The evolutionary conserved proteins CEP90, FOPNL, and OFD1 recruit centriolar distal appendage proteins to initiate their assembly.

In metazoa, cilia assembly is a cellular process that starts with centriole to basal body maturation, migration to the cell surface, and docking to the plasma membrane. Basal body docking involves the interaction of both the distal end of the basal body and the transition fibers/distal appendages, with the plasma membrane. Mutations in numerous genes involved in basal body docking and transition zone assembly are associated with the most severe ciliopathies, highlighting the importance of these events in cilium biogenesis. In this context, the ciliate Paramecium has been widely used as a model system to study basal body and cilia assembly. However, despite the evolutionary conservation of cilia assembly events across phyla, whether the same molecular players are functionally conserved, is not fully known. Here, we demonstrated that CEP90, FOPNL, and OFD1 are evolutionary conserved proteins crucial for ciliogenesis. Using ultrastructure expansion microscopy, we unveiled that these proteins localize at the distal end of both centrioles/basal bodies in Paramecium and mammalian cells. Moreover, we found that these proteins are recruited early during centriole duplication on the external surface of the procentriole. Functional analysis performed both in Paramecium and mammalian cells demonstrate the requirement of these proteins for distal appendage assembly and basal body docking. Finally, we show that mammalian centrioles require another component, Moonraker (MNR), to recruit OFD1, FOPNL, and CEP90, which will then recruit the distal appendage proteins CEP83, CEP89, and CEP164. Altogether, we propose that this OFD1, FOPNL, and CEP90 functional module is required to determine in mammalian cells the future position of distal appendage proteins.

Polarization grating based on diffraction phase microscopy for quantitative phase imaging of paramecia.

This study presents a polarization grating based diffraction phase microscopy (PG-DPM) and its application in bio-imaging. Compared with traditional diffraction phase microscopy (DPM) of which the fringe contrast is sample-dependent, the fringe contrast of PG-DPM is adjustable by changing the polarization of the illumination beam. Moreover, PG-DPM has been applied to real-time phase imaging of live paramecia for the first time. The study reveals that paramecium has self-helical forward motion characteristics, or more specifically, 77% clockwise and 23% anti-clockwise rotation when moving forward. We can envisage that PG-DPM will be applied to many different fields.

Paramecium Biology.

Imagine that in 1678 you are Christiaan Huygens or Antonie van Leeuwenhoek seeing paramecia swim gracefully across the field of view of your new microscope. These unicellular, free-living, and swimming cells might have remained a curiosity if not for the ability of H.S. Jennings (Behavior of the lower organisms. Indiana University Press, Bloomington, 1906) and T.M. Sonneborn (Proc Natl Acad Sci USA 23:378-385, 1937) to recognize them for their behavior and genetics, both Mendelian and non-Mendelian. Following many years of painstaking work by Sonneborn and other researchers, Paramecium now serves as a modern model organism that has made specific contributions to cell and molecular biology and development. We will review the continuing usefulness and contributions of Paramecium species in this chapter.Even without a microscope, Paramecium species is visible to the naked eye because of their size (50-300 µ long). Paramecia are holotrichous ciliates, that is, unicellular organisms in the phylum Ciliophora that are covered with cilia. It was the beating of these cilia that propelled them across the slides of the first microscopes and continue to fascinate us today. Over time, Paramecium became a favorite model organism for a large variety of studies. Denis Lyn has called Paramecium the "white rat" of the Ciliophora for their manipulability and amenity to research. We will touch upon the use of Paramecium species to examine swimming behavior, ciliary structure and function, ion channel function, basal body duplication and patterning, non-Mendelian cortical inheritance, programmed DNA rearrangements, regulated secretion and exocytosis, and cell trafficking. In particular, we will focus on the use of P. tetraurelia and P. caudatum.

Short-pulse neodymium:yttrium-aluminium garnet (Nd:YAG 1064nm) laser irradiation photobiomodulates mitochondria activity and cellular multiplication of Paramecium primaurelia (Protozoa).

Few studies exist to explore the potential photobiomodulation (PBM) effect of neodymium:yttrium-aluminium garnet (Nd:YAG) laser irradiation using a flat-top handpiece delivery system. In this study, we explored the photobiomodulation effect of that laser, on Paramecium primaurelia. The parameters for the different study groups were: 0.50W, 10Hz, 100msp, 30J/cm2; 0.75W, 10Hz, 100msp, 45J/cm2; 1.00W, 10Hz, 100msp, 60J/cm2; 1.25W, 10Hz, 100msp, 75J/cm2 and 1.50W, 10Hz, 100msp, 90J/cm2. Our results suggest that only the parameter 0.5W, 10Hz, 100msp, 30J/cm2 positively photobiomodulates the Paramecium cells inducing an increment in oxygen consumption, endogenous ATP synthesis and fission rate rhythm. Applying the laser energy with parameters of 1.25W, 10Hz, 100msp, 75J/cm2 and 1.50W, 10Hz, 100msp, 90J/cm2, induce adverse effect on the Paramecium cells, which protect themselves through the increase in Heat Shock Protein-70 (HSP70). The data presented in our work support our assumption that, when using appropriate parameters of irradiation, the 1064nm Nd:YAG laser with flat-top handpiece could be a valuable aid for effective clinical application of PBM.

Flow cytometry sorting of nuclei enables the first global characterization of Paramecium germline DNA and transposable elements.

BACKGROUND: DNA elimination is developmentally programmed in a wide variety of eukaryotes, including unicellular ciliates, and leads to the generation of distinct germline and somatic genomes. The ciliate Paramecium tetraurelia harbors two types of nuclei with different functions and genome structures. The transcriptionally inactive micronucleus contains the complete germline genome, while the somatic macronucleus contains a reduced genome streamlined for gene expression. During development of the somatic macronucleus, the germline genome undergoes massive and reproducible DNA elimination events. Availability of both the somatic and germline genomes is essential to examine the genome changes that occur during programmed DNA elimination and ultimately decipher the mechanisms underlying the specific removal of germline-limited sequences. RESULTS: We developed a novel experimental approach that uses flow cell imaging and flow cytometry to sort subpopulations of nuclei to high purity. We sorted vegetative micronuclei and macronuclei during development of P. tetraurelia. We validated the method by flow cell imaging and by high throughput DNA sequencing. Our work establishes the proof of principle that developing somatic macronuclei can be sorted from a complex biological sample to high purity based on their size, shape and DNA content. This method enabled us to sequence, for the first time, the germline DNA from pure micronuclei and to identify novel transposable elements. Sequencing the germline DNA confirms that the Pgm domesticated transposase is required for the excision of all ~45,000 Internal Eliminated Sequences. Comparison of the germline DNA and unrearranged DNA obtained from PGM-silenced cells reveals that the latter does not provide a faithful representation of the germline genome. CONCLUSIONS: We developed a flow cytometry-based method to purify P. tetraurelia nuclei to high purity and provided quality control with flow cell imaging and high throughput DNA sequencing. We identified 61 germline transposable elements including the first Paramecium retrotransposons. This approach paves the way to sequence the germline genomes of P. aurelia sibling species for future comparative genomic studies.

Trichocysts-Paramecium's Projectile-like Secretory Organelles: Reappraisal of their Biogenesis, Composition, Intracellular Transport, and Possible Functions.

This review summarizes biogenesis, composition, intracellular transport, and possible functions of trichocysts. Trichocyst release by Paramecium is the fastest dense core-secretory vesicle exocytosis known. This is enabled by the crystalline nature of the trichocyst "body" whose matrix proteins (tmp), upon contact with extracellular Ca2+ , undergo explosive recrystallization that propagates cooperatively throughout the organelle. Membrane fusion during stimulated trichocyst exocytosis involves Ca2+ mobilization from alveolar sacs and tightly coupled store-operated Ca2+ -influx, initiated by activation of ryanodine receptor-like Ca2+ -release channels. Particularly, aminoethyldextran perfectly mimics a physiological function of trichocysts, i.e. defense against predators, by vigorous, local trichocyst discharge. The tmp's contained in the main "body" of a trichocyst are arranged in a defined pattern, resulting in crossstriation, whose period expands upon expulsion. The second part of a trichocyst, the "tip", contains secretory lectins which diffuse upon discharge. Repulsion from predators may not be the only function of trichocysts. We consider ciliary reversal accompanying stimulated trichocyst exocytosis (also in mutants devoid of depolarization-activated Ca2+ channels) a second, automatically superimposed defense mechanism. A third defensive mechanism may be effectuated by the secretory lectins of the trichocyst tip; they may inhibit toxicyst exocytosis in Dileptus by crosslinking surface proteins (an effect mimicked in Paramecium by antibodies against cell surface components). Some of the proteins, body and tip, are glycosylated as visualized by binding of exogenous lectins. This reflects the biogenetic pathway, from the endoplasmic reticulum via the Golgi apparatus, which is also supported by details from molecular biology. There are fragile links connecting the matrix of a trichocyst with its membrane; these may signal the filling state, full or empty, before and after tmp release upon exocytosis, respectively. This is supported by experimentally produced "frustrated exocytosis", i.e. membrane fusion without contents release, followed by membrane resealing and entry in a new cycle of reattachment for stimulated exocytosis. There are some more puzzles to be solved: Considering the absence of any detectable Ca2+ and of acidity in the organelle, what causes the striking effects of silencing the genes of some specific Ca2+ -release channels and of subunits of the H+ -ATPase? What determines the inherent polarity of a trichocyst? What precisely causes the inability of trichocyst mutants to dock at the cell membrane? Many details now call for further experimental work to unravel more secrets about these fascinating organelles.

Simulation of Paramecium Chemotaxis Exposed to Calcium Gradients.

Paramecium or other ciliates have the potential to be utilized for minimally invasive surgery systems, making internal body organs accessible. Paramecium shows interesting responses to changes in the concentration of specific ions such as K(+), Mg(2+), and Ca(2+) in the ambient fluid. Some specific responses are observed as, changes in beat pattern of cilia and swimming toward or apart from the ion source. Therefore developing a model for chemotactic motility of small organisms is necessary in order to control the directional movements of these microorganisms before testing them. In this article, we have developed a numerical model, investigating the effects of Ca(2+) on swimming trajectory of Paramecium. Results for Ca(2+)-dependent chemotactic motility show that calcium gradients are efficient actuators for controlling the Paramecium swimming trajectory. After applying a very low Ca(2+) gradient, a directional chemotaxis of swimming Paramecium is observable in this model. As a result, chemotaxis is shown to be an efficient method for controlling the propulsion of these small organisms.

Ciliary metachronal wave propagation on the compliant surface of Paramecium cells.

Ciliary movements in protozoa exhibit metachronal wave-like coordination, in which a constant phase difference is maintained between adjacent cilia. It is at present generally thought that metachronal waves require hydrodynamic coupling between adjacent cilia and the extracellular fluid. To test this hypothesis, we aspirated a Paramecium cell using a micropipette which completely sealed the surface of the cell such that no fluid could pass through the micropipette. Thus, the anterior and the posterior regions of the cell were hydrodynamically decoupled. Nevertheless, we still observed that metachronal waves continued to propagate from the anterior to the posterior ends of the cell, suggesting that in addition to hydrodynamic coupling, there are other mechanisms that can also transmit the metachronal waves. Such transmission was also observed in computational modeling where the fluid was fully decoupled between two partitions of a beating ciliary array. We also imposed cyclic stretching on the surface of live Paramecium cells and found that metachronal waves persisted in the presence of cyclic stretching. This demonstrated that, in addition to hydrodynamic coupling, a compliant substrate can also play a critical role in mediating the propagation of metachronal waves.

Mitigation of copper toxicity by DNA oligomers in green paramecia.

Impact of transition metals which catalyze the generation of reactive oxygen species (ROS), on activation of cell death signaling in plant cells have been documented to date. Similarly in green paramecia (Paramecium bursaria), an aquatic protozoan species harboring symbiotic green algae in the cytoplasm, toxicities of various metallic ions have been documented. We have recently examined the effects of double-stranded GC-rich DNA fragments with copper-binding nature and ROS removal catalytic activity as novel plant cell-protecting agents, using the suspension-cultured tobacco cells. Here, we show that above DNA oligomers protect the cells of green paramecia from copper-induced cell death, suggesting that the phenomenon firstly observed in tobacco cells is not limited only within higher plants but it could be universally observable in wider range of organisms.

Use of dyes to increase phase contrast for biological holographic microscopy.

Holographic microscopy is an emerging biological technique that provides amplitude and quantitative phase imaging, though the contrast provided by many cell types and organelles is low, and until now no dyes were known that increased contrast. Here we show that the metallocorrole Ga(tpfc)(SO3)2, which has a strong Soret band absorption, increases contrast in both amplitude and phase and facilitates tracking of Escherichia coli with minimal toxicity. The change in phase contrast may be calculated from the dye-absorbance spectrum using the Kramers-Kronig relations, and represents a general principle that may be applied to any dye or cell type. This enables the use of holographic microscopy for all applications in which specific labeling is desired.

Delimiting Species Boundaries within a Paraphyletic Species Complex: Insights from Morphological, Genetic, and Molecular Data on Paramecium sonneborni (Paramecium aurelia species complex, Ciliophora, Protozoa).

The demarcation of boundaries between protist species is often problematic because of the absence of a uniform species definition, the abundance of cryptic diversity, and the occurrence of convergent morphology. The ciliates belonging to the Paramecium aurelia complex, consisting of 15 species, are a good model for such systematic and evolutionary studies. One member of the complex is P. sonneborni, previously known only from one stand in Texas (USA), but recently found in two new sampling sites in Cyprus (creeks running to Salt Lake and Oroklini Lake near Larnaca). The studied Paramecium sonneborni strains (from the USA and Cyprus) reveal low viability in the F1 and F2 generations of interstrain hybrids and may be an example of ongoing allopatric speciation. Despite its molecular distinctiveness, we postulate that P. sonneborni should remain in the P. aurelia complex, making it a paraphyletic taxon. Morphological studies have revealed that some features of the nuclear apparatus of P. sonneborni correspond to the P. aurelia spp. complex, while others are similar to P. jenningsi and P. schewiakoffi. The observed discordance indicates rapid splitting of the P. aurelia-P. jenningsi-P. schewiakoffi group, in which genetic, morphological, and molecular boundaries between species are not congruent.

A mechanical microcompressor for high resolution imaging of motile specimens.

In order to obtain fine details in 3 dimensions (3D) over time, it is critical for motile biological specimens to be appropriately immobilized. Of the many immobilization options available, the mechanical microcompressor offers many benefits. Our device, previously described, achieves gentle flattening of a cell, allowing us to image finely detailed structures of numerous organelles and physiological processes in living cells. We have imaged protozoa and other small metazoans using differential interference contrast (DIC) microscopy, orientation-independent (OI) DIC, and real-time birefringence imaging using a video-enhanced polychromatic polscope. We also describe an enhancement of our previous design by engineering a new device where the coverslip mount is fashioned onto the top of the base; so the entire apparatus is accessible on top of the stage. The new location allows for easier manipulation of the mount when compressing or releasing a specimen on an inverted microscope. Using this improved design, we imaged immobilized bacteria, yeast, paramecia, and nematode worms and obtained an unprecedented view of cell and specimen details. A variety of microscopic techniques were used to obtain high resolution images of static and dynamic cellular and physiological events.

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.

The first European stand of Paramecium sonneborni (P. aurelia complex), a species known only from North America (Texas, USA).

P. aurelia is currently defined as a complex of 15 sibling species including 14 species designated by Sonneborn (1975) and one, P. sonneborni, by Aufderheide et al. (1983). The latter was known from only one stand (Texas, USA). The main reason for the present study was a new stand of Paramecium in Cyprus, with strains recognized as P. sonneborni based on the results of strain crosses, cytological slides, and molecular analyses of three loci (ITS1-5.8S-ITS2-5'LSU rDNA, COI, CytB). The new stand of P. sonneborni in Europe shows that the species, previously considered endemic, may have a wider range. This demonstrates the impact of under-sampling on the knowledge of the biogeography of microbial eukaryotes. Phylogenetic trees based on all the studied fragments revealed that P. sonneborni forms a separate cluster that is closer to P. jenningsi and P. schewiakoffi than to the other members of the P. aurelia complex.

Multiple lines of evidence shed light on the occurrence of paramecium (ciliophora, oligohymenophorea) in bromeliad tank water.

Phytotelmata are vegetal structures that hold water from the rain, and organic matter from the forest and the soil, resulting in small, compartmentalized bodies of water, which provide an essential environment for the establishment and development of many organisms. These microenvironments generally harbor endemic species, but many organisms that are found in lakes and rivers, are also present. Here, we report, for the first time, the occurrence of the ciliate genus Paramecium in the tank of the bromeliad species Aechmaea distichantha. The identification of the Paramecium species was performed based on live observations, protargol impregnation, scanning electronic microscopy, and sequencing of the 18s rRNA. The absence of Paramecium from bromeliad tank water was highlighted in several earlier investigations, and may be due to the fact that this species is unable to make cysts. The occurrence of Paramecium multimicronucleatum in our samples may be explained by the proximity between the bromeliads and the river, a potential source of the species. Further, we also believe that the counting methodology used in our study provides a more accurate analysis of the species diversity, since we investigated all samples within a maximum period of 6 h after sampling, allowing minimum loss of specimens.

Synchronous induction of detachment and reattachment of symbiotic Chlorella spp. from the cell cortex of the host Paramecium bursaria.

Paramecium bursaria harbor several hundred symbiotic Chlorella spp. Each alga is enclosed in a perialgal vacuole membrane, which can attach to the host cell cortex. How the perialgal vacuole attaches beneath the host cell cortex remains unknown. High-speed centrifugation (> 1000×g) for 1min induces rapid detachment of the algae from the host cell cortex and concentrates the algae to the posterior half of the host cell. Simultaneously, most of the host acidosomes and lysosomes accumulate in the anterior half of the host cell. Both the detached algae and the dislocated acidic vesicles recover their original positions by host cyclosis within 10min after centrifugation. These recoveries were inhibited if the host cytoplasmic streaming was arrested by nocodazole. Endosymbiotic algae during the early reinfection process also show the capability of desorption after centrifugation. These results demonstrate that adhesion of the perialgal vacuole beneath the host cell cortex is repeatedly inducible, and that host cytoplasmic streaming facilitates recovery of the algal attachment. This study is the first report to illuminate the mechanism of the induction to desorb for symbiotic algae and acidic vesicles, and will contribute to the understanding of the mechanism of algal and organelle arrangements in Paramecium.

Epiplasmins and epiplasm in paramecium: the building of a submembraneous cytoskeleton.

In ciliates, basal bodies and associated appendages are bound to a submembrane cytoskeleton. In Paramecium, this cytoskeleton takes the form of a thin dense layer, the epiplasm, segmented into regular territories, the units where basal bodies are inserted. Epiplasmins, the main component of the epiplasm, constitute a large family of 51 proteins distributed in 5 phylogenetic groups, each characterized by a specific molecular design. By GFP-tagging, we analyzed their differential localisation and role in epiplasm building and demonstrated that: 1) The epiplasmins display a low turnover, in agreement with the maintenance of an epiplasm layer throughout the cell cycle; 2) Regionalisation of proteins from different groups allows us to define rim, core, ring and basal body epiplasmins in the interphase cell; 3) Their dynamics allows definition of early and late epiplasmins, detected early versus late in the duplication process of the units. Epiplasmins from each group exhibit a specific combination of properties. Core and rim epiplasmins are required to build a unit; ring and basal body epiplasmins seem more dispensable, suggesting that they are not required for basal body docking. We propose a model of epiplasm unit assembly highlighting its implication in structural heredity in agreement with the evolutionary history of epiplasmins.

Single-cell elemental analysis via high irradiance femtosecond laser ionization time-of-flight mass spectrometry.

In modern bioanalytics, elemental analysis of single cells is important yet challenging due to the complicated biological matrices and elemental contents. We have developed the high irradiance femtosecond laser ionization orthogonal time-of-flight mass spectrometry (fs-LI-O-TOFMS) to determine the elemental composition of individual cells. The sample preparation procedure is simple and fast through heating and drying the cells. Under typical operating conditions, elements above femtogram levels in a single cell can be clearly observed in the spectrum with reasonable isotope ratios. Some of the nonmetallic elements that are difficult to measure by ICPMS, such as P, S, and Cl, can be easily determined by fs-LI-O-TOFMS. Replicate analyses show that signal variations are 15-35% for metallic elements and 25-50% for nonmetallic elements. The results demonstrate that fs-LI-O-TOFMS is a simple, rapid, and practical tool for the elemental determination of single cells.

Scaling body size fluctuations.

The size of an organism matters for its metabolic, growth, mortality, and other vital rates. Scale-free community size spectra (i.e., size distributions regardless of species) are routinely observed in natural ecosystems and are the product of intra- and interspecies regulation of the relative abundance of organisms of different sizes. Intra- and interspecies distributions of body sizes are thus major determinants of ecosystems' structure and function. We show experimentally that single-species mass distributions of unicellular eukaryotes covering different phyla exhibit both characteristic sizes and universal features over more than four orders of magnitude in mass. Remarkably, we find that the mean size of a species is sufficient to characterize its size distribution fully and that the latter has a universal form across all species. We show that an analytical physiological model accounts for the observed universality, which can be synthesized in a log-normal form for the intraspecies size distributions. We also propose how ecological and physiological processes should interact to produce scale-invariant community size spectra and discuss the implications of our results on allometric scaling laws involving body mass.

Stage-specific appearance of cytoplasmic microtubules around the surviving nuclei during the third prezygotic division of Paramecium.

There are six micronuclear divisions during conjugation of Paramecium caudatum: three prezygotic and three postzygotic divisions. Four haploid nuclei are formed during the first two meiotic prezygotic divisions. Usually only one meiotic product is located in the paroral cone (PC) region at the completion of meiosis, which survives and divides mitotically to complete the third prezygotic division to yield a stationary and a migratory pronucleus. The remaining three located outside of the PC degenerate. The migratory pronuclei are then exchanged between two conjugants and fuse with the stationary pronuclei to form synkarya, which undergo three successive divisions (postzygotic divisions). However, little is known about the surviving mechanism of the PC nuclei. In the current study, stage-specific appearance of cytoplasmic microtubules (cMTs) was indicated during the third prezygotic division by immunofluorescence labeling with anti-alpha tubulin antibodies surrounding the surviving nuclei, including the PC nuclei and the two types of prospective pronuclei. This suggested that cMTs were involved in the formation of a physical barrier, whose function may relate to sequestering and protecting the surviving nuclei from the major cytoplasm, where degeneration of extra-meiotic products occurs, another important nuclear event during the third prezygotic division.