Abstracts 91 - 120
120
Evidence of central and peripheral gravireception in the ciliate Loxodes.
Effects of the density of the external medium on gravireception in Loxodes
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/cm3 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 at external
densities corresponding to cellular density (±0.02 g/cm3). Negative
gravikinesis decreased gradually to -9 µm/s with the density difference rising
from 0.020 to 0.036 g/cm3 (= 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, with the transmembrane density difference exceeding 0.02 g/cm3.
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.
119
Relaxation and activation of graviresponses in Paramecium.
The kinetics of gravitaxis and gravikinesis of Paramecium caudatum were
investigated employing (1) step transitions from normal gravity (1 g) to
weightlessness (microgravity) and (2) turns of experimental chambers from the
horizontal to the vertical position at 1 g. The transition to
microgravity left existing cell orientations unchanged. Relaxation of negative
gravitaxis under microgravity exceeded 10 s and may be described by the time
constant of the decay of orientation coefficients. Gravitaxis was started at 1 g
by turning the experimental chamber from horizontal to vertical. Gravitaxis
rapidly activated during the turning procedure and relaxed to an intermediate
level after the turning had come to a standstill. Gravity-induced speed
regulation (gravikinesis) at 1 g was at steady-state after 1 min; here,
gravikinesis counteracted the effects of sedimentation (negative gravikinesis).
A step transition to microgravity initially reversed the sign of the
gravikinesis (positive gravikinesis). The relaxation of this kinetic response
was not completed during 10 s of microgravity. The data suggest that
gravikinesis is functionally unrelated to gravitaxis and is strongly affected
by the rate of change in acceleration. We present a model explaining why
gravikinesis reverses sign upon the onset of a step from 1 g to
microgravity.
118
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 slower 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.
117
The linking of extrinsic stimuli to behaviour: roles of cilia in ciliates.
More than three decades of physiological and structural investigation have lead
to a general picture of sensorimotor properties in ciliates. Each of the key areas
of this research - ultrastructural analysis and modelling, biochemistry,
electrophysiology, and behaviour - contributes to an image of high complexity
relating to the contributing organelles. Examination of the situation from
events of the incoming stimulus to events of the ciliary motor output and
locomotion behaviour shows that fine-tuned ciliary responses are often a direct
reflection of stimulus intensities, and such analog processing of signals is
beneficial to the cell. The regenerative excitability of the membrane, a rapid
bimodal regulation of the ciliary beating rate, and an equally rapid monomodal
regulation of beat direction by the major intracellular messenger, Ca2+,
are the basis of more spectacular events such as action potentials and reversals
of the ciliary cycle, which are nevertheless stimulus-graded and represent the
extreme ends of a wide scale of cellular processing of stimuli.
116
Unicellular responses to gravity transitions.
An overview is given of the current state of research and discussion on
graviresponses in free swimming unicellular organisms. Two major chapters
dealing with orientational and kinetic responses are introduced by general
biological and biophysical considerations of graviresponses, and by a
discussion of the wide array of tools and methods.
1 INTRODUCTION. – 1.1 Why graviresponses of cells are meaningful. – 1.2
Evolutionary considerations. – 1.3 Physical mechanisms of gravitaxis. – 1.4
Physiological basis of gravitransduction. – 2 LIMITS to GRAVITRANSDUCTION and
GRAVIRESPONSES. – 2.1 Thermal noise and signal-to-noise ratio. – 2.2 Cell size.
– 2.3 Membrane channel properties. – 3 TOOLS and METHODS. – 3.1 Sounding
rocket: microgravity following preacceleration. – 3.2 Drop facilities: step
transitions to microgravity. – 3.3 Orbiting crafts: long-term microgravity. –
3.4 Determination of the sedimentation rate. – 3.5 Manipulation of density. –
3.6 Ions and drugs. – 3.7 UV-irradiation. – 3.8 DC-field stimulation. – 3.9
Data processing. – 4 ORIENTATIONAL RESPONSES of CELLS: GRAVITAXIS. – 4.1
Characteristics of orientational responses. – 4.2 Track orientation and cell
orientation. – 4.3 1g-orientations in the horizontal and vertical plane. – 4.4
Loss of orientation in microgravity. – 4.5 Threshold and saturation of gravitaxis.
– 4.6 Does gravitaxis adapt? – 4.7 Effects of raised density. – 4.8 Ionic and
drug effects. – 4.9 UV-irradiation. – 5 SPEED REGULATION of CELLS:
GRAVIKINESIS. – 5.1 Gravikinesis concept deduced from ciliate mechanosensory
organization. – 5.2 Role of sedimentation rate. – 5.3 Orientation dependence of
gravikinesis. – 5.4 Hypergravity: stimulus-response coupling. – 5.5 Is
gravikinesis coupled to swimming rate? – 5.6 Prestimulation and time effects. –
5.7 Electric potentiation of graviresponses. – 5.8 Density manipulation:
Different effects on polar gravireceptors. – 5.9 Separation between central and
peripheral gravireceptor in Loxodes. – 5.10 Relaxation of gravikinesis
upon step transition. – 6 CONCLUSIONS and PERSPECTIVE.
115
Graviresponses of iron-fed Paramecium under hypergravity.
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 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 (roC = 0.36; controls: = 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.
114
Analysis of sedimentation of immobilized cells under normal and
hyper-gravity.
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 includes speed regulation - Graviresponses: linking kinesis and taxis -
The making of the world of protists.
113
Sedimentation velocity of Loxodes striatus immobilized by MnCl2.
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.
112
The dealing with gravity at the unicellular level: concepts and data.
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 g to 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.
111
Mechano-sensorimotor organization in the ciliate cell: basics.
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, were 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.
110
Mechanisms of gravireception and responses in unicellular systems.
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.
109
Electric potentiation of gravikinesis in Paramecium is possibly
mediated by filaments.
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.
108
Assessment of g-dependent cellular gravitaxis: determination of cell
orientation from locomotion track.
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.
107
Motility of cilia and flagella (16-mm film for scientific instruction; 16
min, English sound track).
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.
106
Ciliary and cellular motor responses near threshold stimulation.
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.
105
Gravitaxis screened for physical mechanisms using g-modulated
cellular orientational behaviour. Advanced methods of recording cellular
orientation with respect to the gravity vector are yielding increasingly
well-founded data on gravitaxis. The
present study introduces a quantitative method which allows to predict the
precision of orientational behaviour as a function of acceleration (g) assuming static buoyancy as a
hypothetical physical principle of gravitaxis.
The precision of orientation is expressed by the orientation coefficient (ro) as derived from circular statistics.
Orientation coefficients calculated from experimental data at various g-values are tested for fit with a
sigmoidal ro-g transfer function including a proportionality factor (k). Residual orientation values in the
low-hypogravity range obey a reciprocal function between k and g.
Intersection of this residual-g
function with the ro-g relationship gives the minimal
acceleration to generate cellular orientation.
Those data which clearly diverge from the ro-g
curve bear some probability that the observed gravitaxis was guided in part by
a physiological mechanism of gravireception and active graviorientation. Data which fit the ro-g
curve qualify as being in agreement with a mechanical basis of cellular
gravitaxis. Examples from the literature
are presented and discussed in the light of our scheme of gravitaxis screening.
104
Is there an orientation-dependent excursion of the Müller body in the
"statocystoid" of Loxodes?
The ciliate Loxodes possesses a number of vesicles at its anterior
dorsal margin. These so-called Müller
vesicles contain a spherical inclusion (Müller body) in which lie crystals of
barium salts. The Müller body is
connected via a stalk to the wall of its vesicle. It is presumed to function as a
stato-organelle and to respond by visible motions to changes of the direction
of gravity. We have attempted to
document the motion of the Müller body with respect to the direction of the
gravity vector. Living cells moving in a
horizontal or a vertical plane have been viewed under the light microscope with
differential interference contrast, documented on video film, and sequences of
single frames have been evaluated.
Apparent excursions of the Müller body by about 1.5 µm, corresponding to
a deviation of 10° at the base of the stalk, are observed in cells moving in a
horizontal plane. No larger excursions
have been seen in the vertical plane.
Implications of this result for a model of the stato-organelle are
discussed.
103
Electric control of ciliary beat direction and curvature of gliding in Loxodes
(Ciliata).
The gliding locomotion of Loxodes on solid surfaces is commonly curved
in the counterclockwise direction. Experiments using K+-dependent modulation
of the membrane potential showed that depolarization induced gliding tracks of
clockwise curvature, whereas hyperpolarization induced an increase of track
curvature in the counterclockwise direction.
Galvanotaxis of Loxodes in a
linear DC-field is unorthodox in that cells become oriented parallel to the
isopotentials with their oral sides facing the cathode. The experimental data were combined with a
novel geometric model which predicts locomotion from differential ciliary
activity. The model accounts for the
body shape, distribution and density of ciliation, and beat direction of the
cilia. Ciliary beat direction shifts in
the counterclockwise direction (cilia seen tip-to-base) from posteriad to
anteriad upon membrane depolarization.
During hyperpolarization, beat direction is reoriented in the clockwise
direction toward the posterior cell end.
The characteristics of electromotor coupling with respect to the beat
direction are in accordance with those which were previously documented in Paramecium.
102
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.
101
Is gravikinesis in Paramecium affected by swimming velocity?
Negative gravikinesis is an acceleration-activated modulation of vertical
swimming velocity which can neutralize the passive sedimentation of ciliates.
This paper investigates effects of swimming velocity levels on
gravikinesis. Evaluations of a large
number of data of free swimming cells with velocities ranging from 500 to 2000
µm/s show that the ratio of vertically upward (and downward) swimming cells
over horizontally swimming cells is represented by linear functions with slopes
of 1. This is equivalent to a constant
value of gravikinesis (∆) in upward swimming cells (∆U)
and downward swimming cells (∆D) irrespective of the swimming
rate. Mechanoresponses due to
velocity-dependent membrane deformations (rheokinesis) do not affect
determinations of gravikinesis and may not exist at all. DC-field stimulation interferes with the
slopes of swimming rates depressing vertical velocities as referred to
horizontal velocity. The reduction of
slopes (<1) in the presence of an external voltage gradient implies a
feedback of voltage-dependent activation of gravireceptor channels on the
electric motor control of the cilia.
100
Electric-field stimulation elucidates gravireception in Paramecium.
Paramecium and other unicellular microorganisms exposed to linear
DC-fields are under extracellular voltage clamp conditions. Because Paramecium
swims toward the cathode, the clamp is topographically defined (anterior end:
depolarization; posterior end: hyperpolarization). Shifts of the membrane
potential under DC-field stimulation modify graviresponses following
deformation of the mechanically sensitive membrane. Voltage gradients between
0.3 and 0.8 V/cm (7.5 to 20 mV/cell length) raised the swimming velocity up to
175% as compared to unstimulated cells. With the field lines oriented parallel
to the gravity vector, a large proportion of cells was restricted to
orientations of ±15° from vertical. A gradient of 0.3 V/cm was most effective
in modulation of gravikinesis: the downward gravikinesis rose threefold from
-66 µm/s to -202 µm/s; the upward gravikinesis reversed polarity from negative
(-43 µm/s) to positive (+33 µm/s). This depolarizing bias of the membrane
potential is unrelated to direct voltage-sensitivity of mechanoreceptors. It is
presumably due to Ca2+-entry and Ca2+-dependent
contraction of the cell cortex, which interferes with filament-mediated gating
of the gravireceptor channels.
99
Voltage dependence of ciliary activity in the ciliate Didinium nasutum.
In the gymnostome ciliate Didinium nasutum, swimming behaviour depends upon the
cyclic activity of about 3000 cilia. The normal beating mode, resulting in
forward swimming of the cell, is characterized by a posteriad effective beat
(18° left of the longitudinal axis) at a frequency of approximately 15 Hz. Activation of depolarization-sensitive
ciliary Ca2+ channels leads to an increase in intracellular Ca2+
concentration and a change in the beating mode. Following rapid reorientation,
the effective stroke is anteriad (24 degree right to the longitudinal axis) and the beating frequency is about 26 Hz, resulting
in fast backward swimming of the cell.
In response to minor depolarizations, and hence small increases in
cytoplasmic Ca2+ concentration, the cilia inactivate. Frequency
increase and reversal in beat orientation share a single threshold level of
membrane potential, since both changes of the beating mode occur
simultaneously.
98
Fluorometric measurement of the intracellular free Ca2+-concentration
in the ciliate Didinium nasutum using fura-2.
CONTENTS. We developed an experimental approach to measure somatic and ciliary
Ca2+-signals in the ciliate Didinium under voltage clamp conditions
using the dye Fura-2. Intracellular pressure injection of Fura-2 molecules did
not alter electrophysiological membrane properties besides an expected
buffering effect. The intracellular free Ca-concentration was determined at
2x10-7M. During membrane excitation, this resting value increased in
the cilia; a quantification was not feasible. Within the cell soma, however,
the Ca2+-level was unchanged within the physiological range of the
membrane potential (-70 mV to 0 mV). Increasing the driving force for Ca-ions
via strong hyperpolarization (potentials negative to -200 mV) a centripetal
increase in the somatic Ca concentration was found. Our results support the
hypothesis that Ca2+ is the intracellular messenger in rapid
electromotor coupling in ciliates.
97
Behavior. In: (Hausmann K) Protozoology, p 260-271.
Reports on protozoan behaviour deal with motor
or growth responses which are easily observed and recorded. The behavioural
output often results from both internal and external
factors. Complex forms of behaviour are
generated from integration of various inputs. Stimuli may be one-dimensional (such as “strength” or “intensity”) or vector-type (as given in the case of
gravity or radiant light). Stimulus-dependent responses are subdivided into kineses
and taxes, the latter including an Orientational component.
Time-dependent changes in behaviour are: adaptation, sensitization
(desensitization), and habituation. The classification of behavioural responses often
occurs according to stimulus modality: DC voltage
generating galvanotaxis; mechanical force
(touch or persistent load) leading to escape responses, gravikinesis,
gravitaxis, rheotaxis, thigmotaxis; temperature; chemical substance inducing chemokinesis, chemotaxis; light
generating photoaccumulation, photokinesis, photoshock, phototaxis.
96
Electrophysiology of Ciliates. In: (Dentler WL, Witman GB, eds) Methods in
Cell Biology.
INTRODUCTION. Dealing with cell excitability differs from research devoted
to identification of cellular building blocks. The target of electrophysiology
is immaterial: a rapid communications and integration system between
membrane-associated organelles, based on the separation of electric charges
across lipid bilayers. The growth of eukaryote cells to large size or intricate
ramifications, and the multiplication of specialized cells in different tissues
require communication and integration at and beyond the cell level; in
addition, a persistent flow of extrinsic and intrinsic signals asks for rapid
responses. In ciliates, receptor-effector coupling is obvious in the
behavioural responses to stimulation, in particular in those responses which
are mediated motile cilia. Typical time domains are 10-3 to 10-2
s involving cellular currents of the order of 10-8 A and potential
changes about 10-2 V. At the level of single organelles, such as a
cilium, a few V-sensitive channels of, say, 10-11 S conductance
each, together with Ca2+-pumping molecules and/or membraneous or
intraciliary Ca2+ binding sites shuffle Ca2+ ions between
the axoneme and the outer world with high precision. Thus, the ciliary Ca
channel, albeit V-sensitive, serves one goal in the first line: maintaining
limited amounts, 101 to 103 ions per cilium, of the free
messenger substance, Ca2+. An advantage of electric recording from
whole cells or from single channels is that the experimenter is witnessing the
living system rather than sorting out heaps of fragments from that system. A
disadvantage is that electrophysiology is hard to teach in the cookbook style;
on-site training on cell manipulation and hardware troubleshooting,
supplemented by basic theory on DC and AC circuitry, are common procedures. A
typical approach is that the researcher in one field of expertise feels that
electrophysiology might answer so far unsolved questions. Electrophysiology
rests on basic rules of physics, and ciliary electromotor coupling follows
quite simple lines of cause and effect; therefore, ciliary motion and even the
cellular motile behaviour reflect the electrophysiological state of the cell
and, implicitly, intracellular signalling by Ca2+. An observer may
judge an electrophysiological status from behavioural characteristics. For the
building of thorough electrophysiological expertise, time is an important
factor. Even the trained scientist can rarely promise to quickly answer a
question just by sticking electrodes into Paramecium. It may be
realistic to seek cooperation between two labs in making very substantial
mutual commitments in staff and strategic effort.
95
A theory of gravikinesis in Paramecium.
The archaic eukaryote unicellular microorganism, Paramecium, is propelled by thousands of cilia, which are regulated
by modulation of the membrane potential.
Ciliates can successfully cope with gravity, which is the
phylogenetically oldest stimulus for living things. One mechanism for overcoming sedimentation is
negative gravitaxis, an orientational response antiparallel to the gravity
vector. We have postulated the existence
of a negative gravikinesis in Paramecium,
i.e. a modulation of swimming speed as a function of cellular orientation in
space. With negative gravikinesis, an
upward oriented cell actively augments the rate of forward swimming and
depresses active locomotion during downward orientation. A brief outline of the gravikinesis
hypothesis is given on a quantitative basis and experimental data are presented
which have confirmed the major assumptions.
94
Graviresponses in Paramecium and Didinium examined under varied
hypergravity conditions.
The swimming behaviour of two species of ciliates, Paramecium caudatum and Didinium nasutum, characterized by
different mechanosensory and ciliary motor properties were investigated under
hypergravity up to 5.4 g. The experiments
were designed to examine large numbers of cells using video recording, digital
data processing, and statistics for the documentation of the rates and
orientations of swimming. The gravikinetic responses (change in active swimming
rates) were calculated from (1) the velocities of vertical swimming in the
gravity field, (2) sedimentation of Ni2+-immobilized cells and (3)
the intrinsic rate of propulsion, independent of gravity. Propulsion was determined from the
intersection of regression lines of the gravity-dependent upward and downward
swimming velocities. The rates of swimming and sedimentation, and consequently
the gravikineses, were linear functions of gravitational acceleration.
Comparisons of cell populations from different cultures suggest an age-dependent
change in gravikinesis. In starved Paramecium
(7-day cultures), the kinetic responses antagonizing sedimentation
(negative gravikinesis) increased with acceleration. In Didinium negative gravikinesis was documented at 1 g in downward-swimming specimens only,
which agrees with the mechanosensory organization of this cell. Hypergravity induced the gravikinesis of Didinium to change sign. In both species, and at all accelerations
tested, a neutral gravitaxis was documented.
Such behaviour incorporates distinct acceleration-dependent
orientational and velocity responses keeping populations of cells stationary in
the gravity field (taxis coefficients close to zero).
93
Analysis of three-dimensional ciliary beating by means of high-speed stereo-microscopy.
Results are presented on the analysis of three-dimensional motion of
compound cilia or cirri in voltage-clamped specimens of the protozoan Stylonychia
mytilus. Time series of three-dimensional data were obtained by using the
anaxial illumination method for simultaneous recording of stereoscopic video
images.
Data processing involved the following steps: determination of a reference
coordinate system based solely on features present in each stereo-pair; tracing
of cirral axes in digitized images, conversion to parameter curves by means of
least-squares polynomial approximation, conversion of pairs of two-dimensional
data to a series of three-dimensional data; correction for distortion due to
projective shortening and conversion to a series of polynomial triplets, and
analysis of the periodical components of the motion pattern in the frequency
domain.
Reconstructed beating cycles show typical differences between
hyperpolarization-induced ciliary activity and depolarization-induced ciliary
activity. Reconstructions of the motion of the basal segment of a cirrus are in
agreement with existing data. Analysis of the curvature and torsion of a cirral
axis during beating does not reveal any simple pattern of propagated activity
within the axoneme.
The return stroke may be subdivided into two phases. First, a curvature peak
develops proximally. Secondly, a region with increased torsion arises more
distally and spreads out in proximal direction. Both curvature and torsion
return to minimal values by the beginning of the power stroke.
92
Ciliate Sensory Physiology.
Ciliates know to interpret the direction and spectral composition of light,
and they walk along temperature and chemical gradients. Our present understanding of the sensory
physiology of these organisms is still very limited. Ciliates are useful to elucidate the sensory
organization and excitability of eukaryotes in the early history of life. Regarding the marvellous mechanosensory
organization in ciliates, we have begun to consider the idea that our
conventional pulse stimulation and recording of phasic responses in ciliates
may have veiled a more meaningful and phylogenetically old achievement of the
eukaryote: coping with gravitational pull.
Our data suggest that metazoan cells, too, not only successfully meet
the challenge posed by gravity for maintaining their architecture but also can
actively neutralize less spectacular adverse effects of gravity.
91
Gravity-dependent modulation of swimming rate in ciliates.
In upward swimming Paramecium, gravity acts to open gravireceptor K
channels at the posterior cell end, the cell hyperpolarizes and the cilia, by
raising the frequency of their beating, generate an increment in upward
swimming rate, ∆U, which antagonizes sedimentation. In
horizontally swimming Paramecium, both gravireceptor K and Ca channels
are activated with a conductance ratio which corresponds to that of the resting
potential. Hence, no change in ciliary
frequency occurs. In downward swimming Paramecium,
anterior gravireceptor Ca channels activate; the membrane depolarizes
depressing the rate of ciliary beating. This decrement in downward swimming
rate, ∆D, antagonizes sedimentation.
Our data in Didinium agree with the general scheme of Paramecium
with two exceptions: (1) there exist no hyperpolarizing gravireceptor channels;
(2) gravity-induced inward as well as outward deformation of the membrane can
generate depolarizing gravireceptor potentials.
Thus, with the pull of gravity, swimming in any direction is slightly
inhibited. Only under conditions of weightlessness is this inhibition of
ciliary activity removed so that the Didinium
cell swims faster than at terrestrial gravity.