CELL STRUCTURE AND FUNCTION 25: 263–267 (2000)
© 2000 by Japan Society for Cell Biology
ADP-dependent Microtubule Translocation by Flagellar Inner-Arm Dyneins
Toshiki Yagi
Department of Biological Sciences, Graduate School of Science, University of Tokyo, Tokyo 113-0033, Japan
ABSTRACT. Previous studies have shown that the motility of flagellar and ciliary axonemes in many organisms
are influenced by the concentration of both ATP and ADP. Detergent-extracted cell models of Chlamydomonas
oda1, a mutant lacking flagellar outer-arm dynein, displayed slightly lower flagellar beating frequencies when reactivated with ATP in the presence of an ATP-regenerating system, composed of creatine phosphate and creatine
phosphokinase, than when reactivated with ATP alone. Thus, presence of a low concentration of ADP may somehow stimulate axonemal motility. To see if this motility stimulation is due to a direct effect on dynein, we analyzed
the effect of ADP on the in vitro microtubule translocation caused by isolated inner-arm dyneins in the presence of
ATP. Of the seven inner-arm dyneins (species a–g) fractionated by ion-exchange chromatography, most species
translocated microtubules at faster speed in the presence of 0.1 mM ATP and 0.1 mM ADP than in the presence of
0.1 mM ATP alone. Most notably, species a and e did not translocate microtubules at all in the presence of the
ATP-regenerating system, indicating that a trace amount of ADP is necessary for their motility. This regulation
may be effected through binding of ADP to some of the four nucleotide binding sites in each dynein heavy chain.
Key words:
Chlamydomonas/in vitro motility assay/beat frequency
although it has not yet been experimentally confirmed.
Chemical regulation, on the other hand, has been shown to
actually take place under various experimental conditions.
One type of chemical regulation is through phosphorylation
of dynein. Recent studies have indicated that specific subunits are involved in this type of regulation. In Paramecium
cilia, for example, phosphorylation of an outer-arm light
chain has been shown to increase the velocity of microtubule translocation in an in vitro motility assay system
(Hamasaki et al., 1991). In Chlamydomonas flagella,
phosphorylation of an inner-arm intermediate chain decreases sliding rates in the axoneme (Habermacher and
Sale, 1996). This phosphorylation-based regulation of
dynein activity appears to be important for regulation of
cells’ tactic behavior (King and Dutcher, 1996).
Axonemal dyneins also appear to be subject to a second
type of chemical regulation – a regulation dependent on
ATP and ADP concentrations. In experiments on reactivation of demembranated axonemes, it was often observed
that lower ATP concentrations or presence of ADP resulted
in a higher degree of reactivated motility (Lindemann and
Rikmenspoel, 1972; Brokaw and Gibbons, 1973). Even mutant axonemes that are normally non-motile can beat at low
ATP concentrations or in the simultaneous presence of ATP
and ADP (Omoto et al., 1996; Yagi and Kamiya, 1995;
Wakabayashi et al., 1997; Frey et al., 1997). Experiments
The beating of cilia and flagella is generated by organized
sliding between outer doublet microtubules. Outer and
inner dynein arms produce the force for this sliding. In
Chlamydomonas, the outer dynein arm exists as a single
protein complex containing three different dynein heavy
chains (DHCs), whereas the inner dynein arm exists as seven discrete subspecies containing at least eight different
DHCs (see Porter, 1996). Analysis of flagellar movements
in mutants lacking specific dynein species (Brokaw and Kamiya, 1987; Minoura and Kamiya, 1995), as well as in vitro
motility assays on isolated dynein (Kagami and Kamiya,
1992; Sakakibara and Nakayama, 1998), has indicated that
different dyneins cause microtubule sliding at significantly
different speed and power. For an axoneme to produce regular beating, force production by each dynein must be critically regulated.
The activity of each dynein molecule may be controlled
through both mechanical and chemical processes. Mechanical regulation of dynein activity has been postulated by
theoretical studies (see Lindemann and Kanous, 1997),
Department of Biological Sciences, Graduate School of Science,
University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
Tel/Fax: +81-3-5800-6842
E-mail: [email protected]
Abbreviations: DHC, dynein heavy chain; TAP, Tris-acetate-phosphate;
FFT, fast Fourier transform.
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T. Yagi
appropriate concentration of microtubules, and then with HMDE
containing ATP and an ATP-regenerating system (5 mM creatine
phosphate and 70 units/ml of creatine phosphokinase (Roche
Diagonostics, Tokyo, Japan)) or a combination of ATP and ADP.
The translocation of microtubules was observed with a dark-field
microscope and recorded with an SIT camera on video tapes.
Average translocation velocity was calculated by measuring
velocities of those microtubules that continued moving for at least
five seconds without interruption. More than 30 microtubules were
measured for each sample.
that monitored sliding disintegration in protease-treated axonemes have also demonstrated stimulation of sliding at
lower ATP concentrations (Warner and Zanetti, 1980;
Tanaka and Miki-Noumura, 1988; Okagaki and Kamiya,
1986) or in the presence of ADP or its analogs (Kinoshita et
al., 1995). Although the exact mechanism that causes these
nucleotide effects remains to be established, the discovery
that each dynein heavy chain has three putative nucleotide
binding sites in addition to a unique catalytic site (Gibbons
et al., 1991; Ogawa, 1991) raises the possibility that these
phenomena arise because the activity of each dynein is regulated by nucleotides bound to some of these additional
binding sites. However, few studies have been done to examine the effect of ATP/ADP concentrations on the activity
of individual dynein species. In this study, we therefore examined the effect of nucleotide concentrations on the motile
activity of isolated inner-arm dyneins. The data obtained
clearly showed that some dyneins are strongly inhibited under ADP-free conditions, suggesting that their activities are
in fact regulated by bound nucleotides.
Results
In our previous experiments with detergent-extracted cell
models, we frequently used an ATP-regenerating system
consisting of creatine phosphate (CP) and creatine phosphokinase (CPK), which enabled us to examine motility at low
ATP concentrations. However, we found that the addition
of CP/CPK to oda1 cell models reactivated at 0.1–1 mM
ATP resulted in a lower beat frequency. Although the decrease in beat frequency is only 10–20%, it was always
clearly observed when the frequency was measured by an
FFT-based method that yields the average beat frequency in
a large number of cell models (Kamiya and Hasegawa,
1987). The frequency decrease did not appear to be due to
the presence of CP or CPK itself, since these substances,
when added singly, did not lower the beat frequency so
much (Fig. 1). In addition, another ATP-regenerating system consisting of phosphoenolpyruvate and pyruvate kinase
had a similar inhibitory effect on the beat frequency (data
not shown). From these results, we concluded that the decrease in beat frequency was due to the absence of a product
Materials and Methods
Cell culture
A Chlamydomonas reinhardtii mutant lacking the entire outer arm
(oda1) (Kamiya, 1988) was used. Cells were cultured in liquid
TAP medium on a 12h/12h light/dark cycle. Cell models were
prepared by extracting the cells with 0.1% NP40 (Kamiya and
Witman, 1984).
Preparation of dynein
Flagella were isolated and demembranated as described (Witman
et al., 1978). Inner-arm dyneins were isolated by extracting the
oda1 axonemes with 0.6 M KCl. After ten-fold dilution, the sample was subjected to ion-exchange chromatography on a MonoQ
column to yield discrete subspecies (Kagami and Kamiya, 1992).
The composition of dynein heavy chains in each fraction was examined by SDS-PAGE using 3–5% polyacrylamide gels.
In vitro motility assay
Cell models were reactivated in HMDEKP (30 mM Hepes
(pH 7.4), 5 mM MgSO4, 1 mM DTT, 1 mM EGTA, and 50 mM
K-acetate, 1% polyethylene glycol (Mr 20,000)) containing ATP.
Their flagellar beat frequencies were analyzed by a Fast-FourierTransform method as described (Kamiya and Hasegawa, 1987;
Takada and Kamiya, 1997). In vitro motility assay of dynein
activity was carried out essentially according to Kagami and
Kamiya (1992). Briefly, each dynein sample was perfused into a
flow chamber made of a glass slide, a cover slip, and spacers. After
being settled for 2 min, the chamber was first perfused with
HMDE solution (30 mM Hepes (pH 7.4), 5 mM MgSO4, 1 mM
DTT, 1 mM EGTA) containing 0.5 mg/ml bovine serum albumin,
followed by perfusion with the same solution containing an
Fig. 1. Axonemal beat frequency of the oda1 cell models reactivated in
the presence of 0.5 mM ATP with (+) and without (-) creatine phosphate
(CP) and creatine phosphokinase (CPK). Each beat frequency value was
measured with an FFT analyzer using a large number of cell models and
thus can be regarded as an average frequency in a population of cells. The
bars show the standard deviation in the frequency distribution, as estimated
from the shape of the power spectrum peak (Takada and Kamiya, 1997).
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ADP Stimulation of Axonemal Dynein
of 0.1 mM potassium phosphate. These observations indicate that the inner-arm dyneins require a low concentration
of ADP for their optimal motility.
Strikingly, fractions a and e did not translocate microtubules at all when the ATP-regenerating system was present.
Since the fraction a is only negligibly contaminated by other dyneins, and the fraction e is contaminated only by species d, it is likely that the lack of motility in the presence of
the ATP-regenerating system in fractions a and e is the intrinsic feature of dyneins a and e. Furthermore, fraction a
displayed the highest velocity in the presence of 0.5 mM
ADP in addition to 0.1 mM ATP, while fraction b, d, e, g
displayed better motility with 0.1 mM ADP than with 0.5
mM ADP in accordance with the possible substrate inhibition by ADP. In the presence of ATP and CP/CPK, microtubules readily stuck to the glass surface coated with fraction
a. Under these conditions, the microtubules displayed no
noticeable back-and-forth, random movements, such as
those observed with sea urchin sperm outer-arm dyneins in
the presence of ATP and vanadate (Vale et al., 1989). The
same kind of firm attachment was observed in the absence
of ATP, i.e., under conditions where dynein presumably
forms a rigor crossbridge with the microtubule. When a solution containing 0.1 mmM ATP without CP/CPK was perfused into the same chamber, a small fraction (less than
10%) of microtubules started to display irregular movements, at velocities varying between 0.01 and 0.1 m/sec.
When the perfusion solution contained 0.1 mM ADP in addition to 0.1 mM ATP, about 50% of the microtubules became motile, with the velocity ranging from 0.05 to 4 mm/
sec. Interestingly, under the conditions where only a small
fraction of microtubules displayed translocation, short (< 10
mm) microtubules displayed movements more frequently
than long (> 40 mm) microtubules. The movements displayed by short microtubules are irregular, such that any
single microtubule moves and stops in a random manner. In
the presence of 0.1 mM ATP and 0.5 mM ADP, > 80% of
microtubules displayed translocation. The movement in this
case appeared smooth. The movement stopped again 1–2
min after the sample was perfused with 0.1 mM ATP solution with CP/CPK, while it did not stop when perfused with
the same ATP solution without CP/CPK.
In the above experiments, ATP concentration was fixed
at 0.1 mM. Naturally, however, the optimal ADP concentration for microtubule translocation depended also on the
ATP concentration. A preliminary experiment has suggested that the ADP/ATP concentration ratio is an important
factor. For example, dynein a displayed optimal motility in
the presence of 0.1 mM ATP and 0.5 mM ADP (average velocity: 2.3±1.0 mm/sec), whereas it displayed only poor motility (about 0.2 mm/sec) when the ATP concentration was
increased five-fold without changing the ADP concentration; however, it displayed good motility (1.9±1.5 mm/sec)
when the ADP concentration was also increased five-fold to
2.5 mM.
of ATP hydrolysis, possibly ADP. Such a frequency decrease by CP/CPK was observed only with mutant axonemes lacking the entire outer arm, and not with the axonemes of wild type or mutants that lack various inner-arm
dyneins. We thus speculated that this phenomenon is caused
by some change in the activity of inner-arm dynein, which
may be masked in the presence of outer arm dynein.
To see if CP/CPK and ADP cause any change in the activity of inner-arm dynein, we examined their effects on in
vitro translocation of microtubules by isolated dyneins. As
previously shown (Kagami and Kamiya, 1992), the innerarm dynein of Chlamydomonas contains seven subspecies,
designated a–g, of which all but f has a single heavy chain
while f has two heavy chains. These subspecies can be
separated by ion-exchange chromatography on a Mono-Q
column as discrete peak fractions (Fig. 2). Although some
fractions are contaminated by other species, separate experiments indicated that significant contamination occurs only
between adjacent peaks; for example, the fraction of dynein
c is contaminated by dynein d, but not by dynein a or e
(unpublished results). All these subspecies but f have been
shown to translocate microtubules in vitro (Kagami and
Kamiya, 1992). Following the previous study, we coated the
cover slip with each sample and then introduced microtubules and ATP. We found that, in all the peak fractions
but f, which showed no or extremely slow microtubule
translocation, the velocity of microtubule translocation at
0.1 mM ATP greatly decreased when CP/CPK was present.
In addition, in most cases, the velocity in the presence of 0.1
mM ATP without CP/CPK increased when 0.1 mM ADP
was added. No such increase was observed with the addition
Fig. 2. Chromatographic separation of inner-arm dyneins. High-salt extract from oda1 axonemes was diluted and subjected to a MonoQ column
(Kagami and Kamiya, 1992). Inset: a portion of SDS-PAGE pattern showing the dynein heavy chain region. Each of the 7 peak fractions (a–g) contains one (all but f) or two (f) heavy chains as a major component, although
separation was incomplete in some fractions. In particular, note that fraction a contains almost dynein a only, and fraction e contains only dynein e
and dynein d. CE denotes the crude high-salt extract.
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T. Yagi
Fig. 3. Velocity of microtubule translocation induced by different inner-arm dynein species. Each sample was perfused with a test solution containing one
of the following in addition to 0.1 mM ATP: CP/CPK and no ADP (labeled 0 (CP)); no ADP; 0.1 mM ADP, and 0.5 mM ADP. Note that microtubule translocation did not take place with the samples of dynein a and dynein e when CP and CPK were present in the medium.
quired is so small in dynein species a and e, that the transition can be inhibited only by enzymatic removal of ADP. It
is tempting to speculate that the multiple nucleotide binding
sites on dynein heavy chains are responsible for the transition between these two states. To prove this possibility, we
will need to examine mutant dyneins that lack particular nucleotide binding sites. We can expect that such mutant dyneins will soon become available since several studies have
shown that dynein heavy chains can be expressed from
cloned genes (Mazumdar et al., 1996; Koonce and Samso,
1996).
Discussion
The above observation indicates that the activity of innerarm dyneins to translocate microtubules in vitro depends on
both ATP and ADP concentrations. It is surprising that dynein a and dynein e do not display motility at all in the presence of CP/CPK, i.e., in the complete absence of ADP. The
observation that, under these conditions, the microtubules
appeared to firmly attach to the dynein-coated glass surface
suggests that dyneins form strongly-bound crossbridges that
do not move. Another observation that, at low ADP concentrations, shorter microtubules displayed translocation more
frequently than longer microtubules further suggests that
some minor population of dynein present on the glass surface inhibits translocation by forming strong crossbridges;
shorter microtubules can frequently move possibly because
they encounter these inhibitory dyneins infrequently, and
because the inhibitory state of dynein is released with time
as the kinetic state of the dynein slowly turns over; longer
microtubules cannot move possibly because they tend to be
attached by multiple numbers of inhibitory dyneins at any
instance.
Although the exact mechanism through which the absence of ADP inhibits the dynein activity remains to be elucidated, the above observations thus suggest that the dynein
can assume two states, an active state and an inhibitory
state, depending on the concentration of ATP and ADP. The
transition from the inactive state to the active requires the
presence of a trace amount of ADP; the concentration re-
Acknowledgments. The author would like to thank Professor Ritsu Kamiya for encouragement and critically reading the manuscript. This study
has been supported by a grant-in-aid from the Ministry of Education, Science, Sports and Culture of Japan.
References
Brokaw, C.J. and Gibbons, I.R. 1973. Localized activation of beating in
proximal, medial and distal regions of sea urchin sperm flagella. J. Cell
Sci., 13: 1–10.
Brokaw, C.J. and Kamiya, R. 1987. Bending patterns of Chlamydomonas
flagella: IV. Mutants with defects in inner and outer dynein arms indicate differences in dynein arm function. Cell Motil. Cytoskeleton, 8: 68–
75.
Frey, E., Brokaw, C.J., and Omoto, C.K. 1997. Reactivation at low ATP
distinguishes among classes of paralyzed flagella mutants. Cell Motil.
Cytoskeleton, 38: 91–99.
Gibbons, I.R., Gibbons, B.H., Mocz, G., and Asai, D.J. 1991. Multiple nu-
266
ADP Stimulation of Axonemal Dynein
generated by different dynein deficient Chlamydomonas mutants in viscous media Cell Motil. Cytoskeleton, 31: 130–139.
Ogawa, K. 1991. Four ATP-binding sites in the midregion of the beta
heavy chain of dynein. Nature, 352: 643–645.
Okagaki, T. and Kamiya, R. 1986. Microtubule sliding in mutant
Chlamydomonas axonemes devoid of outer or inner dynein arms. J. Cell
Biol., 103: 1895–1902.
Omoto, C.K., Yagi, T., Kurimoto, E., and Kamiya, R. 1996. Ability of paralyzed flagella mutants of Chlamydomonas to move. Cell Motil.
Cytoskeleton, 33: 88–94.
Porter, M.E. 1996. Axonemal dyneins: assembly, organization, and regulation. Curr. Opin. Cell Biol., 8: 10–17.
Sakakibara, H. and Nakayama, H. 1998. Translocation of microtubules
caused by the alphabeta, beta and gamma outer arm dynein subparticles
of Chlamydomonas. J. Cell Sci., 111: 1155–1164.
Takada, S. and Kamiya, R. 1997. Beat frequency difference between the
two flagella of Chlamydomonas depends on the attachment site of outer
dynein arms on the outerdoublet microtubules. Cell Motil. Cytoskeleton,
36: 68–75.
Tanaka, M. and Miki-Noumura, T. 1988. Stepwise sliding disintegration
of Tetrahymena ciliary axonemes at higher concentrations of ATP. Cell
Motil. Cytoskeleton, 9: 191–204.
Vale, R.D., Soll, D.R., and Gibbons, I.R. 1989. One-dimensional diffusion
of microtubules bound to flagellar dynein. Cell, 59: 915–925.
Wakabayashi, K., Yagi, T., and Kamiya, R. 1997. Ca2+-dependent waveform conversion in the flagellar axoneme of Chlamydomonas mutants
lacking the central pair/radial spoke system. Cell Motil. Cytoskeleton,
38: 22–28.
Warner, F. and Zanetti, N.C. 1980. Properties of microtubule sliding disintegration in isolated Tetrahymena cilia. J. Cell Biol., 86: 436–445.
Witman, G.B., Plummer, J., and Sander, G. 1978. Chlamydomonas flagellar mutants lacking radial spokes and central tubules. J. Cell Biol., 76:
729–747.
Yagi, T. and Kamiya, R. 1995. Novel mode of hyper-oscillation in the paralyzed axoneme of a Chlamydomonas mutant lacking the central-pair
microtubules. Cell Motil. Cytoskeleton, 31: 207–214.
cleotide-binding sites in the sequence of dynein beta heavy chain.
Nature, 352: 640–643.
Habermacher, G. and Sale, W. 1996. Regulation of flagellar dynein by
phosphorylation of a 138-kD inner arm dynein intermediate chain. J.
Cell Biol., 136: 167–176.
Hamasaki, T., Barkalow, K., Richmond, J., and Satir, P. 1991. cAMPstimulated phosphorylation of an axonemal polypeptide that copurifies
with the 22S dynein arm regulates microtubule translocation velocity
and swimming speed in Paramecium. Proc. Natl. Acad. Sci. USA, 88:
7918–7922.
Kagami, O. and Kamiya, R. 1992. Translocation and rotation of microtubules caused by multiple species of Chlamydomonas inner-arm dynein.
J. Cell. Sci., 103: 653–664.
Kamiya, R. 1988. Mutations at twelve independent loci result in absence
of outer dynein arms in Chlamydomonas reinhardtii. J. Cell Biol., 107:
2253–2258.
Kamiya, R. and Hasegawa, E. 1987. Intrinsic difference in beat frequency
between the two flagella of Chlamydomonas reinhardtii. Exp. Cell Res.,
173: 299–304.
Kamiya, R. and Witman, G.B. 1984. Submicromolar levels of calcium
control the balance of beating between the two flagella in demembranated models of Chlamydomonas. J. Cell Biol., 98: 97–107.
King, S.J. and Dutcher, S.K. 1996. Phosphoregulation of an inner dynein
arm complex in Chlamydomonas reinhardtii is altered in phototactic
mutant strains. J. Cell Biol., 136:177–191.
Kinoshita, S., Miki-Noumura, T., and Omoto, C.K. 1995. Regulatory role
of nucleotides in axonemal function. Cell Motil. Cytoskeleton, 32: 46–
54.
Koonce, M.P. and Samso, M. 1996. Overexpression of cytoplasmic
dynein’s globular head causes a collapse of the interphase microtubule
network in Dictyostelium. Mol. Biol. Cell, 7:935–948.
Lindemann, C.B. and Rikmenspoel, R. 1972. Sperm flagellar motion
maintained by ADP. Exp. Cell Res., 73: 255–259.
Lindemann, C.B. and Kanous, K.S. 1997. A model for flagellar motility.
Int. Rev. Cytol., 173: 1–72.
Mazumdar, M., Mikami, A., Gee, M.A., and Vallee, R.B. 1996. In vitro
motility from recombinant dynein heavy chain. Proc. Natl. Acad. Sci.
USA, 93: 6552–6556.
Minoura, I. and Kamiya, R. 1995. Strikingly different propulsive forces
(Received for publication, August 21, 2000
and in revised form, October 4, 2000)
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