Quantitative analysis of trichocysts in Paramecium bursaria following artificial removal and infection with the symbiotic Chlorella variabilis.

The ciliate Paramecium bursaria possesses cell organelles called trichocysts that have defensive functions. Paramecium bursaria is capable of symbiosis with Chlorella variabilis, and the symbiotic algae are situated in close proximity to the trichocysts. To clarify the relationship between trichocysts in P. bursaria and the presence or absence of the intracellular symbiotic C. variabilis, this study compared the regeneration capacity of trichocysts in alga-free and algae-bearing P. bursaria. In addition, trichocyst protein abundance was measured when alga-free P. bursaria specimens were artificially infected with Chlorella. After completely removing trichocysts from P. bursaria cells by treatment with lysozyme and observing them after 24 h, the percentage of regenerating trichocysts in the entire cell was significantly higher in alga-free cells than that in algae-bearing cells. We also developed a simple method for the isolation of high-purity trichocysts to quantify trichocyst protein amounts. There was a significant difference in the trichocyst protein abundance of P. bursaria before and one week after mixing with Chlorella (i.e., after the establishment of symbiosis with algae). This study shows the importance of trichocysts in alga-free P. bursaria as well as their competition with symbiotic C. variabilis for attachment sites during the algal infection process.

Membrane traffic and Ca2+ signals in ciliates.

A Paramecium cell has as many types of membrane interactions as mammalian cells, as established with monoclonal antibodies by R. Allen and A. Fok. Since then, we have identified key players, such as SNARE proteins, Ca2+ -regulating proteins, including Ca2+ -channels, Ca2+ -pumps, Ca2+ -binding proteins of different affinity, etc., at the molecular level, probed their function and localized them at the light and electron microscopy level. SNARE proteins, in conjunction with a synaptotagmin-like Ca2+ -sensor protein, mediate membrane fusion. This interaction is additionally regulated by monomeric GTPases whose spectrum in Tetrahymena and Paramecium has been established by A. Turkewitz. As known from mammalian cells, GTPases are activated on membranes in conjunction with lumenal acidification by an H+ -ATPase. For these complex molecules, we found in Paramecium an unsurpassed number of 17 a-subunit paralogs which connect the polymeric head and basis part, V1 and V0. (This multitude may reflect different local functional requirements.) Together with plasmalemmal Ca2+ -influx channels, locally enriched intracellular InsP3 -type (InsP3 R, mainly in osmoregulatory system) and ryanodine receptor-like Ca2+ -release channels (ryanodine receptor-like proteins, RyR-LP), this complexity mediates Ca2+ signals for most flexible local membrane-to-membrane interactions. As we found, the latter channel types miss a substantial portion of the N-terminal part. Caffeine and 4-chloro-meta-cresol (the agent used to probe mutations of RyRs in man during surgery in malignant insomnia patients) initiate trichocyst exocytosis by activating Ca2+ -release channels type CRC-IV in the peripheral part of alveolar sacs. This is superimposed by Ca2+ -influx, that is, a mechanism called "store-operated Ca2+ -entry" (SOCE). For the majority of key players, we have mapped paralogs throughout the Paramecium cell, with features in common or at variance in the different organelles participating in vesicle trafficking. Local values of free Ca2+ -concentration, [Ca2+ ]i , and their change, for example, upon exocytosis stimulation, have been registered by flurochromes and chelator effects. In parallel, we have registered release of Ca2+ from alveolar sacs by quenched-flow analysis combined with cryofixation and X-ray microanalysis.

RhopH3, rhoptry gene conserved in the free-living alveolate flagellate Colpodella sp. (Apicomplexa).

In this study, we investigated morphological, immunological and molecular characteristics of Colpodella sp. (American Type Culture Collection 50594) in a diprotist culture containing Bodo caudatus as prey using Plasmodium rhoptry specific antibodies and oligonucleotide primers targeting Plasmodium falciparum rhoptry genes. In culture, Colpodella sp. attached to its prey using the apical end with attachment lasting for approximately 20 min while the cytoplasmic contents of the prey were aspirated into the posterior food vacuole of Colpodella sp. Encystment of Colpodella sp. was observed following feeding. Indirect immunofluorescence assay (IFA) and confocal microscopy using P. falciparum rhoptry specific antibodies showed intense reactivity with cytoplasmic vesicles of Colpodella sp. Bodo caudatus from diprotist and monoprotist (ATCC 30395) cultures showed weak background reactivity. Giemsa staining permitted differentiation of both protists. Genomic DNA isolated from the diprotist culture was used in polymerase chain reaction (PCR) with oligonucleotide primers targeting the P. falciparum rhoptry genes RhopH3, RhopH1/Clag3.2 and RAMA. Primers targeting exon 7 of the P. falciparum RhopH3 gene amplified an approximately 2 kb DNA fragment from the diprotist DNA template. DNA sequence and BLAST search analysis of the amplified product from diprotist DNA identified the RhopH3 gene demonstrating that the RhopH3 gene is conserved in Colpodella sp.

Loss of a Fragile Chromosome Region leads to the Screwy Phenotype in Paramecium tetraurelia.

A conspicuous cell-shape phenotype known as "screwy" was reported to result from mutations at two or three uncharacterized loci in the ciliate Paramecium tetraurelia. Here, we describe a new screwy mutation, Spinning Top, which appeared spontaneously in the cross of an unrelated mutant with reference strain 51. The macronuclear (MAC) genome of the Spinning Top mutant is shown to lack a ~28.5-kb segment containing 18 genes at the end of one chromosome, which appears to result from a collinear deletion in the micronuclear (MIC) genome. We tested several candidate genes from the deleted locus by dsRNA-induced silencing in wild-type cells, and identified a single gene responsible for the phenotype. This gene, named Spade, encodes a 566-aa glutamine-rich protein with a C2HC zinc finger. Its silencing leads to a fast phenotype switch during vegetative growth, but cells recover a wild-type phenotype only 5-6 divisions after silencing is stopped. We analyzed 5 independently-obtained mutant alleles of the Sc1 locus, and concluded that all of them also lack the Spade gene and a number of neighboring genes in the MAC and MIC genomes. Mapping of the MAC deletion breakpoints revealed two different positions among the 5 alleles, both of which differ from the Spinning Top breakpoint. These results suggest that this MIC chromosome region is intrinsically unstable in strain 51.

On the morphology and predatory behavior of the dinoflagellate Oxyrrhis marina exposed to reduced salinity.

Changes in salinity are known to alter the morphology of protists, and we hypothesized that these changes subsequently alter also the predatory behavior of the dinoflagellate Oxyrrhis marina. Oxyrrhis was grown in media of 33, 25, 20, and 10% of the regular salinity of f/2 medium (31-32‰). In all cases, the cells discharged trichocysts and swelled. Cell surfaces and volumes increased with decreasing salinity, such that cell surface area at least doubled at 10% and the cell volume increased approximately fourfold. After 1 h, the cells started to regain their regular shape, which was almost completed after 24 h. Oxyrrhis immediately regained its regular shape when culture medium was added 5-10 min after the osmotic stress. When incubated with Pyramimonas grossii as prey, those short-term stressed cells showed no significant different prey uptake in comparison to non-stressed cells. In contrast, 24 h after the addition of prey, short-term stressed Oxyrrhis cells had, with weak statistical significance, more Pyramimonas cells engulfed than non-stressed cells. These results indicated that (1) trichocysts were most likely not involved in prey capture and (2) salinity-stressed Oxyrrhis either enhanced its capability to capture more prey, or its digestion apparatus was hampered.

Lectin-binding and dissociation/reconstitution studies on the trichocysts of the dinoflagellate Oxyrrhis marina.

Trichocyst-enriched fractions were isolated from the dinoflagellate Oxyrrhis marina and subjected to protein staining and lectin-binding studies, to dissociation experiments using heat, and to solubilization/reconstitution experiments using 6 M guanidine hydrochloride. The trichocysts could be stained by Alcian Blue and became labeled by the lectin Concanavalin A, but not by wheat germ agglutinin. The trichocysts did not dissociate when the fractions were heated for 5 min at 40 or 50 °C. Heating at 60 °C resulted in the dissociation of trichocysts into irregular filamentous structures. These filaments were still present when the fractions were incubated for 5 min at temperatures of 70 and 80 °C. Reassembly was not achieved by subsequent cooling steps. The disintegration of trichocysts was also achieved in 6 M guanidine hydrochloride, and reassembly into filamentous structures, similar to those obtained by heat, occurred after dialysis against distilled water. Electron microscopy revealed that the filaments created either by heat or using guanidine hydrochloride by far did not resemble native trichocysts. They were much thinner (5-7 nm in width), missed the characteristic striation of electron-dense and -transparent lines along the longitudinal axes, and showed much more bending. Furthermore, they tend to merge to thicker shapeless structures and blob-like aggregates. Sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed that fractions enriched either in trichocysts or in reconstituted filamentous structures obtained in the guanidine hydrochloride solubilization experiments were dominated by proteins with relative molecular weights in the range of approximately 15 to 29 kDa. Minor amounts of larger proteins were also detected. Tryptic in gel digestion followed by mass spectrometry confirmed the presence of almost the same set of proteins within the both, the trichocyst-enriched fractions, and the fractions of filaments reconstituted thereafter. These proteins were previously proposed to represent the matrix polypeptides of the trichocysts of Oxyrrhis marina (Rhiel et al., Protoplasma 255: 217-230, 2018).

Developmental stages identified in the trophozoite of the free-living Alveolate flagellate Colpodella sp. (Apicomplexa).

In this study we performed light, immunofluorescent and transmission electron microscopy of Colpodella trophozoites to characterize trophozoite morphology and protein distribution. The use of Giemsa staining and antibodies to distinguish Colpodella life cycle stages has not been performed previously. Rhoptry and ß-tubulin antibodies were used in immunofluorescent assays (IFA) to identify protein localization and distribution in the trophozoite stage of Colpodella (ATCC 50594). We report novel data identifying "doughnut-shaped" vesicles in the cytoplasm and apical end of Colpodella trophozoites reactive with antibodies specific to Plasmodium merozoite rhoptry proteins. Giemsa staining and immunofluorescent microscopy identified different developmental stages of Colpodella trophozoites, with the presence or absence of vesicles corresponding to maturity of the trophozoite. These data demonstrate for the first time evidence of rhoptry protein conservation between Plasmodium and Colpodella and provide further evidence that Colpodella trophozoites can be used as a heterologous model to investigate rhoptry biogenesis and function. Staining and antibody reactivity will facilitate phylogenetic, biochemical and molecular investigations of Colpodella sp. Developmental stages can be distinguished by Giemsa staining and antibody reactivity.