Forcevelocity relationships in actinmyosin interactions causing cytoplasmic streaming in algal cells
1 Department of Physiology, School of Medicine, Teikyo University,
Itabashi-ku, Tokyo 173-8605, Japan
2 Department of Applied Physics, College of Humanities and Science, Nihon
University, Setagaya-ku,Tokyo 156-8550, Japan
* Author for correspondence (e-mail: sugi{at}med.teikyo-u.ac.jp)
Accepted 15 January 2003
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: cytoplasmic streaming, actinmyosin sliding, cytoplasmic myosin, actin cables, centrifuge microscope, Nitellopsis obtusa, Chara corallina
![]() |
Characteristic features of ATP-dependent actinmyosin interaction causing cytoplasmic streaming |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Although both cytoplasmic streaming and muscle contraction are caused by
ATP-dependent actinmyosin interaction, they differ from each other in
some properties. Contraction and relaxation in skeletal muscle is regulated by
changes in intracellular Ca2+ concentration via regulatory
proteins on actin filaments (Ebashi and
Endo, 1968); a muscle is relaxed at pCa
7 and fully contracts
at pCa
5. By contrast, cytoplasmic streaming occurs continuously even at
pCa
7 and stops at pCa
6
(Williamson, 1975
;
Williamson and Ashley, 1982
;
Tominaga et al., 1983
). Latex
beads coated with skeletal muscle myosin slide along actin cables when they
are introduced into an internodal cell with Mg-ATP, but the bead movement is
insensitive to pCa (Shimmen and Yano,
1984
), indicating that regulatory proteins are absent on actin
cables, while cytoplasmic myosin is responsible for the
Ca2+-induced stoppage of cytoplasmic streaming. Using the
centrifuge microscope, Chaen et al.
(1995
) showed that the
Ca2+-induced actincytoplasmic myosin linkages are stronger
than their rigor linkages, suggesting that a cytoplasmic myosin head can bind
with actin at two different sites. Higashi-Fujime et al.
(1995
) reported that
proteolytic cleavage of actin impaired ATP-dependent actin filament sliding on
skeletal muscle myosin but not on cytoplasmic myosin, suggesting that
cytoplasmic myosin may interact with actin at sites different from those of
muscle myosin.
The most striking feature of cytoplasmic streaming is that its velocity
(50 µm s-1; Kamiya and
Kuroda, 1956
) is many times larger than the maximum velocity of
actinmyosin sliding in muscle (approximately 3 µm s-1;
Oiwa et al., 1990
). In the
presence of Mg-ATP, muscle myosin-coated beads slide on actin cables with
velocities similar to the maximum unloaded velocity of actinmyosin
sliding in muscle (Sheetz and Spudich,
1983
; Oiwa et al.,
1990
), whereas muscle actin filaments move on cytoplasmic
myosin-coated glass surface with velocities similar to those of native
cytoplasmic streaming (Higashi-Fujime et
al., 1995
). These results indicate that it is cytoplasmic myosin
that is responsible for the rapid cytoplasmic streaming. Meanwhile, the
ultrastructure of cytoplasmic myosin molecules, consisting of two heads
connected to one tail, is similar to that of skeletal muscle myosin, except
that the tail length is much shorter in the former than in the latter
(Yamamoto et al., 1999
).
![]() |
Steady-state forcevelocity relationships of ATP-dependent actinmyosin sliding causing muscle contraction and cytoplasmic streaming |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() | (1) |
|
Under a constant centrifugal force serving as a positive load, skeletal
muscle myosin-coated beads moved with a constant velocity over a large
distance (Fig. 3A), indicating
the definite steady-state relationship between the load (=force generated by
myosin molecules on the bead) and the velocity of actinmyosin sliding.
The maximum unloaded velocity of bead movement (Vmax) was
1.63.6 µm s-1. The bead stopped moving when the load
reached the maximum isometric force (P0) generated by myosin
molecules. The forcevelocity (PV) curve obtained
was hyperbolic in shape in the low force range but deviated from the hyperbola
in the high force range (Fig.
3B). The shape of the PV curve was nearly
similar to that obtained with tetanized single skeletal muscle fibres
(Edman, 1988), indicating that
muscle myosin molecules retain their basic kinetic properties of contracting
muscle fibres despite their random orientation on the bead
(Oiwa et al., 1990
).
|
During the course of the above experiments, we made an incidental observation that, when uncoated beads suspended in Mg-ATP solution were introduced into Chara internodal cells, the bead moved along actin cables with velocities and directions similar to those of native cytoplasmic streaming. The beads stopped moving at pCa <6 or by removal of ATP from the surrounding medium. These features of bead movement indicate that it is caused by cytoplasmic myosin molecules, which remain in the cell and attach spontaneously to the bead surface to interact with actin cables. We therefore took the opportunity to determine PV characteristics of ATP-dependent actinmyosin sliding causing cytoplasmic streaming with the centrifuge microscope system.
Under a constant centrifugal force serving as a positive load, the beads
moved with a constant velocity, indicating the presence of a steady-state
PV relationship in actinmyosin interaction causing
cytoplasmic streaming (Fig.
4A). Vmax was 3261 µm s-1
(mean ± S.D., 46±8.7 µm s-1;
N=14) at 2426°C. The beads stopped moving when the load
was increased to P0, which showed a wide variation from 1.0
pN to 13 pN. The PV curve constructed from six different
beads with large P0 (8.613 pN) was nearly linear
(Fig. 4B) in contrast to the
hyperbolic PV curve of actinmyosin sliding causing
muscle contraction (Fig. 3B).
Correction of the data points for the viscous drag force against the bead
movement did not alter the shape of the PV curve
(Chaen et al., 1995). The
PV curve constructed from six different beads with small
P0 (1.02.4 pN) was also nearly linear, although the
data points showed a large scatter due to difficulties in measuring the
velocity of bead movement under small centrifugal forces
(Fig. 4C). Despite a large
variation of P0 from 1.0 pN to 13 pN, Vmax
of the beads with small P0 values ranged from 36 µm
s-1 to 60 µm s-1, indicating that
Vmax was independent of P0.
|
On the other hand, when centrifugal forces in the same direction as the
bead movement were applied as negative loads, the velocity of bead movement
first decreased under small negative loads and then increased with increasing
negative load until the bead was finally detached from the actin cables
(Fig. 4D). This puzzling
phenomenon has also been seen in actinskeletal muscle myosin sliding
(Oiwa et al., 1990) and
microtubulekinesin sliding (Hall et
al., 1993
). As the centre of gravity of a polystyrene bead
(diameter, 2.8 µm) is 1.4 µm above the actinmyosin contact,
substantial torque forces around the bead would compress myosin molecules to
inhibit actinmyosin sliding.
![]() |
Kinetic properties of cytoplasmic actinmyosin interaction causing cytoplasmic streaming |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() | (2) |
The minimum value of P0 generated by cytoplasmic myosin
molecules on the bead was 1.0 pN, which is smaller than the reported values of
force spikes of single muscle myosin molecules (17 pN,
Finer et al., 1994; 56
pN, Ishijima et al., 1994
,
1996
). In our experiments,
however, the forces measured are time-averaged values against continuously
applied centrifugal forces and do not contradict reported values of transient
force spikes. On this basis at least, a few myosin molecules may be involved
even in generating a P0 of 1 pN if random orientation of
myosin molecules on the bead is taken into consideration. On the other hand,
the large Vmax in actincytoplasmic myosin sliding
indicates a large cycling rate of actincytoplasmic myosin interaction.
Contrary to this expectation, however, the actin-activated ATPase activity of
Chara cytoplasmic myosin in solution is similar to that of muscle
myosin (Tanimura and Higashi-Fujime, in press). This paradox will be
considered later.
In the Huxley contraction model
(Huxley, 1957), in which actin
and myosin exhibit repeating attachmentdetachment cycles, each coupled
with ATP hydrolysis, the probability that all myosin heads are detached from
actin (Poff) is written as:
![]() | (3) |
![]() |
Possible mechanism producing the rapid cytoplasmic streaming |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() | (4) |
In ATP-dependent actinmyosin interaction in skeletal muscle,
p is approximately 0.05 (Uyeda et
al., 1990; Finer et al.,
1994
), A is approximately 20 s-1
head-1 (Toyoshima et al.,
1987
) and Vmax is approximately 3 µm
s-1 (Oiwa et al.,
1990
). Putting these values into equation 4, we obtain a
d of approximately 8 nm, a value consistent with the contraction
model, in which unitary actinmyosin sliding is produced by rotation of
myosin heads (Huxley and Simmons,
1971
). The small duty ratio in skeletal muscle myosin heads
implies that at least 20 heads, working together, are necessary for continuous
actinmyosin sliding.
In ATP-dependent actinmyosin interaction in cytoplasmic streaming,
on the other hand, p is assumed to be 0.25, while
Vmax is approximately 50 µm s-1
(Chaen et al., 1995). The value
of d is at present unknown but, on the basis of the
HuxleySimmons scheme, it is constrained by the size of the myosin head
and may not be more than about 20 nm. In order to make d realistic
(
20 nm), it follows from equation 3 that A should be
approximately 425 s-1 head-1. This extremely high
cycling rate of actinmyosin interaction, which is likely to be
associated with a very low efficiency of chemo-mechanical energy conversion,
is consistent with the very large value of a/P0 of
the straight PV curve (Fig.
4B,C;
Chaen et al., 1995
). This idea,
however, contradicts the result that the actin-activated ATPase rate of
cytoplasmic myosin in solution is similar to that of skeletal muscle myosin
(20 s-1 head-1; Tanimura and Higashi-Fujime, in press).
This paradox may be solved at least qualitatively in the following way. In the
movement of cytoplasmic organelles or cytoplasmic myosin-coated beads along
actin filaments, there seems to be a mechanically coupled interaction between
the heads of myosin molecules attached to the same bead. Such interactions are
absent in solutions where myosin molecules diffuse freely. Due to its large
value of p, each cytoplasmic myosin head interacting with actin would
have many chances of being `pushed forward' by sliding forces generated by the
head(s) of other myosin molecules attached to the same bead. When a head
interacting with actin is pushed forward, it would immediately be detached
from actin to restart its sliding force generation. This would considerably
reduce the time of attachment of each myosin head to actin without changing
its duty ratio and would therefore result in a marked increase in its ATPase
rate compared with that in solution.
The above idea of the mechanically coupled myosin head interaction comes
from the report that, in single tetanized skeletal muscle fibres, the value of
Vmax measured by the slack test increases more than twofold
in the presence of a small resting tension (7% of P0) at
a sarcomere length of approximately 3 µm
(Edman, 1988
). In the slack
test, a tetanized fibre is subjected to a quick release of up to 30% of the
initial fibre length. As the resting tension decreases steeply to a negligible
value with decreasing sarcomere length for the first approximately 6% of
sarcomere shortening following release, this indicates that the effect of
resting tension to compress the stretched sarcomere (and therefore to push the
myosin heads attached to actin forward) is only transient. This implies that
if a myosin head is pushed forward by a transient force, the rate of
actinmyosin interaction coupled with ATP hydrolysis can be markedly
increased.
Meanwhile, the increase of A (from 20 s-1
head-1 to 425 s-1 head-1) by the mechanically
coupled myosin head interaction can be reduced if the value of d is
not actually constrained by the size of the myosin head and could be as large
as approximately 100 nm by a biased Brownian ratchet mechanism
(Vale and Oosawa, 1990). In
this case, the increase of A is reduced from 20 s-1
head-1 to approximately 85 s-1 head-1. Much
more experimental work is necessary to further understand the mechanisms of
cytoplasmic streaming. In the Huxley contraction model
(Huxley, 1957
), the value of
Vmax is determined by the balance between positive forces
generated by myosin heads and negative forces due to negative strain of myosin
heads. If cytoplasmic myosin heads are assumed to detach from actin without
generating appreciable negative strain forces, this would also result in a
marked increase in the velocity of actinmyosin sliding in cytoplasmic
streaming compared with that in muscle contraction. The myosin head negative
strain is not explicitly taken into consideration in the concept of duty ratio
but is implicitly included in the value of d, and in this sense
d may not necessarily be constrained by the size of the myosin
head.
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Chaen, S., Inoue, J. and Sugi, H. (1995). The forcevelocity relationship of the ATP-dependent actinmyosin sliding causing cytoplasmic streaming in algal cells, studied using a centrifuge microscope. J. Exp. Biol. 198,1021 -1027.[Medline]
Ebashi, S. and Endo, M. (1968). Calcium ion and muscle contraction. Prog. Biophys. Mol. Biol. 18,125 -183.
Edman, K. A. P. (1979). The velocity of unloaded shortening and its relation to sarcomere length. J. Physiol. Lond. 291,143 -159.[Abstract]
Edman, K. A. P. (1988). Double-hyperbolic forcevelocity relation in frog muscle fibres. J. Physiol. Lond. 494,301 -321.
Finer, J. T., Summons, R. M. and Spudich, J. A. (1994). Single myosin molecule mechanics: piconewton forces and nanometre steps. Nature 368,113 -119.[CrossRef][Medline]
Hall, K., Cole, D. G., Yen, Y., Scholey, J. M. and Baskin, R. J. (1993). Forcevelocity relationships in kinesin-driven motility. Nature 364,457 -459.[Medline]
Higashi-Fujime, S., Ishikawa, R., Iwasawa, H., Kagami, O., Kurimoto, E., Kohama, K. and Hozumi, T. (1995). The fastest actin-based motor protein from the green algae, Chara, and its distinct mode of interaction with actin. FEBS Lett. 375,151 -154.[CrossRef][Medline]
Hill, A. V. (1938). The heat of shortening and the dynamic constants of muscle. Proc. R. Soc. Lond. B. Biol. Sci. 126,136 -195.
Howard, J. (1997). Molecular motors: structural adaptations to cellular functions. Nature 389,561 -567.[CrossRef][Medline]
Huxley, A. F. (1957). Muscle structure and theories of contraction. Prog. Biophys. Biophys. Chem. 7, 255-318.
Huxley, A. F. and Simmons, R. M. (1971). Proposed mechanism of force generation in striated muscle. Nature 233,533 -538.[Medline]
Ishijima, A., Harada, Y., Kojima, H., Funatsu, T., Higuchi, H. and Yanagida, T. (1994). Single-molecule analysis of the actomyosin motor using nano-manipulation. Biophys. Biophys. Res. Commun. 199,1057 -1063.
Ishijima, A., Kojima, H., Higuchi, H., Harada, Y., Funatsu, T. and Yanagida, T. (1996). Multiple- and single-molecule analysis of the actomyosin motor by nanometer-piconewton manipulation with a microneedle; unitary steps and forces. Biophys. J. 70,383 -400.[Abstract]
Kamitsubo, E. (1966). Motile protoplasmic fibrils in cells of Characeae II. Linear fibrillar structure and its bearing on protoplasmic streaming. Proc. Jpn. Acad. 42,640 -643.
Kamitsubo, E., Ohashi, Y. and Kikuyama, M. (1989). Cytoplasmic streaming in internodal cells of Nitella under centrifugal acceleration: a study done with a newly constructed centrifuge microscope. Protoplasma 152,148 -155.
Kamiya, N. and Kuroda, K. (1956). Velocity distribution of the protoplasmic streaming in Nitella cells. Bot. Mag. 69,544 -554.
Kato, T. and Tonomura, Y. (1977). Identification of myosin in Nitella flexilis. J. Biochem. 82,777 -782.
Kersey, Y. M. and Wessels, N. K. (1976). Localization of actin filaments in internodal cells of Characean algae. J. Cell Biol. 68,264 -275.[Abstract]
Nagai, R. and Hayama, T. (1979). Ultrastructure of the endoplasmic factor responsible for cytoplasmic streaming in Chara internodal cells. J. Cell Sci. 36,121 -136.[Abstract]
Nagai, R. and Rebham, L. (1966). Cytoplasmic microfilaments in streaming Nitella cells. J. Ultrastruct. Res. 14,571 -589.[Medline]
Oiwa, K., Chaen, S., Kamitsubo, E., Shimmen, T. and Sugi, H. (1990). Steady-state forcevelocity relation in the ATP-dependent sliding movement of myosin-coated beads on actin cables in vitro. Proc. Natl. Acad. Sci. USA 87,7893 -7897.[Abstract]
Sheetz, M. P. and Spudich, J. A. (1983). Movement of myosin-coated fluorescent beads on actin cables in vitro. Nature 303,31 -35.[Medline]
Shimmen, T. and Yano, M. (1984). Active sliding movement of latex beads coated with skeletal muscle myosin on Chara actin bundles. Protoplasma 121,132 -137.
Tanimura, A. and Higashi-Fujime, S. (2002). Characteristic motility of Chara myosin. J. Muscle Res. Cell Motil. 23,185 .
Tominaga, Y., Shimmen, T. and Tazawa, M. (1983). Control of cytoplasmic streaming by extracellular Ca2+ in permealized Nitella cells. Protoplasma 116,75 -77.
Toyoshima, Y. Y., Kron, S. J., McNally, E. M., Niebling, K. R., Toyoshima, C. and Spudich, J. A. (1987). Myosin subfragment-1 is sufficient to move actin filaments in vitro. Nature 328,536 -539.[CrossRef][Medline]
Toyoshima, Y., Kron, S. J. and Spudich, J. A. (1990). The myosin step size. Proc. Natl. Acad. Sci. USA 87,7130 -7134.[Abstract]
Uyeda, T. Q. P., Kron, S. J. and Spudich, J. A. (1990). Myosin step size estimation from slow sliding movement of actin over low densities of heavy meromyosin. J. Mol. Biol. 214,699 -710.[Medline]
Vale, R. D. and Oosawa, F. (1990). Protein motors and Maxwell's demons: does mechanochemical transduction involve a thermal rachet. Adv. Biophys. 26, 97-134.[Medline]
Williamson, R. E. (1975). Cytoplasmic streaming in Chara: a cell model activated by ATP and inhibited by cytochalasin B. J. Cell Sci. 17,655 -668.[Abstract]
Williamson, R. E. and Ashley, C. C. (1982). Free Ca2+ and cytoplasmic streaming in the alga Chara Nature 296,647 -651.[Medline]
Woledge, R. C. (1968). The energetics of tortoise muscle. J. Physiol. Lond. 197,685 -707.[Medline]
Yamamoto, K., Hamada, S. and Kashiyama, T. (1999). Myosins from plants. Cell. Mol. Life Sci. 56,227 -232.[CrossRef][Medline]