Three-dimensional image reconstruction of reconstituted smooth muscle thin filaments: effects of caldesmon

Caldesmon inhibits actomyosin ATPase and filament sliding in vitro, and therefore may play a role in modulating smooth and non-muscle motile activities. A bacterially expressed caldesmon fragment, 606C, which consists of the C-terminal 150 amino acids of the intact molecule, possesses the same inhibitory properties as full-length caldesmon and was used in our structural studies to examine caldesmon function. Three-dimensional image reconstruction was carried out from electron micrographs of negatively stained, reconstituted thin filaments consisting of actin and smooth muscle tropomyosin both with and without added 606C. Helically arranged actin monomers and tropomyosin strands were observed in both cases. In the absence of 606C, tropomyosin adopted a position on the inner edge of the outer domain of actin monomers, with an apparent connection to sub-domain 1 of actin. In 606C-containing filaments that inhibited acto-HMM ATPase activity, tropomyosin was found in a different position, in association with the inner domain of actin, away from the majority of strong myosin binding sites. The effect of caldesmon on tropomyosin position therefore differs from that of troponin on skeletal muscle filaments, implying that caldesmon and troponin act by different structural mechanisms.     

Three-dimensional reconstruction of caldesmon-containing smooth muscle thin filaments

Caldesmon is known to inhibit actomyosin ATPase and filament sliding in vitro, and may play a role in modulating smooth muscle contraction as well as in diverse cellular processes including cytokinesis and exocytosis. However, the structural basis of caldesmon action has not previously been apparent. We have recorded electron microscope images of negatively stained thin filaments containing caldesmon and tropomyosin which were isolated from chicken gizzard smooth muscle in EGTA. Three-dimensional helical reconstructions of these filaments show actin monomers whose bilobed shape and connectivity are very similar to those previously seen in reconstructions of frozen-hydrated skeletal muscle thin filaments. In addition, a continuous thin strand of density follows the long-pitch actin helices, in contact with the inner domain of each actin monomer. Gizzard thin filaments treated with Ca2+/calmodulin, which dissociated caldesmon but not tropomyosin, have also been reconstructed. Under these conditions, reconstructions also reveal a bilobed actin monomer, as well as a continuous surface strand that appears to have moved to a position closer to the outer domain of actin. The strands seen in both EGTA- and Ca2+/calmodulin-treated filaments thus presumably represent tropomyosin. It appears that caldesmon can fix tropomyosin in a particular position on actin in the absence of calcium. An influence of caldesmon on tropomyosin position might, in principle, account for caldesmon's ability to modulate actomyosin interaction in both smooth muscles and non-muscle cells.     

The role of tropomyosin in the interactions of F-actin with caldesmon and actin-binding protein (or filamin)

The interactions of actin filaments with actin-binding protein (filamin) and caldesmon under the influence of tropomyosin were studied in detail using falling-ball viscometry, binding assay and electron microscopy. Caldesmon decreased the binding constant of filamin with F-actin. In contrast, the maximum binding ability of filamin to F-actin was decreased by tropomyosin. The filamin-induced gelation of actin filaments was inhibited by caldesmon. Tropomyosin also inhibited this gelation. The effect of caldesmon became stronger under the influence of tropomyosin. Furthermore, both caldesmon and tropomyosin additionally decreased the filamin binding to F-actin. From these results, caldesmon and tropomyosin appeared to influence filamin binding to F-actin with different modes of actin. In addition, there was no sign of direct interactions between filamin, caldesmon and tropomyosin as judged from gel filtration. Under the influence of caldesmon and tropomyosin, calmodulin conferred Ca2+ sensitivity on the filamin-induced gelation of actin filaments.     

The effect of caldesmon on dynamic properties of F-actin alone and bound to heavy meromyosin and/or tropomyosin

The effect of caldesmon on the rotational dynamics of actin filaments alone or conjugated with heavy meromyosin and/or tropomyosin has been measured by the electron paramagnetic resonance (EPR) technique using a maleimide spin label rigidly bound to Cys374 of actin. The rotation of actin protomers in filaments and the angular distribution of spin probes on actin were determined by conventional EPR spectroscopy, while torsional motions within actin filaments were detected by saturation transfer EPR measurements. Binding of caldesmon to F-actin resulted in the reduction of torsional mobility of actin filaments. The maximum effect was produced at a ratio of about one molecule of caldesmon/seven actin protomers. Smooth muscle tropomyosin enhanced the effect of caldesmon, i.e. caused further slowing down of internal motions within actin filaments. Caldesmon increased the degree of order of spin labels on F-actin in macroscopically oriented pellets in the presence of tropomyosin but not in its absence. Computer analysis of the spectra revealed that caldesmon alone slightly changed the orientation of spin probes relative to the long axis of the filament. In the presence of tropomyosin this effect of caldesmon was potentiated and then approximately every twentieth protomer along the actin filament was affected. Caldesmon weakened the effect of heavy meromyosin both on the polarity of environment of the spin label attached to F-actin and on the degree of order of labels on actin in macroscopically oriented pellets. Whereas the former effect of caldesmon was independent of tropomyosin, the latter one was observed only in the absence of tropomyosin.     

Actin-Binding Proteins in Plant Cells

Abstract: Actinoccurs in all plant cells, as monomers, filaments and filament assemblies. In interphase, actin filaments form a cortical network, co-align with cortical microtubules, and extend throughout the cytoplasm functioning in cytoplasmic streaming. During mitosis, they co-align with microtubules in the preprophase band and phragmoplast and are indispensa ble for cell division. Actin filaments continually polymerise and depolymerise from a pool of monomers, and signal transduction pathways affecting cell morphogenesis modify the actin cytoskeleton. The interactions of actin monomers and filaments with actin-binding proteins (ABP5) control actin dynamics. By binding to actin monomers, ABPs, such as profilin, regulate the pool of monomers available for polymerisation. By breaking filaments or capping filament ends, ABPs, such as actin depoly-merising factor (ADF), prevent actin filament elongation or loss of monomers from filament ends. By bivalent cross-linking to actin filaments, ABPs, such as fimbrin and other members of the spectrin family, produce a variety of higher order assemblies, from bundles to networks. The motor protein ABPs,. which are not covered in this review, move organelles along ac tin filaments. The large variety of ABPs share a number of functional modules. A plant representative of ABPs with particular modules, and therefore particular functions, is treated in this review.     

Caldesmon phosphorylation in actin cytoskeletal remodeling

Caldesmon is an actin-binding protein that is capable of stabilizing actin filaments against actin-severing proteins, inhibiting actomyosin ATPase activity, and inhibiting Arp2/3-mediated actin polymerization in vitro. Caldesmon is a substrate of cdc2 kinase and Erk1/2 MAPK, and phosphorylation by either of these kinases reverses the inhibitory effects of caldesmon. Cdc2-mediated caldesmon phosphorylation and the resulting dissociation of caldesmon from actin filaments are essential for M-phase progression during mitosis. Cells overexpressing the actin-binding carboxyterminal fragment of caldesmon fail to release the fragment completely from actin filaments during mitosis, resulting in a higher frequency of multinucleated cells. PKC-mediated MEK/Erk/caldesmon phosphorylation is an important signaling cascade in the regulation of smooth muscle contraction. Furthermore, PKC activation has been shown to remodel actin stress fibers into F-actin-enriched podosome columns in cultured vascular smooth muscle cells. Podosomes are cytoskeletal adhesion structures associated with the release of metalloproteases and degradation of extracellular matrix during cell invasion. Interestingly, caldesmon is one of the few actin-binding proteins that is associated with podosomes but excluded from focal adhesions. Caldesmon also inhibits the function of gelsolin and Arp2/3 complex that are essential for the formation of podosomes. Thus, caldesmon appears to be well positioned for playing a modulatory role in the formation of podosomes. Defining the roles of actin filament-stabilizing proteins such as caldesmon and tropomyosin in the formation of podosomes should provide a more complete understanding of molecular systems that regulate the remodeling of the actin cytoskeleton in cell transformation and invasion.     

Stoichiometry and stability of caldesmon in native thin filaments from sheep aorta smooth muscle.

Ca2(+)-regulated native thin filaments were extracted from sheep aorta smooth muscle. The caldesmon content determined by quantitative gel electrophoresis was 0.06 caldesmon molecule/actin monomer (1 caldesmon molecule per 16.3 actin monomers). Dissociation of caldesmon and tropomyosin from the thin filament and the depolymerization of actin was measured by sedimenting diluted thin filaments. Actin critical concentration was 0.05 microM at 10.1 and 0.13 at 10.05 compared with 0.5 microM for pure F-actin. Tropomyosin was tightly bound, with half-maximal dissociation at less than 0.3 microM thin filaments (actin monomer) under all conditions. Caldesmon dissociation was independent of tropomyosin and not co-operative. The concentration of thin filaments where 50% of the caldesmon was dissociated (CD50) ranged from 0.2 microM (actin monomer) at 10.03 to 8 microM at 10.16 in a 5 mM-MgCl2, pH 7.1, buffer. Mg2+, 25 mM at constant I, increased CD50 4-fold. CD50 was 4-fold greater at 10(-4) M-Ca2+ than at 10(-9) M-Ca2+. Aorta heavy meromyosin (HMM).ADP.Pi complex (2.5 microM excess over thin filaments) strongly antagonized caldesmon dissociation, but skeletal-muscle HMM.ADP.Pi did not. The behaviour of caldesmon in native thin filaments was indistinguishable from caldesmon in reconstituted synthetic thin filaments. The variability of Ca2(+)-sensitivity with conditions observed in thin filament preparations was shown to be related to dissociation of regulatory caldesmon from the thin filament.     

The effects of smooth muscle caldesmon on actin filament motility.

The movement of reconstituted thin filaments over an immobilized surface of thiophosphorylated smooth muscle myosin was examined using an in vitro motility assay. Reconstituted thin filaments contained actin, tropomyosin, and either purified chicken gizzard caldesmon or the purified COOH-terminal actin-binding fragment of caldesmon. Control actin-tropomyosin filaments moved at a velocity of 2.3 +/- 0.5 microns/s. Neither intact caldesmon nor the COOH-terminal fragment, when maintained in the monomeric form by treatment with 10 mM dithiothreitol, had any effect on filament velocity; and yet both were potent inhibitors of actin-activated myosin ATPase activity, indicating that caldesmon primarily inhibits myosin binding as reported by Chalovich et al. (Chalovich, J. M., Hemric, M. E., and Velaz, L. (1990) Ann. N. Y. Acad. Sci. 599, 85-99). Inhibition of filament motion was, however, observed under conditions where cross-linking of caldesmon via disulfide bridges was present. To determine if monomeric caldesmon could "tether" actin filaments to the myosin surface by forming an actin-caldesmon-myosin complex as suggested by Chalovich et al., we looked for caldesmon-dependent filament binding and motility under conditions (80 mM KCl) where filament binding to myosin is weak and motility is not normally seen. At caldesmon concentrations > or = 0.26 microM, actin filament binding was increased and filament motion (2.6 +/- 0.6 microns/s) was observed. The enhanced motility seen with intact caldesmon was not observed with the addition of up to 26 microM COOH-terminal fragment. Moreover, a molar excess of the COOH-terminal fragment competitively reversed the enhanced binding seen with intact caldesmon. These results show that tethering of actin filaments to myosin by the formation of an actin-caldesmon-myosin complex enhanced productive acto-myosin interaction without placing a significant mechanical load on the moving filaments.     

Molecular dynamics study of interactions between polymorphic actin filaments and gelsolin segment-1

The assembly of protein actin into double-helical filaments promotes many eukaryotic cellular processes that are regulated by actin-binding proteins (ABPs). Actin filaments can adopt multiple conformations, known as structural polymorphism, which possibly influences the interaction between filaments and ABPs. Gelsolin is a Ca²⁺-regulated ABP that severs and caps actin filaments. Gelsolin binding modulates filament structure; however, it is not known how polymorphic actin filament structures influence an interaction of gelsolin S1 with the barbed-end of filament. Herein, we investigated how polymorphic structures of actin filaments affect the interactions near interfaces between the gelsolin segment 1 (S1) domain and the filament barbed-end. Using all-atom molecular dynamics simulations, we demonstrate that different tilted states of subunits modulate gelsolin S1 interactions with the barbed-end of polymorphic filaments. Hydrogen bonding and interaction energy at the filament-gelsolin S1 interface indicate distinct conformations of filament barbed ends, resulting in different interactions of gelsolin S1. This study demonstrates that filament's structural multiplicity plays important roles in the interactions of actin with ABPs.     

Interaction between caldesmon and tropomyosin in the presence and absence of smooth muscle actin

Cysteine residues of caldesmon were labeled with the fluorescent reagent N-(1-pyrenyl)maleimide. The number of sulfhydryl (SH) groups in caldesmon was around 3.5 on the basis of reactivity to 5,5'-dithiobis(2-nitrobenzoate); 80% of the SH groups were labeled with pyrene. The fluorescence spectrum from pyrene-caldesmon showed the presence of excited monomer and dimer (excimer). As the ionic strength increased, excimer fluorescence decreased, disappearing at salt concentrations higher than around 50 mM. The labeling of caldesmon with pyrene did not affect its ability to inhibit actin activation of heavy meromyosin Mg-ATPase and the release of this inhibition in the presence of Ca2+-calmodulin. Tropomyosin induced a change in the fluorescence spectrum of pyrene-caldesmon, indicating a conformational change associated with the interaction between caldesmon and tropomyosin. The affinity of caldesmon to tropomyosin was dependent on ionic strength. The binding constant was 5 x 10(6) M-1 in low salt, and the affinity was 20-fold less at ionic strengths close to physiological conditions. In the presence of actin, the affinity of caldesmon to tropomyosin was increased 5-fold. The addition of tropomyosin also changed the fluorescence spectrum of pyrene-caldesmon bound to actin filaments. The change in the conformation of tropomyosin, caused by the interaction between caldesmon and tropomyosin, was studied with pyrene-labeled tropomyosin. Fluorescence change was evident when unlabeled caldesmon was added to pyrene-tropomyosin bound to actin. These data suggest that the interaction between caldesmon and tropomyosin on the actin filament is associated with conformational changes on these thin filament associated proteins. These conformational changes may modulate the ability of thin filament to interact with myosin heads.     

Properties of actin from the fission yeast Schizosaccharomyces pombe and interaction with fission yeast profilin

The fission yeast Schizosaccharomyces pombe serves as a model system for studying role of actin cytoskeleton, since it has simple actin cytoskeletons and is genetically tractable. In contrast, biochemical approaches using this organism are still developing; fission yeast actin has so far not been isolated in its native form and characterized, and therefore, biochemical assays of fission yeast actin-binding proteins (ABPs) or myosin have been performed using rabbit skeletal muscle actin that may interact with the fission yeast ABPs in a manner different from fission yeast actin. Here, we report a novel method for isolating functionally active actin from fission yeast cells. The highly purified fission yeast actin polymerized with kinetics somewhat different from those of muscle actin and forms filaments that are structurally indistinguishable from skeletal muscle actin filaments. The fission yeast actin was a significantly weaker activator of Mg(2+)-ATPase of HMM of skeletal muscle myosin than muscle actin. The fission yeast profilin Cdc3 suppressed polymerization of fission yeast actin more effectively than that of muscle actin and showed an affinity for fission yeast actin higher than for muscle actin. The establishment of purification of fission yeast actin will enable reconstruction of physiologically relevant interactions between the actin and fission yeast ABPs or myosins and contribute to clarification of function of actin cytoskeleton in various cellular activities.     

Differential Effects of Caldesmon on the Intermediate Conformational States of Polymerizing Actin

The actin-binding protein caldesmon (CaD) reversibly inhibits smooth muscle contraction. In non-muscle cells, a shorter CaD isoform co-exists with microfilaments in the stress fibers at the quiescent state, but the phosphorylated CaD is found at the leading edge of migrating cells where dynamic actin filament remodeling occurs. We have studied the effect of a C-terminal fragment of CaD (H32K) on the kinetics of the in vitro actin polymerization by monitoring the fluorescence of pyrene-labeled actin. Addition of H32K or its phosphorylated form either attenuated or accelerated the pyrene emission enhancement, depending on whether it was added at the early or the late phase of actin polymerization. However, the CaD fragment had no effect on the yield of sedimentable actin, nor did it affect the actin ATPase activity. Our findings can be explained by a model in which nascent actin filaments undergo a maturation process that involves at least two intermediate conformational states. If present at early stages of actin polymerization, CaD stabilizes one of the intermediate states and blocks the subsequent filament maturation. Addition of CaD at a later phase accelerates F-actin formation. The fact that CaD is capable of inhibiting actin filament maturation provides a novel function for CaD and suggests an active role in the dynamic reorganization of the actin cytoskeleton.     

X-ray diffraction studies on oriented gels of vertebrate smooth muscle thin filaments.

The ATPase activity of acto-myosin subfragment 1 (S1) at low ratios of S1 to actin in the presence of tropomyosin is dependent on the tropomyosin source and ionic conditions. Whereas skeletal muscle tropomyosin causes a 60% inhibitory effect at all ionic strengths, the effect of smooth muscle tropomyosin was found to be dependent on the ionic strength. At low ionic strength (20 mM) smooth muscle tropomyosin inhibits the ATPase activity by 60%, while at high ionic strength (120 mM) it potentiates the ATPase activity three- to five-fold. Therefore, the difference in the effect of smooth muscle and skeletal muscle tropomyosin on the acto-S1 ATPase activity was due to a greater fraction of the tropomyosin-actin complex being turned on in the absence of S1 with smooth muscle tropomyosin than with skeletal muscle tropomyosin. Using well-oriented gels of actin and of reconstituted specimens from vertebrate smooth muscle thin filament proteins suitable for X-ray diffraction, we localized the position of tropomyosin on actin under different levels of acto-S1 ATPase activity. By analysing the equatorial X-ray pattern of the oriented specimens in combination with solution scattering experiments, we conclude that tropomyosin is located at a binding radius of about 3.5 nm on the f-actin helix under all conditions studied. Furthermore, we find no evidence that the azimuthal position of tropomyosin is different for smooth muscle tropomyosin at various ionic strengths, or vertebrate tropomyosin, since the second actin layer-line intensity (at 17.9 nm axial and 4.3 nm radial spacing), which was shown in skeletal muscle to be a sensitive measure of this parameter, remains strong and unchanged. Differences in the ATPase activity are not necessarily correlated with different positions of tropomyosin on f-actin. The same conclusion is drawn from our observations that, although the regulatory protein caldesmon inhibits the ATPase activity in native and reconstituted vertebrate smooth muscle thin filaments at a molar ratio of actin/tropomyosin/caldesmon of 28:7:1, the second actin layer-line remains strong. Only adding caldesmon in excess reduces the intensity of the second actin layer-line, from which the binding radius of caldesmon can be estimated to be about 4 nm. The lack of predominant meridional reflections in oriented specimens, with caldesmon present, suggests that caldesmon does not project away from the thin filament as troponin molecules in vertebrate striated muscle in agreement with electron micrographs of smooth muscle thin filaments. In freshly prepared native smooth muscle thin filaments we observed a Ca(2+)-sensitive reversible bundling effect.(ABSTRACT TRUNCATED AT 400 WORDS)     

Immunocytochemistry of cytoskeletal proteins in adult Schistosoma mansoni

Antisera to vertebrate actin and actin-binding proteins were used to characterize the cytoskeleton of adult Schistosoma mansoni. Actin, alpha-actinin and tropomyosin immunoreactivities were detected in the cytoplasm of the apical tegument. Antiserum to alpha-actinin bound to the tegumental spines and this protein may be involved in cross-linking of spine actin filaments. Actin, alpha-actinin and tropomyosin antisera bound to the musculature. Strongest immunoreactivity was seen in the parenchyma. Antisera to actin, alpha-actinin, tropomyosin and spectrin bound to parenchyma cells including those of the tubercles, suggesting that these proteins are located in muscle cell bodies. The distribution of cytoskeletal proteins is discussed in relation to tegumental repair processes.     

Cooperative regulation of myosin-actin interactions by a continuous flexible chain I: actin-tropomyosin systems

We present a model for cooperative myosin binding to the regulated actin filament, where tropomyosins are treated as a weakly-confined continuous flexible chain covering myosin binding sites. Thermal fluctuations in chain orientation are initially required for myosin binding, leaving kinked regions under which subsequent myosins may bind without further distortion of the chain. Statistical mechanics predicts the fraction of sites with bound myosin-S1 as a function of their affinities. Published S1 binding curves to regulated filaments with different tropomyosin isoforms are fitted by varying the binding constant, chain persistence length nu (in actin monomers), and chain kink energy A from a single bound S1. With skeletal tropomyosin, we find an S1 actin-binding constant of 2.2 x 10(7) M(-1), A = 1.6 k(B)T and nu = 2.7. Similar persistence lengths are found with yeast tropomyosin. Larger values are found for tropomyosin-troponin in the presence of calcium (nu = 3.7) and tropomyosins from smooth muscle and fibroblasts (nu = 4.5). The relationship of these results to structural information and the rigid-unit model of McKillop and Geeves is discussed.     

The mechanism of Ca2+ regulation of vascular smooth muscle thin filaments by caldesmon and calmodulin

The interactions of vascular smooth muscle caldesmon with actin, tropomyosin, and calmodulin were determined under conditions in which the four proteins can form reconstituted Ca2+-sensitive smooth muscle thin filaments. Caldesmon bound to actin in a complex fashion with high affinity sites (K = 10(7) M-1) saturating at a stoichiometry of 1 per 28 actins, and lower affinity sites at 1 per 7 actins. The affinity of binding was increased in the presence of tropomyosin, and this could be attributed to a direct interaction between caldesmon and tropomyosin which was demonstrated using caldesmon cross-linked to Sepharose. In the presence of tropomyosin, occupancy of the high affinity sites was associated with inhibition of actin-activated myosin MgATPase activity. Caldesmon was found to bind to calmodulin in the presence of Ca2+, with an affinity of 10(6) M-1. The binding of Ca2+ X calmodulin to caldesmon was associated with the neutralization of inhibition of actin-tropomyosin. Ca2+ X calmodulin binding reduced but did not abolish the binding of caldesmon to actin-tropomyosin. From this data we have proposed a model for smooth muscle thin filaments in which Ca2+ regulates activity by converting the inhibited actin-tropomyosin-caldesmon complex to the active complexes, actin-tropomyosin-caldesmon-calmodulin X Ca2+ and actin-tropomyosin.     

The utrophin actin-binding domain binds F-actin in two different modes: implications for the spectrin superfamily of proteins

Utrophin, like its homologue dystrophin, forms a link between the actin cytoskeleton and the extracellular matrix. We have used a new method of image analysis to reconstruct actin filaments decorated with the actin-binding domain of utrophin, which contains two calponin homology domains. We find two different modes of binding, with either one or two calponin-homology (CH) domains bound per actin subunit, and these modes are also distinguishable by their very different effects on F-actin rigidity. Both modes involve an extended conformation of the CH domains, as predicted by a previous crystal structure. The separation of these two modes has been largely dependent upon the use of our new approach to reconstruction of helical filaments. When existing information about tropomyosin, myosin, actin-depolymerizing factor, and nebulin is considered, these results suggest that many actin-binding proteins may have multiple binding sites on F-actin. The cell may use the modular CH domains found in the spectrin superfamily of actin-binding proteins to bind actin in manifold ways, allowing for complexity to arise from the interactions of a relatively few simple modules with actin.     

Troponin is a potential regulator for actomyosin interactions

Troponin extracted from rabbit skeletal muscle directly binds to an actin filament in a molar ratio of 1:1 even in the absence of tropomyosin. An actin filament decorated with troponin did not exhibit significant difference from pure actin filaments in the maximum rate of actomyosin ATP hydrolysis and the sliding velocity of the filament examined by means of an in vitro motility assay. However, the relative number of troponin-bound actin filaments moving in the absence of calcium ions decreased to half that in their presence. The amount of HMM bound to the filaments was less than 4% of actin monomers in the presence of TNs. In addition, actin filaments could not move when Tn molecules were bound in the molar ratio of about 1:1 although they sufficiently bind to myosin heads. These results indicate that troponin can transform an actin monomer within a filament into an Off-state without sterically blocking of the myosin-binding sites with tropomyosin molecules.     

Modes of caldesmon binding to actin: sites of caldesmon contact and modulation of interactions by phosphorylation

Smooth muscle caldesmon binds actin and inhibits actomyosin ATPase activity. Phosphorylation of caldesmon by extracellular signal-regulated kinase (ERK) reverses this inhibitory effect and weakens actin binding. To better understand this function, we have examined the phosphorylation-dependent contact sites of caldesmon on actin by low dose electron microscopy and three-dimensional reconstruction of actin filaments decorated with a C-terminal fragment, hH32K, of human caldesmon containing the principal actin-binding domains. Helical reconstruction of negatively stained filaments demonstrated that hH32K is located on the inner portion of actin subdomain 1, traversing its upper surface toward the C-terminal segment of actin, and forms a bridge to the neighboring actin monomer of the adjacent long pitch helical strand by connecting to its subdomain 3. Such lateral binding was supported by cross-linking experiments using a mutant isoform, which was capable of cross-linking actin subunits. Upon ERK phosphorylation, however, the mutant no longer cross-linked actin to polymers. Three-dimensional reconstruction of ERK-phosphorylated hH32K indeed indicated loss of the interstrand connectivity. These results, together with fluorescence quenching data, are consistent with a phosphorylation-dependent conformational change that moves the C-terminal end segment of caldesmon near the phosphorylation site but not the upstream region around Cys(595), away from F-actin, thus neutralizing its inhibitory effect on actomyosin interactions. The binding pattern of hH32K suggests a mechanism by which unphosphorylated, but not ERK-phosphorylated, caldesmon could stabilize actin filaments and resist F-actin severing or depolymerization in both smooth muscle and nonmuscle cells.