Aligning Paramecium caudatum with static magnetic fields

As they negotiate their environs, unicellular organisms adjust their swimming in response to various physical fields such as temperature, chemical gradients, and electric fields. Because of the weak magnetic properties of most biological materials, however, they do not respond to the earth's magnetic field (5 x 10(-5) Tesla) except in rare cases. Here, we show that the trajectories of Paramecium caudatum align with intense static magnetic fields >3 Tesla. Otherwise straight trajectories curve in magnetic fields and eventually orient parallel or antiparallel to the applied field direction. Neutrally buoyant immobilized paramecia also align with their long axis in the direction of the field. We model this magneto-orientation as a strictly passive, nonphysiological response to a magnetic torque exerted on the diamagnetically anisotropic components of the paramecia. We have determined the average net anisotropy of the diamagnetic susceptibility, Deltachi(p), of a whole Paramecium: Deltachi(p) = (6.7+/- 0.7) x 10(-23) m(3). We show how the measured Deltachi(p) compares to the anisotropy of the diamagnetic susceptibilities of the components in the cell. We suggest that magnetic fields can be exploited as a novel, noninvasive, quantitative means to manipulate swimming populations of unicellular organisms.     

Orientation of paramecium swimming in a static magnetic field: Dependence on membrane lipid fluidity

We studied the swimming orientation of the ciliated protozoan Paramecium aurelia in a static magnetic field (0.78 T). P. aurelia is a complex of species termed syngens, whose cell morphology appears similar on microscopic examination. In the magnetic field, the cells of some syngens gradually changed their swimming orientation so that they were swimming perpendicular or parallel to the magnetic field, although such sensitivity to magnetic fields differs between syngens. When the temperature of the cell suspension was raised, the magnetic sensitivity of the cells was decreased. On the other hand, when the cells were cultured beforehand at a high temperature, their magnetic sensitivity was increased. These results raise the possibility that membrane lipid fluidity, which is inversely proportional to the membrane lipid order, contributes to the magnetic orientation of syngens. In this study, measurements of membrane lipid fluidity obtained using fluorescence image analysis with the lipophilic dye, laurdan (6-lauroyl-2-dimethylaminonaphtalene), showed that the degree of membrane lipid fluidity was correlated with the differences in magnetic orientation between syngens. That is, the syngens with decreased membrane fluidity showed an increased degree of magnetic orientation. Therefore, the membrane lipid order is a key factor in the magnetic orientation of Paramecium swimming.     

Magneto‐optical characteristics of human sperms: normal and deformed

In this study we report on magnetic orientation of human sperms. Samples were taken from 17 donors. Normal human sperms became oriented with their long axis perpendicular to the magnetic field (1 T maximum). Total orientation was achieved with magnetic field of about 1 T, while for abnormal sperms the magnetic behavior was different. The dependence of the measured degree of orientation on the intensity of the magnetic field was in good agreement with the theoretical equation for the magnetic orientation of diamagnetic substances. As a result of a numerical analysis based on the equation, the anisotropic diamagnetic susceptibility of normal sperm was found to be Δ_χ = 8 × 10^–20 J/T². The degree of orientation was influenced by the alterations in the shape of the head, body or the tail. It has been suggested that the DNA in the sperm head retain the strong magnetic anisotropy to counterbalance the magnetic anisotropy retained by flagellum microtubules. Recent studies demonstrated a well‐defined nuclear architecture in human sperm nucleus, where the head morphology has significant correlation with sperm chromatin structure assay SCSA. Then, as the methods to evaluate SCSA can be difficult and expensive our simple magnetic orientation technique can be an alternative to diagnose alteration in DNA. (© 2008 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

The role of cortical orientation in the control of the direction of ciliary beat in Paramecium

The swimming behavior of many ciliate protozoans depends on graded changes in the direction of the ciliary effective stroke in response to depolarizing stimuli (i.e., the avoiding reaction of Paramecium). We investigated the problem of whether the directional response of cilia with a variable plane of beat is related to the polarity of the cell as a whole or to the orientation of the cortical structures themselves. To do this, we used a stock of Paramecium aurelia with part of the cortex reversed 180 degrees. We determined the relation of the orientation of the kineties (ciliary rows) to the direction of beat in these mosaic paramecia by cinemicrography of particle movements near living cells and by scanning electron microscopy of instantaneously fixed material. We found that the cilia of the inverted rows always beat in the direction opposite to that of normally oriented cilia during both forward and backward swimming. In addition, metachronal waves of ciliary coordination were present on the inverted patch, travelling in the direction opposite to those on the normal cortex. The reference point for the directional response of Paramecium cilia to stimuli thus resides within the cilia or their immediate cortical surroundings.     

Effect of a 60 Hz magnetic field on the behavior of Paramecium

Paramecium multimicronucleatum was used as a model cell to study the effects of 60 Hz magnetic fields on swimming behavior. When exposed to a vertical field of 0.6 T, the cells accumulated at the upper end of the cuvette. An analysis of the swimming behavior revealed that the exposure to the field increased the number of cells swimming upwards maximally at 1 min after onset of the exposure. This effect of the magnetic field was transient, disappearing within a few minutes during the exposure. It is suggested that the magnetic field may amplify to a large extent the negative gravitaxis of Paramecium. Effects of an induced electric field on the swimming behavior are also discussed.     

Small GTP-binding proteins associated with secretory vesicles of Paramecium

GTP-binding proteins act as molecular switches in a variety of membrane-associated processes, including secretion. One group of GTP-binding proteins, 20-30 kDa, is related to the product of the ras proto-oncogene. In Saccharomyces cerevisiae, ras-like GTP-binding proteins regulate vesicular traffic in secretion. The ciliate protist Paramecium tetraurelia contains secretory vesicles (trichocysts) whose protein contents are released by regulated exocytosis. Using [alpha-32P]GTP and an on-blot assay for GTP-binding, we detected at least seven GTP-binding proteins of low molecular mass (22-31 kDa) in extracts of Paramecium tetraurelia. Subcellular fractions contained characteristic subsets of these seven; cilia were enriched for the smallest (22 kDa). The pattern of GTP-binding proteins was altered in two mutants defective in the formation or discharge of trichocysts. Trichocysts isolated with their surrounding membranes intact contained two minor GTP-binding proteins (23.5 and 29 kDa) and one major GTP-binding protein (23 kDa) that were absent from demembranated trichocysts. This differential localization of GTP-binding proteins suggests functional specialization of specific GTP-binding proteins in ciliary motility and exocytosis.     

Small GTP-binding Proteins Associated with Secretory Vesicles of Paramecium

GTP-binding proteins act as molecular switches in a variety of membrane-associated processes, including secretion. One group of GTP-binding proteins, 20–30 kDa, is related to the product of the ras proto-oncogene. In Saccharomyces cerevisiae, ras -like GTP-binding proteins regulate vesicular traffic in secretion. The ciliate protist Paramecium tetraurelia contains secretory vesicles (trichocysts) whose protein contents are released by regulated exocytosis. Using [α-³²P]GTP and an on-blot assay for GTP-binding, we detected at least seven GTP-binding proteins of low molecular mass (22–31 kDa) in extracts of Paramecium tetraurelia. Subcellular fractions contained characteristic subsets of these seven; cilia were enriched for the smallest (22 kDa). The pattern of GTP-binding proteins was altered in two mutants defective in the formation or discharge of trichocysts. Trichocysts isolated with their surrounding membranes intact contained two minor GTP-binding proteins (23.5 and 29 kDa) and one major GTP-binding protein (23 kDa) that were absent from demembranated trichocysts. This differential localization of GTP-binding proteins suggests functional specialization of specific GTP-binding proteins in ciliary motility and exocytosis.     

Donnè Structurales et Ultrastructurales Nouvelles sur la Conjugaison de Paramecium caudatum.

The morphologic phenomena of the conjugation of Paramecium caudatum are analysed by transverse sectioning of couples at the level of the junction zone. This orientation allows exact determination of the adjacent surfaces (which strongly suggests the absence of a paroral cone) and their relation to the ciliary fields. The modifications of the outer pellicle are studied with the electron microscope. It is shown that cytoplasmic communications occur at the top of the ridges which limit the periciliary depressions. The kinetosomes remain apparently intact but cilia and trichocysts disappear. An active role by the latter organelles is suggested for the union of the two conjugants.     

Analysis of anisotropic diamagnetic susceptibility of a bull sperm

Bull sperm and paramecium cilium were exposed to uniform static magnetic fields to observe their magnetic orientations and measure their anisotropic diamagnetic susceptibility (deltachi) for each. The prepared samples were whole bull sperm, bull sperm flat heads, and paramecium cilia, because bull sperm tails in a perfect condition could not be prepared. The whole bull sperm and the bull sperm heads became oriented perpendicular to the magnetic fields (1.7 Tesla maximum), while the paramecium cilia became parallel to the magnetic fields (8 Tesla maximum). A whole bull sperm, a bull sperm head, and a paramecium cilium were photometrically studied to obtain deltachi for each, which were estimated to be 1 x 10(-19), 3 x 10(-19), and 2 x 10(-20) J/T(2), respectively. deltachi of a sperm flagellum was estimated from the measured value of deltachi of the paramecium cilium, which agrees well with the difference between deltachi of the whole sperm and the sperm head. Additionally, this difference of deltachi almost coincides with the deltachi values calculated from deltachi of tubulin, as well. If the magnetic effect on biological systems is solved and the magnetic orientation correlates with it, deltachi will become the quantitative index of the effect.     

Orientation of bull sperms in static magnetic fields

The orientation of bull sperm cells in static magnetic fields was investigated by microscopic observation. The bull sperm, which has a very flat head, was fixed and its motion was stopped by glutaraldehyde. It was oriented with the whole body and the flat head perpendicular to the direction of the magnetic field. The diamagnetic cell components, such as the cell membrane, the DNA in the head, and the microtubule in the tail, were thought to contribute to this orientation, because bull sperm does not have paramagnetic components. For quantitative measurement of the orientation, the intensity of transmitted light through glutaraldehyde-fixed bull sperm suspension in a photometric cell was determined. The intensity changed slightly in proportion to the mean degree of orientation of the sperms. It increased sigmoidally depending on the intensity of the magnetic field and reached 100% at just below 1 T. The magnetic orientation is very strong in comparison to that of erythrocytes or platelets. By studying the response of the bull sperm to the magnetic field, it will be possible to understand its microstructure in more detail.     

Changes in Paramecium caudatum (protozoa) near a switched-on GSM telephone

The protozoan Paramecium caudatum was examined under normal conditions versus aside a switched-on GSM telephone (900?MHz; 2 Watts). Exposed individuals moved more slowly and more sinuously than usual. Their physiology was affected: they became broader, their cytopharynx appeared broader, their pulse vesicles had difficult in expelling their content outside the cell, their cilia less efficiently moved, and trichocysts became more visible. All these effects might result from some bad functioning or damage of the cellular membrane. The first target of communication electromagnetic waves might thus be the cellular membrane.     

Does Paramecium sense gravity?

In order to get an insight into the cellular mechanisms for the integration of the effects of gravity, we investigated the gravitactic behaviour in Paramecium. There are two main categories for the model of the mechanism of gravitaxis; one is derived on the basis of the mechanistic properties of the cell (physical model) and the other of the physiological properties including cellular gravireception (physiological model). In this review article, we criticized the physical models and introduced a new physiological model. Physical models postulated so far can be divided into two; one explaining the negative gravitactic orientation of the cell in terms of the static torque generated by the structural properties of the cell (gravity-buoyancy model by Verworn, 1889 and drag-gravity model by Roberts, 1970), and the other explaining it in terms of the dynamic torque generated by the helical swimming of the cell (propulsion-gravity model by Winet and Jahn, 1974 and lifting-force model by Nowakowska and Grebecki, 1977). Among those we excluded the possibility of dynamic-torque models because of their incorrect theoretical assumptions. According to the passive orientation of Ni(2+)-immobilized cells, the physical effect of the static torque should be inevitable for the gravitactic orientation. Downward orientation of the immobilized cells in the course of floating up in the hyper-density medium demonstrated the gravitactic orientation is not resulted by the nonuniform distribution of cellular mass (gravity-buoyancy model) but by the fore-aft asymmetry of the cell (drag-gravity model). A new model explaining the gravitactic behaviour is derived on the basis of the cellular gravity sensation through mechanoreceptor channels of the cell membrane. Paramecium is known to have depolarizing receptor channels in the anterior and hyperpolarizing receptors in the posterior of the cell. The uneven distribution of the receptor may lead to the bidirectional changes of the membrane potential by the selective deformation of the anterior and posterior cell membrane responding to the orientation of the cell in the gravity field; i.e. negative- and positive-going shift of the potential due to the upward and downward orientation, respectively. The orientation dependent changes in membrane potential with respect to gravity, in combination with the close coupling of the membrane potential and the ciliary locomotor activity, may allow the changes in swimming direction along with those in the helical nature of the swimming path; upward shift of axis of helix by decreasing the pitch angle due to hyperpolarization in the upward-orienting cell, and also the upward shift by increasing the pitch angle due to depolarization in the downward-orienting cell. Computer simulation of the model demonstrated that the cell can swim upward along the "super-helical" trajectory consisting of a small helix winding helically an axis parallel to the gravity vector, after which the model was named as "Super-helix model". Three-dimensional recording of the trajectories of the swimming cells demonstrated that about a quarter of the cell population drew super-helical trajectory under the unbounded, thermal convection-free conditions. In addition, quantitative analysis of the orientation rate of the swimming cell indicated that gravity-dependent orientation of the swimming trajectory could not be explained solely by the physical static torque but complementarily by the physiological mechanism as proposed in the super-helix model.     

Effects of static magnetic fields on growth of Paramecium caudatum

Little is known about the influence of magnetic fields on growth of primitive eukaryotes such as the ciliate Paramecium. The latter are known to exhibit interesting characteristics such as electrotaxis, gravitaxis, and membrane excitability not commonly encountered in higher organisms. This preliminary study reports the effects of static magnetic fields on growth of Paramecium caudatum. The microorganisms were either permanently or 24 h on-and-off exposed to North and South polarity magnetic fields of average field gradient 4.3 T/m, for a period of 96 h. The growth rate and lag phase of all exposed populations were not significantly different from control ones exposed to normal geomagnetic field (P > .05). However, a significant negative shift in t(max) (time taken for maximum growth) of 10.5%-12.2% and a significant decrease (P < .05) in population size of 10.2%-15.1% during the 96 h of experimental conditions were recorded for exposed populations compared to control. Our results suggest that magnetic fields, irrespective of polarity and exposure period reduce Paramecium growth by triggering early senescence of the population. The mechanisms underlying the small changes in population growth are unknown at this level, but various hypotheses have been suggested, including disorganization of swimming patterns resulting from (i) changes in cell membrane electric potential due to high speed movement through a gradient magnetic field and (ii) thermodynamic effect of anisotropic magnetic energies on cell membrane components affecting functioning of calcium channels. Altered swimming movements could in turn affect highly orchestrated processes such as conjugation, essential for survival of the organisms during development of adverse environmental conditions as thought to occur in the closed culture system used in this study.     

Magnetic orientation of sphingomyelin-lecithin bilayers.

Phospholipid bilayers consisting of a 60:40 mixture of N-palmitoylsphingomyelin and dimyristoylphosphatidylcholine orient in a strong magnetic field. The orientation is easily observed in 31P- and 2H-nuclear magnetic resonance spectra where the intensity of the perpendicular edges of the powder lineshapes are enhanced. The lineshapes indicate that the long axis of the molecule is perpendicular to the magnetic field.     

Divalent Cation-dependent ATPase Activities Associated with Cilia and Other Subcellular Fractions of Paramecium: An Electrophoretic Characterization on Triton-polyacrylamide Gels1

Several divalent cation-dependent ATP phosphohydrolases associated with cilia, ciliary axonemes, ciliary membranes, pellicles, trichocysts, nuclei, mitochondria, microsomes, and soluble peripheral cell surface fractions of Paramecium tetraurelia were resolved in this study. Fifteen different activity bands were detected in whole cell sonicates or subcellular fractions by Triton polyacrylamide gel electrophoresis and ATPase activity staining. The ciliary surface membrane contained two major ATPase activities that were distinct from the enzymes associated with all other cell fractions.     

Synchronous exocytosis in Paramecium cells. I. A novel approach

From a total number of approximately 1100-1300 secretory organelles ("trichocysts") in a Paramecium tetraurelia cell, approximately 90% are docked to the cell membrane. Approximately 90% of this subpopulation can be discharged from the cells within seconds, when exposed to the novel trigger agent aminoethyldextran (AED) at a concentration of 10(-6) M. No deleterious side effects were recognized with this trigger agent even over long time periods. By application of AED close to cells with the use of a micropipette we found that triggering of trichocyst release by AED involves a local, non-propagated effect and that all regions of the cell body are equally reactive. It requires exogenous Ca2+. It is independent of ciliary Ca2+ channels, since deciliated cells or ciliary mutations with "Ca2+-tight" cilia respond to AED with normal exocytosis performance. The massive and rapid occurrence of trichocyst release in response to AED allowed for a freeze-fracture analysis of intramembraneous changes (see Olbricht et al., Exp cell res 151 (1984) 14 [23]) which also shows the involvement of exocytosis) as well as for a long-term study of the re-attachment of trichocysts (see Haacke & Plattner, Exp cell res 151 (1984) 21 [10]) under synchronous conditions.     

The Fine Structure of the Mature Tomite of Hyalophysa chattoni

SYNOPSIS. The fine structure of the tomite stage of Hyalophysa chattoni was examined with particular attention to its kinetal apparatus. The pellicle, thick and dense compared with that of other ciliates, is formed of three layers. The inner layer is composed of short fibrils oriented perpendicular to the surface. The cytoplasm around the oral passage and beneath falciform field 8 is crowded with dense inclusion bodies of unknown function. Dorsal to the oral passage is the rosette, a disc-shaped organelle subdivided by septa in the form of incomplete radii about a central chamber containing a tuft of cilia. The septa are composed of 3 membranes enclosing a fine layer of cytoplasm. At their inner ends 20 mμ fibers run dorsally and ventrally. Dense clumps of fibrous material line the luminal surface of the septa. Rows of fusiform trichocysts parallel the kineties. The trichocysts are composed of a finely periodic, moderately electron-dense material surrounded by 20 mμ fibrils oriented along the long axis of the trichocyst. Between and below the kinetosomes and the rows of trichocysts are electron-dense vesicles 300 mμ in diameter and bounded by a loose membrane. The large “trichocysts,” the “gros trichocystes” of Chatton and Lwoff, whose appearance heralds the beginnings of trichocystogenesis, prove to be canaliculi opening to the surface. Four separate ciliary membrane systems—the oral ciliature (XYZ), falciform field 8, falciform field 9, and the ogival field—are located on the ventral surface of the tomite. Each differs from the others and from the somatic kineties in the fibrillar organization around its kinetosomes. In the somatic kineties the kinetodesmos is a dense, periodic fiber which is formed of stacks of up to 18 subfibers, each arising from the base of a kinetosome. The kinetosomes are short (300 mμ) and contain dense central granules. In some kineties, alternating between the kinetosomes, are elliptical kinetosome-like structures which do not bear cilia and perhaps provide a reservoir of kinetosomes for future growth of the kinety.     

How dinoflagellates swim

Dinoflagellates possess two flagella; usually these are directed perpendicular to one another constituting a transversal flagellum and a longitudinal, trailing flagellum, respectively. The transversal flagellum causes the cell to rotate around its length axis. The trailing flagellum is responsible for the translation of the cell; due to its asymmetric insertion it also causes a rotation of the cell around an axis perpendicular to the longitudinal axis. Together, these two rotational components result in a helical swimming path. Cells can vary the two rotational components independently as well as the translational velocity. With these three degrees of freedom, cells can vary the parameters of their helical swimming paths for steering. Dinoflagellates use this mechanism for orientation in chemical concentration gradients (“helical klinotaxis”).     

Magnetic field influence on paramecium motility

The influence of a moderately intense static magnetic field on movement patterns of free swimming Paramecium was studied. When exposed to fields of 0.126 T, these ciliated protozoa exhibited significant reduction in velocity as well as a disorganization of movement pattern. It is suggested that these findings may be explained on the basis of alteration in function of ion specific channels within the cell membrane.     

IMMUNOLOGICAL STUDIES OF ISOLATED PARTICULATES OF PARAMECIUM AURELIA : I. Antigenic Relationships Between Cytoplasmic Organelles and Evidence for Mitochondrial Variations as Demonstrated by Gel Diffusion

Mitochondria and other particulates—cilia, trichocysts, and "small granules"—have been isolated from several stocks of Paramecium aurelia, syngen 2. Antisera against these particles and against breis have been used to characterize the fractions by diffusion in gel. Evidence is presented for the relationship of particles, as demonstrated by immunologic cross-reactivity of the soluble antigens extracted from them. Although some antigens are unique for a fraction, cross-reacting antigens in two or more fractions, as determined by "spur" formation in agar, suggest a relationship between morphologically diverse particles. A procedure for studying cross-reactions in gels is described using the specific immobilization antigens as a model. The localization of these antigens within cilia, and perhaps trichocysts, has been confirmed. Other organelles, specifically mitochondria and "small granules," appear to alter their specificity spontaneously and reversibly during cell reproduction, a pattern reminiscent of the immobilization serotypes which can transform to one another during clonal growth.