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Striated muscle is activated by myosin- and actin-linked processes, with the latter being regulated through changes in the position of tropomyosin relative to the actin surface. The C-terminal region of cardiac troponin T (TnT), a tropomyosin-associated protein, is required for full TnT inactivation at low Ca(2+) and for limiting its activation at saturating Ca(2+). Here, we investigated whether basic residues in this TnT region are involved in these activities, whether the TnT C terminus undergoes Ca(2+)-dependent conformational changes, and whether these residues affect cardiac muscle contraction. We generated a human cardiac TnT variant in which we replaced seven C-terminal Lys and Arg residues with Ala and added a Cys residue at either position 289 or 275 to affix a fluorescent probe. At pCa 3.7, actin filaments containing high-alanine TnT had an elevated ATPase rate like that obtained when the last TnT 14 residues were deleted. Acrylodan-tropomyosin fluorescence changes and S1-actin binding kinetics revealed that at pCa 8, the high-alanine TnT-containing filaments did not enter the first inactive state. FRET analyses indicated that the C-terminal TnT region approached Cys-190 of tropomyosin as actin filaments transitioned to the inactive B state; that transition was abolished with high-alanine TnT. High-alanine TnT-containing cardiac muscle preparations had increased Ca(2+) sensitivity of both steady-state isometric force and sinusoidal stiffness as well as increased maximum steady-state isometric force and sinusoidal stiffness. We conclude that C-terminal basic residues in cardiac TnT are critical for the regulation of cardiac muscle contraction.

It has been shown that not only calcium but also strong binding myosin heads contribute to thin filament activation in isometrically contracting animal fast-twitch and cardiac muscle preparations. This behavior has not been studied in human muscle fibers or animal slow-twitch fibers. Human slow-twitch fibers are interesting since they contain the same myosin heavy chain isoform as the human heart. To explore myosin-induced activation of the thin filament in isometrically contracting human slow-twitch fibers, the endogenous troponin complex was exchanged for a well-characterized fast-twitch skeletal troponin complex labeled with the fluorescent dye N-((2-(Iodoacetoxy)ethyl)-N-methyl)amino-7-nitrobenz-2-oxa-1,3-diazole (fsTn-IANBD). The exchange was ≈70% complete (n = 8). The relative contributions of calcium and strong binding cross-bridges to thin filament activation were dissected by increasing the concentration of calcium from relaxing (pCa 7.5) to saturating levels (pCa 4.5) before and after incubating the exchanged fibers in the myosin inhibitor para-aminoblebbistatin (AmBleb). At pCa 4.5, the relative contributions of calcium and strong binding cross-bridges to thin filament activation were ≈69 and ≈31%, respectively. Additionally, switching from isometric to isotonic contraction at pCa 4.5 revealed that strong binding cross-bridges contributed ≈29% to thin filament activation (i.e., virtually the same magnitude obtained with AmBleb). Thus, we showed through two different approaches that lowering the number of strong binding cross-bridges, at saturating calcium, significantly reduced the activation of the thin filament in human slow-twitch fibers. The contribution of myosin to activation resembled that which was previously reported in rat cardiac and rabbit fast-twitch muscle preparations. This method could be applied to slow-twitch human fibers obtained from the soleus muscle of cardiomyopathy patients. Such studies could lead to a better understanding of the effect of point mutations of the cardiac myosin head on the regulation of muscle contraction and could lead to better management by pharmacological approaches.

Activation of striated muscle contraction occurs in response to Ca(2+) binding to troponin C. The resulting reorganization of troponin repositions tropomyosin on actin and permits activation of myosin-catalyzed ATP hydrolysis. It now appears that the C-terminal 14 amino acids of cardiac troponin T (TnT) control the level of activity at both low and high Ca(2+). We made a series of C-terminal truncation mutants of human cardiac troponin T, isoform 2, to determine if the same residues of TnT are involved in the low and high Ca(2+) effects. We measured the effect of these mutations on the normalized ATPase activity at saturating Ca(2+). Changes in acrylodan tropomyosin fluorescence and the degree of Ca(2+) stimulation of the rate of binding of rigor myosin subfragment 1 to pyrene-labeled actin-tropomyosin-troponin were measured at low Ca(2+). These measurements define the distribution of actin-tropomyosin-troponin among the three regulatory states. Residues SKTR and GRWK of TnT were required for the functioning of TnT at both low and high Ca(2+). Thus, the effects on forming the inactive B-state and in retarding formation of the active M-state require the same regions of TnT. We also observed that the rate of binding of rigor subfragment 1 to pyrene-labeled regulated actin at saturating Ca(2+) was higher for the truncation mutants than for wild-type TnT. This violated an assumption necessary for determining the B-state population by this kinetic method.

Activation of striated muscle contraction occurs in response to Ca2+binding to troponin C (TnC). The resulting reorganization of troponin repositions tropomyosin on actin and permits activation of myosin catalyzed ATP hydrolysis. It now appears that the levels of activity at both low and saturating Ca2+are modulated by the C-terminal 14 amino acids of cardiac troponin T (TnT). We made a series of mutants of human cardiac troponin T, isoform 2, with deletions from the C-terminal end: Δ4, Δ6, Δ8, Δ10 and Δ14. We measured the effect of these mutations on the normalized ATPase activity at saturating Ca2+, the change in acrylodan tropomyosin fluorescence at low Ca2+, and the degree of Ca2+stimulation of the rate of binding of rigor myosin S1 to pyrene-labeled actin-tropomyosin-troponin. Together, these measurements define the distribution of actin-tropomyosin-troponin among the 3 regulatory states. Results from rates of rigor S1 binding deviated from other measurements when > 8 residues of TnT were deleted. That deviation was due to increased rates of binding of rigor S1 to pyrene-labeled actin with truncated TnT at saturating Ca2+. Such behavior violated a key assumption in the determination of the B state by this method. Nevertheless, all methods show that as residues were removed from the C-terminus of TnT there was approximately a proportional loss of the inactive B state at low Ca2+and an increase in the active M state at saturating Ca2+. Most of the C-terminal 14 residues of human cardiac troponin T are essential for forming the inactive B state at low Ca2+and for limiting the formation of the active M state at saturating Ca2+.

Striated muscle contraction is regulated by the actin-associated proteins tropomyosin and troponin. The extent of activation of myosin ATPase activity is lowest in the absence of both Ca(2+) and activating cross-bridges (i.e., S1-ADP or rigor S1). Binding of activating species of myosin to actin at a saturating Ca(2+) concentration stabilizes the most active state (M state) of the actin–tropomyosin–troponin complex (regulated actin). Ca(2+) binding alone produces partial stabilization of the active state. The extent of stabilization at a saturating Ca(2+) concentration depends on the isoform of the troponin subunits, the phosphorylation state of troponin, and, in the case of cardiac muscle, the presence of hypertrophic cardiomyopathy-producing mutants of troponin T and troponin I. Cardiac dysfunction is also associated with mutations of troponin C (TnC). Troponin C mutants A8V, C84Y, and D145E increase the Ca(2+) sensitivity of ATPase activity. We show that these mutants change the distribution of regulated actin states. The A8V and C84Y TnC mutants decreased the inactive B state distribution slightly at low Ca(2+) concentrations, but the D145E mutants had no effect on that state. All TnC mutants increased the level of the active M state compared to that of the wild type, at a saturating Ca(2+) concentration. Troponin complexes that contained two mutations that stabilize the active M state, A8V TnC and Δ14 TnT, appeared to be completely in the active state in the presence of only Ca(2+). Because Ca(2+) gives full activation, in this situation, troponin must be capable of positioning tropomyosin in the active M state without the need for rigor myosin binding.

Smooth muscle cells maintain filaments of actin and myosin in the presence of ATP, although dephosphorylated myosin filaments and actin-myosin interactions are unstable under those conditions in vitro. Several proteins that stabilize myosin filaments and that stabilize actin-myosin interactions have been identified. Fesselin or synaptopodin 2 appears to be another such protein. Rapid kinetic measurements and electron microscopy demonstrated that fesselin, isolated from turkey gizzard muscle, reduced the rate of dissociation of myosin filaments. Addition of fesselin increased both the length and thickness of myosin filaments. The rate of detachment of myosin, but not heavy meromyosin, from actin was also greatly reduced by fesselin. Data from this study suggest that fesselin stabilizes myosin filaments and tethers myosin to actin. These results support the view that one role of fesselin is to organize contractile units of myosin and actin.

Fesselin or avian synaptopodin 2 is a member of the synaptopodin family of actin binding proteins. Fesselin promotes G-actin polymerization and the formation of large actin complexes that can be collected by low-speed centrifugation. Because of the potential role of fesselin in some cancers and its effects on actin, we further investigated the effect of fesselin on actin. Fesselin initiated actin polymerization under a variety of conditions, including the virtual absence of salt. Actin filaments formed at low salt concentrations in the presence of fesselin were similar to filaments polymerized in the presence of 100 mM KCl. In both cases, the filaments were long and straight with a common orientation. Highly ordered actin bundles formed with increasing times of incubation. Blockers of actin growth at the barbed end (cytochalasin D and CapZ) did not prevent fesselin from polymerizing actin. Low concentrations of fesselin increased the critical concentration of actin. Both observations are consistent with preferential growth at the pointed end of actin filaments. These results indicate a role of fesselin in organizing cellular actin. These and other results indicate that fesselin is part of a cellular actin organizing center.

Loss of strength in human and animal models of aging can be partially attributed to a well-recognized decrease in muscle mass; however, starting at middle-age, the normalized force (force/muscle cross-sectional area) in the knee extensors and single muscle fibers declines in a curvilinear manner. Strength is lost faster than muscle mass and is a more consistent risk factor for disability and death. Reduced expression of the voltage sensor Ca2+ channel α1 subunit (Cav1.1) with aging leads to excitation-contraction uncoupling, which accounts for a significant fraction of the decrease in skeletal muscle function. We recently reported that in addition to its classical cytoplasmic location, fast skeletal muscle troponin T3 (TnT3) is fragmented in aging mice, and both full-length TnT3 (FL-TnT3) and its carboxyl-terminal (CT-TnT3) fragment shuttle to the nucleus. Here, we demonstrate that it regulates transcription of Cacna1s, the gene encoding Cav1.1. Knocking down TnT3 in vivo downregulated Cav1.1. TnT3 downregulation or overexpression decreased or increased, respectively, Cacna1s promoter activity, and the effect was ablated by truncating the TnT3 nuclear localization sequence. Further, we mapped the Cacna1s promoter region and established the consensus sequence for TnT3 binding to Cacna1s promoter. Systemic administration of BDA-410, a specific calpain inhibitor, prevented TnT3 fragmentation, and Cacna1s and Cav1.1 downregulation and improved muscle force generation in sedentary old mice., Instituto de Investigaciones Bioquímicas de La Plata

Striated muscle contraction is regulated primarily through the action of tropomyosin and troponin that are bound to actin. Activation requires Ca(2+) binding to troponin and/or binding of high affinity myosin complexes to actin. Mutations within components of the regulatory complex may lead to familial cardiomyopathies and myopathies. In several cases examined, either physiological or pathological changes in troponin alter the distribution among states of actin-tropomyosin-troponin that differ in their abilities to stimulate myosin ATPase activity. These observations open possibilities for managing disorders of the troponin complex. Furthermore, analyses of mutant forms of troponin give insights into the regulation of striated muscle contraction.

Changes in the muscle regulatory protein complex, troponin, are important for modulation of activity and may occur as a result of disease-causing mutations. Both increases and decreases in the rate of ATP hydrolysis by myosin may occur as dictated by changes in the distribution of actin-tropomyosin-troponin among its different states. It is important to measure the rates of transition among these states to study physiological adaptation and disease processes. We show here that acrylodan or pyrene probes on tropomyosin can be used to monitor the transition from active to intermediate and inactive states of actin-tropomyosin-troponin. Transitions measured in the absence of calcium had two phases, as previously reported for some other probes on troponin and actin. The first step was a rapid equilibrium that favored the formation of the intermediate state and had an apparent rate constant less than that of S1-ATP dissociation. The second fluorescence transition was slower, with an apparent constant that increased from approximately 5 to 80/s over a range of 1-37 degrees C. Only the initial rapid transition was seen in the presence of saturating calcium. The acrylodan probe had the advantage of yielding a larger signal than the pyrene probe. Furthermore, the acrylodan signal decreased in going from the active state to the intermediate state, and then increased upon going to the inactive state.

We examined four cardiomyopathy-causing mutations of troponin I that appear to disturb function by altering the distribution of thin filament states. The R193H (mouse) troponin I mutant had greater than normal actin-activated myosin-S1 ATPase activity in both the presence and absence of calcium. The rate of ATPase activity was the same as that of the wild-type at near-saturating concentrations of the activator, N-ethylmaleimide-S1. This mutant appeared to function by stabilizing the active state of thin filaments. Mutations D191H, R146G, and R146W had lower ATPase activities in the presence of calcium, but higher activities in the absence of calcium. These effects were most pronounced with mutations at position 146. For all three mutants the rates were similar to those of the wild-type at near-saturating concentrations of N-ethylmaleimide-S1. These results, combined with previous results, show that any alteration in the normal distribution of actomyosin states is capable of producing cardiomyopathy. The results of the D191H, R146G, and R146W mutations are most readily explained if the intermediate state of regulated actin has a unique function. The intermediate state appears to have an ability to accelerate the rate of ATP hydrolysis by myosin that exceeds that of the inactive state.

An analysis of the primary structure of the actin-binding protein fesselin revealed it to be the avian homologue of mammalian synaptopodin 2 [Schroeter, Beall, Heid, and Chalovich (2008) Biochem. Biophys. Res. Commun. 371, 582–586]. We isolated two synaptopodin 2 isoforms from rabbit stomach that corresponded to known types of human synaptopodin 2. The purification scheme used was that developed for avian fesselin. These synaptopodin 2 forms shared several key functions with fesselin. Both avian fesselin and mammalian synaptopodin 2 bound to Ca2+–calmodulin, α-actinin and smooth-muscle myosin. In addition, both proteins stimulated the polymerization of actin in a Ca2+–calmodulin-dependent manner. Synaptopodin 2 has never before been shown to polymerize actin in the absence of α-actinin, to polymerize actin in a Ca2+–calmodulin-dependent manner, or to bind to Ca2+–calmodulin or myosin. These properties are consistent with the proposed function of synaptopodin 2 in organizing the cytoskeleton.

Fesselin is a natively unfolded protein that is abundant in avian smooth muscle. Like many natively unfolded proteins, fesselin has multiple binding partners including actin, myosin, calmodulin and alpha-actinin. Fesselin accelerates actin polymerization and bundles actin. These and other observations suggest that fesselin is a component of the cytoskeleton. We have now cloned fesselin and have determined the cDNA derived amino acid sequence. We verified parts of the sequence by Edman analysis and by mass spectroscopy. Our results confirmed fesselin is homologous to human synaptopodin 2 and belongs to the synaptopodin family of proteins.

Alterations in the troponin complex can lead to increases or decreases in contractile activity. Most mutations of troponin that cause hypertrophic cardiomyopathy increase the activity of cardiac muscle fibers. In at least some cases these mutants stabilize the active state of regulated actin. In contrast, phosphorylation of troponin I at residues 43, 45, and 144 inhibits muscle contractility. To determine if alterations of troponin I that reduce activity do stabilize the inactive state of actin, we introduced negative charges at residues 43, 45, and 144 of troponin I to mimic a constitutively phosphorylated state. At saturating calcium, all mutants decreased ATPase rates relative to wild-type actin-tropomyosin-troponin. Reduced activation of ATPase activity was seen with a single mutation at S45E and was not further altered by mutating the other two sites. In the presence of low concentrations of NEM-S1, wild-type troponin was more active than the mutants. At high NEM-S1, the rates of wild-type and mutants approached the same limiting value. Changes in Ca2+ affinity also support the idea that the equilibrium between states of actin-tropomyosin-troponin was shifted to the inactive state by mutations that mimic troponin I phosphorylation.

Fesselin is a heat stable proline-rich actin binding protein. The stability, amino acid composition, and ability to bind to several proteins suggested that fesselin may be unfolded under native conditions. While the complete sequence of fesselin is unknown an analysis of a closely related protein, synaptopodin 2 from Gallus gallus, indicates that fesselin consists of a series of unstructured regions interspersed between short folded regions. To determine if fesselin is natively unfolded, we compared fesselin to a known globular protein (myosin S1) and a known unfolded protein Cad22 (the COOH terminal 22 kDa fragment of caldesmon). Fesselin, and Cad22, had larger Stokes radii than globular proteins of equivalent mass. The environments of tryptophan residues of fesselin and Cad22 were the same in the presence and absence of 6 M guanidine hydrochloride. Fesselin had a circular dichroism spectrum that was primarily random coil. Changes in pH over the range of 1.5-11.5 did not alter that spectrum. Increasing the temperature to 85 degrees C caused an increase in the degree of secondary structure. Calmodulin binding to fesselin altered the environment of the tryptophan residues so that they became less sensitive to the quencher acrylamide. These results show that fesselin is a natively unfolded protein.

Fesselin (or avian synaptopodin 2) is an actin binding protein that nucleates actin filament formation (Leinweber et al. 1999; Beall et al. 2001) and bundles actin filaments (Schroeter et al. 2013). The nucleating activity is inhibited by Ca2+-calmodulin (Schroeter et al. 2004). Fesselin also binds myosin with an affinity of 2 x 106 M−1 at 50 mM ionic strength (Schroeter & Chalovich 2005). That binding increases the length and width of myosin filaments, and reduces the rate of dissociation of myosin filaments and actin-myosin complexes by ATP (Kingsbury et al. 2013). Fesselin labeled with IANBD produced a fluorescence increase upon binding to smooth muscle myosin or S1. The apparent rate constant for the fluorescence change increased with increasing concentrations of myosin or S1 in a hyperbolic fashion (105 mM ionic strength, 10 deg C). Binding to myosin began with the rapid formation of a low affinity intermediate followed by a second step having an observed rate constant (k2 + k-2) of 30/sec. Several observations suggested that the binding of fesselin to myosin was affected by calmodulin and calcium. The apparent rate constants for both processes in the binding of IANBD-fesselin to S1 were 2-fold faster in the absence of calcium. Binding of IANBD fesselin to intact myosin was complex in the presence of calcium with a rapid increase in fluorescence followed by a slower decrease; that behavior was not observed in the presence of calcium but absence of calmodulin. Ca2+-calmodulin may play roles in the formation of actin filaments, myosin filaments and actin-myosin complexes through fesselin.

Fesselin is an actin binding protein that bundles actin filaments and accelerates nucleation of actin polymerization. The effect of fesselin on actin polymerization is regulated by Ca(++)-calmodulin. Because actin filaments serve both structural and contractile functions we also examined the effect of fesselin on activation of myosin S1 ATPase activity. Fesselin inhibited the activation of S1-catalyzed ATP hydrolysis in a similar manner in both the presence and absence of tropomyosin. This inhibition was unaffected by Ca(++)-calmodulin. Fesselin inhibited the binding of myosin-S1 to actin during steady-state ATP hydrolysis. Fesselin also displaced caldesmon from actin. S1 displaced fesselin from actin in the absence of nucleotide when the affinity of S1 for actin was much greater than the affinity of fesselin for actin. It is likely that fesselin and S1 share common binding sites on F-actin. We also observed that fesselin could bind to smooth muscle myosin with muM affinity. Fesselin shares some similarities to caldesmon in binding to several other proteins and having multiple potential functions.

The complex of tropomyosin and troponin binds to actin and inhibits activation of myosin ATPase activity and force production of striated muscles at low free Ca2+ concentrations. Ca2+ stimulates ATP activity and at subsaturating actin concentrations the binding of NEM-modified S1 to actin– tropomyosin–troponin increases the rate of ATP hydrolysis even further. We show here that the ∆14 mutation of troponin T associated with familial hypertrophic cardiomyopathy results in an increase in ATPase rate like that seen with wild-type troponin in the presence of NEM-S1. The enhanced ATPase activity was not due to a decreased incorporation of mutant troponin T with roponin I and troponin C to form an active troponin complex. The activating effect was more prominent with a hybrid troponin (skeletal TnI TnC and cardiac TnT) than with all cardiac troponin. Thus it appears that changes in the troponin–troponin contacts that result from mutations or from forming hybrids stabilize a more active state of regulated actin. An analysis of the effect of the ∆14 mutation on the equilibrium binding of S1-ADP to actin was consistent with stabilization of an active state of actin. This change in activation may be important in the development of cardiac disease. Originally published Biochemistry Vol. 43 No. 48 Dec 2004

There is no consensus on the mechanism of inhibition of actin-myosin ATPase activity by caldesmon. Various models are based on different assumptions for the number of actin monomers that constitute a caldesmon binding site. Differences in binding behavior may be due to variations in the assay, the range of caldesmon concentrations, the type of caldesmon, and the method of data analysis used. We have evaluated these factors by measuring binding in the presence and absence of tropomyosin with both intact caldesmon and a recombinant 35 kDa actin binding fragment and with actin initially in the polymerized state or monomeric state. In all cases caldesmon binding could be simulated with a model having one class of binding sites. However, the number of actin monomers constituting a site was variable. Binding to F-actin at 165 mM ionic strength was best described with 7 actin monomers per site. When caldesmon bound to actin during the polymerization of G-actin, the size of the binding site was 3. Binding of the expressed truncated fragment, Cad35, could be described with 3 monomers per site. A simple interpretation of the data is that caldesmon binds tightly to 2-3 actin monomers. Additional parts of caldesmon bind less tightly to actin, causing caldesmon to cover approximately 7 actin monomers. The appendix contains an analysis of several binding curves with multiple binding site models. There is no compelling evidence for two classes of binding sites.

Several laboratories have reported cooperative binding of S1 to actin in the presence of caldesmon. This cooperative binding has been interpreted with a model similar to that proposed for the binding of S 1 to regulated actins in which the binding affinity of S 1 is controlled by the position of the tropomyosin filaments. In a recent paper [Sen, A., Chen, Y., Yan, B., and Chalovich, J. M. (2001) Biochemistry 40, 5757-64], we showed qualitatively that Sl binding resulted in rapid dissociation of caldesmon from actin or actin-tropomyosin. This suggests that the cooperativity observed in the case of caldesmon is not due to a conformational change in actin-caldesmon but to the displacement of caldesmon. We show in this paper that the pure competitive binding model, in which both Sl and caldesmon are competing for the same binding sites on actin, can simulate quantitatively the effect of caldesmon on both the equilibrium and the kinetics of Sl binding to actin. This model successfully predicts an apparent cooperativity for the binding of S 1 to actin-caldesmon without the need to assume multiple actin-caldesmon structures and produces a decreased rate of S 1 binding to actin in the presence of caldesmon. This suggests that the inhibitory action of caldesmon on the actin-activated ATPase activity of myosin in solution and on the generation of active force in a contracting muscle may be simply due to the blocking of myosin binding sites on actin by caldesmon.

Actin stimulates the ATPase activity of myosin-S1. The regulatory proteins tropomyosin and troponin prevent this stimulation at conditions of low Ca2+ and low high-affinity myosin binding. At saturating Ca2+ and with strong actin-binding species of myosin (S1-ADP or NEM-S1) the enhancement of ATPase activity exceeds that seen in the absence of regulatory proteins. Inhibition and potentiation of actin activated ATPase activity are dependent on the position of tropomyosin on actin filaments. One position of tropomyosin (M state) leads to high activity whereas the other two positions (B and the intermediate C state) are inactive.We recently showed that the C-terminal 14 residues of troponin T are critical for formation of the inactive B state at low Ca2+. Furthermore, that same region of troponin T limits the formation of the M state at saturating Ca2+. Our present goal is to map the C-terminal region of troponin T to determine if specific residues in the sequence are responsible for each of these activities. The B state was monitored by changes in acrylodan tropomyosin fluorescence and by the rate of rigor S1 binding to regulated actin. The M state was monitored by an ATPase assay. Stepwise deletions within the C-terminal 14 amino acids of Troponin T progressively reduced the population of the inactive B state at low Ca2+. In contrast, the population of the M state was progressively increased at high Ca2+. The net positive charge of the last 14 residues appeared to be a key determinant of activity. Conclusion: The C-terminal region of troponin T plays essential roles in inactivation and in limiting activation. Both of these activities require basic amino acid residues within the COOH terminus.

The troponin complex in a muscle fiber can be replaced with exogenous troponin by using a gentle exchange procedure in which the actin–tropomyosin complex is never devoid of a full complement of troponin (Brenner et al. (1999) Biophys J 77: 2677–2691). The mechanism of this exchange process and the factors that influence this exchange are poorly understood. In this study, the exchange process has now been examined in myofibrils and in solution. In myofibrils under rigor conditions, troponin exchange occurred preferentially in the region of overlap between actin and myosin when the free Ca2+ concentration was low. At higher concentrations of Ca2+, the exchange occurred uniformly along the actin. Ca2+ also accelerated troponin exchange in solution but the effect of S1 could not be confirmed in solution experiments. The rate of exchange in solution was insensitive to moderate changes in pH or ionic strength. Increasing the temperature resulted in a two-fold increase in rate with each 10°C increase in temperature. A sequential two step model of troponin binding to actin–tropomyosin could simulate the observed association and dissociation transients. In the absence of Ca2+ or rigor S1, the following rate constants could describe the binding process: k 1 = 7.12 μM−1s−1, k −1 = 0.65 s−1, k 2 = 0.07 s−1, k −2 = 0.0014 s−1. The slow rate of detachment of troponin from actin (k −2) limits the rate of exchange in solution and most likely contributes to the slow rate of exchange in fibers.

The purpose of this study was to address the paradox of calponin localization with alpha-actinin and filamin, two proteins with tandem calponin homology (CH) domains, by determining the effect of these proteins on the binding of calponin to actin. The results show that actin can accommodate near-saturating concentrations of either calponin and alpha-actinin or calponin and filamin with little change or no change in ligand affinity. Little direct interaction occurred between alpha-actinin and calponin in the absence of actin, so this effect is not likely to explain the co-distribution of these proteins. Calponin, like alpha-actinin, induced elastic gel formation when added to actin. When alpha-actinin was added to newly formed calponin/actin gels, no change was seen in the mechanical properties of the gel compared to calponin and actin alone. However, when calponin was added to newly formed alpha-actinin/actin gels, the resulting gel was much stronger than the gels formed by either ligand alone. Furthermore, gels formed by the addition of calponin to alpha-actinin/actin exhibited a phenomenon known as strain hardening, a characteristic of mechanically resilient gels. These results add weight to the concept that one of the functions of calponin is to stabilize the actin cytoskeleton.

A novel actin binding protein has been isolated from chicken gizzard muscle. When isolated, a pair of proteins with apparent molecular weights of 79 kDa and 103 kDa are obtained; both proteins have a pI near 9.3. Peptide mapping indicates that these proteins are related. Antibodies against this protein cross-reacted with proteins from other smooth muscle containing tissues as well as skeletal and heart muscle. Traces of cross-reactive material were also detected in brain and kidney tissue. The affinity of this protein for actin is ca. 1 × 106 M−1. Interestingly, this actin binding protein is a potent actin-bundling agent. A partial sequence analysis confirmed that there were no previously reported homologues in smooth muscle. However, considerable homology was found with the protein synaptopodin that is found in nervous tissue and kidney but is absent from muscle tissue. It is likely that the new protein is a member of the synaptopodin family. We call the smooth muscle actin binding protein fesselin.

Intact caldesmon and particularly the actin-binding C-terminal fragment (20-kDa) of caldesmon have been shown in skeletal muscle fibers to selectively displace low affinity, weakly bound cross-bridges from actin without significantly altering the actin attachment of force producing, strong binding cross-bridges (Brenner et al., 1991; Kraft et al., 1995a). However, the sarcomeric distribution and the specific binding of externally added caldesmon to the myofilaments of skeletal muscle fibers was not known. It was e.g., unclear whether caldesmon binds along actin in a manner similar to tropomyosin or whether it also binds to myosin. In this study, we determined the binding pattern of exogenously added intact caldesmon and its C-terminal 20-kDa fragment, respectively, in MgATP-relaxed rabbit skeletal muscle fibers using electron (EM) and confocal fluorescence microscopy (CFM). EM showed that similar to what has been demonstrated earlier for smooth muscle thin filaments (Lehman et al., 1989), intact caldesmon binds periodically every 38nm along the thin filaments. CFM revealed that rhodamine-labeled intact caldesmon and the 20-kDa caldesmon fragment bind along nearly the entire length of the thin filaments. A portion of the I-band near the Z-line appears unlabeled, both when equilibrated at normal and long sarcomere lengths. The width of the unlabeled region seems to depend on ionic strength. The 20-kDa C-terminal caldesmon fragment binds in essentially the same pattern as intact caldesmon. This indicates that the high fluorescence intensity in the overlap region seen with intact caldesmon does not depend on caldesmon binding to myosin. X-ray diffraction was used to monitor the effects on filament lattice. Intact caldesmon at >0.3mg/ml induced disorder in the myofilament lattice. No such disordering was observed, however, when fibers were equilibrated with up to 0.8mg/ml of the 20-kDa caldesmon fragment.

Fesselin or avian synaptopodin 2 stimulates actin polymerization in a Ca2+-calmodulin dependent manner. Fesselin binding to F-actin inhibits myosin S1 binding and yet fesselin binds with moderate affinity to smooth muscle myosin. These properties suggest that fesselin could tether actin and myosin together in an inactive complex as caldesmon does. This possibility was tested by observing the effect of fesselin on the rate of dissociation of actin-myosin by ATP in a stopped-flow device. Dissociation was measured by light scattering (a measure of particle size) and by pyrene actin fluorescence (a specific measure of actin-myosin binding). Fesselin reduced the multi-exponential rates of change of light scattering and pyrene fluorescence in concentration dependent manners. Each light scattering trace had a rapid initial transition that was not present in pyrene fluorescence traces. That rapid light scattering change was likely due to dissociation of filamentous myosin. The reduction in rate of that rapid process could mean that fesselin alters the structure and dissociation kinetics of smooth myosin filaments. We examined changes in smooth muscle myosin with electron microscopy. ATP caused dissociation of myosin filaments in both the presence and absence of fesselin. In the absence of ATP, fesselin increased the size of myosin filaments. Furthermore, the filaments were arranged in parallel arrays. These results indicate that fesselin cross-links myosin to actin and also organizes myosin filaments in solution.

The slowly hydrolyzed ATP analog adenosine 5'-(gamma-thiotriphosphate) (ATP gamma S) has been used in many studies of the muscle motor protein myosin in order to form a stable "weak binding" state analogous to the actin-S1-ATP complex However, the results from studies using ATP gamma S do not always agree with the results of experiments using ATP. The binding of myosin subfragment-1-ATP gamma S to actin has now been studied in some detail to determine its relationship to the actin-S1-ATP state. The binding of myosin subfragment-1-ATP gamma S to actin-troponin-tropomyosin is similar in affinity to the binding of myosin subfragment-1-ATP. Like myosin subfragment-1-ATP, the binding is not Ca(2+)-dependent, and most importantly, myosin subfragment-1-ATP gamma S does not stabilize the active configuration of actin-troponin-tropomyosin. Thus, myosin subfragment-1-ATP gamma S is an analog of myosin subfragment-1-ATP but must be used with caution for two reasons: (1) The binding of ATP gamma S to regulated actomyosin subfragment-1 is Ca(2+)-sensitive, and errors can be made in the interpretation of results if proteins are not fully saturated with nucleotide and a mixture of weak and strong binding states is present. (2) At the high concentrations of myosin subfragment-1 used in some experiments, significant amounts of ADP may form.(ABSTRACT TRUNCATED AT 250 WORDS)

Calponin, a protein isolated from smooth muscle and nonmuscle cells, has previously been shown to inhibit the actin-activated ATPase activity of myosin. Reports of the stoichiometry of binding range from 1 calponin per actin to 1 calponin per 3 actin monomers. We now report a detailed study of the binding of [14C]iodoacetamide-labeled calponin to actin. The labeling procedure did not significantly alter the binding constant of calponin to actin. The stoichiometry of binding was variable and dependent on ionic strength. Below 110 mM ionic strength, the stoichiometry of binding was 1:1. As the ionic strength was increased above 110 mM ionic strength, the stoichiometry shifted from 1:1 to 1 calponin per 2 actin monomers. At physiological ionic strength, the binding exhibited a small degree of positive cooperativity and was adequately described by a single class of binding sites with an association constant of 6 x 10(6) M-1. The affinity decreased to 20% of this value in the presence of ATP. Irrespective of the ionic strength, actin formed bundles when saturation with calponin exceeded about 30%. Measurements of the rate of association were complicated by this bundling, but the upper limit for this reaction was placed at 10(6) M-1 s-1. The addition of calponin to actin-caldesmon complexes caused displacement of the caldesmon.

Previously we showed that stiffness of relaxed fibers and active force generated in single skinned fibers of rabbit psoas muscle are inhibited in parallel by actin-binding fragments of caldesmon, an actin-associated protein of smooth muscle, under conditions in which a large fraction of cross-bridges is weakly attached to actin (ionic strength of 50 mM and temperature of 5 degrees C). These results suggested that weak cross-bridge attachment to actin is essential for force generation. The present study provides evidence that this is also true for physiological ionic strength (170 mM) at temperatures up to 30 degrees C, suggesting that weak cross-bridge binding to actin is generally required for force generation. In addition, we show that the inhibition of active force is not a result of changes in cross-bridge cycling kinetics but apparently results from selective inhibition of weak cross-bridge binding to actin. Together with our previous biochemical, mechanical, and structural studies, these findings support the proposal that weak cross-bridge attachment to actin is an essential intermediate on the path to force generation and are consistent with the concept that isometric force mainly results from an increase in strain of the attached cross-bridge as a result of a structural change associated with the transition from a weakly bound to a strongly bound actomyosin complex. This mechanism is different from the processes responsible for quick tension recovery that were proposed by Huxley and Simmons (Proposed mechanism of force generation in striated muscle. Nature. 233:533-538.) to represent the elementary mechanism of force generation.

We have reported earlier that ATP causes both an increase in the affinity of caldesmon for smooth muscle myosin and a change in stoichiometry from 2 caldesmon molecules per myosin to 1:1 (Hemric & Chalovich, 1990). We now show that this ATP effect does not occur with skeletal muscle myosin, indicating that ATP has a specific effect on the structure of filamentous smooth muscle myosin. This ATP effect does not appear to be due to stabilization of a 10S type of filamentous smooth muscle myosin like that reported earlier (Ikebe & Hartshorne, 1984) since neither phosphorylation nor extensive modification of myosin with MalNEt (both which stabilize the 6S state of monomeric myosin) eliminates the effect of ATP. Caldesmon does bind more tightly to a form of smooth muscle myosin which is resistant to papain digestion. These results suggest that the ATP effect is due to stabilization of a local conformation of smooth muscle myosin which is independent of the larger 10S/6S conformational change (Suzuki et al., 1988). In the presence of ATP, the two heads of smooth muscle muscle myosin and the S-2 region form a single, higher affinity binding region for caldesmon.

A multi-site, steady-state Förster resonance energy transfer (FRET) approach was used to quantify Ca(2+)-induced changes in proximity between donor loci on human cardiac troponin I (cTnI), and acceptor loci on human cardiac tropomyosin (cTm) and F-actin within functional thin filaments. A fluorescent donor probe was introduced to unique and key cysteine residues on the C- and N-termini of cTnI. A FRET acceptor probe was introduced to one of three sites located on the inner or outer domain of F-actin, namely Cys-374 and the phalloidin-binding site on F-actin, and Cys-190 of cTm. Unlike earlier FRET analyses of protein dynamics within the thin filament, this study considered the effects of non-random distribution of dipoles for the donor and acceptor probes. The major conclusion drawn from this study is that Ca(2+) and myosin S1-binding to the thin filament results in movement of the C-terminal domain of cTnI from the outer domain of F-actin towards the inner domain, which is associated with the myosin-binding. A hinge-linkage model is used to best-describe the finding of a Ca(2+)-induced movement of the C-terminus of cTnI with a stationary N-terminus. This dynamic model of the activation of the thin filament is discussed in the context of other structural and biochemical studies on normal and mutant cTnI found in hypertrophic cardiomyopathies.

The regulation of muscle contraction is a complex process that involves changes in both the organization of the troponin subunits and the orientation of tropomyosin on actin. The changes in tropomyosin may alter the manner in which myosin binds to actin, but, in our view, the more important change is an allosteric alteration of the ability of actin to participate in the catalysis of ATP hydrolysis. Because the ATPase activity of the system is closely coupled to muscle contraction, we have used the prediction of ATPase activity as our guide to successful modeling.

Actin-based regulation of striated mammalian muscle contraction is dependent on the position of tropomyosin on actin. Troponin stabilizes tropomyosin in the inactive state at low free Ca2+ concentrations. There is little or no activation of the ATPase activity of myosin S1 when actin filaments are in that state. Calcium binding to troponin releases tropomyosin from the inhibitory position so that it rapidly samples the active state (greatest activation of ATPase activity), the intermediate state and the inactive state. The intermediate state allows modest stimulation of myosin ATPase activity and is the most highly populated state at this condition. Binding of rigor myosin S1 to actin stabilizes the active state (1), (2) where tropomyosin occupies a unique position on actin (3). The inhibitory component of troponin, TnI, competes for binding with myosin-ATP in the absence of tropomyosin. However, that competition of binding is largely eliminated in the presence of tropomyosin (4). The actin-troponin-tropomyosin complex is stable; troponin is not displaced even by the high affinity binding of myosin in the presence of ADP. Smooth muscle also contains an actin-linked regulatory system (5) consisting of tropomyosin and the actin binding protein caldesmon (6). Smooth muscle tropomyosin differs from skeletal tropomyosin in several ways. Skeletal tropomyosin inhibits actin activation of myosin S1-ATPase activity over a wide range of conditions. Smooth muscle tropomyosin produces a steep increase in ATPase rate with increasing ionic strength with a crossover point from inhibition to activation near 0.05 M ionic strength (7). The head to tail interactions of smooth tropomyosin are stronger than those of the skeletal variety (8). There is a greater degree of stabilization of the active state of actin-tropomyosin per myosin S1 bound to actin for the smooth muscle variety of tropomyosin (9). Skeletal muscle tropomyosin is a mixture of αα homodimers and αβ heterodimers; smooth muscle tropomyosin appears to be 100% αβ heterodimer (10). Caldesmon is an actin binding protein (6) that appears to participate in regulation of non-muscle and smooth muscle contraction (11), (12). Caldesmon inhibits both actin activation of the rate of ATP hydrolysis by myosin and also myosin S1 binding to actin. Tropomyosin enhances the ability of caldesmon to inhibit actin activated ATPase activity (13), (14), (15). Caldesmon differs from troponin in that it remains competitive with S1-ATP binding in the presence of tropomyosin (16), (17), (18). Our data suggest that this inhibition of binding is biologically relevant (16), (17), (18), (19) and proportional to the extent of inhibition of ATPase activity (20). Other studies suggest that the inhibition of S1-ATP binding occurs at higher concentrations of caldesmon than necessary to inhibit the rate of actin-activated ATP hydrolysis (21). Because tropomyosin is a component of actin filaments of both smooth and striated muscle it is interesting to know the extent to which the inhibitory activity of caldesmon can be attributed to movement of tropomyosin on actin. Pyrene labeled smooth muscle tropomyosin bound to actin undergoes a change in fluorescence in the presence of caldesmon that has been attributed to movement of tropomyosin into an inhibitory state (22). However, image reconstructions of actin filaments containing smooth muscle tropomyosin and an actin binding caldesmon fragment show that tropomyosin does not occupy the same inhibitory position that is stabilized by troponin in the absence of calcium (23). We reexamined the question of tropomyosin movement using an acrylodan probe on smooth muscle tropomyosin that has certain advantages over pyrene tropomyosin for measuring transitions of actin-tropomyosin-troponin (24). Skeletal muscle troponin stabilizes the inactive state of skeletal tropomyosin-actin in the absence of calcium. We found that skeletal muscle troponin had a similar effect with smooth muscle acrylodan-tropomyosin bound to actin. The transition from the active state to the intermediate state occurred with a very rapid decrease in fluorescence followed by a slower increase in fluorescence as the inactive state became populated. A similar pattern was observed with caldesmon except that the fluorescence increase was faster than with troponin and of smaller amplitude. The fluorescence increase was correlated with caldesmon binding to actin-tropomyosin following S1-ATP detachment. With both troponin and caldesmon the slow increase in acrylodan-tropomyosin fluorescence appeared to reflect the rate constant for transition to the inactive state. In the case of troponin the change in acrylodan-tropomyosin fluorescence was attributed to movement of tropomyosin. In the case of caldesmon, the change in fluorescence was due either to caldesmon binding or to some subsequent process. Other results confirm the importance of competition of binding between caldesmon and myosin S1. We previously used the notation 1(0) for the inactive state, 1(2) for the intermediate state and 2(0), 2(1) or 2(2) for the active state of regulated actin (25). The first digit indicates the activity: 1 for low activity and 2 for high activity. The second digit, shown here in parentheses, is the number of bound calcium ions per troponin. The more recent nomenclature, blocked (inactive), closed (intermediate) and open (active), assumes a model of regulation that involves blocking myosin binding sites on actin (26). Because of different views of regulation of striated muscle (27) and uncertainties in regulation of smooth muscle addressed in the present paper we use the model-independent descriptors based on the measured levels of ATPase activity: inactive, intermediate and active.

We examined the cardiomyopathy-causing tropomyosin mutations E180G, D175N, and V95A to determine their effects on actomyosin regulation. V95A reduced the ATPase rate when filaments were saturated with regulatory proteins both in the presence and absence of calcium, indicating either a stabilization of the inactive state or an inability to fully populate the active state. Effects of E180G and D175N were more complex. These two mutations increased ATPase rates at sub-saturating concentrations of troponin and tropomyosin as compared to wild type tropomyosin. At higher concentrations of regulatory proteins, ATPase rates became similar to wild type. Normal activation was achieved with the tight-binding myosin analog N-ethylmaleimide-S1, at saturating regulatory protein concentrations. These results suggest that the E180G and D175N mutations reduce the affinity of tropomyosin for actin and also destabilize troponin binding to the actin thin filaments.

Caldesmon, an actin-binding protein from smooth muscle and non-muscle cells, has previously been shown to bind stoichiometrically to smooth muscle myosin in an ATP-dependent manner. We now show quantitatively the effects of Ca(2+)-calmodulin and phosphorylation on the binding of caldesmon to myosin. Ca(2+)-calmodulin reduces the binding of caldesmon to myosin with the same effectiveness as it does the binding of caldesmon to actin. However, Ca(2+)-calmodulin is ineffective in antagonizing the binding of the purified myosin-binding region of caldesmon to myosin. These and other results suggest that Ca(2+)-calmodulin binding to the COOH-terminal region of caldesmon is responsible for reversal of binding to myosin. Phosphorylation of the NH2-terminal region of caldesmon by the co-purifying kinase, calmodulin-dependent protein kinase II, weakens but does not eliminate the binding of caldesmon to smooth muscle myosin. Finally, phosphorylation of smooth muscle myosin by smooth muscle myosin light chain kinase has no effect on the binding of caldesmon to myosin. Since Ca(2+)-calmodulin and phosphorylation of caldesmon weaken the binding of caldesmon to both actin and myosin, these events may be coordinately regulated.

Caldesmon is known to inhibit actin-activated myosin ATPase activity in solution, to inhibit force production when added to skeletal muscle fibers, and to alter actin movement in the in vitro cell motility assay. It is less clear that caldesmon can inhibit contraction in smooth muscle cells in which caldesmon is abundant. We now show that caldesmon and its 20-kDa actin-binding fragment are able to inhibit force in chemically skinned gizzard fiber bundles, which are activated by a constitutively active myosin light-chain kinase in the presence and absence of okadaic acid. This inhibitory effect is reversed by high concentrations of Ca2+ and calmodulin. Therefore, caldesmon may act by increasing the level of myosin phosphorylation required to obtain full activation. Our results also suggest that caldesmon does not act to maintain force in smooth muscle by cross-linking myosin with actin since competition of binding of caldesmon with myosin does not cause a reduction in tension.

Previous immunological studies have indicated that the molecular structure of hamster relaxin is quite different from that of porcine relaxin. In the present study, hamster relaxin was purified from placentas and characterized in order to investigate its biochemical properties. Placentas from Days 14 and 15 of gestation were homogenized in 0.26 N HCl-62.5% acetone containing protease inhibitors. After centrifugation, soluble proteins were acetone precipitated. Soluble proteins were applied to a carboxymethyl cellulose ion-exchange column and bound proteins were eluted with 0.1 and 0.3 M NaCl. Western blot analysis detected 16.5-, 18.7-, and 36.0-kDa relaxin-immunoreactive (IR) proteins within the 0.1 M NaCl eluant and detected a 5.6-kDa relaxin-IR protein within the 0.3 M NaCl eluant. The 5.6-kDa protein was purified to homogeneity by gel filtration (Sephadex G-50), ion-exchange HPLC, and C18 -HPLC. Reduction of the 5.6-kDa protein prior to electrophoresis resulted in a single band of lower molecular mass, suggesting that hamster relaxin consists of two chains of approximately equal molecular mass. Isoelectric point of the 5.6-kDa protein was 7.78. The 16.5- and 18.7-kDa IR proteins were copurified by gel filtration and ion-exchange HPLC. At least five isoelectric point variants were observed for the 16.5- and 18.7-kDa proteins. The N-terminal amino acid for the 5.6 and 18.7 relaxin-IR proteins was arginine, and subsequent cycles indicated an identical partial sequence that was consistent with that for relaxins from other species.

The synaptopodin family of proteins consists of at least 3 members: synaptopodin, the synaptopodin 2 proteins, and the synaptopodin 2-like proteins. Each family member has at least 3 isoforms that are produced by alternative splicing. Synaptopodin family members are basic proteins that are rich in proline and have little regular 2° or 3° structure at physiological temperature, pH and ionic strength. Like other natively unfolded proteins, synaptopodin family members have multiple binding partners including actin and other actin-binding proteins. Several members of the synaptopodin family have been shown to stimulate actin polymerization and to bundle actin filaments either on their own or in collaboration with other proteins. Synaptopodin 2 has been shown to accelerate nucleation of actin filament formation and to induce actin bundling. The actin polymerization activity is inhibited by Ca2+-calmodulin. Synaptopodin 2 proteins are localized in Z-bands of striated and heart muscle and dense bodies of smooth muscle cells. Depending on the developmental status and stress, at least one member of the synaptopodin family can occupy nuclei of some cells. Members of the synaptopodin 2 subfamily have been implicated in cancers.

Mutations of any subunit of the troponin complex may lead to serious disorders. Rational approaches to managing these disorders require knowledge of the complex interactions among the three subunits that are required for proper function. Molecular dynamics (MD) simulations were performed for both skeletal (sTn) and cardiac (cTn) troponin. The interactions and correlated motions among the three components of the troponin complex were analyzed using both Molecular Mechanics-Generalized Born Surface Area (MMGBSA) and cross-correlation techniques. The TnTH2 helix was strongly positively correlated with the two long helices of TnI. The C domain of TnC was positively correlated with TnI and TnT. The N domain of TnC was negatively correlated with TnI and TnT in cTn, but not in sTn. The two C-domain calcium-binding sites of TnC were dynamically correlated. The two regulatory N-domain calcium-binding sites of TnC were dynamically correlated, even though the calcium-binding site I is dysfunctional. The strong interaction residue pairs and the strong dynamically correlated residues pairs among the three components of troponin complexes were identified. These correlated motions are consistent with the idea that there is a high degree of cooperativity among the components of the regulatory complex in response to Ca(2+) and other effectors. This approach may give insight into the mechanism by which mutations of troponin cause disease. It is interesting that some observed disease causing mutations fall within regions of troponin that are strongly correlated or interacted.

Caldesmon is an actin- and myosin-binding protein that is rich in smooth muscle. Caldesmon inhibits the actin activation of myosin catalyzed ATPase activity and may have additional functions in smooth muscle. The activity of caldesmon is controlled by phosphorylation and by binding to other factors such as Ca++-calmodulin. Caldesmon is a substrate for p21-activated kinase, PAK, which is reported to phosphorylate chicken gizzard caldesmon at two sites, Ser672 and Ser702. We investigated PAK phosphorylation of caldesmon using a 22kDa C-terminal caldesmon fragment. We also substituted Ser672 and Ser702 with either alanine or aspartic acid residues to mimic non-phosphorylated and constitutively phosphorylated states of caldesmon, respectively. We found that the aspartic acid mutation of caldesmon weakened calmodulin binding but had no effect on the inhibitory activity of caldesmon. Phosphorylation of the aspartic acid double mutant with recombinant PAK resulted in additional phosphorylation at Thr627, Ser631, Ser635 and Ser642. Phosphorylation at these sites by PAK was slow, but produced further weakening of calmodulin binding and reduced the inhibitory activity of caldesmon in the absence of calmodulin. Phosphorylation at the additional sites was without effect on Ca++-Calmodulin binding if Ser672 and Ser702 were not phosphorylated, but was sufficient to release inhibition of actomyosin ATPase activity. This work raises the possibility that phosphorylation in the region of residues 627-642 significantly alters the activity of caldesmon.

The synaptopodin 2 gene can be differently spliced, resulting in three mRNAs with varying 3′ ends coding for different C-termini (De Ganck et al 2008). The calculated molecular weights of these proteins are 117, 119, and 136 kDa. These isoforms require the expression of exons 1–3. However, only a single protein product has been detected in mammalian muscle lacking the product of these first exons.We extracted four fesselin isoforms from avian smooth muscle tissue. These include the first isolated 79 and 103 kDa isoforms (Leinweber et al. 1999). The newly detected isoforms migrated on SDS gels with apparent molecular masses of 140 and 160 kDa. In contrast to our initial assumption that the 79 kDa was a proteolytic product of the 103 kDa protein we now show that they are different spliceforms. The 79 kDa isoform forms the core of synaptopodin 2. The other isoforms have different extensions at either the N- or C-terminal regions.The different isoforms were differentially extracted by different buffers. Extractions were most complete under conditions that depolymerized both actin and intermediate filaments. Surprisingly although fesselin binds to actin and myosin none of the four different isoforms was extracted with the acto-myosin-complex. The extraction data suggest that fesselin functions in actin filament organization rather than in regulation of actin-myosin interactions. In contrast to smooth muscle tissue we detected one isoform of fesselin in skeletal and heart muscle tissue in agreement with the findings in mammalian tissue. In avian skeletal muscle we observed a 79 kDa isoform and in heart muscle a 170 kDa isoform. The reason for the differential expression of fesselin in different muscle types is unknown.

Caldesmon interacts with the NH2-terminal region of actin. It is now shown in airfuge centrifugation experiments that modification of the penultimate cysteine residue of actin significantly weakens its binding to caldesmon both in the presence and absence of tropomyosin. Furthermore, as revealed by fluorescence measurements, caldesmon increases the exposure of the COOH-terminal region of actin to the solvent. This effect of caldesmon, like its inhibitory effect on actomyosin ATPase activity, is enhanced in the presence of tropomyosin. Proteolytic removal of the last three COOH-terminal residues of actin, containing the modified cysteine residue, restores the normal binding between caldesmon and actin. These results establish a correlation between the binding of caldesmon to actin and the conformation of the COOH-terminal region of actin and suggest an indirect rather than direct interaction between caldesmon and this part of actin.

This report compares cellular localization of fesselin in chicken smooth, skeletal and cardiac muscle tissues using affinity purified polyclonal fesselin antibodies. Western blot analyses revealed large amounts of fesselin in gizzard smooth muscle with lower amounts in skeletal and cardiac muscle. In gizzard, fesselin was detected by immunofluorescence as discrete cytoplasmic structures. Fesselin did not co-localize with talin, vinculin or caveolin indicating that fesselin is not associated with dense plaques or caveolar regions of the cell membrane. Immunoelectron microscopy established localization of fesselin within dense bodies. Since dense bodies function as anchorage points for actin and desmin in smooth muscle cells, fesselin may be involved in establishing cytoskeletal structure in this tissue. In skeletal muscle, fesselin was associated with desmin in regularly spaced bands distributed along the length of muscle fibers suggesting localization to the Z-line. Infrequently, this banding pattern was observed in heart tissue as well. Localization at the Z-line of skeletal and cardiac muscle suggests a role in contraction of these tissues.

The myosin 2 family of molecular motors includes isoforms regulated in different ways. Vertebrate smooth-muscle myosin is activated by phosphorylation of the regulatory light chain, whereas scallop striated adductor-muscle myosin is activated by direct calcium binding to its essential light chain. The paired heads of inhibited molecules from myosins regulated by phosphorylation have an asymmetric arrangement with motor–motor interactions. It was unknown whether such interactions were a common motif for inactivation used in other forms of myosin-linked regulation. Using electron microscopy and single-particle image processing, we show that indistinguishable structures are indeed found in myosins and heavy meromyosins isolated from scallop striated adductor muscle and turkey gizzard smooth muscle. The similarities extend beyond the shapes of the heads and interactions between them: In both myosins, the tail folds into three segments, apparently at identical sites; all three segments are in close association outside the head region; and two segments are associated in the same way with one head in the asymmetric arrangement. Thus, these organisms, which have different regulatory mechanisms and diverged from a common ancestor >600 Myr ago, have the same quaternary structure. Conservation across such a large evolutionary distance suggests that this conformation is of fundamental functional importance.

We have proposed earlier that caldesmon inhibits the actin-activated ATPase activity of smooth muscle heavy meromyosin (HMM) by inhibiting the binding of the HMM.ATP complex to the productive site of actin (Hemric, M. E., and Chalovich, J. M. (1988) J. Biol. Chem. 263, 1868-1885). This has been difficult to prove directly because caldesmon also binds to HMM and it is difficult to distinguish the actin-caldesmon-HMM complex from the actin-caldesmon complex in binding studies. We have eliminated the interaction between caldesmon and smooth HMM by digestion of caldesmon with chymotrypsin. This cleaved caldesmon inhibits the actin-activated ATPase rate of smooth HMM and this inhibition is correlated with a decrease in the binding of HMM.ATP to actin. Therefore, caldesmon functions by inhibiting the binding of the myosin-ATP complex to actin regardless of the source of myosin. We have also isolated the myosin-binding region of caldesmon and have performed a partial sequence. Comparison of this sequence with the derived sequence of caldesmon demonstrates, unequivocally, that the myosin-binding region of caldesmon begins at the amino terminus and extends beyond the first Cys residue.

Fesselin is a proline-rich actin-binding protein that was isolated from avian smooth muscle. Fesselin bundles actin and accelerates actin polymerization by facilitating nucleation. We now show that this polymerization of actin can be regulated by Ca(2+)-calmodulin. Fesselin was shown to bind to immobilized calmodulin in the presence of Ca(2+). The fesselin-calmodulin interaction was confirmed by a Ca(2+)-dependent increase in 2-(4-maleimidoanilino)naphthalene-6-sulfonic acid (MIANS) fluorescence upon addition of fesselin to MIANS-labeled wheat germ calmodulin. The affinity was estimated to be approximately 10(9) M(-1). The affinity of Ca(2+)-calmodulin to the fesselin F-actin complex was approximately 10(8) M(-1). Calmodulin binding to fesselin appeared to be functionally significant. In the presence of fesselin and calmodulin, the polymerization of actin was Ca(2+)-dependent. Ca(2+)-free calmodulin either had no effect or enhanced the ability of fesselin to accelerate actin polymerization. Ca(2+)-calmodulin not only reversed the stimulatory effect of fesselin but reduced the rate of polymerization below that observed in the absence of fesselin. While Ca(2+)-calmodulin had a large effect on the interaction of fesselin with G-actin, the effect on F-actin was small. Neither the binding of fesselin to F-actin nor the subsequent bundling of F-actin was greatly affected by Ca(2+)-calmodulin. Fesselin may function as an actin-polymerizing factor that is regulated by Ca(2+) levels.

The p21-activated protein kinases (PAKs) have been implicated in cytoskeletal rearrangements and modulation of non-muscle contractility. Little, however, is known about the role of the PAK family members in smooth muscle contraction. Therefore, we investigated the effect of the predominant isoform in vascular smooth muscle cells, PAK1, on contraction and phosphorylation of the regulatory light chains of myosin (r-MLC) in Triton-skinned guinea-pig smooth muscle. We also investigated which of the three putative substrates at the contractile apparatus - MLCK, caldesmon or r-MLC - is phosphorylated by PAK1 in smooth muscle tissue. Incubation of Triton-skinned carotid artery and taenia coli from guinea-pig with an active mutant of PAK1 in relaxing solution for 30-60 min resulted in inhibition of submaximal force by about 50 %. The mechanism of inhibition of force was studied in the Triton-skinned taenia coli. In this preparation, inhibition of force was associated with a respective inhibition of r-MLC phosphorylation. In the presence of the myosin phosphatase inhibitor, microcystin-LR (10 microM), the rate of contraction and r-MLC phosphorylation elicited at pCa 6.79 were both decreased. Because under these conditions the rate of r-MLC phosphorylation is solely dependent on MLCK activity, this result suggests that the inhibitory effect of PAK1 on steady-state force and r-MLC phosphorylation is due to inhibition of MLCK. In line with this, we found that MLCK was significantly phosphorylated by PAK1 while there was very little 32P incorporation into caldesmon. PAK1 phosphorylated isolated r-MLC but not those in the skinned fibres or in purified smooth muscle myosin II. In conclusion, these results suggest that PAK1 attenuates contraction of skinned smooth muscle by phosphorylating and inhibiting MLCK.

The interaction of myosin subfragment 1 (S1) with actin-tropomyosin-troponin (regulated actin) is highly nucleotide dependent. The binding of S1 or S1-ADP (but not S1-ATP nor N,N'-rho-phenylenedimaleimide-modified S1-ATP) to regulated actin activates ATP hydrolysis even in the absence of Ca(2+). Investigations with S1 and S1-ADP have led to the idea that some actin sites are directly blocked toward the binding of S1 either by tropomyosin or troponin. The blocked state is thought to occur only at ionic strengths greater than 50 mM. The question is whether nonactivating S1 binding is blocked under the same conditions. We show that troponin inhibits binding of the nonactivating state, N,N'-rho-phenylenedimaleimide-S1-ATP, to actin but only when tropomyosin is absent. A lag in the rate of binding of activating S1 to actin (an indicator of the blocked state) occurs only in the presence of tropomyosin. Thus, tropomyosin inhibits binding of rigor S1 but not S1-ATP-like states. No evidence for an ionic strength-dependent change in the mechanism of regulation was observed either from measurements of the rate of activating S1 binding or from the equilibrium binding of nonactivating S1 to actin. At all conditions examined, N,N'-rho-phenylenedimaleimide-S1-ATP bound to regulated actin in the absence of Ca(2+). These results support the view of regulation in which tropomyosin movement is an allosteric switch that is modulated by activating myosin binding but that does not function solely by regulating myosin binding.