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.

MATERIALS AND METHODS

Cell Culture

Preparation and Microinjection of Fluorescent Analogs

Fluorescence Spectra

Myosin Light Chain Kinase Assay

FRAP

-Escin Permeabilization

-Toxin Permeabilization

Titration of Free Ca

Fluorescence Ratio Imaging

RESULTS

Mobility of Dextran and Calmodulin in Unstimulated SMC

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).

Calmodulin Diffusion in -Escin-permeabilized SMC

Ca Dependence of LRB-CaM Binding

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.

Sustained Elevation of Intracellular Ca Results in Translocation of Calmodulin into the Nucleus

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).

Effect of CaM-binding Peptides on Quantum Yield of LRB-CaM

DISCUSSION

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.

FOOTNOTES

*

This work was supported by National Science Foundation Grant MCB-9304603 and American Heart Association Grant-in-aid 93011130 (to K. L.-P.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§

American Heart Association Established Investigator. To whom correspondence should be addressed. Tel.: 214-648-2190; Fax: 214-648-8685.

()

The abbreviations used are: CaM, calmodulin; [Ca], concentration of free calcium; CaM Kinase II, multifunctional Ca/calmodulin-dependent protein kinase II; MLCK, myosin light chain kinase; K, Ca/calmodulin concentration required for half-maximal activity; FRAP, fluorescence recovery after photobleaching; DMEM, Dulbecco's modified Eagle's medium; LRB-CaM, lissamine rhodamine B calmodulin; FTC-CaM, fluorescein thiocarbamoyl-calmodulin; AFCys3CaM, acetamido-fluorescein calmodulin labeled on a cysteine substituted for amino acid residue number three; FTC-dex10, 20 or 40, fluorescein-thiocarbamoyl dextran of 10, 20, or 40 kDa; LRB-dex10, lissamine rhodamine B dextran of 10 kDa; CFB, calcium-free buffer; SMC, cultured bovine tracheal smooth muscle cells; HPLC, high performance liquid chromatography; PIPES, 1,4-piperazinediethanesulfonic acid.

ACKNOWLEDGEMENTS

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