Paramecium Cells Research Articles

Novel Insights into the Development and Function of Cilia Using the Advantages of the Paramecium Cell and Its Many Cilia.

Paramecium species, especially P. tetraurelia and caudatum, are model organisms for modern research into the form and function of cilia. In this review, we focus on the ciliary ion channels and other transmembrane proteins that control the beat frequency and wave form of the cilium by controlling the signaling within the cilium. We put these discussions in the context of the advantages that Paramecium brings to the understanding of ciliary motility: mutants for genetic dissections of swimming behavior, electrophysiology, structural analysis, abundant cilia for biochemistry and modern proteomics, genomics and molecular biology. We review the connection between behavior and physiology, which allows the cells to broadcast the function of their ciliary channels in real time. We build a case for the important insights and advantages that this model organism continues to bring to the study of cilia.

Room Temperature Cation Exchange Reaction in Nanocrystals for Ultrasensitive Speciation Analysis of Silver Ions and Silver Nanoparticles.

To evaluate the toxicity of silver nanoparticles (AgNPs) and Ag(+) and gain deep insight into the transformation of AgNPs in the environment or organisms, ultrasensitive analytical methods are needed for their speciation analysis. About 40-fold of Cd(2+) in CdTe ionic nanocrystals can be "bombarded-and-exploded" (exchanged) in less than 1 min simply by mixing the nanocrystals with Ag(+) solution at room temperature, while this cation exchange reaction did not occur when only silver nanoparticles were present. On the basis of this striking difference, an ultrasensitive method was developed for speciation analysis of Ag(+) and AgNPs in complex matrices. The released Cd(2+) was reduced to its volatile species by sodium tetrahydroborate, which was separated and swept to an inductively coupled plasma mass spectrometer (ICPMS) or an atomic fluorescence spectrometer (AFS) for the indirect but ultrasensitive detection of Ag(+). Owing to the remarkable signal amplification via the cation exchange reaction and the advantages of chemical vapor generation for sampling, the limit of detection was 0.0003 μg L(-1) for Ag(+) by ICPMS, which was improved by 100-fold compared to the conventional method. Relative standard deviations are better than 2.5% at a concentration of 0.5 μg L(-1) Ag(+) or AgNPs regardless of the detector. The proposed method retains several unique advantages, including ultrahigh sensitivity, speciation analysis, simplicity and being organic reagent-free, and has been successfully utilized for speciation analysis of Ag(+) and AgNPs in environmental water samples and paramecium cells.

Real-time contrast-enhanced holographic imaging using phase coherent photorefractive quantum wells.

We demonstrate wide-field real-time and depth-resolved contrast enhanced holographic imaging (CEHI) using the all-optical phase coherent photorefractive effect in ZnSe quantum wells. Moving objects are imaged at large depth-of-field by the local enhancement of a static reference hologram. The high refresh rate of the holographic films enables direct-to-video monitoring of floating glass beads and of living Paramecium and Euglena cells moving in water. Depth resolution is achieved by tilting the incident laser beam with respect to the normal of the cuvette. This creates double images of the objects, which are analyzed geometrically and with Fresnel diffraction theory. A two-color CEHI set-up further enables the visualization of a concealed 95 µm thick wire behind a thin layer of chicken skin.

Cypermethrin-Induces Oxidative Stress to the Freshwater Ciliate Model: Paramecium tetraurelia

Synthetic Pyrethroids are considered to be safe over other insecticides; however, data indicate that their use may pose risk to environmental biota, especially, aquatic organisms. Therefore, the aim of the present study was to investigate the effect of cypermethrin, a widely used insecticide and one of the most common contaminants in freshwater aquatic system on the oxidative stress biomarkers of the freshwater ciliate Paramecium tetraurelia. After the treatment of paramecium cells with increasing concentrations of cypermethrin (0.05, 0.5,1, 2�g/l), we followed up the growth kinetics, generation time and the response percentage. Also, we studied the variation in biomarkers of stress such as: Proteins, GSH content, GST and CAT activities. Our results showed a significant decrease in the proliferation of cell, we denote a difference of

Single drop solution electrode glow discharge for plasma assisted-chemical vapor generation: sensitive detection of zinc and cadmium in limited amounts of samples.

A simple and sensitive approach is proposed and evaluated for determination of ultratrace Zn and Cd in limited amounts of samples or tens of cells based on a novel single drop (5-20 μL) solution electrode glow discharge assisted-chemical vapor generation technique. Volatile species of Zn and Cd were immediately generated and separated from the liquid phase for transporting to atomic fluorescence or atomic mass spectrometric detectors for their determination only using hydrogen when the glow discharge was ignited between the surface of a liquid drop and the tip of a tungsten electrode. Limits of detection are better than 0.01 μg L(-1) (0.2 pg) for Cd and 0.1 μg L(-1) (2 pg) for Zn, respectively, and comparable or better than the previously reported results due to only a 20 μL sampling volume required, which makes the proposed technique convenient for the determination of Zn and Cd in limited amounts of samples or even only tens of cells. The proposed method not only retains the advantages of conventional chemical vapor generation but also provides several unique advantages, including better sensitivity, lower sample and power consumption, higher chemical vapor generation efficiencies and simpler setup, as well as greener analytical chemistry. The utility of this technique was demonstrated by the determination of ultratrace Cd and Zn in several single human hair samples, Certified Reference Materials GBW07601a (human hair powder) and paramecium cells.

Paramecium swimming and ciliary beating patterns: a study on four RNA interference mutations.

Paramecium cells swim and feed by beating their thousands of cilia in coordinated patterns. The organization of these patterns and its relationship with cell motility has been the subject of a large body of work, particularly as a model for ciliary beating in human organs where similar organization is seen. However the rapid motion of the cells makes quantitative measurements very challenging. Here we provide detailed measurements of the swimming of Paramecium cells from high-speed video at high magnification, as they move in microfluidic channels. An image analysis protocol allows us to decouple the cell movement from the motion of the cilia, thus allowing us to measure the ciliary beat frequency (CBF) and the spatio-temporal organization into metachronal waves along the cell periphery. Two distinct values of the CBF appear at different regions of the cell: most of the cilia beat in the range of 15 to 45 Hz, while the cilia in the peristomal region beat at almost double the frequency. The body and peristomal CBF display a nearly linear relation with the swimming velocity. Moreover the measurements do not display a measurable correlation between the swimming velocity and the metachronal wave velocity on the cell periphery. These measurements are repeated for four RNAi silenced mutants, where proteins specific to the cilia or to their connection to the cell base are depleted. We find that the mutants whose ciliary structure is affected display similar swimming to the control cells albeit with a reduced efficiency, while the mutations that affect the cilia's anchoring to the cell lead to strongly reduced ability to swim. This reduction in motility can be related to a loss of coordination between the ciliary beating in different parts of the cell.

Impact of a high magnetic field on the orientation of gravitactic unicellular organisms--a critical consideration about the application of magnetic fields to mimic functional weightlessness.

The gravity-dependent behavior of Paramecium biaurelia and Euglena gracilis have previously been studied on ground and in real microgravity. To validate whether high magnetic field exposure indeed provides a ground-based facility to mimic functional weightlessness, as has been suggested earlier, both cell types were observed during exposure in a strong homogeneous magnetic field (up to 30 T) and a strong magnetic field gradient. While swimming, Paramecium cells were aligned along the magnetic field lines; orientation of Euglena was perpendicular, demonstrating that the magnetic field determines the orientation and thus prevents the organisms from the random swimming known to occur in real microgravity. Exposing Astasia longa, a flagellate that is closely related to Euglena but lacks chloroplasts and the photoreceptor, as well as the chloroplast-free mutant E. gracilis 1F, to a high magnetic field revealed no reorientation to the perpendicular direction as in the case of wild-type E. gracilis, indicating the existence of an anisotropic structure (chloroplasts) that determines the direction of passive orientation. Immobilized Euglena and Paramecium cells could not be levitated even in the highest available magnetic field gradient as sedimentation persisted with little impact of the field on the sedimentation velocities. We conclude that magnetic fields are not suited as a microgravity simulation for gravitactic unicellular organisms due to the strong effect of the magnetic field itself, which masks the effects known from experiments in real microgravity.

Single Cell Traffic of Swimming Green Paramecia on Microchips with Micro-Flow Channels Fabricated by Micro-Casting

Members of Paramecium species are often referred to as “swimming neurons or sensory cells” applicable to micro-biorobotics or BioMEMS (biological micro-electro-mechanical systems). Paramecium bursaria known as green paramecia is an unicellular organism that lives widely in fresh water environments such as rivers and ponds. Recent studies have suggested that in vivo cellular robotics using the living cells of green paramecia as micro-machines controllable under electrical, optical and magnetic signals, has a variety of engineering applications such as transportation of micro-sized particles (ingested within the cells) in the capillary systems. In the present study, we aimed to test if the swimming environment of green paramecia can be implementable on microchips. For this purpose, the series of microchips were prepared for cellular swimming platform for green paramecia through fabrication of poly(methyl methacrylate) master plates using the programmable micro-milling system followed by polydimethylsiloxane-based micro-casting. Finally, microchips equipped with optimally sized micro-flow channels for allowing the single cell traffic by swimming green paramecia were successfully prepared, and thus further studies for application of green paramecium cells in BioMEMS are encouraged.

1P200 伸縮性の表層を伝わるゾウリムシのメタクロナールウェーブ(12. 細胞生物的課題,ポスター,第52回日本生物物理学会年会(2014年度))

1P200 伸縮性の表層を伝わるゾウリムシのメタクロナールウェーブ(12. 細胞生物的課題,ポスター,第52回日本生物物理学会年会(2014年度))

Calcium Regulation in the Protozoan Model, Paramecium tetraurelia

Early in eukaryotic evolution, the cell has evolved a considerable inventory of proteins engaged in the regulation of intracellular Ca(2+) concentrations, not only to avoid toxic effects but beyond that to exploit the signaling capacity of Ca(2+) by small changes in local concentration. Among protozoa, the ciliate Paramecium may now be one of the best analyzed models. Ciliary activity and exo-/endocytosis are governed by Ca(2+) , the latter by Ca(2+) mobilization from alveolar sacs and a superimposed store-operated Ca(2+) -influx. Paramecium cells possess plasma membrane- and endoplasmic reticulum-resident Ca(2+) -ATPases/pumps (PMCA, SERCA), a variety of Ca(2+) influx channels, including mechanosensitive and voltage-dependent channels in the plasma membrane, furthermore a plethora of Ca(2+) -release channels (CRC) of the inositol 1,4,5-trisphosphate and ryanodine receptor type in different compartments, notably the contractile vacuole complex and the alveolar sacs, as well as in vesicles participating in vesicular trafficking. Additional types of CRC probably also occur but they have not been identified at a molecular level as yet, as is the equivalent of synaptotagmin as a Ca(2+) sensor for exocytosis. Among established targets and sensors of Ca(2+) in Paramecium are calmodulin, calcineurin, as well as Ca(2+) /calmodulin-dependent protein kinases, all with multiple functions. Thus, basic elements of Ca(2+) signaling are available for Paramecium.

2P177 ゾウリムシのメタクロナールウェーブは外液の粘性だけでなく細胞表層の弾性も使って伝播できる(12.細胞生物的課題,ポスター,日本生物物理学会年会第51回(2013年度))

2P177 ゾウリムシのメタクロナールウェーブは外液の粘性だけでなく細胞表層の弾性も使って伝播できる(12.細胞生物的課題,ポスター,日本生物物理学会年会第51回(2013年度))

Anesthetic action of volatile anesthetics by using Paramecium as a model

Although empirically well understood in their clinical administration, volatile anesthetics are not yet well comprehended in their mechanism studies. A major conundrum emerging from these studies is that there is no validated model to assess the presumed candidate sites of the anesthetics. We undertook this study to test the hypothesis that the single-celled Paramecium could be anesthetized and served as a model organism in the study of anesthetics. We assessed the motion of Paramecium cells with Expert Vision system and the chemoresponse of Paramecium cells with T-maze assays in the presence of four different volatile anesthetics, including isoflurane, sevoflurane, enflurane and ether. Each of those volatiles was dissolved in buffers to give drug concentrations equal to 0.8, 1.0, and 1.2 EC50, respectively, in clinical practice. We could see that after application of volatile anesthetics, the swimming of the Paramecium cells was accelerated and then suppressed, or even stopped eventually, and the index of the chemoresponse of the Paramecium cells (denoted as I ( che )) was decreased. All of the above impacts were found in a concentration-dependent fashion. The biphasic effects of the clinical concentrations of volatile anesthetics on Paramecium simulated the situation of high species in anesthesia, and the inhibition of the chemoresponse also indicated anesthetized. In conclusion, the findings in our studies suggested that the single-celled Paramecium could be anesthetized with clinical concentrations of volatile anesthetics and therefore be utilized as a model organism to study the mechanisms of volatile anesthetics.

Growth Retardation of Paramecium and Mouse Cells by Shielding Them from Background Radiation

In the 1970s and 1980s, Planel et al. reported that the growth of paramecia was decreased by shielding them from background radiation. In the 1990s, Takizawa et al. found that mouse cells displayed a decreased growth rate under shielded conditions. The purpose of the present study was to confirm that growth is impaired in organisms that have been shielded from background radiation. Radioprotection was produced with a shielding chamber surrounded by a 15 cm thick iron wall and a 10 cm thick paraffin wall that reduced the γ ray and neutron levels in the chamber to 2% and 25% of the background levels, respectively. Although the growth of Paramecium tetraurelia was not impaired by short-term radioprotection (around 10 days), which disagreed with the findings of Planel et al., decreased growth was observed after long-term (40-50 days) radiation shielding. When mouse lymphoma L5178Y cells were incubated inside or outside of the shielding chamber for 7 days, the number of cells present on the 6th and 7th days under the shielding conditions was significantly lower than that present under the non-shielding conditions. These inhibitory effects on cell growth were abrogated by the addition of a ¹³⁷Cs γ-ray source disk to the chamber. Furthermore, no growth retardation was observed in XRCC4-deficient mouse M10 cells, which display impaired DNA double strand break repair.

Hierarchical organization of noise generates spontaneous signal in Paramecium cell

In many cellular processes, spontaneous activities are often the basis for their functioning. Paramecium cells change their swimming direction under a homogeneous environment, which is induced by a spontaneous signal generation in the membrane electric potential. For such a spontaneous activity, a theoretical model has been proposed by Oosawa (2007) [Biosystems 88, 191–201.], in which intracellular noise is hierarchically organized from thermal fluctuations to spike-like large fluctuations, which induces a signal to change spontaneously the swimming direction. Our analysis of the model shows that the system is a kind of excitable media, in which a spike is induced by a stochastic fluctuation. We show conditions of channels properties to have a spike train.

Calmodulation of Voltage-Gated Calcium Channels: Frontiers of Biological Impact and Mechanistic Elegance

Calmodulin (CaM) regulation of mammalian CaV channels is both biologically critical and mechanistically rich, rendering this system a central prototype for the decoding of Ca2+ signals and the modulation of channels. This system showcases the remarkable ability of the N- and C-terminal lobes of CaM to function as semiautonomous Ca2+ sensors and effectors, a theme initially recognized in Paramecium (Cell 62:165). In mammalian CaV channels, Ca2+-free CaM (apoCaM) starts off already preassociated with a host channel, and then the C-lobe turns out to respond preferentially to the strong Ca2+ influx through the host channel to induce one form of channel modulation (local selectivity), whereas the N-lobe frequently prefers the far weaker Ca2+ signal emanating from Ca2+ sources at a distance (global selectivity). These striking contrasts in spatial Ca2+ selectivity, crucial to biological Ca2+ signaling, can be simply explained by mechanisms involving the translocation of CaM among an apoCaM preassociation locus and multiple Ca2+/CaM effector sites (Cell 133:1228). However, beyond apoCaM preassociation at an IQ domain on the carboxy-terminus of channels, little has been definitively established regarding the structural identity of Ca2+/CaM effector sites, outside of an NSCaTE element in the channel amino terminus (Nature 451:830). Here, we outline new evidence identifying dominant Ca2+/CaM effector sites outside of the IQ and NSCaTE regions (Ben Johnny et al, Bazzazi et al, this meeting), thereby bolstering a modulatory mechanism wherein CaM departs from an initial IQ preassociation locus, then interacts with structurally distinct effector sites. This intricate translocating dance of CaM complexed with a target molecule may be a general scheme enabling high-order Ca2+ decoding in many signaling complexes throughout biology; indeed, an analogous hypothesis of migratory CaM has been proposed for NaV channels (J. Biol. Chem. 284:6436).

A new laboratory cultivation of Paramecium bursaria using non-pathogenic bacteria strains.

In most studies dealing with the laboratory cultivation of paramecia (Paramecium bursaria), Klebsiella pneumoniae bacteria are used to inoculate the medium. However, Klebsiella pneumoniae is a typical pathogen, and its use is always associated with a risk of infection. The aim of the present research was to examine non-pathogenic bacteria strains as components of the medium for Paramecium bursaria. The paramecia were incubated on lettuce infusions bacterized with different bacteria strains: Bacillus subtilis DSM 10, Bacillus megaterium DSM 32, Escherichia coli DSM 498, Micrococcus luteus DSM 348. A strain derived from the natural habitat of Paramecium bursaria was used as the control one. Experiments were conducted under constant light and in the dark. Paramecia cells were counted under a stereomicroscope on consecutive days of incubation. The obtained results show that the most intensive growth of Paramecium bursaria occurs in the presence of Escherichia coli DSM 498. The use of this strain as a component of the medium allows one to obtain a high number of ciliates regardless of the light conditions. It can be concluded that the Paramecium bursaria cultivation procedure can be modified by using the non-pathogenic bacteria strain Escherichia coli DSM 498 instead of Klebsiella pneumoniae.

The conjugation-specific Die5 protein is required for development of the somatic nucleus in both Paramecium and Tetrahymena.

Development in ciliated protozoa involves extensive genome reorganization within differentiating macronuclei, which shapes the somatic genome of the next vegetative generation. Major events of macronuclear differentiation include excision of internal eliminated sequences (IESs), chromosome fragmentation, and genome amplification. Proteins required for these events include those with homology throughout eukaryotes as well as proteins apparently unique to ciliates. In this study, we identified the ciliate-specific Defective in IES Excision 5 (DIE5) genes of Paramecium tetraurelia (PtDIE5) and Tetrahymena thermophila (TtDIE5) as orthologs that encode nuclear proteins expressed exclusively during development. Abrogation of PtDie5 protein (PtDie5p) function by RNA interference (RNAi)-mediated silencing or TtDie5p by gene disruption resulted in the failure of developing macronuclei to differentiate into new somatic nuclei. Tetrahymena DeltaDIE5 cells arrested late in development and failed to complete genome amplification, whereas RNAi-treated Paramecium cells highly amplified new macronuclear DNA before the failure in differentiation, findings that highlight clear differences in the biology of these distantly related species. Nevertheless, IES excision and chromosome fragmentation failed to occur in either ciliate, which strongly supports that Die5p is a critical player in these processes. In Tetrahymena, loss of zygotic expression during development was sufficient to block nuclear differentiation. This observation, together with the finding that knockdown of Die5p in Paramecium still allows genome amplification, indicates that this protein acts late in macronuclear development. Even though DNA rearrangements in these two ciliates look to be quite distinct, analysis of DIE5 establishes the action of a conserved mechanism within the genome reorganization pathway.

The actin subfamily PtAct4, out of many subfamilies, is differentially localized for specific local functions in Paramecium tetraurelia cells

Paramecium tetraurelia possesses more actin isoforms than most other cells. With monospecific antibodies against actin subfamily 4 members, we could label cleavage furrow, nascent food vacuoles, oral apparatus, cilia, cell surface and macronucleus. Expression as green fluorescent protein- (GFP-) fusion protein now allowed us to localize more stringently actin4, e.g., in the macronucleus, particularly when enhanced with anti-GFP antibodies. Posttranscriptional gene silencing of actin4 resulted in disturbances at sites where actin4 has been localized. Cell division was impaired already early on, occasionally resulting in deformed cells. Both micro- and macronuclear development during vegetative cell fission were disturbed. Over longer periods, actin4 silencing entailed reduced phagocytotic activity, paralleled by accumulation of “acidosomes” (late endosomes) near the cytopharynx where they normally fuse with nascent phagosomes. In addition, near the cell surface, extensively misshapen “terminal cisternae” (early endosomes) occurred. In deformed cells, both constitutive endocytosis and stimulated trichocyst exocytosis were impaired. Thus, actin4 exerts pleiotropic effects at widely different sites of the Paramecium cell and disturbances generally coincide with sites where actin4 is normally enriched. Evidently the loss of actin4 cannot easily be compensated for by any other of the large number of actin isoforms occurring in a Paramecium cell.

The Infl uence of Hypergravity on the Paramecium bursaria- Chlorella sp. Symbiotic Association

The aim of the research was to determine the influence of strong hypergravity on the Paramecium bursaria-Chlorella sp. symbiotic association, which is considered to be a model example of symbiosis between a heterotroph and an autotroph. The paramecia cells were exposed to 1073 x g, 4293 x g, and 9658 x g hypergravity for 15 min. Then they were incubated for 21 d on a standard lettuce medium. The experiments were conducted in parallel under constant white light and in the dark. The changes in the number of paramecia cells during incubation were determined. Measurements of the number of Chlorella sp. endosymbionts inside host cells were also made. The results showed that a 15-min exposure to hypergravity attenuates the subsequent growth of Paramecium bursaria in the dark, but it may stimulate the growth of paramecia under constant light. Moreover, it causes an increase in the number of algae inside the paramecia cells. Presumably, the influence of hypergravity on the studied symbiotic complex is connected with its effect on the endosymbiotic Chlorella sp. cells. This subject requires further research, focused on the influence of hypergravity on the physiology and growth of the Chlorella sp. endosymbionts living inside the Paramecium bursaria cells.

Micro-Particle Transporting System Using Galvanotactically Stimulated Apo-Symbiotic Cells of Paramecium bursaria

It is well known that Paramecium species including green paramecia (Paramecium bursaria) migrate towards the anode when exposed to an electric field in a medium. This type of a cellular movement is known as galvanotaxis. Our previous study revealed that an electric stimulus given to P bursaria is converted to a galvanotactic cellular movement by involvement of T-type calcium channel on the plasma membrane [Aonuma et al. (2007), Z. Naturforsch. 62c, 93-102]. This phenomenon has attracted the attention of bioengineers in the fields of biorobotics or micro-robotics in order to develop electrically controllable micromachineries. Here, we demonstrate the galvanotactic controls of the cellular migration of P bursaria in capillary tubes (diameter, 1-2 mm; length, 30-240 mm). Since the Paramecium cells take up particles of various sizes, we attempted to use the electrically stimulated cells of P bursaria as the vehicle for transportation of micro-particles in the capillary system. By using apo-symbiotic cells of P bursaria obtained after forced removal of symbiotic algae, the uptake of the particles could be maximized and visualized. Then, electrically controlled transportations of particle-filled apo-symbiotic P bursaria cells were manifested. The particles transported by electrically controlled cells (varying in size from nm to /m levels) included re-introduced green algae, fluorescence-labeled polystyrene beads, magnetic microspheres, emerald green fluorescent protein (EmGFP)-labeled cells of E. coli, Indian ink, and crystals of zeolite (hydrated aluminosilicate minerals with a micro-porous structure) and some metal oxides. Since the above demonstrations were successful, we concluded that P bursaria has a potential to be employed as one of the micro-biorobotic devices used in BioMEMS (biological micro-electro-mechanical systems).