(Received for publication, February 3, 1994; and in revised form, November 14, 1994)
From the
Smooth muscle myosin filaments are much less stable than the
skeletal muscle counterpart. Smooth myosin requires higher
concentration of Mg than skeletal myosin to form
thick filaments and addition of ATP disassembles the dephosphorylated
smooth muscle myosin filaments into monomers but not phosphorylated
ones. We found that the addition of caldesmon to dephosphorylated
myosin induced the formation of the filaments under the conditions
where myosin by itself is soluble or disassembled. Although the induced
filaments were short at 1 mM Mg
, they became
medium sized and seemed like side polar filaments with prominent 14 nm
periodicity at higher Mg
conditions (8 mM).
In the presence of F-actin, myosin filaments induced by caldesmon were
associated along actin filaments to form large structures. The
association of actin and myosin filaments was observed only in the
presence of caldesmon, suggesting that caldesmon cross-linked actin and
myosin filaments. This cross-linking was disrupted by the addition of
calmodulin. Caldesmon-induced filament formation of dephosphorylated
myosin in the presence of Mg
-ATP may explain the
existence of myosin filaments in relaxed smooth muscle fibers. A
similar effect of telokin on myosin filament assembly was also examined
and is discussed.
It is well accepted that the initial regulation of the smooth
muscle contractile machinery is through the reversible phosphorylation
of the 20-kDa light chain of myosin. This is catalyzed by
Ca/calmodulin-dependent myosin light chain kinase and
myosin light chain phosphatase (Hartshorne, 1987; Sellers and
Adelstein, 1987; Kamm and Stull, 1989). Although phosphorylation of
myosin is necessary and sufficient for the initiation of muscle
shortening (Ito et al., 1989b), the response of smooth muscle
to various stimulants is complex. It has been suggested that secondary
regulatory systems might be involved in smooth muscle regulation in
addition to myosin phosphorylation (Hartshorne, 1987; Marston and
Redwood, 1991).
Because of its ability to modify actomyosin ATPase,
caldesmon has been implicated as a potential regulator of contraction,
secondary to myosin phosphorylation. In support of a modulatory role
for caldesmon, Katsuyama et al.(1992) reported that a
synthetic peptide of caldesmon
(Gly-Ser
) which can bind to
calmodulin and actin raises the basic tone of skinned smooth muscle
cells.
Caldesmon is an actin and calmodulin-binding protein isolated from smooth and non-muscle cells. In addition to actin and calmodulin (Sobue et al., 1981), caldesmon also has a specific affinity to tropomyosin (Graceffa, 1987; Fujii et al., 1987) and myosin (Ikebe and Reardon, 1988).
Three actin binding regions were mapped to the COOH-terminal domain (Mornet et al., 1988; Wang et al., 1991), and the calmodulin binding region lies between residues 659-665 (Bartegi et al., 1990; Wang et al., 1991; Hayashi et al., 1991). The COOH-terminal domain of caldesmon is also involved in the binding to tropomyosin (Fujii et al., 1987; Dabrowska et al., 1985; Katayama et al., 1989; Hayashi et al., 1991), whereas myosin binding is restricted to the amino-terminal domain of caldesmon (Sutherland and Walsh, 1989; Katayama et al., 1989; Katayama, 1989a; Hemric and Chalovich, 1990). Although the primary structure of caldesmon and its domain mapping has been studied in detail, the physiological functions of caldesmon have not yet been clarified.
A
number of studies have shown that caldesmon can inhibit actomyosin
ATPase activity in vitro, suggesting its involvement in the
regulation of cross-bridge cycling (Marston and Redwood, 1991; Sobue
and Sellers, 1991). Ca/calmodulin not only reverses
the inhibition induced by caldesmon but also reduces the binding of
caldesmon to actin (Sobue et al., 1981; Bretcher, 1984; Furst et al., 1986; Dingus et al., 1986). However, a much
higher calmodulin concentration is required for the dissociation of
caldesmon from actin than that for the reversal of inhibition. These
results suggest that the reversal of the inhibition might not be due to
the dissociation of caldesmon from F-actin but rather due to the change
in the interaction of caldesmon with actin induced by
Ca
/calmodulin.
The physiological significance of caldesmon-myosin interaction is not clear. Localization of caldesmon in smooth muscle cells was reported to be in the thin filament-thick filament domain but not at the thin filament-intermediate filament domain (Furst et al., 1986), suggesting a role for caldesmon interaction with the contractile machinery.
Under physiological salt
conditions in vitro dephosphorylated monomeric myosin adopts
an unconventional conformation in which the tail portion of myosin is
bent back toward the head-rod junction so as to form a folded structure
(Trybus et al., 1982; Onishi and Wakabayashi, 1982; Craig et al., 1983). Upon phosphorylation, myosin changes to an
extended conformation which is conceivably more suitable for filament
formation. Dephosphorylated smooth muscle myosin can form thick
filaments in the presence of high Mg concentration;
however, the filaments are readily disassembled by addition of ATP
(Suzuki et al., 1978). Phosphorylated myosin filaments are
more stable and are not disassembled in the presence of
Mg
-ATP (Suzuki et al., 1978). Although this
may suggest that in smooth muscle, myosin filament formation may be
regulated by phosphorylation, in vivo studies using
quick-freeze electron microscopy have demonstrated that thick filaments
are unambiguously present in resting smooth muscle cells where the
majority of myosin molecules are assumed to be dephosphorylated (Somlyo et al., 1981). This discrepancy in the stability of smooth
muscle myosin thick filaments in vitro and in vivo is
unresolved. It has been suggested that high concentrations of myosin
such as those found in smooth muscle cells could be sufficient to
produce thick filaments even though myosin is dephosphorylated
(Kendrick-Jones et al., 1987). Another possibility is that
myosin-binding proteins might stabilize myosin thick filaments. We have
explored this latter possibility and in this study, we show that
caldesmon induces filaments of smooth muscle myosin.
Smooth muscle myosin was prepared from turkey gizzard as
described previously (Ikebe and Hartshorne, 1985b). Caldesmon was
prepared according to Bretcher(1984). Actin was prepared from rabbit
skeletal muscle acetone powder by the method of Spudich and Watt
(1971). Telokin was prepared according to Ito et al. (1989a).
Tropomyosin was prepared from turkey gizzard as follows. Muscle mince
was homogenized with 4 volumes of 0.1 M KCl, 0.2 mM DTT, ()and 30 mM Tris-HCl, pH 7.5, then
centrifuged for 5 min at 4000
g. The pellet was washed
four times with the same buffer. To the pellet, 4 volumes of 0.3 M KCl, 0.15 M KP
, pH 6.5, was added and
homogenized. After 30 min at 4 °C, the homogenate was centrifuged
at 10,000
g for 15 min. The pellet was washed five
times with 4 volumes of 1 mM NaHCO
. Following the
homogenization with 3 volumes of 2 mM NaHCO
, pH
was adjusted to 8.3 with Tris base. Tropomyosin was extracted for 2 h
at room temperature. The extract was collected by centrifugation at
10,000
g for 5 min, then subjected to ammonium sulfate
fractionation. 250-500 g/liter ammonium sulfate fraction was
collected, suspended with and dialyzed against (50 mM CaCl
, 10 mM Tris-HCl, pH 7.5, and 0.2 mM DTT). The produced pellet was dissolved in 5 volumes of 1 M KCl. Tropomyosin was precipitated by adjusting the pH to 4.6 with
acetic acid. After standing for 1 h, the precipitate was collected and
resuspended in 1 M KCl, and the pH was adjusted to 7.5. Any
undissolved materials were removed by centrifugation, and the final
supernatant was dialyzed against 10 mM Tris-HCl, pH 7.5, and
0.5 mM DTT and stored at -80 °C. The final product
contained no contaminant proteins as judged by SDS-PAGE analysis.
Myosin Mg-ATPase activity was measured in solution
containing 1 mM MgCl
, 0.5 mg/ml dephosphorylated
myosin, 30 mM Tris-HCl, pH 7.5, and 0.2 mM [
-
P]ATP at 25 °C. The ATPase
activity was also measured in the presence and absence of 0.5 mg/ml
F-actin and 0.2 mg/ml caldesmon. The liberated
P was
quantitated as described previously (Ikebe and Hartshorne, 1985a).
SDS-gel electrophoresis was carried out in a 7.5-20% polyacrylamide gradient slab gel using the discontinuous buffer system of Laemmli (1970).
Various combinations of protein mixture samples
were examined by negative staining electron microscopy. To obtain clear
images of small protein aggregates, samples of various protein mixtures
(0.5 mg/ml myosin, 0.14 mg/ml caldesmon, and/or 0.1 mg/ml telokin or
0.14 mg/ml myosin 0.2 mg/ml actin, 0.07 mg/ml tropomyosin, 0.15 mg/ml
caldesmon) in buffer (50 mM KCl, 1 or 8 mM MgCl, 0.5 mM ATP, 1 mM EGTA, 5
mM sodium phosphate, pH 7.5) were applied to uncoated number
400 copper grids followed by 1% uranyl acetate containing bacitracin as
described previously (Katayama, 1989b). When the sample was too thick,
it was diluted five times with the same buffer prior to the negative
staining. Electron micrographs were taken by JEOL 100CX or JEOL 2000ES
with an acceleration voltage of 80 kV. The advantages and disadvantages
for the use of this staining method have been described previously
(Katayama, 1989b). Myosin-caldesmon-actin complexes in the presence of
ATP formed large aggregates of thick and thin filaments, and the
structural details were difficult to resolve by negative staining. In
order to visualize the structural components of such a complex, we
subjected the mixture to harsh mechanical mixing or ultrasonic
agitation followed by quick dilution and immediate negative staining.
Optical transforms of thick filaments were taken using Luzex-F
real-time image analyzer (Nireco, Japan). The statistics on the
distribution of the filament length was done with the same equipment.
Myosin with phosphorylated light chain has high
Mg-ATPase activity and can form thick filaments in
the presence of ATP, initiating superprecipitation with actin filaments
(Ikebe et al., 1977; Suzuki et al., 1978). The
structure of thick filaments formed by phosphorylated myosin is stable
irrespective of the presence of ATP under high (10 mM Mg
) or low (1 mM) Mg
concentrations (data not shown). On the other hand, the activity
of myosin with dephosphorylated light chain is kept low in terms not
only of ATPase but also its ability to form filaments. Although
dephosphorylated myosin forms side polar filaments (Craig and Meyerman,
1977) at high Mg
concentration (Fig. 1a), addition of small amounts of ATP readily
disassembles the filaments almost completely to its soluble form (data
not shown).
Figure 1:
Negatively stained
images of dephosphorylated myosin. a, under 10 mM MgCl in the absence of ATP, many side polar filaments
were observed in the field; b, under 1 mM MgCl
in the presence of ATP myosin molecules remained soluble without
forming any filaments; c, under 1 mM MgCl
in the absence of ATP, addition of caldesmon induced numerous
filament-like aggregates, whereas myosin by itself was soluble as in b; d, under 1 mM MgCl
but with
ATP, many filament-like aggregates were observed when caldesmon was
added. The size of the aggregates was almost the same or slightly
larger than that in the absence of ATP. Scale bars indicate
0.2 µm throughout all electron
micrographs.
Although caldesmon was first described as a thin
filament component, it was later reported that caldesmon binds to
myosin at S-2 region (Ikebe and Reardon, 1988). We examined the effects
of caldesmon on the assembly properties of myosin. As shown in Fig. 1c, many short filament-like aggregates were
observed upon addition of caldesmon, under the conditions where myosin
by itself hardly formed filaments. Very similar filamentous aggregates
were also induced when caldesmon was added to myosin solution in the
presence of Mg-ATP (Fig. 1d), although their length
distribution seemed somewhat larger than those in the absence of ATP.
Under 8 mM Mg concentration,
dephosphorylated myosin could not yet form any filamentous structure if
ATP was present (Fig. 2a). However caldesmon induced
decent medium sized filaments (Fig. 2b) well comparable
with those produced with phosphorylated myosin or with dephosphorylated
myosin in the absence of ATP. The filaments seemed straight and stiff
showing prominent axial periodicity corresponding to 14 nm throughout
the whole length (Fig. 2c) and characteristic of side
polar filaments. Because caldesmon is known to associate with thin
filaments, we examined the effects of caldesmon-F-actin complex on
myosin filament formation.
Figure 2:
Negatively stained images of
dephosphorylated myosin under 8 mM MgCl conditions
in the presence of 0.5 mM ATP. a, most myosin
molecules remain disassembled even under such high Mg
conditions; b, when caldesmon was added, myosin forms
rigid medium sized filaments with apparent 14 nm axial periodicity
throughout its whole length. The inset in c is an
optical transform of one of such filaments as enlarged on the right with prominent 14 nm banding pattern. Arrows indicate the
meridional reflections for that
periodicity.
Fig. 3a shows that the
turbidity of the solution increased upon the addition of caldesmon to
actomyosin in the presence of Mg-ATP. The marked
increase in the turbidity was not observed in the absence of myosin
(data not shown). Similar turbidity increase was also observed when
F-actin was added to myosin/caldesmon solution (data not shown). The
increase in turbidity was dependent on caldesmon concentration and
reached a plateau at 0.15 mg/ml caldesmon if actin concentration was
fixed at 0.5 mg/ml. This corresponds to approximately 1 caldesmon/16
actin monomers.
Figure 3:
Increase in the turbidity of smooth muscle
actomyosin by caldesmon and the reversal effect by calmodulin. a, various concentrations of caldesmon were added to a
solution containing 0.5 mg/ml myosin, 0.5 mg/ml actin, 5 mM sodium phosphate, pH 7.5, 1 mM EGTA, 1 mM MgCl, 50 mM KCl, and 0.5 mM ATP. The
mixture was incubated for 5 min at 25 °C, and the turbidity was
measured by Perkin-Elmer UV/VIS spectrophotometer at a wavelength of
400 nm. The turbidity was stable for at least 30 min at room
temperature.
, without tropomyosin;
, with 0.12 mg/ml
gizzard tropomyosin. b, effect of calmodulin on the
caldesmon-induced increase in the turbidity of smooth muscle
actomyosin. Conditions are the same as for a, except that 0.15
mg/ml caldesmon and 0.1 mM CaCl
were
used.
The results suggest that caldesmon can cross-link
myosin and actin and supports our earlier report (Ikebe and Reardon,
1988). Tropomyosin which is also known to bind caldesmon did not
significantly affect the increase in the turbidity of actin/myosin
solution induced by caldesmon (Fig. 3a). Fig. 3b shows that the turbidity of actomyosin induced
by caldesmon declined by a further addition of
Ca/calmodulin. The decrease in the turbidity occurred
in calmodulin dose-dependent manner and more than 6 µM was required
to completely reverse the caldesmon-induced increase in turbidity. This
was consistent with the notion that calmodulin can compete with the
binding of caldesmon to actin.
The structure responsible for the large increase in turbidity was examined by electron microscopy (Fig. 4). In the absence of caldesmon the majority of myosin was dissociated from actin filaments and did not form filamentous aggregates (Fig. 4a). Addition of caldesmon induced filamentous aggregates of myosin as described above and these aggregates were associated along F-actin filaments to form large structures (Fig. 4b). This large actin-caldesmon-myosin filament complex tended to become entangled with each other and harsh mechanical mixing of the solution was needed to obtain clear image of each structural component by negative staining.
Figure 4:
Negatively stained images of the mixture
of unphosphorylated myosin and F-actin in the presence of ATP. a, most myosin oligomers were dissociated from
actin-filaments; b, under 1 mM MgCl conditions, filamentous myosin aggregates induced by caldesmon
were all closely associated to actin filaments forming large
light-scattering clumps. Micrograph was taken after vigorous mechanical
agitation; c, under 8 mM MgCl
in the
presence of ATP, actomyosin with added caldesmon formed very solid
filament bundles whose appearance was similar to stress fibers.
Micrograph shows the association of filaments in the complex which was
so tight that a substantial fraction remained still in bundles even
after ultrasonic treatment; d, on the same specimen grid was
found an image showing the structural constituents of filament bundles
which was dispersed by the above treatment. Several thick filaments
were observed together with many actin filaments. Note the presence of
clear periodic banding pattern throughout the thick filament surface,
suggesting that thick filaments are side polar myosin
filaments.
To examine further
the constitution of these complexes, actin, myosin, caldesmon, and
tropomyosin were mixed in various combinations and were spun by low
speed centrifugation at 10,000 g for 5 min. The
protein composition of the supernatant and the pellets was analyzed by
SDS-PAGE (Fig. 5). Caldesmon, myosin, and actin coprecipitated.
The molar amounts of myosin and actin which coprecipitated with
caldesmon were higher than that of caldesmon. This is consistent with
the observation by electron microscopy that the formation of large
aggregates is not due to the cross-linking of F-actin and individual
myosin molecules by caldesmon, but involves myosin in aggregated form.
Caldesmon and myosin coprecipitated under the conditions where myosin
by itself did not precipitate, although the amount of precipitated
proteins were less than in the presence of F-actin (Fig. 5).
Under these conditions the smaller size of each myosin filamentous
aggregate was similar to aggregates formed under low Mg
conditions in the presence of ATP. If caldesmon was added to the
mixture of actin and dephosphorylated myosin in the presence of ATP but
with 8 mM MgCl
, very thick bundles composed of a
parallel array of thick and thin filaments were formed and were
reminiscent of stress fiber in the cell. The filaments involved in such
bundles associated with each other so tightly that the harsh mixing,
which was effective under 1 mM Mg
conditions, hardly altered the final image of the complex. It was
necessary to subject the entire mixture to ultrasonic agitation to
effect partial dissociation (Fig. 4, c and d).
Negatively stained images of forcibly dissociated bundles showed the
parallel array of actin filaments together with myosin filaments with
periodic banding pattern throughout the length, indicating a side polar
assembly.
Figure 5:
Cosedimentation analysis of
myosin-actin-caldesmon complex. Various combination of myosin (0.5
mg/ml), actin (0.5 mg/ml), caldesmon (0.15 mg/ml), and tropomyosin
(0.12 mg/ml) were mixed in the buffer containing 5 mM sodium
phosphate, pH 7.5, 1 mM EGTA, 1 mM MgCl,
0.5 mM ATP, and 50 mM KCl and then centrifuged at low
speed (10,000
g) for 5 min. The precipitate (lanes
2-5 and 10) and the supernatant (lanes
6-9 and 11) were analyzed by SDS-PAGE: lanes 2 and 6, actin and caldesmon; lanes 3 and 7, myosin, actin, and caldesmon; lanes 4 and 8, myosin and caldesmon; lanes 5 and 9,
myosin, actin, caldesmon, and tropomyosin; lanes 10 and 11, actin and myosin; lane 1, molecular mass
standards, molecular masses in kilodaltons are indicated on the far
left.
Monomeric smooth muscle myosin forms a folded (10 S
myosin) or an extended structure (6 S myosin), depending on its
environment. Thick filaments are formed only from the latter conformers
(Hartshorne, 1987). Since the two conformations are characterized by
distinct enzyme activities (Ikebe et al., 1983), we examined
the effect of caldesmon on the equilibrium between 10 S and 6 S
myosins. The KCl dependence of Mg-ATPase of
dephosphorylated myosin was measured in the presence and absence of
caldesmon and/or actin (Fig. 6). The depression of the
Mg
-ATPase activity below 0.3 M KCl, which
reflects the myosin conformational transition from 6 to 10 S, was
unaffected by caldesmon and/or actin. At lower KCl conditions where
caldesmon induced formation of filamentous myosin as well as
actin-myosin filament cross-linking, the Mg
-ATPase
activity was not influenced by caldesmon. These results suggest that
the formation of the small myosin filamentous aggregate might not be
due to the change in the myosin conformation.
Figure 6:
KCl dependence of
Mg-ATPase activity of myosin in the presence of
caldesmon. ATPase activity was measured as described under
``Materials and Methods.''
, myosin alone;
, with
0.2 mg/ml caldesmon;
, with 0.5 mg/ml actin;
, with 0.2
mg/ml caldesmon and 0.5 mg/ml actin.
It was recently
reported that telokin, a protein whose primary amino acid sequence is
identical to the tail part of myosin light chain kinase, might
contribute to the stabilization of the thick filament structure of
dephosphorylated myosin in the presence of ATP (Shirinsky et
al., 1993). We have found that dephosphorylated myosin filaments
were induced by the addition of telokin. However, the size and shapes
of the filaments observed in our hands were less homogenous than those
induced by caldesmon, including the slender ones and some aggregates
under 1 mM Mg conditions (Fig. 7b). In the presence of 8 mM Mg
, myosin aggregates consisted of a mixture of
very long and medium to small sized filaments. The filaments in the
very long population often showed a curved contour with a tendency to
merge with each other giving a somewhat fragile appearance. When
caldesmon and telokin were simultaneously added to the solution of
dephosphorylated myosin, the observed filaments were shorter than the
longest population in the latter case but were more homogenous in shape
and size (length distribution was 0.95 + 0.25 µm for
100
filaments in some selected fields) as compared with telokin alone. The
tendency for filaments to merge was less, although they sometimes
aligned themselves side-by-side. The density of myosin molecules in the
background seemed less than the other cases, indicating that most
molecules were efficiently taken up into filamentous form. These
filaments also showed prominent 14 nm periodicity, indicating an
ordered structure. Myosin light chain kinase did not induce thick
filament formation at all, in spite of the existence of the amino acid
sequence identical to telokin at its COOH-terminal domain (data not
shown).
Figure 7:
Negatively stained images showing the
effect of telokin on the assembly of dephosphorylated myosin. a, addition of telokin induces slender filaments under 1
mM MgCl and 0.5 mM ATP conditions,
whereas control myosin by itself did not form any filaments; b, when telokin alone was added to dephosphorylated myosin
under 8 mM MgCl
and 0.5 mM ATP
conditions, induced filaments are variable in size and shape, including
very long ones (densely stained across the field) and medium sized but
flexible filaments. Some periodic banding pattern was recognized in
very long filaments; c, by addition of caldesmon together with
telokin, the size and shape of thick filaments became less variable and
each component showed prominent 14 nm periodicity. The inset of d indicates the optical transform of one of such filaments as
enlarged on the right. Arrows indicate the meridional
reflections for that periodicity.
Caldesmon can bind various smooth muscle contractile proteins
such as actin (Sobue et al., 1981), tropomyosin (Graceffa et al., 1987), and myosin (Ikebe and Reardon, 1988) within
distinct domains (Marston and Redwood, 1991; Sobue and Sellers, 1991).
It was suggested previously that caldesmon can cross-link F-actin and
myosin (Ikebe and Reardon, 1988; Marston et al., 1992; Hemric
and Chalovich, 1988), but detailed structures of such myosin aggregates
have not been investigated. The present study confirmed the
cross-linking of F-actin and myosin by caldesmon, but more importantly,
this study demonstrated that caldesmon induced the assembly of myosin
filaments in the presence of Mg-ATP.
Smooth muscle
myosin requires a higher Mg concentration
(
8
15 mM) for thick filament formation as compared
with the concentration required by skeletal myosin. Even at high
Mg
concentration the dephosphorylated myosin
filaments are disassembled by the addition of Mg
-ATP
(Suzuki et al., 1978). Under these conditions, myosin has been
shown to be in a monomeric, 10 S conformation as determined by
analytical ultracentrifugation (Trybus et al., 1982; Ikebe et al., 1983; Suzuki et al., 1978; Onishi and
Wakabayashi, 1982). In the present study we confirmed that freshly
isolated smooth muscle dephosphorylated myosin did not form filaments
under lower Mg
conditions (
1 mM). At
higher Mg
concentration (
8 mM)
dephosphorylated myosin formed thick filaments (Fig. 1a), but this was readily disassembled by the
addition of Mg
-ATP (Fig. 1b). By
raising Mg
concentration to 8 mM, such small
aggregates of myosin grew into the filaments whose size was comparable
with that of dephosphorylated myosin in the absence of ATP or
phosphorylated myosin in the absence or presence of ATP. The small
aggregates of myosin induced by caldesmon at low Mg
condition might act as a precursor or ``seed.'' Larger
myosin filaments induced by caldesmon can be produced either from small
myosin oligomer or monomer, but in either case caldesmon seems to shift
the equilibrium between monomeric myosin and filamentous myosin toward
the formation of filaments.
Under low Mg conditions in which myosin alone exists as monomer or small
oligomers but not large filaments, caldesmon induced formation of
filamentous aggregates of myosin though these filaments were smaller
than that observed with myosin alone at higher Mg
concentration. It is known that caldesmon itself forms aggregate
due to oxidation. However, we do not think that caldesmon aggregation
is involved in the observed myosin filament formation because: 1)
caldesmon was treated with 5 mM DTT to reduce any oxidized
species of caldesmon; 2) caldesmon by itself was not precipitated
during the sedimentation analysis; 3) electron microscopic observation
failed to detect a large caldesmon aggregate; 4) the effect of
caldesmon to induce the turbidity increase of actin/myosin mixture was
saturated within the range of expected molar ratio of
actin/caldesmon/myosin. It remains obscure as to how caldesmon
stabilizes myosin filaments in the presence of Mg
-ATP
where dephosphorylated myosin alone is in the 10 S conformation and
does not form filaments. One possibility is that a folded conformation
of myosin is destabilized by binding of caldesmon at S-2 moiety (Ikebe
and Reardon, 1988), and the tail portion is consequently extended and
induces filament formation. Supporting this notion, we recently
observed that native smooth muscle thin filaments show thin whisker
like projections which are labeled with an antibody recognizing the
NH
-terminal myosin binding domain of caldesmon. (
)It is plausible, therefore, that the
NH
-terminal domain of caldesmon protrudes from the thin
filaments and binds the S-2 portion of myosin so as to stabilize the
myosin filaments.
The results of cosedimentation experiment where
much more myosin molecules coprecipitated with caldesmon molecules
suggests that caldesmon may exert a cooperative effect on filament
formation by inducing myosin into some conformation that will
polymerize. This may be less likely since 10 S myosin does not
polymerize with 6 S myosin to form copolymer (Trybus and Lowey, 1987).
Alternatively, caldesmon may bind to the S-2 region of several myosin
molecules. This hypothesis is supported by previous findings by
Katayama and co-workers (Katayama et al., 1989; Katayama,
1989a) that caldesmon contains more than two S-2 binding sites within
the NH-terminal and central domains. It has also been shown
that the caldesmon molecule can bind several molecules of heavy
meromyosin (Marston, 1989). Recent reports on the properties of
caldesmon's COOH-terminal domain (Huber et al., 1993)
also indicated its affinity to myosin. We also observed by electron
microscopy that clusters of heavy meromyosin bind periodically to the
thin filaments via their S-2 region.
These results support
the idea that caldesmon can bind several myosin molecules and stabilize
its filamentous structure. It has been shown in two different types of
smooth muscle tissue that the thick filaments are present in relaxed
smooth muscle fiber (Somlyo et al., 1981; Tsukita et
al., 1982), even though isolated dephosphorylated myosin fails to
form stable thick filaments under physiological ionic conditions. As
shown in the present study, caldesmon may help stabilize myosin
filaments under physiological ionic conditions and resolve the apparent
discrepancy between in vitro and in vivo reports.
After completion of our experiments on the effects of caldesmon on
myosin filament formation, a paper appeared concerning very similar
issues to ours (Shirinsky et al., 1993). They attributed the
stability of dephosphorylated myosin filaments in relaxed muscle, to
the presence of telokin, an interesting protein whose function is not
fully elucidated. We confirmed their results except in the distribution
of the filaments in terms of size and shape. These differences may be
attributed to the differences in our experimental conditions from
theirs. In addition, we checked the effect of simultaneous addition of
caldesmon and telokin on dephosphorylated myosin filament formation.
Under such conditions, thick filaments were shorter but seemed more
homogenous in size and shape as compared with those with telokin alone.
It is notable that these long filaments showed clear 14 nm periodicity,
giving rise to the appearance of native thick filaments (Cooke et
al., 1989). Caldesmon and telokin are both the abundant
constituents of smooth muscle cells. These results suggest they might
work cooperatively to stabilize the organization of dephosphorylated
myosin filament under relaxed conditions. It has been reported that
telokin is not necessarily a universal component of all smooth muscle
tissues and is deficient in aorta, trachea, and non-muscle cells
(Gallagher and Herring, 1991). Caldesmon, on the other hand,
distributes more widely among various tissues, including those above.
Therefore, caldesmon might be a more universal factor in stabilizing
dephosphorylated myosin thick filament structure in relaxed smooth
muscle cells. Furthermore, we observed that caldesmon-induced myosin
filaments were tethered to actin filaments in the presence of
Mg-ATP. This is consistent with the intracellular
localization of caldesmon in the actomyosin domain (Furst et
al., 1986) and suggests an important structural role in smooth
muscle organization.
In non-muscle cells, myosin colocalizes with actin in stress fibers. During mitosis, microfilament organization changes dramatically and stress fibers are disassembled during prophase. It was shown recently (Yamashiro et al., 1990, 1991) that caldesmon dissociates from microfilaments during mitosis probably due to the phosphorylation by cdc2 kinase. These results suggest that caldesmon may play an important role in stabilizing stress fibers in non-muscle cells. The present results show that caldesmon stabilizes myosin filaments and furthermore cross-links actin and myosin filaments to form actomyosin bundles that resemble stress fibers (Fig. 4c). These findings predict roles for caldesmon in the stabilization of stress fibers consisting of actin filament bundles and myosin molecules.