Abstracts 61 - 90
90
Cationic inhibition of Ca current
and Ca-dependent ciliary responses in Paramecium.
The Paramecium cell membrane was voltage-clamped under K+
current suppression conditions. Ciliary
beating was registered using high-speed video microscopy. Depolarizing step
pulses activated a transient inward current and induced reversed ciliary
beating. Very strong positive steps inhibited ciliary reversal during the pulse
suggesting inhibition of the Ca2+ influx. We call the potential,
which is sufficiently positive to induce transition from reversed to normal
ciliary beating, the transition potential. The transition potential rose with
increasing external Ca2+ showing saturation beyond 1 mM Ca2+. Addition of Mg2+, Ba2+
or K+ to the 1 mM CaCl2 bathing solution depressed the
transition potential in a concentration-dependent manner. The depolarization-activated
inward Ca current increased with rising external Ca2+ concentration,
and addition of either Mg2+, Ba2+ or K+
diminished the inward Ca2+ current.
The diverging results of Ca2+-dependent
positive shifts, and Mg2+- (Ba2+-, K+-)
dependent negative shifts in transition potential are compared with similar
shifts of VImax. It is concluded
that external cations bind competitively - in addition to membrane surface
charges - to external charged sites of the ciliary Ca channel, where they
specifically modulate permeation of calcium.
89
Velocity and graviresponses in Paramecium during adaptation and
varied oxygen concentration.
Populations of Paramecium caudatum
adapted to new O2- 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 O2 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 O2 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 O2 tension. No
correlation was found between the kinesis and orientation coefficients. The data suggest that gravikinesis in Paramecium is largely independent of O2
levels and adaptation time.
Graviorientation declines with adaptation, but is not a direct function
of O2 saturation of the medium.
88
Gravireception and graviresponses in ciliates.
An account is given of approaches to gravireception, terminology,
mechanisms of responses to gravity as investigated and documented in the
literature, and sensori-motor coupling properties in ciliates. Current theories and methods are discussed,
and previously published experimental data on graviresponses are reviewed.
Contents
1. Introduction
2. Approaches to Gravireception
2.1 Energetic
Considerations
2.2 Structures
Suitable for Gravireception
2.3 Behavioural
Analysis
3. Terminology
4. Mechanisms in Graviresponses
4.1 Static
Hypothesis (Gravity-Buoyancy Model)
4.2 Hydrostatic
Hypothesis
4.3 General
Statocyst Hypothesis
4.4 Resistance
Hypothesis
4.5 Hydrodynamic
Hypothesis
4.6 Propulsion
Hypothesis
4.7 Lifting
Force Hypothesis
4.8 Special
Statocyst Hypothesis (Electrophysiological Model)
4.9 Conclusions
Regarding Various Hypotheses on Graviresponses
5. Ciliates as Excitable Cells
6. Mechanoreception in Ciliates
7. Electromotor Coupling
8. Physiology of
Gravistimulation and Response
8.1 Basic
Mechanisms
8.1.1 Gravikinesis
8.1.2 Gravitaxis
8.2 Implications
of the Electrophysiological Model
8.2.1 Masking of the Graviresponse
8.2.2 Effects of Cell Orientation
8.3 Circumstantial
Experimental Conditions
8.3.1 Cell Cultures and Experimental Solutions
8.3.2 Equilibration Time
8.3.3 Proportions of Chamber
8.3.4 Mechanical Disturbances
8.3.5 Temperature
8.3.6 Illumination
8.3.7 Aeration
8.3.8 Determination of Sedimentation Rates
8.3.9 Acquisition of Data
8.3.10 Processing and Presentation of Data
8.3.10.1 Behavioural Variety
8.3.10.2 Statistical Treatment
8.3.10.3 Circular Histograms
8.3.10.4 Direction Coefficient (r-value)
8.3.10.5 Orientation Coefficient
8.3.10.6 Taxis Coefficient
8.3.10.7 Kinesis Coefficient
8.4 Current
Experiments
8.4.1 Experiments under Normal Gravity
8.4.1.1 Free Locomotion
8.4.1.2 Velocity-dependence of Gravikinesis?
8.4.1.3 Cells under Galvanotactic Alignment
8.4.1.4 Swimming in Solutions of Adjusted Density
8.4.1.5 Nonswimming Locomotion
8.4.2 Microgravity Experiments
8.4.3 Hypergravity Experiments
9. Conclusion
and Perspective.
87
Real-time 3D-tracking of fast moving microscopic objects.
Anaxial illumination has been used so far for either enhancement of visual acuity,
or three-dimensional (3-D) measurements of static objects. Here we report the
use of anaxial illumination for 3-D tracking of fast-moving objects, with only
slight modifications to the standard microscope. To achieve this, we have investigated high-speed
recording of stereoscopic images in two different set-ups.
(1) Alternating-beam illumination; alternating left and right offset
illumination by a vibrating mirror, synchronized with the video sync, so that
alternating fields of left and right partial images result.
(2) Split-screen recording of simultaneously illuminated left and right
partial images, separated with the use of polarizing filters.
To reconstruct the motion of an object through space maximum depth
resolution is required; at the same time high recording speed is desirable to
enable recording of rapid movements.
Compared with split-screen recording, alternating-beam illumination
provides better spatial resolution, and a brighter image, but only at the
expense of speed and depth resolution.
For measuring and reconstruction purposes the split-screen method is
better.
Excellent depth resolution (>23° angular disparity in our
electrophysiological set-up), at full recording speed (200 fields/s in our
set-up), can be acquired with two small circular apertures at extreme lateral
positions.
These methods allow us to observe cells in a standard electrophysiological
set-up, under voltage clamp conditions, for maximum reproducibility of
voltage-dependent motor responses of cellular organelles, such as cilia.
86
Short-term microgravity to isolate graviperception in cells.
In the fall of 1991 a series of drop-tower experiments in Zarm (Bremen) was devoted to
behavioural responses of unicellular organisms to step-type transition from
normal gravity to microgravity. Modules
for simultaneous 4-fold video-recording were incorporated into the flight
capsule. In the course of 25 flights, 100 sets of experiments, each holding 100
to 200 cells, were flown under various conditions with a technical success rate
of 94% and about 80% of the cells accessible to evaluation in the
laboratory. A major goal of the
experiments was the assessment of parameters of locomotion (velocity,
orientation) in the absence of the gravity vector. The data show that in two species,
Paramecium and Loxodes, the
properties of steady-state µg-swimming correspond to horizontal swimming under
1g-conditions. In a third species, Didinium,
µg-swimming velocity exceeds 1g-horizontal
rates. The data are in agreement with an
electrophysiological hypothesis of graviperception in cells.
85
Neutral gravitaxis of gliding Loxodes exposed to normal and raised
gravity.
Vertical gliding of Loxodes was investigated at g-values
between 1 g and 5.4 g in a centrifuge microscope. Videorecordings of a large number of a large
cell population were processed with an automated analysis procedure. At 1 g,
sedimentation was fully compensated by a gravikinetic response, and vertical
gain was neutralized because upward and downward orientations of the cells
occur at equal proportions (neutral gravitaxis). With rising gravity, the
upward gravikinetic response increased in proportion to the sedimentation rate,
whereas the downward gravikinetic response increasingly trailed behind
sedimentation. Long-term exposure to hypergravity did not generate a
perceptible adaptive response. An
increase in gravity shifted the bipolar orientation diagram of gliding Loxodes
in the counterclockwise direction. The data suggest that both gravikinesis and
graviorientation fully neutralize sedimentation so that the cell is in a
favourable condition for sensing and response to chemical gradients.
84
Graviperception in unicellular organisms: a comparative behavioural study
under short-term microgravity.
Three species of unicellular ciliated organisms, Paramecium, Didinium and Loxodes,
were adjusted to defined culturing state, experimental solution, O2-supply
and temperature and subjected to step-type transition from terrestrial gravity to
4.5 seconds of microgravity (near 10-4 g) in a drop tower.
For a quantitative approach to cellular behaviour under microgravity, four
identical modules designed for video-tape recording of cellular locomotion
(velocity, orientation) were incubated at one time in the drop capsule, and
each module held an experimental chamber including 100 to 200 cells. Image analysis of the data shows that the
normal orientational bias of vertically swimming cells of Paramecium and
Didinium (negative gravitaxis) was
missing under microgravity; so were gravity-induced changes in velocity of
these cells (gravikinesis), which tend to compensate passive sedimentation
rates at 1g. The rates of µg-locomotion in Paramecium and Loxodes correspond to horizontal
locomotion at 1g. In Didinium,
swimming at µg exceeds horizontal swimming at 1g. These data are
interpreted in terms of the mechanosensory organization of these cells and of a
previously published model of electrophysiologically regulated gravisensory
transduction.
83
Photobehaviour of Fabrea salina: responses to directional and
diffused gradient-type illumination.
Under the light intensities and gradients used, swimming specimens of Fabrea salina respond to light direction
with a mechanism that is unrelated to sensation of a gradient. The results obtained with bilateral and
unilateral illumination suggest that Fabrea salina cells can detect and
track a light direction. The biased
random-walk hypothesis proposed in a previous work (Colombetti et al., 1992) can explain these results assuming the
existence of a direction-sensitive light detector in Fabrea.
82
Phototaxis in Fabrea salina.
Some coloured ciliated protozoa (Stentor, Blepharisma) exhibit a change
in their swimming pattern upon light Stimulation. The action spectra of these
light responses are very similar to the absorption spectra of their endogenous
pigments. We have, therefore, investigated the photomotile reactions of the
coloured salt-marsh dweller heterotrich ciliate Fabrea salina Henneguy. Fabrea apparently respond to light
showing both phobic and tactic reactions.
In particular, the cells show a clear step-down response at light levels
of the order of 1017 to 1019 photons/m2s in
the visible range (400-700 nm). Under unilateral illumination, Fabrea swim
following light direction (positive phototaxis), with the directness of their
path strongly depending on light intensity and wavelength. Phototaxis in Fabrea can be simulated
by a biased random walk mechanism.
81
Gravity-controlled gliding velocity in Loxodes.
1. The locomotion of Loxodes as
controlled by the natural gravity vector was investigated employing a mass-cell
approach. Samples of cells were incubated for 4 hours in a 1.6 mm deep well (41
mm x 85mm) filled with defined experimental solution. Their gliding locomotion
on surfaces inclined between 0° and 90° was recorded by video camera. Steady gliding rates (median of total: 206
µm/s; n = 12.711) were evaluated by calculating the vertical component from
observed tracks. At a given inclination the rates of downward and upward
gliding were similar. The sedimentation
rate of freely suspended nickel-immobilized specimens (S = 49 µm/s), and
vertical rates of displacement at 6 differently inclined planes, and with cells
in "sitting" posture as well as "hanging" (upside down),
were used to determine the gravity-dependent component (∆ = -49
µm/s) during gliding motion. Numerical
equality of the gravity force to produce S and of the counterforce to produce ∆
follows from the observed constancy of gliding rates and identical medians of
vertical locomotion up and down (169 µm/s).
It is concluded that neutralization of sedimentation is a precondition
for Loxodes to migrate along a vertical O2 gradient in its freshwater
habitat.
80
Physiological basis of gravitaxis
in protozoa.
The topographical organization of mechanosensitivity in ciliates is
presumably instrumental in gravisensation.
Because modulations of ciliary activity and the rate of locomotion in
ciliates reflect shifts in membrane potential, analysis of the direction and
rate of steady locomotion may reveal a physiological response of the cell to
the gravity vector. This report summarizes a theoretical approach to, and
initial experimental results on, gravisensation in two ciliates, Paramecium
and Loxodes.
79
A novel type of ciliary activity in Stylonychia: Potential-coupled
motor responses in the transverse cirri.
The motor responses of the transverse cirri of Stylonychia mytilus were
investigated by applying high-speed microcinematography and step voltage
-clamp. As a response to hyperpolarization, the transverse cirri began to swing
posteriorly from an inactive upright posture at rest. The deeply inclined
posture was maintained as long as the hyperpolarizing
pulse was on. Upon depolarization, the cirri began to swing towards the
anterior end of the cell and continued regular cyclic beating, orienting
the effective stroke anteriorly. Motor responses of the transverse cirri
occurred in quasi-planar motion, allowing analysis of the bend configuration
along the full length of the cirri. Beating activity induced during sustained depolarization was virtually stable,
with different oscillation profiles at base and tip. Cyclic movement of
the distal region was enhanced at large amplitudes of depolarization. Termination of a hyperpolarizing voltage step
induced a transient depolarization-type anterior beating, and termination of a depolarizing step induced a transient posteriad
inclination of the transverse cirri. In both hyperpolarization- and
depolarization-induced motor responses, a shear angle analysis of the initiation of the response indicated that sliding
displacement of doublet microtubules
was initiated at the base and propagated towards the tip. The discovery in a ciliary organelle of a very
distinct response to hyperpolarizing and
depolarizing stimulation is highly useful for the analysis of ciliary
electromotor coupling. The
functions of intraciliary Ca2+ in the regulation of the motor responses are discussed.
78
In-vivo activation of cirral movement in Stylonychia by calcium.
Motor responses of cirri (= organelles consisting of bundles of cilia) in the
protozoan Stylonychia, are
elicited by positive or negative shifts of the membrane voltage from its
resting state. The same responses are
evoked at voltages near the Ca2+ equilibrium potential (ECa)
applying extremely positive steps under voltage-clamp. Motor responses recorded
at large positive voltages approaching ECa from the negative side corresponded
to cirral activation following physiological depolarization from the resting
potential (DCA). The hyperpolarization-induced activation of the cirri (HCA)
was documented during step potentials positive to ECa suggesting
that the observed HCA of the cirri resulted from an efflux of Ca2+
from the ciliary space, as compared to DCA being related to Ca2+
influx. The ciliary responses were
graded functions of the rising outward or inward driving force for Ca2+. Slopes of reciprocal plots of response
latencies near ECa as a function of membrane potential indicate a
removal of Ca2+ during HCA which exceeds the free intraciliary Ca2+
content at rest. It is suggested that this excess Ca2+ is released
from axonemal binding sites.
77
Sensorimotor coupling and motor responses. In (Hausmann K, Bradbury PC)
Ciliates: Cells as Organisms, p 379-402.
Introduction. The movements of ciliate organisms seen under the microscope
have amazed generations of nature's admirers and workers in science. A basic
question is how single cells can behave like highly complex multicellular
organisms, animals possessing sensory organs, brains and sophisticated motor
systems. Because the world of eucaryotic microbes is small, often invisible to
the unaided eye, and has been inaccessible to scientific analysis for a long
time, we have grown accustomed to judging ciliates from the perspective of the
metazoon. "Swimming sensory cell" (Machemer and de Peyer 1977) or
"swimming neuron" (Naitoh 1982) are terms born out of this point of
view. As modern tools in cell biology and physiology increasingly expand
spatial and temporal dimensions for exploration, ciliates are now viewed
differently: cells of unprecedented universality, evolved at an early
eucaryotic level to set the standards for most phylogenetically later cellular
achievements. Thus, ciliates, being organisms, are sensitive to stimuli,
transduce these to cellular signals, amplify, integrate, accumulate or
"forget", and transport the intracellular message and ultimately
generate various responses in a unique manner. The present essay intends to
concentrate on principles of ciliate sensory-motor organization using examples
rather than offering a comprehensive description. In-depth reviews on stimulus
reception and/or motor control in ciliates have been published elsewhere
(Machemer and Deitmer 1985; Machemer 1986, 1988a,b, 1989, 1990; Hennessey 1990;
Preston and Saimi 1990; Van Houten 1992, 1994).
Contents:
1 Introduction
2 Structural concepts
3 Functional organization
4 Resting membrane and excitation
5 Responses to stimuli
5.1 Transduction sites
5.2 Electric correlates of transduction
5.3 Stimulus integration
5.4 Action potentials
6 The cilium: elementary unit of motor response
6.1 Ciliary steady-state
6.2
Ciliary stimulus-response coupling
6.3 Intraciliary signalling
7 Time-dependent changes in responsiveness
7.1 Adaptation
7.2 Sensitization - desensitization
7.3 Habituation
8 Stimuli, cell migration and orientation
8.1 Nature of stimuli
8.2 Classes of responses
8.3 Graviresponses
8.4 Photoresponses
8.5 Thermoresponses
8.6 Chemoresponses
9 Conclusion and Perspective.
76
Ciliary beating in three dimensions: steps of a quantitative description.
We document a novel approach for quantitative assessment of ciliary
activity, exemplified in rapid three-dimensional cyclic motion of the frontal
cirri of Stylonychia. Cells held
under voltage-clamp control are stimulated by step pulses to elicit
reproducible hyperpolarization- or depolarization-induced ciliary motor
responses. High-speed video recording at 200 fields per second is used for
imaging ciliary organelles of the same cell in two perspectives: the axial view
and, following cell rotation by 90°, the lateral view. From video sequences of
typically 1s duration, the contours of the cirral images are determined and
digitized. Computer programs are established to (1) reduce an observed image to
a "ciliary axis", (2) sort series of axes by template to generate an
averaged ciliary cycle in 2D-projection, and (3) to associate the generalized
axial and lateral 2D-images for generation of a sequence of three-dimensional
images, which quantitatively represent the cycle in space and time. The method allows us to produce predetermined
perspectives of images selected from the ciliary cycle, and to generate
stereo-views for graphical representation of ciliary motion. The approach includes a potential for
extraction of the complete microtubular sliding program of a cilium under reproducible
electric stimulation of the ciliary membrane.
75
Messenger role of calcium in ciliary electromotor coupling: a reassessment.
Electrophysiological and cell reactivation studies in Paramecium and other ciliates have established that depolarizing
stimulation opens voltage-sensitive ciliary Ca channels leading to an elevation
in intraciliary Ca2+, a rapid "reversal" in
sliding-microtubule based ciliary activity and backward swimming. Regulation of cilia by hyperpolarization
modulates the pitch and rate of forward locomotion. The control of this predominant behaviour has
been a matter of controversy because ciliary conductances do not change with
negative shifts from the resting potential. Recordings of ciliary responses
during electrophysiological manipulation of the Ca driving force in the
ciliates Stylonychia and Didinium
now suggests that a crucial step in hyperpolarization-induced ciliary
activation (HCA) is a reduction in intraciliary Ca2+ from a resting
steady-state level. The data are
discussed with respect to previous hypotheses of the regulation of HCA.
74
Gravikinesis in Paramecium: Theory and isolation of a physiological
response to the natural gravity vector.
1. 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 (∆), 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 ∆. ∆ is supposed to be the arithmetic
mean of two subcomponents, ∆a and ∆p,
from gravistimulation of the anterior and posterior cell ends,
respectively.
4. A negative value of ∆ (0.038 mm/s) was isolated with ∆a
(0.070 mm/s) subtracting from the downward swimming rate, and ∆p
(0.005 mm/s) adding to rate of 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 ∆a and ∆p
antagonize sedimentation.
73
Depolarization-controlled parameters of the ciliary cycle and axonemal
function. Depolarization-induced cycles of a frontal cirrus of Stylonychia
were investigated applying methods of axial-view analysis of the cilia,
high-speed microcinématography, and step voltage-clamp. Rising depolarization
(from 3 mV to >30 mV) increased the rate of beating from zero to maximally
58 Hz. During cyclic activity, the axis of the beat cone of a proximal segment
of the cirrus was inclined by 60° (0° = perpendicular to cell surface), and was
always oriented 90° counterclockwise to the power stroke. With the stimulus
amplitude rising, the orientations of the power stroke and inclination were
increasingly shifted in more counterclockwise directions by up to 80°. After
correction for inclination (= normalization), and following planification of
the track of the segment, we determined the following properties of the cycle
during depolarization: The course of the cycle tended to be rounded, i.e., the
ratio of major over minor amplitudes (= spatial polarity) approximated a value
of 1.6 which is only two thirds of maximal spatial polarity observed during
hyperpolarization. The angular velocity generally increased with rising steps
of depolarization; up to +5 mV (and comparable to hyperpolarization-induced
responses), the velocity maximum occurred during the return stroke. With
depolarizations >7 mV the angular velocity maximum shifted to the power
stroke so that the temporal polarity (rates of power stroke over rates of
return stroke) increased from 0.4 to 1.6. Calculations of the angular velocity
as referred to the proximal ciliary segment level suggest active sliding rates
(between 5 and 30 nm/ms) of identified pairs of doublet microtubules. Ciliary
frequency is directly proportional to the rate of reorientation of the cyclic
track; this parameter, which corresponds to the translocation rate of active
sliding between pairs of doublets, grew with the amplitude of depolarization.
Translocation rates were high during transitions between the beat phases (power
stroke, return stroke), and were reduced during these phases. Orientational
polarograms of the mean rates of both active sliding and sliding translocation
show properties of discreteness as well as continuity. The
depolarization-induced changes in inclination, and the inferred patterns of
sliding rate and sliding translocation rate, are compared with previous results
from hyperpolarization-dependent activation of the same motor organelle.
72
Cilia in cell motility: membrane-controlled rotary engines.
Since the late fifties, when the sliding microtubule hypothesis began to
successfully explain the principle of ciliary motion [1-4], our understanding
of the design of the 9+2 machine has increased due to a variety of techniques
which became available to the experimenter, among the electrophysiology and
high-speed motion analysis. In this chapter, five topics will be discussed: the
role of cilia in general, their control by the membrane potential, the types of
ciliary activities and the messenger role of ionic calcium, properties of the
cycle and, in conclusion, an approach to isolate functional elements of the
cycle.
[1] Afzelius, B. (1959) J.Biophys.Biochem.Cytol.5, 269-278. [2] Brokaw, C. (1959)
J.Exp.Biol.43, 155-169. [3] Gibbons,
I.R. & Rowe, A.J. (1965) Science 149, 424-426. [4] Satir, P. (1965) J.Cell Biol. 26,
805-834.
71
Role of cell membranes and cytoskeletal organization in cell motility.
The symposium was divided into two subsessions
based on the topics of the papers. The first three papers are concerned with
ciliary movement, and the last three papers with non-ciliary movement. Ciliate
protozoans, such as Paramecium, Tetrahymena and many others provide model
systems excellent for the studies on the mechanism by which their ciliary
activity is controlled at various different structural hierarchy levels. The first paper by Machemer and
Machemer-Röhnisch dealt with the control of a whole cilium of Paramecium by
its membrane electrogenesis (cellular level), the second paper by Hamasaki,
Barkalow and Satir in vitro phosphorylation of Paramecium
axonemes with endogenous protein kinase in relation to ciliary activity
(organelle level), and the third paper by Shimizu, Furusawa, Ohashi, Okuno and
Toyoshima dealt with molecular mechanisms of ATP-energization in ciliary
movement of Tetrahymena (molecular
level). The fourth paper by Hauser dealt
with control of the stability of microtubules in tyronisation of tubulin in
relation to cytoplasmic streaming and to pseudopod formation in Reticulomyxa, the fifth paper by Butler,
Evans and McCrohan described control of the tentacle contraction by
intracellularly released Ca2+ ions in suctorians such as Trichophrya and Heliophrya, and the last paper by Naitoh and Oami dealt with
bioelectric control of the tentacle movement by the Ca2+-mediated
intracellular release of H+ ions in a marine dinoflagellate Noctiluca.
70
Cilia and flagella (Overview).
Principles of the ciliary sliding machine have been unravelling during the
last 30 years, but the detailed motor functions and controls continue to be
difficult to isolate for several reasons: 1. The rapid
response of the diminutive axoneme (200nm in diameter) is modulated temporally
as well as spatially. 2. Recording of ciliary beating meets technical and interpretation
problems (framing rates, illumination, two-dimensionality of image). 3. Because
external stimuli can alter the ciliary motor response via modulation of
the Ca membrane conductance and intraciliary ionic composition, experiments
designed to characterize the intrinsic signalling chain include
electrophysiological controls of the live cell, or monitoring of the components
of aqueous solutions applied to demembranated ciliary models. A synopsis of geometry, mechanisms and
controls of ciliary motion suggests that the axonemal molecular architecture
incorporates crucial elements of function in a unique structural framework. In
view of this central result of ciliary physiology and the existence of
spatially and temporally diverse motion in cilia and flagella, axonemal
chemo-mechanical transduction promises to reveal an unforeseen topological
diversity.
69
Ca-Mg control of ciliary motion: a quantitative model study.
We have developed a generalized model for Ca-mediated control of ciliary
beating using established data from the literature. According to the model, both direction and
frequency of beating are controlled by membrane-regulated Ca2+ as the intraciliary messenger including a
modulatory function of Mg2+ which competes with Ca2+ for
binding to an axonemal protein.
68
Bioelectric control of the ciliary cycle.
Electrophysiological research in ciliates has
established the central role of ionic Ca2+ in membrane excitation
and ciliary electromotor coupling. Ca2+ passes
depolarization-sensitive ciliary channels and is thought to bind to axonemal
proteins. During hyperpolarization, the concentration of axonemally bound Ca2+
is presumably reduced. The ciliary motor response - frequency and beat
direction - is a monotonous function of the intensity of a stimulus impinging
on the cell. Intermediate steps in sensory-motor coupling: potentials of either
polarity, concentration of the messenger substance Ca2+, and the
binding of Ca2+ to axonemal target proteins reflect the transmission
of gradedness to the ciliary motor response.
67
Effects of cyclic nucleotides and intracellular Ca2+ on
voltage-activated ciliary beating in Paramecium.
1. Coupling mechanisms between ciliary beat and the membrane potential in Paramecium
were investigated under voltage clamp applying intracellular pressure
injection of cAMP, cGMP and Ca-EGTA buffer. Ciliary responses following step
changes in membrane potential were recorded by high-speed video on magnetic
tape.
2. Injections of cAMP and cGMP up to millimolar concentrations caused no
detectable changes in the frequency-voltage relationship. A minor effect was
that the reorientation towards the anterior cell end (reversal) tended to be
inhibited with depolarization up to 10mV.
3. Injection of Ca2+ into the cell clamped at the resting potential
caused a transient anteriad ciliary reorientation and a simultaneous increase
in the beating frequency.
4. Injection of EGTA (to buffer Ca2+ below 10-8 M) was
ineffective in relation to frequency for several minutes. After this time,
hyperpolarization- and depolarization-activated frequency responses of
EGTA-injected cells were increasingly inhibited. The reorientation following
depolarization was not affected by EGTA.
5. A posterior contraction of the cell diameter was noticed upon membrane
hyperpolarization. The contraction coincided in time with the increase in
beating frequency.
6. The results support the view that the voltage-dependent augmentation of the
ciliary beating rate is not directly mediated by an intracellular increase in
either cAMP or cGMP.
7. The role of Ca2+ as intracellular messenger in the ciliary and
somatic compartments is discussed.
66
Cellular behaviour modulated by ions: electrophysiological implications.
This essay considers the responses of Paramecium and other ciliates
to the inorganic ion environment from an electrophysiological point of view. In
reviewing data from published and unpublished sources it is shown that ions
affect the cellular behaviour in multiple ways because the transmembrane
potential can change due to the alteration of equilibrium potentials, ion
conductances and surface charges of the membrane. Sensory input including
effects from the ionic environment converge upon the membrane potential which
has a temporal and spatial summing function.
Hyperpolarizing and depolarizing potential shifts from the set point are
near-simultaneously and omnidirectionally transmitted along the membrane
including the ciliary boundaries. The membrane potential regulates ciliary
motility via an intraciliary messenger, Ca2+, which can enter, and
presumably leave, the cytosol directly adjacent to the ciliary motor.
Integration of the responses of thousands of cilia occurs in accordance with
the electrical and structural provisions of the cell. Potential-regulated motor
and behavioural responses attenuate with time. This phenomenon, which has been
loosely termed adaptation, has an electrophysiological basis in analogy to
membrane accomrnodation following sustained stimulus input. The mechanisms of
adaptation serve to restore, in principle, the membrane resting state and,
thereby, the sensitivity to depolarizing and hyperpolarizing shifts of the
membrane potential and the cell's responsiveness to environmental stimuli,
respectively. For the inorganic ions involved in chemosensation the terms attractant
and repellent are not applicable.
They should be reserved to signalling substances which per se can define
the behaviour of the cell.
CONTENTS.
1. Introduction
2. Behaviour of Cells in Equilibrium
2.1 Media
2.2 Normal Behaviour
2.2.1 Forward swimming
2.2.2 Reversals
2.3 Effects of Swimming
and Turning on Cell Distribution
3. Electrophysiology at steady ionic environments
3.1 Definitions
3.2 Membrane-associated
Potentials
3.2.1 Ions and bulk-phase potential
3.2.2 Ions and transmembrane potential
3.3 Fluctuations of the
Membrane Potential
3.4 Repetitive
Excitation
4. Membrane
Control of Motility
4.1 Three Components of
Ciliary Motor Response
4.2 Hyperpolarization-induced
Ciliary Activity (HCA)
4.3 Depolarization-induced
Ciliary Activity (DCA)
5. Changing
the ionic Environment
5.1 Methodical
considerations
5.1.1 State of
5.1.2 Mechanical Disturbance
5.1.3 Potential Artefacts
5.1.4 Effects of pH
5.2 Potassium Changes
5.3 Calcium Changes
5.4 Ion Competition,
Behaviour and Membrane Properties
6. Behavioural
Adaptation
6.1 Definition
6.2 Phenomena
6.3 Electrophysiological
Correlates
6.4 Mechanisms
6.4.1 Adaptation following DCA
6.4.2 Adaptation following HCA
6.4.3 Bipolarity of adaptation
6.4.4 I/V shift
7. chemical
Gradients and Behaviour
7.1 Jennings Chemical Trap
7.2 T-Maze Experiments
7.3 Mechanisms
7.3.1 Behaviour in a chemical trap
7.3.2 Behaviour in a hyperpolarizing gradient
7.3.3 Behaviour in a depolarizing gradient
8. Perspective.
65
A Ca paradox: electric and behavioural responses of Paramecium to changes
in cation concentration of the medium.
1. Raising [Ca2+]o depolarized the membrane and caused
augmented forward swimming which is characteristic of hyperpolarizing
stimulation.
2. Lowering [Ca2+]o hyperpolarized the membrane and
induced backward swimming or reduction in forward swimming; these motor
responses are known to follow depolarizing stimulation.
3. The apparent inconsistencies in responses ("Ca paradox") were
suspended when changes in Ca2+ concentration had been compensated by
equivalent amounts of Mg2+.
4. Mechanical disturbance of the cells during solution transfer affected
the early electrical and behavioural responses of Paramecium for up to
5min.
5. External Ca2+ modulated the rates of forward swimming, but
not the frequency rates of reversals. Swimming was elevated in high-Ca
solutions; peak rates of reversals occurred in solutions of high Mg2+
plus low Ca2+.
6. The analysis of the data suggests that ionically-induced changes of
observed membrane potentials do not always reflect changes of the transmembrane
potential which controls the behaviour via modulation of
voltage-sensitive membrane channels.
7. The data are discussed using a model which resolves the Ca paradox by
incorporation of three instantaneous effects on the membrane potential of [Ca2+]o
(and other divalent cations): (1) external surface charge neutralization, (2)
shifts in equilibrium potential, and (3) changes in ion conductance.
8. Three observed parameters: Ca-dependent
shifts in current-voltage relation, changes in input resistance, and
behavioural adaptation suggest that the transmembrane potential accommodates
(i.e. returns to rest in a time-dependent manner).
64
Electrophysiological control of ciliary beating: A basis of motile behaviour
in ciliated protozoa.
1. The motor behaviour of viable ciliates is regulated by depolarizing and
hyperpolarizing shifts from the membrane resting potential.
2. Modulations of the velocity and form of the helical swimming tracks in Paramecium reflect changes in
transmembrane potential. These patterns may be used for approximations of the
electrophysiological state of the membrane.
3. Continuous transitions exist between ciliary responses elicited by
membrane potentials from hyperpolarization to depolarization.
4. Ciliary responses in Paramecium are thought to be regulated by a
comparatively narrow range of intraciliary Ca concentrations serving as
intracellular messenger.
5. In Stylonychia a voltage-dependent static ciliary
response, a proximal bend, is separable from the voltage-dependent cyclic response of the cilium.
6. Cyclic ciliary responses, depolarization-induced or
hyperpolarization-induced, are superimposed by a component of non-cyclic
proximal bending.
63
Depolarization-induced membrane current components in Didinium.
1. Properties of the membrane currents of Didinium nasutum have been investigated under voltage-clamp in
different solutions and after deciliation.
2. The early transient Ca2+ inward current activates in a voltage-dependent
manner. Inactivation is both Ca2+-dependent and voltage-dependent.
3. A late Ca2+ current rises with time-to-peak of >50ms and
decays in the order of seconds.
4. Activation and inactivation of the late Ca2+ current is
voltage-dependent.
5. The delayed outward current is activated by voltage. The kinetics of this K+ current,
but not its amplitude, are enhanced in the presence of intracellular EGTA.
6. The two voltage-dependent Ca2+
channels are located in the cilia, whereas all K+ channels are
restricted to the somatic membrane.
62
Das Experiment: Können Einzeller lernen? Prüfung am Habituationsexperiment.
(Do unicells learn? A test applying habituation experiments)
The capacity for habituation of stimuli is important for the survival of
organisms so that habituation appears to have been adopted at an early level of
evolution. In this paper the question is raised and experimentally tested,
whether or not unicellular organisms can habituate. A positive answer leads to
the conclusion that a simple form of learning, such as habituation, is not
necessarily associated with nervous systems, which do not exist in unicellular
eukaryotes. At the same time, evidence for higher forms of learning, such as
the association of two stimuli, has failed to be established experimentally in
unicellular organisms.
61
The ciliary cycle during hyperpolarization-induced activity: an analysis of
axonemal functional parameters.
Motor responses of the frontal cirri of the ciliate Stylonychia were
recorded at the axial view of the ciliary base with high-speed
cinematography. Voltage-clamp applying
sustained hyperpolarizing voltage steps was used to explore the properties of
the ciliary cycle modulated by the membrane potential. Upon hyperpolarization
between -1 and -13mV, a previously inactive frontal cirrus reoriented from a
neutral posture and started beating so that the axis of the beating cone of a
proximal cirral segment assumed an orientation near 100° (proceeding
counterclockwise from posterior = 0°) and inclination near 60° (0° =
perpendicular to the cell surface). The major beating amplitude was limited to
about 150°. Increasing hyperpolarization increased the spatial polarity
of the cycle (ratio of major over minor amplitude, from 2 to 2.4). Rates of the
power stroke increased with hyperpolarizations up to -4mV but were consistently
smaller than those of the return stroke during the ciliary cycle (ratio: 0.4 to
0.6; = temporal polarity). Comparison of different hypothetical beat
forms (O-shape, D-shape, and egg-shape) showed that the orientation-time data
are the major determinants of the angular velocity and rate of reorientation of
the cilium during the cycle. Geometric
transformation of these data led to descriptions of the cycle of a proximal
ciliary segment in terms of active sliding velocities and rates of
unidirectional sliding translocation between identified doublets. Three
voltage-sensitive functional parameters of the cilium - the inclination (which
is non-cyclic) and the rates of active sliding and sliding
translocation (both of which are cyclic in nature) - are discussed as
generating the spatial and temporal properties of the ciliary beat.