RESUMEN
X-ray structural determination of segments of the myosin rod has proved difficult because of the strong salt-dependent aggregation properties and repeating pattern of charges on the surface of the coiled-coil that lead to the formation of paracrystals. This problem has been resolved in part through the use of globular assembly domains that improve protein folding and prevent aggregation. The primary consideration now in designing coiled-coil fusion constructs for myosin is deciding where to truncate the coiled-coil and which amino acid residues to include from the folding domain. This is especially important for myosin that contains numerous regions of low predicted coiled-coil propensity. Here we describe the strategy adopted to determine the structure of the region that extends from Arg1677 - Leu1797 that included two areas that do not show a strong sequence signature of a conventional left-handed coiled coil or canonical heptad repeat. This demonstrates again that, with careful choice of fusion constructs, overlapping structures exhibit very similar conformations for the myosin rod fragments in the canonical regions. However, conformational variability is seen around Leu1706 which is a hot spot for cardiomyopathy mutations suggesting that this might be important for function.
Asunto(s)
Subfragmentos de Miosina/química , Miosinas Ventriculares/química , Cristalografía por Rayos X , Humanos , Leucina/genética , Modelos Moleculares , Subfragmentos de Miosina/genética , Conformación Proteica , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Miosinas Ventriculares/genéticaRESUMEN
Non-muscle cell contractility is critical for tissues to adopt shape changes. Although, the non-muscle myosin II holoenzyme (myosin) is a molecular motor that powers contraction of actin cytoskeleton networks, recent studies have questioned the importance of myosin motor activity cell and tissue shape changes. Here, combining the biochemical analysis of enzymatic and motile properties for purified myosin mutants with in vivo measurements of apical constriction for the same mutants, we show that in vivo constriction rate scales with myosin motor activity. We show that so-called phosphomimetic mutants of the Drosophila regulatory light chain (RLC) do not mimic the phosphorylated RLC state in vitro. The defect in the myosin motor activity in these mutants is evident in developing Drosophila embryos where tissue recoil following laser ablation is decreased compared to wild-type tissue. Overall, our data highlights that myosin activity is required for rapid cell contraction and tissue folding in developing Drosophila embryos.
Asunto(s)
Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Cadenas Ligeras de Miosina/genética , Subfragmentos de Miosina/genética , Miosina Tipo IIA no Muscular/genética , Secuencia de Aminoácidos , Animales , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/crecimiento & desarrollo , Drosophila melanogaster/metabolismo , Embrión no Mamífero , Expresión Génica , Humanos , Cinética , Ratones , Morfogénesis/genética , Movimiento (Física) , Cadenas Ligeras de Miosina/metabolismo , Subfragmentos de Miosina/metabolismo , Miosina Tipo IIA no Muscular/metabolismo , Fosforilación , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Homología de Secuencia de AminoácidoRESUMEN
Cytokinesis requires the spatio-temporal coordination of membrane deposition and primary septum (PS) formation at the division site to drive acto-myosin ring (AMR) constriction. It has been demonstrated that AMR constriction invariably occurs only after the mitotic spindle disassembly. It has also been established that Chitin Synthase II (Chs2p) neck localization precedes mitotic spindle disassembly during mitotic exit. As AMR constriction depends upon PS formation, the question arises as to how chitin deposition is regulated so as to prevent premature AMR constriction and mitotic spindle breakage. In this study, we propose that cells regulate the coordination between spindle disassembly and AMR constriction via timely endocytosis of cytokinetic enzymes, Chs2p, Chs3p, and Fks1p. Inhibition of endocytosis leads to over accumulation of cytokinetic enzymes during mitotic exit, which accelerates the constriction of the AMR, and causes spindle breakage that eventually could contribute to monopolar spindle formation in the subsequent round of cell division. Intriguingly, the mitotic spindle breakage observed in endocytosis mutants can be rescued either by deleting or inhibiting the activities of, CHS2, CHS3 and FKS1, which are involved in septum formation. The findings from our study highlight the importance of timely endocytosis of cytokinetic enzymes at the division site in safeguarding mitotic spindle integrity during mitotic exit.
Asunto(s)
Quitina Sintasa/genética , Equinocandinas/genética , Endocitosis/genética , Glucosiltransferasas/genética , Proteínas de la Membrana/genética , Mitosis/genética , Proteínas de Saccharomyces cerevisiae/genética , Ciclo Celular/genética , Membrana Celular/genética , Quitina/genética , Citocinesis/genética , Microscopía Fluorescente , Subfragmentos de Miosina/genética , Saccharomyces cerevisiae/genética , Huso Acromático/genéticaRESUMEN
An "invariant proline" separates the myosin S1 head from its S2 tail and is proposed to be critical for orienting S1 during its interaction with actin, a process that leads to muscle contraction. Mutation of the invariant proline to leucine (P838L) caused dominant restrictive cardiomyopathy in a pediatric patient (Karam et al., Congenit. Heart Dis. 3:138-43, 2008). Here, we use Drosophila melanogaster to model this mutation and dissect its effects on the biochemical and biophysical properties of myosin, as well as on the structure and physiology of skeletal and cardiac muscles. P838L mutant myosin isolated from indirect flight muscles of transgenic Drosophila showed elevated ATPase and actin sliding velocity in vitro. Furthermore, the mutant heads exhibited increased rotational flexibility, and there was an increase in the average angle between the two heads. Indirect flight muscle myofibril assembly was minimally affected in mutant homozygotes, and isolated fibers displayed normal mechanical properties. However, myofibrils degraded during aging, correlating with reduced flight abilities. In contrast, hearts from homozygotes and heterozygotes showed normal morphology, myofibrillar arrays, and contractile parameters. When P838L was placed in trans to Mhc(5), an allele known to cause cardiac restriction in flies, it did not yield the constricted phenotype. Overall, our studies suggest that increased rotational flexibility of myosin S1 enhances myosin ATPase and actin sliding. Moreover, instability of P838L myofibrils leads to decreased function during aging of Drosophila skeletal muscle, but not cardiac muscle, despite the strong evolutionary conservation of the P838 residue.
Asunto(s)
Cardiomiopatía Restrictiva/genética , Drosophila melanogaster/genética , Mutación/genética , Subfragmentos de Miosina/genética , Prolina/genética , Actinas/genética , Animales , Drosophila melanogaster/metabolismo , Vuelo Animal/fisiología , Contracción Muscular/genética , Músculo Esquelético/metabolismo , Miocardio/metabolismo , Miofibrillas/genética , Cadenas Pesadas de Miosina/genética , Miosinas/genética , FenotipoRESUMEN
The embryonic myosin isoform is expressed during fetal development and rapidly down-regulated after birth. Freeman-Sheldon syndrome (FSS) is a disease associated with missense mutations in the motor domain of this myosin. It is the most severe form of distal arthrogryposis, leading to overcontraction of the hands, feet, and orofacial muscles and other joints of the body. Availability of human embryonic muscle tissue has been a limiting factor in investigating the properties of this isoform and its mutations. Using a recombinant expression system, we have studied homogeneous samples of human motors for the WT and three of the most common FSS mutants: R672H, R672C, and T178I. Our data suggest that the WT embryonic myosin motor is similar in contractile speed to the slow type I/ß cardiac based on the rate constant for ADP release and ADP affinity for actin-myosin. All three FSS mutations show dramatic changes in kinetic properties, most notably the slowing of the apparent ATP hydrolysis step (reduced 5-9-fold), leading to a longer lived detached state and a slowed Vmax of the ATPase (2-35-fold), indicating a slower cycling time. These mutations therefore seriously disrupt myosin function.
Asunto(s)
Adenosina Trifosfato/metabolismo , Disostosis Craneofacial/genética , Disostosis Craneofacial/patología , Proteínas del Citoesqueleto/genética , Contracción Muscular/fisiología , Mutación/genética , Subfragmentos de Miosina/genética , Adenosina Trifosfatasas/metabolismo , Células Cultivadas , Disostosis Craneofacial/metabolismo , Proteínas del Citoesqueleto/metabolismo , Células Germinales Embrionarias/citología , Células Germinales Embrionarias/metabolismo , Humanos , Hidrólisis , Subfragmentos de Miosina/metabolismo , Isoformas de ProteínasRESUMEN
Sarcomeric myosins have the remarkable ability to form regular bipolar thick filaments that, together with actin thin filaments, constitute the fundamental contractile unit of skeletal and cardiac muscle. This has been established for over 50 years and yet a molecular model for the thick filament has not been attained. In part this is due to the lack of a detailed molecular model for the coiled-coil that constitutes the myosin rod. The ability to self-assemble resides in the C-terminal section of myosin known as light meromyosin (LMM) which exhibits strong salt-dependent aggregation that has inhibited structural studies. Here we evaluate the feasibility of generating a complete model for the myosin rod by combining overlapping structures of five sections of coiled-coil covering 164 amino acid residues which constitute 20% of LMM. Each section contains â¼ 7-9 heptads of myosin. The problem of aggregation was overcome by incorporating the globular folding domains, Gp7 and Xrcc4 which enhance crystallization. The effect of these domains on the stability and conformation of the myosin rod was examined through biophysical studies and overlapping structures. In addition, a computational approach was developed to combine the sections into a contiguous model. The structures were aligned, trimmed to form a contiguous model, and simulated for >700 ns to remove the discontinuities and achieve an equilibrated conformation that represents the native state. This experimental and computational strategy lays the foundation for building a model for the entire myosin rod.
Asunto(s)
Subfragmentos de Miosina/química , Secuencia de Aminoácidos , Cardiomiopatías/genética , Cristalografía por Rayos X , Humanos , Simulación de Dinámica Molecular , Datos de Secuencia Molecular , Mutación , Subfragmentos de Miosina/genética , Conformación Proteica , Estabilidad Proteica , Estructura Secundaria de Proteína , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , TemperaturaRESUMEN
In skeletal muscle cells, myofibrillar proteins are highly organized into sarcomeres in which thick filaments interdigitate with thin filaments to generate contractile force. The size of thick filaments, which consist mainly of myosin molecules, is strictly controlled. However, little is known about the mechanisms by which myosin molecules assemble into thick filaments. Here, we assessed the ability of each domain of myosin heavy chain (Myh) to form thick filaments. We showed that exogenously expressed subfragment 2 (S2) + light meromyosin (LMM) of Myh was efficiently incorporated into thick filaments in muscle cells, although neither solely expressed S2 nor LMM targeted to thick filaments properly. In nonmuscle COS7 cells, S2+LMM formed more enlarged filaments/speckles than LMM. These results suggest that Myh filament formation is induced by S2 accompanying LMM. We further examined the effects of Myh C-terminus on thick filament assembly. C-terminal deletion mutants were incorporated not into entire thick filaments but rather into restricted regions of thick filaments. Our findings suggest that the elongation of myosin filaments to form thick filaments is regulated by S2 as well as C-terminus of LMM.
Asunto(s)
Citoesqueleto/metabolismo , Citoesqueleto/fisiología , Músculo Esquelético/citología , Subfragmentos de Miosina/metabolismo , Miosinas del Músculo Esquelético/metabolismo , Animales , Células Cultivadas , Ratones , Proteínas Mutantes/metabolismo , Subfragmentos de Miosina/genética , Subfragmentos de Miosina/fisiología , SarcómerosRESUMEN
The triangle sail mussel, Hyriopsis cumingii, is the most important freshwater pearl mussel in China. However, the mechanisms underlying its chitin-mediated shell and nacre formation remain largely unknown. Here, we characterized a chitin synthase (CS) gene (HcCS1) in H. cumingii, and analyzed its possible physiological function. The complete ORF sequence of HcCS1 contained 6903 bp, encoding a 2300-amino acid protein (theoretical molecular mass = 264 kDa; isoelectric point = 6.22), and no putative signal peptide was predicted. A myosin motor head domain, a CS domain, and 12 transmembrane domains were found. The predicted spatial structures of the myosin head and CS domains were similar to the electron microscopic structure of the heavy meromyosin subfragment of chicken smooth muscle myosin and the crystal structure of bacterial cellulose synthase, respectively. This structural similarity indicates that the functions of these two domains might be conserved. Quantitative reverse transcription PCR results showed that HcCS1 was present in all detected tissues, with the highest expression levels detected in the mantle. The HcCS1 transcripts in the mantle were upregulated following shell damage from 12 to 24 h post-damage, and they peaked (approximately 1.5-fold increase) at 12 h after shell damage. These findings suggest that HcCS1 was involved in shell regeneration, and that it might participate in shell and nacre formation in this species via chitin synthesis. HcCS1 might also dynamically regulate chitin deposition during the process of shell and nacre formation with the help of its conserved myosin head domain.
Asunto(s)
Exoesqueleto/metabolismo , Bivalvos/genética , Quitina Sintasa/genética , Quitina/biosíntesis , Nácar/metabolismo , Secuencia de Aminoácidos , Animales , Bivalvos/clasificación , Bivalvos/enzimología , Pollos , Quitina Sintasa/química , Quitina Sintasa/metabolismo , Agua Dulce , Expresión Génica , Glucosiltransferasas/química , Glucosiltransferasas/genética , Punto Isoeléctrico , Modelos Moleculares , Datos de Secuencia Molecular , Peso Molecular , Subfragmentos de Miosina/química , Subfragmentos de Miosina/genética , Sistemas de Lectura Abierta , Filogenia , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Alineación de Secuencia , Homología Estructural de ProteínaRESUMEN
Unconventional myosin 15 is a molecular motor expressed in inner ear hair cells that transports protein cargos within developing mechanosensory stereocilia. Mutations of myosin 15 cause profound hearing loss in humans and mice; however, the properties of this motor and its regulation within the stereocilia organelle are unknown. To address these questions, we expressed a subfragment 1-like (S1) truncation of mouse myosin 15, comprising the predicted motor domain plus three light-chain binding sites. Following unsuccessful attempts to express functional myosin 15-S1 using the Spodoptera frugiperda (Sf9)-baculovirus system, we discovered that coexpression of the muscle-myosin-specific chaperone UNC45B, in addition to the chaperone heat-shock protein 90 (HSP90) significantly increased the yield of functional protein. Surprisingly, myosin 15-S1 did not bind calmodulin with high affinity. Instead, the IQ domains bound essential and regulatory light chains that are normally associated with class II myosins. We show that myosin 15-S1 is a barbed-end-directed motor that moves actin filaments in a gliding assay (â¼ 430 nm · s(-1) at 30 °C), using a power stroke of 7.9 nm. The maximum ATPase rate (k(cat) â¼ 6 s(-1)) was similar to the actin-detachment rate (k(det) = 6.2 s(-1)) determined in single molecule optical trapping experiments, indicating that myosin 15-S1 was rate limited by transit through strongly actin-bound states, similar to other processive myosin motors. Our data further indicate that in addition to folding muscle myosin, UNC45B facilitates maturation of an unconventional myosin. We speculate that chaperone coexpression may be a simple method to optimize the purification of other myosin motors from Sf9 insect cells.
Asunto(s)
Miosinas/aislamiento & purificación , Miosinas/metabolismo , Estereocilios/metabolismo , Actinas/metabolismo , Secuencia de Aminoácidos , Animales , Calmodulina/metabolismo , Proteínas HSP90 de Choque Térmico/genética , Proteínas HSP90 de Choque Térmico/metabolismo , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Ratones , Chaperonas Moleculares , Datos de Secuencia Molecular , Cadenas Ligeras de Miosina/metabolismo , Subfragmentos de Miosina/genética , Subfragmentos de Miosina/aislamiento & purificación , Subfragmentos de Miosina/metabolismo , Miosinas/genética , Pinzas Ópticas , Pliegue de Proteína , Transporte de Proteínas , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo , Homología de Secuencia de Aminoácido , Células Sf9 , SpodopteraRESUMEN
Myosin-10 is an actin-based molecular motor that participates in essential intracellular processes such as filopodia formation/extension, phagocytosis, cell migration, and mitotic spindle maintenance. To study this motor protein's mechano-chemical properties, we used a recombinant, truncated form of myosin-10 consisting of the first 936 amino acids, followed by a GCN4 leucine zipper motif, to force dimerization. Negative-stain electron microscopy reveals that the majority of molecules are dimeric with a head-to-head contour distance of â¼50 nm. In vitro motility assays show that myosin-10 moves actin filaments smoothly with a velocity of â¼310 nm/s. Steady-state and transient kinetic analysis of the ATPase cycle shows that the ADP release rate (â¼13 s(-1)) is similar to the maximum ATPase activity (â¼12-14 s(-1)) and therefore contributes to rate limitation of the enzymatic cycle. Single molecule optical tweezers experiments show that under intermediate load (â¼0.5 pN), myosin-10 interacts intermittently with actin and produces a power stroke of â¼17 nm, composed of an initial 15-nm and subsequent 2-nm movement. At low optical trap loads, we observed staircase-like processive movements of myosin-10 interacting with the actin filament, consisting of up to six â¼35-nm steps per binding interaction. We discuss the implications of this load-dependent processivity of myosin-10 as a filopodial transport motor.
Asunto(s)
Actinas/fisiología , Cadenas Pesadas de Miosina/fisiología , Actinas/química , Adenosina Difosfato/metabolismo , Secuencia de Aminoácidos , Animales , Fenómenos Biomecánicos , Bovinos , Técnicas In Vitro , Cinética , Microscopía Electrónica , Microscopía Fluorescente , Modelos Biológicos , Modelos Moleculares , Datos de Secuencia Molecular , Cadenas Pesadas de Miosina/química , Cadenas Pesadas de Miosina/genética , Subfragmentos de Miosina/química , Subfragmentos de Miosina/genética , Subfragmentos de Miosina/fisiología , Pinzas Ópticas , Dominios y Motivos de Interacción de Proteínas , Estructura Cuaternaria de Proteína , Seudópodos/fisiología , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
FRET was used to investigate the structural and kinetic effects that PKC phosphorylations exert on Ca(2+) and myosin subfragment-1 dependent conformational transitions of the cardiac thin filament. PKC phosphorylations of cTnT were mimicked by glutamate substitution. Ca(2+) and S1-induced distance changes between the central linker of cTnC and the switch region of cTnI (cTnI-Sr) were monitored in reconstituted thin filaments using steady state and time resolved FRET, while kinetics of structural transitions were determined using stopped flow. Thin filament Ca(2+) sensitivity was found to be significantly blunted by the presence of the cTnT(T204E) mutant, whereas pseudo-phosphorylation at additional sites increased the Ca(2+)-sensitivity. The rate of Ca(2+)-dissociation induced structural changes was decreased in the C-terminal end of cTnI-Sr in the presence of pseudo-phosphorylations while remaining unchanged at the N-terminal end of this region. Additionally, the distance between cTnI-Sr and cTnC was decreased significantly for the triple and quadruple phosphomimetic mutants cTnT(T195E/S199E/T204E) and cTnT(T195E/S199E/T204E/T285E), which correlated with the Ca(2+)-sensitivity increase seen in these same mutants. We conclude that significant changes in thin filament Ca(2+)-sensitivity, structure and kinetics are brought about through PKC phosphorylation of cTnT. These changes can either decrease or increase Ca(2+)-sensitivity and likely play an important role in cardiac regulation.
Asunto(s)
Calcio/metabolismo , Miofibrillas/metabolismo , Subfragmentos de Miosina/metabolismo , Proteína Quinasa C/metabolismo , Troponina T/metabolismo , Sustitución de Aminoácidos , Animales , Bovinos , Escherichia coli/genética , Escherichia coli/metabolismo , Transferencia Resonante de Energía de Fluorescencia , Regulación de la Expresión Génica , Ácido Glutámico/metabolismo , Cinética , Imitación Molecular , Mutagénesis Sitio-Dirigida , Miocardio/metabolismo , Miofibrillas/genética , Subfragmentos de Miosina/genética , Fosforilación , Conformación Proteica , Proteína Quinasa C/genética , Conejos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transducción de Señal , Troponina T/genéticaRESUMEN
Mutations in cardiac troponin I (cTnI) that cause hypertrophic cardiomyopathy (HCM) have been reported to change the contractility of cardiac myofilaments, but the underlying molecular mechanism remains elusive. In this study, Förster resonance energy transfer (FRET) was used to investigate the specific structural and kinetic effects that HCM related rat cTnI mutations R146G/Q and R163W exert on Ca(2+) and myosin S1 dependent conformational transitions in rat cTn structure. Ca(2+)-induced changes in interactions between cTnC and cTnI were individually monitored in reconstituted thin filaments using steady state and time resolved FRET, and kinetics were determined using stopped flow. R146G/Q and R163W all changed the FRET distances between cTnC and cTnI in unique and various ways. However, kinetic rates of conformational transitions induced by Ca(2+)-dissociation were universally slowed when R146G/Q and R163W were present. Interestingly, the kinetic rates of changes in the inhibitory region of cTnI were always slower than that of the regulatory region, suggesting that the fly casting mechanism that normally underlies deactivation is preserved in spite of mutation. In situ rat myocardial fiber studies also revealed that FRET distance changes indicating mutation specific disruption of the cTnIIR-actin interaction were consistent with increased passive tension.
Asunto(s)
Cardiomiopatía Hipertrófica/genética , Mutación , Miofibrillas/metabolismo , Troponina I/metabolismo , Actinas/metabolismo , Animales , Calcio/metabolismo , Cardiomiopatía Hipertrófica/metabolismo , Cisteína/genética , Cisteína/metabolismo , Transferencia Resonante de Energía de Fluorescencia , Ventrículos Cardíacos/metabolismo , Cinética , Masculino , Contracción Muscular , Miofibrillas/genética , Subfragmentos de Miosina/genética , Subfragmentos de Miosina/metabolismo , Unión Proteica , Conformación Proteica , Mapeo de Interacción de Proteínas , Ratas , Ratas Long-Evans , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Troponina C/genética , Troponina C/metabolismo , Troponina I/genéticaRESUMEN
We successfully synthesized full-length and the mutant Physarum myosin and heavy meromyosin (HMM) constructs associated with Physarum regulatory light chain and essential light chain (PhELC) using Physarum myosin heavy chain in Sf-9 cells, and examined their Ca(2+)-mediated regulation. Ca(2+) inhibited the motility and ATPase activities of Physarum myosin and HMM. The Ca(2+) effect is also reversible at the in vitro motility of Physarum myosin. We demonstrated that full-length myosin increases the Ca(2+) inhibition more effectively than HMM. Furthermore, Ca(2+) did not affect the motility and ATPase activities of the mutant Physarum myosin with PhELC that lost Ca(2+)-binding ability. Therefore, we conclude that PhELC plays a critical role in Ca(2+)-dependent regulation of Physarum myosin.
Asunto(s)
Calcio/metabolismo , Miosinas/metabolismo , Physarum/metabolismo , Animales , Calcio/farmacología , Células Cultivadas , Mutación , Cadenas Ligeras de Miosina/genética , Cadenas Ligeras de Miosina/metabolismo , Subfragmentos de Miosina/genética , Subfragmentos de Miosina/metabolismo , Miosinas/genética , Physarum/efectos de los fármacos , Physarum/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismoRESUMEN
Myosin has an intrinsic ability to organize into ordered thick filaments that mediate muscle contraction. Here, we use surface plasmon resonance and light scattering analysis to further characterize the molecular determinants that guide myosin filament assembly. Both assays identify a cluster of lysine and arginine residues as important for myosin polymerization in vitro. Moreover, in cardiomyocytes, replacement of these charged residues by alanine severely affects the incorporation of myosin into the distal ends of the sarcomere. Our findings show that a novel assembly element with a distinct charge profile is present at the C-terminus of sarcomeric myosins.
Asunto(s)
Miosinas Ventriculares/química , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Electroquímica , Luz , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Miocitos Cardíacos/química , Miocitos Cardíacos/metabolismo , Subfragmentos de Miosina/química , Subfragmentos de Miosina/genética , Subfragmentos de Miosina/metabolismo , Multimerización de Proteína , Estructura Terciaria de Proteína , Ratas , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Dispersión de Radiación , Resonancia por Plasmón de Superficie , Transfección , Miosinas Ventriculares/genética , Miosinas Ventriculares/metabolismoRESUMEN
The roles of myosin during muscle contraction are well studied, but how different domains of this protein are involved in myofibril assembly in vivo is far less understood. The indirect flight muscles (IFMs) of Drosophila melanogaster provide a good model for understanding muscle development and function in vivo. We show that two missense mutations in the rod region of the myosin heavy-chain gene, Mhc, give rise to IFM defects and abnormal myofibrils. These defects likely result from thick filament abnormalities that manifest during early sarcomere development or later by hypercontraction. The thick filament defects are accompanied by marked reduction in accumulation of flightin, a myosin binding protein, and its phosphorylated forms, which are required to stabilise thick filaments. We investigated with purified rod fragments whether the mutations affect the coiled-coil structure, rod aggregate size or rod stability. No significant changes in these parameters were detected, except for rod thermodynamic stability in one mutation. Molecular dynamics simulations suggest that these mutations may produce localised rod instabilities. We conclude that the aberrant myofibrils are a result of thick filament defects, but that these in vivo effects cannot be detected in vitro using the biophysical techniques employed. The in vivo investigation of these mutant phenotypes in IFM development and function provides a useful platform for studying myosin rod and thick filament formation generically, with application to the aetiology of human myosin rod myopathies.
Asunto(s)
Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Mutación Missense , Miofibrillas/metabolismo , Subfragmentos de Miosina/genética , Subfragmentos de Miosina/metabolismo , Secuencia de Aminoácidos , Animales , Proteínas de Drosophila/química , Drosophila melanogaster , Filaminas , Vuelo Animal/fisiología , Simulación de Dinámica Molecular , Datos de Secuencia Molecular , Contracción Muscular , Proteínas Musculares/química , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Miofibrillas/química , Miofibrillas/genética , Miofibrillas/ultraestructura , Cadenas Pesadas de Miosina/química , Cadenas Pesadas de Miosina/genética , Cadenas Pesadas de Miosina/metabolismo , Subfragmentos de Miosina/química , Fenotipo , Fosforilación/genéticaRESUMEN
While mutations in the myosin subfragment 1 motor domain can directly disrupt the generation and transmission of force along myofibrils and lead to myopathy, the mechanism whereby mutations in the myosin rod influences mechanical function is less clear. Here, we used a combination of various imaging techniques and molecular dynamics simulations to test the hypothesis that perturbations in the myosin rod can disturb normal sarcomeric uniformity and, like motor domain lesions, would influence force production and propagation. We show that disrupting the rod can alter its nanomechanical properties and, in vivo, can drive asymmetric myofilament and sarcomere formation. Our imaging results indicate that myosin rod mutations likely disturb production and/or propagation of contractile force. This provides a unifying theory where common pathological cascades accompany both myosin motor and specific rod domain mutations. Finally, we suggest that sarcomeric inhomogeneity, caused by asymmetric thick filaments, could be a useful index of myopathic dysfunction.
Asunto(s)
Placa Motora/fisiología , Enfermedades Musculares/fisiopatología , Subfragmentos de Miosina/fisiología , Sarcómeros/fisiología , Humanos , Modelos Moleculares , Placa Motora/genética , Contracción Muscular , Enfermedades Musculares/genética , Enfermedades Musculares/patología , Mutación , Subfragmentos de Miosina/química , Subfragmentos de Miosina/genética , Subfragmentos de Miosina/ultraestructura , Sarcómeros/química , Sarcómeros/genética , Sarcómeros/ultraestructuraRESUMEN
Cytokinesis in animal and fungal cells utilizes a contractile actomyosin ring (AMR). However, how myosin II is targeted to the division site and promotes AMR assembly, and how the AMR coordinates with membrane trafficking during cytokinesis, remains poorly understood. Here we show that Myo1 is a two-headed myosin II in Saccharomyces cerevisiae, and that Myo1 localizes to the division site via two distinct targeting signals in its tail that act sequentially during the cell cycle. Before cytokinesis, Myo1 localization depends on the septin-binding protein Bni5. During cytokinesis, Myo1 localization depends on the IQGAP Iqg1. We also show that the Myo1 tail is sufficient for promoting the assembly of a "headless" AMR, which guides membrane deposition and extracellular matrix remodeling at the division site. Our study establishes a biphasic targeting mechanism for myosin II and highlights an underappreciated role of the AMR in cytokinesis beyond force generation.
Asunto(s)
Actomiosina/metabolismo , Citocinesis/fisiología , Cadenas Pesadas de Miosina/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Ciclo Celular/fisiología , Cinética , Cadenas Pesadas de Miosina/genética , Cadenas Pesadas de Miosina/ultraestructura , Cadenas Ligeras de Miosina/genética , Subfragmentos de Miosina/genética , Subfragmentos de Miosina/metabolismo , Subfragmentos de Miosina/ultraestructura , Unión Proteica/fisiología , Dominios y Motivos de Interacción de Proteínas/fisiología , Estructura Cuaternaria de Proteína , Transporte de Proteínas/fisiología , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes de Fusión/ultraestructura , Saccharomyces cerevisiae/fisiología , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/ultraestructura , Proteínas Activadoras de ras GTPasa/genéticaRESUMEN
Tropomyosin is a ubiquitous actin-binding protein with an extended coiled-coil structure. Tropomyosin-actin interactions are weak and loosely specific, but they potently influence myosin. One such influence is inhibitory and is due to tropomyosin's statistically preferred positions on actin that sterically interfere with actin's strong attachment site for myosin. Contrastingly, tropomyosin's other influence is activating. It increases myosin's overall actin affinity â¼4-fold. Stoichiometric considerations cause this activating effect to equate to an â¼4(7)-fold effect of myosin on the actin affinity of tropomyosin. These positive, mutual, myosin-tropomyosin effects are absent if Saccharomyces cerevisiae tropomyosin replaces mammalian tropomyosin. To investigate these phenomena, chimeric tropomyosins were generated in which 38-residue muscle tropomyosin segments replaced a natural duplication within S. cerevisiae tropomyosin TPM1. Two such chimeric tropomyosins were sufficiently folded coiled coils to allow functional study. The two chimeras differed from TPM1 but in opposite ways. Consistent with steric interference, myosin greatly decreased the actin affinity of chimera 7, which contained muscle tropomyosin residues 228-265. On the other hand, myosin S1 increased by an order of magnitude the actin affinity of chimera 3, which contained muscle tropomyosin residues 74-111. Similarly, myosin S1-ADP binding to actin was strengthened 2-fold by substitution of chimera 3 tropomyosin for wild-type TPM1. Thus, a yeast tropomyosin was induced to mimic the activating behavior of mammalian tropomyosin by inserting a mammalian tropomyosin sequence. The data were not consistent with direct tropomyosin-myosin binding. Rather, they suggest an allosteric mechanism, in which myosin and tropomyosin share an effect on the actin filament.
Asunto(s)
Actinas/metabolismo , Proteínas Musculares/metabolismo , Miosinas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Tropomiosina/metabolismo , Secuencia de Aminoácidos , Animales , Bovinos , Proteínas Musculares/química , Proteínas Musculares/genética , Proteínas Mutantes Quiméricas/genética , Proteínas Mutantes Quiméricas/metabolismo , Subfragmentos de Miosina/genética , Unión Proteica , Pliegue de Proteína , Estabilidad Proteica , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Tropomiosina/genéticaRESUMEN
Striated muscles are relaxed under low Ca(2+) concentration conditions due to actions of the thin filament protein troponin. To investigate this regulatory mechanism, an 11-residue segment of cardiac troponin I previously termed the inhibitory peptide region was studied by mutagenesis. Several mutant troponin complexes were characterized in which specific effects of the inhibitory peptide region were abrogated by replacements of 4-10 residues with Gly-Ala linkers. The mutations greatly impaired two of troponin's actions under low Ca(2+) concentration conditions: inhibition of myosin subfragment 1 (S1)-thin filament MgATPase activity and cooperative suppression of myosin S1-ADP binding to thin filaments with low myosin saturation. Inhibitory peptide replacement diminished but did not abolish the Ca(2+) dependence of the ATPase rate; ATPase rates were at least 2-fold greater when Ca(2+) rather than EGTA was present. This residual regulation was highly cooperative as a function of Ca(2+) concentration, similar to the degree of cooperativity observed with WT troponin present. Other effects of the mutations included 2-fold or less increases in the apparent affinity of the thin filament regulatory Ca(2+) sites, similar decreases in the affinity of troponin for actin-tropomyosin regardless of Ca(2+), and increases in myosin S1-thin filament ATPase rates in the presence of saturating Ca(2+). The overall results indicate that cooperative myosin binding to Ca(2+)-free thin filaments depends upon the inhibitory peptide region but that a cooperatively activating effect of Ca(2+) binding does not. The findings suggest that these two processes are separable and involve different conformational changes in the thin filament.
Asunto(s)
Calcio/química , Subfragmentos de Miosina/química , Péptidos/química , Tropomiosina/química , Troponina I/antagonistas & inhibidores , Troponina I/química , Animales , Sitios de Unión , Calcio/metabolismo , Ácido Egtácico/química , Humanos , Músculo Estriado/química , Músculo Estriado/metabolismo , Mutación , Subfragmentos de Miosina/genética , Subfragmentos de Miosina/metabolismo , Péptidos/genética , Péptidos/metabolismo , Conejos , Tropomiosina/genética , Tropomiosina/metabolismo , Troponina I/genética , Troponina I/metabolismoRESUMEN
To date, more than 230 disease-causing mutations have been linked to the slow/cardiac muscle myosin gene, beta-MyHC (MYH7). Most of these mutations are located in the globular head region of the protein and result in cardiomyopathies. Recently, however, a number of novel disease-causing mutations have been described in the long, alpha-helical, coiled coil tail region of the beta-MyHC protein. Mutations in this region are of particular interest because they are associated with a multitude of human diseases, including both cardiac and skeletal myopathies. Here, we attempt to dissect the mechanism(s) by which mutations in the rod region of beta-MyHC can cause a variety of diseases by analyzing two mutations at a single amino acid (R1500P and R1500W) which cause two distinct diseases (Laing-type early-onset distal myopathy and dilated cardiomyopathy, respectively). For diseases linked to the R1500 residue, we find that each mutation displays distinct structural, thermodynamic, and functional properties. Both R1500P and R1500W cause a decrease in thermodynamic stability, although the R1500W phenotype is more severe. Both mutations also affect filament assembly, with R1500P causing an additional decrease in filament stability. In addition to furthering our understanding of the mechanism of pathogenesis for each of these diseases, these data also suggest how the variance in molecular phenotype may be associated with the variance in clinical phenotype present with mutations in the beta-MyHC rod.