RESUMEN
Mavacamten is a FDA-approved small-molecule therapeutic designed to regulate cardiac function at the sarcomere level by selectively but reversibly inhibiting the enzymatic activity of myosin. It shifts myosin toward ordered off states close to the thick filament backbone. It remains elusive whether these myosin heads in the off state(s) can be recruited in response to physiological stimuli when required to boost cardiac output. We show that cardiac myosins stabilized in these off state(s) by mavacamten are recruitable by 1) Ca2+, 2) increased chronotropy [heart rate (HR)], 3) stretch, and 4) ß-adrenergic (ß-AR) stimulation, all known physiological inotropic interventions. At the molecular level, we show that Ca2+ increases myosin ATPase activity by shifting mavacamten-stabilized myosin heads from the inactive super-relaxed state to the active disordered relaxed state. At the myofilament level, both Ca2+ and passive lengthening can shift mavacamten-ordered off myosin heads from positions close to the thick filament backbone to disordered on states closer to the thin filaments. In isolated rat cardiomyocytes, increased stimulation rates enhanced shortening fraction in mavacamten-treated cells. This observation was confirmed in vivo in telemetered rats, where left-ventricular dP/dtmax, an index of inotropy, increased with HR in mavacamten-treated animals. Finally, we show that ß-AR stimulation in vivo increases left-ventricular function and stroke volume in the setting of mavacamten. Our data demonstrate that the mavacamten-promoted off states of myosin in the thick filament are at least partially activable, thus preserving cardiac reserve mechanisms.
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Miocitos Cardíacos , Miosinas , Uracilo/análogos & derivados , Animales , Ratas , Bencilaminas/farmacología , Contracción MuscularRESUMEN
Chitin is a structural polymer in many eukaryotes. Many organisms can degrade chitin to defend against chitinous pathogens or use chitin oligomers as food. Beneficial microorganisms like nitrogen-fixing symbiotic rhizobia and mycorrhizal fungi produce chitin-based signal molecules called lipo-chitooligosaccharides (LCOs) and short chitin oligomers to initiate a symbiotic relationship with their compatible hosts and exchange nutrients. A recent study revealed that a broad range of fungi produce LCOs and chitooligosaccharides (COs), suggesting that these signaling molecules are not limited to beneficial microbes. The fungal LCOs also affect fungal growth and development, indicating that the roles of LCOs beyond symbiosis and LCO production may predate mycorrhizal symbiosis. This review describes the diverse structures of chitin; their perception by eukaryotes and prokaryotes; and their roles in symbiotic interactions, defense, and microbe-microbe interactions. We also discuss potential strategies of fungi to synthesize LCOs and their roles in fungi with different lifestyles.
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Micorrizas , Simbiosis , Quitina/metabolismo , Micorrizas/metabolismo , Transducción de SeñalRESUMEN
Contraction in striated muscle is initiated by calcium binding to troponin complexes, but it is now understood that dynamic transition of myosin between resting, ordered OFF states on thick filaments and active, disordered ON states that can bind to thin filaments is critical in regulating muscle contractility. These structural OFF to ON transitions of myosin are widely assumed to correspond to transitions from the biochemically defined, energy-sparing, super-relaxed (SRX) state to the higher ATPase disordered-relaxed (DRX) state. Here we examined the effect of 2'-deoxy-ATP (dATP), a naturally occurring energy substrate for myosin, on the structural OFF to ON transitions of myosin motors in porcine cardiac muscle thick filaments. Small-angle X-ray diffraction revealed that titrating dATP in relaxation solutions progressively moves the myosin heads from ordered OFF states on the thick filament backbone to disordered ON states closer to thin filaments. Importantly, we found that the structural OFF to ON transitions are not equivalent to the biochemically defined SRX to DRX transitions and that the dATP-induced structural OFF to ON transitions of myosin motors in relaxed muscle are strongly correlated with submaximal force augmentation by dATP. These results indicate that structural OFF to ON transitions of myosin in relaxed muscle can predict the level of force attained in calcium-activated cardiac muscle. Computational modeling and stiffness measurements suggest a final step in the OFF to ON transition may involve a subset of DRX myosins that form weakly bound cross-bridges prior to becoming active force-producing cross-bridges.
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Calcio , Músculo Estriado , Animales , Porcinos , Calcio/metabolismo , Miocardio/metabolismo , Miosinas/metabolismo , Músculo Esquelético/metabolismo , Músculo Estriado/metabolismo , Calcio de la DietaRESUMEN
Missense variant Ile79Asn in human cardiac troponin T (cTnT-I79N) has been associated with hypertrophic cardiomyopathy and sudden cardiac arrest in juveniles. cTnT-I79N is located in the cTnT N-terminal (TnT1) loop region and is known for its pathological and prognostic relevance. A recent structural study revealed that I79 is part of a hydrophobic interface between the TnT1 loop and actin, which stabilizes the relaxed (OFF) state of the cardiac thin filament. Given the importance of understanding the role of TnT1 loop region in Ca2+ regulation of the cardiac thin filament along with the underlying mechanisms of cTnT-I79N-linked pathogenesis, we investigated the effects of cTnT-I79N on cardiac myofilament function. Transgenic I79N (Tg-I79N) muscle bundles displayed increased myofilament Ca2+ sensitivity, smaller myofilament lattice spacing, and slower crossbridge kinetics. These findings can be attributed to destabilization of the cardiac thin filament's relaxed state resulting in an increased number of crossbridges during Ca2+ activation. Additionally, in the low Ca2+-relaxed state (pCa8), we showed that more myosin heads are in the disordered-relaxed state (DRX) that are more likely to interact with actin in cTnT-I79N muscle bundles. Dysregulation of the myosin super-relaxed state (SRX) and the SRX/DRX equilibrium in cTnT-I79N muscle bundles likely result in increased mobility of myosin heads at pCa8, enhanced actomyosin interactions as evidenced by increased active force at low Ca2+, and increased sinusoidal stiffness. These findings point to a mechanism whereby cTnT-I79N weakens the interaction of the TnT1 loop with the actin filament, which in turn destabilizes the relaxed state of the cardiac thin filament.
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Miofibrillas , Troponina T , Humanos , Miofibrillas/genética , Miofibrillas/patología , Troponina T/genética , Troponina T/química , Actinas/genética , Mutación , Citoesqueleto de Actina/genética , Miosinas , CalcioRESUMEN
BACKGROUND: Modulating myosin function is a novel therapeutic approach in patients with cardiomyopathy. Danicamtiv is a novel myosin activator with promising preclinical data that is currently in clinical trials. While it is known that danicamtiv increases force and cardiomyocyte contractility without affecting calcium levels, detailed mechanistic studies regarding its mode of action are lacking. METHODS: Permeabilized porcine cardiac tissue and myofibrils were used for X-ray diffraction and mechanical measurements. A mouse model of genetic dilated cardiomyopathy was used to evaluate the ability of danicamtiv to correct the contractile deficit. RESULTS: Danicamtiv increased force and calcium sensitivity via increasing the number of myosins in the ON state and slowing cross-bridge turnover. Our detailed analysis showed that inhibition of ADP release results in decreased cross-bridge turnover with cross bridges staying attached longer and prolonging myofibril relaxation. Danicamtiv corrected decreased calcium sensitivity in demembranated tissue, abnormal twitch magnitude and kinetics in intact cardiac tissue, and reduced ejection fraction in the whole organ. CONCLUSIONS: As demonstrated by the detailed studies of Danicamtiv, increasing myosin recruitment and altering cross-bridge cycling are 2 mechanisms to increase force and calcium sensitivity in cardiac muscle. Myosin activators such as Danicamtiv can treat the causative hypocontractile phenotype in genetic dilated cardiomyopathy.
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Cardiomiopatía Dilatada , Ratones , Animales , Porcinos , Cardiomiopatía Dilatada/tratamiento farmacológico , Calcio/fisiología , Miocardio , Miosinas , Miocitos Cardíacos , CardiotónicosRESUMEN
In this study, we investigated the role of the super-relaxed (SRX) state of myosin in the structure-function relationship of sarcomeres in the hearts of mouse models of cardiomyopathy-bearing mutations in the human ventricular regulatory light chain (RLC, MYL2 gene). Skinned papillary muscles from hypertrophic (HCM-D166V) and dilated (DCM-D94A) cardiomyopathy models were subjected to small-angle X-ray diffraction simultaneously with isometric force measurements to obtain the interfilament lattice spacing and equatorial intensity ratios (I11/I10) together with the force-pCa relationship over a full range of [Ca2+] and at a sarcomere length of 2.1 µm. In parallel, we studied the effect of mutations on the ATP-dependent myosin energetic states. Compared with wild-type (WT) and DCM-D94A mice, HCM-D166V significantly increased the Ca2+ sensitivity of force and left shifted the I11/I10-pCa relationship, indicating an apparent movement of HCM-D166V cross-bridges closer to actin-containing thin filaments, thereby allowing for their premature Ca2+ activation. The HCM-D166V model also disrupted the SRX state and promoted an SRX-to-DRX (super-relaxed to disordered relaxed) transition that correlated with an HCM-linked phenotype of hypercontractility. While this dysregulation of SRX â DRX equilibrium was consistent with repositioning of myosin motors closer to the thin filaments and with increased force-pCa dependence for HCM-D166V, the DCM-D94A model favored the energy-conserving SRX state, but the structure/function-pCa data were similar to WT. Our results suggest that the mutation-induced redistribution of myosin energetic states is one of the key mechanisms contributing to the development of complex clinical phenotypes associated with human HCM-D166V and DCM-D94A mutations.
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Miosinas Cardíacas/genética , Cardiomiopatías/metabolismo , Cadenas Ligeras de Miosina/genética , Actinas/metabolismo , Animales , Miosinas Cardíacas/metabolismo , Cardiomiopatías/genética , Cardiomiopatía Hipertrófica/genética , Modelos Animales de Enfermedad , Femenino , Humanos , Hipertrofia/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Mutación , Contracción Miocárdica/genética , Cadenas Ligeras de Miosina/metabolismo , Miosinas/metabolismo , Miosinas/fisiología , Fenotipo , Fosforilación , Sarcómeros/metabolismo , Relación Estructura-Actividad , Difracción de Rayos X/métodosRESUMEN
Skeletal muscle force production is increased at longer compared to shorter muscle lengths because of length-dependent priming of thick filament proteins in the contractile unit before contraction. Using small-angle X-ray diffraction in combination with a mouse model that specifically cleaves the stretch-sensitive titin protein, we found that titin cleavage diminished the length-dependent priming of the thick filament. Strikingly, a titin-sensitive, length-dependent priming was also present in thin filaments, which seems only possible via bridge proteins between thick and thin filaments in resting muscle, potentially myosin-binding protein C. We further show that these bridges can be forcibly ruptured via high-speed stretches. Our results advance a paradigm shift to the fundamental regulation of length-dependent priming, with titin as the key driver.
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Citoesqueleto de Actina , Sarcómeros , Ratones , Animales , Conectina/metabolismo , Sarcómeros/metabolismo , Citoesqueleto de Actina/metabolismo , Contracción Muscular/fisiología , Músculo Esquelético/metabolismo , Proteínas Quinasas/metabolismoRESUMEN
There is a growing appreciation that regulation of muscle contraction requires both thin filament and thick filament activation in order to fully activate the sarcomere. The prevailing mechano-sensing model for thick filament activation was derived from experiments on fast-twitch muscle. We address the question whether, or to what extent, this mechanism can be extrapolated to the slow muscle in the hearts of large mammals, including humans. We investigated the similarities and differences in structural signatures of thick filament activation in porcine myocardium as compared to fast rat extensor digitorum longus (EDL) skeletal muscle under relaxed conditions and sub-maximal contraction using small angle X-ray diffraction. Thick and thin filaments were found to adopt different structural configurations under relaxing conditions, and myosin heads showed different changes in configuration upon sub-maximal activation, when comparing the two muscle types. Titin was found to have an X-ray diffraction signature distinct from those of the overall thick filament backbone, and its spacing change appeared to be positively correlated to the force exerted on the thick filament. Structural changes in fast EDL muscle were found to be consistent with the mechano-sensing model. In porcine myocardium, however, the structural basis of mechano-sensing is blunted suggesting the need for additional activation mechanism(s) in slow cardiac muscle. These differences in thick filament regulation can be related to their different physiological roles where fast muscle is optimized for rapid, burst-like, contractions, and the slow cardiac muscle in large mammalian hearts adopts a more finely tuned, graded response to allow for their substantial functional reserve. KEY POINTS: Both thin filament and thick filament activation are required to fully activate the sarcomere. Thick and thin filaments adopt different structural configurations under relaxing conditions, and myosin heads show different changes in configuration upon sub-maximal activation in fast extensor digitorum longus muscle and slow porcine cardiac muscle. Titin has an X-ray diffraction signature distinct from those of the overall thick filament backbone and this titin reflection spacing change appeared to be directly proportional to the force exerted on the thick filament. Mechano-sensing is blunted in porcine myocardium suggesting the need for additional activation mechanism(s) in slow cardiac muscle. Fast skeletal muscle is optimized for rapid, burst-like contractions, and the slow cardiac muscle in large mammalian hearts adopts a more finely tuned graded response to allow for their substantial functional reserve.
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Miocardio , Animales , Porcinos , Miocardio/metabolismo , Conectina/metabolismo , Ratas , Masculino , Fibras Musculares de Contracción Rápida/fisiología , Fibras Musculares de Contracción Rápida/metabolismo , Sarcómeros/fisiología , Sarcómeros/metabolismo , Fibras Musculares de Contracción Lenta/fisiología , Fibras Musculares de Contracción Lenta/metabolismo , Músculo Esquelético/fisiología , Músculo Esquelético/metabolismo , Difracción de Rayos X , Contracción Muscular/fisiología , Miosinas/metabolismo , Miosinas/fisiologíaRESUMEN
BACKGROUND: Right ventricular (RV) contractile dysfunction commonly occurs and worsens outcomes in patients with heart failure with reduced ejection fraction and pulmonary hypertension (HFrEF-PH). However, such dysfunction often goes undetected by standard clinical RV indices, raising concerns that they may not reflect aspects of underlying myocyte dysfunction. We thus sought to characterize RV myocyte contractile depression in HFrEF-PH, identify those components reflected by clinical RV indices, and uncover underlying biophysical mechanisms. METHODS: Resting, calcium-, and load-dependent mechanics were prospectively studied in permeabilized RV cardiomyocytes isolated from explanted hearts from 23 patients with HFrEF-PH undergoing cardiac transplantation and 9 organ donor controls. RESULTS: Unsupervised machine learning using myocyte mechanical data with the highest variance yielded 2 HFrEF-PH subgroups that in turn mapped to patients with decompensated or compensated clinical RV function. This correspondence was driven by reduced calcium-activated isometric tension in decompensated clinical RV function, whereas surprisingly, many other major myocyte contractile measures including peak power and myocyte active stiffness were similarly depressed in both groups. Similar results were obtained when subgroups were first defined by clinical indices, and then myocyte mechanical properties in each group compared. To test the role of thick filament defects, myofibrillar structure was assessed by x-ray diffraction of muscle fibers. This revealed more myosin heads associated with the thick filament backbone in decompensated clinical RV function, but not compensated clinical RV function, as compared with controls. This corresponded to reduced myosin ATP turnover in decompensated clinical RV function myocytes, indicating less myosin in a crossbridge-ready disordered-relaxed (DRX) state. Altering DRX proportion (%DRX) affected peak calcium-activated tension in the patient groups differently, depending on their basal %DRX, highlighting potential roles for precision-guided therapeutics. Last, increasing myocyte preload (sarcomere length) increased %DRX 1.5-fold in controls but only 1.2-fold in both HFrEF-PH groups, revealing a novel mechanism for reduced myocyte active stiffness and by extension Frank-Starling reserve in human heart failure. CONCLUSIONS: Although there are many RV myocyte contractile deficits in HFrEF-PH, commonly used clinical indices only detect reduced isometric calcium-stimulated force, which is related to deficits in basal and recruitable %DRX myosin. Our results support use of therapies to increase %DRX and enhance length-dependent recruitment of DRX myosin heads in such patients.
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Insuficiencia Cardíaca , Hipertensión Pulmonar , Disfunción Ventricular Derecha , Humanos , Sarcómeros , Calcio , Depresión , Volumen Sistólico , Miocitos Cardíacos , Función Ventricular Derecha/fisiologíaRESUMEN
MuscleX is an integrated, open-source computer software suite for data reduction of X-ray fiber diffraction patterns from striated muscle and other fibrous systems. It is written in Python and runs on Linux, Microsoft Windows or macOS. Most modules can be run either from a graphical user interface or in a `headless mode' from the command line, suitable for incorporation into beamline control systems. Here, we provide an overview of the general structure of the MuscleX software package and describe the specific features of the individual modules as well as examples of applications.
RESUMEN
BACKGROUND: Altered kinase localization is gaining appreciation as a mechanism of cardiovascular disease. Previous work suggests GSK-3ß (glycogen synthase kinase 3ß) localizes to and regulates contractile function of the myofilament. We aimed to discover GSK-3ß's in vivo role in regulating myofilament function, the mechanisms involved, and the translational relevance. METHODS: Inducible cardiomyocyte-specific GSK-3ß knockout mice and left ventricular myocardium from nonfailing and failing human hearts were studied. RESULTS: Skinned cardiomyocytes from knockout mice failed to exhibit calcium sensitization with stretch indicating a loss of length-dependent activation (LDA), the mechanism underlying the Frank-Starling Law. Titin acts as a length sensor for LDA, and knockout mice had decreased titin stiffness compared with control mice, explaining the lack of LDA. Knockout mice exhibited no changes in titin isoforms, titin phosphorylation, or other thin filament phosphorylation sites known to affect passive tension or LDA. Mass spectrometry identified several z-disc proteins as myofilament phospho-substrates of GSK-3ß. Agreeing with the localization of its targets, GSK-3ß that is phosphorylated at Y216 binds to the z-disc. We showed pY216 was necessary and sufficient for z-disc binding using adenoviruses for wild-type, Y216F, and Y216E GSK-3ß in neonatal rat ventricular cardiomyocytes. One of GSK-3ß's z-disc targets, abLIM-1 (actin-binding LIM protein 1), binds to the z-disc domains of titin that are important for maintaining passive tension. Genetic knockdown of abLIM-1 via siRNA in human engineered heart tissues resulted in enhancement of LDA, indicating abLIM-1 may act as a negative regulator that is modulated by GSK-3ß. Last, GSK-3ß myofilament localization was reduced in left ventricular myocardium from failing human hearts, which correlated with depressed LDA. CONCLUSIONS: We identified a novel mechanism by which GSK-3ß localizes to the myofilament to modulate LDA. Importantly, z-disc GSK-3ß levels were reduced in patients with heart failure, indicating z-disc localized GSK-3ß is a possible therapeutic target to restore the Frank-Starling mechanism in patients with heart failure.
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Insuficiencia Cardíaca , Miocitos Cardíacos , Animales , Conectina/genética , Conectina/metabolismo , Glucógeno Sintasa Quinasa 3 beta/metabolismo , Insuficiencia Cardíaca/genética , Insuficiencia Cardíaca/metabolismo , Humanos , Ratones , Ratones Noqueados , Miocitos Cardíacos/metabolismo , Fosforilación , RatasRESUMEN
The atomic structure of the complete myosin tail within thick filaments isolated from Lethocerus indicus flight muscle is described and compared to crystal structures of recombinant, human cardiac myosin tail segments. Overall, the agreement is good with three exceptions: the proximal S2, in which the filament has heads attached but the crystal structure doesn't, and skip regions 2 and 4. At the head-tail junction, the tail α-helices are asymmetrically structured encompassing well-defined unfolding of 12 residues for one myosin tail, â¼4 residues of the other, and different degrees of α-helix unwinding for both tail α-helices, thereby providing an atomic resolution description of coiled-coil "uncoiling" at the head-tail junction. Asymmetry is observed in the nonhelical C termini; one C-terminal segment is intercalated between ribbons of myosin tails, the other apparently terminating at Skip 4 of another myosin tail. Between skip residues, crystal and filament structures agree well. Skips 1 and 3 also agree well and show the expected α-helix unwinding and coiled-coil untwisting in response to skip residue insertion. Skips 2 and 4 are different. Skip 2 is accommodated in an unusual manner through an increase in α-helix radius and corresponding reduction in rise/residue. Skip 4 remains helical in one chain, with the other chain unfolded, apparently influenced by the acidic myosin C terminus. The atomic model may shed some light on thick filament mechanosensing and is a step in understanding the complex roles that thick filaments of all species undergo during muscle contraction.
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Proteínas de Insectos/química , Miosina Tipo II/química , Animales , Microscopía por Crioelectrón , Hemípteros , Simulación de Dinámica Molecular , Músculo Esquelético/química , Músculo Esquelético/ultraestructura , Conformación Proteica en Hélice alfaRESUMEN
Fast skeletal myosin-binding protein-C (fMyBP-C) is one of three MyBP-C paralogs and is predominantly expressed in fast skeletal muscle. Mutations in the gene that encodes fMyBP-C, MYBPC2, are associated with distal arthrogryposis, while loss of fMyBP-C protein is associated with diseased muscle. However, the functional and structural roles of fMyBP-C in skeletal muscle remain unclear. To address this gap, we generated a homozygous fMyBP-C knockout mouse (C2-/-) and characterized it both in vivo and in vitro compared to wild-type mice. Ablation of fMyBP-C was benign in terms of muscle weight, fiber type, cross-sectional area, and sarcomere ultrastructure. However, grip strength and plantar flexor muscle strength were significantly decreased in C2-/- mice. Peak isometric tetanic force and isotonic speed of contraction were significantly reduced in isolated extensor digitorum longus (EDL) from C2-/- mice. Small-angle X-ray diffraction of C2-/- EDL muscle showed significantly increased equatorial intensity ratio during contraction, indicating a greater shift of myosin heads toward actin, while MLL4 layer line intensity was decreased at rest, indicating less ordered myosin heads. Interfilament lattice spacing increased significantly in C2-/- EDL muscle. Consistent with these findings, we observed a significant reduction of steady-state isometric force during Ca2+-activation, decreased myofilament calcium sensitivity, and sinusoidal stiffness in skinned EDL muscle fibers from C2-/- mice. Finally, C2-/- muscles displayed disruption of inflammatory and regenerative pathways, along with increased muscle damage upon mechanical overload. Together, our data suggest that fMyBP-C is essential for maximal speed and force of contraction, sarcomere integrity, and calcium sensitivity in fast-twitch muscle.
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Proteínas Portadoras/metabolismo , Contracción Muscular/fisiología , Fibras Musculares de Contracción Rápida/metabolismo , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Animales , Calcio/metabolismo , Contracción Isométrica/fisiología , Ratones , Fuerza Muscular , Músculo Esquelético/metabolismo , Miofibrillas/metabolismo , Miosinas/metabolismo , Sarcómeros/metabolismoRESUMEN
Arbuscular mycorrhizal fungi help their host plant in the acquisition of nutrients, and this association is itself impacted by soil nutrient levels. High phosphorus levels inhibit the symbiosis, whereas high nitrogen levels enhance it. The genetic mechanisms regulating the symbiosis in response to soil nutrients are poorly understood. Here, we characterised the symbiotic phenotypes in four Medicago truncatula Tnt1-insertion mutants affected in arbuscular mycorrhizal colonisation. We located their Tnt1 insertions and identified alleles for two genes known to be involved in mycorrhization, RAM1 and KIN3. We compared the effects of the kin3-2 and ram1-4 mutations on gene expression, revealing that the two genes alter the expression of overlapping but not identical gene sets, suggesting that RAM1 acts upstream of KIN3. Additionally, KIN3 appears to be involved in the suppression of plant defences in response to the fungal symbiont. KIN3 is located on the endoplasmic reticulum of arbuscule-containing cortical cells, and kin3-2 mutants plants hosted significantly fewer arbuscules than the wild type. KIN3 plays an essential role in the symbiotic response to soil nitrogen levels, as, contrary to wild-type plants, the kin3-2 mutant did not exhibit increased root colonisation under high nitrogen.
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Medicago truncatula , Micorrizas , Regulación de la Expresión Génica de las Plantas , Medicago truncatula/metabolismo , Micorrizas/metabolismo , Nitrógeno/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Raíces de Plantas/metabolismo , Suelo , Simbiosis/fisiologíaRESUMEN
The transcription factor NODULE INCEPTION (NIN) has been studied extensively for its multiple roles in root nodule symbiosis within plants of the nitrogen-fixing clade (NFC) that associate with soil bacteria, such as rhizobia and Frankia. However, NIN homologs are present in plants outside the NFC, suggesting a role in other developmental processes. Here, we show that the biofuel crop Populus sp., which is not part of the NFC, contains eight copies of NIN with diversified protein sequence and expression patterns. Lipo-chitooligosaccharides (LCOs) are produced by rhizobia and a wide range of fungi, including mycorrhizal ones, and act as symbiotic signals that promote lateral root formation. RNAseq analysis of Populus sp. treated with purified LCO showed induction of the PtNIN2 subfamily. Moreover, the expression of PtNIN2b correlated with the formation of lateral roots and was suppressed by cytokinin treatment. Constitutive expression of PtNIN2b overcame the inhibition of lateral root development by cytokinin under high nitrate conditions. Lateral root induction in response to LCOs likely represents an ancestral function of NIN retained and repurposed in nodulating plants, as we demonstrate that the role of NIN in LCO-induced root branching is conserved in both Populus sp. and legumes. We further established a visual marker of LCO perception in Populus sp. roots, the putative sulfotransferase PtSS1 that can be used to study symbiotic interactions with the bacterial and fungal symbionts of Populus sp.
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Populus , Rhizobium , Populus/genética , Populus/metabolismo , Regulación de la Expresión Génica de las Plantas , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Organogénesis de las Plantas , Simbiosis , Quitina/metabolismo , Citocininas , Raíces de Plantas/metabolismoRESUMEN
Most legumes can establish a symbiotic association with soil rhizobia that trigger the development of root nodules. These nodules host the rhizobia and allow them to fix nitrogen efficiently. The perception of bacterial lipo-chitooligosaccharides (LCOs) in the epidermis initiates a signaling cascade that allows rhizobial intracellular infection in the root and de-differentiation and activation of cell division that gives rise to the nodule. Thus, nodule organogenesis and rhizobial infection need to be coupled in space and time for successful nodulation. The plant hormone cytokinin (CK) contributes to the coordination of this process, acting as an essential positive regulator of nodule organogenesis. However, the temporal regulation of tissue-specific CK signaling and biosynthesis in response to LCOs or Sinorhizobium meliloti inoculation in Medicago truncatula remains poorly understood. In this study, using a fluorescence-based CK sensor (pTCSn::nls:tGFP), we performed a high-resolution tissue-specific temporal characterization of the sequential activation of CK response during root infection and nodule development in M. truncatula after inoculation with S. meliloti. Loss-of-function mutants of the CK-biosynthetic gene ISOPENTENYLTRANSFERASE 3 (IPT3) showed impairment of nodulation, suggesting that IPT3 is required for nodule development in M. truncatula. Simultaneous live imaging of pIPT3::nls:tdTOMATO and the CK sensor showed that IPT3 induction in the pericycle at the base of nodule primordium contributes to CK biosynthesis, which in turn promotes expression of positive regulators of nodule organogenesis in M. truncatula.
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Transferasas Alquil y Aril/metabolismo , Citocininas/genética , Citocininas/metabolismo , Medicago truncatula/genética , Medicago truncatula/fisiología , Nodulación de la Raíz de la Planta/genética , Nódulos de las Raíces de las Plantas/metabolismo , Simbiosis/genética , Transferasas Alquil y Aril/genética , Regulación de la Expresión Génica de las Plantas , Genes de Plantas , Fijación del Nitrógeno/genética , Fijación del Nitrógeno/fisiología , Organogénesis/genética , Raíces de Plantas/genética , Raíces de Plantas/metabolismo , Nódulos de las Raíces de las Plantas/genética , Nódulos de las Raíces de las Plantas/crecimiento & desarrollo , Sinorhizobium meliloti/fisiología , Simbiosis/fisiologíaRESUMEN
[Figure: see text].
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Diástole , Sarcómeros/fisiología , Adenosina Trifosfato/metabolismo , Animales , Masculino , Sarcómeros/metabolismo , Porcinos , Porcinos EnanosRESUMEN
Striated muscle contraction involves sliding of actin thin filaments along myosin thick filaments, controlled by calcium through thin filament activation. In relaxed muscle, the two heads of myosin interact with each other on the filament surface to form the interacting-heads motif (IHM). A key question is how both heads are released from the surface to approach actin and produce force. We used time-resolved synchrotron X-ray diffraction to study tarantula muscle before and after tetani. The patterns showed that the IHM is present in live relaxed muscle. Tetanic contraction produced only a very small backbone elongation, implying that mechanosensing-proposed in vertebrate muscle-is not of primary importance in tarantula. Rather, thick filament activation results from increases in myosin phosphorylation that release a fraction of heads to produce force, with the remainder staying in the ordered IHM configuration. After the tetanus, the released heads slowly recover toward the resting, helically ordered state. During this time the released heads remain close to actin and can quickly rebind, enhancing the force produced by posttetanic twitches, structurally explaining posttetanic potentiation. Taken together, these results suggest that, in addition to stretch activation in insects, two other mechanisms for thick filament activation have evolved to disrupt the interactions that establish the relaxed helices of IHMs: one in invertebrates, by either regulatory light-chain phosphorylation (as in arthropods) or Ca2+-binding (in mollusks, lacking phosphorylation), and another in vertebrates, by mechanosensing.
Asunto(s)
Músculo Estriado/fisiología , Miosinas/metabolismo , Fosforilación/fisiología , Citoesqueleto de Actina/química , Citoesqueleto de Actina/metabolismo , Animales , Artrópodos/fisiología , Evolución Molecular , Invertebrados/fisiología , Modelos Moleculares , Contracción Muscular , Relajación Muscular , Miosinas/química , Estructura Secundaria de Proteína , Arañas/fisiología , Vertebrados/fisiologíaRESUMEN
BACKGROUND: Symbiotic associations between bacteria and leguminous plants lead to the formation of root nodules that fix nitrogen needed for sustainable agricultural systems. Symbiosis triggers extensive genome and transcriptome remodeling in the plant, yet an integrated understanding of the extent of chromatin changes and transcriptional networks that functionally regulate gene expression associated with symbiosis remains poorly understood. In particular, analyses of early temporal events driving this symbiosis have only captured correlative relationships between regulators and targets at mRNA level. Here, we characterize changes in transcriptome and chromatin accessibility in the model legume Medicago truncatula, in response to rhizobial signals that trigger the formation of root nodules. RESULTS: We profiled the temporal chromatin accessibility (ATAC-seq) and transcriptome (RNA-seq) dynamics of M. truncatula roots treated with bacterial small molecules called lipo-chitooligosaccharides that trigger host symbiotic pathways of nodule development. Using a novel approach, dynamic regulatory module networks, we integrated ATAC-seq and RNA-seq time courses to predict cis-regulatory elements and transcription factors that most significantly contribute to transcriptomic changes associated with symbiosis. Regulators involved in auxin (IAA4-5, SHY2), ethylene (EIN3, ERF1), and abscisic acid (ABI5) hormone response, as well as histone and DNA methylation (IBM1), emerged among those most predictive of transcriptome dynamics. RNAi-based knockdown of EIN3 and ERF1 reduced nodule number in M. truncatula validating the role of these predicted regulators in symbiosis between legumes and rhizobia. CONCLUSIONS: Our transcriptomic and chromatin accessibility datasets provide a valuable resource to understand the gene regulatory programs controlling the early stages of the dynamic process of symbiosis. The regulators identified provide potential targets for future experimental validation, and the engineering of nodulation in species is unable to establish that symbiosis naturally.
Asunto(s)
Medicago truncatula , Medicago truncatula/genética , Medicago truncatula/metabolismo , Medicago truncatula/microbiología , Cromatina/genética , Cromatina/metabolismo , Regulación de la Expresión Génica de las Plantas , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Simbiosis/fisiologíaRESUMEN
Synchrotron small-angle X-ray diffraction is the method of choice for nm-scale structural studies of striated muscle under physiological conditions and on millisecond time scales. The lack of generally applicable computational tools for modeling X-ray diffraction patterns from intact muscles has been a significant barrier to exploiting the full potential of this technique. Here, we report a novel "forward problem" approach using the spatially explicit computational simulation platform MUSICO to predict equatorial small-angle X-ray diffraction patterns and the force output simultaneously from resting and isometrically contracting rat skeletal muscle that can be compared to experimental data. The simulation generates families of thick-thin filament repeating units, each with their individually predicted occupancies of different populations of active and inactive myosin heads that can be used to generate 2D-projected electron density models based on known Protein Data Bank structures. We show how, by adjusting only a few selected parameters, we can achieve a good correspondence between experimental and predicted X-ray intensities. The developments presented here demonstrate the feasibility of combining X-ray diffraction and spatially explicit modeling to form a powerful hypothesis-generating tool that can be used to motivate experiments that can reveal emergent properties of muscle.