Tether-scanning the kinesin motor domain reveals a core mechanical action.

Natural kinesin motors are tethered to their cargoes via short C-terminal or N-terminal linkers, whose docking against the core motor domain generates directional force. It remains unclear whether linker docking is the only process contributing directional force or whether linker docking is coupled to and amplifies an underlying, more fundamental force-generating mechanical cycle of the kinesin motor domain. Here, we show that kinesin motor domains tethered via double-stranded DNAs (dsDNAs) attached to surface loops drive robust microtubule (MT) gliding. Tethering using dsDNA attached to surface loops disconnects the C-terminal neck-linker and the N-terminal cover strand so that their dock-undock cycle cannot exert force. The most effective attachment positions for the dsDNA tether are loop 2 or loop 10, which lie closest to the MT plus and minus ends, respectively. In three cases, we observed minus-end-directed motility. Our findings demonstrate an underlying, potentially ancient, force-generating core mechanical action of the kinesin motor domain, which drives, and is amplified by, linker docking.

Cost-Efficient Expression of Human Cardiac Myosin Heavy Chain in C2C12 Cells with a Non-Viral Transfection Reagent.

Production of functional myosin heavy chain (MHC) of striated muscle myosin II for studies of isolated proteins requires mature muscle (e.g., C2C12) cells for expression. This is important both for fundamental studies of molecular mechanisms and for investigations of deleterious diseases like cardiomyopathies due to mutations in the MHC gene (MYH7). Generally, an adenovirus vector is used for transfection, but recently we demonstrated transfection by a non-viral polymer reagent, JetPrime. Due to the rather high costs of JetPrime and for the sustainability of the virus-free expression method, access to more than one transfection reagent is important. Here, we therefore evaluate such a candidate substance, GenJet. Using the human cardiac ß-myosin heavy chain (ß-MHC) as a model system, we found effective transfection of C2C12 cells showing a transfection efficiency nearly as good as with the JetPrime reagent. This was achieved following a protocol developed for JetPrime because a manufacturer-recommended application protocol for GenJet to transfect cells in suspension did not perform well. We demonstrate, using in vitro motility assays and single-molecule ATP turnover assays, that the protein expressed and purified from cells transfected with the GenJet reagent is functional. The purification yields reached were slightly lower than in JetPrime-based purifications, but they were achieved at a significantly lower cost. Our results demonstrate the sustainability of the virus-free method by showing that more than one polymer-based transfection reagent can generate useful amounts of active MHC. Particularly, we suggest that GenJet, due to its current ~4-fold lower cost, is useful for applications requiring larger amounts of a given MHC variant.

N-Terminal Fragment of Cardiac Myosin Binding Protein C Modulates Cooperative Mechanisms of Thin Filament Activation in Atria and Ventricles.

Cardiac myosin binding protein C (cMyBP-C) is one of the essential control components of the myosin cross-bridge cycle. The C-terminal part of cMyBP-C is located on the surface of the thick filament, and its N-terminal part interacts with actin, myosin, and tropomyosin, affecting both kinetics of the ATP hydrolysis cycle and lifetime of the cross-bridge, as well as calcium regulation of the actin-myosin interaction, thereby modulating contractile function of myocardium. The role of cMyBP-C in atrial contraction has not been practically studied. We examined effect of the N-terminal C0-C1-m-C2 (C0-C2) fragment of cMyBP-C on actin-myosin interaction using ventricular and atrial myosin in an in vitro motility assay. The C0-C2 fragment of cMyBP-C significantly reduced the maximum sliding velocity of thin filaments on both myosin isoforms and increased the calcium sensitivity of the actin-myosin interaction. The C0-C2 fragment had different effects on the kinetics of ATP and ADP exchange, increasing the affinity of ventricular myosin for ADP and decreasing the affinity of atrial myosin. The effect of the C0-C2 fragment on the activation of the thin filament depended on the myosin isoforms. Atrial myosin activates the thin filament less than ventricular myosin, and the C0-C2 fragment makes these differences in the myosin isoforms more pronounced.

N-Terminal Fragment of Cardiac Myosin Binding Protein-C Increases the Duration of Actin-Myosin Interaction.

Cardiac myosin binding protein-C (cMyBP-C) located in the C-zone of myocyte sarcomere is involved in the regulation of myocardial contraction. Its N-terminal domains C0, C1, C2, and the m-motif between C1 and C2 can bind to the myosin head and actin of the thin filament and affect the characteristics of their interaction. Measurements using an optical trap showed that the C0-C2 fragment of cMyBP-C increases the interaction time of cardiac myosin with the actin filament, while in an in vitro motility assay, it dose-dependently reduces the sliding velocity of actin filaments. Thus, it was found that the N-terminal part of cMyBP-C affects the kinetics of the myosin cross-bridge.

Myopathy-causing mutation R91P in the TPM3 gene drastically impairs structural and functional properties of slow skeletal muscle tropomyosin γß-heterodimer.

Tropomyosin (Tpm) is a regulatory actin-binding protein involved in Ca2+ activation of contraction of striated muscle. In human slow skeletal muscles, two distinct Tpm isoforms, γ and ß, are present. They interact to form three types of dimeric Tpm molecules: γγ-homodimers, γß-heterodimers, or ßß-homodimers, and a majority of the molecules are present as γß-Tpm heterodimers. Point mutation R91P within the TPM3 gene encoding γ-Tpm is linked to the condition known as congenital fiber-type disproportion (CFTD), which is characterized by severe muscle weakness. Here, we investigated the influence of the R91P mutation in the γ-chain on the properties of the γß-Tpm heterodimer. We found that the R91P mutation impairs the functional properties of γß-Tpm heterodimer more severely than those of earlier studied γγ-Tpm homodimer carrying this mutation in both γ-chains. Since a significant part of Tpm molecules in slow skeletal muscle is present as γß-heterodimers, our results explain why this mutation leads to muscle weakness in CFTD.

Direct observation of oriented behavior of actin filaments interacting with desmin intermediate filaments.

BACKGROUND: Associations between actin filaments (AFs) and intermediate filaments (IFs) are frequently observed in living cells. The crosstalk between these cytoskeletal components underpins cellular organization and dynamics; however, the molecular basis of filamentous interactions is not fully understood. Here, we describe the mode of interaction between AFs and desmin IFs (DIFs) in a reconstituted in vitro system. METHODS: AFs (rabbit skeletal muscle) and DIFs (chicken gizzard) were labeled with fluorescent dyes. DIFs were immobilized on a heavy meromyosin (HMM)-coated collodion surface. HMM-driven AFs with ATP hydrolysis was assessed in the presence of DIFs. Images of single filaments were obtained using fluorescence microscopy. Vector changes in the trajectories of single AFs were calculated from microscopy images. RESULTS: AF speed transiently decreased upon contact with DIF. The difference between the incoming and outgoing angles of a moving AF broadened upon contact with a DIF. A smaller incoming angle tended to result in a smaller outgoing angle in a nematic manner. The percentage of moving AFs decreased with an increasing DIF density, but the speed of the moving AFs was similar to that in the no-desmin control. An abundance of DIFs tended to exclude AFs from the HMM-coated surfaces. CONCLUSIONS: DIFs agitate the movement of AFs with the orientation. DIFs can bind to HMMs and weaken actin-myosin interactions. GENERAL SIGNIFICANCE: The study indicates that apart from the binding strength, the accumulation of weak interactions characteristic of filamentous structures may affect the dynamic organization of cell architecture.

Novel Mutation Glu98Lys in Cardiac Tropomyosin Alters Its Structure and Impairs Myocardial Relaxation.

We characterized a novel genetic variant c.292G > A (p.E98K) in the TPM1 gene encoding cardiac tropomyosin 1.1 isoform (Tpm1.1), found in a proband with a phenotype of complex cardiomyopathy with conduction dysfunction and slow progressive neuromuscular involvement. To understand the molecular mechanism by which this mutation impairs cardiac function, we produced recombinant Tpm1.1 carrying an E98K substitution and studied how this substitution affects the structure of the Tpm1.1 molecule and its functional properties. The results showed that the E98K substitution in the N-terminal part of the Tpm molecule significantly destabilizes the C-terminal part of Tpm, thus indicating a long-distance destabilizing effect of the substitution on the Tpm coiled-coil structure. The E98K substitution did not noticeably affect Tpm's affinity for F-actin but significantly impaired Tpm's regulatory properties. It increased the Ca2+ sensitivity of the sliding velocity of regulated thin filaments over cardiac myosin in an in vitro motility assay and caused an incomplete block of the thin filament sliding at low Ca2+ concentrations. The incomplete motility block in the absence of Ca2+ can be explained by the loosening of the Tpm interaction with troponin I (TnI), thus increasing Tpm mobility on the surface of an actin filament that partially unlocks the myosin binding sites. This hypothesis is supported by the molecular dynamics (MD) simulation that showed that the E98 Tpm residue is involved in hydrogen bonding with the C-terminal part of TnI. Thus, the results allowed us to explain the mechanism by which the E98K Tpm mutation impairs sarcomeric function and myocardial relaxation.

Structural and Functional Properties of Kappa Tropomyosin.

In the myocardium, the TPM1 gene expresses two isoforms of tropomyosin (Tpm), alpha (αTpm; Tpm 1.1) and kappa (κTpm; Tpm 1.2). κTpm is the result of alternative splicing of the TPM1 gene. We studied the structural features of κTpm and its regulatory function in the atrial and ventricular myocardium using an in vitro motility assay. We tested the possibility of Tpm heterodimer formation from α- and κ-chains. Our result shows that the formation of ακTpm heterodimer is thermodynamically favorable, and in the myocardium, κTpm most likely exists as ακTpm heterodimer. Using circular dichroism, we compared the thermal unfolding of ααTpm, ακTpm, and κκTpm. κκTpm had the lowest stability, while the ακTpm was more stable than ααTpm. The differential scanning calorimetry results indicated that the thermal stability of the N-terminal part of κκTpm is much lower than that of ααTpm. The affinity of ααTpm and κκTpm to F-actin did not differ, and ακTpm interacted with F-actin significantly worse. The troponin T1 fragment enhanced the κκTpm and ακTpm affinity to F-actin. κκTpm differently affected the calcium regulation of the interaction of pig and rat ventricular myosin with the thin filament. With rat myosin, calcium sensitivity of thin filaments containing κκTpm was significantly lower than that with ααTpm and with pig myosin, and the sensitivity did not differ. Thin filaments containing κκTpm and ακTpm were better activated by pig atrial myosin than those containing ααTpm.

The effects of the tropomyosin cardiomyopathy mutations on the calcium regulation of actin-myosin interaction in the atrium and ventricle differ.

The molecular mechanisms of pathogenesis of atrial myopathy associated with hypertrophic (HCM) and dilated (DCM) mutations of sarcomeric proteins are still poorly understood. For this, one needs to investigate the effects of the mutations on actin-myosin interaction in the atria separately from ventricles. We compared the impact of the HCM and DCM mutations of tropomyosin (Tpm) on the calcium regulation of the thin filament interaction with atrial and ventricular myosin using an in vitro motility assay. We found that the mutations differently affect the calcium regulation of actin-myosin interaction in the atria and ventricles. The DCM E40K Tpm mutation significantly reduced the maximum sliding velocity of thin filaments with ventricular myosin and its Ca2+-sensitivity. With atrial myosin, its effects were less pronounced. The HCM I172T mutation reduced the Ca2+-sensitivity of the sliding velocity of filaments with ventricular myosin but increased it with the atrial one. The HCM L185R mutation did not affect actin-myosin interaction in the atria. The results indicate that the difference in the effects of Tpm mutations on the actin-myosin interaction in the atria and ventricles may be responsible for the difference in pathological changes in the atrial and ventricular myocardium.

De Novo Asp219Val Mutation in Cardiac Tropomyosin Associated with Hypertrophic Cardiomyopathy.

Hypertrophic cardiomyopathy (HCM), caused by mutations in thin filament proteins, manifests as moderate cardiac hypertrophy and is associated with sudden cardiac death (SCD). We identified a new de novo variant, c.656A>T (p.D219V), in the TPM1 gene encoding cardiac tropomyosin 1.1 (Tpm) in a young SCD victim with post-mortem-diagnosed HCM. We produced recombinant D219V Tpm1.1 and studied its structural and functional properties using various biochemical and biophysical methods. The D219V mutation did not affect the Tpm affinity for F-actin but increased the thermal stability of the Tpm molecule and Tpm-F-actin complex. The D219V mutation significantly increased the Ca2+ sensitivity of the sliding velocity of thin filaments over cardiac myosin in an in vitro motility assay and impaired the inhibition of the filament sliding at low Ca2+ concentration. The molecular dynamics (MD) simulation provided insight into a possible molecular mechanism of the effect of the mutation that is most likely a cause of the weakening of the Tpm interaction with actin in the "closed" state and so makes it an easier transition to the "open" state. The changes in the Ca2+ regulation of the actin-myosin interaction characteristic of genetic HCM suggest that the mutation is likely pathogenic.

Investigation of the molecular motor of muscle: from generating life in a test tube to myosin structure over beers.

This article traces 60 years of investigation of the molecular motor of skeletal muscle from the 1940s through the 1990s. It started with the discovery that myosin interaction with actin in the presence of ATP caused shortening of threads of actin and myosin. In 1957, structures protruding from myosin filaments were seen for the first time and called "cross bridges." A combination of techniques led to the proposal in 1969 of the "swinging-tilting cross bridge" model of contraction. In the early 1980s, a major problem arose when it was shown that a probe attached to the cross bridges did not move during contraction. A spectacular breakthrough came when it was discovered that only the cross bridge was required to support movement in an in vitro motility assay. Next it was determined that single myosin molecules caused the movement of actin filaments in 10-nm steps. The atomic structure of the cross bridge was published in 1993, and this discovery supercharged the muscle field. The cross bridge contained a globular head or motor domain that bound actin and ATP. But the most striking feature was the long tail of the cross bridge surrounded by two subunits of the myosin molecule. This structure suggested that the tail might act as a lever arm magnifying head movement. Consistent with this proposal, genetic techniques that lengthened the lever arm resulted in larger myosin steps. Thus the molecular motor of muscle operated not by the tilting of the globular head of myosin but by tilting of the lever arm generating the driving force for contraction.

Cardioinotropic Effects in Subchronic Intoxication of Rats with Lead and/or Cadmium Oxide Nanoparticles.

Subchronic intoxication was induced in outbred male rats by repeated intraperitoneal injections with lead oxide (PbO) and/or cadmium oxide (CdO) nanoparticles (NPs) 3 times a week during 6 weeks for the purpose of examining its effects on the contractile characteristics of isolated right ventricle trabeculae and papillary muscles in isometric and afterload contractions. Isolated and combined intoxication with these NPs was observed to reduce the mechanical work produced by both types of myocardial preparation. Using the in vitro motility assay, we showed that the sliding velocity of regulated thin filaments drops under both isolated and combined intoxication with CdO-NP and PbO-NP. These results correlate with a shift in the expression of myosin heavy chain (MHC) isoforms towards slowly cycling ß-MHC. The type of CdO-NP + PbO-NP combined cardiotoxicity depends on the effect of the toxic impact, the extent of this effect, the ratio of toxicant doses, and the degree of stretching of cardiomyocytes and muscle type studied. Some indices of combined Pb-NP and CdO-NP cardiotoxicity and general toxicity (genotoxicity included) became fully or partly normalized if intoxication developed against background administration of a bioprotective complex.

The formin inhibitor SMIFH2 inhibits members of the myosin superfamily.

The small molecular inhibitor of formin FH2 domains, SMIFH2, is widely used in cell biological studies. It inhibits formin-driven actin polymerization in vitro, but not polymerization of pure actin. It is active against several types of formin from different species. Here, we found that SMIFH2 inhibits retrograde flow of myosin 2 filaments and contraction of stress fibers. We further checked the effect of SMIFH2 on non-muscle myosin 2A and skeletal muscle myosin 2 in vitro, and found that SMIFH2 inhibits activity of myosin ATPase and the ability to translocate actin filaments in the gliding actin in vitro motility assay. Inhibition of non-muscle myosin 2A in vitro required a higher concentration of SMIFH2 compared with that needed to inhibit retrograde flow and stress fiber contraction in cells. We also found that SMIFH2 inhibits several other non-muscle myosin types, including bovine myosin 10, Drosophila myosin 7a and Drosophila myosin 5, more efficiently than it inhibits formins. These off-target inhibitions demand additional careful analysis in each case when solely SMIFH2 is used to probe formin functions. This article has an associated First Person interview with Yukako Nishimura, joint first author of the paper.

Acidosis modifies effects of phosphorylated tropomyosin on the actin-myosin interaction in the myocardium.

Phosphorylation of α-tropomyosin (Tpm1.1), a predominant Tpm isoform in the myocardium, is one of the regulatory mechanisms of the heart contractility. The Tpm 1.1 molecule has one site of phosphorylation, Ser283. The degree of the Tpm phosphorylation decreases with age and also changes in heart pathologies. Myocardial pathologies, in particular ischemia, are usually accompanied by pH lowering in the cardiomyocyte cytosol. We studied the effects of acidosis on the structural and functional properties of the pseudo-phosphorylated form of Tpm1.1 with the S283D substitution. We found that in acidosis, the interaction of the N- and C-ends of the S283D Tpm molecules decreases, whereas that of WT Tpm does not change. The pH lowering increased thermostability of the complex of F-actin with S283D Tpm to a greater extent than with WT Tpm. Using an in vitro motility assay with NEM- modified myosin as a load, we assessed the effect of the Tpm pseudo-phosphorylation on the force of the actin-myosin interaction. In acidosis, the force generated by myosin in the interaction with thin filaments containing S283D Tpm was higher than with those containing WT Tpm. Also, the pseudo-phosphorylation increased the myosin ability to resist a load. We conclude that ischemia changes the effect of the phosphorylated Tpm on the contractile function of the myocardium.

Myosin and gelsolin cooperate in actin filament severing and actomyosin motor activity.

Actin is a major intracellular protein with key functions in cellular motility, signaling, and structural rearrangements. Its dynamic behavior, such as polymerization and depolymerization of actin filaments in response to intracellular and extracellular cues, is regulated by an abundance of actin binding proteins. Out of these, gelsolin is one of the most potent for filament severing. However, myosin motor activity also fragments actin filaments through motor-induced forces, suggesting that these two proteins could cooperate to regulate filament dynamics and motility. To test this idea, we used an in vitro motility assay, where actin filaments are propelled by surface-adsorbed heavy meromyosin (HMM) motor fragments. This allows studies of both motility and filament dynamics using isolated proteins. Gelsolin, at both nanomolar and micromolar Ca2+ concentration, appreciably enhanced actin filament severing caused by HMM-induced forces at 1 mM MgATP, an effect that was increased at higher HMM motor density. This finding is consistent with cooperativity between actin filament severing by myosin-induced forces and by gelsolin. We also observed reduced sliding velocity of the HMM-propelled filaments in the presence of gelsolin, providing further support of myosin-gelsolin cooperativity. Total internal reflection fluorescence microscopy-based single molecule studies corroborated that the velocity reduction was a direct effect of gelsolin binding to the filament and revealed different filament severing pattern of stationary and HMM propelled filaments. Overall, the results corroborate cooperative effects between gelsolin-induced alterations in the actin filaments and changes due to myosin motor activity leading to enhanced F-actin severing of possible physiological relevance.

Effects of myopathy-causing mutations R91P and R245G in the TPM3 gene on structural and functional properties of slow skeletal muscle tropomyosin.

Tropomyosin (Tpm) is an actin-binding protein that plays a crucial role in the regulation of muscle contraction. Numerous point mutations in the TPM3 gene encoding Tpm of slow skeletal muscles (Tpm 3.12 or γ-Tpm) are associated with the genesis of various congenital myopathies. Two of these mutations, R91P and R245G, are associated with congenital fiber-type disproportion (CFTD) characterized by hypotonia and generalized muscle weakness. We applied various methods to investigate how these mutations affect the structural and functional properties of γγ-Tpm homodimers. The results show that both these mutations lead to strong structural changes in the γγ-Tpm molecule and significantly impaired its functional properties. These changes in the Tpm properties caused by R91P and R245G mutations give insight into the molecular mechanism of the CFTD development and the weakness of slow skeletal muscles observed in this inherited disease.

Mechanisms of disturbance of the contractile function of slow skeletal muscles induced by myopathic mutations in the tropomyosin TPM3 gene.

Several congenital myopathies of slow skeletal muscles are associated with mutations in the tropomyosin (Tpm) TPM3 gene. Tropomyosin is an actin-binding protein that plays a crucial role in the regulation of muscle contraction. Two Tpm isoforms, γ (Tpm3.12) and ß (Tpm2.2) are expressed in human slow skeletal muscles forming γγ-homodimers and γß-heterodimers of Tpm molecules. We applied various methods to investigate how myopathy-causing mutations M9R, E151A, and K169E in the Tpm γ-chain modify the structure-functional properties of Tpm dimers, and how this affects the muscle functioning. The results show that the features of γγ-Tpm and γß-Tpm with substitutions in the Tpm γ-chain vary significantly. The characteristics of the γγ-Tpm depend on whether these mutations located in only one or both γ-chains. The mechanism of the development of nemaline myopathy associated with the M9R mutation was revealed. At the molecular level, a cause-and-effect relationship has been established for the development of myopathy by the K169E mutation. Also, we described the structure-functional properties of the Tpm dimers with the E151A mutation, which explain muscle weakness linked to this substitution. The results demonstrate a diversity of the molecular mechanisms of myopathy pathogenesis induced by studied Tpm mutations.

Comparison of Functional Characteristics of Myosin in Fast and Slow Skeletal Muscles.

Myosins of fast and slow skeletal muscles differ by the isoform composition of the heavy and light chains. We compared functional characteristics of myosin from the fast (m. psoas) and slow (m. soleus) muscles of rabbits. The parameters of single actin-myosin interaction were measured in an optical trap, and the characteristics of the Ca2+ regulation of actin-myosin interaction were studied using an in vitro motility assay. The duration of interaction of myosin from the fast muscle with actin was shorter and the filament sliding velocity over this myosin was higher than the corresponding parameters for myosin from the slow muscle. The dependence pCa-velocity for myosin from the fast muscle was less sensitive to Ca2+ than that of slow muscle myosin. Thus, functional properties of myosin determine not only mechanical and kinetic characteristics of muscle contraction, but also the peculiarities of its Ca2+ regulation.

Lysozyme-induced suppression of enzymatic and motile activities of actin-myosin: Impact of basic proteins.

Electrostatic interactions between actin filaments and myosin molecules, which are ubiquitous proteins in eukaryotes, are crucial for their enzymatic activity and motility. Nonspecific electrostatic interactions between proteins are unavoidable in cells; therefore, it is worth exploring how ambient proteins, such as polyelectrolytes, affect actin-myosin functions. To understand the effect of counterionic proteins on actin-myosin, we examined ATPase activity and sliding velocity via actin-myosin interactions in the presence of the basic model protein hen egg lysozyme. In an in vitro motility assay with ATP, the sliding velocity of actin filaments on heavy meromyosin (HMM) decreased with increasing lysozyme concentrations. Actin filaments were completely stalled at a lysozyme concentration above 0.08 mg/mL. Lysozyme decreased the ATP hydrolysis rate of the actin-HMM complex but not that HMM alone. Co-sedimentation assays revealed that lysozyme enhanced the binding of HMM to actin filaments in the presence of ATP. Additionally, lysozyme could bind to actin and myosin filaments. The inhibitory effect of poly-l-lysine, histone mixture, and lactoferrin on the motility of actin-myosin was higher than that of lysozyme. Thus, nonspecific electrostatic interactions of basic proteins are involved in the bundling of actin filaments and modulation of essential functions of the actomyosin complex.

Myosin from the ventricle is more sensitive to omecamtiv mecarbil than myosin from the atrium.

Omecamtiv mecarbil (OM), an activator of cardiac myosin, strongly affects contractile characteristics of the ventricles and, to a much lesser extent, the characteristics of atrial contraction. We compared the molecular mechanism of action of OM on the interaction of atrial and ventricular myosin with actin using an optical trap and an in vitro motility assay. In concentrations up to 0.5 µM, OM did not affect the step size of a myosin molecule but reduced it at a higher OM level. OM substantially prolonged the interaction of both isoforms of myosin with actin. However, the interaction characteristics of ventricular myosin with actin were more sensitive to OM than those of atrial myosin. Our results, obtained at the level of isolated proteins, can explain why the impact of OM in therapeutic concentrations on the contractile function of the atrium is less significant as compared to those of the ventricle.