the effect of partial extraction of dynein arms on the movement of

J. Cell Sd. 13, 337-357 (i973)
337
Printed in Great Britain
THE EFFECT OF PARTIAL EXTRACTION OF
DYNEIN ARMS ON THE MOVEMENT OF
REACTIVATED SEA-URCHIN SPERM
BARBARA H. GIBBONS AND I. R. GIBBONS
Pacific Biomedical Research Center, University of Hawaii,
41 Ahui Street, Honolulu, Hawaii 96813, U.S.A.
SUMMARY
Sea-urchin sperm were extracted with 0-5 M KC1 for 45 s at room temperature in the
presence of Triton X-ioo, and then transferred to reactivating solution containing 1 mM ATP.
The flagellar beat frequency of these KCl-extracted sperm (16 beats/s) was only about half
that of control Triton-extracted sperm that had not been exposed to 0-5 M KC1 (31 beats/s),
although the form of their bending waves was not significantly altered. Examination by electron microscopy showed that the extraction with 0-5 M KC1 removed the majority of the
outer arms from the doublet tubules, leaving the inner arms apparently intact. By varying
the duration of the KCl-extraction, it was shown that the rate of decrease in beat frequency
paralleled the rate of disappearance of the arms. Prolonging the extraction time beyond 45 3
at room temperature, or 4 min at o CC, had little further effect on beat frequency. ATPase
measurements suggested that 60—65 % of th e dynein in the original axonemes had been solubilized
when the extraction with KC1 was permitted to go to completion. These results indicate that
the generation and propagation of flagellar bending waves of essentially typical form are not
prevented by the removal of the outer row of dynein arms from the doublet tubules. In terms
of the sliding filament model of flagellar bending, the results suggest that the rate of sliding
between tubules under these conditions is proportional to the number of dynein arms present.
The lack of significant change in wave form implies that the total amount of sliding that occurs
during each bending cycle is not affected by the reduced number of dynein arms, but is regulated
independently in some manner by the elastic forces generated by other structures in the bent
axoneme.
INTRODUCTION
It has recently been demonstrated that ATP induces active sliding movements
between adjacent doublet tubules in trypsin-treated axonemes of sea-urchin sperm
flagella (Summers & Gibbons, 1971). Since the chemical specificity requirements for
generating this sliding closely resemble those for reactivating normal bending waves
in demembranated sperm that have not been exposed to trypsin (Gibbons, B. H. &
Gibbons, 1972), it seems likely that the same ATP-induced shearing force is involved
in both cases. These findings, together with the earlier report of Satir (1968) that
the overall length of the tubules appears to remain constant during bending, strongly
support a sliding-tubule model offlagellarmovement, in which the normal propagated
bending waves are the result of localized sliding movements between tubules (Summers & Gibbons, 1971). Several theoretical models have been proposed to explain
how such localized sliding might generate propagated bending waves (Brokaw, 1971,
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5. H. Gibbons and I. R. Gibbons
1972; Rikmenspoel, 1971; Lubliner & Blum, 1972). However, although the experimental basis of the sliding-tubule model is now well established, there has been
little direct evidence to indicate how ATP generates the sliding movements between
tubules, and how these movements are coordinated to produce propagated bending
waves.
Several lines of indirect evidence have suggested that the arms on the doublet
tubules may be involved in the transduction of chemical to mechanical energy. The
ATPase protein, dynein, has been localized in the arms (Gibbons, I. R., 1965 a). The
conditions necessary for dynein ATPase activity resemble those for the reactivation
of motility in demembranated cilia andflagella(Brokaw, 1961; Gibbons, I. R., 1965A;
Raff & Blum, 1969; Gibbons, B. H. & Gibbons, 1972; Gibbons, I. R. & Fronk, 1972;
Ogawa & Mohri, 1972). Motility of demembranated sperm is accompanied by a rapid
hydrolysis of the ATP in the medium, with about 70% of this ATPase activity
being tightly coupled to motility (Brokaw & Benedict, 1968a, b; Gibbons, B. H. &
Gibbons, 1972), and treatment of the sperm with agents that uncouple ATP hydrolysis from motility results in a substantial change in the enzymic properties of dynein
(Brokaw & Benedict, 1971; Gibbons, I. R. & Fronk, 1972). The above evidence all
tends to implicate the dynein arms in the mechanism of motility, but it is insufficient
to define the nature of their role.
In this paper we report the effects of partial removal of dynein arms on the beat
frequency and waveform of reactivated sea-urchin sperm. Our results provide direct
evidence for an active role of the dynein arms in generating the sliding movements
between tubules.
MATERIALS AND METHODS
Sperm of the sea urchin Colobocentrotus atratus were collected by injecting 0-5 M KCI
into the body of the animals. The undiluted semen with a covering layer of seawater was
stored at 4 °C. For use, semen was diluted with 0-5-2 vol. of seawater to give a protein concentration of 15-30 mg/ml, and stored at o °C.
All preparations of sperm were extracted with a solution containing Triton X-100 in order
to remove their membranes, as described in detail below. The concentration of KCI in this
extraction solution was made 0-5 M to obtain preparations of' KCl-extracted sperm' from which
the dynein had been partially extracted, and was 0-15 M in control preparations where no
extraction of the dynein was desired.
The standard preparations of KCl-extracted sperm were made by adding a 50-fil sample
of the diluted sperm suspension in seawater to o-6 ml of extracting solution containing 0-04 %
(w/v) Triton X-100, 0-5 M KCI, 4 mM MgSO 4 , 0-5 mM ethylenediaminetetra-acetate (EDTA)
1 mM dithiothreitol (DTT), and 2 mM tris-hydroxymethylaminomethane (Tris)-HCl buffer,
p H 8-o, at room temperature, and gently mixing for 45 8. The extraction was then terminated
by diluting the sperm suspension into a relatively large volume of reactivating solution containing 0-15 M KCI, 2 mM MgSO 4 , 0-5 mM EDTA, 1 mM D T T , 1 mM ATP, 2 % polyethylene
glycol (mol. wt. = 2 0 0 0 0 ) , 20 mM Tris-HCl buffer, p H 8-o, at room temperature. When
required by particular experiments, the conditions of extraction were varied as described in
the Results. On some occasions, the extraction was terminated by centrifuging the sperm
suspension, and resuspending the pellet in reactivating solution.
In control preparations, the composition of the extracting solution was the same as above
except that the concentration of KCI was reduced to 0-15 M. Extraction was performed for
about 30 s at room temperature. The conditions of extraction and reactivation are essentially
the same as those described previously (Gibbons, B. H. & Gibbons, 1972).
Partial extraction of dynein arms
3 39
Sperm were observed at room temperature (23-26 °C) by dark-field light microscopy in
buffered reactivating solution, in glass Petri dishes, or in trough slides 1 mm deep. The beat
frequencies of the sperm tails were measured with a stroboscopic flash unit (ChadwickHelmuth Co., Monrovia, California). Most measurements were performed on sperm which
were attached to the bottom of the dish by the tip of their heads. Measured values of beat
frequency were corrected to a standard temperature of 25 °C as described earlier (Gibbons,
B. H. & Gibbons, 1972). Each value given represents the average for at least 15 sperm in a
preparation. Michaelis constants for frequency were calculated by the weighted least squares
procedure of Wilkinson (1961).
Samples of sperm to be examined by electron microscopy were collected by centrifuging
the suspension of sperm in reactivating solution at 12000 g for 5 min. The resultant pellets
were fixed in a solution containing 0-15 M KCl, 2 mil MgSO 4 , 10 mM phosphate buffer and
2 % glutaraldehyde, p H 7-8. After 1 h, the pellets were washed 3 times in the same buffer
without glutaraldehyde, and then postfixed with 1 % osmium tetroxide. The pellets were
dehydrated with acetone, embedded in Araldite epoxy resin, and thin-sectioned. Sections
were stained first with uranyl acetate, and then with lead citrate. Electron micrographs were
taken with a Philips EM 300 at 80 kV.
An enzymic method was used to estimate what fraction of the dynein was removed from the
axonemes by the standard KCl extraction. Previous work has shown that the enzymic properties of dynein change when it is extracted from the axonemes into solution. However, if the
ATPase assays are performed in 0-05 M KCl at pH 8-o in the absence of Triton, the specific
activity of dynein remains unchanged upon extraction, so that the relative ATPase activity
in different fractions provides an approximate measure of the quantity of dynein present
(Gibbons, I. R. & Fronk, 1972). Since this assay requires the absence of Triton, the standard
KCl-extraction procedure was modified by first treating the sperm with a solution containing
0 0 4 % Triton, 0-15 M KCl, 4 mM MgSO 4 , 1 mM D T T , and 0 5 mM E D T A to remove the
membranes, and then centrifuging at 800 g for 5 min. The pellet of demembranated sperm
was suspended in modified KCl-extracting solution containing 0-5 M KCl, 2 mM MgSO 4 ,
1 mM D T T , 0-5 mM EDTA, 0 1 mM A T P and 2 mM Tris-HCl buffer, pH 8 0 . After 1 min,
the extraction was terminated by centrifuging at 3000 g for 5 min, and the resulting supernatant and pellet assayed for ATPase activity using an assay solution containing 0 0 5 M KCl,
2 mM MgSO 4 , 1 mM ATP, 0-5 mM EDTA, and 100 fig oligomycin in the pH-stat at pH 8 0
(Gibbons, B. H. & Gibbons, 1972; Gibbons, I. R. & Fronk, 1972). Appropriate controls were
performed by diluting a small sample of the KCl-extracted sperm into regular reactivating
solution and measuring their beat frequency, and by fixing a pellet of the extracted sperm for
electron microscopy, in order to ensure that the extraction had proceeded normally. Sources
of chemicals and the preparation of seawater were the same as given in our earlier paper
(Gibbons, B. H. & Gibbons, 1972).
RESULTS
Structure of control and KCl-extracted sperm flagella
As reported earlier, treatment of the sperm with 0-04% Triton X-100 removes
the flagellar membrane, leaving the resultant naked axoneme accessible to exogenous
ATP. In the control sperm (Fig. 3), the structure of the axoneme appears essentially
intact, and the 2 dynein arms are present on almost all of the outer doublet tubules.
However, brief extraction of the sperm with 0-5 M KCl causes a characteristic change
in the structure of the axoneme, with the outer dynein arms being removed from
most of the doublet tubules, while the inner dynein arms remain apparently intact.
This change in structure is seen most clearly when the KCl-extraction is terminated
by centrifugation, for in this case the solubilized dynein has no opportunity to become
rebound when the extracted sperm are suspended in reactivating solution prior to
fixation. In such centrifuged preparations almost all the outer dynein arms appear
340
B. H. Gibbons and I. R. Gibbons
to have been removed (Fig. 4). The KCl-extraction appears to cause no significant
disruption or extraction of the other structures of the axoneme, such as the inner
arms, the radial spokes, the central sheath, and the central and outer tubules. This
effect of KCl-extraction on the axonemal structure is essentially the same as that
reported previously by Gibbons, I. R. & Fronk (1972), although a much longer
extraction time of 30 min was used in this earlier work.
In preparations where the KCl-extraction is terminated by dilution, about 75 %
of the solubilized dynein becomes rebound to the sperm when the salt concentration
is lowered (see below). However, under our conditions, most of this dynein appears
to be bound with little specificity. Electron micrographs of these preparations show
(Fig. 5) an average of only about one normally positioned outer arm per axonemal
cross-section. Most axonemes appear to possess, in addition, some outer arms of
low density that point in an abnormal direction away from the axonemal cylinder
and there is also irregular flurry material associated with some of the tubules. This
fluffy material and the abnormally directed arms are not present in the control or the
centrifuged preparations of sperm (Figs. 3, 4), and they presumably represent dynein
that has rebound non-specifically or in an incorrect location. The results presented
in the next section indicate that this non-specifically bound dynein is functionally
inactive.
Motility of KCl-extracted sperm
The sperm that have been extracted with 0-5 M KC1 can be reactivated with ATP
in a manner similar to that of the control sperm. After being transferred to reactivating solution containing 1 mM ATP, the KCl-extracted sperm immediately begin
swimming through the medium, and soon accumulate at the bottom of the dish and
at the meniscus where they mostly move in repeated circles. Many of the sperm
become attached to the glass by the tip of their head, while their tail continues to
beat, parallel and close to the glass surface. This general pattern of movement is
similar to that of live sperm (Gray, 1955) and of control reactivated sperm (Gibbons,
B. H. & Gibbons, 1972). Both the control and the KCl-extracted preparations show
a high degree of uniformity, with 95-100% of the sperm becoming motile.
The most immediately apparent and striking difference between the movement
of the KCl-extracted and the control sperm is in beat frequency. The flagella of the
KCl-extracted sperm beat at an average frequency of about 16 beats/s in reactivating
solution containing 1 mM ATP at 25 °C, while those of the control sperm beat at
31 beats/s under the same conditions. The reduction in beat frequency resulting from
KCl-extraction is highly uniform, and the frequencies of individual sperm within a
preparation show a scatter of only about ±10%, which is about the same as that in
preparations of control sperm. The reduction is also highly reproducible from one
preparation to another, and in 13 preparations the average frequency has ranged
from 14-8 to 16-8 beats/s, while that of the corresponding preparations of control
sperm ranged from 28 to 32 beats/s. This uniformity and reproducibility result from
the fact that the beat frequency of the KCl-extracted sperm is not sensitive to small
variations in the extraction conditions. For example, prolonging the duration of
Partial extraction of dynein arms
341
extraction from the usual 45 s up to 5 min did not cause any significant further
decrease in frequency.
In spite of the considerable difference in beat frequency between KCl-extracted
and control sperm, there is remarkably little difference in the form of their bending
waves (Figs. 6, 7). The slight differences between them do not exceed the magnitude
of the differences between individual sperm in a preparation of a given type.
This similarity in waveform is consistently observed so long as the duration of
the KCl-extraction is kept brief (i.e. less than 1 min). After longer extraction times,
however, the waveform of KCl-extracted sperm tends to deteriorate, with the bending
waves dying away before they propagate the full length of the axoneme. After 2 min
extraction, the waves usually die away about half way along the axoneme, and the
distal portion appears stiff. A similar abnormal waveform has been observed previously in some preparations of reactivated sperm which have not been extracted
with 0-5 M KCl, and it is believed to be due partly to oxidation of axonemal sulphydryl
groups, since it develops more quickly when DTT is omitted from the reactivating
solution (Gibbons, B. H. & Gibbons, 1972). Whether the apparent distal stiffness
that develops during the longer KCl extraction is also due to oxidation is not known,
but it was not prevented by using more highly purified KCl or by increasing the DTT
concentration to 50 mM. Whatever may be the cause of this change in waveform,
its effect can be avoided by using a suitably short extraction time, 45 s in our standard
procedure.
Although prolonged KCl extraction causes a pronounced decrease in wave amplitude toward the distal region of the axoneme, this change in waveform does not appear
to result in a significant change in beat frequency. The frequency of sperm which
have been extracted for 45 s and have normal waveform is essentially the same as
the frequency of sperm extracted for 5 min which have quite abnormal waveforms.
We have compared the beating of KCl-extracted sperm in which the extraction
was terminated by centrifugation with that of sperm in which the extraction was
terminated by dilution. When the durations of the extraction were matched as
nearly a? possible, the waveforms of the 2 groups of sperm appeared identical, while
the beat frequency of the centrifuged sperm averaged 14-8 beats/s as compared to
16-0 beats/s for the diluted sperm. This small difference in frequency (8%) was
only marginally significant, indicating that only a minor fraction of the dynein that
becomes rebound on dilution is functionally active. In our standard procedure, we
chose to terminate the extraction with KCl by dilution because this permitted us to
use shorter and more accurately defined extraction times.
Effect of variation in KCl-extraction time upon beat frequency and axonemal structure
We wished to make detailed studies of the changes in beat frequency and axonemal
structure as a function of the time of extraction with 0-5 M KCl. For these experiments, we slowed the process down by extracting at o °C because at room temperature
the extraction process was largely complete within 20-25 s> a n ^ it was not feasible
to make reliable observations at shorter times. In order to compensate for the change
in pH that results from the temperature coefficient of Tris-buffer, the pH of the
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B. H. Gibbons and I. R. Gibbons
2
3
4
5
6
7
8
Extraction time, min
Fig. i. Effect of time of KCl-extraction at o °C on the fiagellar beat frequency
(O
O), and on the number of arms remaining on the outer doublet tubules
(0
%). For both curves, the values obtained with control reactivated sperm
were taken as the zero extraction time points, i.e. 31 beats/s for frequency, and
18 arms. The data for other times were then expressed as percentages of these values.
All frequencies were measured at room temperature and corrected to 25 °C. Each
point on the curve for number of arms remaining represents the average count for
30-50 axonemal cross-sections, except the 4-min point which represents the average
of 12 sections.
extracting solution, measured at room temperature, was lowered from 8-o to 7-2.
Control experiments showed that demembranation of the sperm flagella by Triton
was complete within 30 s at o °C.
Fig. 1 gives the results of an experiment in which samples of sperm were extracted
with 0-5 M KC1 at o °C for times ranging from 25 s to 8 min. At the end of the
appropriate extraction time, the sperm were diluted into reactivating solution at
room temperature. A portion of the diluted suspension was examined by light microscopy for measurement of beat frequency; the remainder of the suspension was
centrifuged, and the resultant pellet fixed immediately for electron microscopy.
The observations show that at o °C about 4 min extraction are required for the beat
frequency to reach its lower level of about 16 beats/s, and that the frequency then
remains approximately constant with longer extraction times up to 8 min. As before,
the beat frequency was not affected by the gradual change in waveform at the distal
ends of the flagella after prolonged extraction. It is notable that although the speed
of extraction is considerably slower at o °C than at room temperature, the final
frequency of 16 beats/s is the same in both cases, with more prolonged extraction
causing little further decrease in frequency.
Examination of electron micrographs of these same preparations showed that
Partial extraction of dynein arms
343
20
1/ATP, mri
Fig. 2. Double-reciprocal plots for the variation of beat frequency with ATP concentration for control reactivated sperm ( # ) , and standard KCl-extracted sperm (O).
Values for the effective 'Michaelis constant' and maximal frequency were calculated
using the weighted least-squares procedure of Wilkinson (1961).
there is a rapid decrease in the number of outer arms present on the doublet tubules
as the KCl-extraction progresses (Fig. 8). In order to obtain unbiased counts of the
number of outer arms remaining after different extraction times, the micrographs
were all mixed together and identified by code numbers, before the arms were
counted. An arm of normal position and density was counted as one; an arm of
normal position but of lower density than normal was counted as half; arms that
were misplaced or misdirected were not counted at all. Stubby arms of about half
normal length were observed quite frequently in partially extracted axonemes, and
these also were not counted. The results of these counts were tabulated, and plotted
together with the frequency data derived from the same preparation of extracted
sperm (Fig. 1). Within the limits of experimental error, the rate of disappearance
of the arms parallels the rate of decrease in beat frequency. In both cases, the process
is about half complete after 1 min, and essentially fully complete after 4 min, with
no significant change occurring during the next 4 min. A repeat of this whole experiment with another batch of sperm gave essentially the same result.
Determination of the effective Michaelis constant for frequency, Klllf
The beat frequency of reactivated sperm varies with the concentration of ATP
in the medium, and double-reciprocal plots of beat frequency against ATP concentration are linear (Brokaw, 1967). The effective 'Michaelis constant' thus obtained
for beat frequency has been denoted Kmf, and is about 0-2 mM for reactivated sperm
(Gibbons, B. H. & Gibbons, 1972). In the present work we have determined Km/
for standard KCl-extracted sperm (Fig. 2) and also repeated measurements of Kinf
for control reactivated sperm. For KCl-extracted sperm, the average values from 2
344
B- H. Gibbons and I. R. Gibbons
separate preparations were o-i4mM + o-oi and 17-7 beats/s + o-3, for Kmj and frequencymax, respectively. For the control reactivated sperm, equivalent values were
0-21 mM + o-oi and 36-2 beats/s + o-6. Thus, at high ATP concentration the beat
frequency of KCl-extracted sperm is half that of control sperm, while at low ATP
concentration the KCl-extraction causes a relatively smaller decrease in frequency
(Fig. 2).
As the ATP concentration was decreased below o-i mM, the shape of the bending
wave of the KCl-extracted sperm showed less resemblance to that of the control
reactivated sperm than at the higher ATP concentrations. Under these conditions
the speed of forward progression of the sperm head became less uniform than usual,
with a marked forward surge at one particular stage of the beat cycle and little forward
movement occurring during the rest of the cycle.
Estimation of the fraction of dynein extracted by KCl
The fraction of dynein solubilized by 0-5 M KCl was estimated by determining
the relative ATPase activity of the supernatant and pellet obtained by centrifugation
of the extracted sperm suspension. The supernatant of solubilized dynein was
assayed directly in the pH-stat as described in Methods. The pellet of KCl-extracted
sperm was resuspended in KCl-extraction solution, homogenized to prevent motility,
and then assayed for ATPase activity by the same procedure. The results obtained
from 2 of these experiments showed that 60-65 % °f the total ATPase activity was
in the supernatant of the KCl-extraction, with the other 35-40 % remaining associated
with the pellet.
A similar procedure has also been used to estimate how much of the ATPase
activity that is solubilized by the KCl extraction becomes rebound to the sperm
when the KCl concentration is lowered by dilution. In this case, the KCl extraction
was terminated by diluting the suspension into a relatively large volume of pH-stat
assay solution with a final KCl concentration of 0-05 M. After about 10 min, the
suspension was centrifuged at 27 000 g for 5 min, and the ATPase activity remaining
in the supernatant was assayed. This ATPase activity was less than the supernatant
activity when the KCl-extraction was terminated by centrifugation as described
above, and the difference provided a measure of the amount of ATPase activity that
was rebound to the axonemes upon dilution. The average results from 2 experiments
indicated that about 75 % of the solubilized ATPase activity became rebound on
dilution.
To the extent that the amount of ATPase activity can be equated with the quantity
of dynein present, these results indicate that 60—65 % °f the dynein is present in
the outer arms and is solubilized by KCl extraction. The rebinding experiments
indicate that about 75 % of this solubilized dynein becomes rebound to the sperm
when the KCl extraction is terminated by dilution. However, the total number of
enzyme units present did not remain constant upon extraction in these experiments as
they did in the earlier experiments with isolated axonemes (Gibbons, I. R. & Fronk, 1972).
For this reason, the above figures for dynein must be regarded only as approximate,
and they require confirmation by other methods. (See note added in proof, p. 351.)
Partial extraction of dynein arms
345
Effects of changes in extraction conditions
Our standard extraction conditions, using 0-5 M KC1 for 45 s at room temperature,
are such as to mask 2 factors which appear to be necessary for a successful extraction.
In our early experiments, we obtained occasional preparations of KCl-extracted
sperm in which the flagellar bending waves were highly asymmetric. These sperm
swam in circles of such narrow diameter as to make the measurement of beat frequency and observation of waveform impossible. This abnormal movement was
traced to a requirement for a minimum level of 1 mM Ca2+ during the KC1 extraction.
For reasons that remain to be determined, the absence of a sufficient concentration
of Ca2+ during the extraction leads to formation of highly asymmetric bending waves
when the sperm are subsequently reactivated. When the relative volumes of sperm in
seawater and of KCl-extraction solution are as given above, an adequate concentration
of Ca2+ is provided by the seawater. Under other extraction conditions, it may be
necessary to add additional CaCla to the extraction solution to bring the Ca2+ concentration up to 1 mM. The presence or absence of Ca2+ in the extracting solution
containing 0-15 M KC1 had no apparent effect on the waveform of the control reactivated sperm.
A second factor which appears necessary for a good extraction is the presence of
ATP in the extracting solution. This requirement was revealed in the experiments
in which it was necessary to remove the Triton before the dynein was extracted. In
these experiments, the sperm were first demembranated with Triton solution containing 0-1511 KC1, then centrifuged, and resuspended in extracting solution containing 0-5 M KC1 but no Triton. After extraction for 1 min at room temperature
under such conditions we found that the reactivated sperm had frequencies of about
25 beats/s, about 80% that of the controls. Examination of electron micrographs
showed that more than half of the outer arms were still present on the doublet tubules,
indicating incomplete extraction of the dynein. We found that this problem could
be cured by adding o-i mM ATP to the extraction solution. With this addition, the
dynein extracted as readily as in the standard one-step procedure where the initial
extracting solution contains both Triton and 0-5 M KC1. The requirement is not
apparent in this standard procedure because the ATP contained within the live
sperm contributes a concentration of about 0-05 M ATP to the extracting solution
(Gibbons, B. H. & Gibbons, 1972), and this is apparently sufficient to facilitate the
extraction.
In addition to investigating the effects of Ca2+ and of ATP, we have performed a
limited number of experiments to determine the effects of other changes in extraction
conditions.
There is a fairly sharply defined minimum effective concentration for the KC1 in
the extraction solution. If this concentration is lowered from the usual 0-5 M to
0-4 M, then little extraction of dynein occurs within a i-min extraction period, and
the frequency of the reactivated sperm is only slightly lower than that of the controls.
On the other hand, increasing the KC1 within the range 0-5-0-6 M does not result in
any significant increase in the amount of dynein extracted, and the frequency remains
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B. H. Gibbons and I. R. Gibbons
constant at about half the value of control sperm. The fact that increasing the KC1
concentration above 0-5 M, and prolonging the duration of extraction in 0-5 M KC1
beyond 45 s at room temperature, have no further effect on beat frequency or on the
number of dynein arms removed indicates that the inner and outer dynein arms
differ considerably in their extractability, with the inner arms being almost completely resistant to solubilization by KC1 under these conditions. The use of higher
concentrations of KC1 than o-6 M proved impractical, because they caused extraction
of DNA from the sperm heads.
Tests of the extraction efficiency of other salts indicated that NaCl behaved essentially identically to KC1. The minimum effective concentration was about 0-5 M,
and extraction for 45 s at room temperature reduced the frequency by half without
changing the form of the bending waves. The minimum effective concentration of
LiCl was also about 0-5 M, but slight modification of the extraction procedure was
required to get optimum results in this case. The proportion of seawater to extracting
solution had to be increased, so that o-i ml of sperm suspension in seawater was
added to o-6 ml of extracting solution, and the time of extraction had to be limited
to 30 s at room temperature. Under these conditions, the frequency was halved and
the waveform remained normal. If the extraction was prolonged to 2 min, the sperm
were only 50% motile and had an average frequency of about 10 beats/s when initially
diluted into reactivating solution, but the percentage of motility increased up to nearly
100% within, a few minutes. The third salt tested was Na2SO4, but it appeared to
give inferior results; a concentration of only about 0-35 M was necessary to reduce
the beat frequency of the sperm, but even this concentration caused some extraction
of DNA from the sperm heads.
We have made some preliminary experiments using more vigorous extracting
conditions in an attempt to solubilize the inner dynein arms and obtain frequencies
less than half those of the control sperm. As mentioned above, we obtained frequencies of about 10 beats/s, 30% that of the controls, after extracting with 0-5 M
LiCl for 2 min at room temperature (24 °C). Approximately the same results were
obtained by extracting with 0-5 M LiCl for 30 s at 33 °C, and by extracting with
0-5 M NaCl, pH 9-5 for 1 min at 24 °C. However, in all cases, the frequencies were
less uniform than normal, and many sperm showed abnormal waveforms. More
detailed experiments will be needed to determine whether this further decrease in
frequency results from partial extraction of the inner dynein arms.
Dynein arms as cross-bridges between doublet tubules
In electron micrographs of flagella and cilia, the dynein arms appear as a double
row of projections extending from the A-tubule of each outer doublet toward the
B-tubule of the adjacent doublet. In most previously published micrographs, the arms
do not usually appear to bridge completely the space between tubules, and there
is a gap 5-7 nm wide between the end of an arm and the B-tubule of the next doublet
(Gibbons, I. R. & Grimstone, i960; Allen, 1968; Warner, 1970). Only in a few cases
have the arms been observed to form a complete cross-bridge between tubules, and
this bridge was present at only one particular position, between tubules numbers
Partial extraction of dynein arms
347
5 and 6 (Afzelius, 1959; Gibbons, I. R., 1961; the system of numbering the tubules
is given in these references).
During the present studies, we have incidentally observed that in many of our
preparations the axonemes show a substantial number of dynein arms forming
complete cross-bridges between tubules. In a typical preparation (Fig. 9), counts of
12 randomly selected sections showed an average of 4-5 cross-bridges per axonemal
cross-section. The occurrence of the bridges was not equally frequent at all positions,
but most commonly there would be 2 groups of tubules bridged together, one group
consisting of about 4 tubules around positions 4, 5, 6, 7, and the other group of about
3 tubules around positions 1, 2, 3. However, this pattern was not invariant, and 1
or 3 groups of bridged tubules were observed in some sections. The most common
position for a bridge was between tubules 5 and 6, where it was present in almost
all sections. The least common positions were between tubules 3 and 4 and between
tubules 7, 8, and 9. The average spacing between tubules appears to be rather less
when a bridge is present than when it is not, suggesting that formation of a crossbridge is associated with a moving together of the affected doublet tubules. However,
the measurements show appreciable scatter, possibly as a result of the distortions
introduced during sectioning of the specimen for microscopy, and the possibility of
some change in the length of the dynein arms cannot be excluded.
It must be noted that the average number of cross-bridges varied significantly
from one preparation of sperm to another. In occasional preparations, such as the
one illustrated in Fig. 10, there was only an average of about o-6 cross-bridge per
axonemal cross-section. We have not yet been able to determine whether this variation results from differences in physiological state of the sperm at the time of fixation,
or from small inadvertent changes in the technique of fixation. Until the factors
causing this variability are resolved, any interpretation of the significance of the
cross-bridges must be made with considerable caution. Nevertheless, we believe that
these preliminary results constitute evidence that the dynein arms do form crossbridges between tubules at some stage of their cyclic activity, and that, in favourable
conditions, these cross-bridges can be preserved by fixation for examination by
electron microscopy.
DISCUSSION
Our results confirm the previous report (Gibbons, I. R. & Fronk, 1972) that the
dynein from axonemes of sea-urchin sperm consists of 2 distinct fractions which
differ substantially in extractability, the more readily extracted fraction being located
in the outer arm3 on the doublet tubules, and the less readily extracted fraction
presumably being located in the inner arms. When the extraction of the axonemes
with 0-5 M KC1 is permitted to go to completion, essentially all the outer arms are
removed from the doublet tubules, while the inner arms and all the other axonemal
structures remain apparently intact. Preliminary enzymic measurements suggest
that about 60 % of the dynein is extracted from the axonemes under these conditions, although this value needs to be confirmed by other more direct methods. The
348
B. H. Gibbons and I. R. Gibbons
relationship of these two solubility fractions of dynein to those obtained by dialysis of
scallop gill cilia (Linck, 1970), and to the 14s and 30s dynein fractions obtained from
Tetrahymena cilia (Gibbons, I. R., 1966), remains to be determined.
The most striking and important of our new observations is that the KCl-extracted
sperm, with their outer dynein arms removed, can be reactivated with 1 mM ATP to
produce bending waves whose shape is essentially the same as that of control demembranated sperm, but whose frequency is only half that of the controls.
When the KC1 extraction is terminated before completion, the decrease in beat
frequency is approximately proportional to the fraction of total number of arms that
has been removed. After complete extraction, both the beat frequency and the
number of arms are reduced to about half of their original values. Prolonging the
extraction time beyond 45 s at room temperature, or 4 min at o °C, has little further
effect on the beat frequency or on the number of arms remaining. This parallel
relationship of beat frequency and number of dynein arms, together with the fact
that no other structural component of the axoneme appears to be affected by the
KC1 extraction, and that dynein appears to be the only protein present in significant
quantity in electrophoresis gels of the fraction solubilized by KC1 under these
conditions (H. L. Kincaid, B. H. Gibbons & I. R. Gibbons, unpublished results),
strongly suggests that the decrease in beat frequency is a direct result of the reduced
number of dynein arms on the tubules. When interpreted in terms of the slidingtubule model of motility, it implies that the rate of sliding between tubules, under
these conditions, is proportional to the number of functional dynein arms present.
The inner and outer dynein arms have been shown to differ in fine structure
(Allen, 1968), and as we have seen they differ substantially in their extractability,
but in spite of these differences, our results suggest that the inner and outer arms
are functionally equivalent in their role of inducing sliding between tubules. Since
bending waves of normal form can be produced when only the inner arms are present,
it is probable that any polarity which the arms may possess with respect to the
proximal-distal axis of the tubules lies in the same direction for both inner and outer
arms.
The formation and propagation of planar bending waves implies that some mechanism exists to control and coordinate the amount of sliding that occurs between
particular doublet tubules at different stages of the bending cycle. The geometrical
relationships are such that, if the tubules are inextensible, the amount of sliding
between tubules in a bent region of the axoneme is proportional to the angle of the
bend, and is independent of the curvature. The bend angles usually observed are
about 1-5-2-0 radians, and bends of tiiis magnitude require a sliding of about 200300 nm between tubules on opposite sides of the axoneme, with at least 50-75 nm
between adjacent doublets. The production of bending waves of normal form when
the outer arms have been removed indicates that the total amount of sliding between
tubules that occurs during each cycle of bending is not affected by the removal of
these arms, or by the reduced speed of sliding. This finding suggests that the mechanism regulating the amount of sliding involves elastic forces resulting from the
strain of some axonemal component other than the dynein arms, and also suggests
Partial extraction of dynein arms
349
that the mechanism is relatively independent of forces resulting from the internal
and external viscous resistances. The latter conclusion is further supported by the
fact that the waveform undergoes little change when the beat frequency is changed
10-fold by varying the ATP concentration (Gibbons, B. H. & Gibbons, 1972).
The details of the mechanism regulating the sliding between tubules are not yet
known, but since the plane of the bending waves is correlated with and perpendicular
to the plane of the 2 central tubules (Gibbons, I. R., 1961; Tamm & Horridge, 1970),
it seems possible that the central tubules and sheath, together with the spokes that
connect them to the doublet tubules, are involved in the coordination process.
The proportionality between beat frequency and number of arms is observed only
at high ATP concentrations, such as the 1 mM used in our standard reactivating
solution. At low concentrations of ATP, the decrease in the number of dynein arms
has relatively less effect on the frequency (Fig. 2). It is not known whether the
diminished effect of the decreased number of arms results from the fact that the
amount of viscous mechanical work performed by the flagellum per cycle of bending
decreases as the beat frequency falls at low ATP concentrations, or whether it results
from the changed waveform under these conditions. The former hypothesis would
be analogous to the situation in a muscle sarcomere, where the number of ATP
molecules hydrolysed for a given amount of shortening decreases as the load on the
muscle is decreased (Kushmerick & Davies, 1969).
When the KC1 extractions were terminated by dilution, about 75 % of the extracted
dynein became rebound to the axonemes. However, very little of this rebound dynein
regained its functional activity, for the beat frequency of sperm in which the extraction was terminated by dilution was only about 8 % greater than when the extraction
was terminated by centrifugation to remove the solubilized dynein. This slight
difference in beat frequency is consistent with the fact that the diluted sperm contained an average of about one normally positioned outer arm per axonemal crosssection, while the centrifuged sperm contained almost none. The greater part of
the dynein which rebinds upon dilution presumably does so in a relatively nonspecific manner, and it probably accounts for the misplaced arms and fluffy material
visible around the axonemes in these preparations. It is also possible that some
dynein becomes bound to the sperm heads. The overall percentage of dynein that
becomes rebound is similar to that reported previously for sea-urchin sperm by
Ogawa & Mohri (1972) and by Hayashi & Higashi-Fujime (1972), but these workers
did not examine the functional activity of the rebound dynein. The rebinding of
dynein to flagella of sea-urchin sperm appears to occur with less specificity than it
does to cilia of Tetrahymena, where about 60% of the arms are restored to their
original positions, and functional activity is substantially regained (Gibbons, I. R.,
1965 a, c). The lower specificity of rebinding to sea-urchin flagella may result from
the fact that this dynein is solubilized wholly in the 14s monomeric form (Gibbons,
I. R., 1965a; Gibbons, I. R. & Fronk, 1972), while it is the 30s polymeric form that
binds best in Tetrahymena cilia (Gibbons, I. R., 1965 a). However, the possibility
that greater specificity of rebinding to sea-urchin flagella could be achieved by
varying the experimental conditions merits further study.
350
B. H. Gibbons and I. R. Gibbons
Quite apart from these detailed considerations, our general finding of a relationship
between the removal of dynein arms and the decrease in beat frequency provides
direct evidence for a functional role of the arms in the mechanism of motility. Taken
together with the previous evidence for the similarity of the conditions necessary
for enzymic activity of dynein to the conditions necessary for reactivating motility
in demembranated cilia and flagella (Brokaw, 1961; Gibbons, I. R., 19656, 1966;
Raff & Blum, 1969; Gibbons, B. H. & Gibbons, 1972; Gibbons, I. R. & Fronk,
1972; Ogawa & Mohri, 1972) and for inducing sliding between tubules in trypsintreated flagella (Summers & Gibbons, 1971), it strongly suggests that the dynein
arms are responsible for generating the shearing force that leads to sliding between
tubules. Such a role for the dynein arms implies that the arms on one doublet are
capable of binding chemically to specific sites on the B-tubule of the adjacent
doublet, and thus forming cross-bridges between the 2 doublets. Our electronmicroscopic evidence indicates that it is possible, under favourable circumstances,
to preserve these cross-bridges for examination by electron microscopy. Further
study of the factors controlling and coordinating the formation of these crossbridges may provide information concerning the mechanism by which the sliding
between tubules is coordinated to produce and propagate the bending waves characteristic of normal motility.
We are grateful to Mrs Lina Guillory for her skilful help with the electron microscopy and
to Dr Charles Lindemann for his comments on the manuscript. This work has been supported
in part by NIH grants GM 15090 and HD 06565.
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Biol. 25, 400—402.
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GIBBONS, I. R. (1965 c). An effect of A T P on light scattered by suspensions of cilia. J. Cell Biol.
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GIBBONS, I. R. (1966). Studies on the adenosine triphosphatase activity of 14S and 30S dynein
from cilia of Tetrahymena. J. biol. Chem. 241, 5590—5596.
GIBBONS, I. R. & FRONK, E. (1972). Some properties of bound and soluble dynein from sea
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{Received 3 January 1973)
NOTE ADDED IN PROOF
Recent quantitative analysis by gel electrophoresis has shown that extraction of
isolated flagellar axonemes with 0-5 M salt solubilizes about hah0 of the A-electrophoretic component of the dynein, while the other half of the A-component and all
of the B-component remain bound to the extracted axonemes. Thus, the fraction of
dynein solubilized does not appear simply to parallel the solubilization of ATPase
activity (KINCAID, H. L., GIBBONS, B. H. & GIBBONS, I. R. (1973). The salt-extractable fraction of dynein from sea-urchin sperm flagella: an analysis by gel electrophoresis and by ATPase activity. J. Supramol. Struct, (in Press).)
B, H. Gibbons and I. R. Gibbons
Fig. 3. Electron micrograph of control reactivated sperm. This field was selected to
show cross-sections of flagellar axonemes, and it does not happen to contain any of
the sperm heads in the preparation, x 100 000.
Partial extraction of dynein arms
353
Fig. 4. Electron micrograph of KCl-extracted sperm from a preparation in which the
extraction was terminated by centrifugation. x 100000.
Fig. 5. Electron micrograph of KCl-extracted sperm from a preparation in which the
extraction was terminated by dilution. The distorted axoneme near the centre of the
field probably represents a developmental abnormality, x 100000.
23
c EL 13
354
B. H. Gibbons and I. R. Gibbons
Fig. 6. Dark-field light micrographs of a control reactivated sperm in buffered reactivating solution. The micrographs, which are all of the same sperm, have been
arranged into a series to illustrate the form of the bending waves as they propagate.
The sperm was circling at the underside of the coverglass with its tail beating
parallel and close to the glass surface. Flash exposure; magnification x iooo. Beat
frequency was 31/s.
Fig. 7. Same as Fig. 6, but showing a KCl-extracted sperm. Beat frequency was 16/s.
Partial extraction of iyfiein arms
355
23-2
356
B. H. Gibbons and I. R. Gibbons
Fig. 8. Typical examples of the cross-sections of sperm axonemes which were used
for counting the number of arms remaining after extraction with 0-5 M KC1 at o °C.
The top row show control reactivated sperm, which represent zero extraction time.
The sections shown in the middle row are from sperm which have been extracted
with KC1 for 1 min, and those in the bottom row have been extracted for 8 min.
x 110000.
Fig. 9. Electron micrographs showing cross-sections of 2 flagella in a preparation of
control reactivated sperm The demembranated sperm were suspended in reactivating
solution containing 1 mM ATP, and centrifuged to form a pellet. Fixation was accomplished by layering 2 % glutaraldehyde solution over the pellet (see Methods),
x 110000.
Fig. 10. Same as Fig. 9, but a different preparation of sperm. The demembranated
sperm were centrifuged into a pellet from Triton-extraction solution, x 110000.
Partial extraction of dynein arms
\
8
10