Sequence and Properties of Cagein,a Coiled-Coil Scaffold Protein Linking Basal Bodies in the Polykinetids of the Ciliate Euplotes aediculatus

In the hypotrich ciliate Euplotes, many individual basal bodies are grouped together in tightly packed clusters, forming ventral polykinetids. These groups of basal bodies (which produce compound ciliary organelles such as cirri and oral membranelles) are cross-linked into ordered arrays by scaffold structures known as “basal-body cages.” The major protein comprising Euplotes cages has been previously identified and termed “cagein.” Screening a E. aediculatus cDNA expression library with anti-cagein antisera identified a DNA insert containing most of a putative cagein gene; standard PCR techniques were used to complete the sequence. Probes designed from this gene identified a macronuclear “nanochromosome” of ca. 1.5 kb in Southern blots against whole-cell DNA. The protein derived from this sequence (463 residues) is predicted to be hydrophilic and highly charged; however, the native cage structures are highly resistant to salt/detergent extraction. This insolubility could be explained by the coiled-coil regions predicted to extend over much of the length of the derived cagein polypeptide. One frameshift sequence is found within the gene, as well as a short intron. BLAST searches find many ciliates with evident homologues to cagein within their derived genomic sequences.     

DISTINCTIVE LECTIN LABELING OF THE FLAGELLAR APPARATUS OF TETRASELMIS (PRASINOPHYCEAE) AND DUNALIELLA (CHLOROPHYCEAE)1

Fluorescent labeling of the flagellar apparatus of Tetraselmis (Prasinophyceae) and Dunaliella (Polyblepharidaceae, Chlorophyceae) were successfully performed using fluorescein isothiocyanate–labeled lectins from Arachis hypogaea and Glycine maxima. These lectins specifically bound to the flagella and kinetosome of the cell but did not bind to the cell surface. Lectin binding on the flagellar apparatus remained constant under different culture media, temperatures, irradiances, cell division cycle, and culture aging. All the Tetraselmis and Dunaliella analyzed (five species, 20 clones) showed intense labeling of the flagellar apparatus. In contrast, no other species analyzed (46 clones of 25 species from four classes) showed binding to their flagellar apparatus. After the lectin treatment, many cells remained alive, and they were able to swim with the flagellar apparatus intensely labeled. The lectin binding indicates that the flagella and kinetosome of Tetraselmis are rich in Gal and GalNH₂ moieties and that the flagella of Dunaliella are rich in Gal and GalNAc moieties. Apparently, this feature seems to be specific to these species.     

A TEM study on pre—excystment cellular structures of Euplotes encysticus

Right before the excystment of an Euplotes encysticus sawtooth-like folds appeared among the pellicle plasmalemma,the inner and outer alveolar membranes were still sticking together,and were not distinguishable.Microtubular layers already formed at the sites beneath the dorsal cortical pellicle corresponding to vegetative cells,but they still proceed to be organized on the ventral structures.Cristae,highly-tangled with tubular-type structures,appeared on the mitochondria,and were morphologically similar to that of vegetative cells.In the cortical ciliatures,such as ciliary shafts,kinetosomes,surrounding fibrillar cirral baskets,and attached structures of ciliatures,etc.,they are different from those in resting cysts which are degenerated or lost.All the ciliature microtubules of ciliary shafts are of the 9 2 pattern,but the microtubule-like structure aggregates at tripletmicrotubule centers of many kinetosmes,are still under various stages of differentiation.Microtubules beneath the kinetosomal rows are of a developmentally elongated stage;crowded chromatins of various shapes and sizes are found in macronucleus,but there are no nuclear pores (formed by nuclear membrane as in resting cysts) on the nuclear membrane where these chromatins attached.     

Ultrastructure of the Oral Apparatus of Mesodinium rubrum from Korea

Mesodinium rubrum Lohmann is a photosynthetic marine ciliate that has functional chloroplasts of cryptophyte origin. Little is known about the oral ultrastructure of M. rubrum compared with several reports on the sequestration of nuclei and plastids from prey organisms, such as Geminigera cryophila and Teleaulax species. Here, we describe the fine structure of the oral apparatus of a M. rubrum strain from Gomso Bay, Korea. The cytopharynx was cone‐shaped and supported by 20–22 ribbons of triplet microtubules. At the anterior end of the cytopharynx, an annulus anchored small cylinders composed of 11 microtubules. The small cylinders were spaced at regular intervals, each reinforced by one set of the triplet microtubules. At the opening of the cytostome, larger 14‐membered microtubular cylinders were set adjacent to the small, 11‐membered microtubular cylinders, each pair surrounded by separate membranes, however, only the large cylinders extended into the oral tentacles. There were 20–22 oral tentacles each having one to five extrusomes at its tip. At the anterior end of the oral apparatus, microtubular bands supporting the cytostome curved posteriad, extending beneath the cell cortex to the kinetosomes of the somatic cirri. The microtubular bands were connected by striated fibers and originated from kinetosomes anchored by fibers. Each cirrus consisted of eight cilia associated with 16 kinetosomes. The ultrastructure of M. rubrum from Korea provides information useful for taxonomic characterization of the genus Mesodinium and relevant to developing a better understanding of the acquisition of foreign organelles through phagocytosis by M. rubrum.     

The Ultrastructure of Sanchytrium tribonematis (Sanchytriaceae,Fungi incertae sedis) Confirms its Close Relationship to Amoeboradix

Fungi encompass, in addition to classically well‐studied lineages, an ever‐expanding diversity of poorly known lineages that include, among others, zoosporic chytrid‐like parasites. According to recent phylogenetic analysis based on 18S + 28S rRNA concatenated genes two unusual chytrid‐like fungi Amoeboradix gromovi and Sanchytrium tribonematis form a monophyletic group, the family Sanchytriaceae, which represents a new divergent taxon that remains incertae sedis within Fungi. Zoospores of Amoeboradix gromovi contain one of the longest kinetosomes known in eukaryotic cells, which are composed of microtubular singlets or doublets. However, the ultrastructure of S. tribonematis, the type species of the genus had not been yet studied. Here, we provide the results of TEM investigations of zoospores and sporangia from two strains of S. tribonematis. The two strains are endowed with unusual features. Like in A. gromovi, amoeboid zoospores of S. tribonematis contain a long kinetosome composed of microtubular singlets, and the two orthogonal centrioles in their sporangia have nine microtubular singlets with an internal ring. The morphological and ultrastructural features of S. tribonematis are now included in the improved taxonomic diagnosis for this species.     

Structure and Protein Composition of a Basal‐Body Scaffold (“Cage”) in the Hypotrich Ciliate Euplotes

Cilia on the ventral surface of the hypotrich ciliate Euplotes are clustered into polykinetids or compound ciliary organelles, such as cirri or oral membranelles, used in locomotion and prey capture. A single polykinetid may contain more than 150 individual cilia; these emerge from basal bodies held in a closely spaced array within a scaffold or framework structure that has been referred to as a basal‐body “cage”. Cage structures were isolated free of cilia and basal bodies; the predominant component of such cages was found on polyacrylamide gels to be a 45‐kDa polypeptide. Antisera were raised against this protein band and used for immunolocalizations at the light and electron microscope levels. Indirect immunofluorescence revealed the 45‐kDa polypeptide to be localized exclusively to the bases of the ventral polykinetids. Immunogold staining of thin sections of intact cells further localized this reactivity to filaments of a double‐layered dense lattice that appears to link adjoining basal bodies into ordered arrays within each polykinetid. Scanning electron microscopy of isolated cages reveals the lower or “basal” cage layer to be a fine lacey meshwork supporting the basal bodies at their proximal ends; adjoining basal bodies are held at their characteristic spacing by filaments of an upper or “medial” cage layer. The isolated cage thus resembles a miniature test‐tube rack, able to accommodate varying arrangements of basal‐body rows, depending on the particular type of polykinetid. Because of its clear and specific localization to the basal‐body cages in Euplotes, we have termed this novel 45‐kDa protein “cagein”.     

The amoebae of Idionectes vortex (Cutosea,Amoebozoa): Motility,cytoskeleton architecture and extracellular scales

The Cutosea represent a deep-branching lineage within the phylum Amoebozoa that is still relatively poorly explored. Currently, there are four cutosean representatives known – the monotypic genera Armaparvus, Idionectes, Sapocribrum, and Squamamoeba – with marked genetic distances. Idionectes vortex is the deepest-branching species and differs markedly from the other Cutosea in ecology, life history, and most importantly, in its ability to form a flagellated swarmer with an exceptional swimming mechanism. As far as we know, the other Cutosea lack flagella and rather represent small, marine amoebae with a characteristic cell coat. The present paper focuses on the amoeboid life history stage of the algivorous amoeboflagellate Idionectes vortex to provide data for a first in-depth comparison with other Cutosea and to document structural specialties. The amoeboid stage of Idionectes is mainly associated with the specific feeding process, that is, the interaction with algal prey cells and phagocytosis of protoplast material. Yet, the present data from time-lapse microscopy, cytochemical stainings, and electron microscopy demonstrate clear similarities with the other cutosean species concerning amoeboid locomotion and cell coat ultrastructure. Furthermore, Idionectes amoebae exhibit a well-developed microtubular cytoskeleton, and an unusual basal apparatus that seems to undergo marked changes during the life history of this exceptional amoebozoan.