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First published online March 17, 2006
Journal of Experimental Biology 209, 1336-1343 (2006)
Published by The Company of Biologists 2006
doi: 10.1242/jeb.02131
Requirement of the fixed end for spontaneous beating in flagella
Department of Life Sciences, Graduate School of Arts and Sciences, University of Tokyo, Meguro-ku, Tokyo 153-8902, Japan
* Author for correspondence (e-mail: cokuno{at}mail.ecc.u-tokyo.ac.jp)
Accepted 26 January 2006
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Summary |
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 TOP Summary IntroductionMaterials and methodsResultsDiscussionReferences |
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Key words: flagellar movement, local inhibition, PRODAN, sea urchin
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Introduction |
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Several experiments have been performed in order to gain deeper
understanding of these mechanisms. When an actively beating sperm flagellum
was cut by a laser beam, pre-existing waves in the distal part continued to
propagate. The distal part, however, lost the ability to produce a new wave,
whereas the proximal part of flagellum connected to the head continued beating
(Goldstein, 1969
). Similar
results were obtained in short flagella dissected mechanically by
homogenization (Gibbons, 1974)
and in starfish sperm flagella arrested by a glass needle
(Okuno and Hiramoto, 1976).
These results suggest that the basal part of flagella is necessary to produce
spontaneous beating.
Every part of demembranated flagella could bend when ATP was applied
locally to flagella in the rigor state (see below) by iontophoresis
(Shingyoji et al., 1977).
Although successive pulse application of ATP did cause successive progression
of the waveform all along the flagella
(Shingyoji and Takahashi,
1995), it was not concluded that spontaneous beating can occur at
any position on the flagella because the preparation used was very different
from intact sperm flagella. In contrast to live sperm flagella, demembranated
flagella stayed in the `rigor state', in which dynein arms build tight
cross-bridges among microtubules because of the lack of ATP, except in the
area exposed to ATP in that experiment. How is the bending wave spontaneously
generated, propagated through the flagellum, and maintained stably?
In his series of computer simulation works, Brokaw demonstrated that
simulated flagella continued to develop, propagate and maintain the bending
waves under various conditions if the microtubules were tied together at one
end (Brokaw, 1986). If this is
also true for real flagella, spontaneous beating could occur at any site on
the flagellar axoneme when one end is fixed. This `basal anchoring' model is
supported by Lindemann in his `Geometric Clutch' hypothesis
(Lindemann, 1994;
Lindemann and Kanous, 1995).
Lindemann considered that basal anchoring of the flagellum provides the
tension on the doublet, and this tension is necessary to provide the
transverse force required for switching the activity of dynein. Woolley and
Bozkurt reported that dissected sperm flagella produced beating
(Woolley and Bozkurt, 1995),
although it lasted for only a short period. They also found that compression
of the proximal end of the dissected flagellum made it possible to produce
bends. Thus, they confirmed the basal region of flagellum as the fixed end
that generates resistance against sliding of microtubules. Their experiments,
however, failed to maintain the stable beating for a long time. In addition,
it was not evident that the compression of the axoneme by the microneedle
could tie 9+2 microtubules together. The bending wave shown in their paper was
of small amplitude and low beat frequency, as we previously observed with
amputated starfish sperm flagella (Okuno
and Hiramoto, 1976) compared with reactivated intact flagella. So
far, it has not been established whether any part of the flagellum has the
potential to generate and maintain a normal bending wave of large amplitude
spontaneously and stably. The necessity for the basal region of the flagellum
to act as `pacemaker-like machinery' for normal beating with high beat
frequency and large amplitude cannot be eliminated.
To solve this problem, we wished to inhibit flagellar movement locally. We aimed to develop a new technique that would introduce a fixed narrow region where sliding between microtubules could not occur. If successful, we could, for example, examine whether movement of the distal region was arrested or maintained when movement of the basal region was inhibited. If movement was arrested, then spontaneous beating must require some kind of `signal' transmitted from the `pacemaker-like machinery' at the base of the flagellum. By contrast, if movement was maintained, then there would be no need for the `pacemaker-like machinery'. Until now, however, it has been very difficult to test this idea since application of inhibitors to a limited area of flagella is technically difficult because of diffusion of the inhibitor itself.
In the present study, we identified a fluorescent reagent, PRODAN (6-propionyl-2-dimethylamino-naphtalene), which inhibits flagellar movement only after excitation by UV irradiation. PRODAN treatment and spot irradiation after excitation with UV light successfully caused a local inhibition of flagellar movement.
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Materials and methods |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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) with
a little modification. Briefly, sea urchin spermatozoa were suspended in 4
volumes of Ca2+-free artificial seawater containing 465 mmol
l–1 NaCl, 10 mmol l–1 KCl, 25 mmol
l–1 MgSO4, 0.2 mmol l–1 EDTA and
2 mmol l–1 Tris-HCl, pH 8.2. The suspended spermatozoa were
demembranated with 40 volumes of demembranating solution and reactivated with
the reactivating solution. The demembranating solution contained 0.04% (w/v)
Triton X-100, 0.2 mol l–1 potassium acetate, 2 mmol
l–1 MgCl2, 5 mmol l–1
CaCl2, 2 mmol l–1 EGTA, 2 mmol
l–1 Tris-HCl, pH 8.2), and 2 mmol l–1
dithiothreitol (DTT). The reactivating solution contained 0.2 mol
l–1 potassium acetate, 2 mmol l–1
MgCl2, 2 mmol l–1 EGTA, 20 mmol
l–1 Tris-HCl, pH 8.2, 2% (w/v) polyethylene glycol, 1 mmol
l–1 DTT and various concentrations of ATP. PRODAN (6-propionyl-2-dimethylamino-naphtalene; Molecular Probes, Eugene, OR, USA) was applied to the demembranated spermatozoa, by transferring them into the reactivation solution without ATP, then incubating with 10 µmol l–1 dye for 2 min on ice. The spermatozoa were then reactivated by adding an appropriate volume of solution containing ATP.
Observation and analysis of flagellar movement
Reactivated sperm suspension was poured into the observation chamber, which
consisted of two strips of vinyl tape on the slide glass covered with a cover
glass. The depth of the chamber was changed according to the experiment. Thin
tape was used for observation only or for perfusion experiments. Thick tape
was used for the micromanipulation experiment described later, in which a
glass microneedle was inserted through the side openings of the chamber.
Reactivated spermatozoa were observed and recorded using phase-contrast or dark-field, and fluorescence microscopy (BX-51, Olympus, Tokyo, Japan). The microscope was equipped with a video camera (63V1N, Mintron, or CR-20, Video-device, Tokyo, Japan) and a video tape recorder (BR-S800, Victor, Yokohama, Japan). The objective lens was UplanFl (40x, NA 0.75, Olympus, Tokyo, Japan). Video-recorded images were captured by Storm Video Version 1.00 (Canopus, Kobe, Japan). Shear angle was analyzed from video recordings by `Bohboh', a flagellar movement auto-analyzing software kindly provided by Dr Baba. Shear angle was defined as the angle between the tangent at the base of the flagellum and that at any point along the flagellum, and was assumed to be proportional to the amount of microtubule sliding.
Local irradiation of UV
Local UV irradiation was applied to the demembranated flagellum using
fluorescence microscopy (Olympus BX-51). The microscope was equipped with a
100 W ultra high-pressure mercury lamp (USH102D, Ushio, Tokyo, Japan) and
U-MWU2 (Olympus, Tokyo, Japan) filter-box for irradiation of UV light
(330–385 nm) to the specimen and for observation. We put a pinhole at
the position of the iris on the optical path for the mercury lamp and could
thereby irradiate UV light to a very restricted area (minimum 2.5 µm in
diameter). The strength of UV was adjusted by ND filters (U-25ND6, U-25ND25,
U-25ND50, Olympus, Tokyo, Japan).
The irradiated area was marked on a flat TV-monitor before the inhibition experiment was carried out, and the PRODAN-treated flagellum was moved to the marked area for UV irradiation of the intended portion.
Trypsin treatment of flagella
Spermatozoa were demembranated and reactivated in the presence of PRODAN.
The reactivated sperm suspension was poured into the observation chamber for
UV irradiation, after ensuring that sperm were beating in the focal plane with
the head attached to the glass surface. Then, the chamber was perfused with
reactivating solution containing 0.2 µg ml–1 trypsin.
After an appropriate time, when microtubules had completed sliding out from
the axoneme, the trypsin was washed out by reactivating solution without
trypsin, and photographs were taken in order to assess the microtubule
disintegration patterns.
Dissection of flagella
Glass microneedles were made using a pipette puller (PG-1, Narishige,
Tokyo, Japan) from thin glass rods about 1 mm in diameter, and were held by a
micromanipulator (MO-102, Narishige, Tokyo, Japan). A head-attached and
PRODAN-treated spermatozoon beating in the focal plane was displaced to almost
the center of the microscope field. After spot UV treatment, the middle of the
UV-irradiated area (the motility-inhibited region) of the flagellum was cut by
pressing a glass microneedle onto the coverslip.
Temperature
All experiments were carried out at room temperature 23±2°C.
Reagents
PRODAN was purchased from Molecular Probes; all other chemicals were from
Wako Pure Chemical Industries, Ltd (Osaka, Japan).
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Results |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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PRODAN was introduced by Weber and Farris in 1979
(Weber and Farris, 1979) and
has generally been used as a membrane surface marker. It has also been used as
a non-covalently interacting probe for proteins
(Hiratsuka, 1999). When the
solvent is water, the excitation wavelength of the dye is 361 nm and the
emission wavelength 531 nm. For UV irradiation, we employed a fluorescence
microscope. A pinhole was put at the position of the iris on the optical path
of the high pressure-mercury lamp to control the irradiation area, so that we
could change the irradiation area by exchanging the pinhole with one of a
different diameter.
Spermatozoa of the sea urchin Anthocidaris crassispina were demembranated with Triton X-100 and reactivated. The beat frequency and other features of the reactivated flagellar movement in Triton-extracted sperm were not affected by incubation with PRODAN alone (at concentrations of 10 µmol l–1 or less), since the motility of the reactivated sperm was maintained for more than 20 min without any change in wave parameters (data not shown).
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Inhibition of microtubule sliding by PRODAN-UV
Flagella lost their motility after PRODAN-UV treatment. We assumed this
loss of motility was due to inhibition of the sliding between microtubules. To
test this, we locally activated PRODAN by UV irradiation through a pinhole,
and then treated the flagellum with trypsin. Brief treatment with trypsin
should cause non-irradiated flagella to disintegrate by disrupting
cross-linking proteins such as nexin among microtubules. However, trypsin
should fail to cause this disintegration if the proteins cross-linked by
PRODAN were dynein rather than nexin, since dynein is resistant to
trypsin.
Fig. 2 shows the results of the experiment where PRODAN-UV treatment was carried out on the basal (Fig. 2A) or distal (Fig. 2B) regions of the flagellum, followed by perfusion by trypsin-containing reactivating solution. Arrowheads mark PRODAN-UV treated regions and arrows the microtubule that disintegrated.
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In both the basal and distal regions, disintegration or spreading out was not observed at PRODAN-UV treated regions. By contrast, disintegration of microtubules was observed in the non-treated regions. When the whole flagellum was treated with PRODAN-UV, no disintegration or spreading out of microtubule(s) was observed at any part of the flagellum (data not shown), which often maintained the bent shape of `rigor bends'. Therefore, we suggest that PRODAN-UV treatment inhibits sliding between microtubules by producing a tight cross-linker between microtubules.
Local inhibition of flagellar movement
We next investigated the effects of local UV irradiation on flagellar
movement. To assess the inhibition, we measured the shear angle along the
flagellum from video recordings. Shear curves represent the degree of bend in
the flagellum at various distances along its length, and thus indicate the
amount of microtubule sliding at each position (see Materials and methods).
Fig. 3 shows photographs of
wave-form and shear-curve analysis of a typical reactivated spermatozoon after
the local UV irradiation, in the presence of 15 µmol l–1
ATP. The flagellum presented symmetrical bending waves before irradiation
(Fig. 3A). When UV radiation
was localized to the 5 µm region proximal to the flagellar base, no bend
was observed in the irradiated area while the continuous beating was observed
in the distal region (Fig. 3B).
When the area of irradiation was extended to 12 µm in the same spermatozoon
(Fig. 3C), the distal intact
region still continued to generate the bending wave. The corresponding shear
curves are shown in Fig.
3D–F, respectively.
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The relationship between beat frequency and length of the beating area of flagella is shown in Fig. 4. Flagella were gradually inhibited from their basal to distal regions and the beat frequency was measured as shown in Fig. 3. We defined `movable length of flagellum' as the distance between the total length and inhibited length of the flagellum, in the presence of 10 µmol l–1 ATP. This experiment showed that the shorter the length of active flagellum, the higher the beat frequency. Therefore, it was likely that the beat frequency was determined not only by the ATP concentration but also the `movable length' of flagella.
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When the distal part of a flagellum was subjected to UV irradiation, spontaneous beating continued in the intact area between the base and the irradiated distal part of the flagellum (data not shown).
The above experiments were carried out at low ATP concentration. When the ATP concentration was increased up to about 50 µmol l–1 or more, the flagellum features were dramatically changed. Typical results are shown in Fig. 5, in which ATP concentration was 0.2 mmol l–1. When the distal part of flagella was subjected to UV irradiation, that area lost motility while the proximal region maintained motility (Fig. 5A). The result was almost equivalent to that observed at low concentrations of ATP. By contrast, when UV irradiation was performed at the proximal region, flagella lost motility in the intact distal region, as shown in Fig. 5B. Therefore, it was likely that the basal region of flagellum was necessary to generate and maintain the bending wave at high ATP concentrations.
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Mechanical dissection of basal region reveals requirement of fixed end for spontaneous beating
The above experiments clearly demonstrate that any part of a flagellum has
the potential to generate and maintain the spontaneous bending wave. However,
some previous experiments (Goldstein,
1969; Okuno and Hiramoto,
1976) have suggested the possibility that a kind of `control
center' or `pacemaker' exists at the base of flagellar axis that initiates the
periodical beating, since the proximal part of flagellum is able to continue
beating under various conditions such as cut-short or arrested flagella. In
addition, it is also possible that a signal from the base of flagellum is
transmitted to the distal area even when motility of the proximal part of
flagellum is inhibited by PRODAN-UV. We therefore performed further
experiments in which the base of the flagellum (including the head) was
dissected out from the principal part of the flagellum by a glass microneedle
in order to examine whether the fixed end is necessary for generating and
propagating the spontaneous bending wave.
Fig. 7 shows a typical result
in which the base of the flagellum was dissected out by pressing a glass
microneedle onto the coverslip. When the basal region of demembranated
flagellum was dissected out without PRODAN-UV treatment, we did not observe
spontaneous generation of sinusoidal waves (data not shown). By contrast, when
the basal region of flagella was subjected to PRODAN-UV treatment followed by
dissection at the middle of its treated region, generation and propagation of
spontaneous bending wave was maintained in the distal intact part. In
Fig. 7B, the amplitude of the
`dissected' flagellar movement was a little smaller than of the intact one
(see Fig. 3A). However, this
phenomenon was commonly seen when the basal region was inhibited as shown in
Fig. 3B,C, since the intact
length of flagella had become shorter.
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We therefore concluded that every part of a flagellum has the potential to generate and maintain the bending wave spontaneously when one end of the axoneme is fixed tightly, i.e. the bundle of microtubules was tightly cross-linked to provide an anchor point where the microtubules cannot slide.
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Discussion |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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Fluorescent reagent PRODAN as an inhibitor for flagellar movement
To achieve this local inhibition, we examined several fluorescent dyes that
inhibited flagellar movement only on excitation of the dye. Some reagents
other than PRODAN, such as 2,6-TNS (2-(p-toluidinyl)naphthalene-6-sulfonic
acid) and 1,8-ANS (1-aminonaphthalene-8-sulfonic acid), had imilar features as
inhibitors of flagellar movement. TNS and ANS are both naphthalene sulfonates
and bind to protein or membranes, like PRODAN. PRODAN, however, was the best
inhibitor because of its strong inhibition and the fact that it was harmless
to flagellar movement without UV irradiation.
Walker demonstrated a similar effect
(Walker, 1961), the
suppression of flagellar movement of a trypanosome by light irradiation and a
fluorescent dye (acriflavine). However, his experiment was done with the
intact trypanosome. Therefore, damage to the cell membrane might have caused
inhibition of motility, since he reported local inhibition only on application
of weak irradiation. High power irradiation caused complete suppression of
flagellar motility. In the present study, by contrast, we succeeded in
introducing the inhibition only at the restricted area of the axoneme
subjected to UV irradiation.
It was likely that PRODAN-UV treatment inhibited active sliding by
constructing some tight cross-linking among microtubules. This conclusion was
supported by observations that the inhibited flagella maintained a bent shape
like `rigor bends' and that the trypsinated flagella failed to disintegrate
the microtubules either with extrusion or with spreading out in the presence
of ATP (Fig. 2). What
component(s) of flagella contributed to the formation of the tight connection
among microtubules? The most plausible candidate at present could be dynein
arms since the rigor state is introduced by cross-bridged dynein arms
(Gibbons, 1975;
Okuno, 1980). Other possible
candidates might be nexin, radial spokes, and so on. If they were the cause,
however, trypsinated flagella must have disintegrated since nexin and radial
spokes are digested earlier than dynein so that dynein could work to promote
active sliding of microtubules (Summers
and Gibbons, 1971), and this should be explored using molecular
level approaches.
Local inhibition of flagellar movement reveals the necessity of a tied end in the axoneme for spontaneous flagellar movement
In the present study, we demonstrate that any part of a flagellum has the
potential to generate and maintain a bending wave when a tied end exists in
the flagellar axoneme, although cAMP was also required at high concentrations
of ATP. The basal body of the intact flagella could be substituted for the
tied end introduced by PRODAN-UV in the present experiments. These results
agree with Brokaw's provision in his computer simulation work
(Brokaw, 1986).
When the ATP concentration is high, the beat frequency of flagellar
movement is high. It is likely that flagellar beating at high frequency
requires `a coordination mechanism' that functions with the assistance of
cAMP. The requirement of cAMP for reactivation at high ATP concentrations has
been demonstrated in salmonid fish sperm
(Okuno and Morisawa, 1982) and
sea urchin sperm (Ishiguro et al.,
1982). The coordination mechanism should be distributed along the
entire length of flagellum, not at the base of it, as shown in the present
study. It could be assumed that cAMP works via A-kinase, resulting in
phosphorylation of proteins such as dynein light chains in the flagellar
axoneme (Inaba, 2003). In the
present study, however, the concentration of cAMP required for spontaneous
beating at high ATP concentration was much higher
(Fig. 6). Therefore, the effect
of cAMP might be different from the activation of A-kinase, and this requires
further investigation.
It has been discussed whether the basal region of flagellum has a
`pacemaker-like' function or acts only as a fixed end to generate basal
resistance that evokes bending (Brokaw,
1986; Lindemann,
1994). The pacemaker hypothesis is based on the results of
experiments indicating that the flagellum shows no sinusoidal beating without
its basal region. By contrast, the basal anchoring hypothesis was originally
proposed from computer simulation, since experimental evidence was not
available because of technical difficulties. In the present study, we
introduced an `artificial fixed end' into the flagellum and dissected out the
middle of it. If the basal region of a flagellum has `pacemaker-like'
function, the dissected flagellum could not maintain a bending wave because it
could not receive any kind of `signal' transmitted from the `pacemaker'. If
the basal region of a flagellum bears only a fixed end function that generates
basal resistance, the dissected flagellum should maintain its bending wave
without its basal region.
The results of the present experiments show that the latter is the answer.
A flagellum with an introduced artificial fixed end generated and maintained a
spontaneous bending wave like an `intact' demembranated flagellum. This result
agrees well with the Brokaw's computer simulation
(Brokaw, 1986) and Lindemann's
`Geometric Clutch' hypothesis (Lindemann,
1994; Lindemann and Kanous,
1995).
Factors for determining the beat frequency of flagella
It was thought that the beat frequency of flagellar movement is determined
predominantly by the ATP concentration
(Okuno and Brokaw, 1979).
However, micromanipulation studies demonstrated that the beat frequency of
flagellar movement could be regulated within the approximate range of
30–80 Hz by vibrating the micropipette holding the head of the
demembranated sea urchin sperm (Gibbons et
al., 1987; Eshel et al.,
1990). These results implied that beat frequency is not determined
only by ATP concentration. In the present study, we directly demonstrated that
beat frequency is also determined by the `movable length' of the flagellum as
well as the ATP concentration. In one flagellum, the shorter the `movable
length', the higher becomes the beat frequency. Furthermore, the sliding
velocity seemed to be approximately preserved during this procedure.
Therefore, we concluded that ATP concentration determined the sliding
velocity, not the beat frequency, which is probably determined by the sliding
velocity of microtubules, the length of the flagellum and, presumably, some
other external factor such as viscosity of the medium.
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Acknowledgments |
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