THE IONIC BASIS OF THE PHOTOSENSORY TRANSDUCTION
IN STENTOR COERULEUS
by
TAEK-HYOUN CHANG, B. S.
A THESIS
IN
CHEMISTRY
Submitted to the Graduate Faculty
of Texas Tech University in
Partial Fulfillment of
the Requirements for
the Degree of
MASTER OF SCIENCE
Approved
Accepted
-.OP'
-<>'
ACKNOWLEDGMENTS
I am deeply indebted to Professor Pill-Soon Song for
his direction of this thesis and to the other members of
my committee, Professors John A. Anderson and Jerry Mills,
for their helpful criticism.
I also greatly appreciate the financial support received
from Texas Tech University, the National Institute of Health
and the Robert A, Welch Foundation.
11
TABLE OF CONTENTS
ACKNOWLEDGMENTS
ii
LIST OF FIGURES
iv
I.
II.
INTRODUCTION AND STATEMENT OF PURPOSE
1
MATERIALS AND METHODS
6
Materials
6
Chemicals
6
Stentor Culture
6
Methods
8
Negative Phototaxis of Stentor coeruleus
Electron Microscopy
III.
8
11
RESULTS
13
Photoresponse of Stentor coeruleus
13
Effects of Protonophores on Photoresponse
16
Effects of Ca
Ca
-blocking agents and
-ionophores on Photoresponse . .
l6
Effects of other Ionophores on Photoresponse .. 37
Electron Microscopy
IV.
V.
37
DISCUSSION
^2
CONCLUSIONS
^6
LIST OF REFERENCES
^7
111
LIST OF FIGURES
1.
Step-up photophobic response of Stentor coeruleus ....
2.
Diagram of partitioned photomovement channel
3.
Time-dependent
^.
Effect of FCCP on the phototaxis of
Stentor coeruleus
18
Effect of CCCP on the phototaxis of
Stentor coeruleus
20
Effect of SF-86^7 on the phototaxis of
Stentor coeruleus
22
Effect of nigericin on the phototaxis of
Stentor coeruleus
2^
Effect of A23187 on the phototaxis of
Stentor coeruleus
26
Effect of Ruthenium Red on the phototaxis
of Stentor coeruleus
28
Effect of Ln^ on the phototaxis of
Stentor coeruleus
30
Effect of 3,4-diaminopyridine on the phototaxis
of Stentor coeruleus
32
Effect of valinomycin on the phototaxis
of Stentor coeruleus
3^
Effect of ouabain on the phototaxis of
Stentor coeruleus
36
Electron micrograph of the frontal field and
the basal body of Stentor coeruleus
39
Electron micrograph of the pigment granule
of Stentor coeruleus
^1
Relative ATP concentrations in CPM of Stentor
coeruleus as a function of illumination
^5
5.
6.
7.
8.
9.
10.
11.
12.
13.
1^-.
15.
16,
3
10
photoresponse of Stentor coeruleus ... 15
IV
CHAPTER I
INTRODUCTION AND STATEMENT OF PURPOSE
The heterotrich Stentor coeruleus is a large blue-green
ciliate of the class of Polymenophora and order of Hymenostomanita (1). One of the most interesting- aspects of Stentor
coeruleus is its light avoiding behavior.
This organism
swims away from the light source, when subjected to the
external light stimulus.
This light avoiding behavior of
Stentor coeruleus is caused by a step-up photophobic response
as well as a negative phototaxis (2, 3 ) . Stentor coeruleus
provides an ideal case for the study of photoreception in
protozoa, as it exhibits well defined photophobic and negative phototactic responses (Figure 1).
Although the biology of Stentor coeruleus has been well
described (4) and the action spectra for the light-induced
ciliary reversal and photophobic responses have been determined, the molecular mechanism of Stentor's ability to
respond to changes in light intensity and to reorient itself
away from the light stimulus have not been studied until
recently (2-6).
These responses are mediated by a photoreceptor pigment
which has an absorption maximum at 6lO nm.
The photoreceptor
pigment is enclosed in pigment granules, which are in ordered
rows near the cilia.
Each of the pigment granules is sur-
rouned by a membrane and appears to be held tightly against
1
Figure 1.
The step-up photophobic response of Stentor
coeruleus, as it encounters the lighted area (7^0 W/m ).
The position of the cells has been traced every 1/6 second
from a video recording (ref. 2).
•'m
•;';•/.
• ' r \ ' / \ . ' ' -
,v.6r,Vv.
. • ; ' • " - ' • / ^
.• . . . ' j . ; • • s . : .
•^o'-r;'
v'
'••.?•
.V.^y-y'T^.J.-';
'-'^y-•!:-••••:'••\
.
'
•
' . ' • .•
•.•V.^.
, '••
• •
'
the inside of the cytoplasmic membrane, near the surface of
the organism (7. 8, 9 ) . The photoreceptor has been identified
to be the protein bound form of the neurotherapeutic agent,
hypericin (10). Wood (5) suggested that ciliary reversal
of the Stentor coeruleus was due to an intracellular photic
receptor potential generated in the vacules.
A similar photic
potential was recorded in the cytoplasm by means of KClmicroelectrode across the cell membrane of Stentor coeruleus
(3).
From the spectroscopic studies of pigment granules.
Song and coworkers have reported that the stimulus light
triggers the release of protons from the photoreceptors
embedded in pigment granules and this proton flux is the
primary signal for the photosensory transduction mechanism
in Stentor coeruleus (11, 12, 13).
The purpose of this work is to study the mechanism by
which the Stentor coeruleus cells perceive stimulus light
and transduces it into the mechanical response.
This thesis
reports the results from my investigation on the ionic fluxes
across the cell membrane induced by photoexcitation.
Photo-
responses of Stentor coeruleus are tested in the presence of
various ionophores and ion blockers.
The results from these
experiments reveal the fact that proton flux and calcium ion
flux across the membrane are the critical factors in the
photosensory transduction mechanism of Stentor coeruleus.
Secondly, the electron microscopic study of the ultrastructure of Stentor cell provides some information about
5
the fine structure of the pigment granules and the distribution of chromoprotein in the Stentor coeruleus cell.
CHAPTER II
MATERIALS AND METHODS
Materials
Chemicals
Valinomycin and nigericin were obtained from Ely Lilly
Co. and A23187 was purchased from Calbiochem-Behring Corp.
SF-6847 was a gift from Professor T. Ozawa at the University
of Nagoya, Japan.
All other ionophores and ion blockers were
purchased from Sigma Chemical Company and they were used as
received except Ruthenium Red.
Ruthenium Red was purified
according to Luft (26). All materials used in electron
microscopy were purchased from Ted Pella Inc..
Stentor Culture
The heavily-pigmented strain, "stella" of Stentor
coeruleus was used for this study.
They were grown in large,
discontinuous cultures as described previously (10). The
growth medium was consisted of 2.5 mM CaClp, 1.0 mM NaNOoi
0.1 mM KHpPO^^, 1.0 mM MgSOj^and 1.0 mM Trizma base buffer;
the pH was adjusted to 7.8*0.2 with concentrated HCl.
As
the carbon source, 80 wheat grains were added to 2 liters of
culture medium in a 6.5 gallon bottle.
About 10,000 cells
in approximately 100 ml of old growth medium were inoculated into this fresh growth medium.
6
From one week after
inculation, 100 ml of fresh growth medium was added to the
7
culture every other day.
The temperature of the culture room was kept at 19*'-20''C.
It is very critical to keep this temperature.
At the tem-
perature above 20°C, growth of bacteria and fungi take over
the entire culture in one to two days.
The culture was illu-
minated with the normal room lighting (white fluorescence
lamp) during daytime and it was kept in the dark during the
night.
Stentor cultures reach a maximum population in about
7-10 days.
After first harvest, the culture repopulates to
the maximum population in 2-3 days and these cultures can be
harvested for about a month.
experiments, 10-12 day
For the negative phototaxis
old Stentor cells were used.
Methods
Negative Phototaxis of Stentor coeruleus
A number of different methods had been tried in order
to measure the photoresponse of Stentor coeruleus quantitatively (2, 12, 1^). Among these various methods, the visual
counting method using narrow channel turned out to be the
most suitable technique for this study.
The advantage of
using this method is the fact that it gives reasonably reproducible results without employing sophisticated equipment.
Figure 2 shows the apparatus which was used for measuring
the negative phototactic response of Stentor coeruleus.
The
channel was cut into a Plexiglass block 9.0 mm deep, 6.2 mm
wide and 75 mm long, and fitted on one end with a hinged lid.
Four thin pieces of glass were inserted into the clear Plexiglass lid, such that when the lid was closed the channel was
devided into five equal sections.
One end of the channel
was covered with a quartz, non-birefrigent plate.
The light
stimulus was presented through this plate directly into the
suspension of the Stentor cells, after passing through the
appropriate filters.
After the Stentor cells had responded
to the stimulating light for a given amount of time, the lid
was closed, and the number of the Stentor cells in each
compartments were counted.
The Nicolas microscope illuminator was used as a stimulating light source and the Corning CS2-63 colored glass filter
Figure 2.
1.
Partitioned photomovement channel.
Nicolas Microscope Illuminator lamp, which fits securely
into the chasis of the apparatus.
2.
Filter holder, allows up to four filters of various
wavelengths and neutral density to be placed in the
stimulating beam.
3-
Rigid Plexiglass chassis, which maintains conformation
of the device.
4-.
Removable channel block, which may be viewed under the
microscope.
The channel is open while- Stentor cells are responding
to the light. After a given period of time, the hinged lid
is closed, thus lowering four partitions into the channel
and trapping the cells.
Actual photographs of the parti-
tioned channel show the response of cells to the light as a
function of time.
UIUJ
min
C^J
min
10
o
—
<N
II
II
II
H
1-
H
1 A 1 B 1 C 1 D1 E 1
to
^
m •
.tr
;i-^.,r;
«
. . I"
11
and an IR-absorbing filter were used to limit the wavelength
of the stimulating light from 'S15 nm to 750 nm.
ating light intensity was 1.6 W/m
The stimul-
and it was measured with
a Y. S. I. Kettering Radiometer, Model 65A.
Stentor cells used for this experiment were prepared in
the following way.
10-12 day old Stentor cells were harvested
and washed with fresh growth medium.
These Stentor cells
were resuspended in a new medium and dark adapted for two
hours before the experiments.
For each experiment, an ion-
ophore solution was added to the 2 ml Stentor suspension
containing 120-130 cells and allowed 5 minutes incubation
time in the darkness, followed by the illumination for one
minute.
Then lid was closed and the number of cells in
each section of the channel was
counted.
Control experiments
in the standard culture medium were done for each individual
batch of Stentor cultures and each experiment was repeated
at least five times with different batches of Stentor
cultures.
Electron Microscopy
During the course of this electron microscopic work,
two different fixation procedures were tried.
However,
they did not produce any detectable differences in the
ultrastructure of Stentor coeruleus.
The first method was
the fixation in 3 ^ buffered glutaraldehyde for one hour
followed by 1 %
buffered osmium tetraoxide post fixation for
12
3 0 minutes.
Entire fixation procedure was carried out at
room temperature and the fixatives were buffered with 0.1 M
cacodylate to pH 7.2-7.4 (15). The second method was the
fixation by osmium tetraoxide vapor.
A small drop of culture
medium containing Stentor cells was placed at the center of
a coverslip, which was then inverted over the mouth of a
container of aqueous 2^ osmium tetraoxide.
The usual fixa-
tion time was five minutes at room temperature.
The fixed
cells were dehydrated in a graded series of ethanol (70^,
95?^» lOOfo, respectively) followed by propylene oxide, then
infiltrated and embedded in Epon 821 mixture.
After poly-
merization for 12-l4 hours at 80 C, individual cells were
selected and sectioned with a glass knife mounted on a
Reichert ultramicrotome.
These sections were stained by
uranyl acetate and lead citrate, and examined in a Hitachi
HS-8 electron microscope.
This electron microscopic study
was carried out at the Department of Biology, Texas Tech
University.
CHAPTER III
RESULTS
Photoresponse of Stentor coeruleus
Negative phototaxis of Stentor coeruleus was observed
at the stimulating light intensity as low as 0.1 W/m
2
while
2
maximum response was reached at 0.8 to 1.0 W/m . However,
much higher light intensity did not affect the swimming velocity or photoresponse of the Stentor cells and Stentor cells
did not show any significant adaptation to the light in 10
to 20 minutes.
The swimming velocity of Stentor coeruleus
reaches to 2-3 body length per second at the optimum condition.
Figure 3 shows time-dependent phototaxis of Stentor
2
coeruleus.
Light intensity was 1.6 W/m
at the point where
light entered the channel and the wavelength of the light
stimulus was '^'I'j to 750 nm.
Percent response reached the
saturated level after two minutes illumination (fluence is
192 W/m^).
To quantitate the photoresponses, the percent responses
were calculated by the following equation,
(N/N, - 0.4)
% Response =
x 100
(N/N;
- 0.4)
where N and N, are the numbers of Stentor cells in compartments A and B and the total number of cells in the channel
respectively.
The denominator is the result from the control
13
14
Figure 3-
Time-dependent photoresponse of Stentor
coeruleus.
% Response is defined in the text. Light
2
intensity was 1.6 W/m at the point where light entered
the channel.
The wavelength of the light was 515-75'^
nm.
15
LIGHT FLUENCE (W/m^)
48
96
T
l44
30
60
90
TIME (SEC)
120
150
16
experiments which were done for every new batch of culture.
Effects of Protonophores on Photoresponse
In order to confirm the involvement of proton flux in
the photosensory transduction mechanism, the photoresponse of
Stentor coeruleus was tested in the presence of various protonophores.
All of the protonophores tested showed very strong
inhibitory effects on the phototaxis.
No more than 2 (iM con-
centration of FCCP, CCCP or SF-8647 was needed to see the
total inhibition of phototaxis ( Figures 4, 5 and 6 ) .
Nigericin caused total inhibition of phototaxis at 4 (iM concentration (Figure 7)- DMSO was used as the solvent for
these protonophores.
When DMSO alone was added to the sus-
pension, there was no effect on the photoresponse of Stentor
coeruleus.
These experiments were carried out in pH 7*8
growth media.
Effects of Ca
-blocking agents and Ca
-ionophores on
Photoreponse
To study the mechanism of the calcium movement in the
photosensory transduction of Stentor coeruleus, the photoresponses of Stentor coeruleus were measured in the presence
of calcium blockers and calcium ionophores.
A23187, a calcium
ionophore, shows strong inhibitory effect on the photoresponse
of Stentor coeruleus in the magnesium free medium.
However,
it has no effect on the photoresponse in the normal growth
17
Figure 4.
coeruleus.
Effect of FCCP on phototaxis of Stentor
External pH was 7.8 in growth medium and FCCP
(2 mM stock solution in DMSO) was added in |j,l aliquots.
When DMSO alone was added to the suspension in equal concentrations, there was no effect on response.
2
Fluence of the stimulating light was 96 W/m .
18
100
w
O
m
w
0.5
1.0
1.5
FCCP CONCENTRATION (^LM)
2.0
19
Figure 5.
coeruleus.
Effect of CCCP on phototaxis of Stentor
External pH was 7•8 in growth medium and CCCP
(2mM stock solution in DMSO) was added in |j,l aliquots.
When DMSO alone was added to the suspension in equal concentration, there was no effect on response.
Fluence of the stimulating light was 96 W/m .
20
100
80
60
w
OQ
o
PH
m
w
40
-
20
_
0.5
1.0
1.5
CCCP CONCENTRATION (^iM)
21
Figure 6.
Effect of SF-8647 on phototaxis of Stentor
coeruleus. External pH was 7•8 in growth medium and SF-8647
(2 mM stock solution in DMSO) was added in |j.l aliquots.
When DMSO alone was added to the suspension in equal concentration, there was no effect on response.
2
Fluence of the stimulating light was 96 W/m .
22
100
m
o
PH
m
0.5
1.0
1.5
SF-8647 CONCENTRATION (^iM)
2.0
23
Figure 7.
Effect of nigericin on phototaxis of Stentor
coeruleus. External pH was 7.8 in growth medium and 2 mM
DMSO solution of nigericin was added in |j.l aliquots.
When DMSO alone was added to the suspension in equal concentration, there was no effect on response.
2
Fluence of the stimulating light was 96 W/m .
24
100
80
w 60
O
PH
OQ
^
40
20
-
0
1
2
3
NIGERICIN CONCENTRATION (^iM)
^
^5
Figure 8.
Effects of A23187 on the phototaxis of
Stentor coeruleus.
70 ^ ethanol was used as a solvent for
A23187 and same concentration of ethanol caused no effect
on photoresponse.
-•—•—
-O—O—
Effect of A23187 in normal growth medium
Effect of A23187 in a medium with no Mg"^"^
2
Fluence of the stimulating light was 96 W/m .
26
1
2
3
A23187 CONCENTRATION (^iM)
^
27
Figure 9.
Effect of Ruthenium Red on phototaxis of
Stentor coeruleus. External pH was 7•8 in growth medium.
2 mM aqueous solution of Ruthenium Red was used.
'
2
Fluence of the stimulating light was 96 W/m .
28
o
o
o
o
CO
vO
o
asMOdsaa %
o
29
Figure 10. Effect of Lanthanum on the phototaxis of
Stentor coeruleus.
External pH was 7.8 in growth medium,
and 2 mM aqueous solution of LnCl^ was used.
2
Fluence of the stimulating light was 96 W/m .
30
o
o
o
<
cc
2
O
o
__
O
o
o
00
o
o
o
CO
asMOdsan °J>
O
(TA
31
Figure 11. Effect of 3t^-diaminopyridine on phototaxis
of Stentor coeruleus. External pH was 7•8 in growth medium
and 2 mM DMSO solution of 3»^-diaminopyridine was used.
When DMSO alone was added to the suspension in equal
concentration, there was no effect on photorespnose.
2
Fluence of the stimulating light was 96 W/m .
32
100
80
w 60
OQ
2
O
PH
02
W
PC
40
20
J_
1
2
1
±
3
^
1
5
1
6
3,4-DAP CONCENTRATION (^iM)
33
Figure 12.
Effect of valinomycin on phototaxis of
Stentor coeruleus. External pH was 7•8 in growth medium and
2 mM DMSO solution of valinomycin was used.
When DMSO alone was added to the suspension in equal concentration, there was no effect on photoresponse.
2
Fluence of the stimulating light was 96 W/m .
34
100
80
_
60
w
OQ
2
O
PH
OQ
W
CC
40
20
-
0
1
2
3
^
5
VALINOMYCIN CONCENTRATION
6
(^iM)
35
Figure 13.
Effect of ouabain on phototaxis of Stentor
coeruleus. External pH was 7•8 in growth medium and 2 mM
DMSO solution of ouabain was used.
When DMSO alone was added to the suspension in equal concentration, there was no effect on response.
2
Fluence of the stimulating light was 96 W/m .
36
o
M
EH
<
PC
EH
2
W
o
2
O
o
pq
<
o
asMOdsan 2^
37
medium (Figure 8). Ruthenium Red, a calcium channel blocker/
calcium ATPase inhibitor, strongly inhibits the phototactic
response of Stentor coeruleus (Figure 9) and the inhibitory
effect of lanthanum ion on the photoresponse of Stentor
coeruleus is shown in Figure 10,
Effects of Other Ionophores on Photoresponse
The effects of potassium ionophores, valinomycin and
3,4-diaminopyridine, are shown in Figure 11 and Figure 12,
respectively.
Both of them have no inhibitory effect on
the photoresponse of Stentor coeruleus.
Also, sodium iono-
phore, ouabain, does not inhibit the photoresponse (Figure 13).
Electron Microscopy
Electron micrograph-of the frontal field of Stentor
coeruleus is shown in Figure l4. The frontal field is
surrounded by a band of membranelles and the cilia of the
membranelles are connected to the basal body and km fiber
system inside the cell.
of pigment granules.
Figure 15 shows the ultrastructure
Most of the pigment granules are
attached to the inside surface of the cortex.
pigment granule is about 0.5 to 0.7 m
The size of
in diameter and some
fine structure is shown in the inside of the pigment granule.
38
Figure l4. Electron micrograph of the frontal field and
the basal body of Stentor coeruleus.
bb; basal body
km; km fiber
my; myoneme
P; pigment granule
M; mitochondria
12,000 X (1), 45,ooo X (2).
»
**••'**»
'.'."-•
J~*-,
*
'
« . .
<t> - i ; ;
*
V*^'
l^
4o
Figure 15-
Electron micrograph of the pigment granule of
Stentor coeruleus.
P; pigment granule
M; mitochondria
45,000 X (3, 4 ) , 35,000 X (5).
V
CHAPTER IV
DISCUSSION
The proton release from the excited state of the photoreceptor was proposed to be the primary triggering signal
in the photosensory transduction mechanism of Stentor
coeruleus (13)«
The strong inhibitory effects of proto-
nophores on the photoresponses also support this proposal.
These protonophores inhibit the photoresponse of Stentor
coeruleus by abolishing the proton gradient across the
cytoplasmic membrane.
Valinomycin and 3>^-diaminopyridine showed no inhibition
on the photoresponses of Stentor coeruleus.
These results
and the previous observation of the effect of TPMP
on the
light induced pH change in Stentor coeruleus (12) strongly
indicate that the K -diffusion potential and the membrane
potential term of the proton motive force are not likely to
be involved in the photosensory transduction mechanism.
Result from the experiment with ouabain shows that sodium ion
flux is not involved in this mechanism.
On the other hand, calcium ion flux across the membrane
plays
a very important role in the photosensory transduction
mechanism of Stentor coeruleus.
Calcium ionophore, A23187,
is a mobile carrier of calcium ions in the biological membrane (17), and it makes 1:2 complexes with divalent
cations in the membrane (18, 19). The lack of inhibitory
42
43
effect of A23187 in the presence of extracellular magnesium
ions is probably due to the binding of the antibiotic with
magnesium ions (20, 21). Lanthanum ion and Ruthenium Red gave
the same results as A23187 and the effect of Verapamil on
the photoresponse of Stentor coeruleus is also consistent
with these results (22). Though very preliminary, the ATP
level in Stentor cell depends on the actinic irradiation
(Figure I6). Lanthanum is a calcium antagonist.
Ruthenium
Red is an inhibitor for calcium transport as well as an inhibitor for calcium ATPase (23) and it is also reported to inhibit the ciliary membrane ATPase in Paramecium (24). Verapamil
has been reported to effect the total supression of voltagedependent inward calcium ion movement across several excitable
membranes (25). These results probably reflect the existence
of two different calcium channels; one for the calcium influx
and the other for the calcium efflux.
On the basis of these observations in addition to the
previous results (10, 12, 13)f the following mechanism is proposed for the photosensory transduction in Stentor coeruleus.
The light signal induces proton release from the photoreceptor.
This transient high concentration of proton inside the cell
triggers the opening of the calcium channel.
The influx of
calcium ions causes the ciliary reversal and activates the
calcium ATPase in the ciliary membrane.
44
Figure l6.
Relative ATP concentrations in CPM of
Stentor coeruleus as a function of illumination.
ATP concentrations were determined by the LuciferinLuciferase bioluminescence method.
45
2
o
Q
W
2
CC
D
EH
EH
X
o
o
o
o
CM
MO
2-
0 1 X MdO
CHAPTER V
CONCLUSIONS
1.
A proton flux from the excited state of the photoreceptor
is the primary triggering signal in the photosensory transduction mechanism of Stentor coeruleus.
2.
A transient membrane potential produced by the proton flux
triggers calcium influx across the cell membrane.
3.
The K -diffusion potential and the membrane potential term
of the proton motive force are not likely to play a direct
role in the photosensory transduction in Stentor coeruleus.
4.
Sodium is not directly involved in the photosensory trans-
duction in Stentor coeruleus.
5.
Ca
-ATPase in the ciliary membrane might be involved in
the process of the light-induced ciliary reversal in Stentor
coeruleus.
46
LIST OF REFERENCES
1.
Corliss, J. 0., (1979) The Ciliated Protozoa, sec, ed.,
Pergamon Press, United Kingdom p. 455.
2.
Song, P. S., Hader, D. P. and Poff, K. L., (I98O) Arch.
Microbiol. 1_26, I8I-I86,
3.
Song, P. S., Hader, D. P. and Poff, K, L., (1980)
Photochem, Photobiol. ^2. 781-786.
4.
Tartar, V., (I961) The Biology of Stentor, Pergamon Press
New York.
5.
Wood, D. C , (1976) Photochem. Photobiol. 24, 261-266
6.
Song, P. S., (1980) in "Photoreception and Sensory
Transduction in Aneural Organisms" (Lenci, F. and
Colombetti, G., eds.), pp. 189-210, Plenum Press
New York.
7.
Blumberg, S., Propst, S., Honjo, S., Otoka, T.,
Antanavage, J., Banerjee, S. and Margulis, L., (1973)
Trans. Amer. Micros. Soc. 22. 551-5^9'
8.
Newman, E., (197^) J. Protozool. 2A_ (5), 729-737.
9.
Small, E, D, and Marszalek, D. S., (I969) Science 163,
1064-1065.
10.
Walker, E. B,, Lee, T, Y. and Song, P. S., (1979)
Biochim. Biophys. Acta ^82., 129-144.
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