Journal of Muscle Research and Cell Motility 13, 206--218 (1992)

Caldesmon binds fo smooth muscle myosin and myosin rod and crosslinks thick filaments fo actin filaments STEVEN

MARSTON

TM, K A T A L I N

P I N T E R I and P A U L I N E

BENNETT z

~ Departrnent of Cardiac Medicine, National Heart and Lung Institute, Dovehouse StreeL London SW3 6L Y, UK 2 M R C Muscle and Cell Motility Unit, Kings College, 26--29 Drury Lane, London WC2B 5RL, UK Received 17 October 1991; revised and accepted 20 October 1991

Summary It is well established that caldesmon binds fo actin (Kb = I07-108 M -z) and to tropomyosin (Kb = 106 M -1) and that if is a potent inhibitor of actomyosin ATPase. Caldesmon can also bind tightly fo myosin. We investigated the binding of smooth muscle and nonmusde caldesmon isoforms (CDh and CDI respectively) fo myosin using proteins from sheep aorta. Both caldesmon isoforms bind to myosin with indistinguishable affinity. The affinity is about 106 M-1 in low salt buffer, but is weakened by increasing [KC1] reaching 1087M-1 in 100 mM KC1. The stoichiometry of binding is about three caldesmon per myosin molecule. Stoichiometry and affinity are not dependent on whether myosin is phosphorylated nor on the presence of Mg z+ and ATP, provided the ionic strength is maintained constant. The caldesmon binding site of smooth muscle myosin is located in the S-2 region, consequently both HMM and myosin rod bind fo caldesmon. Over a range of conditions myosin and myosin rod binding fo caldesmon were indistinguishable. Skeletal muscle myosin has no caldesmon binding site. Smooth muscle myosin rods form side-polar filaments in low salt buffer in which the backbone packing of LMM into the filament ihaft is clearly visible in negatively-stained electron microscopic images. Sometimes the S-2 portions can be seen 'ffayed' ffom the filament shaft. When caldesmon is bound the filament shaft appears to be about 20% thicker and the frayed effect is dramatically increased; long filamentous 'whiskers' are often seen curving out from the filament shaft. Similar structures are observed with smooth muscle and with non-muscle caldesmon. Myosin also binds to caldesmon when if is incorporated into the rhin filament; however, this interaction is qualitatively different. Measurements of smooth muscle HMM binding to native rhin filaments in the presence of 3 mM MgATP shows there is a high affinity binding (Kb = 106 M-~) which is independent of [Ca2+] and of the level of myosin phosphorylation. The stoichiometry is one HMM molecule per actin monomer which is equivalent to up fo I4 HMM bound af high affinity per caldesmon. Negatively stained electron microscopic images of the HMM.ADP.Pi-thin filament complex have failed fo show any attachment of HMM to the rhin filaments. When rod filaments are added fo actin plus caldesmon or to native rhin filaments the rod filaments are strongly associated with the actin filament bundles. The majority of rod filaments are lined up parallel and in close proximity to actin filaments. Similar crosslinking is observed with non-muscle caldesmon. In the smooth muscle cell, caldesmon-containing thin filaments are found together with myosin filaments in the 'contractile domain' in parallel arrays not unlike those shown in our synthetic systems. Thus caldesmon ought fo be able to crosslink thick and rhin filaments in vivo.

Introduction Caldesmon is an 87 000 molecular weight protein which is a component of all smooth muscle cells. A smaller isoform, Mr 65 000 is present in most non-muscle contractile cells (reviewed by Marston & Redwood, 1991). The most studied property of caldesmon in vitro is ifs high affinity binding fo actin filaments and consequent potent inhibition of actin activation of myosin MgATPase (Smith et al., 1987; Velaz et al., 1989). In the smooth muscle cell caldesmon is present af a concen*To whom correspondenceshould be addressed. 0142-4319 9

1992 Chapman & Hall

tration around 10 ~,M (Walsh & Sutherland, 1989) and has been located on the rhin filaments of the 'contractile domain' (Furst et al., 1986). Native thin filaments extracted from vascular smooth muscle contain actin, tropomyosin and caldesmon in molar ratios 1 4 : 2 : 1 (Marston & Lehman, 1985; Lehman et al., 1990; Marston, 1990) and a calcium sensitizing protein (Pritchard & Marston, 1988). We have shown that caldesmon is the major component of the Ca2+-dependent regulation of smooth muscle rhin filaments in vitro (Marston et al., 1988; Pritchard & Marston, 1989). Consequently, it has been proposed that caldesmon is involved in Ca 2+dependent control of smooth muscle contractility, an

207

Smooth muscle myosin-caldesmon-actin interaction hypothesis that is yet fo be proven (Marston & Smith, 1985; Taggart & Marston, 1988; Kamm & Stull, 1989; Marston & Redwoocl, 1991). Recently caldesmon has been shown to have another property; if can bind to smooth muscle myosin (Ikebe & Reardon, 1988) and thus it could bind fo actin and myosin simultaneously (Hemric & Chalovich, 1988; Ikebe & Reardon, 1988; Marston, 1989a). This is an unusual property which might be important to both smooth and non-muscle cells. In this paper we bave made a quantitative investigation of myosin binding fo both smooth muscle and non-muscle caldesmon isoforms and we bave established that caldesmon binds fo the S-2 component of myosin. We have sought to confirm the binding interaction and determine the structure of the myosincontaining complexes of caldesmon by electron microscopy. To do this we bave made synthetic filaments from smooth muscle myosin rod, these contain the caldesmon binding sites but lack the S-1 fragment. Rod filaments have enabled us fo obtain clear images of the myosin-caldesmon complex and to demonstrate crosslinking between thick and rhin filaments by caldesmon in the absence of any crosslinking by myosin crossbridges.

Materials and methods

Preparation of caldesmon Caldesmon was prepared from sheep aorta as described by Taggart and Marston (1988). The end product of this procedure is a mixture of 80% caldesmonh, the smooth muscle isoform and 20% caldesmon/, the non-muscle isoform. These were separated by chromatography on a Q Sepharose column in 20 mM Tris HC1, pli 7.5, 2.5 mM DTT with a linear gradient of NaC1 from 0 to 400 mM. CD/eluted at 140--170 mM NaC1 and CDh eluted af 180-200 mM NaC1 (Fig. lA).

Under these conditions 100% of myosin and rod sedimented while no caldesmon sedimented by itself. In the presence of myosin and rod up to 95% of caldesmon co-sedimented. The quantity of caldesmon remaining in the supernatant was assayed by quantitative gel electrophoresis. This was necessary because we could not specifically label our caldesmon. We round that all the standard SH-directed protein labelling agents (iodoacetamide, N-ethyl maleimeide and N-hydroxysuccinimide) abolished the myosin-binding capacity of aorta caldesmon. Aliquots (20 I.tl) of supematant were mixed with an equal volume of 10% glycerol/5% SDS/5% mercaptoethanol/ 20 mM pli 8.0 Tris and boiled for 3 min. Then 15 Ixl portions were separated on 4-30% polyacrylamide gradient/0.1% SDS slab gels, 10 x 10 x 0.3 cm. Figure 1C and D shows typical results. The stained gels were scanned as described in Fig. lB. The relationship between peak area and caldesmon concentration was linear in the range used. Each set of gels was separately calibrated.

Electron microscopy Mixtures of myosin rod filaments af 2 I,tM with caldesmon af 6-8 IXM were made up by diluting stock solutions into 5 mM PIPES. K87pli 7.1, 2.5 mM MgC1z, I mM dithiothreitol. After 10 rein equilibration the mixture was diluted fivefold in the same buffer and immediately placed on carbon-coated electron microscope grids. Aorta myosin rod filament or aorta myosin (final concentration 1 I.tM) was mixed with actin, actincaldesmon or rhin filaments (final actin concentration 2 laM) in 5 mM PIPES.K2,pH 7.1, 40 mM KC1, 2.5 mM MgC12, I mM dithiothreitol, 3 mM MgATP, and incubated for up fo I h at room temperature. The mixtures were then diluted fivefold and immediately applied to grids as described above. Grids were negatively stained with 1% uranyl acetate. In some cases qaoley' grids were used to improve contrast; these were coated with a rhin carbon film before viewing. The samples were viewed with a JEOL 200CX microscope and photographs were taken at both normal and low electron dosage.

Preparation of actin filaments

Results

Native sheep aorta thin filaments and pure F-actin were prepared as described by Marston and Smith (1984).

We measured the interactions between H M M and caldesmon incorporated into rhin filaments or actin-tropomyosin-caldesmon complex and between caldesmon and myosin or myosin rod. Measurement of caldesmonH M M and myosin-thin filaments interaction was not possible by sedimentation assays as if requires one component fo be soluble and the other sedimentable. However, the unmeasured interactions may be predicted from the interactions that were measured.

Preparation of myosin and myosin rod Sheep aorta myosin was isolated according fo Sellers and colleagues (1981). Myosin was cleaved into rod and S-1 by digestion of a 10 mg ml -I solution in 0.2 M KC1, 5 mM PIPES buffer, pli 7 with 50 ~tg ml -1 papain for 20 min at 25 ~ C. The reaction was stopped with I mM iodoacetate. Rod was purified from the digest by the ethanol precipitation method (Margossian & Lowey, 1982). Myosin filaments were formed by dialysis of 3 mg ml -~ myosin dissolved in 0.6 M KC1 against 0.3 M KC1, 10 mM MES, pli 6.1 af 4 ~ C (Craig & Megerman, 1977). Rod filarnents were formed by dialysis of a 1.5 mg ml -~ or 5 mg ml -~ solution of rods against either 0.3 M KCL 10 mM MES, pli 6.1 or 5 mM PIPES, pli 7.1, 2.5 mM MgC12 at 4 ~ C.

Myosin-caldesmon binding Caldesmon binding to myosin or rod filaments was determined by sedimentation assay. Caldesmon-myosin mixtures (60 lxl) were centrifuged for 30 min at 50 O00g (Sorvall SM24 rotor).

HMM-thin filament interaction In previous work we round that sheep aorta H M M bound fo native rhin filaments in the presence of MgATP with an affinity > 106 M -~ which was largely indepenclent of ionic strength in the range 17-100 mM with a stoichiometry of one H M M molecule per actin (0.07 caldesmon) (Marston, 1989a). We attempted to determine the structure of this complex in the electron microscope. In mixtures of 4.5 I.tM aorta H M M and 1.5 ~tM thin filaments (actin monomer concentration) bincling assay showed that

208

MARSTON, PINTER and BENNETT

Fig. 1. (A) SDS PAGE of the proteins used in this study. M, aorta myosin; R, aorta rod; CDh, aorta caldesmonh; CDI, aorta caldesmonl; TF, aorta thin filaments. Apparent molecular rnass, kDa on left of figure. (B) Determination of caldesmon concentration frorn area of bands on SDS PAGE. Samples (15 Ixl) of caldesrnon at a range of concentrations were applied to Pharmacia 4-30% polyacrylamide slab gels. Gels were stained in PAGE Blue 83 (BDH). Each caldesrnon band was scanned three tirnes using an LKB 2202 laser scanning densitometer and areas were integrated by a HP cornputing integrator. Mean scan area is plotted against caldesmon concentration. The line is a best fit linear regression curve, r z= 0.986. (C and D) Exarnples of binding assay. In (C) samples contained a constant 3 IxM caldesmonh and a range of myosin concentrations from 0 to 6 I.tMin 5 mM PIPES buffer, pli 7, 2.5 mM MgClz, 30 mM KC1. The samples were sedimented for 30 rein at 50 000g and aliquots of the supematant prepared for PAGE. The caldesmon band decreases in area as rnyosin concentration increases. In (D) samples contained 4.5 I.tMcaldesmonl and 0--4 ~tM myosin. The binding curves obtained flore these experiments appear in Fig. SA.

the rhin filaments were 100% occupied by HMM both in the presence and absence of 3 mM MgATP. When these mixtures were applied fo microscope grids and stained with uranyl acetate without washing, the rhin filaments were observed to bind a full complement of H M M in the absence of MgATP (rigor crossbridges) but no binding of H M M fo the filaments could be observed in the presence of MgATP. Re-assay of the mixtures the following day showed that the H M M still bound fo the thin filaments in the presence of MgATP in the test tube. We concluded that the stain had destroyed the HMM-thin filam~.~flr contact region. The same result was obtained using phosphotungstate af pli 7.0, silico-tungstate and ammonium molybdate stains.

Myosin-caldesmon interaction Caldesmon binding fo filamentous smooth muscle myosin was measured by a direct sedimentation assay. Af low

ionic strength caldesmon bound with an affinity around 106M -1 (Figs. 1C and 2). Affinity was reduced by increasing ionic strength (Fig. 2), reaching 10 87 M -a af 90 mM. We could not detect any changes in affinity due to Mg 2+ in the range 0-5 mM (data not shown) or fo MgATP (1 mM) when measurements were ruade af constant ionic strength (Fig. 3), despite a report that MgATP caused a 20-fold increase in affinity of myosin for chicken gizzard caldesmon (Hemric & Chalovich, 1990). The stoichiometry of caldesmon binding fo myosin was determined by titrating caldesmon at a constant myosin concentration (Fig. 4). This procedure is subject fo more random error than the titration at constant caldesmon concentration (Figs. 2 and 3). At low ionic strength we round stoichiometry to be 2.5-3.5 caldesmon bound per molecule (450 kDa) of myosin. This did not change in the presence of I mM MgATP (data not shown). Neither assay procedure showed any differences

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Caldesmon binds to smooth muscle myosin and myosin rod and crosslinks thick filaments to actin filaments.

It is well established that caldesmon binds to actin (Kb = 10(7) - 10(-8) M-1) and to tropomyosin (Kb = 10(6) M-1) and that it is a potent inhibitor o...
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