciliary cycle, and to generate stereo views for graphical representation of ciliary motion. The approach includes a potential for extraction of the complete microtubular sliding program of a cilium under reproducible electric stimulation of the ciliary membrane.     

SYMMETRY AND DEVELOPMENT OF LIMNOBIUM SPONGIA (HYDROCHARITACEAE)

Limnobium spongia produces upright vegetative axes and prostrate stolons. The upright axes bear new stolons, whereas the stolons bear new upright axes and fertile and sterile branching systems. Upright axes and fertile and sterile branching systems are all interpreted to have sympodial growth. However, it was not determined whether growth of stolons is monopodial or sympodial. Both stolons and upright axes branch in alternate plastochrons, and branching is achieved solely by the bifurcation of apical meristems. Each meristematic bifurcation is interpreted to represent the formation of a precocious lateral bud. The upright axes develop from presumed precocious lateral buds on stolons, whereas such buds on upright axes produce renewal shoots. Limnobium spongia exhibits a marked degree of mirror-image symmetry.     

Versatile ciliary behaviour in capture of particles by the bryozoan cyphonautes larva

Cyphonautes larvae of a bryozoan, Membranipora membranacea, used several ciliary mechanisms to capture algal cells upstream from the lateral band of cilia that produces a feeding current. (1) Lateral cilia changed beat and a backcurrent occurred at the time and place that particles were retained. (2) Algal cells were sieved and held stationary at the upstream (frontal) side of a row of laterofrontal cilia that were not beating. (3) Localized extension of cilia toward the inhalant chamber, coincident with particle captures, indicated that laterofrontal cilia flick toward the inhalant chamber. These flicks may aid transport of captured particles toward the mouth. Thus my earlier report that larvae only sieve, in contrast to the adults (which have an active ciliary response) was in error. The similar ciliary bands in adult and larval bryozoans and in other lophophorates (brachiopods, and phoronids) suggest that these animals share a core repertoire of ciliary behaviours in the capture and concentration of suspended food particles.     

Activities of auxin polar transport in inflorescence axes of flower mutants ofArabidopsis thaliana: Relevance to flower formation and growth

Relationships between the activity of auxin polar transport and flower formation were studied using several flower mutants ofArabidopsis thaliana. The activity of auxin polar transport in the upper portion of inflorescence axis of wildtype plants ofArabidopsis thaliana was significantly lower than that of the basal part. The activities of auxin polar transport in the upper portion of inflorescence axes ofap1 andclv1 mutants were significantly higher than that of wild-type plant. However, those of other flower mutants tested,ap3-1, ag, pi, Fl-40, Fl-54, Fl-89 andpin-formed, were extremely low as compared with that of wild one. We got some evidence that the reduction of the activity of auxin polar transport is concerned with the growth and development of plants. We could mimic it by the removal of all flowers and pods including mature or immature seeds. Moreover, artificial pollination inap3-1 andpi mutants, in which no seeds are found naturally, resulted in the partial recovery of the activity of auxin polar transport in inflorescence axis. Considering these results in this study together with the fact that inhibitors of auxin polar transport generated almost same disruptions ofpin-formed orpinoid mutants which normally had no flowers in inflorescence axis (Okadaet al. 1991, Uedaet al. 1992, Bennettet al. 1995), the systern of auxin polar transport and its activity in inflorescence axis seems to be essential for the development of flower bud in early stage ofArabidopsis thaliana, and the activity of auxin polar transport is also regulated by the formation of flowers and seeds in inflorescence axis.     

The ventral epithelium of Trichoplax adhaerens (Placozoa): Cytoskeletal structures,cell contacts and endocytosis

Summary All cilia emerge from ciliary pits supported along their circumference by 22–24 dense rodlets that are connected by filaments to a surrounding sheath of endoplasmic reticulum. The proximal part of the basal body is provided with two short lateral rootlets and one long terminal rootlet, all associated with microtubules. The lateral rootlets are in turn connected by fine fibrous material to the dense supporting rodlets which follow the contour of the ciliary pit and extend along the ciliary membrane beyond the level of the basal plate where the central pair of microtubules originates. The proximal part of the basal body has fine fibrous connections to the endoplasmic reticulum while its distal portion is surrounded by nine curved sheets. The terminal cell contactions are by belt desmosomes that are accompanied by a bundle of microfilaments which encircle the apical region of the cell and insert at the cell membrane. Tight junctions are lacking. Endocytosis was demonstrated by the uptake of cationized ferritin. The structures associated with the ciliary pits are probably associated with the firm anchorage of the ciliary base since Trichoplax adheres to the substrate as it moves propelled by its ventral cilia. The marginal bundle of microfilaments may be involved in folding of the organism during feeding.     

Ultrastructure of cilia and flagella - back to the future!

Eukaryotic cilia and flagella perform motility and sensory functions which are essential for cell survival in protozoans, and to organism development and homoeostasis in metazoans. Their ultrastructure has been studied from the early beginnings of electron microscopy, and these studies continue to contribute to much of our understanding about ciliary biology. In the light of the progress made in the visualization of cellular structures over the last decade, we revisit the ultrastructure of cilia and flagella. We briefly describe the typical features of a 9+2 axoneme before focusing extensively on the transition zone, the ciliary necklace, the singlet zone, the ciliary cap and the ciliary crown. We discuss how the singlet zone is linked to sensory and/or motile function, the contribution of the ciliary crown to ovocyte and mucosal propulsion, and the relationship between the ciliary cap and microtubule growth and shortening, and its relation to ciliary beat. We further examine the involvement of the transition zone/the ciliary necklace in axonemal stabilization, autotomy and as a diffusion barrier.     

Growth and Development of Young Roots of Barley (Hordeum vulgare L.) Genotypes

Barley (Hordeum vulgare L.) genotypes from countries with aMediterranean climate grown in temperature-controlled glasshousein nutrient solution to determine whether the co-ordinationof root branching and growth found by other workers appliedto a wider of up to 14 genotypes. There was substantial variationin the number of seminal axes produced by the genotypes, rangingfrom about seven for Hoshimasari and Swanneck to about fourfor Gerbel 'B'. The number of nodal axes was linearly relatedto the number of leaves and typically between one and two mainstemleaves were required before nodal axes appeared. There weresmall genotypic differences in the number of axes produced perleaf with values ranging from 1·5 to 2·3. Theproduction and growth of lateral roots were coordinated so thatthe mean length of laterals generally increased with time. Landraces(Arabic abiad and Arabic aswad) produced more lateral rootswith a faster rate extension compared with other genotypes.The length and number of primary and secondary lateral rootswere related linearly, but no genotypic differences in thisrelation were evident. Length of primary lateral roots increasedmore rapidly than that of secondary lateral roots throughoutthe three to five leaf stage. The ratio of root weight to totalplant weight decreased with time but there were only small differenceswithin this range of genotypes.Copyright 1995, 1999 AcademicPress Barley, seminal axes, nodal axes, primary lateral roots, relative extension rates, relative multiplication rates     

Ciliary feeding structures and particle capture mechanism in the freshwater bryozoan Plumatella repens (Phylactolaemata)

Abstract. In contrast to marine bryozoans, the lophophore structure and the ciliary filter‐feeding mechanism in freshwater bryozoans have so far been only poorly described. Specimens of the phylactolaemate bryozoan Plumatella repens were studied to clarify the tentacular ciliary structures and the particle capture mechanism. Scanning electron microscopy revealed that the tentacles of the lophophore have a frontal band of densely packed cilia, and on each side a zigzag row of laterofrontal cilia and a band of lateral cilia. Phalloidin‐linked fluorescent dye showed no sign of muscular tissue within the tentacles. Video microscopy was used to describe basic characteristics of particle capture. Suspended particles in the incoming water flow, set up by the lateral ‘pump’ cilia on the tentacles, approach the tentacles with a velocity of 1–2 mm s^‐1. Near the tentacles, the particles are stopped by the stiff sensory laterofrontal cilia acting as a mechanical sieve, as previously seen in marine bryozoans. The particle capture mechanism suggested is based on the assumed ability of the sensory stiff laterofrontal cilia to be triggered by the deflection caused by the drag force of the through‐flowing water on a captured food particle. Thus, when a particle is stopped by the laterofrontal cilia, the otherwise stiff cilia are presumably triggered to make an inward flick which brings the restrained particle back into the downward directed main current, possibly to be captured again further down in the lophophore before being carried to the mouth via the food groove. No tentacle flicks and no transport of captured particles on the frontal side of the tentacles were observed. The velocity of the metachronal wave of the water‐pumping lateral cilia was measured to be ～0.2 mm s^‐1, the wavelength was ～7 μm, and hence the ciliary beat frequency estimated to be ～30 Hz (～20 °C). The filter feeding process in P. repens reported here resembles the ciliary sieving process described for marine bryozoans in recent years, although no tentacle flicks were observed in P. repens. The phylogenetic position of the phylactolaemates is discussed in the light of these findings.     

FRESHWATER HYPHOMYCETES OF THE NORTHERN APPALACHIAN HIGHLAND INCLUDING NEW ENGLAND,AND THREE COASTAL PLAIN STATES

Twenty-seven species of freshwater Hyphomycetes from two five-state areas in the northern Appalachian Highland, New England, and three Coastal Plain states were collected, isolated, and identified. The spores of three fungi which are probably aquatic Moniliales were also studied. Sigmoidea, a new Hyphomycete genus, includes submerged aquatic fungi which produce phialides laterally along the mycelium and sigmoid, multicellular phialospores. Varicosporium giganteum sp. n. produces terminal, hyaline, branched, and multiseptate aleuriospores. The main spore axis has one to four lateral branches which arise successively on one side of this axis. Tricladium marylandicum sp. n. is similar to T. splendens in that the lateral branches are constricted where each branch joins the main spore axis. It differs from this species in that the spore has a blunt apical cell on the main axis and on each of the lateral axes. Three possible methods of dispersal are presented to explain the cosmopolitan distribution of freshwater Hyphomycetes.     

Autoradiographic analysis of amino acid uptake by the gill ofMytilus

Summary Autoradiography was used to examine the influence of lateral ciliary activity on the pattern of leucine uptake into isolated gill tissue from the mussel,Mytilus californianus. Metachronal activity of the lateral cilia, normally absent in the in vitro gill, was reestablished by application of 10 μM 5-hydroxytryptamine (5-HT). This treatment produced a 5–7 fold stimulation in the rate of leucine uptake into isolated gills. The treatment with 5-HT did not, however, affect the fractional incorporation of leucine into alcohol insoluble vs alcohol soluble material. Autoradiograms of gills treated with 5-HT showed extensive labelling of frontal, lateral, and abfrontal surfaces of gill filaments compared to the control condition in which label was largely confined to the frontal region of the gill. Quantitative analyses of the autoradiograms revealed a 4-fold increase in the number of silver grains over lateral and abfrontal surfaces compared to control gills. Autoradiograms of gills from intact mussels exposed to³H-leucine showed a pattern of silver grain deposition similar to that observed in in vitro gills treated with 5-HT. It is concluded that the capacity for amino acid transport exists in cells from the frontal, lateral, and abfrontal surfaces of gill filaments, butaccess to dissolved substrates by transport sites on lateral and abfrontal surfaces is dependent upon lateral ciliary activity.     

Distribution of catecholamine-containing,serotonin-like and neuropeptide FMRFamide-like immunoreactive neurons and processes in the nervous system of the actinotroch larva ofPhoronis muelleri (Phoronida)

Summary Glyoxylic-acid-induced fluorescence of catecholamines and antibodies against serotonin and FMRFamide were used to study the distribution of putative neurotransmitters in the actinotroch larva ofPhoronis muelleri Selys-Longchamps, 1903. Catecholamines occur in the neuropile of the apical ganglion, in the longitudinal median epistome nerves, in the epistome marginal nerves, and in the nerve along the bases of the tentacles. The tentacles have laterofrontal and latero-abfrontal bundles of processes that form two minor nerves along the lateral ciliary band of the tentacles, and a medio-frontal bundle of processes. Monopolar cells are located on the ventro-lateral part of the mesosome. Processes are located along the posterior ciliary band and as a reticulum in the epidermis. Serotonin-like immunoreactive cells and processes are located in the apical ganglion, in the longitudinal median epistome nerves, and as a dorsal and ventral pair of bundles along the tentacle bases. Processes from the latter extend into the tentacles as the medioabfrontal processes. The latero-abfrontal processes form a minor nerve along the ciliary band. The dorsal bundles forms the major nerve ring along the tentacles and processes extend from it to the metasome. Processes are located along the posterior ciliary band. FMRFamide-like immunoreactive cells and processes are found in the apical ganglion, in the longitudinal median epistome nerves and as a pair of lateral epistome processes projecting towards the ring of tentacles. In the tentacles, a pair of latero-frontal processes are found; these form a minor nerve along the ciliary band. A band of cells can be seen along the tentacle ring.     

Unusual patterns of somatic embryogenesis in the domesticated carrot: developmental effects of exogenous auxins and auxin transport inhibitors

The effects of various exogenous auxins and polar auxin transport inhibitors on somatic embryogenesis in carrot cultures were investigated. Indole-3-acetic acid and 2,4-dichlorophenoxyacetic acid do not disrupt the sequence or the polarity of individual stages in embryo development, but tend to cause developing embryos to revert to undifferentiated callus, with increasing frequency in later embryo stages. The transport inhibitors, N-(1-naphthyl)phthalamic acid and 2,3,5-triiodobenzoic acid, block morphological transitions to the subsequent stage; for example, they cause the formation of enlarged globular and oblong embryos. Heart embryos in these treatments usually develop additional lateral growth axes. These results shed light on the role of auxin and its polar transport in somatic embryogenesis.     

Lateral flagella of Aeromonas species are essential for epithelial cell adherence and biofilm formation

Mesophilic Aeromonas strains express a single polar flagellum in all culture conditions and produce lateral flagella on solid media. Such hyperflagellated cells demonstrate increased adherence. Nine lateral flagella genes, lafA-U for Aeromonas hydrophila, and four Aeromonas caviae genes, lafA1, lafA2, lafB and fliU, were isolated. Mutant characterization, nucleotide and N-terminal sequencing demonstrated that the A. hydrophila and A. caviae lateral flagellins were almost identical, but were distinct from their polar flagellum counterparts. The aeromonad lateral flagellins exhibited higher molecular masses on SDS-PAGE, and this aberrant migration was thought to result from post-translational modification through glycosylation. Mutation of the Aeromonas lafB, lafS or both A. caviae lateral flagellins caused the loss of lateral flagella and a reduction in adherence and biofilm formation. Mutations in lafA1, lafA2, fliU or lafT resulted in strains that expressed lateral flagella, but had reduced adherence levels. Mutation of the lateral flagella loci did not affect polar flagellum synthesis, but the polarity of the transposon insertions on the A. hydrophila lafTlU genes resulted in non-motility. However, mutations that abolished polar flagellum production also inhibited lateral flagella expression. We conclude that Aeromonas lateral flagella: (i) play a role in adherence and biofilm formation; (ii) are distinct from the polar flagellum; (iii) synthesis is dependent upon the presence of a polar flagellum filament; and (iv) that the motor proteins of the polar and lateral flagella systems appear to be shared.     

Ultrastructure and membrane topography of special ciliary organelles in the ciliate Eufolliculina uhligi (Protozoa)

Summary In Eufolliculina uhligi and other folliculinid ciliates, a territory has been identified that differs ultrastructurally from other areas of the cell, and that is especially sensitive to mechanical stimuli. This territory is located around the anterior oral apparatus of the loricate trophont and posterior to the membranellar spiral of the swarmer. Each cilium in this territory is closely apposed to a small membrane-covered pin that is supported by transverse microtubules of the cilium. In front of the pin, the base of the cilium bulges out; the ciliary membrane is interconnected with the axoneme by filamentous material. Freeze-fractured cilia show a large rectangular particle array at the site of the basal swelling. Only scattered particles have been observed in the pin membrane. It is suggested that the cilium and the pin act as a unit, which has therefore been named the ciliumpin-complex. Comparison with ciliary organelles of unicellular and multicellular organisms indicates that, because of their polar organization, the complexes are involved in the transduction of oriented, presumably mechanical, stimuli.     

Mechanical sensitivity and cell coupling in the ciliated epithelial cells ofMytilus edulis gill

Summary Transmission electron microscopy has not provided strong evidence for gap junctions inMytilus edulis gill tissue, in spite of extensive physiological evidence for coupled ciliary arrest in lateral cells and coupled activation in abfrontal cells. To investigate the kinds and relative distribution of cell junctions and also to determine whether ciliary membrane particle differences exist in these two types of oppositely mechanically sensitive cells, we analyzed the structure of these and two other ciliated cell types (frontal and laterofrontal) by freeze-fracture replication. Gap junctions occur in all four ciliated cell types, but they are relatively small and of variable morphology, often consisting of elongate, winding complexes of membrane particles. Statistically, such structures rarely would be recognized as gap junctions in thin sections. Gap junctions appear to be most abundant between the highly coupled abfrontal cells, minimal between laterofrontal cells, and not evident in the epithelial cells that separate coupled ciliated cell types. The ciliary necklaces of the mechanically activated abfrontal cilia are typically 4- or 5-stranded while those of the remaining three cell types are mainly 3-stranded. In developing gill tips, ciliated cells have abundant gap junctions and newly formed cilia have a full complement of necklace particles. Nascent lateral cilia are not mechanically sensitive, indicating that the acquisition of mechanosensitivity does not correlate with the presence of ciliary necklace or other membrane particles. Lateral and laterofrontal cells become sensitive to neurotransmitters soon after the appearance of the latter during development, but mechanosensitivity of both lateral and abfrontal cells arises substantially later.