(Received for publication, April 21, 1995; and in revised form, June 26, 1995)
From the
A key assumption of most models for calmodulin regulation of
smooth and non-muscle contractility is that calmodulin is freely
diffusible at resting intracellular concentrations of free
Ca. However, fluorescence recovery after
photobleaching (FRAP) measurements of three different fluorescent
analogs of calmodulin in cultured bovine tracheal smooth muscle cells
suggest that free calmodulin may be limiting in unstimulated cells.
Thirty-seven % of microinjected calmodulin is immobile by FRAP and the
fastest recovering component has an effective diffusion coefficient
7-fold slower than a dextran of equivalent size. Combining the FRAP
data with extraction data reported in a previous paper (Tansey, M.,
Luby-Phelps, K., Kamm, K. E., and Stull, J. T.(1994) J. Biol. Chem. 269, 9912-9920), we estimate that at most 5% of total
endogenous calmodulin in resting smooth muscle cells is unbound (freely
diffusible). Examination of the Ca
dependence of
calmodulin mobility in permeabilized cells reveals that binding
persists even at intracellular Ca
concentrations as
low as 17 nM. When Ca
is elevated to between
450 nM and 3 µM, some of the bound calmodulin is
released, as indicated by an increase in the effective diffusion
coefficient and the percent mobile fraction. At higher
Ca
, calmodulin becomes increasingly immobilized. In
about 50% of the cell population, clamping Ca
at
micromolar levels results in translocation of cytoplasmic calmodulin to
the nucleus. The compartmentalization and complex dynamics of
calmodulin in living smooth muscle cells have profound implications for
understanding how calmodulin regulates contractility in response to
extracellular signals.
Calmodulin plays a central role in the
Ca-dependent regulation of smooth muscle
contractility and is thought to regulate a host of other cellular
processes including non-muscle cell contractility, intracellular
Ca
-homeostasis, calcium signaling, and nitric oxide
production(1) . Most current models of calmodulin-dependent
functions assume that under resting conditions the large intracellular
pool of calmodulin is freely diffusible. Receptor-mediated elevation of
cytoplasmic free Ca
above resting levels is thought
to promote calmodulin binding to and activation of target enzymes such
as myosin light chain kinase(2) , the plasma membrane
Ca
-ATPase(3) , the CaM (
)kinases(4) , and nitric oxide
synthetase(5) . The details of these models are based largely
on what has been learned from studies of calmodulin and its target
enzymes in dilute solution. However, several reports in the literature
suggest that events in the more complex milieu of intact systems cannot
be extrapolated directly from in vitro experiments(6) . Macromolecular crowding and cytoskeletal
elements may impose constraints on diffusion (7) or render some
cytoplasmic compartments inaccessible(8) . Macromolecular
crowding might also promote intermolecular associations that are not
favored in dilute solution(9) . In any case, competition among
multiple calmodulin-binding proteins could complicate the kinetics of
calmodulin activation of a particular target enzyme, especially since a
number of proteins have been identified that bind calmodulin with high
affinity at resting Ca
levels (10, 11) .
Recent experimental evidence weakens the
assumption that calmodulin is freely diffusible in resting smooth
muscle cells. It has been reported that only 50% of endogenous
calmodulin is extracted from smooth muscle fibers that have been
extensively skinned in the absence of
Ca(12) , suggesting that the remainder is
tightly bound, perhaps to the cytoskeleton. Although the calmodulin
remaining in the skinned fibers is in 3-fold excess of myosin light
chain kinase (MLCK) and 10,000-fold greater than the K
for activation of MLCK, exogenous calmodulin must be added to the
skinned fibers to elicit contraction(12) . Smooth muscle cells
(SMC) in culture that have been permeabilized less extensively by
treatment with
-escin appear to retain all their calmodulin, and
phosphorylate MLCK to nearly the same extent as intact cells, even
though the holes in the plasma membrane are sufficiently large to allow
the escape of dextran molecules the size of calmodulin(12) ,
suggesting that even the Triton-extractable fraction is not freely
diffusible.
Fluorescence recovery after photobleaching (FRAP) has
been used to gain further insight into the diffusibility of calmodulin
in intact living cells. This technique involves the use of a focused
laser beam to create a concentration gradient of fluorescent molecules
by the irreversible photolysis of a portion of the fluorophores in the
volume illuminated by the laser beam. The relaxation of this gradient,
leading to recovery of fluorescence in the bleached region, reflects
the translational mobility of the fluorophores. In cases where flow or
active transport is not a factor, the half-life of the fluorescence
recovery in the bleached region is proportional to the effective
diffusion coefficient of the fluorescent molecule, and the plateau
value (percent recovery) is a measure of the fraction of molecules that
are mobile on the timescale of the measurement(13) . We have
previously shown by FRAP that a rhodamine-labeled analog of calmodulin
microinjected into living Swiss 3T3 fibroblasts or SMC shows reduced
mobility when compared with a dextran of equivalent
size(12, 14) . This strongly suggests that calmodulin
is not freely diffusible in intact cells. However, the possibilities
that binding was due to a nonspecific interaction of the fluorophore
with intracellular components or to the presence of the fluorophore on
a particular amino acid residue of the calmodulin were not ruled out.
In addition, the mobility of the analog in permeabilized cells and the
possible Ca dependence of the binding were not
addressed.
In the current report we examine the specificity and
Ca dependence of the calmodulin binding in SMC by
studying improved fluorescent analogs of calmodulin in intact and
permeabilized SMC. The results suggest that endogenous calmodulin is
compartmentalized into several intracellular pools with differing
affinities and dependence on intracellular free Ca
concentration. In addition, there appears to be a pool of
calmodulin that becomes concentrated in the nucleus when intracellular
free Ca
is clamped at elevated levels.
For all three calmodulin analogs, there
was a wide variety in the detailed shape of recovery curves from cell
to cell. Some recoveries were fit well by assuming a single recovering
species, while others were not, indicating multiple classes of binding
sites with differing affinities for the analog (Fig. 1). The
equation that describes the recovery of fluorescence by diffusive
transport is an infinite series that does not converge(13) ,
and no approximations are available in the literature. The algorithm we
use to fit our data is an empirical solution applicable only to single
component recoveries and has no physical meaning(19) . For
these reasons, we are unable to analyze multiple component recoveries
such as those exhibited by calmodulin analogs in living cells. However,
we found that the initial 5 s of each data record were fit well by
assuming a single recovering species. This provided an estimate of the
effective diffusion coefficient for the fastest recovering component (Fig. 1). This parameter was found to vary over nearly an order
of magnitude from cell to cell. For LRB-CaM, the mean value was 4.9
10
cm
/s ± 0.42 S.E.(n = 41). In contrast, all recovery curves for FTC-dex20 were
fit well by assuming a single recovering species, and the mean value of
the diffusion coefficient was approximately 7-fold faster than for
LRB-CaM (34
10
cm
/s ± 2.4
S.E. (n = 48). The average percent recovery for LRB-CaM
was 63 ± 5 S.E., compared with 87 ± 2 S.E. for FTC-dex20,
indicating that about 37% of LRB-CaM is immobile on the 30-s timescale
of the measurements. Immobilization of LRB-CaM following microinjection
was rapid. When FRAP measurements were made within 30 min after
injection, the mean diffusion coefficient for the fastest recovering
component of LRB-CaM was already 4.2
10
cm
/s ± 0.78 S.E. (n = 19) and
the mean percent recovery was 67.5 ± 3.4 S.E. (n = 19).
Figure 1: FRAP recovery kinetics for fluorescent calmodulin analogs in the cytoplasm of living SMC are complex. Data were acquired before the bleach to obtain the initial fluorescence intensity. A 400-ms bleaching pulse was initiated at time 0. The recovery of fluorescence following the bleach was monitored for 30 s. Plateau values (indicated by a point at infinite time) were determined as described under ``Materials and Methods.'' The amount of fluorescence that does not recover (A) is the immobile component (in this case 15%). The extent of recovery (B) is the mobile component. The recovery curve is a weighted average of the contributions from all recovering species. Many curves such as the one depicted here were not well fit by assuming a single recovering species (dashed line). This indicates compartmentalization of calmodulin analogs into more than one intracellular pool with differing characteristic times of recovery. In most cases, the first 5 s of data were well fit to a single component (solid line), allowing estimation of the cytoplasmic diffusion coefficient for the fastest recovering species.
To rule out the possibility that the slow recovery of LRB-CaM in SMC might be an artifact due to recovery of fluorescence during the relatively long time required to bleach lissamine rhodamine, we varied the bleaching time from 100 to 1000 ms. Varying the bleaching time had no significant effect on the diffusion coefficient obtained for LRB-CaM. In addition, the cytoplasmic diffusion coefficients for LRB-dex10 and FTC-dex10 measured in SMC were virtually identical (Table 1). Besides ruling out recovery during the bleaching pulse, these data indicate that the differences in recovery kinetics between FTC-dex20 and LRB-CaM do not result from differential binding of the two fluorophores to cytoplasmic components. This was also demonstrated by the observation that the fastest recovering components of AFCys3CaM and FTC-CaM in SMC have mean diffusion coefficients very similar to that of LRB-CaM (Table 1).
To see whether binding of LRB-CaM was
Ca sensitive, we raised intracellular free
Ca
in a step-wise manner by perfusing the
permeabilized cultures with Ca
-EGTA buffer solutions
as described under ``Materials and Methods.'' FRAP
measurements were made on the same group of cells at each
Ca
concentration to control for cell to cell
variability. We found that the mobility of LRB-CaM rose as
Ca
was increased, passed through a maximum between
450 nM and 3 µM free Ca
, and
then declined. This is reflected in both the diffusion coefficient for
the fastest component and in the percent recovery (Fig. 2). The
peak values of both parameters approached the mean value for intact
cells. The recovery kinetics of FTC-dex20 were unaffected by
-toxin permeabilization or by changes in free Ca
(Table 1).
Figure 2:
Ca dependence of the
diffusion coefficient of the fastest recovering component and of
percent recovery for LRB-CaM in
-toxin permeabilized SMC. Cells
were permeabilized in CFB and Ca
was elevated
stepwise by perfusion of permeabilized cultures with Ca-EGTA-buffered
solutions. The free Ca
concentration of these
solutions was measured using the fluorescent Ca
indicator, fluo-3 as described under ``Materials and
Methods.'' Closed circles: D
was
plotted as percent of D
in CFB versus [Ca
]. Open circles: percent
mobile fraction versus [Ca
]. The
mobility of LRB-CaM increases as Ca
is elevated,
passes through a maximum between 450 nM and 3 µM
free Ca
, and then
declines.
We raised intracellular Ca in intact cells by perfusing the cultures with DMEM containing 10
µM ionomycin. FRAP measurements of LRB-CaM in the
cytoplasm of ionomycin-treated cells indicated that the mean percent
recovery decreased to 25% (Table 1), and in some cells LRB-CaM
was completely immobile. In cells that still exhibited measurable
fluorescence recovery, a slight decrease in the diffusion coefficient
for the fastest recovering component was observed (Table 1). The
recovery kinetics of FTC-dex20 were unchanged by treatment of SMC with
ionomycin (Table 1). Representative FRAP curves for LRB-CaM in
SMC before and after ionomycin treatment are shown in Fig. 3.
Figure 3:
Effect of 10 µM ionomycin on
FRAP of LRB-CaM in intact SMC. A, a representative
fluorescence recovery curve for LRB-CaM in the cytoplasm of an intact
SMC before perfusion of the culture with 10 µM ionomycin
in DMEM. B, a representative fluorescence recovery curve for
LRB-CaM after perfusion with ionomycin. The initial rate of recovery is
little changed by the treatment, but the extent of recovery after
ionomycin treatment is significantly reduced. This may either be due to
Ca-mediated binding of the analog or to
Ca
-induced translocation of calmodulin into the
nucleus, resulting in a smaller pool of mobile calmodulin in the
cytoplasm. The two curves shown in this figure were not taken from the
same cell and were normalized for fluorescence intensity. Fluorescence
intensity is plotted as percent of initial prebleach value. RFI, relative fluorescence
intensity.
Figure 4:
The fluorescence intensity of LRB-CaM in
the nucleus becomes elevated upon treatment of SMC with ionomycin in
the presence of mM Ca. Ratio images were
generated by dividing the image of LRB-CaM fluorescence in a single
living SMC by the image of FTC-dex20 fluorescence in the same cell to
normalize LRB-CaM fluorescence intensity for pathlength. A,
before perfusion of the culture with 10 µM ionomycin in
DMEM. B, same cell after perfusion with ionomycin-containing
medium. N = nucleus. In A, the ratio of
nuclear to cytoplasmic LRB-CaM is 1.03. In B, the ratio is
1.26, an increase of 22%. In vitro fluorescence measurements
of LRB-CaM in the presence of calmodulin-binding peptides suggests the
increase represents a real change in concentration rather than an
effect on the quantum yield of fluorescence due to binding of
calmodulin to target proteins (see
``Results'').
It has been shown
previously that treatment of SMC with 10 µM ionomycin in
DMEM elevates intracellular Ca to 4.4
µM(15) , which is greatly in excess of what is
required to initiate smooth muscle contraction. To see whether
calmodulin also translocates into the nucleus at a Ca
concentration in the range evoked by agonists, SMC were
microinjected with LRB-CaM and FTC-dex20 and then permeabilized with
-toxin in Ca-EGTA buffer solutions at defined concentrations of
free Ca
. The normalized fluorescence intensity of
LRB-CaM in the nucleus versus the cytoplasm was quantified by
ratio imaging as for the ionomycin experiments described above. The
data are summarized in Table 2. When cells were permeabilized in
CFB (17 nM free Ca
), the mean LRB-CaM
fluorescence intensity in the nucleus was 11% higher than in the
cytoplasm, similar to intact cells (Table 2). When cells were
permeabilized at a free Ca
concentration of 450
nM, the mean intensity of LRB-CaM in the nucleus was 21%
higher than in the cytoplasm (Table 2). Student's t test showed that the mean values at the two concentrations of
Ca
were significantly different with p <
0.005. At the higher Ca
concentration, 34 of 70 cells
exhibited a ratio of nuclear LRB-CaM to cytoplasmic LRB-CaM that was
more than one standard deviation higher than the mean for all cells in
CFB, while in CFB only four cells were more than one standard deviation
higher than the mean. When the data for the 34 responding cells were
averaged, the mean increase in nuclear fluorescence of LRB-CaM over the
mean value for all cells in CFB was 16% (Table 2).
The diffusion and binding of fluorescent CaM analogs in the cytoplasm of serum-starved SMC were studied by FRAP and compared with the recovery kinetics of FTC-dex20, whose molecular size is similar to CaM(14) . The results indicate that a large fraction of CaM is bound to intracellular binding sites, even at resting intracellular . Since we obtain similar results with two analogs that are fully capable of activating myosin light chain kinase, and since these analogs differ both in the electrostatic properties of the fluorophore and in the amino acid residue that is labeled, we conclude that binding reflects the behavior of endogenous calmodulin rather than some property peculiar to the fluorescent analog.
Our results differ from an earlier report in which no binding of FTC-CaM was detected in unstimulated smooth muscle cells by steady state fluorescence polarization microscopy, although binding was detected when the cells were stimulated(16) . Our FRAP measurements of the same analog used by those authors indicate significant binding even in unstimulated cells. One possible reason for this discrepancy is that steady state polarization of fluorescence may not be a very sensitive measure of calmodulin binding. Depolarization of fluorescence can arise from rotation of the fluorophore about the thiocarbamoyl bond that links it to calmodulin, as well as from segmental motion of the calmodulin itself(24) .
FRAP has been criticized on the basis that such high intensity illumination can cause severing of polymers or cross-linking of macromolecules(25, 26) . However, these effects may be limited to within a few Ångstroms of the bleached fluorophore since no damage to actin filaments in solution was observed from FRAP of fluorophores that were not covalently attached to the actin monomers(26) . The short range of FRAP damage is also indicated by the observation that fluorescein dextrans, which exhibit little if any binding to intracellular components, are nearly 100% mobile in living cells. In the current study, FRAP damage might occur to the bleached molecule of calmodulin itself or to other molecules bound to the analog at the moment it is bleached. Damage to unbound calmodulin during bleaching will not affect our data, since the recovery is due only to diffusion of unbleached molecules. Damage to the fluorescent analog while it is bound to a target protein might result in cross-linking of the complex, artifactually inflating the immobile fraction at the expense of a transiently bound fraction. However, this would not alter our estimates of how much calmodulin is freely diffusible in living SMC.
An upper limit for the fraction of
calmodulin that is freely diffusible in unstimulated SMC can be
estimated by assuming that the fastest recovering component represents
CaM transiently bound to immobile binding sites. In that case, the
ratio of the observed diffusion coefficient to the diffusion
coefficient of free calmodulin is the unbound (freely diffusible)
fraction(13) . The mean value for the diffusion coefficient of
the fast component (averaged over the three analogs used in this study)
is 4.4 10
cm
/s. Taking the
diffusion coefficient measured for FTC-dex20 in SMC as equivalent to
the diffusion coefficient for unbound LRB-CaM, we find that the unbound
fraction of the recovering component is 4.4/34 or 13%. Since the
recovering components represent only 63% of the total LRB-CaM, we
conclude that at most 0.63
0.13 or 8% of the analog is freely
diffusible in living SMC. Since 41% of total calmodulin cannot be
extracted from smooth muscle tissue with Triton and
glycerol(12) , it is likely that the injected analog only
exchanges with the extractable pool, which is 59% of total calmodulin.
Thus at most 0.08
0.59 or 5% of calmodulin is freely diffusible
in living SMC. Five % of the endogenous calmodulin concentration (39
µM) would be 2 µM, which is somewhat less
than the concentration of myosin light chain kinase(12) .
Assuming that the binding is saturable, the freely diffusible fraction
of endogenous calmodulin in uninjected cells would be even smaller.
An alternative explanation for the hindered diffusion of the fastest recovering component is that there is a pool of calmodulin that is tightly bound to (a) diffusible target(s). We can estimate the size of such a complex by comparing the observed mean diffusion coefficient of the fast component of LRB-CaM with the diffusion coefficients of three different sized FTC-dextrans (Fig. 5). In SMC, the cytoplasmic diffusion coefficient of the dextrans decreases almost linearly with the hydrodynamic radius of the particle. Extrapolation of a line fit to these data shows that the observed diffusion coefficient of the fast component of LRB-CaM corresponds to a particle radius of 3.7 nm. This is considerably smaller than expected for a complex of CaM with smooth muscle MLCK, since the complex of calmodulin with skeletal muscle MLCK has a radius of 5.9 nm(27) . However, at least one known calmodulin-binding protein (CaM kinase I) is of the appropriate size (28) .
Figure 5:
D for FTC-dextrans
as a function of hydrodynamic radius. Comparison of D
for the fastest recovering component of LRB-CaM with D
for dextrans was used to estimate the size of
a particle with this diffusion coefficient. The position of D
for LRB-CaM is indicated by the arrow. This corresponds to a hydrodynamic radius of 3.7
nm.
Perhaps the most surprising observation is that so
little calmodulin is freely diffusible under resting conditions, where
the intracellular concentration of Ca is below the
threshold for Ca
-dependent binding of calmodulin. One
possible explanation is that binding of calmodulin to targets can
increase the affinity of calmodulin for
Ca
(10, 11) . However, the data from
cells permeabilized in the presence of EGTA or BAPTA suggest that the
binding we observe by FRAP in unstimulated cells fits the accepted
criteria for Ca
-independent binding.
Unless
calmodulin is already complexed with MLCK in resting cells, free
calmodulin may be limiting for smooth muscle contraction, especially if
other targets, such as CaM kinase II compete with MLCK(12) . In
that case, calmodulin must be released from
Ca-independent binding sites in order for smooth
muscle contraction to occur. Consistent with this idea, we observed an
increase in the mobility of LRB-CaM in
-toxin-permeabilized SMC as
[Ca
] was raised stepwise by perfusion with
Ca-EGTA buffered solutions. The diffusion coefficient of the fastest
recovering component passed through a maximum and then declined as
Ca
was increased further, as would be expected if
Ca
-independent binding sites were being exchanged for
Ca
-dependent binding sites.
The increase in the
immobile fraction of LRB-CaM when intracellular Ca is
elevated by treating SMC with ionomycin in the presence of mM Ca
may also reflect binding of calmodulin to
Ca
-dependent sites or it may reflect the
translocation of a portion of the mobile component into the nucleus. We
noted that in about 50% of cells the fluorescence intensity of LRB-CaM
in the nucleus was elevated with respect to the cytoplasm following
treatment with ionomycin or
-toxin permeabilization in the
presence of 450 nM free Ca
. The excess
LRB-CaM fluorescence in the nucleus cannot be due to a
Ca
-induced increase in quantum yield since
Ca
readily equilibrates between nucleus and
cytoplasm(29, 30) . It is also unlikely that the
elevated LRB-CaM intensity in the nucleus reflects an increase in the
quantum yield of the analog upon binding of calmodulin to target
enzymes, since the quantum yield of LRB-CaM is unaffected by binding to
phosphodiesterase (14) or to peptides mimicking the
calmodulin-binding domains of several target enzymes. Further study
will be required to determine the mechanism by which calmodulin is
translocated into the nucleus at elevated intracellular
Ca
. One possibility is that
Ca
-dependent binding sites in the nucleus act as a
sink for calmodulin. Another possibility is that calmodulin is
transported into the nucleus as part of a complex with a protein having
a nuclear localization signal, such as a transcription factor. In this
regard, it has been reported recently that an alternatively spliced
form of CaM kinase II has a nuclear localization signal(31) .
The possible functions of calmodulin in the nucleus are not well
understood(29) , and at present it is not clear what role
calmodulin translocation into the nucleus might play in smooth muscle
physiology.
In summary, it appears that only a small fraction
(5%) of the 39 µM calmodulin SMC is freely diffusible
in unstimulated cells. Our data indicate the existence of several pools
of bound calmodulin with different binding affinities and
Ca
dependence. From published data on
Triton-glycerol-extracted smooth muscle fibers, we estimate that 23% of
total endogenous calmodulin is non-extractable and
Ca
-insensitive(12) . Another 18% is
non-extractable at low Ca
but is released during a
single contraction cycle, during which free Ca
is
elevated(12) . Our FRAP measurements show that of the remaining
59%, 22% is bound to slowly exchanging sites that do not turnover
within 30 s. The other 37% exhibits an effective diffusion coefficient
7-fold slower than expected for a molecule of this size. These
calculations are summarized in Table 3. A fraction of the 59%
exchangeable calmodulin is mobilized by elevation of intracellular
[Ca
] and may rebind to
Ca
-dependent sites at high
[Ca
]. At high Ca
,
calmodulin also appears to become spatially compartmentalized by
translocation of calmodulin into the nucleus. Thus, regulation of
smooth muscle contraction by calmodulin may be far more complex than
can be inferred from dilute solution assays.