Department of Biological Sciences, Graduate School of Science, University
of Tokyo, Hongo, Tokyo 113-0033, Japan
* Present address: ERATO Kusumi Membrane Organizer Project, JST, Kumazaki bldg.
5-11-33 Chiyoda, Naka-Ku, Nagoya 460-0012, Japan
Author for correspondence (e-mail:
chikako{at}biol.s.u-tokyo.ac.jp)
Accepted 21 December 2002
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Summary |
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Key words: Dynein, Sliding velocity, Sliding pattern, 9+2 structure, Elastase
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Introduction |
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The central pair of microtubules (central apparatus) and the radial spokes
are thought to form a complex (CP/RS) that regulates the activity of dynein
arms. The rotation of the central pair during ciliary and flagellar beating
suggests that the central pair might act as a `distributor' to regulate the
activity of dynein arms (Omoto et al.,
1999). The CP/RS is also implicated in the
Ca2+-dependent conversion of the waveforms. In
Chlamydomonas, the flagella of mutants lacking the CP/RS do not beat,
even though their axonemes are capable of sliding disintegration
(Witman et al., 1978
;
Kamiya, 2002
). At low ATP
concentrations (<20 µM), however, the CP/RS-deficient axonemes are
capable of beating and, in response to Ca2+, convert the waveform
from an asymmetrical (at <106 M Ca2+) to a
symmetrical (at
105 M Ca2+) pattern, although
they are non-beating at high ATP concentrations
(Wakabayashi et al., 1997
). At
physiological concentrations of ATP (
1 mM), the central-pair microtubules
are probably involved in the response to Ca2+ in the wild type
(Hosokawa and Miki-Noumura,
1987
) as well as in the CP/RS mutants of Chlamydomonas
(Smith, 2002b
). The
ATP-dependent role of the CP/RS in the Ca2+ regulation is also
observed in sea urchin sperm flagella. Based on the analysis of flagellar
waveforms of flagella `beating' under imposed head vibration
(Gibbons et al., 1987
), we
showed that Ca2+ decreased the velocity of microtubule sliding
through a trypsin-sensitive regulatory mechanism, which possibly involves the
central-pair microtubules (Bannai et al.,
2000
). This was also supported by the artificially induced
rotation of the Ca2+-induced asymmetrical bending pattern. The
rotation of the beating plane under imposed head vibration occurred only at
high ATP concentrations (
100 µM), indicating that the regulation of
dynein activity through the central pair occurs only at high ATP
concentrations (Bannai et al.,
2000
). These studies suggest that, at least at ATP concentrations
higher than
100 µM, high concentrations of Ca2+ modify the
regulatory signal from the central pair, which is mediated by the radial
spokes to control the activity of the dynein arms.
In this study, we tested the idea that Ca2+ alters the dynein
activity, using a novel microtubule sliding assay. As the protease necessary
to initiate sliding between the doublets, we used elastase
(Brokaw, 1980) instead of
trypsin, although trypsin is more widely used, to digest axonemal structures
that restrict free sliding of the doublets
(Summers and Gibbons, 1971
).
Unlike the trypsin-treated axonemes, which do not show oscillatory bending
movements in response to local, repetitive application of ATP, the
elastase-treated axonemes were capable of oscillatory bending movements,
indicating that they retained certain regulatory mechanisms for producing
local cyclical bending (Shingyoji and
Takahashi, 1995
).
When the elastase-treated axonemes were exposed to physiological concentrations (e.g. 1 mM) of ATP, they split lengthwise into two unequal microtubule bundles: a thicker bundle that contained the central-pair microtubules and a thinner one that did not. We found that 104 M Ca2+ did not affect the velocity at which the two bundles slid along each other during the splitting, although it affected the patterns into which the axonemes split. Electron microscopic analysis of the split bundles showed that the axonemes were split into a pair of bundles at some preferred interdoublet sites that were close to either of the central pair.
To study the effect of the central pair on the dynein activity at high
Ca2+, we used the microtubule sliding assay developed by Yoshimura
and Shingyoji (Yoshimura and Shingyoji,
1999), which was a modification of the method of Shingyoji et al.
(Shingyoji et al., 1998
). In
the present study, we analysed the behaviour of singlet microtubules that were
made to interact with either the thicker or the thinner bundles obtained from
the splitting of the axonemes. We found that the velocity of microtubule
sliding on the thicker bundles, which contained the central-pair microtubules,
was significantly decreased by 107-104 M
Ca2+. The frequency of microtubule sliding on the bundles was
significantly reduced by the presence of the central pair but was not affected
by Ca2+ except at 104 M. These results indicate
that Ca2+ alters the sliding activity of dynein arms in flagella
through the CP/RS complex.
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Materials and Methods |
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The demembranated sperm were fragmented by passing a sperm suspension
through a 22-gauge hypodermic needle (for sliding disintegration experiments
and electron microscopy) or by homogenizing the sperm suspension (for new
sliding assay experiments). Sperm heads were removed by centrifugation at
2,000-4000 g; the supernatant containing axonemal fragments
was centrifuged at 17,000-20,000 g and resuspended in
reactivating solution (without ATP). For observation of the sliding at 1 mM
ATP (0.6-0.9 mM MgATP) and 104 M Ca2+, ATP and
CaCl2 were added to the reactivating solution following
calculations according to Goldstein
(Goldstein, 1979).
For the new sliding assay experiments, demembranated sperm were labelled
with tetramethylrhodamine before fragmentation, and singlet microtubules were
assembled from tetramethylrhodamine-labelled bovine tubulin according to
Yoshimura and Shingyoji (Yoshimura and
Shingyoji, 1999). Strongly labelled singlet microtubules were
distinguished from weakly labelled fragmented axonemes by adjusting the
illumination.
Observation of sliding disintegration and microtubule sliding
Sliding disintegration of doublet microtubules was observed by the method
described by Takahashi et al. (Takahashi
et al., 1982). The axonemal fragments in ATP-free reactivating
solutions containing or not containing CaCl2 were first placed
between two coverslips separated by two strips of plastic adhesive tape used
as spacers, thus forming a chamber that was open on two sides. They were then
perfused with the reactivating solution containing the same concentration of
CaCl2 and 20 µM or 1 mM ATP. Finally, they were perfused with a
reactivating solution containing elastase (5 µg ml1
elastase, Sigma type III and 5 µg ml1 trypsin inhibitor,
Sigma type I-S) as well as the same concentrations of CaCl2 and
ATP.
Observation was made using an inverted microscope (Nikon, TMD) fitted with a 40x objective lens (Nikon), a dark-field condenser (Nikon, NA=0.95-0.80), heat-absorbing filters and a halogen lamp. Sliding disintegration was recorded on videotape using a CCD camera (Hamamatsu Photonics, C2400-77) and a U-matic videocassette recorder (Sony, VO-5800), enhancing the contrast of the image with a control unit (Hamamatsu Photonics, ARGUS-10) and a CCD camera unit (Hamamatsu Photonics, C2400). To analyse the sliding velocity, the recorded images were imported into a personal computer by using LF-3 Scientific Frame Grabber (Scion Corporation) and the displacement of the doublet microtubule was measured by using the NIH Image software. The sliding velocity was determined by plotting the displacement against time and applying the least-squares method to the plots.
For the observation of microtubule sliding on the doublet bundles in the
new sliding assay experiment, we first induced sliding disintegration of
elastase-treated rhodamine-labelled axonemes by the same procedure as used in
the previous study (Yoshimura and
Shingyoji, 1999), except for the following: the two sides of the
slide of the 5 µl chamber were sealed with adhesive plastic tape instead of
enamel in order to obtain a constant depth of the perfusion solution. A
suspension of axonemal fragments was first introduced into the chamber and
treated with elastase (5 µg ml1 elastase, Sigma type III
and 5 µg ml1 trypsin inhibitor, Sigma type I-S, in the
reactivating solution containing 104 M Ca2+
without ATP) for 1-1.5 minutes. The elastase treatment was stopped with
ovoinhibitor (50 µg ml1 ovoinhibitor, Sigma type IV-O, in
reactivating solution containing 104 M Ca2+
without ATP), and followed by perfusion with casein (
1 mg
ml1 in reactivating solution containing
104 M Ca2+ without ATP). The perfusion of
reactivating solution containing 104 M Ca2+ and 1
mM ATP (0.6 mM MgATP) then induced sliding disintegration of the axonemal
fragments into two parts. As the next step, to reduce the Ca2+
concentration, the assay buffer (see below) without EGTA was perfused followed
by perfusion of the assay buffer containing 1 mM ATP (0.9 mM MgATP) with or
without Ca2+. The rhodamine-labelled microtubules were suspended in
the assay buffer to which 2% (v/v) ß-mercaptoethanol, 40 mM glucose, 430
µg ml1 glucose oxidase, 70 µg ml1
catalase, 10 µM Taxol and the same concentration of MgATP and
Ca2+ had been added. The assay buffer contained 70 mM potassium
acetate, 5 mM magnesium acetate, 20 mM HEPES, 2 mM EGTA, 0.1 mM
ethylenediaminetetraacetic acid (EDTA) and 1 mM DTT, pH 7.8. The
concentrations of Ca2+ in the solution used for the observation of
microtubule sliding were <109 M, 107 M,
105 M and 104 M.
Microtubule sliding on doublet bundles was observed under a fluorescent microscope (Olympus, BX 60) with a 100x oil-immersion objective lens (Olympus PlanApo, NA=1.4) and recorded on videotape using a high-sensitivity silicon-intensified target camera (SIT camera, Hamamatsu Photonics, C2400-08) and a VHS videocassette recorder. To analyse the sliding velocity, the video images were traced by hand from the screen of a video monitor onto a sheet of transparent film. The sliding velocity was determined from the time measured by counting the number of video fields and the distance of microtubule movement measured on the traced images.
Electron microscopy
The axonemal fragments were resuspended in the reactivating solution
containing 1 mM ATP (0.6 mM MgATP) and Ca2+ (104
M, 103 M or <109 M) and incubated at
20°C for 5 minutes. After reactivation, the axonemes were digested with
elastase (5 µg ml1 elastase, Sigma type III and 5 µg
ml1 trypsin inhibitor, Sigma type I-S) for 7 minutes. To
stop the digestion, glutaraldehyde was added to a final concentration of 2% on
ice. After 15 minutes fixation at 10°C, the axonemes were pelleted by
centrifugation at 28,000 g for 10 minutes at 4°C. The pelleted
samples were fixed with 2% glutaraldehyde in the rinse buffer (0.15 M
potassium acetate, 25 mM MgSO4 and 10 mM phosphate buffer, pH 8.0)
for 1 hour at 0°C. Then the samples were washed with the rinse buffer,
postfixed in 1% OsO4 in the rinse buffer for 45 minutes at 0°C,
dehydrated in a graded ethanol series and embedded in Epon 812 (TAAB).
Silver-gold sections were cut with a glass knife on Sorvall, MT2-B
ultramicrotome, collected on copper grids, stained with uranyl acetate and
lead citrate, and observed at 80 kV with a JEOL JEM100CX electron
microscope.
When the sliding pattern of disintegrated axonemes was studied, we examined
thicker bundles consisting of doublets and the central pair. We used the
conventional numbering system for the doublets originally proposed by Afzelius
(Afzelius, 1959). The doublet
microtubules in the thicker bundles were identified from the position of the
5-6 bridge and the orientation of the central pair, but we could not identify
all the doublets of the thinner bundles. The identification of microtubules in
the thicker bundles based on the position of the 5-6 bridge and the
orientation of the central pair would be appropriate, because the central pair
of P. depressus flagella does not rotate with respect to the
peripheral doublet microtubules (Takahashi
et al., 1991
). In order to identify the sliding pattern of thicker
bundles, we photographed randomly selected cross sections of thicker bundles
at a magnification of 50,000x. The photographs were labelled with code
numbers and shuffled before the identification to avoid subjectivity in the
judgement.
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Results |
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The effect of Ca2+ on the sliding velocity observed in the
elastase-treated axonemes that slid into paired bundles at 1 mM ATP (0.6 mM
MgATP) is summarized in Fig. 2.
The average sliding velocities were 9.8±2.8 µm
second1 (mean±s.d., n=45) and 9.2±2.5
µm second1 (n=53) at low
(<109 M) (Fig.
2A) and high (104 M)
(Fig. 2B) concentrations of
Ca2+, respectively. The sliding velocities at low and high
Ca2+ concentrations were not significantly different
(P>0.2, MannWhitney U test). This seems to accord
with previous reports that Ca2+ does not affect the sliding
velocity in trypsin-treated axonemes
(Walter and Satir, 1979;
Mogami and Takahashi, 1983
;
Okagaki and Kamiya, 1986
;
Vale and Toyoshima, 1989
). The
following experiments, however, show that microtubule sliding in flagellar
axonemes is regulated by Ca2+. They also give us a clue to
understanding why the velocity of sliding disintegration in the
elastase-treated axonemes is apparently not affected by Ca2+.
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Splitting patterns of doublet bundles
When elastase-treated axonemes split into thinner and thicker bundles, the
thickness of these bundles were clearly distinguished one from the other by
dark-field microscopy, suggesting that the number of doublets in each of the
two kinds of bundles are somewhat fixed. Electron microscopy of thin-sectioned
bundles revealed that the thicker bundles consisted of five or six doublets
and the central pair, whereas the thinner bundles consisted of four or fewer
doublets and were without the central pair. Because the doublet microtubules
do not rotate around the central pair in the species we used
(Takahashi et al., 1991 for
Pseudocentrotus; unpublished data for Clypeaster), we were
able to identify each of the doublets in the thicker bundles from its position
relative to the 5-6 bridge and the orientation of the central pair
(Fig. 3A,B).
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We recognized eight patterns of cross section among the bundles that contained the central pair. Of these, four patterns composed more than 90% of the bundles that were obtained at low concentrations of Ca2+ (Fig. 3C). For simplicity, we will hereafter call, for example, the cross-sectional pattern of a thicker bundle that contains the doublets 8, 9, 1, 2, 3 and the central pair a `8-3 pattern'. According to this convention, the four main patterns of thicker bundles obtained at <109 M Ca2+ were 8-4, 8-3, 4-8 and 3-8 (Fig. 3A,B). The thinner bundles corresponding to the 8-4 and 8-3 patterns were assumed to consist of doublets 5-7 and 4-7, respectively (Fig. 3B). Lacking the central pair, they could be identified by the presence of the 5-6 bridge. The thinner bundles corresponding to the 4-8 and 3-8 patterns were assumed to be doublets 9-3 and 9-2, respectively. There were also bundles of three or four doublets that contained neither the 5-6 bridge nor the central pair. In these bundles, we could not identify the individual doublets.
The splitting pattern was affected by Ca2+. As the concentration of Ca2+ increased, the frequency of 4-8 patterns gradually decreased so that at 106-105 M Ca2+, other three patterns (8-4, 8-3 and 3-8) were mainly observed. Similarly, at Ca2+ concentrations higher than 104 M, the frequency of 3-8 decreased so that at 103 M Ca2+, we found mainly two patterns, 8-4 and 8-3. At Ca2+ concentrations lower than 107 M, the frequencies of 8-4 and 8-3 combined were about the same as those of 4-8 and 3-8 put together but, at 103 M Ca2+, 8-4 plus 8-3 occurred more than seven times as frequently as did 4-8 plus 3-8 (Fig. 3C).
To induce the four patterns of thicker bundles, the dynein arms on the doublets 7, 3 and 2 of the thinner bundles and/or those on the doublets 3, 4 and 8 of the thicker bundles should be active during the sliding separation of the axonemes. To determine the doublets whose dynein arms were active, we examined the sliding direction of the thinner bundles with respect to the thicker bundles along which they slid, using elastase-treated axonemes that still had the sperm heads attached to them (Fig. 4). Demembranated sperm with the heads were placed on the glass surface of the perfusion chamber. Under a dark-field microscope, we cut each flagellum carefully into two or three short lengths using a glass microneedle without changing the orientation of the fragmented flagellum with respect to the head (Fig. 4, upper panel). When reactivating solution containing elastase, 1 mM ATP, and either 103 M or <109 M Ca2+ was perfused, sliding disintegration of the fragments was induced as a bundle of doublets slid over the remaining part of the axoneme.
|
We found that, in most cases, the thinner bundles moved toward the head (or the thicker bundles moved away from the head) (Fig. 4, lower panel). Movements in this direction were observed in 84% of the axonemal fragments examined at <109 M Ca2+ and this rate increased to 92% at 103 M Ca2+ (Table 1). Because the basal end of the axoneme is the `minus', or more stable, end of the microtubule, and dynein is a minus-end-directed motor, the above observation indicates that the dynein arms on the thinner bundles were mainly active to induce the separation of the axonemes, regardless of the concentration of Ca2+. We suggest that, at low concentrations of Ca2+, the dynein arms on doublets 7, 3 and 2 of the thinner bundles are mainly active to induce the 8-4, 8-3, 4-8 and 3-8 patterns, and that, at high concentration of Ca2+ (103-104 M), the dynein arms on doublet 7 of the thinner bundles are mainly active to induce the 8-4 and 8-3 patterns (Fig. 3). This implies that the activity of the dynein arms of the thicker bundles is inhibited.
|
Microtubule sliding on thinner and thicker bundles in an improved
assay system
To understand the role of the central pair in the regulation of the dynein
activity, we examined the effect of Ca2+ on microtubule sliding on
bundles with or without the central pair. The thinner and thicker bundles were
obtained from elastase-treated axonemes by application of 1 mM ATP in the
presence of 104 M Ca2+. When singlet microtubules
were added to the split bundles, we observed sliding of the singlet
microtubules not only on the thinner bundles but also on the thicker bundles
at <109 M-104 M Ca2+
(Fig. 5). This is apparently
inconsistent with the above suggestion that the dynein arms on the thinner
bundles are mainly active when the axonemes slid into two bundles
(Table 1). To see what exactly
happens, we analysed the behaviour of microtubules on the bundles.
|
Before describing the behaviour of microtubules, some improvements in our
assay method should be realized. In the previous study
(Yoshimura and Shingyoji,
1999), in which we developed the new sliding assay system,
microtubule sliding was not always smooth. In the present study, we reduced
the flow rate and kept a constant depth of the perfusion in order to maintain
the activity of dynein arms that were exposed on the doublets. After these
improvements, the behaviour of microtubules on the bundles changed. First, the
number of bundles supporting active microtubule sliding increased (from 30% in
the original method to >50% in the new method). Second, the back-and-forth
movements of microtubules observed in the previous study were not observed in
the present study, whereas a different type of back-and-forth movements was
observed. Third, microtubules slid on thinner and thicker bundles at
significantly different speeds in the previous study, but no such difference
was found in the present study.
Frequency of microtubule sliding on thinner and thicker bundles
Fig. 6A summarizes the
behaviour of microtubules attached on the thinner and thicker bundles. Because
only one of the doublets of the bundles has dynein arms exposed
(Fig. 3), orientations of
doublet bundles on the glass surface are important to determine whether
singlet microtubules are able to interact with the bundles. Thinner bundles
showed straight and curved configuration, while thicker bundles were almost
straight. The number of bundles to which microtubules attached and the number
of microtubules attached to the bundles are shown in the right-hand column of
Fig. 6A. The difference in
configuration of bundles is probably related to the different numbers of
thinner and thicker bundles to which microtubules attached. Comparison of the
number of bundles with the number of microtubules, however, indicates that the
thicker bundles had lower affinity to microtubules than the thinner bundles.
The frequency of microtubule sliding was about 30-40% on the thinner bundles
but was less than 20% (at <109-105 M
Ca2+) and 9% (at 104 M Ca2+) on the
thicker bundles. Fig. 6B shows
ratio of the number of occurrences of microtubule sliding on the thinner
bundles to that on the thicker bundles. At Ca2+ concentration
105 M, the ratio was about 4 but, at
104 M Ca2+, it increased to 9.6, indicating that
the sliding on the thicker bundles occurs one tenth as frequently as that on
the thinner bundles in the presence of 104 M
Ca2+.
|
In the present improved sliding assay system, the microtubule sliding was smooth on both the thinner and the thicker bundles. But a back-and-forth movement with a distance of about 0.5-1 µm was observed only on curved regions along the thinner bundles at fewer than 10% of the microtubules attached to the bundles (hatched boxes in Fig. 6A). We need more detail analysis of the back-and-forth movement to know whether it is related to the regulation of dynein activity.
Velocity of microtubule sliding on thinner and thicker bundles
Most of the microtubules moved smoothly throughout the sliding, and this
smooth movement was not changed by the presence of Ca2+
(Fig. 7). The time course of
microtubule sliding on the thinner bundles and that on the thicker bundles was
similar in the present assay system (Fig.
7A). An increase of Ca2+ concentration, however,
affected the velocity of microtubule sliding on the thicker bundles
(Fig. 7B).
|
Fig. 8 summarizes the velocity of microtubule sliding at 1 mM ATP (0.9 mM MgATP) on the thinner (left panels) and the thicker (right panels) bundles at lower (A) and higher (B) concentrations of Ca2+. The sliding velocities did not show a Gaussian distribution but showed a broad distribution with a peak at around the middle and a longer tail towards higher sliding velocities (Fig. 8). At <109 M Ca2+, the distribution of sliding velocity was similar on the thinner and on the thicker bundles (Fig. 8A). At 107-104 M Ca2+, the distribution of the sliding velocities on the thinner bundles was also similar to that at low concentration of Ca2+, whereas that on the thicker bundles shifted towards the lower velocities. Statistical differences between the sliding velocities on the thinner and the thicker bundles at <109 and at 107-104 M Ca2+ were examined by using the MannWhitney U test. We found that the sliding velocity on the thinner bundles and that on the thicker bundles were not different at low concentrations of Ca2+ but were significantly different in the presence of Ca2+ (P<0.01). At any Ca2+ concentration of 107-104 M, the sliding velocity on the thicker bundles was lower than that on the thinner bundles. This showed clearly that the sliding velocity decreases with Ca2+ and the regulation of dynein activity by Ca2+ requires the presence of the central pair microtubules.
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Discussion |
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The Ca2+-induced changes in the microtubule sliding velocity had
not been demonstrated until our previous work using beating flagella
(Bannai et al., 2000). In
earlier studies, Ca2+ was shown to affect neither the sliding
disintegration of axonemes treated with trypsin (
1 µg
ml1) (Walter and Satir,
1979
; Mogami and Takahashi,
1983
; Okagaki and Kamiya,
1986
) nor the gliding of microtubules on isolated axonemal dynein
(Vale and Toyoshima, 1989
).
More recently, we found that trypsin digested some axonemal structures
responsible for cyclical bending whereas elastase did not
(Shingyoji and Takahashi,
1995
). The present study using elastase-treated axonemes has
demonstrated that the regulatory mechanism, which is necessary for cyclical
bending and is retained in the elastase-treated axonemes, is also essential
for the Ca2+ regulation of the microtubule-sliding activity of
dynein. This regulatory mechanism requires the presence of the central-pair
microtubules and the functional dynein arms that are attached to their natural
location on the doublet. These results are consistent with the previous
studies suggesting involvement of the central-pair microtubules in the
Ca2+ regulation of flagellar bending movement
(Sale, 1986
;
Bannai et al., 2000
).
The present study has shown that Ca2+ causes three major changes in the dynein activity: (1) a decrease in the velocity of microtubule sliding on thicker bundles; (2) a decrease in the frequency of microtubule sliding on thicker bundles; and (3) changes in the splitting patterns of axonemes, which seem to correspond to the sites of active sliding among the axonemal microtubules. These changes were all closely associated with the presence of the central-pair microtubules but were induced at different concentrations of Ca2+. This suggests that the three kinds of response to Ca2+ might be regulated by different mechanisms.
Our previous study has shown that trifluoperazine inhibits the `quiescence'
of sperm flagella, implying that Ca2+-calmodulin is involved in the
regulation of sliding that leads to the quiescence at 104 M
Ca2+ (Bannai et al.,
2000). The present study suggests that the quiescence is caused
not only by the decrease in sliding frequency but also by the decrease in
sliding velocity of dynein arms at one side (probably on doublet 3) of the
central-pair microtubules. Smith (Smith,
2002b
) has recently shown in Chlamydomonas flagella that
the microtubule sliding velocity of axonemes lacking the central pair is
reduced at <108 M Ca2+ compared with that of
the wild-type axonemes, but is restored by 104 M
Ca2+. She has also shown that calmodulin is involved in this
Ca2+ regulation of microtubule sliding. This suggests a similar
regulation of flagellar motility in both the sea urchin sperm and the
Chlamydomonas flagella.
The patterns of splitting of the elastase-treated axonemes into two doublet
bundles with or without the central pair are probably related to the mechanism
regulating the dynein activity. We have found four main patterns of splitting,
and the frequency of their appearance depended on the Ca2+
concentration. The four patterns were the thicker bundles consisting of the
8-4, 8-3, 4-8 and 3-8 doublet groups, with their corresponding thinner bundles
of the remaining four or three doublets. Spontaneous axonemal fracture into a
thinner and a thicker bundle has previously been reported in the quiescent sea
urchin sperm flagella at high Ca2+ concentrations, in which the 8-3
pattern of the thicker bundles was dominant
(Sale, 1986). Holwill and
Satir (Holwill and Satir,
1994
) have developed a physical model of axonemal splitting based
on the observation of splitting of the axonemes, including six-and-three
doublet groups and five-and-four doublet groups. This model provides a
physical basis for the axonemal splitting that was observed in the present
study.
Our analysis of the splitting patterns provided some interesting findings.
The dynein arms on the thinner bundles, but not those on the thicker bundles,
were active to produce microtubule sliding and to split the axonemes into
paired bundles. Furthermore, we found that the occurrence of the 8-4 and 8-3
patterns and of the 4-8 and 3-8 patterns were similar at lower concentrations
of Ca2+, whereas the 8-4 and 8-3 patterns increased and the 4-8 and
3-8 patterns decreased with an increase of the Ca2+ concentration.
These changes in the frequency of occurrence might be related to the waveforms
of reactivated flagella: the flagellar beating is nearly symmetrical at lower
concentrations of Ca2+, becoming increasingly asymmetrical with an
increase of Ca2+ and finally becomes quiescent with a large
principal bend at the base of the flagella at 104 M
Ca2+. The increase in asymmetry and the quiescence are induced by
the inhibition of reverse-bend formation
(Brokaw, 1979
;
Gibbons and Gibbons, 1980
;
Bannai et al., 2000
). Thus, it
is likely that the occurrence of the 4-8 and 3-8 patterns, which decreases
with Ca2+ concentration, is related to the reverse-bend formation.
If this is the case, switching between the group of the 8-4 and 8-3 patterns
and the group of the 4-8 and 3-8 patterns in other words, switching of
the activity of dynein arms between doublet 7 (of thinner bundles) and doublet
3 or 2 (of the thinner bundles) would be the basis for the alternate
formation of the principal and the reverse bends.
The mechanism regulating the dynein activity to split the axoneme into a
thinner and a thicker bundle has been unclear. From the present result, we can
speculate about the roles of the two microtubules of the central pair.
Structural and biochemical differences between the C1 and C2 microtubules with
their associated projections have been described in Chlamydomonas
flagella, suggesting possible different roles of C1 and C2 in the regulatory
mechanism (Smith and Lefebvre,
1997; Mitchell and Sale,
1999
; Porter and Sale,
2000
; Smith,
2002a
). In sea urchin sperm flagella, two microtubules of the
central pair and their associated projections look similar in electron
micrographs, and their biochemical and physiological differences have not been
well documented. If we assume that the microtubules that are near doublets 7
and 3 are C1 and C2, respectively, similar to those of Chlamydomonas,
the alternate activation of dynein might be regulated through the radial
spokes by some signal from C1 and C2. The signal might be associated with a
protein phosphorylation (Yang et al.,
2000
; Roush-Gaillard et al.,
2001
), with Ca2+-calmodulin
(Bannai et al., 2000
;
Smith, 2002b
) or with changes
in the mechanical states (Omoto et al.,
1999
; Bannai et al.,
2000
).
The elastase-treated axonemes showed different sliding features with
varying ATP concentration. Low concentrations of ATP induced sliding
disintegration of the axonemes into individual doublets, indicating that the
dynein arms on any doublet of the axonemes are active. By contrast, high
concentrations of ATP induced splitting of the axonemes into two or three
bundles, indicating that the dynein arms attached to the doublets (3 or 7)
near the central pair are active but those on the remaining doublets are
inactive. These observations show that the dynein activity is inhibited at
high concentrations of ATP but that the inhibition is overridden by the CP/RS.
The inhibitory effect of a physiological concentration of ATP on the number of
doublets that slide has been demonstrated in Tetrahymena cilia by
Kinoshita et al. (Kinoshita et al.,
1995), although, in their study, the sliding was also inhibited at
low concentration of ATP, which is different from the present finding. Similar
inhibition of the outer arms by a high level of ATP has also been reported in
CP/RS-deficient Chlamydomonas mutants, in which lower concentrations
of ATP induce beating of paralysed flagella
(Omoto et al., 1996
). The
CP/RS complex is thought to override the ATP inhibition by activating the
inner arms in a coordinated fashion in Chlamydomonas
(Porter and Sale, 2000
). The
roles of ATP concentrations in the mechanism regulating the dynein activity
might be similar in cilia and flagella of different species and the CP/RS
might release the inhibitory effect of high ATP concentrations
(Bannai et al., 2000
).
The role of the CP/RS in the Ca2+ regulation of dynein activity
also depends on the ATP concentration. At low concentrations of ATP, the
axonemes of the CP/RS-deficient Chlamydomonas mutants are able to
beat and show Ca2+-dependent waveform conversion
(Wakabayashi et al., 1997). At
a high (physiological) level of ATP, however, the CP/RS is suggested to play a
key role in the Ca2+ regulation of waveform and microtubule sliding
(Hosokawa and Miki-Noumura,
1987
; Smith,
2002b
). The present result in sea urchin sperm flagella show that
the CP/RS is essential for the Ca2+ regulation of dynein activity
at the physiological level of ATP.
We thus conclude that the outer and probably also the inner dynein arms are inhibited by physiological levels of ATP and that the CP/RS complex might override the inhibition by activating both the outer and inner dynein arms. When the CP/RS complex is functional, both the C1 and C2 microtubules might alternately activate the dynein arms on the near microtubules. We also postulate that mechanism regulating the activity of dynein by C2 is sensitive to Ca2+.
Finally, we discuss the velocity of sliding of the elastase-treated axonemes to induce thicker and thinner bundles. The velocity of sliding disintegration was not affected by Ca2+ even in the elastase-treated axonemes. This is apparently inconsistent with the Ca2+-induced inhibition of dynein activity on the thicker bundles that was shown in the novel sliding assay. This inconsistency could be explained, however, if we take all results of the present study into consideration. When the elastase-treated axonemes split lengthwise into two bundles, the dynein arms on the thinner bundles are predominantly active at low as well as at high concentrations of Ca2+. This means that the velocity of sliding induced by dynein arms on the thicker bundles could not be measured in the sliding disintegration. Our study also showed that the velocity and the frequency of sliding of singlet microtubules that interacted with the dynein arms exposed on the thicker bundles, but not with those on the thinner bundles, were significantly inhibited by Ca2+. Ca2+ inhibited the activity of the dynein arms on the thicker bundles but not that on the thinner bundles, and the activity of dynein arms on the thinner bundles was required for the sliding disintegration. Thus, it is very plausible that the inhibitory effect of Ca2+ could not be detected in the sliding disintegration of the axonemes.
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