Structural dynamics of actin during active interaction with myosin: different effects of weakly and strongly bound myosin heads

We have used optical spectroscopy (transient phosphorescence anisotropy, TPA, and fluorescence resonance energy transfer, FRET) to detect the effects of weakly bound myosin S1 on actin during the actomyosin ATPase cycle. The changes in actin were reported by (a) a phosphorescent probe (ErIA) attached to Cys 374 and (b) a FRET donor-acceptor pair, IAEDANS attached to Cys 374 and a nucleotide analogue (TNPADP) in the nucleotide-binding cleft. Strong interactions were detected in the absence of ATP, and weak interactions were detected in the presence of ATP or its slowly hydrolyzed analogue ATP-gamma-S, under conditions where a significant fraction of weakly bound acto-S1 complex was present and the rate of nucleotide hydrolysis was low enough to enable steady-state measurements. The results show that actin in the weakly bound complex with S1 assumes a new structural state in which (a) the actin filament has microsecond rotational dynamics intermediate between that of free actin and the strongly bound complex and (b) S1-induced changes are not propagated along the actin filament, in contrast to the highly cooperative changes due to the strongly bound complex. We propose that the transition on the acto-myosin interface from weak to strong binding is accompanied by transitions in the structural dynamics of actin parallel to transitions in the dynamics of interacting myosin heads.     

Site-specific mutations in the myosin binding sites of actin affect structural transitions that control myosin binding.

We have examined the effects of actin mutations on myosin binding, detected by cosedimentation, and actin structural dynamics, detected by spectroscopic probes. Specific mutations were chosen that have been shown to affect the functional interactions of actin and myosin, two mutations (4Ac and E99A/E100A) in the proposed region of weak binding to myosin and one mutation (I341A) in the proposed region of strong binding. In the absence of nucleotide and salt, S1 bound to both wild-type and mutant actins with high affinity (K(d) < microM), but either ADP or increased ionic strength decreased this affinity. This decrease was more pronounced for actins with mutations that inhibit functional interaction with myosin (E99A/E100A and I341A) than for a mutation that enhances the interaction (4Ac). The mutations E99A/E100A and I341A affected the microsecond time scale dynamics of actin in the absence of myosin, but the 4Ac mutation did not have any effect. The binding of myosin eliminated these effects of mutations on structural dynamics; i.e., the spectroscopic signals from mutant actins bound to S1 were the same as those from wild-type actin. These results indicate that mutations in the myosin binding sites affect structural transitions within actin that control strong myosin binding, without affecting the structural dynamics of the strongly bound actomyosin complex.     

Effect of the substitution of muscle actin-specific subdomain 1 and 2 residues in yeast actin on actin function

Muscle and yeast actins display distinct behavioral characteristics. To better understand the allosteric interactions that regulate actin function, we created a muscle/yeast hybrid actin containing a muscle-specific outer domain (subdomains 1 and 2) and a yeast inner domain (subdomains 3 and 4). Actin with muscle subdomain 1 and the two yeast N-terminal negative charges supported viability. The four negative charge muscle N terminus in a muscle subdomain 1 background caused death, but in the same background actin with three N-terminal acidic residues (3Ac/Sub1) led to sick but viable cells. Addition of three muscle subdomain 2 residues (3Ac/Sub12) produced no further deleterious effects. These hybrid actins caused depolarized cytoskeletons, abnormal vacuoles, and mitochondrial and endocytosis defects. 3Ac/Sub1 G-actin exchanged bound epsilonATP more slowly than wild type actin, and the exchange rate for 3Ac/Sub12 was even slower, similar to that for muscle actin. The mutant actins polymerized faster and produced less stable and shorter filaments than yeast actin, the opposite of that expected for muscle actin. Unlike wild type actin, in the absence of unbound ATP, polymerization led to ADP-F-actin, which rapidly depolymerized. Like yeast actin, the hybrid actins activated muscle myosin S1 ATPase activity only about one-eighth as well as muscle actin, despite having essentially a muscle actin-specific myosin-binding site. Finally, the hybrid actins behaved abnormally in a yeast Arp2/3-dependent polymerization assay. Our results demonstrate a unique sensitivity of yeast to actin N-terminal negative charge density. They also provide insight into the role of each domain in the control of the various functions of actin.     

Changes in actin structural transitions associated with oxidative inhibition of muscle contraction

We have used transient phosphorescence anisotropy (TPA) to detect changes in actin structural dynamics associated with oxidative inhibition of muscle contraction. Contractility of skinned rabbit psoas muscle fibers was inhibited by treatment with 50 mM H 2O 2, which induced oxidative modifications in the myosin head and in actin, as previously reported. Using proteins purified from oxidized and unoxidized muscle, we used TPA to measure the effects of weakly (+ATP) and strongly (no ATP) bound myosin heads (S1) on the microsecond dynamics of actin labeled at Cys374 with erythrosine iodoacetamide. Oxidative modification of S1 had no effect on actin dynamics in the absence of ATP (strong binding complex), but restricted the dynamics in the presence of ATP (weakly bound complex). In contrast, oxidative modification of actin did not have a significant effect on the weak-to-strong transitions. Thus, we concluded that (1) the effects of oxidation on the dynamics of actin in the actomyosin complex are predominantly determined by oxidation-induced changes in S1, and (2) changes in weak-to-strong structural transitions in actin and myosin are coupled to each other and are associated with oxidative inhibition of muscle contractility.     

Removal of the amino-terminal acidic residues of yeast actin. Studies in vitro and in vivo.

We have examined the role of the acidic residues Asp2 and Glu4 at the NH2 terminus of Saccharomyces cerevisiae actin through site-directed mutagenesis. In DNEQ actin, these residues have been changed to Asn2 and Gln4, whereas in delta DSE actin, the Asp2-Ser-Glu tripeptide has been deleted. Both mutant actins can replace wild type yeast actin. Peptide mapping studies reveal that DNEQ, like wild type actin, retains the initiator Met and is NH2 terminally acetylated, whereas delta DSE has a free NH2 terminus and has lost the initiator Met. Interestingly, microscopic examination of filaments of these two actins reveal the appearance of bundled filaments. The DNEQ bundles are smaller and more ordered, whereas the delta DSE bundles are larger and more loosely organized. Additionally, both mutant actins activate the ATPase activity of rabbit muscle myosin S1 fragment to a lesser extent than wild type. We have also developed a sensitive assay for actin function in vivo that enabled us to detect a slight defect in the ability of these mutant actins to support secretion, an important function in yeast. Thus, although the mutant actins resulted in no gross phenotypic changes, we were able to detect a defect in actin function through this assay. From these studies we can conclude that 1) although NH2-terminal negative charges are not essential to yeast life, the loss of such charges does result in a slight defect in the actins' ability to support secretion, 2) removal of the NH2-terminal negative charges promotes the bundling of actin filaments, and 3) actins lacking NH2-terminal negative charges are unable to activate the myosin S1 ATPase activity as well as wild type actin.     

Structural Comparisons of Muscle and Nonmuscle Actins Give Insights Into the Evolution of Their Functional Differences

Actin is a highly conserved protein although many isoforms exist. In vertebrates and insects the different actin isoforms can be grouped by their amino acid sequence and tissue-specific gene expression into muscle and nonmuscle actins, suggesting that the different actins may have a functional significance. We ask here whether atomic models for G- and F-actins may help to explain this functional diversity. Using a molecular graphics program we have mapped the few amino acids that differ between isoactins. A small number of residues specific for muscle actins are buried in internal positions and some present a remarkable organization. Within the molecule, the replacements observed between muscle and nonmuscle actins are often accompanied by compensatory changes. The others are dispersed on the protein surface, except for a cluster located at the N-terminus which protrudes outward. Only a few of these residues specific for muscle actins are present in known ligand binding sites except the N-terminus, which has a sequence specific for each isoactin and is directly implicated in the binding to myosin. When we simulated the replacements of side chains of residues specific for muscle actins to those specific for nonmuscle actins, the N-terminus appears to be less compact and more flexible in nonmuscle actins. This would represent the first conformational grounds for proposing that muscle and nonmuscle actins may be functionally distinguishable. The rest of the molecule is very similar or identical in all the actins, except for a possible higher internal flexibility in muscle actins. We propose that muscle actin genes have evolved from genes of nonmuscle actins by substitutions leading to some conformational changes in the protruding N-terminus and the internal dynamics of the main body of the protein. Received: 15 March 1996 / Accepted: 14 July 1996     

Actin from Saccharomyces cerevisiae.

Inhibition of DNase I activity has been used as an assay to purify actin from Saccharomyces cerevisiae (yeast actin). The final fraction, obtained after a 300-fold purification, is approximately 97% pure as judged by sodium dodecyl sulfate-gel electrophoresis. Like rabbit skeletal muscle actin, yeast actin has a molecular weight of about 43,000, forms 7-nm-diameter filaments when polymerization is induced by KCl or Mg2+, and can be decorated with a proteolytic fragment of muscle myosin (heavy meromyosin). Although heavy meromyosin ATPase activity is stimulated by rabbit muscle and yeast actins to approximately the same Vmax (2 mmol of Pi per min per mumol of heavy meromyosin), half-maximal activation (Kapp) is obtained with 14 micro M muscle actin, but requires approximately 135 micro M yeast actin. This difference suggests a low affinity of yeast actin for muscle myosin. Yeast and muscle filamentous actin respond similarly to cytochalasin and phalloidin, although the drugs have no effect on S. cerevisiae cell growth.     

Cofilin increases the torsional flexibility and dynamics of actin filaments

We have measured the effects of cofilin on the conformation and dynamics of actin filaments labeled at Cys374 with erythrosin-iodoacetemide (ErIA), using time-resolved phosphorescence anisotropy (TPA). Cofilin quenches the phosphorescence intensity of actin-bound ErIA, indicating that binding changes the local environment of the probe. The cofilin concentration-dependence of the phosphorescence intensity is sigmoidal, consistent with cooperative actin filament binding. Model-independent analysis of the anisotropies indicates that cofilin increases the rates of the microsecond rotational motions of actin. In contrast to the reduction in phosphorescence intensity, the changes in the rates of rotational motions display non-nearest-neighbor cooperative interactions and saturate at substoichiometric cofilin binding densities. Detailed analysis of the TPA decays indicates that cofilin decreases the torsional rigidity (C) of actin, increasing the thermally driven root-mean-square torsional angle between adjacent filament subunits from approximately 4 degrees (C = 2.30 x 10(-27) Nm2 radian(-1)) to approximately 17 degrees (C = 0.13 x 10(-27) Nm2 radian(-1)) at 25 degrees C. We favor a mechanism in which cofilin binding shifts the equilibrium between thermal ErIA-actin filament conformers, and facilitates two distinct structural changes in actin. One is local in nature, which affects the structure of actin's C terminus and is likely to mediate nearest-neighbor cooperative binding and filament severing. The second is a change in the internal dynamics of actin, which displays non-nearest-neighbor cooperativity and increases the torsional flexibility of filaments. The long-range effects of cofilin on the torsional dynamics of actin may accelerate P(i) release from filaments and modulate interactions with other regulatory actin filament binding proteins.     

Orientation of the myosin light chain region by single molecule total internal reflection fluorescence polarization microscopy

To study the orientation and dynamics of myosin, we measured fluorescence polarization of single molecules and ensembles of myosin decorating actin filaments. Engineered chicken gizzard regulatory light chain (RLC), labeled with bisiodoacetamidorhodamine at cysteine residues 100 and 108 or 104 and 115, was exchanged for endogenous RLC in rabbit skeletal muscle HMM or S1. AEDANS-labeled actin, fully decorated with labeled myosin fragment or a ratio of approximately 1:1000 labeled:unlabeled myosin fragment, was adhered to a quartz slide. Eight polarized fluorescence intensities were combined with the actin orientation from the AEDANS fluorescence to determine the axial angle (relative to actin), the azimuthal angle (around actin), and RLC mobility on the <10 ms timescale. Order parameters of the orientation distributions from heavily labeled filaments agree well with comparable measurements in muscle fibers, verifying the technique. Experiments with HMM provide sufficient angular resolution to detect two orientations corresponding to the two heads in rigor. Experiments with S1 show a single orientation intermediate to the two seen for HMM. The angles measured for HMM are consistent with heads bound on adjacent actin monomers of a filament, under strain, similar to predictions based on ensemble measurements made on muscle fibers with electron microscopy and spectroscopic experiments.     

Actin Isoform-specific Conformational Differences Observed with Hydrogen/Deuterium Exchange and Mass Spectrometry

Actin can exist in multiple conformations necessary for normal function. Actin isoforms, although highly conserved in sequence, exhibit different biochemical properties and cellular roles. We used amide proton hydrogen/deuterium (HD) exchange detected by mass spectrometry to analyze conformational differences between Saccharomyces cerevisiae and muscle actins in the G and F forms to gain insight into these differences. We also utilized HD exchange to study interdomain and allosteric communication in yeast-muscle hybrid actins to better understand the conformational dynamics of actin. Areas showing differences in HD exchange between G- and F-actins are areas of intermonomer contacts, consistent with the current filament models. Our results showed greater exchange for yeast G-actin compared with muscle actin in the barbed end pivot region and areas in subdomains 1 and 2 and for F-actin in monomer-monomer contact areas. These results suggest greater flexibility of the yeast actin monomer and filament compared with muscle actin. For hybrid G-actins, the muscle-like and yeastlike parts of the molecule generally showed exchange characteristics resembling their parent actins. A few exceptions were a peptide on top of subdomain 2 and the pivot region between subdomains 1 and 3 with muscle actin-like exchange characteristics although the areas were yeastlike. These results demonstrate that there is cross-talk between subdomains 1 and 2 and the large and small domains. Hybrid F-actin data showing greater exchange compared with both yeast and muscle actins are consistent with mismatched yeast-muscle interfaces resulting in decreased stability of the hybrid filament contacts.The ability of actin to engage in a wide range of physiological functions requires that it be subject to complex spatial and temporal control by a large array of actin-binding proteins (1–3). Such regulation demands that both the monomeric and filamentous forms of actin be able to exist in a number of different conformations. Some of these may be differentially recognized by different actin-binding proteins, and some might actually be induced by the interaction of actin with these proteins.Actin is highly conserved from yeast to humans. There is 87% sequence identity between yeast and muscle actin and 91% sequence identity between yeast and nonmuscle actins. Even with this high sequence similarity and minor differences in crystal structures, there are some major differences in yeast and muscle actin behavior. Yeast, compared with muscle actin, polymerizes faster and exchanges its bound nucleotide faster. In contrast to muscle actin, the yeast actin filament releases its P_i almost immediately after ATP hydrolysis and the filament fragments more readily (4). Yeast cells cannot survive with muscle actin as the sole actin, and with β-nonmuscle actin as the only actin, yeast cells are very sick (5). A set of biochemical data indicates that the muscle filament is less flexible than yeast (6, 7). However, conformational and structural differences that could explain these behavioral differences are unknown.The actin molecule is divided into two domains, the small domain consisting of subdomains 1 and 2 and the large domain consisting of subdomains 3 and 4. Modeling studies (8, 9) have suggested that each of the subdomains can move, and biochemical studies have suggested allosteric interactions between the subdomains. However, the nature of these interactions has not been elucidated on a molecular level.To try to gain insight into what parts of the actin molecule are important for the behavioral differences observed between yeast and muscle actin, yeast-muscle hybrid actins were constructed (10). Hybrid sub1 actin has muscle-specific residues introduced into the small domain of actin, making it muscle-like in subdomain 1 and yeastlike in subdomains 2, 3, and 4. Sub12 hybrid actin has muscle-specific residues in both subdomains 1 and 2, making it muscle-like in subdomains 1 and 2 and yeastlike in subdomains 3 and 4. These actins showed muscle-like behavior in several biochemical properties: nucleotide exchange rates, thermostability, and the nucleation phase during polymerization. Although most of the residues proposed to be involved in the interaction of actin with myosin are located in subdomains 1 and 2, both hybrids exhibited yeast actin behavior in the activation of myosin ATPase activity. The hybrid actins also exhibited higher critical concentrations than either yeast or muscle actin and formed ADP-F-actin that was less stable than either yeast or muscle actin (10).The experimental results with the hybrid actins indicated that the behavior of actin as a whole is dictated by the interaction and cross-talk of the two halves. One experimental approach to further understand the structural bases of 1) biochemical differences observed with yeast and muscle actins, 2) how introduction of muscle-specific residues affects conformational behavior of the hybrids, and 3) whether there is propagation of change and cross-talk from muscle domains into yeast domains of the hybrid actins in solution is to use NMR. However, NMR as a technique is not suitable here due to the high actin concentrations that would be needed and the propensity of actin to aggregate at these concentrations. Therefore, we utilized hydrogen-deuterium (HD)² exchange detected with mass spectrometry. This is a powerful technique that can be used to obtain information about conformational changes in proteins in solution on a global as well as a local level.A protein sample placed in a deuterated solution will exchange its amide protons of the polypeptide backbone with exchange times varying from seconds to months (11). The amount and rate of deuterium exchange will depend on factors like temperature, pD, accessible surface area, local environment, and conformational flexibility (12–15). Labeled protein samples are digested by pepsin, the protease of choice in HD experiments, because of the requirement for acidic pH in order to minimize back exchange. The resulting peptide fragments are separated by high pressure liquid chromatography and analyzed for deuterium uptake.For a peptide that is undergoing amide proton exchange, the average mass of the peptide will increase with labeling time with an increase in mass greater for the peptides that are more exposed to the solvent. The analysis of deuterium uptake for specific peptides and areas of proteins could potentially provide information about structural and conformational differences between different actin forms. Since changes in conformation among different actin states will affect deuterium uptake, HD exchange coupled with mass spectrometry should provide us with a tool to study the states of actin present in solution. HD exchange detected with mass spectrometry was previously used successfully by Chik and co-workers (16) to assess changes in muscle actin structure due to the G- to F-actin transition and the binding of phallodin to F-actin and of DNase I to G-actin. More recently, the technique has been used to assess conformational changes in the Arp2/3 complex, which contains actin like subunits (17). In this study, we used HD exchange detected with mass spectrometry to analyze differences in solution structures of yeast, muscle, sub1, and sub12 G- and F-actins.     

Orientation of cross-bridges in skeletal muscle measured with a hydrophobic probe.

Cis-parinaric acid (PA) binds to a hydrophobic pocket formed between the heavy chain of myosin subfragment-1 (S1) and the 41-residue N-terminal of essential light chain 1 (A1). The binding is strong (Ka = 5.6 x 10(7) M-1) and rigid (polarization = 0.334). PA does not bind to myofibrils in which A1 has been extracted or replaced with alkali light chain 2 (A2). As in the case of S1 labeled with other probes, polarization of fluorescence of S1-PA added to myofibrils depended on fractional saturation of actin filament with S1, i.e., on whether the filaments were fully or partially saturated with myosin heads. Because fluorescence quantum yield of PA is enhanced manyfold upon binding, and because PA binds weakly to myofibrillar structures other then A1, the dye is a convenient probe of cross-bridge orientation in native muscle fibers. The polarization of a fiber irrigated with PA was equal to the polarization of S1-PA added to fibers at nonsaturating concentration. Cross-linking of S1 added to fibers at nonsaturating concentration showed that each S1 bound to two actin monomers of a thin filament. These results suggest that in rigor rabbit psoas muscle fiber each myosin cross-bridge binds to two actins.     

Control of the ability of profilin to bind and facilitate nucleotide exchange from G-actin

A major factor in profilin regulation of actin cytoskeletal dynamics is its facilitation of G-actin nucleotide exchange. However, the mechanism of this facilitation is unknown. We studied the interaction of yeast (YPF) and human profilin 1 (HPF1) with yeast and mammalian skeletal muscle actins. Homologous pairs (YPF and yeast actin, HPF1 and muscle actin) bound more tightly to one another than heterologous pairs. However, with saturating profilin, HPF1 caused a faster etheno-ATP exchange with both yeast and muscle actins than did YPF. Based on the -fold change in ATP exchange rate/K(d), however, the homologous pairs are more efficient than the heterologous pairs. Thus, strength of binding of profilin to actin and nucleotide exchange rate are not tightly coupled. Actin/HPF interactions were entropically driven, whereas YPF interactions were enthalpically driven. Hybrid yeast actins containing subdomain 1 (sub1) or subdomain 1 and 2 (sub12) muscle actin residues bound more weakly to YPF than did yeast actin (K(d) = 2 microm versus 0.6 microm). These hybrids bound even more weakly to HPF than did yeast actin (K(d) = 5 microm versus 3.2 microm). sub1/YPF interactions were entropically driven, whereas the sub12/YPF binding was enthalpically driven. Compared with WT yeast actin, YPF binding to sub1 occurred with a 5 times faster k(off) and a 2 times faster k(on). sub12 bound with a 3 times faster k(off) and a 1.5 times slower k(on). Profilin controls the energetics of its interaction with nonhybrid actin, but interactions between actin subdomains 1 and 2 affect the topography of the profilin binding site.     

Mechanism of formation of actomyosin interface

Force generation in muscle results from binding of myosin to F-actin. ATP binding to myosin provides energy to dissociate actomyosin complex while the hydrolysis of ATP is needed for re-binding of myosin to F-actin. At the end of each cycle myosin and actin form a tight complex with a substantial interface area. We investigated the dynamics of formation of actomyosin interface in presence and absence of nucleotides by quenched flow cross-linking technique. We showed previously that myosin head (subfragment 1, S1) directly interacts with at least two monomers in the actin filament. The quenched flow cross-linking experiments revealed that the initial contact (in presence or absence of nucleotides) occurs between loop 635-647 of S1 and 1-12 N-terminal residues of one actin and, then, the second contact forms between loop 567-574 of S1 and the N terminus of the second actin. The distance between these two loops in S1 corresponds to the distance between N termini of two actins in the same strand (53 A) but is smaller than that between two actins from the different strands (102 A). The formation of the actomyosin complex proceeds in ordered sequence: S1 initially binds to one actin then binds with the second actin located in the same strand but probably closer to the barbed end of F-actin. The presence of nucleotides slows down the interaction of S1 with the second actin, which correlates with recently proposed cleft movement in a 50 kDa domain of S1. The sequential mechanism of formation of actomyosin interface starting from one end and developing towards the barbed end might be involved in force generation and directional movement in actin-myosin system.     

Identification of amino acid substitutions differentiating actin isoforms in their interaction with myosin

Various aspects of actin--myosin interaction were studied with actin preparations from two types of smooth muscle: bovine aorta and chicken gizzard, and from two types of sarcomeric muscle: bovine cardiac and rabbit skeletal. All four preparations activated the Mg2+-ATPase activity of skeletal muscle myosin to the same Vmax, but the Kapp for the smooth muscle preparations was higher. At low KCl, pH 8.0 and millimolar substrate concentrations the Kapp values differed by a factor of 2.5. This differential behaviour of the four actin preparations correlates with amino acid substitutions at positions 17 and 89 of actin polypeptide chain, differentiating the smooth-muscle-specific gamma and alpha isomers from cardiac and skeletal-muscle-specific alpha isomers. This correlation provides evidence for involvement of the NH2-terminal portion of the actin polypeptide chain in the interaction with myosin. The differences in the activation of myosin ATPase by various actins were sensitive to changes in the substrate and KCl concentration and pH of the assay medium. Addition of myosin subfragment-1 or heavy meromyosin in the absence of nucleotide produced similar changes in the fluorescence of a fluorescent reagent N-(1-pyrenyl)-iodoacetamide, attached at Cys-374, or 1,N6-ethenoadenosine 5'-diphosphate substituted for the bound ADP in actin protomers in gizzard and skeletal muscle F-actin. The results are consistent with an influence of the amino acid substitutions on ionic interactions leading to complex formation between actin and myosin intermediates in the ATPase cycle but not on the associated states.     

Study of structural changes of contractile muscle proteins with the aid of polarization ultraviolet fluorescence microscopy. 1. Conformational changes of F-actin in the muscle fiber caused by ATP and its analogs]

Increase of anisotropy of F-actin fluorescence of balanus and rabbit muscle fibers under the influence of ATP, AMP and pyrophosphate in EGTA presence was detected by means of the polarized ultraviolet (UV) fluorescent microscopy methods. The fluorescence anisotropy changes are assumed to be associated with the conformational changes in the actin. ATP cause more noticeable changes of actin structure, than pyrophosphate and AMP. The conformational changes in the actin of balanus and rabbit muscle fibres were similar. ATP and its analogs induced also decrease of UV fluorescence anisotropy of A-band which appears to be associated with conformational changes in myosin. It was siggested that the changes in fluorescence of anisotropy of A-bands are due to structural changes in both HMM and LMM parts of myosin molecule.     

Electron microscopic study of actin polymerization in airway smooth muscle

Actin polymerization as part of the normal smooth muscle response to various stimuli has been reported. The actin dynamics are believed to be necessary for cytoskeletal remodeling in smooth muscle in its adaptation to external stress and strain and for maintenance of optimal contractility. We have shown in our previous studies in airway smooth muscle that myosins polymerized in response to contractile activation as well as to adaptation at longer cell lengths. We postulated that the same response could be elicited from actins under the same conditions. In the present study, actin filament formation was quantified electron microscopically in cell cross sections. Nanometer resolution allowed us to examine regional distribution of filaments in a cell cross section. Airway smooth muscle bundles were fixed in relaxed and activated states at two lengths; muscle preparations were also fixed after a period of oscillatory strain, a condition known to cause depolymerization of myosin filaments. The results indicate that contractile activation and increased cell length nonsynergistically enhanced actin polymerization; the extent of actin polymerization was substantially less than that of myosin polymerization. Oscillatory strain increased thin filament formation. Although thin filament density was found higher in cytoplasmic areas near dense bodies, contractile activation did not preferentially enhance actin polymerization in these areas. It is concluded that actin thin filaments are dynamic structures whose length and number are regulated by the cell in response to changes in extracellular environment and that polymerization and depolymerization of thin filaments occur uniformly across the whole cell cross section.     

Sequential disassembly of myofibrils induced by myristate acetate in cultured myotubes

The phorbol ester TPA induces the sequential disassembly of myofibrils. First the alpha-actin thin filaments are disrupted and then, hours later, the myosin heavy chain (MHC) thick filaments. TPA does not induce the disassembly of the beta- and gamma-actin thin filaments of stress fibers in presumptive myoblasts or fibroblasts, nor does it block the reemergence of stress fibers in 72-h myosacs that have been depleted of all myofibrillar molecules. There are differences in where, when, and how myofibrillar alpha-actin and MHC are degraded and eliminated from TPA-myosacs. Though the anisodiametric myotubes have begun to retract into isodiametric myosacs after 5 h in TPA, staining with anti-MHC reveals normal tandem A bands. In contrast, staining with mAb to muscle actin fails to reveal tandem I bands. Instead, both mAb to muscle actin and rhophalloidin brilliantly stain numerous disk-like bodies approximately 3.0 micron in diameter. These muscle actin bodies do not fuse with one another, nor do they costain with anti-MHC. All muscle actin bodies and/or molecules disappear in 36-h myosacs. The collapse of A bands is first initiated in 10-h myosacs. Their loss correlates with the appearance of immense, amorphous MHC patches. MHC patches range from a few micrometers to over 60 micron in size. They do not costain with antimuscle actin or rho-phalloidin. While diminishing in number and fluorescence intensity, MHC aggregates are present in 30% of the 72-h myosacs. Myosacs removed from TPA rapidly elongate, and after 48 h display normal newly assembled myofibrils. TPA reversibly blocks incorporation of [35S]methionine into myofibrillar alpha-actin, MHC, myosin light chains 1 and 2, the tropomyosins, and troponin C. It does not block the synthesis of beta- or gamma-actins, the nonmyofibrillar MHC or light chains, tubulin, vimentin, desmin, or most household molecules.     

Chicken-gizzard actin. Interaction with skeletal-muscle myosin

Interaction of actin from chicken gizzard and from rabbit skeletal muscle with rabbit skeletal muscle myosin was compared by measuring the rate of superprecipitation, the activation of the Mg-ATPase and inhibition of K-ATPase activity of myosin and heavy meromyosin, and determination of binding of heavy meromyosin in the absence of ATP. Both the rate of superprecipitation of the hybrid actomyosin and the activation of myosin ATPase by gizzard actin are lower than those obtained with skeletal muscle actin. The activation of myosin Mg-ATPase by the two actin species also shows different dependence on substrate concentration: with gizzard actin the substrate inhibition starts at lower ATP concentration. The double-reciprocal plots of the Mg-ATPase activity of heavy meromyosin versus actin concentration yield the same value of the extrapolated ATPase activity at infinite actin concentration (V) for the two actins and nearly double the actin concentration needed to produce half-maximal activation (Kapp) in the case of gizzard actin. A corresponding difference in the abilities of the two actin species to inhibit the K-ATPase activity of heavy meromyosin in the absence of divalent cations was also observed. The results are discussed in terms of the effect of substitutions in the amino acid sequence of gizzard and skeletal muscle actins on their interaction with myosin.     

Actins from plant and animal sources tend not to form heteropolymers in vitro and function differently in plant cells

Summary. Pollen and skeletal muscle actins were purified and labeled with fluorescent dyes that have different emission wavelengths. Observation by electron microscopy shows that the fluorescent actins are capable to polymerize into filamentous actin in vitro, bind to myosin S-1 fragments, and have a critical concentration similar to unlabeled actin, indicating that they are functionally active. The globular actins from two sources were mixed and polymerized by the addition of ATP and salts. The copolymerization experiment shows that when excited by light of the appropriate wavelength, both red actin filaments (pollen actin) and green actin filaments (muscle actin) can be visualized under the microscope, but no filaments exhibiting both green and red colors are detected. Furthermore, coprecipitations of labeled pollen actin with unlabeled pollen and skeletal muscle actin were performed. Measurements of fluorescent intensity show that the amount of labeled pollen actin precipitating with pollen actin was much higher than that with skeletal muscle actin, indicating that pollen and muscle actin tend not to form heteropolymers. Injection of labeled pollen actin into living stamen hair cells results in the formation of normal actin filaments in transvacuolar strands and the cortical cytoplasm. In contrast, labeled skeletal muscle actin has detrimental effects on the cellular architecture. The results from coinjection of the actin-disrupting reagent cytochalasin D with pollen actin show that overexpression of pollen actin prolongs the displacement of the nucleus and facilitates the recovery of the nuclear position, actin filament architecture, and transvacuolar strands. However, muscle actin perturbs actin filaments when injected into stamen hair cells. Moreover, nuclear displacement occurs more rapidly when cytochalasin D and muscle actin are coinjected into the cell. It is concluded that actins from plant and animal sources behave differently in vitro and in vivo and that they are functionally not interchangeable.     

The effect of caldesmon on actin-myosin interaction in skeletal muscle fibers

The effects of caldesmon on structural and dynamic properties of phalloidin-rhodamine-labeled F-actin in single skeletal muscle fibers were investigated by polarized microphotometry. The binding of caldesmon to F-actin in glycerinated fibers reduced the alterations of thin filaments structure and dynamics that occur upon the transition of the fibers from rigor to relaxing conditions. In fibers devoid of myosin and regulatory proteins (ghost fibers) the binding of caldesmon to F-actin precluded structural changes in actin filaments induced by skeletal muscle myosin subfragment 1 and smooth muscle tropomyosin. These results suggest that the restraint for the alteration of actin structure and dynamics upon binding of myosin heads and/or tropomyosin evoked by caldesmon can be related to its inhibitory effect on actin-myosin interaction.