Abstracts of papers by Hans Machemer and coworkers

Abstracts 121 - 135

135
(see Abstract 97)

133
CECILIA, a versatile research tool for cellular responses to gravity.
We describe a centrifuge designed and constructed according to current demands for a versatile instrument in cellular gravitational research, in particular protists (Ciliates, Flagellates).  The instrument (called CECILIA, centrifuge for ciliates) is suited for videomonitoring, videorecording, and quantitative evaluation of data from large numbers of swimming cells in a ground-based laboratory or in a drop tower/drop shaft under microgravity conditions.  The horizontal rotating platform holds up to six 8mm-camcorders and six chambers holding the experimental cells.  Under hypergravity conditions (up to 15 g) chambers can be rotated about 2 axes to adjust the swimming space at right angles or parallel to the resulting gravity vector.  Evaluations of cellular responses to central acceleration - in the presence of gravitational 1 g - are used for extrapolation of cellular behaviour under hypogravity conditions.  CECILIA is operated and monitored by computer using custom-made software.  Times and slopes of rising and decreasing acceleration are supervised on-line, as well as values and quality of steady acceleration.  CECILIA can serve as an on-ground research instrument for precursor investigations of the behaviour of cells under microgravity conditions such as long-term experiments in the International Space Station.

132
Two essays for a Dictionary of Protozoology on: GALVANOTAXIS: an oriented movement of actively swimming eucaryotic microorganisms in a DC voltage gradient between anode and cathode. GRAVITAXIS (formerly geotaxis): in its general use an accumulation or directional response of motile organisms to gravity. In a more narrow definition: the directional response of the organism toward the centre of gravity (positive gravitaxis) or away from it (negative gravitaxis) due to the gravity stimulus.

131
The swimming cell and its world: structures and mechanisms of orientation in protists.
The properties of motility and sensory organisation of unicellular organisms suggest that cells were able to utilise the resources of their fluid environment before they had developed senses for its exploration. Two principles of locomotion, helical swimming and abrupt responses of turning ("tumbling", "reversal") are used by both prokaryotes and protists to provide access to multiple sites in the biosphere. There is no indication that motility alone can establish any dimensionality to the world of cells. The primary sensorimotor mechanism in bacteria and protists, kinesis, identifies nutrients etc. by their concentration gradients using the dimension of time. Increases in cell sizes of protists necessitated highly sensitive mechanoreceptors to overcome passive sedimentation due to gravity. The polar and/or gradient-type arrangement of membrane receptors, deformed through gravitational forces, allows the assessment of the gravity vector and thereby establishes the most important dimension of the biosphere, its vertical axis. Responses are modulations of swimming speed and frequency of reversals (kineses). Gravikinesis uses, presumably for the first time in evolution, spatial and no temporal information. The rise of physiologically guided, directly orientating taxes in protists (e.g. phototaxis) is associated with spatial assessment of a vector-type stimulus (radiant light) as well. Taxes exploit only a single dimension of space (bright/dark; up/down). The first indication of inclusion of a second spatial dimension comes from advanced ciliates (Paramecium), which are able to fully neutralise gravikinesis in the horizontal position. The world of protists is thereby, at best, a sheet spread between the vertical and horizontal axes.

CONTENTS.   What is a world? – How cells tour the biosphere. – Two principles of active cellular propagation. – How is a stimulus sensed? – Adaptation. – Kinesis or walking along a stimulus gradient. – The challenge of gravity. – How to sense a vector-type stimulus? – Role of the topology of cellular sensing. – Protistan kinesis include speed regulation. – Graviresponses: linking kinesis and taxis. – The making of the world of protists.

130
Effects of gadolinium on electrical membrane properties and behaviour in Paramecium tetraurelia.
Low concentrations (4 to 10 µM) of the trivalent cation gadolinium raised the input resistance and altered the membrane potential of Paramecium tetraurelia suggesting an interference with membrane channels. Current-clamp conditions revealed a concentration-dependent reduction of membrane rectification, in particular in the hyperpolarizing direction. The graded action potential and the early inward current seen under voltage-clamp conditions were depressed. Standardized applications of probes for focal mechanostimulation showed a reduction in mechanoreceptor potentials. Reversal responses of downward swimming cells in gadolinium transiently rose to higher frequency and settled at a reduced level thereafter explaining observations of a transient increase in gravitaxis. Vertical swimming speed was reduced, whereas the horizontal speed was largely unchanged. Gravity-induced modulation of swimming speed (gravikinesis) was depressed. The data indicate that gadolinium at micromolar concentrations effectively interferes with various types of membrane channels in Paramecium and is therefore no specific channel inhibitor to characterize graviresponses in this ciliate.

129
Physical and physiological components in the graviresponses of Paramecium tetraurelia wild-type and mutant.
Wild-type and the morphological mutant kin 241 of Paramecium tetraurelia improved orientation away from the centre of gravity (= negative gravitaxis) at accelerations rising from 1 gto 7 g. The gravitaxis was more pronounced in the mutant. A correlation between the efficiency of orientation and the applied g-value suggests a physical basis of gravitaxis. Transiently enhanced rates of reversals of the swimming direction coincided with transiently enhanced gravitaxis because reversals occurred more often in downward swimmers than in upward swimmers. The results are evidence of a physiological modulation of gravitaxis by means of the randomizing effect of depolarization-dependent reversals. Gravity bimodally altered propulsion rates of the wild-type so that sedimentation was partly antagonized in upward and downward swimmers (= negative gravikinesis). In the mutant only increases in propulsion were observed, although the orientation-dependent sensitivity of the gravikinetic response was the same as in the wild-type. Data of observed speed and sedimentation rates in the wild-type and mutant were linearly related to acceleration allowing the determination of gravikinesis as a linear (and so far nonsaturating) function of gravity.

128
A gravity-induced regulation of swimming speed in Euglena gracilis.
We investigated the autotrophic flagellate Euglena gracilis for gravity-induced modulation of the speed of swimming as previously documented for larger protozoan cells. Methods of video-tracking of swimming and sedimenting cells under 1 g and hypergravity up to 2 g, and computer-assisted data processing were applied. The vertical and horizontal swimming speed, and sedimentation rates of immobilized cells, was found to be linear functions of acceleration. Accounting for sedimentation in the observed upward and downward movements of Euglena, the active component of speed (propulsion) rose in proportion to acceleration. No saturation of gravikinesis was seen within the g-range tested. Gravity-dependent augmentation of speed was maximal in upward swimmers and decreased continuously over horizontal to downward swimmers. Linear extrapolations of the data to zero-g conditions suggest the absence of a threshold of gravikinesis in Euglena. Energetic considerations indicate a high sensitivity of gravitransduction near the level of Brownian molecular motion.

127
A closed artificial ecosystem for ciliates.
MICROPOND is a minimalized ecosystem designed for the investigation of effects of long-term-microgravity in ciliates. The system is controlled automatically by a computer program. With the cell species Stylonychia mytilus and Chlorogonium elongatum, the longest lasting experiment was stable for 48 days so far; a closed stand-alone culture of Chlorogonium has was continued for 197 days. During this time, important physical and chemical parameters of the system were controlled within acceptable limits.

126
Transient graviresponses in Paramecium: Swimming track analysis by free-fall experiments.
Using the drop shaft at the Japan Microgravity Center (JAMIC, Kamisunagawa, Hokkaido), we recorded swimming tracks and transient responses of Paramecium subjected to an abrupt shift of gravity from 1g to µg by free-fall experiments. The results indicate that at least two types of mechanisms that differ in their relaxation rates underlie the responses to the gravity shift. The free-fall facilities provide effective methods to investigate phasic responses of organisms to microgravity.

125
Responses of Tetrahymena pyriformis to the natural gravity vector.
Tetrahymena pyriformis is a small protozoan cell, which has less than 10% of the volume of the well-known Paramecium caudatum. We investigated the graviresponses of Tetrahymena in a search of the lower limits of gravitransduction in ciliates. Equilibrated populations of free swimming cells were enclosed in chambers of 2 mm depth testing the orientational and speed responses with the chambers in horizontal or vertical position. For determinations of gravikinesis, the sedimentation rates of cells immobilized by application of two different procedures were measured. Negative gravitaxis was pronounced after turning the chambers from horizontal to vertical position; it settled, after 1 min, toward an orientation coefficient of 0.2. Gravikinesis did not only neutralize the sedimentation rate (= 22 µm/s, but even exceeded that rate by at least 30%. Tetrahymena is thereby the first cell, in which overcompensation of the sedimentation rate was documented. Biophysical considerations suggest a high gravisensitivity of Tetrahymena with channel gating energy being less than 33 times above the thermic noise level.

124
The gravikinetic response of Paramecium is based on orientation-dependent mechanotransduction.
Paramecium generates persistent shifts of the membrane potential of a few millivolts depending on its orientation with respect to the gravity vector. The resulting potential-induced modulation of the speed of propulsion is called gravikinesis because it acts to neutralize, fully or in part, sedimentation. Gravisensitivity is maximal at neutral orientation, i. e. in horizontally swimming cells, when the gravitational force per unit membrane area is at minimum. Stimulus-response relationships and energetic considerations show that sensing of the gravity vector by a non-specialized, single-cell organism ranks among the most sensitive mechanoreceptors known in nature.

123
The identification of gravikinesis from ciliates: methods and experience.
Recent advances in the gravitational physiology of ciliates are reported: the theoretical and experimental assessment of gravikinesis and sedimentation, calculation of gravikinesis using slopes of observed swimming and sedimentation data under hypergravity, orientational distributions of gravikinesis, central and membrane-associated gravitransduction, and the kinetics of activation and relaxation of gravikinesis.

122
Wie eine Zelle "oben" und "unten" registriert.
(How a cell can deal wit "up" and "down")
CONCLUDING REMARKS. In this chapter on gravisensation and graviresponses by free swimming cells we have selected from the large field of gravitational physiology the section dealing with gravikinesis. First of all we realized that gravity acts on any body we can think of. If a mass is not in free fall, a force results from acceleration. Cells swimming in freshwater and seawater are heavier than their environment, which is the cause of their sedimentation and leads to a pressure exerted by the dense cytoplasm upon the lower cell membrane. During the evolution of life, unicellular organisms have developed a polar organization of mechanoreceptor channels. Opening of these channels modifies the membrane potential and eventually the swimming rate of the cells. Measurements of active swimming rates in Paramecium (increasing in upward swimming cells, decreasing in downward swimming cells) have led to the conclusion, that gravitational acceleration is strong enough to open mechanoreceptor channels. Model considerations of a population of 10 cells have shown that the highly dangerous sedimentation can be overcome employing gravikinesis and/or gravitaxis. Observations show that a pronounced gravitaxis occurs after the impact of strong stimuli (shaking, low temperature). A weak negative gravitaxis combined with a gravikinesis often results in a so-called "neutral gravitaxis" with a vertical gain missing. A neutral gravitaxis is an advantage for cells, which respond to light or chemical attractants without being displaced upward or downward due to effects of gravity.
SCHLUSSBETRACHTUNG. Wir haben in diesem Kapitel über Schwerkraftwahrnehmung und Schwerkraftbeantwortung durch frei schwimmende Zellen nur einen kleinen Ausschnitt aus dem weiten Feld der Gravitationsphysiologie darstellen können: jenen, der sich mit Gravikinesen beschäftigt. Am Anfang stand die Erkenntnis, dass Gravitation auf alle nur denkbaren Körper wirkt. Eine Kraft entsteht aus der Beschleunigung immer dann, wenn Körper am freien Fall gehindert werden. Im Süß- und Seewasser schwimmende Zellen sind schwerer als ihre Umgebung und sedimentieren deswegen. Dabei drückt das dichtere Zellplasma auf die jeweils unten liegende Zellmembran. In der Evolution hat sich bei Einzellern eine polare Anordnung von Mechanorezeptorkanälen herausgebildet, deren Öffnung das Membranpotential verändert und auf die Schwimmgeschwindigkeit einwirkt. Messungen einer erhöhten Schwimmaktivität bei aufwärtsschwimmenden Paramecium-Zellen und einer reduzierten Aktivität bei abwärtsschwimmenden Zellen haben zu dem Schluss geführt, dass die Erdbeschleunigung Mechanorezeptorkanäle zu öffnen vermag. Modellbetrachtungen mit einer Population von 10 Zellen haben uns gelehrt, dass Zellen die lebensbedrohliche Sedimentation mit Hilfe einer Gravikinese, einer Gravitaxis oder durch eine Kombination von Gravikinese und Gravitaxis überwinden können. Beobachtungen und Experimente zeigen immer wieder, dass eine sehr ausgeprägte, negative Gravitaxis bei Paramecium nach starken Reizungen (durch Schütteln, Kälte etc.) auftritt. Eine schwache negative Gravitaxis, verbunden mit einer Gravikinese, führt oft zu einer sog. neutralen Gravitaxis, bei der für die Population insgesamt kein vertikaler "Gewinn" herausspringt. Eine neutrale Gravitaxis macht einen Sinn immer dann, wenn die Beibehaltung eines bestimmten Niveaus im Wasser von Vorteil ist, bzw. wenn die Zellen auf Licht oder chemische Lockstoffe angemessen reagieren wollen, ohne zugleich durch die Schwerkraft abwärts oder aufwärts verfrachtet zu werden.

121
Relaxation of graviresponses of the ciliate Didinium following step transition to the weightless condition.
Equilibrated, free swimming specimens of Didinium nasutum were exposed to a step transition from normal acceleration to 10 s of microgravity in a 500-m drop shaft for free-fall experiments. Prior to microgravity, the orientation and swimming speed of the cells was registered by video microscopy in the horizontal and vertical position of the experimental chamber, and recording continued beyond the end of the weightless condition. At normal gravity horizontally swimming cells showed constant speed and did not prefer any direction. Turning the chamber to the vertical position induced Didinium to be oriented upwards (negative gravitaxis) and to raise speed in the downward direction. Gravitaxis persisted until the end of microgravity. For about 1 s of microgravity upward swimming rates exceeded the downward rates. During the entire weightless period, the median speed of all cells was below the value of cellular propulsion as unaffected by gravity. A detailed analysis of the events during microgravity revealed a gradual rise in downward swimming rates and a decline of upward swimming rates. The apparent gravikinetic paradox (gravikinesis persisting in the weightless condition), and the slow kinetics of gravikinesis are discussed at the basis of the established mechano- and gravisensory organization of Didinium and a model of visco-elastic linkage between the sensitive anterior cell end and the insensitive posterior cell end.