Myosin Filaments Research Articles

Graded Titin Cleavage Uncovers the Protein's Role for Sarcomere Structure and Force Generation of Contracting Muscle

The giant muscle protein titin is a major contributor to passive force; however, its role during active force generation is unresolved. Here, we use a novel titin-cleavage (TC) mouse model that allows specific and rapid cutting of the titin springs to quantify how titin-based forces define myocyte ultrastructure and mechanics. We conduct a series of passive and active mechanical tests on permeabilized psoas fiber bundles from wildtype, heterozygote, and homozygote TC mice. We show that under mechanical strain, as titin cleavage doubles from heterozygous to homozygous TC muscles, Z-disks become increasingly non-linear, while passive and active forces are progressively reduced. Furthermore, interactions of elastic titin with sarcomeric actin filaments are revealed, as cleaved I-band titins only partially recoil to the Z-disk, and instead stick to the thin filaments. Strikingly, when titin-cleaved fibers contract, myosin-containing A-bands quickly stream to the point that they split and adjacent thick filaments move in opposite directions. Cleavage of I-band titin also destabilizes the myosin filaments during contraction, which leads to a shedding of individual myosin rods from the D-zone of thick filaments. Taken together, these results establish intact titin filaments as critical force-transmission networks, buffering the forces between myosin filaments during contraction, while also stabilizing the myosin rods within the thick filament. Based on our calculations, a structural change in titin that increases stiffness compared to passive muscle is critical for titin to perform these buffering tasks, unveiling its fundamental role as an activation-dependent spring in contracting muscle.

Dynamics of change of biochemical and functional-technological properties of lake’s trout meat in storage

The leading role in muscle contraction is played by myofibrils, which, from the substances that make up the sarcoplasm, use the necessary energy to perform their functions. The source of energy, in this case, is only the biochemical processes of enzymatic decomposition (hydrolysis, phosphorolysis) of certain substances, in particular, glycogen nucleoside phosphates, located mainly in the sarcoplasm. In the muscle tissue of fish, the bulk of the energy needed to contract muscles is released as a result of ATP breakdown. By the outward manifestation and chemistry of the processes, post mortem rigour Mortis is almost identical to the motor contraction of the living muscle. The molecular mechanism of muscle contraction is based on the interaction of myosin and actin filaments. Studies have shown that the intense breakdown of muscle glycogen leads to a sharp decrease in the pH of muscle tissue in the acidic direction, which in turn affects the chemical composition and physical and colloidal structure of proteins, resulting in increased resistance of fish meat to the action of putrefactive microorganisms; decreased solubility of muscle proteins, their hydration, water-binding ability; swelling of connective tissue collagen; increased activity causing hydrolysis of proteins in the later stages of autolysis; the bicarbonate system of muscle tissue is destroyed with the release of carbon dioxide; precursors of meat taste and aroma are formed; the process of lipid oxidation is activated. As a result of the accumulation of lactic, phosphoric and other acids in fish meat, the concentration of hydrogen ions increases resulting in a decrease in pH.

Structural Changes in the Myosin Motors Induced by Stretch and Phosphorylation of the Myosin Regulatory Light Chain in Rat Cardiac Trabeculae

The contractility of cardiac muscle is potentiated by phosphorylation of the myosin regulatory light chain (RLC) and by length-dependent activation, but the molecular pathways mediating these effects remain unclear. Here we studied the role of structural changes in the thick filament in the activation of cardiac muscle by stretch and RLC phosphorylation. We used fluorescence polarization from bifunctional sulphorhodamine probes on the N- and C-lobes of the myosin RLC to monitor changes in the orientation of the myosin motors induced by increasing the sarcomere length in the range 2.0-2.3 µm in relaxed and partially calcium-activated demembranated cardiac trabeculae, at different levels of RLC phosphorylation. We show that under relaxing or diastolic conditions at near physiological temperature and lattice spacing the RLC lobe orientations are consistent with about one third of the myosin motors being folded onto the filament surface in the interacting-heads motif seen in isolated filaments. This folded conformation is disrupted by cooling relaxed trabecula, but not by RLC phosphorylation or stretch at diastolic [Ca2+]. These results indicate that in near-physiological conditions the myosin filament is not switched on by RLC phosphorylation or by the titin-based passive tension at longer sarcomere lengths in diastole, as suggested previously. However, stretching cardiac trabecula at near-systolic [Ca2+] triggers a stress-dependent activation of the thick filament and a delayed increase in active force. Physiological levels of RLC phosphorylation potentiate both the stress-dependent release of myosin motors from the folded state and the stretch-activated force. We conclude that the increase in heart contractility induced by RLC phosphorylation and stretch can be explained by an inter-filament signaling pathway involving both thin filament sensitization and thick filament mechano-sensing (Supported by Wellcome Trust, BHF, UK).

Troponin -T as a prognostic and diagnostic marker for myocardial infarction

Over the past decade, there has been a progressive evolution of cardiac marker testing in patients with acute coronary syndromes. Myocardial infarction (MI) is the leading cause of death in the developed world. Biomarkers have an vital role in analysis, risk stratification, administrative running and medical assessment making in the setting of patients presenting with signs and symptoms of MI. Cardiac troponin (cTn) rose to prominence during the 1990s and has evolved to be the cornerstone for diagnosis of MI. Cardiac troponins are released from myocytes following myocardial damage and loss of membrane integrity. Troponin T are member of a group of cardiac regulatory proteins which function to regulate the calcium mediated interaction of muscle filaments actin and myosin resulting in contraction and relaxation of striated muscle. New algorithms integrating Brain natriuretic peptide (BNP), NT-proBNP, and more sensitive cTn assays footing potential for more rapid diagnosis of MI, allowing for appropriate management steps to be initiated and more efficient and effective utilization of healthcare resources.

Evidence for S2 flexibility by direct visualization of quantum dot-labeled myosin heads and rods within smooth muscle myosin filaments moving on actin in vitro.

Myosins in muscle assemble into filaments by interactions between the C-terminal light meromyosin (LMM) subdomains of the coiled-coil rod domain. The two head domains are connected to LMM by the subfragment-2 (S2) subdomain of the rod. Our mixed kinetic model predicts that the flexibility and length of S2 that can be pulled away from the filament affects the maximum distance working heads can move a filament unimpeded by actin-attached heads. It also suggests that it should be possible to observe a head remain stationary relative to the filament backbone while bound to actin (dwell), followed immediately by a measurable jump upon detachment to regain the backbone trajectory. We tested these predictions by observing filaments moving along actin at varying ATP using TIRF microscopy. We simultaneously tracked two different color quantum dots (QDs), one attached to a regulatory light chain on the lever arm and the other attached to an LMM in the filament backbone. We identified events (dwells followed by jumps) by comparing the trajectories of the QDs. The average dwell times were consistent with known kinetics of the actomyosin system, and the distribution of the waiting time between observed events was consistent with a Poisson process and the expected ATPase rate. Geometric constraints suggest a maximum of ∼26 nm of S2 can be unzipped from the filament, presumably involving disruption in the coiled-coil S2, a result consistent with observations by others of S2 protruding from the filament in muscle. We propose that sufficient force is available from the working heads in the filament to overcome the stiffness imposed by filament-S2 interactions.

Myosin 18Aα targets the guanine nucleotide exchange factor β-Pix to the dendritic spines of cerebellar Purkinje neurons and promotes spine maturation.

Myosin 18Aα is a myosin 2-like protein containing unique N- and C-terminal protein interaction domains that co-assembles with myosin 2. One protein known to bind to myosin 18Aα is β-Pix, a guanine nucleotide exchange factor (GEF) for Rac1 and Cdc42 that has been shown to promote dendritic spine maturation by activating the assembly of actin and myosin filaments in spines.Here, we show that myosin 18A⍺ concentrates in the spines of cerebellar Purkinje neurons via co-assembly with myosin 2 and through an actin binding site in its N-terminal extension. miRNA-mediated knockdown of myosin 18A⍺ results in a significant defect in spine maturation that is rescued by an RNAi-immune version of myosin 18A⍺.Importantly, β-Pix co-localizes with myosin 18A⍺ in spines, and its spine localization is lost upon myosin 18A⍺ knockdown or when its myosin 18A⍺ binding site is deleted.Finally, we show that the spines of myosin 18A⍺ knockdown Purkinje neurons contain significantly less F-actin and myosin 2. Together, these data argue that mixed filaments of myosin 2 and myosin 18A⍺ form a complex with β-Pix in Purkinje neuron spines that promotes spine maturation by enhancing the assembly of actin and myosin filaments downstream of β-Pix's GEF activity.

Graded titin cleavage progressively reduces tension and uncovers the source of A-band stability in contracting muscle.

The giant muscle protein titin is a major contributor to passive force; however, its role in active force generation is unresolved. Here, we use a novel titin-cleavage (TC) mouse model that allows specific and rapid cutting of elastic titin to quantify how titin-based forces define myocyte ultrastructure and mechanics. We show that under mechanical strain, as TC doubles from heterozygous to homozygous TC muscles, Z-disks become increasingly out of register while passive and active forces are reduced. Interactions of elastic titin with sarcomeric actin filaments are revealed. Strikingly, when titin-cleaved muscles contract, myosin-containing A-bands become split and adjacent myosin filaments move in opposite directions while also shedding myosins. This establishes intact titin filaments as critical force-transmission networks, buffering the forces observed by myosin filaments during contraction. To perform this function, elastic titin must change stiffness or extensible length, unveiling its fundamental role as an activation-dependent spring in contracting muscle.

Defects in the Existing Theory of Skeletal Muscle Contraction and Postulation of a New Theory

In 1954 , two independent research teams, one consisting of Andrew F. Huxley and Rolf Niedergerke from the University of Cambridge, and the other consisting of Hugh Huxley and Jean Hanson from the Massachusetts Institute of Technology proposed the theory of skeletal muscle contraction [1]. They used electron microscopy to study the details of muscle filaments. The structure was studied in detail by then, but the mechanism of skeletal muscle contraction was not defined. Based on various assumptions about the actin and myosin filaments of muscle, later they postulated a theory called “sliding filament theory”. When this theory is scrutinized in detail, I find that there are a lot of defects in this theory, which I have pointed out and I have made an attempt to postulate a different mechanism for the skeletal muscle contraction.

Sarcomere length non-uniformities dictate force production along the descending limb of the force-length relation.

The force-length relation is one of the most defining features of muscle contraction, and yet a topic of debate in the literature. The sliding filament theory predicts that the force produced by muscle fibres is proportional to the degree of overlap between myosin and actin filaments, producing a linear descending limb of the active force-length relation. However, several studies have shown forces that are larger than predicted, especially at long sarcomere lengths (SLs). Studies have been conducted with muscle fibres, preparations containing thousands of sarcomeres that make measurements of individual SL challenging. The aim of this study was to evaluate force production and sarcomere dynamics in isolated myofibrils and single sarcomeres from the rabbit psoas muscle to enhance our understanding of the theoretically predicted force-length relation. Contractions at varying SLs along the plateau (SL = 2.25-2.39 µm) and the descending limb (SL > 2.39 µm) of the force-length relation were induced in sarcomeres and myofibrils, and different modes of force measurements were used. Our results show that when forces are measured in single sarcomeres, the experimental force-length relation follows theoretical predictions. When forces are measured in myofibrils with large SL dispersions, there is an extension of the plateau and forces elevated above the predicted levels along the descending limb. We also found an increase in SL non-uniformity and slowed rates of force production at long lengths in myofibrils but not in single sarcomere preparations. We conclude that the deviation of the descending limb of the force-length relation is correlated with the degree of SL non-uniformity and slowed force development.

S100A4 is activated by RhoA and catalyses the polymerization of non-muscle myosin, adhesion complex assembly and contraction in airway smooth muscle.

S100A4 is expressed in many tissues, including smooth muscle (SM), but its physiologic function is unknown. S100A4 regulates the motility of metastatic cancer cells by binding to non-muscle (NM) myosin II. Contractile stimulation causes the polymerization of NM myosin in airway SM, which is necessary for tension development. NM myosin regulates the assembly of adhesion junction signalling complexes (adhesomes) that catalyse actin polymerization. In airway SM, ACh (acetylcholine) stimulated the binding of S100A4 to the NM myosin heavy chain, which was catalysed by RhoA GTPase via the RhoA-binding protein, rhotekin. The binding of S100A4 to NM myosin was required for NM myosin polymerization, adhesome assembly and actin polymerization. S100A4 plays a critical function in the regulation of airway SM contraction by catalysing NM myosin filament assembly. The interaction of S100A4 with NM myosin may also play an important role in the physiologic function of other tissues. S100A4 binds to the heavy chain of non-muscle (NM) myosin II and can regulate the motility of crawling cells. S100A4 is widely expressed in many tissues including smooth muscle (SM), although its role in the regulation of their physiologic function is not known. We hypothesized that S100A4 contributes to the regulation of contraction in airway SM by regulating a pool of NM myosin II at the cell cortex. NM myosin II undergoes polymerization in airway SM and regulates contraction by catalysing the assembly of integrin-associated adhesome complexes that activate pathways that catalyse actin polymerization. ACh stimulated the interaction of S100A4 with NM myosin II in airway SM at the cell cortex and catalysed NM myosin filament assembly. RhoA GTPase regulated the activation of S100A4 via rhotekin, which facilitated the formation of a complex between RhoA, S100A4 and NM myosin II. The depletion of S100A4, RhoA or rhotekin from airway SM tissues using short hairpin RNA or small interfering RNA prevented NM myosin II polymerization as well as the recruitment of vinculin and paxillin to adhesome signalling complexes in response to ACh, and inhibited actin polymerization and tension development. S100A4 depletion did not affect ACh-stimulated SM myosin regulatory light chain phosphorylation. The results show that S100A4 plays a critical role in tension development in airway SM tissue by catalysing NM myosin filament assembly, and that the interaction of S100A4 with NM myosin in response to contractile stimulation is activated by RhoA GTPase. These results may be broadly relevant to the physiologic function of S100A4 in other cell and tissue types.

MICAL2 is essential for myogenic lineage commitment

Contractile myofiber units are mainly composed of thick myosin and thin actin (F-actin) filaments. F-Actin interacts with Microtubule Associated Monooxygenase, Calponin And LIM Domain Containing 2 (MICAL2). Indeed, MICAL2 modifies actin subunits and promotes actin filament turnover by severing them and preventing repolymerization. In this study, we found that MICAL2 increases during myogenic differentiation of adult and pluripotent stem cells (PSCs) towards skeletal, smooth and cardiac muscle cells and localizes in the nucleus of acute and chronic regenerating muscle fibers. In vivo delivery of Cas9–Mical2 guide RNA complexes results in muscle actin defects and demonstrates that MICAL2 is essential for skeletal muscle homeostasis and functionality. Conversely, MICAL2 upregulation shows a positive impact on skeletal and cardiac muscle commitments. Taken together these data demonstrate that modulations of MICAL2 have an impact on muscle filament dynamics and its fine-tuned balance is essential for the regeneration of muscle tissues.

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.

Muscle myosins form folded monomers, dimers, and tetramers during filament polymerization in vitro

Muscle contraction depends on the cyclical interaction of myosin and actin filaments. Therefore, it is important to understand the mechanisms of polymerization and depolymerization of muscle myosins. Muscle myosin 2 monomers exist in two states: one with a folded tail that interacts with the heads (10S) and one with an unfolded tail (6S). It has been thought that only unfolded monomers assemble into bipolar and side-polar (smooth muscle myosin) filaments. We now show by electron microscopy that, after 4 s of polymerization in vitro in both the presence (smooth muscle myosin) and absence of ATP, skeletal, cardiac, and smooth muscle myosins form tail-folded monomers without tail-head interaction, tail-folded antiparallel dimers, tail-folded antiparallel tetramers, unfolded bipolar tetramers, and small filaments. After 4 h, the myosins form thick bipolar and, for smooth muscle myosin, side-polar filaments. Nonphosphorylated smooth muscle myosin polymerizes in the presence of ATP but with a higher critical concentration than in the absence of ATP and forms only bipolar filaments with bare zones. Partial depolymerization in vitro of nonphosphorylated smooth muscle myosin filaments by the addition of MgATP is the reverse of polymerization.

Deconstructing sarcomeric structure\u2013function relations in titin-BioID knock-in mice

Proximity proteomics has greatly advanced the analysis of native protein complexes and subcellular structures in culture, but has not been amenable to study development and disease in vivo. Here, we have generated a knock-in mouse with the biotin ligase (BioID) inserted at titin’s Z-disc region to identify protein networks that connect the sarcomere to signal transduction and metabolism. Our census of the sarcomeric proteome from neonatal to adult heart and quadriceps reveals how perinatal signaling, protein homeostasis and the shift to adult energy metabolism shape the properties of striated muscle cells. Mapping biotinylation sites to sarcomere structures refines our understanding of myofilament dynamics and supports the hypothesis that myosin filaments penetrate Z-discs to dampen contraction. Extending this proof of concept study to BioID fusion proteins generated with Crispr/CAS9 in animal models recapitulating human pathology will facilitate the future analysis of molecular machines and signaling hubs in physiological, pharmacological, and disease context.

Bioengineered Skeletal Muscle as a Model of Muscle Aging and Regeneration.

With age, adult skeletal muscle (SkM) is known to decrease in muscle mass, strength, and functional capacity, a state known as sarcopenia. Here we developed an in vitro three-dimensional (3D) bioengineered senescent SkM tissue using primary human myoblasts. These tissues exhibited the characteristics of atrophied muscle, including expression of senescent genes, decreased number of satellite cells, reduced number and size of myofibers, and compromised metabolism and calcium flux. As a result, senescent SkM tissues showed impaired ability to generate force in response to electrical stimulation compared with young tissues. Furthermore, in contrast to young SkM tissues, senescent tissues failed to regenerate in response to injury, possibly as a result of persistent apoptosis and failure to initiate a proliferation program. Our findings suggest that 3D senescent SkM may provide a powerful model for studying aging and a platform for drug testing and discovery of therapeutic compounds to improve the function of sarcopenic muscle. Impact statement Skeletal muscle (SkM) plays important physiological roles and has significant regenerative capacity. However, aged SkM lose their functionality and regeneration ability. In this article, we present a senescent human bioengineering SkM tissue model that can be used to investigate senescence, metabolic or genetic diseases that inflict SkM, and to test various strategies including novel small molecules that restore muscle function and promote regeneration. One key limitation of two-dimensional cell culture system is the detachment of contractile myotubes from the surface over time, thereby limiting the evaluation of myogenic function. Here we use primary human myoblasts, which exhibit all major hallmarks of aging to mimic the organization and function of native muscle. Using this system, we were able to measure the contractile function, calcium transients, and regeneration capacity of SkM tissues. We also evaluated the response of senescent SkM tissues to injury and their ability to regenerate and recover, compared with "young" tissues. Our results suggest that three-dimensional constructs enable organization of contractile units including myosin and actin filaments, thereby providing a powerful platform for the quantitative assessment of muscle myotubes in response to injury, genetic or metabolic disorders, or pharmacological testing.

Regulation of Actin Filament Length by Muscle Isoforms of Tropomyosin and Cofilin.

In striated muscle the extent of the overlap between actin and myosin filaments contributes to the development of force. In slow twitch muscle fibers actin filaments are longer than in fast twitch fibers, but the mechanism which determines this difference is not well understood. We hypothesized that tropomyosin isoforms Tpm1.1 and Tpm3.12, the actin regulatory proteins, which are specific respectively for fast and slow muscle fibers, differently stabilize actin filaments and regulate severing of the filaments by cofilin-2. Using in vitro assays, we showed that Tpm3.12 bound to F-actin with almost 2-fold higher apparent binding constant (Kapp) than Tpm1.1. Cofilin2 reduced Kapp of both tropomyosin isoforms. In the presence of Tpm1.1 and Tpm3.12 the filaments were longer than unregulated F-actin by 25% and 40%, respectively. None of the tropomyosins affected the affinity of cofilin-2 for F-actin, but according to the linear lattice model both isoforms increased cofilin-2 binding to an isolated site and reduced binding cooperativity. The filaments decorated with Tpm1.1 and Tpm3.12 were severed by cofilin-2 more often than unregulated filaments, but depolymerization of the severed filaments was inhibited. The stabilization of the filaments by Tpm3.12 was more efficient, which can be attributed to lower dynamics of Tpm3.12 binding to actin.

Tyrosine Phosphorylation of the Myosin Regulatory Light Chain Controls Non-muscle Myosin II Assembly and Function in Migrating Cells

Active non-muscle myosin II (NMII) enables migratory cell polarization and controls dynamic cellular processes, such as focal adhesion formation and turnover and cell division. Filament assembly and force generation depend on NMII activation through the phosphorylation of Ser19 of the regulatory light chain (RLC). Here, we identify amino acid Tyr (Y) 155 of the RLC as a novel regulatory site that spatially controls NMII function. We show that Y155 is phosphorylated invitro by the Tyr kinase domain of epidermal growth factor (EGF) receptor. In cells, phosphorylation of Y155, or its phospho-mimetic mutation (Glu), prevents the interaction of RLC with the myosin heavy chain (MHCII) to form functional NMII units. Conversely, Y155 mutation to a structurally similar but non-phosphorylatable amino acid (Phe) restores the more dynamic cellular functions of NMII, such as myosin filament formation and nascent adhesion assembly, but not those requiring stable actomyosin bundles, e.g., focal adhesion elongation or migratory front-back polarization. In live cells, phospho-Y155 RLC is prominently featured in protrusions, where it prevents NMII assembly. Our data indicate that Y155 phosphorylation constitutes a novel regulatory mechanism that contributes to the compartmentalization of NMII assembly and function in live cells.

Evolution of Histology Lab with Interactive Panel

IntroductionTechnology assisted learning has gained popularity among medical educators and students for various beneficial reasons. It provides a quick way of assessing student knowledge, provides instant feedback, and has the potential to modify student learning strategies. The objective of this study is to examine the implementation of an Interactive Flat Panel into a student‐centered virtual histology lab environment, while encouraging involvement of all types of leaners with student accustomed technology.MethodologyA 75” Interactive Flat Panel was used to create an interactive forum between groups of students learning virtual histology. Students were asked to sit at different tables as groups marked with a specific color. Content on the Interactive Flat Panel was projected to individual student devices via a URL with a room code or QR code. Prompts were assigned by a faculty member for images displayed on the Interactive Flat Panel. Groups were asked to identify different structures while other groups were asked to comment.ResultsDuring the musculoskeletal system course, a muscle histology lab session was conducted with one faculty and thirty‐six students organized into six groups. The lab session was designed into three segments. The first segment (15 minutes) consisted of faculty instructions on an Interactive Flat Panel using various histology websites. During the next segment, faculty displayed saved light and electron microscopic images on the Interactive Flat Panel’s white board app and instructed each group to identify and outline important microscopic structures in muscles: sarcomere, actin filaments, myosin filaments , Z‐lines, T‐Tubules, H, A, I bands.Identification markers were placed by group leaders on histology photos that was displayed on the Interactive Flat Panel for other groups to view. The other groups were instructed to post questions or comments via a digital sticky note on the right‐hand side of the screen. All the annotations, comments and images were instantly projected back on the Interactive Flat Panel for the whole lab to view. This interaction encouraged student involvement and interactivity. Faculty were able to instantly address all questions displayed. The comment‐initiated group discussions were easily facilitated by a single faculty member.This pedagogy helped in identifying student misconceptions and engaged sidelined students by forming a new communication setting between groups of students and one faculty. Initial survey results of faculty and ITTS staff are encouraging regarding the learning experience, training and instructional benefits. Initial surveys were taken, and students’ perception were found favorable to using the Interactive Flat Panel when learning histology in a lab environment. We are also in contact with the manufacturer BenQ to configure certain applications to enhance education in a medical school laboratory setting.ConclusionWith the use of an Interactive Flat Panel, students were able to manipulate content and clarify misconceptions of histology, which shifted the paradigm from teaching to learning. This form of technology is an opportunity for sideliners and outliers to be involved with group discussions.

Myosin filament-based regulation of the dynamics of contraction in heart muscle

Myosin-based mechanisms are increasingly recognized as supplementing their better-known actin-based counterparts to control the strength and time course of contraction in both skeletal and heart muscle. Here we use synchrotron small-angle X-ray diffraction to determine the structural dynamics of local domains of the myosin filament during contraction of heart muscle. We show that, although myosin motors throughout the filament contribute to force development, only about 10% of the motors in each filament bear the peak force, and these are confined to the filament domain containing myosin binding protein-C, the "C-zone." Myosin motors in domains further from the filament midpoint are likely to be activated and inactivated first in each contraction. Inactivated myosin motors are folded against the filament core, and a subset of folded motors lie on the helical tracks described previously. These helically ordered motors are also likely to be confined to the C-zone, and the associated motor conformation reforms only slowly during relaxation. Myosin filament stress-sensing determines the strength and time course of contraction in conjunction with actin-based regulation. These results establish the fundamental roles of myosin filament domains and the associated motor conformations in controlling the strength and dynamics of contraction in heart muscle, enabling those structures to be targeted to develop new therapies for heart disease.

SUMO system - a key regulator in sarcomere organization.

Skeletal muscles constitute roughly 40% of human body mass. Muscles are specialized tissues that generate force to drive movements through ATP-driven cyclic interactions between the protein filaments, namely actin and myosin filaments. The filaments are organized in an intricate structure called the 'sarcomere', which is a fundamental contractile unit of striated skeletal and cardiac muscle, hosting a fine assembly of macromolecular protein complexes. The micrometer-sized sarcomere units are arranged in a reiterated array within myofibrils of muscle cells. The precise spatial organization of sarcomere is tightly controlled by several molecular mechanisms, indispensable for its force-generating function. Disorganized sarcomeres, either due to erroneous molecular signaling or due to mutations in the sarcomeric proteins, lead to human diseases such as cardiomyopathies and muscle atrophic conditions prevalent in cachexia. Protein post-translational modifications (PTMs) of the sarcomeric proteins serve a critical role in sarcomere formation (sarcomerogenesis), as well as in the steady-state maintenance of sarcomeres. PTMs such as phosphorylation, acetylation, ubiquitination, and SUMOylation provide cells with a swift and reversible means to adapt to an altered molecular and therefore cellular environment. Over the past years, SUMOylation has emerged as a crucial modification with implications for different aspects of cell function, including organizing higher-order protein assemblies. In this review, we highlight the fundamentals of the small ubiquitin-like modifiers (SUMO) pathway and its link specifically to the mechanisms of sarcomere assembly. Furthermore, we discuss recent studies connecting the SUMO pathway-modulated protein homeostasis with sarcomere organization and muscle-related pathologies.