Food Vacuole Formation Research Articles

The Tetrahymena Paradigm: Genetic Insights and Ecotoxicity Assessment

Tetrahymena, a ciliated protozoan, thrives in freshwater habitats across diverse climates. Its importance as a genetic model organism lies in its cellular complexity, with cells comparable to human cells and a genome size between yeast and human. Tetrahymena’s germline genome undergoes unique chromosome breakage and DNA elimination, shedding light on genome rearrangement mechanisms. In 2020, the macronuclear genome was fully sequenced, offering improved insights into chromosome structure and gene models. Tetrahymena’s molecular tools enable gene characterization, knockouts, and overexpression. Beyond genetics, Tetrahymena serves as an ecotoxicity model, particularly Tetrahymena pyriformis. Changes in its morphology, food vacuole formation, and contractile vacuole response serve as toxicity indicators. Tetrahymena’s unique nuclear dimorphism (micronucleus and macronucleus) adds depth to ecotoxicological research. Additionally, Tetrahymena’s behavioral endpoints, including chemotaxis, phagocytosis, and motility, offer sensitive toxicity markers. Researchers have developed a T-maze toxotactic assay, a quick tool for studying Tetrahymena’s behavioral responses to different chemicals. Tetrahymena’s phagocytosis and motility are influenced by exposure to toxicants, reflecting changes in ion channels. Tetrahymena’s rapid movement makes it ideal for studying motility alterations caused by toxic substances. AI-driven tools enhance Tetrahymena behavior studies, allowing for behavior classification, feature extraction, and predictive modeling. This interdisciplinary approach aids in understanding Tetrahymena’s intricate behaviors, offering potential applications in gene discovery and pharmacological interventions. In conclusion, Tetrahymena stands as an invaluable model organism for genetic and ecotoxicological research. Its advantages, including genetic tractability and behavioral sensitivity, contribute to our understanding of fundamental biological processes and chemical safety assessment, with AI playing a growing role in advancing this field.

Uptake of plastic microbeads by ciliate Paramecium aurelia

Microplastics (MPs) are small fraction of plastics that are less than 5 mm in length. They are bountiful and widespread pollutants in the aquatic environment. A wide range of organisms which play an important role in the food web, ingest microplastic particles and transfer them to the higher trophic levels. In this work, ingestion of fluorescent polystyrene beads 2 µm of diameter by ciliated protozoa Paramecium aurelia in different concentrations and times of exposure was studied. We studied also the ingestion and clearance rate as well as formation of food vacuoles. The highest uptake of beads by ciliates reached 1047.2 ± 414.46 particles after 10 min of incubation. Food vacuoles formation reflected the ingestion rate of P. aurelia, which increased at higher beads concentration up to the10th minute of incubation and decreased afterwards. On the contrary, the clearance rate persisted to be higher at low concentration. These findings showed that maximum capacity of microplastics ingestion by paramecia depended on beads concentration and on time of exposure.

Rosette Colonies of Choanoflagellates (Salpingoeca rosetta) Show Increased Food Vacuole Formation Compared with Single Swimming Cells.

Choanoflagellates exist as both single-celled and colonial forms and filter-feed by generating water currents using a single apical flagellum. Hydrodynamic modeling studies have differed in predictions of whether single cells or colonies produce greater fluid flow to enhance feeding, and a recent study reported no increase in feeding efficiency of stalked colonies of choanoflagellates compared with single cells. We report that rosette colonies of Salpingoeca rosetta demonstrate higher rates of food vacuole formation compared with unicellular, slow swimmers.

Longer Exposure to 2% India Ink Increases Average Number of Vacuoles in Tetrahymena pyriformis

Tetrahymena pyriformis are single-celled phagocytes that feed by forming food vacuoles in a process known as phagocytosis. The goal of this experiment was to examine to what extent exposure of Tetrahymena to India ink affects the average number of vacuoles formed. We hypothesized that the increase of feeding time would increase the amount of India ink food vacuoles per Tetrahymena in a linear fashion. By observing Tetrahymena vacuole formation in response to India ink over a 60-minute period, we found that that vacuole formation in Tetrahymena initially increased linearly with increased India ink exposure time. After a certain time, however, vacuole formation ceased due to either a lack of remaining ink or a deficiency in the energy or resources necessary to continue performing phagocytosis. The relationship between India ink exposure time and food vacuole formation also provides insight into correlated changes in metabolic rate in Tetrahymena and other organisms for which Tetrahymena serves as a model. The way in which Tetrahymena budget their energy over time between vacuole formation, metabolism, and the countless other biological processes necessary for survival, particularly when supplied a non-nutritional particle such as India ink, could also be important for further studies on various organisms’ reactions to food shortages.

Kaolin Alleviates Graphene Oxide Toxicity

With increased interest in the practical use of graphene-based materials, concerns about the remediation of the environmental nanotoxicity of graphene and graphene-related materials have grown. In this study, we report that kaolin nanoclay significantly alleviates the toxicity of graphene oxide in aqueous environments. We employed the Paramecium caudatum protozoan to demonstrate the effects of equal concentrations of kaolin on the remediation of graphene oxide toxicity on survival and growth rates, chemotaxis, galvanotaxis, DNA complexation, and food vacuole formation. Importantly, the toxicity of graphene oxide coagulated with kaolin is reduced without the aggregated particles being removed from the environment.

ADF/Cofilin Is Not Essential but Is Critically Important for Actin Activities during Phagocytosis in Tetrahymena thermophila

ADF/cofilin is a highly conserved actin-modulating protein. Reorganization of the actin cytoskeleton in vivo through severing and depolymerizing of F-actin by this protein is essential for various cellular events, such as endocytosis, phagocytosis, cytokinesis, and cell migration. We show that in the ciliate Tetrahymena thermophila, the ADF/cofilin homologue Adf73p associates with actin on nascent food vacuoles. Overexpression of Adf73p disrupted the proper localization of actin and inhibited the formation of food vacuoles. In vitro, recombinant Adf73p promoted the depolymerization of filaments made of T. thermophila actin (Act1p). Knockout cells lacking the ADF73 gene are viable but grow extremely slowly and have a severely decreased rate of food vacuole formation. Knockout cells have abnormal aggregates of actin in the cytoplasm. Surprisingly, unlike the case in animals and yeasts, in Tetrahymena, ADF/cofilin is not required for cytokinesis. Thus, the Tetrahymena model shows promise for future studies of the role of ADF/cofilin in vivo.

Is It Time to Consider Photobiomodulation As a Drug Equivalent?

The question of whether photobiomodulation should be used as a drug equivalent arose in my mind after listening to presentations at the recent conference of the World Association for Laser Therapy (WALT)-2012 (Gold Cost City, Australia), and later at home when searching MEDLINE for the years 2009–2012. Photobiomodulation (earlier terms: low level laser therapy, LLLT, laser biostimulation) has been used in clinical practice for >40 years by now, and its action mechanisms on cellular and molecular levels have been studied for > 30 years. Enthusiastic medical specialists successfully used photobiomodulation in treating healing-resistant wounds and ulcers (e.g., chronic diabetic ulcers), in pain management, and in spinal cord and nervous system injuries when other methods had had limited success. However, photobiomodulation is still not a part of mainstream medicine. The goal of the present Editorial is to highlight some important recent developments in clinical applications and in studies of cellular and molecular mechanisms behind the clinical findings. One of the impressive and perspective challenges for photobiomodulation is its use in cases of Parkinson’s disease. Research in recent years evidenced that neuroprotective treatment with red and near infrared radiation (NIR) prevented mitochondrial dysfunction and dopamine loss in Parkinson’s disease patients. In another set of experiments, NIR normalized mitochondrial movement and axon transport, as well as stimulating respiration in cytoplasmic hybrid (‘‘cybrid’’) neurons. It is important to recall that reduced axonal transport contributes substantially to the degeneration of neuronal processes in Parkinson’s disease. Another development in recent years is the successful stimulation of stem cells with red and NIR radiation. One example is the treatment of myocardial infarction. The heart has been considered a post-mitotic organ lacking the capacity for self-renewal after injury. Surprisingly enough, human cardiac stem cells, in combination with bone marrow mesenchymal stem cells, were found to reduce infarct size and restore cardiac functions after myocardial infarction. This positive effect can even be increased by irradiation of stem cells. Mesenchymal stem cells were derived from bone marrow and adipose tissue, and stimulated by irradiation at k = 810 nm. Implantation of irradiated cells into the infarcted rat heart resulted in an *50% decrease in cardiac infarct size. An increase in proliferation rates and membrane potential was established after 532 nm irradiation of adipose tissue-derived stem cells. A recent review summarized data about enhancement of the proliferation of various cultured cell lines, including stem cells, as well as cell lines used for the production of viral vaccines and hybrid cell lines. The review underlined that photobiomodulation improves the proliferation of cells without causing any cytotoxic effects. One has to emphasize that laser therapy shares none of the risks associated with stem cell therapy, requires no anesthesia, and is painless. The optimal light parameters in this review were found to be as follows: doses were 0.5–4.0 J/ cm and wavelengths ranged from 600 to 700 nm. It is important to recall that, in this particular wavelength range, two peaks in absorption and action spectra connected with activation of cytochrome c oxidase (the primary photoacceptor for photobiomodulation effects) are situated. The peak at 620 nm belongs to reduced CuA, and that at 680 nm, to oxidized CuB atoms in cytochrome c oxidase molecule. The treatment of vitiligo (a depigmentary disorder) remains a challenge for clinical dermatologists. He-Ne laser irradiation was found to stimulate melanocyte proliferation. The expression of phosphorylated cyclic-adenosine monophosphate (AMP) response element-binding protein, an important regulator of melanocyte growth, was upregulated by He-Ne laser treatment. He-Ne laser irradiation imparted a growth stimulatory effect on functional melanocytes via mitochondria-related pathways. Irradiation with red light caused gene and noncoding RNA regulation for photoacceptor protection in the retina. This finding may open a new challenge for photobiomodulation. One of the major dose-limiting effects of chemotherapy drugs is oral mucositis of treated patients. Oral mucositis can affect up to 100% of patients undergoing high-dose chemotherapy and hematopoietic stem cell transplantation. Photobiomodulation can improve tissue repair and immune response in these patients. Photobiomodulation has been shown to improve functional outcome after surgical intervention to repair injured nerves. LED irradiation at 810 nm accelerated functional recovery and improved the quality of nerve regeneration after autograft repair of severely injured peripheral nerves. Forehead treatments with NIR reversed major depression and anxiety. Transcranial NIR laser therapy was investigated

Identification, Localization, and Functional Implications of the Microdomain-Forming Stomatin Family in the Ciliated Protozoan Paramecium tetraurelia

The SPFH protein superfamily is assumed to occur universally in eukaryotes, but information from protozoa is scarce. In the Paramecium genome, we found only Stomatins, 20 paralogs grouped in 8 families, STO1 to STO8. According to cDNA analysis, all are expressed, and molecular modeling shows the typical SPFH domain structure for all subgroups. For further analysis we used family-specific sequences for fluorescence and immunogold labeling, gene silencing, and functional tests. With all family members tested, we found a patchy localization at/near the cell surface and on vesicles. The Sto1p and Sto4p families are also associated with the contractile vacuole complex. Sto4p also makes puncta on some food vacuoles and is abundant on vesicles recycling from the release site of spent food vacuoles to the site of nascent food vacuole formation. Silencing of the STO1 family reduces mechanosensitivity (ciliary reversal upon touching an obstacle), thus suggesting relevance for positioning of mechanosensitive channels in the plasmalemma. Silencing of STO4 members increases pulsation frequency of the contractile vacuole complex and reduces phagocytotic activity of Paramecium cells. In summary, Sto1p and Sto4p members seem to be involved in positioning specific superficial and intracellular microdomain-based membrane components whose functions may depend on mechanosensation (extracellular stimuli and internal osmotic pressure).

Plasmodium falciparum enolase: stage-specific expression and sub-cellular localization

BackgroundIn an earlier study, it was observed that the vaccination with Plasmodium falciparum enolase can confer partial protection against malaria in mice. Evidence has also build up to indicate that enolases may perform several non-glycolytic functions in pathogens. Investigating the stage-specific expression and sub-cellular localization of a protein may provide insights into its moonlighting functions.MethodsSub-cellular localization of P. falciparum enolase was examined using immunofluorescence assay, immuno-gold electron microscopy and western blotting.ResultsEnolase protein was detected at every stage in parasite life cycle examined. In asexual stages, enolase was predominantly (≥85–90%) present in soluble fraction, while in sexual stages it was mostly associated with particulate fraction. Apart from cytosol, enolase was found to be associated with nucleus, food vacuole, cytoskeleton and plasma membrane.ConclusionDiverse localization of enolase suggests that apart from catalyzing the conversion of 2-phosphoglycericacid into phosphoenolpyruvate in glycolysis, enolase may be involved in a host of other biological functions. For instance, enolase localized on the merozoite surface may be involved in red blood cell invasion; vacuolar enolase may be involved in food vacuole formation and/or development; nuclear enolase may play a role in transcription.

Inactivation of the Bacteriophage MS2 by the Ciliated Protozoan, Tetrahymena thermophila

Abstract Because the range of biological mechanisms responsible for the inactivation of viruses in man-made and natural water systems is poorly understood, the involvement of the free-living ciliated protozoan, Tetrahymena thermophila, in viral inactivation was investigated. The ciliate was found to remove the bacteriophage MS2 when the phage and ciliate were co-incubated in a simple salt solution. MS2 was enumerated as plaque forming units (pfus). MS2 removal was achieved only by living and not formalin-fixed ciliates, and was blocked by treatments that impaired the formation of food vacuoles. These treatments were cytochalasin B and low temperature. When fluorescently labelled with SYBR gold prior to co-incubation, MS2 were seen inside Tetrahymena within vesicles that had the shape and size of food vacuoles. The number of pfus associated with Tetrahymena was low. This suggests that the engulfment of the phage into food vacuoles led to the inactivation of MS2, which is frequently used as a surrogate for poliovirus in environmental microbiology. In the future, a broader understanding of the capacity of ciliates to inactivate viruses could lead to methods for improving water quality through the manipulation of ciliate populations and activities.

Effect of the bioactive metabolite euplotin C on phagocytosis and fluid-phase endocytosis in the single-celled eukaryote Paramecium

The effect of euplotin C – a lipophilic bioactive metabolite produced by the ciliate Euplotes crassus – on the kinetics of both phagocytosis of latex particles and fluid-phase uptake of dextran, was studied in the single-cell ciliate Paramecium primaurelia. The inhibition of food vacuole formation was concentration- and time-dependent ( p < 0.001), even if euplotin C did not completely block the phagocytosis. Following a 15 min treatment with a euplotin C (0.5 μg/ml), the latex particle uptake was inhibited up to 25%. Furthermore, the pretreatment of cells with taxol strongly counteracted euplotin C effect. The amount of extracellularly provided dextran, which is internalized exclusively by fluid-phase uptake, was quantified in cells whose phagocytic activity was blocked by trifluoperazine. The amount of the internalized dextran was about 50% of that in controls after 15 min incubation in the presence of euplotin C. Fluorescence confocal images showed that no endosomes were formed on the surface of these cells. The effect of euplotin C on the food vacuole formation and fluid-phase endocytosis is apparently mediated by a modification of microtubule network.

Low cost microbioassay test for assessing cytopathological and physiological responses of ciliate model Paramecium caudatum to carbofuran pesticide

One of the major changes that have occurred over the past few years is the re-examination of conventional methods and the global demand for innovative low cost bioassay tests for assessing water quality, toxicity evaluation and bioremediation. In the present study acute toxicity, physiological and cytotoxic impact of carbofuran to a commonly occurring fresh water ciliate was measured. Intrinsic cytotoxicity was evident on macro nuclear apparatus that exhibited deformities such as fragmentation, deep incision, vacuolization and degenerative macronucleus. Depletion in the food vacuole formation of Paramecia was evident. Changes in the pulsatory vacuole activity of Paramecium caudatum, highlighted dose dependent response by the test compound. The simplicity of handling this ciliate makes unicellular eukaryote, an alternative organism for the toxicity assessment of pollutants. The tests carried in this study are simple and fast, a bioassay that gives overall information about the physiological and cytotoxic effects of carbofuran to P. caudatum. Such bioassay tests using ciliates are more suitable for risk assessment of water quality, early detection of water pollution and possible role of ciliates as bioindicators and strategic tools in the bioremediation of water bodies.

Lanthanum affects proliferation and motility of Tetrahymena

Lanthanum (La 3+ ), a Ca-channel blocker, affected proliferation of Tetrahymena concentration-dependently in 2% proteose-peptone (PP) medium. The effect increased with decreasing amount of organic matter (2–0.5% PP). Cell motility was affected slightly in low La 3+ concentrations but in high concentrations the cells became motionless with rocking movements; however, most cells regained motility after some hours (restored Ca-homeostasis). Addition of La 3+ to the PP media caused formation of a fluffy precipitate which was engulfed by the cells. The capacity to form food vacuoles, was normal in proliferating cells but decreased, or ceased, in growth-inhibited cells. Small refractive granules appeared in all treated cells and most abundantly in growth-inhibited cells. The fine structure of treated cells related to whether they were capable, or not, of proliferation. Electron-dense material (La 3+ ) was abundant in food vacuoles, seen on the surface of cells early after exposure to high concentrations, and present in the dense (small refractive) granules. Hence, La 3+ apparently enters the cells.

Effects of nutrient limitation on food uptake in the toxic haptophyte Prymnesium parvum

AME Aquatic Microbial Ecology Contact the journal Facebook Twitter RSS Mailing List Subscribe to our mailing list via Mailchimp HomeLatest VolumeAbout the JournalEditorsSpecials AME 31:259-265 (2003) - doi:10.3354/ame031259 Effects of nutrient limitation on food uptake in the toxic haptophyte Prymnesium parvum Alf Skovgaard1,3,*, Catherine Legrand2, Per J. Hansen1, Edna Granéli2 1Marine Biological Laboratory, University of Copenhagen, Strandpromenaden 5, 3000 Helsingør, Denmark 2Department of Marine Sciences, University of Kalmar, Barlastgatan 1, 391 82 Kalmar, Sweden 3Present address: Departament de Biologia Marina i Oceanografia, Institute de Ciències del Mar, CMIMA-CSIC, Passeig Marítim de la Barceloneta 37-49, 08003 Barcelona, Spain *Email: skovgaard@icm.csic.es ABSTRACT: The haptophyte Prymnesium parvum Carter is toxic and frequently responsible for harmful algal blooms in coastal waters. It is a mixotrophic species having the capability to feed on various planktonic microorganisms. It is frequently suggested that mixotrophic algae may obtain inorganic nutrients through phagotrophy and that nutrient depletion should then lead to increased food uptake. To study this, we investigated the feeding activity of P. parvum in semi-continuous, nutrient-limited cultures, using the cryptophyte Rhodomonas baltica as prey. P. parvum showed to be an active predator under all conditions investigated. After 2 h of incubation with prey, 40% of P. parvum cells were either feeding or contained recently formed food vacuoles. However, under the conditions used, no difference in feeding activity was found between treatments. On the contrary, the feeding activity was similar in P. parvum cultures that had been grown under N-limiting, P-limiting, N- and P-limiting, as well as under nutrient-replete conditions. It cannot be excluded that P. parvum under limiting nutrient conditions may acquire nutrients to be used in photosynthetic growth through phagotrophy. It is evident, however, that the species also feeds when inorganic nutrients are present in concentrations sufficient to support maximum phototrophic growth. KEY WORDS: Food uptake · Phagotrophy · Mixotrophy · Toxic algae · Prymnesiophyceae · Nutrient limitation · Semi-continuous cultures · Harmful algal blooms Full text in pdf format PreviousNextExport citation RSS - Facebook - Tweet - linkedIn Cited by Published in AME Vol. 31, No. 3. Online publication date: April 03, 2003 Print ISSN: 0948-3055; Online ISSN: 1616-1564 Copyright © 2003 Inter-Research.

Mapping cholesteryl ester analogue uptake and intracellular flow in Paramecium by confocal fluorescence microscopy.

In Paramecium primaurelia the uptake and intracellular flow of cholesteryl ester was studied by fluorescence confocal laser scanning optical microscopy and by the fluorescent analogue cholesteryl-BODIPY FL C12 (BODIPY-CE). The BODIPY FL fluorophore has the characteristic of emitting green fluorescence, which is red-shifted as the probe concentrates. In cells incubated with 25 microm BODIPY-CE for 30 s, fluorescence is found in vesicles located around the cytopharynx in the posterior half of the cell. Successively, the lipid is internalized by food vacuoles, the fluorescent vesicles are distributed throughout the cell and the intracellular membranes are labelled. The food vacuole number is maximum after 10-15 min of continuous labelling, then it decreases until no food vacuoles are found in 30-min fed cells. BODIPY-CE accumulates in red-labelled cytoplasmic droplets located in the anterior half of the cell. When food vacuole formation is inhibited by trifluoperazine, fluorescence is found on cellular membranes and in small green-labelled vesicles at the apical pole. The inhibition of clathrin-mediated endocytosis does not interfere in P. primaurelia with BODIPY-CE intracellular flow: intracellular membranes and storage droplets in the cell anterior part are dyed. Conversely, the use of sterol-binding drugs prevents the lipid accumulation in droplets, stopping the lipid within the cytoplasmic membranes. Furthermore, the cells treated with monensin and cytochalasin B show a labelling of the cellular membranes and lipid droplets, whereas NH4Cl reduces the lipid storage. Low temperature (4 degrees C) does not prevent the internalization of BODIPY-CE that, however, is localized at the cytoplasmic membrane level and does not accumulate in storage droplets. In addition, BODIPY-CE inhibits phagocytosis, as evidenced by comparing the kinetics of food vacuole formation of control cells, only fed with latex particles, with that of cells fed with latex particles and BODIPY-CE. In conclusion, this study points out that in P. primaurelia the cholesteryl ester enters the cell via food vacuoles and through the plasma membrane and, inside the cell, it alters cell functions.

Fluid phase and receptor-mediated endocytosis in Paramecium primaurelia by fluorescence confocal laser scanning microscopy.

In ciliated protozoa, most nutrients are internalized via phagocytosis by food vacuole formation at the posterior end of the buccal cavity. The uptake of small-sized molecules and external fluid through the plasma membrane is a localized process. That is because most of the cell surface is internally covered by an alveolar system and a fibrous epiplasm, so that only defined areas of the cell surface are potential substance uptake sites. The purpose of this study is to analyze, by fluorescence confocal laser scanning microscopy, the relationship between WGA (Triticum vulgaris agglutinin) and dextran internalization in Paramecium primaurelia cells blocked in the phagocytic process, so that markers could not be internalized via food vacuole formation. WGA, which binds to surface constituents of fixed and living cells, was used as a marker for membrane transport and dextran as a marker for fluid phase endocytosis. After 3 min incubation, WGA-FITC is found on plasma membrane and cilia, and successively within small cytoplasmic vesicles. After a 10-15 min chase in unlabeled medium, the marked vesicles decrease in number, increase in size and fuse with food vacuoles. This fusion was evidenced by labeling food vacuoles with BSA-Texas red. Dextran enters the cell via endocytic vesicles which first localize in the cortical region, under the plasma membrane, and then migrate in the cytoplasm and fuse with other endocytic vesicles and food vacuoles. When cells are fed with WGA-FITC and dextran-Texas red at the same time, two differently labeled vesicle populations are found. Cytosol acidification and incubation in sucrose medium or in chlorpromazine showed that WGA is internalized via clathrin vesicles, whereas fluid phase endocytosis is a clathrin-independent process.

Age-associated survival and extinction in mixed cultures of Paramecium

When Paramecium tetraurelia (Pt) and P. multimicronucleatum (Pm) were cultured together, the outcome was either survival of Pt and extinction of Pm, or coexistence of Pt and Pm. We have shown that these fates were experimentally controllable by changing external culture conditions or by selecting stocks of Pm. However, we have so far failed to observe survival of Pm and extinction of Pt. We showed previously that the survival or extinction of Pm was associated with its ability or inability to form food vacuoles (FVs). An inability to form FVs is one of the characteristics of senescence in Paramecium. The extinction of Pm was observed only when stock CH312 of Pm was used, and this stock shows some senescent characteristics. Therefore, we studied the effects of clonal aging on the fate in mixed culture. Here, we report that all three possible fates including survival of Pm and extinction of Pt are controllable by changing the clonal age of Pt. Survival or extinction is also controllable between different age groups of the same species (Pt), indicating decisively that the phenomenon is not an inter-species problem. Cells without the ability to form FVs were found prior to the occurrence of extinction.

Effects of low dose of ketotifen and chloroquine combination on the infrastructure of chloroquine resistant strain of Plasmodium yoelii

46 inbred NIH mice were infected by chloroquine resistant strain of Plasmodium yoelii. The ultrastructure changes were observed under the administration of ketotifen(10mg·kg−1·d−1) and chloroquine(10mg·kg−1·d−1) combination and one after another respectively. The effect of taking ketotifen and chloroquine combination showed that parasites died rapidly with a few of intermittent membranes and vacuoles. The effect of taking two kinds of drugs one after another showed that there were exceedingly rich membranes, concentric arrangement structures similar to rough endo-reticulum and conspiciously blocking of the formation of food vacuoles.

MYO1, a novel, unconventional myosin gene affects endocytosis and macronuclear elongation in Tetrahymena thermophila.

Targeted gene disruption was used to investigate the function of MYO1, an unconventional myosin gene in Tetrahymena thermophila. Phenotypic analysis of a transformed strain that lacked a functional MYO1 gene was conducted at both 20 degrees C and 35 degrees C. At either temperature the delta MYO1 strain had a smaller cytoplasm/nucleus ratio than wild type. At 20 degrees C, delta MYO1 populations had a longer doubling time than wild type, lower saturation density, and a reduced rate of food vacuole formation. However, at 35 degrees C, these characteristics were comparable to wild type. Although micronuclear division and cytokinesis appeared normal in delta MYO1 cells, failure of the macronucleus to elongate properly resulted in unequal segregation of macronuclear DNA in cells maintained at either 20 degrees C or 35 degrees C.

Calmodulin and Ca2+/Calmodulin-Binding Proteins are Involved in Tetrahymena thermophila Phagocytosis.

The ciliated protist, Tetrahymena thermophila, possesses one oral apparatus for phagocytosis, one of the most important cell functions, in the anterior cell cortex. The apparatus comprises four membrane structures which consist of ciliated and unciliated basal bodies, a cytostome where food is collected by oral ciliary motility, and a cytopharynx where food vacuoles are formed. The food vacuole is thought to be transported into the cytoplasm by a deep fiber which connects with the oral apparatus. Although a large number of studies have been done on the structure of the oral apparatus, the molecular mechanisms of phagocytosis in Tetrahymena thermophila are not well understood. In this study, using indirect immunofluorescence, we demonstrated that the deep fiber consisted of actin, CaM, and Ca2+/CaM-binding proteins, p85 and EF-1alpha, which are closely involved in cytokinesis. Moreover, we showed that CaM, p85, and EF-1alpha are colocalized in the cytostome and the cytopharynx of the oral apparatus. Next, we examined whether Ca2+/CaM signal regulates Tetrahymena thermophila phagocytosis, using Ca2+/CaM inhibitors chlorpromazine, trifluoperazine, N-(6-aminohexyl)-1-naphthalenesulfonamide, and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide HCI. In Tetrahymena, it is known that Ca2+/CaM signal is closely involved in ciliary motility and cytokinesis. The results showed that one of the inhibitors, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide HCl, inhibited the food vacuole formation rather than the ciliary motility, while the other three inhibitors effectively prevented the ciliary motility. Considering the colocalization of CaM, p85, and EF-1alpha to the cytopharynx, these results suggest that the Ca2+/CaM signal plays a pivotal role in Tetrahymena thermophila food vacuole formation.