Photolysis of caged calcium in cilia induces ciliary reversal in Paramecium caudatum
Department of Life Science, Faculty of Integrated Arts and Sciences, The University of Tokushima, Tokushima 770-8502, Japan
* e-mail: iwadate.yoshiaki{at}nifty.ne.jp
Accepted 10 January 2003
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Summary |
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We injected caged calcium into living P. caudatum cells and applied ultraviolet (UV) light to portions of the injected cells to raise artificially the intracellular Ca2+ level ([Ca2+]i). UV application to the upper ciliary region above the basal body induced ciliary reversal in injected cells. Furthermore, UV application to the tips of cilia induced weak ciliary reversal. Larger areas of photolysis in the cilium gave rise to greater angles of ciliary reversal. These results strongly suggest that the Ca2+-sensitive region for ciliary reversal is distributed all over the cilium, above the basal body.
Key words: Ca2+, cilia, flagella, Paramecium caudatum
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Introduction |
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Using permeabilized P. caudatum cells, Nakaoka and Ooi
(1985) found that cAMP
inhibits Ca2+-induced ciliary reversal. Several investigators
identified a 29 kDa axonemal protein that copurified with 22S outer-arm
dynein, which is phosphorylated in a Ca2+- and cAMP-dependent
manner (Hamasaki et al., 1989
,
1991
;
Bonini and Nelson, 1990
). Brief
digestion of demembranated cilia with trypsin suppressed not only the
inhibitory effect of cAMP on Ca2+-induced ciliary reversal but also
the phosphorylation of the 29 kDa axonemal protein
(Noguchi et al., 2000
). These
results suggest that the 29 kDa protein may be a mediator of the effect of
Ca2+ on ciliary reversal. If this were the case, Ca2+
would control the direction of ciliary beat not by acting on the ciliary base,
but throughout the cilium, because the 29 kDa dynein-associated protein is
present throughout the cilium.
To clarify the region of Ca2+ sensitivity with respect to
ciliary reversal, Hamasaki and Naitoh
(1985) iontophoretically
applied Ca2+ to detergent-permeabilized cilia of P.
caudatum. Their study revealed that the basal region of the permeabilized
cilium is the most sensitive to Ca2+ in inducing ciliary reversal.
In contrast, Tamm and Tamm
(1989
) showed that the
Ca2+ sensitivity extended the length of cilia, using
detergent-permeabilized macrocilia of Beroë mitrata. In an
effort to more faithfully represent the natural conditions of cilia, however,
we used living Paramecia, rather than a detergent-permeabilized
system in the present study, to prevent any possible loss of Ca2+
sensitivity through the permeabilization procedure. We adopted a photolysis
method, instead, in which caged calcium (o-nitrophenyl EGTA
[NP-EGTA]) was first injected into intact P. caudatum cells, followed
by application of a UV-light pulse to a restricted area to release
Ca2+ from the NP-EGTA. The results of this study suggest strongly
that Ca2+ causes ciliary reversal by acting upon the entire cilium
above the basal body.
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Materials and methods |
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Micromanipulation
The method of micromanipulation was described previously (Iwadate et al.,
1997,
1999b
). Briefly, standard
saline medium dispersed with Paramecium cells was mixed with the same
volume of a Ca2+-containing, highly viscous medium (1% methyl
cellulose, 1 mmol l1 KCl, 1 mmol l1
CaCl2 and 20 mmol l1 Pipes-Tris, pH 7.0) or a
Ca2+-chelating, highly viscous medium (1% methyl cellulose, 1 mmol
l1 KCl, 20 mmol l1 EGTA and 20 mmol
l1 Pipes-Tris, pH 7.0) before microinjection of NP-EGTA.
When Ca2+-chelating medium was used, the pH of the mixed medium was
6.95. The free Ca2+ concentration in the mixed medium was estimated
to be below 107.5 mol l1
(Iwadate et al., 1999b
). A
Paramecium cell swimming slowly in the medium was caught at the tip
of a suction pipette (about 35 µm in inner diameter).
NP-EGTA medium containing 60 mmol l1 Ca(OH)2,
100 mmol l1 NP-EGTA, 20 mmol l1
dithiothreitol (DTT) and 100 mmol l1 Hepes-KOH, pH 7.2, or
control medium containing 40 mmol l1 Ca(OH)2, 100 mmol
l1 ethylene
glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic
acid (EGTA), 20 mmol l1 DTT, 120 mmol l1
Hepes-KOH, pH 7.2, was injected into the Paramecium cell according to
the method of braking micropipette
(Hiramoto, 1974). The free
Ca2+ concentration in each medium was estimated to be
106.9 mol l1
(Iwadate et al., 1999a
). The
injected volume of each medium was approximately 8 pl, corresponding to
approx. 3% of the whole cell volume.
Micromanipulation of the suction pipette and micropipette was carried out using two micromanipulators (MO-102N and WR-60, Narishige, Tokyo, Japan) and injection through the micropipette was performed using a microinjector (IM-50A, Narishige, Tokyo, Japan).
Photolysis of caged calcium
UV application to the restricted area was performed according to the
methods described previously (Iwadate et
al., 1999a), with slight modifications. An inverted microscope
(TE300; Nikon, Tokyo, Japan) was used throughout the experiment. A mercury
short arc lamp (USH102D; Ushio, Tokyo, Japan) was attached to the side light
path of the microscope and used as the UV light source. A pinhole 400 µm in
diameter (43-5305, Coherent, Tokyo, Japan) was arranged at the field plane
conjugated to the plane of the specimen. The UV light through the pinhole was
filtered through a band-pass filter of 270400 nm (U340; Hoya, Tokyo,
Japan) and a cutoff filter of 300 nm (UV30; Hoya, Tokyo, Japan) to pass
300400 nm UV light, as described by Funatsu et al.
(1993
). The light was collected
by a UV-transmitting objective lens (CFI S Fluor 40x H, NA 1.30; Nikon,
Tokyo, Japan) to form a small image of the pinhole on the plane of the
specimen (Fig. 1). Thus, the
300400 nm UV light was applied to the restricted portion of the
Paramecium cell, about 2 min after microinjection of NP-EGTA. The
application time was controlled with an electromagnetic shutter (No. 0; Copal,
Tokyo, Japan). UV light applied to the inner portion of the
Paramecium cell was scattered by cytoplasmic particles. We determined
the area where the rise in [Ca2+]i
(
[Ca2+]i) occurred as described below.
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Determination of [Ca2+]i
As illustrated in Fig. 2,
the basal body is located just under the terminal plate. The diameter of
cilium is about 200 nm. Ciliary beating is always highly active, so imaging
methods using a Ca2+ indicator such as Calcium Green are predicted
to suffer from poor spatial and temporal resolution and are unlikely to allow
accurate determination of [Ca2+]i.
|
A Paramecium cell contains several thousands of secretory vesicles
named trichocysts, which in the resting state are located just beneath the
plasma membrane (Fig. 2). The
release of Ca2+ from cortical Ca2+ stores, called
alveolar sacs (reviewed by Plattner and
Klauke, 2001), triggers the fusion of trichocyst membranes with
the plasma membrane. Just after the membrane fusion, released trichocysts
expand 25 µm in length instantaneously (<1 ms) if the external medium
contains Ca2+. Thus, the released trichocysts are easily observed
by optical microscopy, as shown by an arrowhead in Figs
7A and
8A, for example. Klauke and
Plattner (1997
) indicated that
the threshold level of [Ca2+]i for fusion of trichocyst
membranes with the plasma membrane is >1 µmol l1,
which is the threshold level of [Ca2+]i for ciliary
reversal (Naitoh and Kaneko,
1972
). Iwadate et al.
(1999a
) directly demonstrated
that the threshold level of [Ca2+]i for membrane fusion
is higher than that necessary for ciliary reversal. Thus, trichocyst
exocytosis upon photolysis of NP-EGTA can be used as a marker of whether the
[Ca2+]i is greater than the threshold level for ciliary
reversal. Electron microscopy studies revealed that the three structures
(basal body, trichocyst and alveolar sac) are positioned very close to each
other (Stelly et al., 1990; Knoll et al.,
1993
) (Fig. 2) and
that Ca2+ release from the alveolar sacs reaches the neighboring
basal bodies (Knoll et al.,
1993
). According to this logic, we judged
[Ca2+]i to have occurred at the basal bodies when
trichocyst exocytosis was observed as shown in Figs
7A and
8A.
|
|
Estimation of [Ca2+]i
A photomultiplier tube (PMT) (R374; Hamamatsu Photonics, Hamamatsu, Japan),
equipped with an electromagnetic shutter (No. 0; Copal, Tokyo, Japan), a
high-pass filter of 500 nm (SC-50, Fuji Photo Film, Tokyo, Japan) and
band-pass filter of 530 nm (35-3607, Coherent, Tokyo, Japan), was attached to
the top light path of the microscope.
Calcium Green medium (2 pl) containing 1 mmol l1 Calcium
Green-1 dextran 10 000 MW (Molecular Probes, Eugene, OR, USA) and 120 mmol
l1 Hepes-KOH, pH 7.2, was injected simultaneously with
NP-EGTA medium or control medium into the Paramecium cell. We used a
blue light-emitting diode (blue LED, max=473 nm; LSPB500S,
Nichia, Tokushima, Japan) to excite the Calcium Green. Details of the method
have been previously described (Iwadate
and Kikuyama, 2001
). The fluorescence intensity of Calcium Green
was detected with the PMT.
Observation of ciliary direction
To detect images of ciliary reversal, the PMT at the top light path of the
microscope was removed and a CCD camera (XC-ST50, Sony, Tokyo, Japan) was
attached in its place. The images were recorded on VHS videotape with a
videotape recorder (HR-VX200, Victor, Tokyo, Japan). The images were then
transferred to a computer (PC-9821Nr13, NEC, Tokyo, Japan) and analyzed with
NIH-image version 1.62.
In highly viscous medium containing 0.5% methyl cellulose, ciliary movement
composed of the effective stroke and the recovery stroke is altered. Under
these conditions, the cilia remain relatively straight, and the apical end
moves in a circular path as if drawing a small circle, such that the movement
of each cilium traces a cone-like path. The ciliary angle () was
measured as shown in Fig. 3. A
cilium was selected randomly, within (or outside of) the UV application area.
Then, its angle (
) was measured against the line (the line with one
arrowhead in Fig. 3) perpendicular to the cell surface. If the cilium was positioned in an apical
portion of the cell to the perpendicular line, the angle (
) was shown
as positive.
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Results |
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We applied a UV light for 125 ms to an entire Paramecium cell that had been previously injected with NP-EGTA medium and Calcium Green medium. UV application caused a significant increase in fluorescence of Calcium Green (N=4) and caused both trichocyst exocytosis and ciliary reversal at the whole cell surface in all Paramecium cells tested (N=4). Representative results are shown in Fig. 4. By contrast, in cells injected with control medium alone, the fluorescence intensity did not change in response to UV application (N=3), nor did the cells show any trichocyst exocytosis or ciliary reversal (N=3). The duration of UV application was set as 125 ms in all subsequent experiments.
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Photolysis of caged calcium at cilia in Ca2+-chelating
medium
To determine whether Ca2+ influx through the Ca2+
channels in the ciliary membrane was contributing to ciliary reversal, we
applied UV light to cilia of Paramecium cells that had been first
immersed in a Ca2+-chelating medium and injected with NP-EGTA
medium. It is known that a low-Ca2+ condition is critical for
Paramecia, so all experiments were carried out within 2 min after
Paramecium cells were mixed with the Ca2+-chelating
medium.
Application of UV light to cilia at various portions in the anterior of cells treated in this manner led to ciliary reversal, indicating that it was the increase in [Ca2+]i caused by the photolysis of NP-EGTA, and not the Ca2+ influx through the Ca2+ channels that directly induced ciliary reversal (N=16). Typical results are shown in Fig. 5A (Movie 1). Ciliary reversal occurred strongly only in the area of UV application. The angle of ciliary reversal was 2.2 rad.
|
The relationship between the distance from the rim of the UV-applied area to the cell surface and the angle of ciliary reversal is summarized in Fig. 5B. Smaller distances led to greater angles of ciliary reversal.
Photolysis of caged calcium at cilia in Ca2+-containing
medium
We next immersed a Paramecium cell that had been injected with
NP-EGTA medium in Ca2+-containing medium and applied UV light to
selected cilia at the anterior of the cell.
Photolysis at cilia above basal body
UV light was first applied outside the cell body. In these cases, only the
cilia in the area of UV application reversed their direction. It is important
to note that no trichocyst exocytosis took place (N=22), even though
Ca2+ was present outside the cell. Typical results are shown in
Figs 6A (Movie 2) and
6B (Movie 3). In the example
shown in Fig. 6A, UV light was
applied to the entire cilium above the basal body. Ciliary reversal took place
strongly only in the area of UV application. The angle of ciliary reversal was
1.9 rad. When the distance from the rim of the UV-irradiated area to the cell
surface was increased to 6 µm, UV application induced only weak ciliary
reversal in the area (Fig. 6B,
Movie 3). In this case, the angle of ciliary reversal was 0.9 rad. The
relationship between the distance of UV irradiation and the angle of ciliary
reversal is summarized in Fig.
6C. Smaller distances led to greater angles of ciliary reversal,
although the ciliary angle before UV application was independent of the
distance of the irradiated area from the cell surface.
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These results are similar to those obtained when ciliary reversal was induced by the photolysis of NP-EGTA in the Ca2+-chelating medium. The following experiments were carried out in Ca2+-containing medium.
Photolysis at cilia including the basal body
When UV light was applied to the portion of the cilium including both the
basal body and the upper ciliary region in the anterior of Paramecium
cell, ciliary reversal and trichocyst exocytosis took place in response to UV
application in the following order. At first, ciliary reversal took place only
in the area of UV application. Then, after a slight delay equivalent to about
one video frame (33 ms), trichocyst exocytosis also occurred but only in the
immediate area, indicating that [Ca2+]i had reached the
threshold level for ciliary reversal. After a small further delay (one video
frame) or concomitant with the exocytosis, ciliary reversal at the whole cell
surface took place (N=13). Typical results are shown in
Fig. 7 and Movie 4. As shown in
the image at 0.23 s in Fig. 7A,
cilia in the area of UV application reversed their direction, although cilia
outside that area did not. After trichocyst exocytosis in the area of UV
application (arrowheads in Fig.
7A), cilia outside the application area reversed their direction
(at 0.3 and 0.37 s in Fig. 7A).
Fig. 7B shows that ciliary
reversal in the area of UV application (solid line) took place before
trichocyst exocytosis, whereas ciliary reversal outside the application area
(broken line) took place after the trichocyst exocytosis (triangle).
When UV light was applied to the inside at the anterior of the
Paramecium cell, trichocyst exocytosis took place just outside the
area of UV application, indicating that [Ca2+]i had
reached the threshold level for the ciliary reversal. Then, ciliary reversal
at the entire cell surface occurred after a short delay (one video frame) or
concomitant with trichocyst exocytosis (N=16). Typical results are
shown in Fig. 8 and Movie 5. As
shown in the 0.23 s image in Fig.
8A, cilia within and outside the area of trichocyst exocytosis
reversed their direction simultaneously after trichocyst exocytosis
(arrowheads in Fig. 8A).
Fig. 8B shows that ciliary
reversal within (solid line) and outside (broken line) the area of trichocyst
exocytosis occurred simultaneously just after trichocyst exocytosis. It should
be noted that, even in the area where trichocyst exocytosis took place (in
response to sufficient [Ca2+]i at the neighboring
basal bodies), ciliary reversal never occurred prior to trichocyst exocytosis
(triangle in Fig. 8B).
All of these results are summarized in Fig. 9, as described in the Discussion.
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Discussion |
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When the experiment was repeated in a Ca2+-containing medium,
ciliary reversal also took place (Fig.
6) at levels similar to that seen in Ca2+-chelating
medium. In both cases, the larger the area of UV application in cilia, the
greater the maximum angle of ciliary reversal (Figs
5B,
6C). These results strongly
suggest that, even when the external medium contains Ca2+, the
[Ca2+]i induced by the photolysis of NP-EGTA is
directly responsible for ciliary reversal.
The relationship between the cellular localization of the photolysis of
NP-EGTA (=[Ca2+]i) and the induction of ciliary
reversal is summarized in Fig.
9. (i) When
[Ca2+]i took place at the
cilia but not at the basal bodies, ciliary reversal was restricted to the area
of UV application (Figs 5 and
6, cf.
Fig. 9A,B). In such cases,
ciliary reversal was probably induced directly by the
[Ca2+]i in each cilium, because the reversal took
place only in cilia subjected to UV application. (ii) When
[Ca2+]i took place at the basal bodies,
trichocyst exocytosis, but not ciliary reversal, was evident in the area of UV
application, and after a slight delay ciliary reversal occurred over the
entire cell surface (Fig. 8,
cf. Fig. 9D). This ciliary
reversal over the whole cell surface was probably not induced directly by the
[Ca2+]i upon UV application, but through a signal
transduction cascade that begins with membrane depolarization due to the
trichocyst exocytosis. Trichocyst exocytosis from the anterior of the cell is
known to bring about a membrane depolarization
(Erxleben and Plattner, 1994
).
This idea is supported by the following observations: (iii) When UV light was
applied to the area containing both the basal bodies and the upper ciliary
region, cilia in the area of UV application reversed their direction prior to
trichocyst exocytosis, whereas cilia outside the area reversed their direction
concomitantly with, or just after, trichocyst exocytosis
(Fig. 7, cf.
Fig. 9C). This suggests that
ciliary reversal in the UV application area was induced directly by
[Ca2+]i, while ciliary reversal outside the area
was secondarily induced by membrane depolarization accompanying trichocyst
exocytosis. Thus it is likely that the basal bodies do not receive the
Ca2+ signal for ciliary reversal. We note that larger areas of
photolysis were associated with greater angles of ciliary reversal (Figs
5B,
6C). There are two possible
explanations for these observations. (1) The Ca2+ sensitive region
for ciliary reversal may not be restricted to the ciliary base, but may lie
over the entire cilium above the basal body. (2) Ca2+ released from
the NP-EGTA in the upper portion of the cilium may diffuse to the base,
causing
[Ca2+]i around the ciliary base, leading
subsequently to ciliary reversal. As shown in Figs
5B and
6C, ciliary reversal occurred
even when the distance between the region of the cilium irradiated with UV
light and the cell surface (l) was >6 µm. When l=6
µm, the concentration of Ca2+ at the ciliary base was estimated
to be 106.7 mol l1, assuming that all
NP-EGTA molecules in the target area released their Ca2+ upon UV
application, and that the released Ca2+ distributed itself evenly
throughout the whole cilium, including the ciliary base. This Ca2+
concentration is much lower than that required to induce ciliary reversal
(106 mol l1; Naitoh and Kaneko,
1972
,
1973
). Thus, we propose that
the Ca2+ sensitive region for ciliary reversal exists over the
entire cilium (above the basal body).
In the present study, ciliary reversal occurred in response to
[Ca2+]i even when the latter took place only at
the tip of the cilia. This result coincides well with observations that local
iontophoretic application of Ca2+ to any site along the length of
demembranated macrocilia of B. mitrata elicits oscillatory bending,
indicating that Ca2+ sensitivity extends the length of the cilia
(Tamm and Tamm, 1989
), that
Ca2+ inward current decreases upon removal of cilia and recovers
accompanying ciliary regrowth, indicating that voltage-sensitive
Ca2+ channels are present throughout the cilia of P.
caudatum (Machemer and Ogura,
1979
), and that fluorescence of Calcium Green rises at a similar
rate along the ciliary length during ciliary reversal in Mnemiopsis
leidyi (Tamm and Terasaki,
1994
). Our findings are not necessarily consistent, however, with
the fact that iontophoretic application of Ca2+ to the ciliary base
of a permeabilized P. caudatum cell induces ciliary reversal, though
the application to the ciliary tip does not
(Hamasaki and Naitoh, 1985
).
Hamasaki and Naitoh (1985
) also
found that the ciliary reversal is always preceded by tremulous beating,
indicating that tremulous beating and ciliary reversal are probably part of a
single chain of events. They found that Ca2+ sensitivity in
producing tremulous beating is roughly equivalent between the base and the tip
in the permeabilized cilium. Thus, Ca2+ sensitivity in producing
tremulous beating, but not in ciliary reversal, in permeabilized cilia is
similar to that in ciliary reversal in living Paramecium. Thus, the
sensitivity should extend the length of the cilium beyond the basal body.
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Acknowledgments |
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Footnotes |
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