J. Cell Sci. 80, 75-89 (1986)
75
Printed in Great Britain © The Company of Biologists Limited 1986
REACTIVATION OF EUGLENOID MOVEMENT AND
FLAGELLAR BEATING IN DETERGENT-EXTRACTED
CELLS OF ASTASIA LONGA: DIFFERENT
MECHANISMS OF FORCE GENERATION ARE
INVOLVED
T. SUZAKI AND R. E. WILLIAMSON*
Department of Developmental Biology, Research School of Biological Sciences, The
Australian National University, PO Box 475, Canberra City, ACT 2601, Australia
SUMMARY
Detergent-extracted cell models of the euglenoid flagellate, Astasia longa, were obtained that
rounded-up on addition of calcium. Treatment with 4% Triton X-100 and Nonidet P-40 removed
the flagellar membrane, all membranous structures inside the cell body and the plasma membrane
at groove regions of the cell surface. Maximum rounding-up was induced when the concentration
of free calcium was raised to ^10~ 7 M, and ATP strongly enhanced this response. The ionic
requirements and sensitivity to vanadate were different from those for the reactivation of flagellar
movement. The results suggest that the mechanism of force generation is different from the
dynein-based system of the flagellum and that a rise in cytoplasmic free Ca 2+ concentration might
cause euglenoid movement in vivo. The mechanism of euglenoid movement is discussed in relation
to other protozoan motile systems.
INTRODUCTION
Euglenoid movement or 'metaboly' is a unique and characteristic motility of the
euglenoid flagellates, and was denned to include all the kinds of cell body movements
or shape changes that they exhibit (Pringsheim, 1956; Mikolajczyk & Kufnicki,
1981). In spite of many attempts to elucidate the mechanism of euglenoid
movement, little is known about it and even the site and the structures generating the
active force are still open questions (Bovee, 1982). Although the involvement of an
actomyosin system (Pringsheim, 1948, 1956; Leedale, 1964, 1966; Leedale, Meeuse
& Pringsheim, 1965) or a microtubule-dynein system (Hofmann & Bouck, 1976;
Gallo & SchreVel, 1982) has been suggested, both of these hypotheses lack
supporting evidence. We demonstrated recently that pellicular strips slide relative to
each other during euglenoid movement in Euglena fusca, and we proposed a
hypothesis that envisages an active sliding mechanism between adjacent pellicular
strips (Suzaki & Williamson, 1985).
Techniques that permeabilize cells and allow their motile system to be
manipulated by the experimenter have often been useful. Such 'cell model'
•Author for correspondence.
Key words: motility, Astasia longa, Caz+, vanadate, euglenoid movement,flagellarbeating.
76
T. Suzaki and R. E. Williamson
techniques were successfully applied to various euglenoid flagellates by Murray
(1981) who used Ca2+ to induce rounding of elongated cells treated with a divalent
cation ionophore and/or detergent. Cell model experiments are most informative
when the cell is highly permeable and lacks its own energy supply and Ca 2+ sequestering system. The situation with the Astasia cell models is not completely
clear since, while not requiring exogenous ATP for rounding, their sensitivity to
cyanide and dinitrophenol (Murray, 1981) suggests that ATP generation was still
occurring and was needed for rounding up. In such a situation, an intracellular
membrane system sequestering Ca2+ (Murray, 1981) might also still be functional
and capable of maintaining an intracellular free Ca2+ concentration that differed
from that in the external solution.
We have therefore prepared more thoroughly extracted cell models lacking the
organelles involved in ATP generation and Ca2+ sequestration. Such cells rounded
up at much lower concentrations of free Ca2+ than the cells in Murray's (1981) study
and are stimulated by exogenous ATP. In addition, because flagellar beating was
reactivated concurrently, we investigated the degree of similarity between the
microtubule—dynein system powering the flagellum and the largely uncharacterized
mechanism generating the force for cell rounding.
MATERIALS AND METHODS
Cells
Astasia longa (strain no. 512) was purchased from the Culture Collection of Algae at the
University of Texas at Austin, U.S.A., and cultured as previously described (Suzaki &
Williamson, 1983). Cells were washed and kept for 2-4 days in lOOmM-iV-2-hydroxyethylpiperazineW-2-ethanesulphonic acid (Hepes) at pH7-0.
Preparation and reactivation of cell models
For each experiment, 10 /d of cell suspension in lOOmM-Hepes-KOH (pH 7-0) was mixed with
40^1 of an extraction solution (ES) consisting of lOOmM-Hepes-KOH, 20mM-piperazine-N^V'bis(2-ethanesulphonic acid) (Pipes), 10mM-ethyleneglycol-bis(/S-aminoethyl
ctheT)-N,NJ^'J^'tetraacetic acid (EGTA), SOmM-sucrose, 1 mM-dithiothreitol (DTT), 7-5% (v/v) glycerol, 4 %
(v/v) Triton X-100, and 4 % (v/v) Nonidet P-40 at pH 7-0 adjusted with KOH. Extraction of the
cells was carried out at room temperature (20 ± 3 deg. C) in a small centrifuge tube. At 2-5 min
after the addition of ES, the above mixture was then added to 30^1 of a reactivation solution (RS)
consisting of lOOmM-Tris-HCl, 20mM-Pipes-KOH, lOmM-EGTA, 50mM-sucrose, 1 mM-DTT,
and various concentrations of MgC^, CaCl2 and ATP (pH7-0). Reactivation was carried out at
room temperature. The final concentrations were: Hepes, 62-5 mM; Pipes, 17-5 mM; Tris,
37-5 mM; EGTA, 8-75 mM; sucrose, 43-75 mM; D T T , 0-88 mM; glycerol, 3-75%; Triton X-100,
2 % ; and Nonidet P-40, 2 % . (A pH change of <0-l pH unit occurred on mixing ES and RS to
achieve a final free Ca + concentration of 5xl0~ 7 M, sufficient to saturate the response.) The
percentage of the total volume occupied by the cell bodies was less than 0-1 % of the final mixture.
Cells were then fixed by adding about 200^1 of a fixative (1:1 (v/v) mixture of methanol and a
commercial 37% formaldehyde solution). Cells were fixed very rapidly without changes in cell
shape. After 5 min, the cells were spun down by a gentle centrifugation (about 100 jf) for 1 min, and
numbers of elongated and rounded cells were counted using a light microscope. Cell shape
categories were determined using an egg shape (length: width = 3:2) as a critical standard. About
2000 cells were examined in each experiment.
Euglenoid movement andflagellar beating in Astasia
77
Light and electron microscopy
Light micrographs were taken with a Zeiss photomicroscope equipped with Nomarski
differential interference optics. For electron microscopy, the cells were fixed either before or after
treatments for reactivation by mixing with an equal volume of a fixative (6 % (v/v) glutaraldehyde,
3 % (w/v) paraformaldehyde, 2mM-MgSO4, and SOmM-potassium phosphate buffer at pH7-0).
After 20 min at room temperature, the cells were centrifuged, rinsed with 25 mM-phosphate buffer,
and then post-fixed with 1% (w/v) OSO4 in 25 mM-phosphate buffer (pH7-0) for 2h at room
temperature. The fixed cells were rinsed, dehydrated in a graded acetone series, and embedded in
Spurr's resin (Spurr, 1969). Thin sections were stained with 10% (w/v) uranyl acetate in ethanol
for 10 min and lead citrate (Reynolds, 1963) for 5 min, and examined in a Hitachi H600 electron
microscope.
Miscellaneous
Hepes, Pipes, Tris, EGTA and cytochalasin B were purchased from Sigma Chemical Co. (St
Louis, U.S.A.). Sodium metavanadate (NaVCy was obtained from BDH Chemicals Ltd (Poole,
U.K.), and recrystallized from methanol/water (Gibbons et al. 1978) before use. Apparent
association constants for EGTA to calcium and magnesium were calculated from the absolute
association constants (Martell & Smith, 1974). The values obtained at pH7-0 (2-66X106 for
calcium and 29-5 for magnesium) are in accordance with values used by many other investigators
(Marban, Rink, Tsien & Tsien, 1980; Murray, 1981; Blinks, Wier, Hess & Prendergast, 1982;
Keller, Jemiolo, Burgess & Rebhun, 1982). Concentrations of free calcium and magnesium ions
were then calculated by an iterative procedure (Walz, 1982) using a computer.
RESULTS
Within 2min of treatment with ES, all cells stopped flagellar movement and
became completely elongated (Fig. 1). The elongated cells showed no responses to
blue light or mechanical stimulation, which usually cause rounding-up movement of
living Astasia cells (Suzaki & Williamson, 1983). Almost 100% of the cells rounded
up (Fig. 2) following the subsequent addition of RS containing 5 mM-CaCl2 and
SmM-ATP (final concentrations were: 1 X 1 0 ~ 7 M free Ca 2+ , 2mM-ATP). Figs 3-5
show detergent-treated cell models before (Fig. 3) and after addition of RS (Fig. 4,
partially rounded cell during movement; Fig. 5, completely rounded one). Flagella
did not detach from the cell body (arrows). It took about 5 s to complete the cell
shape change after the cell started rounding-up (Figs 3—5), and all parts of the cell
increased in diameter simultaneously (Fig. 4). The cells completely rounded up into
ball shapes and no surface infoldings were observed on the rounded cells. Treatment
with ES removed the plasma membrane from the groove region of the cell body
surface (compare Figs 6 and 7) and from the flagellum (compare Figs 8 and 9). After
the treatment with ES, membranous structures such as endoplasmic reticulum and
mitochondria were not observed inside the cell body (Fig. 7). Microtubules,
pellicular strips and associated projections (p in Fig. 7) remained intact, and new
electron-dense structures were found (arrowhead in Fig. 7), which were associated
with both the microtubules and the inner surface of the pellicular strips. Traversing
fibres connecting adjacent grooves (Mikolajczyk & Ku£nicki, 1981) could not be
observed in either the intact (Fig. 6) or the model cell (Fig. 7). Permeability of the
cell models was tested by using dyes of different molecular weight (Table 1). None of
the dyes used in this experiment entered the living cells, but the dyes neutral red
T. Suzaki and R. E. Williamson
Figs 1, 2. Detergent-extracted cell models of A. longa. Cells were treated with ES for
2 min, and photographs were taken before (Fig. 1) and 3 min after (Fig. 2) the addition of
RM containing 5mM-CaCl2 and 5mM-ATP (final concentrations are: free Ca z + ,
1X1O"7M; ATP, 2mM). Bar, 100fan.
(Mr 289) and fluorescein (Mr 376) entered the extracted models. Almost half of the
extracted cells were stained with eosin Y (MT 692), while erythrosine B (MT 880)
failed to enter either living cells or cell models.
The kinetics for the reactivation of rounding-up movement are shown in Fig. 10.
Treatment with ES induced a transient rounding-up (arrow in Fig. 10), but within
2 min every cell became elongated again and flagellar movement stopped. When RS
containing Ca2+ and ATP was added at 2 min, almost all of the cells became rounded
within 5 min. There was a variation in the time required before individual cells
started the movement (10s to 5 min), but once movement was initiated, it was
completed within a short time (about 5 s). Conditions were not found in which the
cell shape was reversible. Re-extension did not take place when the rounded cells
were washed and resuspended in ES (open squares in Fig. 10). Rounded cells did not
re-elongate even when ES contained Mg2"1" and/or ATP (data not shown). When RS
was added later than 2 min, fewer cells changed in shape (open triangles in Fig. 10).
Rounding-up movement of the cell body required free calcium (3=10~7M for
maximum effect; Fig. 11). In the absence of ATP, fewer cells were reactivated than
with ATP, and the reactivation was enhanced by increasing concentrations of ATP
(Fig. 12). ATP itself did not induce rounding-up, but it enhanced the movement in
the presence of calcium. Of different divalent cations tested, calcium was the most
Euglenoid movement and flagellar beating in Astasia
79
effective in inducing the rounding-up movement, while magnesium was the least
effective. Rounding-up movement of the cell body was not inhibited by cytochalasin
B (100 im).
Figs 3-5. Highly magnified light micrographs of extracted cell models before and after
addition of RS containing Ca 2+ and ATP. Arrows indicate the flagella. Fig. 3: cell before
addition of RS showing elongated cell shape. Fig. 4: partially rounded cell undergoing
uniform rounding-up. Fig. 5: completely rounded cell. Bars, 10/an.
Figs 6, 7. Electron micrographs of cross-sections of pellicular structures. Fig. 6, intact
cell showing endoplasmic reticulum (er) and a mitochondrion (m). Fig. 7, detergentextracted cell model. Plasma membrane was removed from the groove regions (arrows)
and no membranous or filamentous structures can be detected inside the cell body.
Arrowhead indicates a newly found structure associated with both microtubules and
inner surface of pellicular strips, p, periodic projections. Traversing fibres cannot be
observed in either of these sections. Bars, 100 nm.
Figs 8, 9. Electron micrographs of an intact (Fig. 8) and a detergent-extracted flagellum
(Fig. 9) showing loss of the plasma membrane, pr, paraflagellar rod. Bars, 100 nm.
T. Suzaki and R. E. Williamson
80
Table 1. Permeability of cell model
Dye
Mr
Concn (%)
Stained cells (%)
Neutral red*
Fluoresceinf
Eosin Yf
Erythrosine B*
289
376
692
880
0-3
0-2
0-3
0-3
100-0
90-9
47-6
0-0
•Observed under a bright-field microscope,
f Observed under a fluorescence microscope.
100
!
£
40
20
10
Time (min)
Fig. 10. Kinetics of reactivation of cell body rounding-up in detergent-extracted cell
models. Abscissa, time after addition of ES. Ordinate, percentage of rounded cells.
About 40 % of cells showed a transient rounding-up movement induced by the addition of
ES (arrow), but within 2min almost all of the cells became elongated and flagellar
movement stopped. When RS containing Ca 2+ and ATP was added at 2 min, the cells
became rounded up within a few min (A), while only 40% of the cells were reactivated
when the same RS was added at 7-5 min (A). Rounded cells did not re-elongate when
the cells were washed and resuspended in ES at 7-5 min ( • ) . Final concentrations of
free Ca 2+ and ATP were 1X10" 7 M and 2 mM, respectively. (O) cells kept in ES; ( • ) RS
without Ca z+ and ATP was added at 2 min.
Euglenoid movement and flagellar beating in Astasia
81
100
80
8 60
'o
o
40
20
Fig. 11. Effect of free calcium concentration on rounding-up movement. Abscissa, free
Ca 2 + concentration (pCa =—log [free calcium concentration]). Ordinate, percentage of
rounded cells. After extraction with ES for 2min, cells were treated with RS containing
various concentrations of CaC^ with ( • and A) and without (O and A) ATP (final
2mM) for 5 min before fixation. Different symbols in each curve represent results from
two independent experiments. Values for pCa = » were determined when RS did not
contain CaCl2.
Flagellar movement could also be reactivated in the detergent-extracted cell
models. Since flagella remained on the cell body, some of the extracted cell models
swam in a manner similar to living cells when flagellar movement was reactivated.
Reactivation of rounding-up was therefore compared with that of flagellar
movement. Addition of Mg^+ was not required for the calcium-induced rounding-up
movement, and high concentrations of magnesium (>10~2M) completely inhibited
the movement (Fig. 13A). On the other hand, flagellar movement required
magnesium and ATP (Fig. 13B). Flagellar reactivation took place even at a high
magnesium concentration and calcium could not substitute for magnesium.
Vanadate, which is an inhibitor of dynein ATPase (Gibbons et al. 1978), inhibited
flagellar reactivation at 10~5 M (Fig. 14). Cell body rounding-up was also inhibited by
vanadate, but only at a higher concentration ( 1 0 ~ 3 M ) .
DISCUSSION
A cell model has been prepared in which euglenoid cell shape changes and flagellar
beating can be reactivated and their force-generation mechanisms compared.
82
T. Suzaki and R. E. Williamson
Differences between rounding-up movement of cell models and living cells
Rounding-up movement of living Euglena cells always starts from either the
anterior tip (Mackinnon & Hawes, 1961; Suzaki & Williamson, 1983) or the
posterior tip (Kamiya, 1939; Mikolajczyk, 1972; Mikolajczyk & Kufnicki, 1981;
Gallo & Schre"vel, 1982) and propagates to the whole cell body. This anteriorposterior gradient was abolished in the cell models, and the shape change took place
1-0
20
Concentration of ATP (miu)
30
Fig. 12. Effect of ATP concentration on rounding-up movement. Abscissa, final
concentration of ATP. Ordinate, percentage of rounded cells. Cells were first extracted
with ES for 2 min, then treated with RS containing various concentrations of ATP and
CaCl2 for 5 min before fixation. (A) Final, 8xlO" 8 M free Ca 2 + ; (O) final, 4xlO~ 8 M free
Ca 2 + ;(»)Ca 2 + -free.
Fig. 13. Effect of magnesium concentration on reactivation of rounding-up movement
(A) and flagellar movement (B). Abscissaes, concentration of free magnesium (pMg =
—log [free magnesium concentration]). Ordinates, percentage of rounded cells (A) and
cells with reactivated flagella (B). Mean values and standard errors for four independent
experiments are shown. Cells were first extracted with ES for 2 min, then treated for
5 min with RS containing various concentrations of MgCl2. Concentrations of free
calcium and ATP were as follows: (O) lXl(T 7 M-Ca 2 + , 2mM-ATP; (A) 1 X 1 0 ~ 7 M Ca 2+ , ATP-free; ( • ) Ca2+-free, 2mM-ATP; (A) Ca2+-free, ATP-free.
Euglenoid movement and flagellar beating in Astasia
83
A. Cell body rounding-up
100
80
60
40
20
B. Flagellar reactivation
100 r
i
Fig. 13
84
T. Suzaki and R. E. Williamson
10"
10
icr3
i(r4
10"
10"
Concentration of vanadatc (M)
Fig. 14. Effect of vanadate (sodium metavanadate, NaVOs) on reactivation of roundingup movement and flagellar movement. Abscissa, concentration of vanadate. Ordinate,
percentage of rounded cells ( • ) or cells with reactivated flagella (O). Cells were first
extracted with ES containing various concentrations of vanadate for 2min, then mixed
with RS containing Ca 2+ , ATP and vanadate for 5 min. Concentrations of vanadate in RS
were prepared to be the same as those in the mixture of cell suspension and ES, and final
7
M and 2mM, respectively.
over all parts of the cell surface at the same time. The cell models also showed no
photoresponse. This suggests that, while activation may be localized in vivo, the
sensory transduction system was probably destroyed in the model cells and the
motile systems directly and uniformly stimulated by the added calcium ions. Subpellicular endoplasmic reticulum is a Ca2+-sequestering structure in A. longa
(Murray, 1981) and was found to be broken down in the model cells. This
observation supports the earlier hypothesis that the endoplasmic reticulum is
involved in the sensory transduction systems (Murray, 1981).
Another difference between cell models and living cells was found in the shape of
the rounded cells. Reactivated rounding-up movement was not accompanied by the
formation of infoldings on the cell surface of the sort usually observed in living cells
(Mikolajczyk & Kuznicki, 1975; Suzaki, 1984). The absence of surface infoldings
Euglenoid movement andflagellarbeating in Astasia
85
may indicate that the volume of the cell model was able to increase when the cells
rounded up, and suggests that the surface infoldings on living cells were probably
passively formed to keep the cell volume constant. The cell model's ability to expand
would result from the reduction in resistance to water inflow caused by detergent
solubilization of the plasma membrane.
Site of active force generation for euglenoid movement
In a previous paper, the generation of force within the pellicle to cause active
sliding between adjacent pellicular strips has been suggested as the basis for cell
shape changes (Suzaki & Williamson, 1985). However, there is still an alternative
explanation for the mechanism of euglenoid movement, which has not been ruled
out. This idea involves an actomyosin contractile system deeper inside the cytoplasm
than the pellicular structures, and has been suggested by Pringsheim (1948),
Gojdics (1953), Leedale (1964, 1966, 1971), Leedale et al. (1965), Gallo et al.
(1982) and Bassi & Donini (1984). According to this hypothesis, the force-generating
system should be similar to that of amoeboid movement, or in other words, "Inside
every Euglena, there is an Amoeba trying to get out" (Leedale, 1966).
Other than pellicular microtubules and traversing filaments within the pellicular
complex (Mikolajczyk & Kuinicki, 1981), no filamentous structures have been
reported inside the cytoplasm. This, however, could be simply because of the
problem of poor fixation for electron microscopy. In the case of amoeba, for instance,
both thin and thick filaments were first observed by using a special procedure (pretreatment with Alcian Blue and fixation with unbuffered OsO4), and these filaments
could not be preserved by conventional fixation (Nachmias, 1964, 1968). Microfilaments have been visualized also in permeabilized cell models of amoeba that could
undergo cytoplasmic contraction with the addition of magnesium and ATP
(Holberton & Preston, 1970). There is a variety of different types of euglenoid
movement, and each type of movement involves differently localized sliding between
adjacent pellicular strips (Suzaki & Williamson, 1985). Therefore, if euglenoid
movement is generated by some kind of motile system deeper in the cytoplasm, the
force-generating structures (probably microfilaments) should be tightly connected to
the pellicular complex at many parts of the cell surface to enable the pellicular strips
to slide in restricted regions.
Actin is clearly present in the euglenoid Distigma proteus (Gallo et al. 1982). Like
some other algal actins (Marano et al. 1982; Williamson, Perkin & Hurley, 1985),
Distigma actin migrates just behind mammalian skeletal muscle actins when
electrophoresed in gels containing sodium dodecyl sulphate. Therefore, the
conclusion of Murray (1981) that Astasia lacks significant quantities of actin is
unreliable, since he considered only material co-migrating with rabbit skeletal muscle
actin. Fluorescent localization techniques have not yet revealed any specific
structural locations for the actin (Gallo et al. 1982; Bassi & Donini, 1984) and its
functions are unknown. In the present study no filamentous structures could be
observed inside the detergent-extracted cell model where visibility should be
particularly favourable, and cytochalasin B did not inhibit rounding-up. In addition,
86
T. Suzaki and R. E. Williamson
Murray (1984) reported partial success in inducing a vibrating movement of isolated
pellicular structures of D. proteus. These observations suggest that the site for force
generation is probably located within the pellicular structures themselves, not in the
deeper cytoplasm.
This study ruled out another possible mechanism for euglenoid movement, an
active sliding between microtubules and plasma membrane (Hofmann & Bouck,
1976; Gallo & Schrevel, 1982). As shown in Fig. 7, treatment with ES removed the
plasma membrane from the groove region. Since this region is the only part of the
cell surface where microtubules are apposed to the plasma membrane, it is clear that
direct interaction between microtubules and plasma membrane cannot be involved in
the movement.
Molecular mechanism of euglenoid movement
Murray (1981) demonstrated by using low concentrations of detergent and the
calcium ionophore A23187 that maximum rounding-up movement of A. longa was
induced when the surrounding medium contained >10~sM free calcium. The
present study showed, however, that a much lower concentration of Ca 2+ (=5lO~7M)
induced full reactivation of euglenoid movement in the detergent-extracted cell
models. The differences may be because the cells in our preparation were more
completely permeabilized and lacked an intracellular system for Ca 2+ sequestration.
(See Williamson, 1984, for some further discussion of models of other plant cells
whose variations in Ca sensitivity may be related to their preparation methods.) This
saturation concentration of free calcium is relatively low compared with those in
other calcium-regulated systems (see Martonosi (1983) for animal cells, and
Williamson (1984) for plant cells), and is one of the unusual characteristics of this
motile system.
It was also demonstrated that ATP enhanced the Caz+-induced response.
However, the exact significance of ATP for the rounding-up movement still needs
clarification, since calcium itself could induce some response. Some protozoan motile
systems do not require ATP and Ca2+ alone induces contractile movements. These
movements include contraction of spasmonemes in Vorticella and Zoothamnium
(Amos, 1971; Weis-Fogh & Amos, 1972; Asai et al. 1978) and contraction of
myonemes in Stentor and Spirostomum (Huang & Pitelka, 1973; Huang & Mazia,
1975; Yogosawa-Ohara & Shigenaka, 1985). However, the cell body movement of
Euglena does not seem to be similar to any of these Ca2+-induced, ATP-independent
systems, since euglenoid movement is based on sliding and not pellicular contraction
(Suzaki & Williamson, 1985). Furthermore, filamentous structures like myonemes
or spasmonemes could not be detected in Euglena cells. The detergent-treated cell
models used in the present study were not completely permeabilized; as shown in
Table 1, molecules bigger than ~500M r seem to have difficulty in penetrating the
cell surface. This is probably because the plasma membrane was removed only at the
groove region where microtubules and other electron-dense materials lie beneath the
plasma membrane and might hinder passage of bigger molecules (see Fig. 7).
Judging from the molecular weight, it might be reasonable to speculate that some
Euglenoid movement andflagellarbeating in Astasia
87
endogenous ATP (MT 507) might still remain after treatment with ES for 2min and
could be used in the reactivation process with the added Ca 2+ . ATP enhanced the
Ca2+-induced reactivation (Fig. 12) and maximum reactivation could not be
achieved without addition of ATP (Fig. 11). These enhancing effects of ATP can be
explained if ATP is required for the mechanism. The ES used in this study
apparently needs improvement, since prolonged treatment with ES reduced the
degree of reactivation (Fig. 10). A more long-lasting model system would probably
help to clarify further the role of ATP.
Since euglenoid cell shape change requires sliding between pellicular strips and the
possible involvement of microtubules has been suggested (Hofmann & Bouck, 1976;
Gallo & SchreVel, 1982), a microtubule—dynein system of the type seen in flagella
could be the force-generating mechanism. Dynein is a microtubule-associated
ATPase (>300xl0 in sub-unit MT), which generates a force for sliding movement
coupled to the hydrolysis of ATP (Johnson, Porter & Shimizu, 1984). The
microtubule-dynein system is not restricted to cilia and flagella, but has also been
strongly suggested to be present in termite flagellates as a force-generating mechanism in the bending movement of axostyles (Bloodgood & Miller, 1974; Bloodgood,
1975). The possible involvement of a microtubule-dynein system has also been
suggested in food ingestion systems in suctorian ciliates (Tucker, 1974) and
cytopharyngeal basket-bearing ciliates (Hausmann & Peck, 1978), and in the reelongation system of contractile ciliates such as Stentor (Huang & Mazia, 1975) and
Spirostomum (Yogosawa-Ohara, Suzaki & Shigenaka, 1985). In Astasia, periodic
projections are associated with the no. 1 microtubule and are located where the
sliding between adjacent pellicular strips might take place (Suzaki, 1984, and
unpublished data). It is therefore possible that these structures are involved in the
force-generating mechanism for the active sliding movement, and the possibility that
they are dynein was examined in the present study. Since flagellar movement was
also reactivated in the detergent-extracted cell models of Astasia, this could be used
as a good internal control for dynein-based motility. Requirements for divalent
cations and sensitivity to vanadate were found to be significantly different (Figs 13,
14) for cell body and flagellar motility. These results clearly indicate that the
molecular mechanism of euglenoid movement is different from that of flagellar
movement and that euglenoid movement does not depend on an ATPase that shares
flagellar dynein's high sensitivity to vanadate.
Thus, euglenoid movement is concluded to be a unique form of protozoan
movement that is probably controlled in vivo by Ca 2+ . A crucial problem still
waiting to be solved is the mechanism of force-generation for the active sliding
between pellicular strips.
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(Received 9 May 1985 -Accepted 22 July 1985)