Richard Bräucker

Gravikinesis in Paramecium: Theory and isolation of a physiological response to the natural gravity vector

Summary1.We have investigated a physiological component of the gravitaxis of Paramecium using established mechanisms of ciliate mechanosensitivity. The horizontal, up and down swimming rates of cells, and the sedimentation of immobilized specimens were determined. Weak DC voltage gradients were applied to predetermine the Paramecium swimming direction.2.An observed steady swimming rate is the vector sum of active propulsion (P), a possible gravity-dependent change in swimming rate (Δ), and rate of sedimentation (S). We approximated P from horizontal swimming. S was measured after cell immobilization.3.Theory predicts that the difference between the down and up swimming rates, divided by two, equals the sum of S and Δ. Δ is supposed to be the arithmetic mean of two subcomponents, Δa and Δp, from gravistimulation of the anterior and posterior cell ends, respectively.4.A negative value of Δ (0.038 mm/s) was isolated with Δa(0.070 mm/s) subtracting from downward swimming, and Δp(0.005 mm/s) adding to upward propulsion. The data agree with one out of three possible ways of gravisensory transduction: outward deformation of the mechanically sensitive ‘lower’ soma membrane. We call the response a negative gravikinesis because both Δa and Δp antagonize sedimentation.

Neutral gravitaxis of gliding Loxodes exposed to normal and raised gravity

SummaryIn the statocystoid-bearing, flat ciliate Loxodes, the peculiar steady locomotion on submersed substrates (called “gliding”) was investigated between 1 g and 5.4 g under controlled environmental conditions in a centrifuge microscope. Videorecordings of the movements of large cell populations were processed with an automated analysis procedure. At 1 g, possible sedimentation was fully compensated, and vertical shifts of the population were neutralized because upward and downward orientations of the cells occurred at equal proportions (“neutral gravitaxis”). With rising gravity the resultant velocity of upward-gliding cells remained unchanged, whereas the velocity of downward-gliding cells increased continuously. Long-term exposure to hypergravity did not generate detectable signs of adaptation. The bipolar orientation of Loxodes persisted even under fivefold normal gravity, but the axis of orientation rotated from the gravity axis in the counterclockwise direction. The data suggest that both gravikinesis and graviorientation of gliding Loxodes are instrumental in perfect neutralization of sedimentation at terrestrial conditions.

Electric-field effects on gravikinesis in Paramecium

Equilibrated Paramecium caudatum cells exposed to a constant DC gradient reorient with their depolarized anterior ends toward the cathode (galvanotaxis). Voltage gradients were applied to cells swimming either horizontally or vertically. Their velocity and orientation were recorded and compared to unstimulated cells. The DC field increased the horizontal velocity (= reference) up to 175% (galvanokinesis). Swimming velocities saturated after 1 min and were unchanged during the following 4 min. The upward and downward swimming velocities of stimulated cells were below those of horizontal swimmers. The difference in vertical rates (determining gravikinesis) was independent of variations in absolute velocity. Normalization of vertical velocities to horizontal velocities (= 100%) separated DC-field dependent changes from gravity-induced changes in velocities. A weak voltage gradient (0.3 V/cm) was most effective in raising downward gravikinesis up to threefold (-202 μm/s) above the unstimulated reference (-66 μm/s) and to change sign of gravikinesis in upward swimmers (-43 μm/s →+33 μm/s). We conclude that DC-field stimulation is equivalent to a depolarizing bias on gravikinetic responses of Paramecium. The stimulation does not directly interfere with mechanoreception, but modulates somatic Ca2+ entry to induce contraction of the cell soma. This presumably affects the gating of gravisensory transduction channels.

Graviperception and Graviresponse at the Cellular Level

The evolution of life on Earth occurred under the persistent influence of gravity. Even protists (unicellular organisms such as flagellates and ciliates) had to fmd and to stay in environments whose chemical and physical parameters fit their needs. Consequently, already unicellular organisms developed organelles for active (oriented) movement (cilia, flagella) and sensors for diverse stimuli. Among environmental parameters, gravitational acceleration is a most reliable reference for orientation, because it is virtually constant in its magnitude and direction. Consequently, graviorientation can be already found on very early, unicellular, stages of development [1-3]. As protists are heavier than water, they had to develop mechanisms to compensate sedimentation. Without graviorientation, a population of Paramecium, for instance, would sink to the ground of a 1m depth pond within 3½ hours.

Evidence of central and peripheral gravireception in the ciliate Loxodes striatus

Abstract Effects of the density of the external medium on gravireception in Loxodes striatus were investigated using Percoll solutions. With increasing density, the swimming rates changed from prevailing in the downward direction to prevailing in the upward direction. A cellular density of 1.036 g cm−3 was determined measuring direction and speed of sedimenting immobilized cells at different accelerations and medium densities. Viscosity increases by Percoll were measured and taken into account. At 30% air saturation Loxodes maintained a negative gravikinesis of approximately −27 μm s−1 at external densities corresponding to cellular density (±0.02 g cm−3). Negative gravikinesis decreased gradually to −9 μm s−1 with the density difference rising from 0.020 to 0.036 g cm−3 (=normal). The data indicate the existence of central gravireception, presumably by the Müller organelle, to generate in swimming Loxodes a constant value of gravikinesis and a bimodal gravitaxis. Peripheral gravireception occurs, in addition to central gravireception, when the transmembrane density difference exceeds 0.02 g cm−3. Peripheral gravireception can neutralize, in part, gravikinesis as raised by the central gravireceptor. We hypothesize that both central and peripheral gravireception of Loxodes guide vertical locomotion in gliding and swimming cells.

Velocity and Graviresponses in Paramecium during Adaptation and Varied Oxygen Concentrations

Summary Populations of Paramecium caudatum adapted to new O 2- and chemical environments within at least 6 hours show two subgroups of cells which are distinguished by their median swimming velocity. “Fast swimmers” prevailed at the beginning, and “normal swimmers” toward the end of the adaptation period. The median swimming rate of these two groups was largely constant throughout 6 hours, apart from initial disturbances following transfer into a closed environment. The degree of air saturation of the experimental solution (5% to 100%) was monitored continuously in a new flow-through chamber. Changes in the O 2 level did not have a consistent effect on the swimming velocity. Negative gravitactic orientation in normal swimmers gradually decreased with incubation time. Gravitaxis was absent in the fast swimmers. Variation in O 2 tension did not produce perceptible effects on gravitaxis. At all air saturations and incubation times negative gravikinesis was found, both in normal and in fast swimmers, centering near −40 µm/s. Gravikinesis correlated with swimming velocity, but not with O 2 tension. No correlation was found between the kinesis and orientation coefficients. The data suggest that gravikinesis in Paramecium is largely independent of O 2 levels and adaptation time. Graviorientation declines with adaptation, but is not a direct function of O 2 saturation of the medium.

Gravikinesis in Stylonychia mytilus is based on membrane potential changes

SUMMARY The graviperception of the hypotrichous ciliate Stylonychia mytilus was investigated using electrophysiological methods and behavioural analysis. It is shown that Stylonychia can sense gravity and thereby compensates sedimentation rate by a negative gravikinesis. The graviresponse consists of a velocity-regulating physiological component (negative gravikinesis) and an additional orientational component. The latter is largely based on a physical mechanism but might, in addition, be affected by the frequency of ciliary reversals, which is under physiological control. We show that the external stimulus of gravity is transformed to a physiological signal, activating mechanosensitive calcium and potassium channels. Earlier electrophysiological experiments revealed that these ion channels are distributed in the manner of two opposing gradients over the surface membrane. Here, we show, for the first time, records of gravireceptor potentials in Stylonychia that are presumably based on this two-gradient system of ion channels. The gravireceptor potentials had maximum amplitudes of approximately 4 mV and slow activation characteristics (0.03 mV s–1). The presumptive number of involved graviperceptive ion channels was calculated and correlates with the analysis of the locomotive behaviour.

Phototaxis in Porpostoma notatum, a marine scuticociliate with a composed crystalline organelle

Summary The histophagous scuticociliate Porpostoma notatum passes through five developmental stages during its life cycle: theronts, trophonts, protomonts, tomonts and tomites. The stages are characterized by both, a different morphology and behaviour. When cells are exposed to radiant white light of 10 klx, negative phototaxis occurs in trophonts, which represent the feeding stage of the reproduction cycle, as well as in protomonts and young tomonts, the two successive stages after food uptake. Tomites, resulting from one or two divisions of a tomont, show no phototactic orientation until they have differentiated into theronts. Theronts may show either a positive or negative phototactic response. Individuals of all life cycle stages, except for young tomites, commonly bear a well-developed cup-like organelle, which faces the oral cavity with its concave side. This organelle is composed of alternating layers of cytoplasm and crystals. By this architecture, the organelle resembles a multilayer interference reflector, a structure which has been described in multicellular organisms several times. A possible function of the crystalline organelle in photo-orientation of P. notatum is discussed.

Graviresponses of iron-fed Paramecium under hypergravity

Summary Paramecium caudatum cells were fed with iron-particles to increase the density of the cytoplasm. The swimming speed and orientation of iron-fed and iron-free control cells were analyzed under terrestrial gravity and raised acceleration up to 6 g in a centrifuge. Iron-fed cells sedimented at increased rates (133 μm · s −1 per g unit) as compared to controls (118 μm · s −1 per g unit). Gravikinesis increased in iron-fed cells with rising acceleration at a rate of 67 μm · s −1 per g unit as compared to 46 μm · s −1 per g unit in control cells. In particular, the gravikinesis of downward swimming iron-fed cells was strongly enhanced, thereby compensating for the sedimentation rate. This was not the case in upward swimming cells, where ingested iron depressed the gravikinetic response. The negative gravitaxis of Paramecium , as being represented by the cell orientation coefficient, was much pronounced in iron-fed cells at 1 g (r oC = 0.36; controls: r oC = 0.13). At 4 g , the orientation coefficient of iron-fed Paramecium rose to 0.80 (controls: 0.54). The effect of artificially raised cytoplasmic density on gravikinesis is explained at the basis of the mechanoreceptor organization of Paramecium . The effect on gravitaxis continues to be uncertain.

Graviresponses of gliding and swimming Loxodes using step transition to weightlessness.

Cells of Loxodes striatus were adjusted to defined culturing, experimental solution, O2‐supply, temperature, and state of equilibration to be subjected to step‐type transition of acceleration from normal gravity (1 g) to the weightless condition (μg) during free fall in a 500‐m drop shaft. Cellular locomotion inside a vertical experimental chamber was recorded preceding transition and during 10 s of μg. Cell tracks from video records were used to separate cells gliding along a solid surface from free swimmers, and to determine gravitaxis and gravikinesis of gliding and swimming cells. With O2 concentrations ≥ 40% air saturation, gliders and swimmers showed a positive gravitaxis. In μg gravitaxis of gliders relaxed within 5 s, whereas gravitaxis relaxation of swimmers was not completed even after 10 s. Rates of horizontal gliders (319 μm/s) exceeded those of horizontal swimmers (275 μm/s). Relaxation of gravikinesis was incomplete after 10 s of μg. Analysis of the locomotion rates during the g‐step transition revealed that gliders sediment more slowly than swimmers (14 versus 45 μm/s). The gravikinesis of gliders cancelled sedimentation effects during upward and downward locomotion tending to maintain cells at a predetermined level inside sediments of a freshwater habitat. At ≥ 40% air saturation, gravikinesis of swimmers augmented the speed of the majority of cells during gravitaxis, which favours fast vertical migrations of Loxodes.