(Received for publication, May 18, 1995; and in revised form, August 23, 1995)
From the
The NH- and COOH-terminal domains of muscle
caldesmon are separated by a long
-helical stretch. Cys-580, in
the COOH-terminal domain, can be rapidly and efficiently
disulfide-cross-linked to Cys-374 of actin by incubation with actin
modified with 5,5`-dithiobis(2-nitrobenzoic acid) (Graceffa, P., and
Jancso, A.(1991) J. Biol. Chem. 266, 20305-20310). Upon
further incubation (±tropomyosin), a second cross-link was
slowly formed between Cys-153 in the NH
-terminal domain and
Cys-374 of another actin monomer. The yield of the second cross-link
was relatively insensitive to increasing ionic strength, whereas the
caldesmon-actin binding strength decreased considerably, suggesting
that the NH
-terminal domain is largely dissociated from
actin and becomes slowly cross-linked to it during collisions with the
actin filament. In support of these conclusions, the yield of
photocross-linking actin to caldesmon specifically labeled with
benzophenonemaleimide at Cys-580 was high, but close to zero for
caldesmon labeled at Cys-153, and the fluorescence intensity and
polarization of tetramethylrhodamine iodoacetamide attached to Cys-580
showed large changes, while there were no changes for the probe at
Cys-153 upon binding caldesmon to actin (±tropomyosin). These
findings are consistent with the knowledge that COOH-terminal fragments
of caldesmon bind to actin, whereas NH
-terminal fragments
do not. Since the NH
-terminal domain of caldesmon binds to
myosin, a dissociated NH
-terminal domain may account for
caldesmon's ability to link myosin and actin filaments.
Smooth muscle contraction is primarily activated by myosin
phosphorylation by a Ca/calmodulin-dependent kinase
and deactivated by dephosphorylation by a phosphatase(2) .
However, physiological evidence indicates that this mechanism cannot
fully explain regulation(3, 4) . For example, tension
can be inhibited although myosin is phosphorylated(5) , and
full tension can be maintained at low levels of myosin phosphorylation
and ATP hydrolysis (6) . This latter, so-called ``latch
state'' is thought to be responsible for smooth muscle tone, i.e. tension maintenance with low energy expenditure.
The
search for an additional regulatory system has focused on the thin
filament protein caldesmon. Chicken gizzard caldesmon is a single
756-residue polypeptide chain forming an elongated molecule with three
domains: a COOH-terminal domain, which binds to actin and inhibits
actomyosin ATPase activity; an NH-terminal domain, which
contains a binding site for the ``neck region'' of myosin;
and a central domain consisting of a long
-helical ``spacer
rod'' (7, 8) separating the other two domains
(for review, see (9, 10, 11) ). The molecule
contains two cysteine residues at position 153 in the
NH
-terminal domain and position 580 in the COOH-terminal
domain (see (8) for a schematic domain map). Since the
inhibition of actomyosin ATPase activity by caldesmon can be reversed
by Ca
/calmodulin binding to caldesmon, it has been
assumed that this reversible inhibition plays a role in regulation.
Physiological evidence lends support to this assumption (12, 13, 14) . Since the NH
- and
COOH-terminal domains of caldesmon bind to myosin and actin,
respectively, it has also been proposed that a simultaneous binding of
caldesmon to myosin in the thick filament and actin in the thin
filament could link the filaments and possibly 1) account for the
passive tension of the latch
state(15, 16, 17, 18) , 2) enhance a
``productive'' interaction between myosin and
actin(19) , or 3) promote the assembly of myosin filaments in
the vicinity of actin filaments(20) . In fact, there is
evidence that thick and thin filaments are linked into parallel arrays
or bundles only in the presence of caldesmon (20, 21) and that caldesmon can form a link between
myosin and actin thin filaments(19, 22) .
It is
well established that COOH-terminal fragments of caldesmon bind to
actin, whereas NH-terminal fragments bind very weakly or
not at
all(23, 24, 25, 26, 27) .
However, the interaction of the NH
-terminal domain of
intact caldesmon with actin cannot be excluded from fragment studies
since proteolytic digestion of caldesmon might directly or indirectly
interfere with actin binding. Furthermore, in the intact molecule, the
actin binding of the COOH-terminal domain might allow for significant
interaction between the NH
-terminal domain and actin by
increasing the effective concentration of the NH
-terminal
domain (see discussion by Goody and Holmes(28) ). If, however,
the NH
-terminal domain of actin-bound caldesmon is
dissociated, then it might be able to interact with the neck region of
myosin and provide the molecular basis for the caldesmon link between
thick and thin filaments. Therefore, it is the object of this study to
investigate the interaction of the NH
-terminal domain of
intact caldesmon with actin by comparing the cross-linking and
fluorescence of the COOH- and NH
-terminal domains of
caldesmon bound to actin.
The basis of this work was our initial
observation that Cys-580 in caldesmon's COOH-terminal domain can
be easily disulfide-cross-linked to Cys-374 in actin's COOH
terminus(29, 30) . Once this cross-link was formed, I
could then follow the disulfide cross-linking between Cys-153 in
caldesmon's NH-terminal domain and another actin
monomer in the actin filament. I also compared the photocross-linking
and fluorescence of probes specifically attached to either Cys-580 or
Cys-153 of caldesmon. Specific labeling was made possible by our
ability to label Cys-153 of caldesmon whose Cys-580 had been
disulfide-cross-linked to actin (29) and by using porcine
stomach caldesmon, which contains a single cysteine at or close to
residue 580 (30) .
Figure 4:
Photocross-linking of BPM-labeled
caldesmon to actin (±tropomyosin) for 0 or 30 min. Shown is 6.7%
SDS-PAGE of the gel region showing caldesmon (CaD) and its
cross-linked product with actin (CaD A). A,
cross-linking to skeletal muscle actin of BPM-labeled heat-treated
chicken gizzard caldesmon (lane 1), BPM/Cys-153-labeled
heat-treated chicken gizzard caldesmon (lanes 2-5), and
BPM/Cys-580-labeled heat-treated porcine stomach caldesmon (lanes
6-9); B, cross-linking to gizzard muscle actin of
BPM/Cys-153-labeled heat-treated chicken gizzard caldesmon (lanes
1-4), BPM/Cys-580-labeled heat-treated porcine stomach
caldesmon (lanes 5 and 6), and BPM/Cys-153-labeled
native chicken gizzard caldesmon (lanes 7 and 8). In B, the bands between CaD and CaD
A are small impurities in the actin. Chicken caldesmon migrates
faster than porcine caldesmon (30) .
Figure 2:
A,
time course of the reaction between chicken gizzard caldesmon and
Nbs-modified actin (±tropomyosin (Tm)) by
6.7% SDS-PAGE. The reaction was carried out at room temperature in 40
mM NaCl, 5 mM Mops, 2 mM MgCl
,
pH 7.5. M indicates the internally disulfide-cross-linked
caldesmon monomer, and D indicates the disulfide-cross-linked
caldesmon dimer(43) , which are present in caldesmon at time 0
before addition to Nbs
-modified actin. X indicates
the disulfide-cross-linked product between Cys-580 of caldesmon and
Cys-374 of actin(29) . B, the X and X1 bands rerun in
the presence of DTT on 10% SDS-PAGE.
The absorption spectrum of BPM has a relatively low
extinction coefficient and overlaps with protein absorption, making
determination of the degree of labeling difficult and uncertain.
Therefore, to confirm that ``Cys-153-labeled'' gizzard
caldesmon was singly labeled at Cys-153 with BPM, use was made of the
fact that the two cysteines of chicken gizzard caldesmon form intra-
and intermolecular disulfide bonds, catalyzed by
Cu-(o-phenanthroline)
, to yield
disulfide-cross-linked monomer, dimer, and oligomers(43) . At
relatively low caldesmon concentrations, the formation of the
intramolecular cross-linked monomer
predominates(29, 30, 43) . For
single-cysteine caldesmons, like that from porcine stomach, no
cross-linked monomer forms, and the dimer is the only cross-linked
product(30) . Chicken gizzard caldesmon specifically labeled at
Cys-153 is also a single-cysteine caldesmon and should not result in
any cross-linked monomer upon oxidation at low caldesmon
concentrations. Oxidation was performed as described previously (29) with the additional step of adding excess N-ethylmaleimide, in order to block all free sulfhydryl
groups, after the oxidation reaction was stopped with EDTA. As
demonstrated in Fig. 1, under conditions where oxidation of
unlabeled caldesmon results in a high yield of cross-linked monomer (lanes A and B), Cys-153-labeled caldesmon forms very
little cross-linked monomer and some cross-linked dimer, with most of
the caldesmon remaining in the uncross-linked state (lanes C and D). This is consistent with most of the caldesmon
being singly labeled at Cys-153. If one assumes that the cross-linked
monomer represents all of the unlabeled caldesmon present, then
densitometry of the gel indicates that 88% of the caldesmon is singly
labeled. Although, hypothetically, the labeled caldesmon migrating as
the uncross-linked species could also represent doubly labeled
caldesmon or caldesmon singly labeled at Cys-580, photocross-linking
experiments described under ``Results'' rule this out.
Figure 1:
Cu-phenanthroline-catalyzed
oxidation of chicken gizzard caldesmon, unlabeled or specifically
labeled at Cys-153 with BPM. Shown is the 6.7% SDS-PAGE of unlabeled
caldesmon before (lane A) and after (lane B)
oxidation and of labeled caldesmon before (lane C) and after (lane D) oxidation. CaD, caldesmon; M,
disulfide-cross-linked monomer; D, disulfide-cross-linked
dimer. The bands above D are disulfide-cross-linked
oligomers(43) . Since lanes A and B and lanes C and D were run on separate gels, there is not
an exact match-up between bands of the same
species.
In the experiments described below, actin refers to rabbit
skeletal muscle actin, and caldesmon refers to that prepared with a
heat treatment step, except where otherwise specified. Cross-linking
and fluorescence experiments were performed at relatively high
actin/caldesmon molar ratios (14-20 for disulfide cross-linking
and 20 for photocross-linking and fluorescence) in order to minimize
any possible interference between caldesmon molecules on the actin
filament, which might reduce the interaction between the caldesmon
NH-terminal domain and actin. The molar ratio of actin to
caldesmon in native thin filaments has been reported to be
16:1(45) , although much lower values were found in
reconstituted thin filaments saturated with caldesmon (see discussion
in (29) ).
Figure 3:
Salt and temperature dependence of X1
formation and actin binding of chicken gizzard caldesmon. A,
SDS-PAGE Coomassie Blue staining intensity ratio of X1 to X after a
60-min reaction between caldesmon (CaD) and
Nbs-modified actin. Triangles, 40 °C; squares, room temperature; circles, 0 °C; open symbols, without tropomyosin; closed symbols,
with tropomyosin. Also present were 5 mM Mops and 2
mM MgCl
, pH 7.5. B, binding of caldesmon
to actin under identical conditions and with the same symbols as
described for A. At 40 mM NaCl, almost all of the
caldesmon was bound to actin, whereas at 500 mM NaCl, very
little was bound, at all temperatures (±tropomyosin); thus, at
these salt concentrations, there is only a single closed
circle.
Since gizzard caldesmon contains a second
cysteine residue at position 153 in the NH-terminal domain,
I suspected that the X1 product was due to a second cross-link between
Cys-153 and another Nbs
-modified actin monomer by disulfide
exchange. Strong support for this came from cutting out and rerunning
the X and X1 bands on SDS-PAGE in the presence of DTT, which reduces
disulfide bonds (Fig. 2B). Reduction of both X and X1
resulted in the formation of a caldesmon and an actin band (Fig. 2B) with the actin/caldesmon intensity ratio of
the X1 band twice that of the X band, as determined by densitometry,
showing that the X1 band is due to a 2:1 molar complex of actin and
caldesmon. The migration of X1 on SDS-PAGE also is consistent with a
2:1 actin/caldesmon product, which should have an apparent M
of
224,000 (M
of actin
= 42,000; M
of caldesmon = 140,000,
which is anomalous(46) ). Indeed, X1 migrates (Fig. 2A) between the X band, which has an M
of 182,000, and the D band, which has an M
of 280,000. For the remainder of this work, the
nature and formation of the unresolved, higher molecular weight species
will not be considered, and I will focus on the X1 species, which is
well resolved and thus amenable to study.
To gain a better
understanding of the disulfide cross-linking of the
NH-terminal domain of caldesmon to actin, the yield of the
X1 product, relative to the X species, was studied as a function of
ionic strength at different temperatures as described under
``Experimental Procedures.'' Essentially all of the caldesmon
was first converted to the X product by incubation of caldesmon with
Nbs
-modified actin (±tropomyosin) for 2 min at room
temperature in 40 mM NaCl (Fig. 2A). The salt
and temperature were then changed or kept the same, and incubation was
continued for another 60 min, whereupon the reaction was stopped with N-ethylmaleimide. SDS-PAGE was run, and the X1/X Coomassie
Blue staining ratio was determined by densitometry (Fig. 3A). (An X1/X staining ratio of 1 is equivalent
to an X1/X molar ratio of
0.8.) To correlate this cross-linking
with the caldesmon-actin interaction strength, the actin binding of
caldesmon was also studied under the same conditions and protein
concentrations as the cross-linking experiments (Fig. 3B). The yield of X1 increased substantially with
increasing temperature, but except for a small decrease in X1 with
increasing salt concentration, was relatively insensitive to ionic
strength and to the presence or absence of tropomyosin (Fig. 3A). In contrast to the yield of X1, the binding
of caldesmon changed dramatically, going from
100% to close to 0%
over the same salt concentration range (Fig. 3B). The
lack of correlation of caldesmon NH
-terminal domain
cross-linking to actin with the caldesmon-actin binding strength
strongly suggests that the NH
-terminal domain of caldesmon
is largely dissociated from actin, while the COOH-terminal domain is
bound and becomes slowly cross-linked to Nbs
-modified actin
during collisions with the actin filament. Further dissociation of the
COOH-terminal domain of caldesmon from actin, except for a disulfide
tether, with high salt would be expected to have only a small effect on
cross-linking of the already dissociated NH
-terminal
domain. The increase in cross-linking of the NH
-terminal
domain to actin with increasing temperature might be due to greater
thermal motion and flexibility of the NH
-terminal domain
and thus a higher collisional frequency with actin. Alternatively, the
increase in cross-linking might result from a thermal increase in the
rate of the disulfide exchange reaction, possibly due to a change in
conformation of caldesmon and/or actin in the region of the involved
cysteines.
It has been shown that photolysis of actin plus chicken gizzard caldesmon, nonspecifically labeled at both cysteines with BPM, results in one major product identified as a 1:1 complex of actin and caldesmon by its apparent molecular weight on SDS-PAGE(48) . I confirmed this finding by also observing one major cross-linked product (Fig. 4A, lane 1), which was identified as a 1:1 complex of actin and caldesmon by the fact that this product comigrated with the X band, the 1:1 caldesmon-actin disulfide-cross-linked complex, on SDS-PAGE (data not shown). When BPM-labeled porcine stomach caldesmon, which contains a single cysteine at/or near position 580(30) , was photolyzed in the presence of (skeletal or smooth muscle) actin (±tropomyosin), similar results were obtained (Fig. 4, A (lanes 6-9) and B (lanes 5 and 6)).
The fact that caldesmon labeled at both
cysteines yields about the same cross-linking results as compared to
when only Cys-580 is labeled suggests the possibility that the extent
of the Cys-153 label cross-linking to actin is low. To test this
directly, I studied the photocross-linking of chicken gizzard caldesmon
specifically labeled at Cys-153 with BPM. The photolysis of
BPM/Cys-153-labeled caldesmon plus actin resulted in only a very faint
cross-linked product (±tropomyosin) (Fig. 4A, lanes 2-5). (The faint band cannot be seen in the gel
photographs.) The same results were obtained when actin from chicken
gizzard muscle was used (Fig. 4B, lanes
1-4) and when caldesmon prepared without a heat treatment
step, i.e. native caldesmon, was used with gizzard muscle (lanes 7 and 8) or skeletal muscle (data not shown)
actin. (The fact that there is no cross-linking indicates that the
caldesmon is neither doubly labeled nor singly labeled at Cys-580 with
BPM (see ``Experimental Procedures'').) Thus, the BPM label
at Cys-153, in the NH-terminal domain of caldesmon, does
not cross-link to actin. The BPM/Cys-153-labeled caldesmon was fully
bound to actin since all of the labeled caldesmon cosedimented with
actin under the conditions of the photocross-linking experiments. Thus,
the lack of cross-linking is not due to a lack of binding. The absence
of cross-linking of BPM at Cys-153, together with a high yield of
cross-linking of the label at Cys-580, is consistent with the
conclusion, drawn above, that the NH
-terminal domain of
caldesmon binds weakly, if at all, to actin, while the COOH-terminal
domain binds strongly.
Caldesmon specifically labeled with BPM at either Cys-580 or Cys-153 was photolyzed in the absence of actin as controls (data not shown). Very little, if any, cross-linked products were obtained with the label at Cys-580. However, for the Cys-153-labeled caldesmon, there was a significant drop in the intensity of the caldesmon SDS-PAGE band with a corresponding increase in the amounts of many higher molecular weight species (which migrated slower than the cross-linked product between caldesmon and actin). For the longest photolysis time (120 min), there was also some cross-linked product that did not enter the stacking gel. These slower migrating species most likely correspond to oligomeric cross-linked caldesmon formed upon very weak interactions between caldesmon molecules. These higher molecular weight bands were not observed when actin was present since the caldesmon is bound to actin and is less likely to interact with other caldesmon molecules. These control experiments indicate that the cross-linked product formed (with Cys-580-labeled caldesmon) in the presence of actin is not due to caldesmon alone (negative control), and they demonstrate that although the BPM label at Cys-153 of caldesmon does not significantly cross-link to actin, it can cross-link to another protein (in this case itself) when it comes in contact with it (positive control).
The fluorescence emission and
polarization of the rhodamine probe at Cys-580 were very sensitive to
interaction with actin (±tropomyosin). The fluorescence
intensity decreased 28% upon interaction with actin and 26% with actin
+ tropomyosin (Fig. 5A), indicating an interaction
between the COOH-terminal domain of caldesmon and actin. The
polarization of rhodamine at Cys-580 changed from a value of 0.21 for
free caldesmon to 0.38 in the presence of actin and 0.40 with the
additional presence of tropomyosin (Fig. 6A).
Fluorescence polarization is sensitive to changes in both probe
mobility and fluorescence intensity(44) . From the decrease in
rhodamine fluorescence intensity upon the addition of actin +
tropomyosin, an increase in polarization of 15% is expected (44) . That the polarization increased by 100% to 0.4, which is
the maximum observable polarization for this rhodamine
probe(49) , indicates a dramatic decrease in the mobility of
the probe upon interaction of the COOH-terminal domain of caldesmon
with actin. In contrast, the emission intensity (Fig. 5B) and polarization (Fig. 6B) of
the rhodamine probe at Cys-153, in the NH-terminal domain
of caldesmon, were unresponsive to the binding of actin
(±tropomyosin), consistent with the NH
-terminal
domain being dissociated from actin. The actin binding of the two
labeled caldesmons was measured under the same conditions as used for
the fluorescence measurements. Essentially 100% of both labeled
caldesmons was bound to actin, indicating that the fluorescence changes
observed were representative of the fully bound protein.
Figure 5: Fluorescence emission spectra of the rhodamine probe attached to caldesmon at either cysteine 580 (A) or cysteine 153 (B). The excitation wavelength was 530 nm. Circles, caldesmon; triangles, caldesmon + actin; squares, caldesmon + actin + tropomyosin.
Figure 6: Fluorescence polarization spectra of the rhodamine probe attached to caldesmon at either cysteine 580 (A) or cysteine 153 (B). The excitation wavelength was 530 nm. Circles, caldesmon; triangles, caldesmon + actin; squares, caldesmon + actin + tropomyosin.
We have shown that, for intact caldesmon, the COOH-terminal
domain is very responsive to the binding of actin ± tropomyosin,
whereas the NH-terminal domain is almost completely
insensitive in reconstituted thin filaments. The COOH-terminal domain
can be disulfide-cross-linked to actin (±tropomyosin) much more
rapidly and with much higher yield than the NH
-terminal
domain. Furthermore, the small amount of NH
-terminal domain
cross-linking, which is already disulfide-cross-linked to actin via
Cys-580, is relatively insensitive to increases in ionic strength,
which greatly reduces the caldesmon-actin interaction. Benzophenone
attached to Cys-580 in the COOH-terminal domain of caldesmon can be
photocross-linked to actin (±tropomyosin) with good yield,
whereas there is almost no cross-linking of the probe attached to
Cys-153 in the NH
-terminal domain, results found for native
or heat-treated caldesmon and with actin from striated or smooth
muscle. Finally, the fluorescence emission and polarization of a
rhodamine probe attached to caldesmon Cys-580 are very sensitive to the
binding of actin (±tropomyosin), whereas the fluorescence of the
probe at Cys-153 does not change. These findings, together with studies
that have shown that COOH-terminal fragments of caldesmon bind to actin
± tropomyosin and that NH
-terminal fragments do
not(23, 24, 25, 26, 27) ,
strongly suggest that in reconstituted thin filaments, the
COOH-terminal domain of caldesmon is bound to actin, while the
NH
-terminal portion is, for the most part, dissociated. If
this interpretation of the results is correct, then the extent of the
NH
-terminal portion of caldesmon dissociated from actin
must extend somewhat beyond Cys-153, but how much beyond cannot be
decided from this work.
Tropomyosin, which binds to caldesmon in the
absence of actin(31, 50, 51, 52, 53, 54) and
increases the actin binding of caldesmon(33, 55) , has
no effect on the cross-linking or fluorescence of the
NH-terminal domain of actin-bound caldesmon. This suggests
that actin-bound tropomyosin neither binds to the
NH
-terminal domain of caldesmon nor promotes the actin
binding of the NH
-terminal domain. This is in contrast to
studies conducted in the absence of actin that show an interaction
between tropomyosin and the NH
-terminal domain of
caldesmon(25, 56, 57) . Thus, any interaction
between actin-bound tropomyosin and caldesmon most likely involves
caldesmon's COOH-terminal domain, which can also interact with
tropomyosin in the absence of
actin(26, 27, 51, 57, 58, 59, 60) and which contains sequence homologies to troponin
T(58, 59, 61, 62) , the
tropomyosin-binding subunit of skeletal muscle troponin.
Three
microscopy studies have addressed the question of the arrangement of
the NH-terminal domain of caldesmon in reconstituted and
native thin filaments. For native thin filaments, all three studies
found no visual evidence for caldesmon
projections(38, 63, 64) , projections that
might be expected for a dissociated NH
-terminal domain.
(However, a recent publication has reported unpublished observations of
NH
-terminal domain projections of caldesmon in native thin
filaments(20) .) Similarly for reconstituted thin filaments,
two of the studies, using negative stain electron microscopy (63) or x-ray diffraction microscopy(38) , concluded
that there are no (38) or occasional (63) molecules
projecting out from the filament. On the contrary, one study on
reconstituted thin filaments, performed with rotary shadow
immunoelectron microscopy(64) , observed that greater than
two-thirds of the caldesmon was bound to actin with either its
NH
- or COOH-terminal end, with the opposite end projecting
out away from the filament. About 30% of the projecting molecules were
bound via their NH
-terminal end, suggesting significant
interaction between the NH
-terminal domain of caldesmon and
actin, even in the absence of COOH-terminal domain binding.
The
basis for these discrepancies is presently unclear. First of all, how
do I reconcile my conclusion of the caldesmon NH-terminal
domain dissociated from actin with the microscopy studies not observing
caldesmon projections. One might expect projections if a significant
portion of the NH
-terminal part of caldesmon was not
interacting with actin. However, the part of the
NH
-terminal domain dissociated from actin may not extend
much beyond Cys-153, and it may not be possible to observe projections
of such a small part of the caldesmon molecule. Even if a larger part
of the NH
-terminal part of caldesmon was dissociated from
actin, only a small part of this might project out, or the entire
dissociated part of caldesmon might be confined to a region close to
the filament and might not project enough to be seen, all depending on
the degree and region of flexibility of bound caldesmon. Thus, the
NH
-terminal domain could be dissociated, but not fully
projecting out from the actin filament. On the other hand, since one
study (64) found a large difference in caldesmon arrangement
between native and reconstituted thin filaments and another study (63) finds some difference, caldesmon may bind differently in
both types of filaments, with a dissociated NH
-terminal
domain more prevalent in reconstituted filaments. Since we have
provided evidence suggesting that the COOH-terminal domain of caldesmon
assembles similarly in native and reconstituted thin
filaments(30) , any difference in assembly is most likely
confined to the rest of the molecule. Finally, the arrangement of
caldesmon on actin might depend on the very different sample conditions
and treatments used for each study. Specifically, this work was
performed on dilute thin filament solutions, x-ray microscopy was
carried out on very concentrated thin filament sols, and electron
microscopy was done with fixed thin filament samples. More work is
necessary to resolve these differences and uncertainties.
The
dissociation of the NH-terminal domain of caldesmon from
actin in reconstituted thin filaments is important, regardless of
whether or not it is dissociated in vivo. If it is not
dissociated in vivo, then knowledge of why it is dissociated in vitro will give a deeper understanding of the
caldesmon-actin interaction. If the myosin-binding
NH
-terminal domain of caldesmon does exist in the
dissociated state in vivo, it might easily link the actin
filament to the myosin filament, a link for which there is in vitro(19, 20, 21) and in situ(22) evidence. Since caldesmon interacts with the neck (i.e. S2 region) of myosin(16, 65) , a region
of myosin that is not thought to come in contact with the actin
filament, it might be necessary for the caldesmon
NH
-terminal domain to be dissociated from actin in order to
reach the S2 part of the myosin molecule. It has been proposed that a
caldesmon link between myosin and actin might enhance a productive
interaction between myosin and actin during contraction (19) or
might aid in the assembly of myosin filaments in the vicinity of actin
filaments(20) . It has also been suggested (15, 16, 17, 18) that such a static
connection between filaments might possibly be a molecular basis for
the latch state(6) , in which tension is maintained at the
expense of very little energy.