Gymnodinium aeruginosum (Dinophyta): A blue-green dinoflagellate with a vestigial,anucleate, cryptophycean endosymbiont

Gymnodinium aeruginosum has the usual fine structure of a dinoflagellate but does not seem to contain a well elaborated peduncle or a microtubular basket. Naked cells are surrounded by a single large amphiesmal vesicle. It houses an endosymbiont with typical blue-green cryptophycean chloroplasts (generally only one), cryptophycean starch grains in the periplastidal cytoplasm without a nucleomorph, and two membranes separating the periplastidal cytoplasm from the cryptophycean cytoplasm which contains mitochondria, ER, vesicles and ribosomes, but no eukaryotic nucleus. The endosymbiont is surrounded by a single membrane. Possible ways of the acquisition of the endosymbiont and the problem of the existence of ribosomes within a compartment without nucleus are discussed.Devoted to Prof. Dr.L. Geitler, the Nestor of phycology and endosymbiosis research, on the occasion of the 90th anniversary of his birthday.     

The bloom-forming ciliate Mesodinium rubrum harbours a single permanent endosymbiont

Whether the red tide Mesodinium rubrum contains a permanent cryptophyte symbiont or whether it only sequesters chloroplasts from cryptophyte prey was addressed using electron microscopy and the dynamics of photosynthesis, chloroplasts and nuclei. Mesodinium rubrum contains a branched cryptophyte symbiont consisting of many chloroplasts, mitochondria, nucleomorphs, an endoplasmic reticulum and one nucleus. The volume of the symbiont constitutes 36% of the consortium and it is separated from its host by a single-cell membrane. The chloroplasts of Mesodium are larger and morphologically different from two Teleaulax species that served as prey. The symbiont nucleus is also much larger than Teleaulax nuclei. Although M. rubrum is functionally a phototroph, sustained growth beyond two to four generations requires ingestion of prey, but less than one prey cell per generation suffices for maximum growth. This suggests that either the ciliate or its symbiont needs an essential growth factor for continuous growth.     

Phylogenomics of non-model ciliates based on transcriptomic analyses

Ciliates are one of the oldest living eukaryotic unicellular organisms, widely distributed in the waters around the world. As a typical marine oligotrich ciliate, Strombidium sulcatum plays an important role in marine food webs and energy flow. Here we report the first deep sequencing and analyses of RNA-Seq data from Strombidium sulcatum. We generated 42,640 unigenes with an N50 of 1,451 bp after de novo assembly and removing rRNA, mitochondrial and bacteria contaminants. We employed SPOCS to detect orthologs from S. sulcatum and 17 other ciliates, and then carried out the phylogenomic reconstruction using 127 single copy orthologs. In phylogenomic analyses, concatenated trees have similar topological structures with concordance tree on the class level. Together with phylogenetic networks analysis, it aroused more doubts about the placement of Protocruzia, Mesodinium and Myrionecta. While epiplasmic proteins are known to be related to morphological characteristics, we found the potential relationship between gene expression of epiplasmic proteins and morphological characteristics. This work supports the use of high throughput approaches for phylogenomic analysis as well as correlation analysis between expression level of target genes and morphological characteristics.     

Motile behaviour of the bloom-forming ciliate Mesodinium rubrum

Mesodinium rubrum (Lohman) (=Myrionecta rubra Jankowsky) swims backwards in jumps of short duration interspersed by longer periods of rest. Cells attain a velocity of up to 1.2 cm s^-1 during jumps and this is probably a speed record for ciliates. The ciliate carries long cirri that serve as mechanoreceptors and for orientating the cell at the initiation of jumps, while the ciliary rows on the posterior part of the cell are responsible for propulsion. The cirri are sensitive to shear so that they can orientate themselves against the current in a siphon flow (such as generated by filter-feeding copepods). Mesodinium cells do not reorientate their body axis during sinking, but they reorientate their direction during the initiation of jumps so that they always tend to move upwards. Because the cells sink between jumps they can regulate their vertical position by modulation of the frequency of jumps. The cells' tendency to drift vertically up or down is light dependent. The jumps are so rapid that these phototrophic organisms can enhance their uptake of dissolved mineral nutrients beyond the limitation of molecular diffusion.     

Ultrastructure of the symbionts and mycetocytes of bird cherry aphid (Rhopalosiphum padi)

As is known for other aphids, the symbiont cytoplasm in Rhopalosiphum padi is differentiated into a central eccentric and a cortical region which contains electron-lucent nucleoplasm and DNA fibrils. The cytoplasm is surrounded by a plasma membrane, cell wall and perisymbiotic membrane which are separated by periplasmic spaces. The periplasmic space between the cell wall and perisymbiotic membrane contains small vesicles. The symbionts are packed into mycetocyte cells which contain a large nucleus and all the normal cell organelles. For the first time, however microtubules and microfilaments have been identified, but only at high magnification and evidence has been found which makes it possible to propose a mechanism of vesicular transport.     

Characterization of nuclear targeting signal of hepatitis delta antigen: nuclear transport as a protein complex.

Hepatitis delta antigen (HDAg) is the only protein encoded by hepatitis delta virus (HDV). HDAg has been demonstrated in the nuclei of HDV-infected hepatocytes, and its nuclear transport may be important for the replication of HDV RNA. In this report, we investigated the mechanism of nuclear transport of HDAg. By expressing fusion proteins consisting of the different portions of HDAg and alpha-globin, we have identified a nuclear localization signal (NLS) within the N-terminal one-third of HDAg. It consists of two stretches of basic amino acid domains separated by a short run of nonbasic amino acids. Both of the basic domains are necessary for the efficient nuclear transport of HDAg. The nonbasic spacer amino acids could be removed without affecting the nuclear targeting of HDAg significantly. Thus, the HDAg NLS belongs to a newly identified class of NLS which consists of two discontiguous stretches of basic amino acids. This NLS is separated from a stretch of steroid receptor NLS-like sequence, which is also present but not functioning as an NLS, in HDAg. Furthermore, we have shown that subfragments of HDAg which do not contain the NLS can be passively transported into the nucleus by a trans-acting full-length HDAg, provided that these subfragments contain the region with a leucine zipper sequence. Thus, our results indicate that HDAg forms aggregates in the cytoplasm and that the HDAg oligomerization is probably mediated by the leucine zipper sequence. Therefore, HDAg is likely transported into the nucleus as a protein complex.     

Pollen ultrastructure in anther cultures of Datura innoxia. III. Incomplete microspore division.

During the microspore division in Datura innoxia, the mitotic spindle is oriented in planes both perpendicular (PE) and oblique (OB) to the spore wall against which the nucleus is situated. However, irrespective of polarity, the usual type of hemispherical wall is laid down at cytokinesis and isolates the generative cell from the rest of the pollen grain (type A). In PE spores the vegetative nucleus initially occupies a central position in the pollen grain, whereas in OB spores the vegetative nucleus lies at the periphery of the grain close to the generative cell. In anther cultures initiated just before the microspore division is due to take place, no marked change can be observed in either orientation or symmetry of the mitotic spindle when the spores divide. In some, however, cytokinesis is disrupted and deposition of the hemispherical wall arrested. In the absence of a complete wall, differentiation of the generative cell cannot take place and binucleate pollen grains are formed having 2 vegetative-type nuclei (type B). The 2 nuclei in the B pollens are always situated against the pollen-grain wall, suggesting that the disruption phenomenon is related to the OB spores. The incomplete wall always makes contact with the intine on the intine-side of the spindle. Wall material may be represented merely as short stubs projecting out from the intine into the cytoplasm, in which event the 2 nuclei lie close to each other and are separated by only a narrow zone of cytoplasm. In other grains the wall is partially developed between the nuclei and terminates at varying distances from the tonoplast; in these, the nuclei are separated by a wider zone of cytoplasm. The significance of these binucleate grains in pollen embryogenesis is discussed.     

Ciliates and their picophytoplankton-feeding activity in a high-altitude warm-monomictic saline lake

The impact of feeding on autotrophic picoplankton (APP) on the ciliate composition of the assemblage was surveyed monthly along a depth gradient in the maar crater, athalassohaline, warm monomictic Lake Alchichica (Puebla, Mexico) from June 2003 to December 2005. Numbers of APP were evaluated from their autofluorescence. DAPI staining and the Fluorescently Labeled Bacteria technique were employed to count ciliates and estimate their feeding rates. A total of 38 taxa of ciliates have been identified using Quantitative Protargol Staining. Peritrichs followed by minute spirotrichs (particularly Halteria grandinella) often numerically dominated the ciliate assemblage and emerged as the most efficient APP feeders. A maximum of 54 ciliate cells ml⁻¹ was observed in the surface layer at the end of the mixing period, during the development of diatoms (Cyclotella alchichicana), the cyanobacterial bloom (Nodularia sp.) and its decay. Vorticellids (Pelagovorticella natans, Vorticella sp.) had the highest APP uptake (median 130 APP cil⁻¹ h⁻¹). Mixotrophic Euplotes cf. daidaleos were important APP grazers near the oxycline. Scuticociliates (Cyclidium glaucoma, Uronema nigricans and an anaerobic cf. Isocyclidium globossum), were numerically dominant within the hypolimnetic assemblages and did not ingest APP. Generally, APP were not an important food source for the majority of the ciliate assemblage, being positively selected by a few species during the APP decay in aerobic and microaerobic conditions.     

Morphogenesis of Ascospores in Saccharomyces cerevisiae

Ultrastructural changes associated with ascospore formation in Saccharomyces cerevisiae were investigated by using freeze-etching and thin-sectioning techniques. The first nuclear division (meiosis I) is indicated by the appearance of spindle fibers within the nucleus. The nucleus subsequently elongates and eventually assumes a barbell shape; the second nuclear division (meiosis II) occurs before nuclear separation. The spindle fibers involved in meiosis II appear to be oriented perpendicular to those observed in meiosis I. A discrete bilaminar structure (forespore wall) progressively delineates each ascospore nucleus and encloses cytoplasmic material including mitochondria and endoplasmic reticulum. The forespores then elongate, close off, and become separated from the ascus cytoplasm by membranes. The ascospores assume a spherical shape as spore coat material is laid down; the latter stages of ascospore formation are characterized by thickening of the ascospore wall and disintegration of the ascus cytoplasm. No structures which could be identified as chromosomes were observed.     

Possible participation of mitochondria in lipid yolk formation in oocytes of paddlefish and sturgeon

The ovary of paddlefish and sturgeons (Acipenseriformes) is composed of discrete units: the ovarian nests and ovarian follicles. The ovarian nests comprise oogonia and numerous early dictyotene oocytes surrounded by somatic prefollicular cells. Each ovarian follicle consists of a spherical oocyte and a layer of follicular cells situated on a thick basal lamina, encompassed by thecal cells. The cytoplasm of previtellogenic oocytes is differentiated into two distinct zones: the homogeneous and granular zones. The homogeneous cytoplasm is organelle-free, whereas the granular cytoplasm contains numerous organelles, including mitochondria and lipid droplets. We have analyzed the cytoplasm of early dictyotene and previtellogenic oocytes ultrastructurally and histologically. In the cytoplasm of early dictyotene oocytes, two morphologically different types of mitochondria can be distinguished: (1) with well-developed cristae and (2) with distorted and fused cristae. In previtellogenic oocytes, the mitochondria of the second type show various stages of cristae distortion; they contain and release material morphologically similar to that of lipid droplets and eventually degenerate. This process of mitochondrial transformation is accompanied by an accumulation of lipid droplets that form a single large accumulation (lipid body) located in the vicinity of the oocyte nucleus (germinal vesicle). The lipid body eventually disperses in the oocyte center. The possible participation of these mitochondria in the formation of oocyte lipid droplets is discussed. This work was supported by funds from the research grant BW/IZ/2005 to M.Ż. An erratum to this article can be found at http://dx.doi.org/. An erratum to this article can be found at     

Phylogeny of trichostome ciliates (Ciliophora, Litostomatea) endosymbiotic in the Yakut horse (Equus caballus)

Ciliates of the subclass Trichostomatia inhabit the fermentative regions of the digestive tract of herbivores. Most available small subunit ribosomal RNA (SSrRNA) gene sequences of trichostomes are from species isolated from the rumen of cattle or sheep and from marsupials. No ciliate species endosymbiotic in horses has yet been analyzed. We have sequenced the SSrRNA genes of five ciliate species, isolated from the cecum and colon of four Yakut horses: Cycloposthium edentatum, Cycloposthium ishikawai, Tripalmaria dogieli, Cochliatoxum periachtum, and Paraisotricha colpoidea.

Based on their morphology, Cycloposthium, Tripalmaria, and Cochliatoxum are classified as Entodiniomorphida, while Paraisotricha is considered a member of the Vestibuliferida. Phylogenetic analyses using Bayesian inference, distance, and parsimony methods confirm these placements. The two Cycloposthium species cluster together with the published Cycloposthium species isolated from a wallaby in Australia. Tripalmaria and Cochliatoxum branch as a sister group to or basal within the Entodiniomorphida. The Vestibuliferida remain paraphyletic with Paraisotricha and Balantidium branching basal to all other trichostome species, but not closely related to Isotricha and Dasytricha.     

Major and minor capsid proteins of human polyomavirus JC cooperatively accumulate to nuclear domain 10 for assembly into virions

The human polyomavirus JC (JCV) replicates in the nuclei of infected cells. Here we report that JCV virions are efficiently assembled at nuclear domain 10 (ND10), which is also known as promyelocytic leukemia (PML) nuclear bodies. The major capsid protein VP1, the minor capsid proteins VP2 and VP3, and a regulatory protein called agnoprotein were coexpressed from a polycistronic expression vector in COS-7 cells. We found that VP1 accumulated to distinct subnuclear domains in the presence of VP2/VP3 and agnoprotein, while VP1 expressed alone was distributed both in the cytoplasm and in the nucleus. Mutation analysis revealed that discrete intranuclear accumulation of VP1 requires the presence of either VP2 or VP3. However, VP2 or VP3 expressed in the absence of VP1 showed diffuse, not discrete, nuclear localization. The C-terminal sequence of VP2/VP3 contains two basic regions, GPNKKKRRK (cluster 1) and KRRSRSSRS (cluster 2). The deletion of cluster 2 abolished the accumulation of VP1 to distinct subnuclear domains. Deletion of the C-terminal 34 residues of VP2/VP3, including both cluster 1 and cluster 2, caused VP1 to localize both in the cytoplasm and in the nucleus. Using immunoelectron microscopy of cells that coexpressed VP1, VP2/VP3, and agnoprotein, we detected the assembly of virus-like particles in discrete locations along the inner nuclear periphery. Both in oligodendrocytes of the human brain and in transfected cells, discrete nuclear domains for VP1 accumulation were identified as ND10, which contains the PML protein. These results indicate that major and minor capsid proteins cooperatively accumulate in ND10, where they are efficiently assembled into virions.     

Ultrastructural Study of an Endosymbiotic Alga and Its Host Ciliate Stentor niger

Stentor niger collected in the suburbs of Hiroshima contained in its cytoplasm several hundreds of endosymbiotic algae and innumerable brownish pigment granules. The body of the ciliate was dark due to a mixture of the green endosymbiotic algae and brown pigment granules. The algae belonged to the genus Chlorella; each was enclosed in a perialgal vacuole and dispersed uniformly in the host cytoplasm from the myoneme layer inward to the center of the ciliate. The cell wall and plasma membrane of the alga enclosed a nucleus, chloroplast, mitochondrion, Golgi complex, accumulation bodies, myelinated vesicles, and many ribosomes. The chloroplast occupied more than half of the volume of the alga and contained a conspicuous pyrenoid. Algal multiplication occurred by two successive divisions of an alga, leading to four autospores within a perialgal vacuole; the walls of the vacuole invaginated to separate the autospores each into its own vacuole. Three types of pigment granules were scattered uniformly throughout the cytoplasm of the ciliate. The ultrastructure of the membranellar region, somatic cortex, and macro- and micronucleus of the ciliate are also described.     

Partial purification and characterization of a protein kinase that is activated by nuclear localization signal peptides

A nuclear localization signal (NLS) is required for the transport of karyophilic proteins from the cytoplasm to the nucleus. In this study, NLS was examined in terms of its effect on diverse cellular functions such as protein phosphorylation reactions. When synthetic peptides containing the NLS of SV40 T-antigen were injected into the cytoplasm of Xenopus oocytes, and the oocytes incubated with [³²P]phosphorus-containing medium, a 32 kDa protein was found to be preferentially phosphorylated in an NLS-dependent manner. The incubation of fractionated cytosolic extracts prepared from mouse Ehrlich ascites tumor cells with [γ-³²P]ATP in the presence of the NLS peptides, results in the stimulation of the phosphorylation of several proteins. Similar in vitro stimulation was observed by other functional NLS peptides such as those of polyoma virus T-antigen and nucleoplasmin. Little or no stimulation, however, was detected for peptides of mutant type and reverse type NLS of SV40 T-antigen, and the C-terminal portion of lamin B. Using an in vitro assay, the phosphorylation activity was fractionated chromatographically and a fraction was obtained which contained a high level of activity. The fraction was found to contain three major proteins having molecular masses of 95, 70, and 43 kDa. The in vivo and in vitro results are consistent with the existence of a protein kinase, called NLS kinase, that is specifically activated by NLS peptides.     

The HIV-1 Rev protein: a model system for coupled RNA transport and translation.

The impact of the Rev protein of the human immunodeficiency virus type 1 (HIV-1) on RNA transport, intranuclear RNA distribution, and gene expression was examined for two Rev-dependent expression systems by means of fluorescence in situ hybridization, immunofluorescence, S1 nuclease protection, and functional assays. In the pgTat expression system, which utilizes authentic HIV-1 splice signals, unspliced mRNA remained entrapped in the nucleus in the absence of Rev and was exported to the cytoplasm in its presence, consistent with published findings. In the pSVAR expression system, significant levels of mRNA were found in the nucleus and cytoplasm in both the presence and absence of Rev, but only in the presence of Rev was mRNA translated into protein. The presence of cytoplasmic untranslated mRNA in the absence of Rev was demonstrated by in situ hybridization analysis of individual cells as well as by S1 nuclease analysis of cell populations. The results indicate that Rev has the potential to affect translation as well as transport, suggesting the possibility that cellular mechanisms exist whereby the translational efficiency of an mRNA may be affected by the manner in which it is transported from the nucleus. Fluorescence hybridization also provided high-resolution visualization of the intranuclear distribution of RNAs containing the Rev response element. This demonstrated for both expression systems that mRNA was not highly localized in tracks or around the nucleolus in the presence or absence of Rev, a nucleolar protein, but was more widely distributed throughout the nucleus. In pgTat transfectants, HIV-1 RNA often became localized in 5 to 20 discrete large intranuclear clusters in the presence of Rev, the potential significance of which is discussed.     

Ultrastructure of the photosynthetic ciliate Mesodinium rubrum

New cellular structures, bifurcated oral tentacles, were observed in many specimens of the photosynthetic ciliate Mesodinium rubrum from the northern Baltic Sea. Cross-sections of tentacles revealed rings (cylinders) of 14 microtubules with spokes. The number of microtubules per ring decreased from 14 to 12 or 11 inside the cell but no true kinetosomes were detected. These "micro-rings" were often associated with extrusomes and the tentacle tips consisted of extrusomes. A nucleus of a symbiotic alga was present, surrounded by algal cytoplasm containing plastids and delimited from the ciliate cytoplasm by two membranes. Each plastid was bounded by four membranes and was associated with one nucleomorph, suggesting a symbiotic origin as a cryptophyte. The unique symbiotic organization and the organelles of 14 microtubules make Mesodinium rubrum an organism of unusual evolutionary interest.