Non-muscle Cells Research Articles

Considering the downregulation of Tpm1.6 and Tpm1.7 in squamous cell carcinoma of esophagus as a potent biomarker.

Squamous cell carcinoma of esophagus (SCCE) is an aggressive disease with a poor prognosis. Tropomyosins attach to actin microfilaments, providing its stability. Nonmuscle cells express Tpm isoforms such as Tpm1.6 and Tpm1.7 which are involved in cytoskeleton functional properties regulation. The expression of Tpm1.6 and Tpm1.7 was analyzed in SCCE tissues and its association with clinicopathological parameters and survival of patients was assessed. Tpm1.6 and Tpm1.7, besides TPM1 mRNA decreased considerably in SCCE tissues relative to normal esophageal tissues (p<0.001). TPM1 downregulation level was significantly associated with the degree of tumor differentiation (p=0.017). Tpm1.6 and Tpm1.7 suppression play a crucial role in esophagus tumorigenesis and could be associated with SCCE poor prognosis.

Cellular homeostatic tension and force transmission measured in human engineered tendon

Tendons transmit contractile muscular force to bone to produce movement, and it is believed cells can generate endogenous forces on the extracellular matrix to maintain tissue homeostasis. However, little is known about the direct mechanical measurement of cell-matrix interaction in cell-generated human tendon constructs. In this study we examined if cell-generated force could be detected and quantified in engineered human tendon constructs, and if glycosaminoglycans (GAGs) contribute to tendon force transmission. Following de-tensioning of the tendon constructs it was possible to quantify an endogenous re-tensioning. Further, it was demonstrated that the endogenous re-tensioning response was markedly blunted after interference with the cytoskeleton (inhibiting non-muscle myosin-dependent cell contraction by blebbistatin), which confirmed that re-tensioning was cell generated. When the constructs were elongated and held at a constant length a stress relaxation response was quantified, and removing 27% of the GAG content of tendon did not alter the relaxation behavior, which indicates that GAGs do not play a meaningful role in force transmission within this system.

Blebbistatin Effects Expose Hidden Secrets in the Force-Generating Cycle of Actin and Myosin

Cyclic interactions between myosin II motors and actin filaments driven by ATP turnover underlie muscle contraction and have key roles in the motility of nonmuscle cells. A remaining enigma in the understanding of this interaction is the relationship between the force-generating structural change and the release of the ATP-hydrolysis product, inorganic phosphate (Pi), from the active site of myosin. Here, we use the small molecular compound blebbistatin to probe otherwise hidden states and transitions in this process. Different hypotheses for the Pi release mechanism are tested by interpreting experimental results from in vitro motility assays and isolated muscle fibers in terms of mechanokinetic actomyosin models. The data fit with ideas that actomyosin force generation is preceded by Pi release, which in turn is preceded by two serial transitions after/coincident with cross-bridge attachment. Blebbistatin changes the rate limitation of the cycle from the first to the second of these transitions, uncovering functional roles of an otherwise short-lived pre-power stroke state that has been implicated by structural data.

Inactivation of glycogen synthase kinase 3β (GSK-3β) enhances mitochondrial biogenesis during myogenesis

BackgroundMitochondrial biogenesis is crucial for myogenic differentiation and regeneration of skeletal muscle tissue and is tightly controlled by the peroxisome proliferator-activated receptor-γ co-activator 1 (PGC-1) signaling network. In the present study, we hypothesized that inactivation of glycogen synthase kinase (GSK)-3β, previously suggested to interfere with PGC-1 in non-muscle cells, potentiates PGC-1 signaling and the development of mitochondrial biogenesis during myogenesis, ultimately resulting in an enhanced myotube oxidative capacity. MethodsGSK-3β was inactivated genetically or pharmacologically during myogenic differentiation of C2C12 muscle cells. In addition, m. gastrocnemius tissue was collected from wild-type and muscle-specific GSK-3β knock-out (KO) mice at different time-points during the reloading/regeneration phase following a 14-day hind-limb suspension period. Subsequently, expression levels of constituents of the PGC-1 signaling network as well as key parameters of mitochondrial oxidative metabolism were investigated. ResultsIn vitro, both knock-down as well as pharmacological inhibition of GSK-3β not only increased expression levels of important constituents of the PGC-1 signaling network, but also potentiated myogenic differentiation-associated increases in mitochondrial respiration, mitochondrial DNA copy number, oxidative phosphorylation (OXPHOS) protein abundance and the activity of key enzymes involved in the Krebs cycle and fatty acid β-oxidation. In addition, GSK-3β KO animals showed augmented reloading-induced increases in skeletal muscle gene expression of constituents of the PGC-1 signaling network as well as sub-units of OXPHOS complexes compared to wild-type animals. ConclusionInactivation of GSK-3β stimulates activation of PGC-1 signaling and mitochondrial biogenesis during myogenic differentiation and reloading of the skeletal musculature.

Ordering of myosin II filaments driven by mechanical forces: experiments and theory

Myosin II filaments form ordered superstructures in both cross-striated muscle and non-muscle cells. In cross-striated muscle, myosin II (thick) filaments, actin (thin) filaments and elastic titin filaments comprise the stereotypical contractile units of muscles called sarcomeres. Linear chains of sarcomeres, called myofibrils, are aligned laterally in registry to form cross-striated muscle cells. The experimentally observed dependence of the registered organization of myofibrils on extracellular matrix elasticity has been proposed to arise from the interactions of sarcomeric contractile elements (considered as force dipoles) through the matrix. Non-muscle cells form small bipolar filaments built of less than 30 myosin II molecules. These filaments are associated in registry forming superstructures ('stacks') orthogonal to actin filament bundles. Formation of myosin II filament stacks requires the myosin II ATPase activity and function of the actin filament crosslinking, polymerizing and depolymerizing proteins. We propose that the myosin II filaments embedded into elastic, intervening actin network (IVN) function as force dipoles that interact attractively through the IVN. This is in analogy with the theoretical picture developed for myofibrils where the elastic medium is now the actin cytoskeleton itself. Myosin stack formation in non-muscle cells provides a novel mechanism for the self-organization of the actin cytoskeleton at the level of the entire cell.This article is part of the theme issue 'Self-organization in cell biology'.

Changing of expression actinbinding proteins in the kidney under dehydration

Actin is related to main structural proteins in eukaryotes. In opposite to muscle alpha-actin, beta-actin is expressing in all types of cells. The constant reorganization of actin cytoskeleton takes place in non-muscle cells. Fibrillar actin, organized by globular monomers, interacts with the actin-binding proteins. Alpha-actinin forms a transverse links in actin fibrillar network, as well as concentrates in the fields of focal contacts. Tropomyosin is related to regulatory components of beta-actin, and due to the expense of longitudinal localization of the molecule in the groove of actin microfilament, stereochemically shields the sites of other actin-binding proteins. The most important function of actin cytoskeleton is the participation in the transportation of vesicles with aquaporins of second type in principal cells of an epithelium of collecting ducts in renal medulla. Vasopressin is stimulating the release of aquaporin tetramers from cytoplasmic store to apical plasmatic membrane. The participation and role of separate cytoskeleton proteins in the process of aquaporin trafficking and forming additional pores for water stays a poorly studied place in the molecular physiology of kidney. We explored the osmoregulatory action of prolonged hydration and dehydration on the protein composition of actin cytoskeleton in rats depending on the presence or absence of the actively expressing vasopressin gene in the genome. We found that the efficiency of the renal concentrating system, controlled by vasopressin, depends on expression of actin-binding proteins in the renal medulla. On the background of a stable level of inner cellular beta-actin, a change of expression an alpha-actinin and tropomyosin is observed. Dehydration of the organism is accompanied by essential reducing of alpha-actinin. In the absence of vasopressin, reduction of alpha-actinin has a smaller amplitude. The presence of the normal vasopressin gene in the genome, regardless of transitory expression level and secretion of hormone, is a factor of lower tropomyosin in the kidney. The most probable molecular mechanism of changing the expression of the genes for alpha-actinin and tropomyosin is transduction of the V2-mediated vasopressin hormonal signal to protein kinase A, phosphorylation of the cAMPresponsible transcriptional factor CREB, and nuclear CREB interaction with gene CRE sites sensitive to it.

Stochastic Ratcheting on a Funneled Energy Landscape Is Necessary for Highly Efficient Contractility of Actomyosin Force Dipoles

New simulations explain how mixing of actin, the most abundant protein in cells of higher organisms, with molecular motors, such as myosins, and cross-linking proteins causes cells to contract. While acto-myosin contractility is well understood for muscle cells, these new results encompass nonmuscle cells as well, providing key insights into a range of biological processes.

Effects of Agmatine Exposure on Skeletal Muscle of Aging Rats

Skeletal muscle from aging individuals is smaller and weaker than muscle from younger adults, which is a condition referred to as “sarcopenia”. The etiology of sarcopenia is complex as many factors and cellular responses are likely to contribute to the onset and progression of the condition. Thus, a strategy that might favorably affect multiple cellular responses may be more likely to impart beneficial effects on aging skeletal muscle. Agmatine is a compound that has potential to lessen age‐related muscle loss. The synthesis of agmatine occurs in the body via the decarboxylation of arginine, a conditionally essential amino acid. Although the formation of agmatine occurs principally in neurons it is detected in several tissues including skeletal muscle. While ample data support the role of agmatine as a neuromodulator and neurotransmitter, other physiological roles of agmatine have been revealed in the literature. Mounting evidence indicates a beneficial role of agmatine administration on various disease states and conditions. Notably, agmatine may produce beneficial effects through the activation or de‐activation of various cell signaling pathways. Collectively, alteration of cell signaling cascades by agmatine shown to occur in non‐muscle cells and tissues could induce responses that may benefit aging skeletal muscle. Thus, the purpose of this pilot experiment was to obtain evidence that agmatine supplementation may attenuate the development of sarcopenia in aging rats. Ten, thirty‐two‐month old male F344xBN F1 rats were singly housed and provided with either agmatine sulfate (0.2–0.25%) dissolved in drinking water, or plain water for a period of 4 weeks. Solutions were replaced with fresh mixtures every 3–4 days. Following the 4‐week supplementation period, plantaris force measures were tested in situ by stimulation of the sciatic nerve. Immediately afterwards the muscle was harvested for biochemical analyses. Body mass, muscle mass, muscle peak tetanic tension, and protein levels of inducible nitric oxide synthase (iNOS), manganese superoxide dismutase (MnSOD), and peroxisome proliferator‐activated receptor gamma coactivator 1‐alpha (PGC1a) were obtained. Due to low sample size only descriptive statistics are reported. All values are reported as mean ± SD for control and agmatine supplemented groups, respectively. Average body mass (g) (467.6 ± 44.4, n=4; 476.0 ± 19.5, n=3), plantaris mass (g) (0.36 ± 0.06, n=4; 0.30 ± 0.02, n=3) and muscle mass/body mass ratio (mg/g) (0.77 ± 0.13, n=4; 0.62 ± 0.01, n=3) values were similar or appeared lower in agmatine supplemented animals. Average maximal force production of the plantaris appeared to be similar (Po/muscle mass) (751.3 ± 198.1, n=4; 776.2 ± 260.8, n=3) between groups. Expression of iNOS (1.11 ± 0.96, n=5; 1.85 ± 1.53, n=4), PGC1a (0.16 ± 0.13, n=5; 0.22 ± 0.22, n=4) and MnSOD (4.82 ± 0.91, n=5; 5.05 ± 1.37, n=3) were either similar between the groups or slightly greater in agmatine supplemented rats, but low sample size likely affected variability of these measures. Our preliminary findings suggest that 4 weeks of agmatine administration does not favorably alter the functional or biochemical response of skeletal muscle in aging rats.Support or Funding InformationSupport by the Syracuse University SOEThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Modulating Cytoskeletal Structure And Cellular Signaling To Target Neuron Cell Membrane Repair

Most cells in the human body have the capacity to reseal their cellular membranes following disruption of the lipid bilayer. The cell signaling response regulating membrane repair is dependent on changes in the mechanical tension on the plasma membrane. Increased mechanical tension on the membrane can lead to membrane disruptions remaining open and thus preventing resealing of the membrane. Disruption of these membrane repair processes can result in a number of diseases including muscular dystrophy, Alzheimer's and neurodegeneration. Conversely, if membrane repair can be elevated in striated muscle or neuronal tissues, this response could potentially minimize cell death in neuromuscular diseases and alleviate the progression of these disease states. We have previously shown that tripartite motif (TRIM) 72, also known as mitsugumin 53 (MG53), can increase plasma membrane repair in skeletal and cardiac muscle and in non‐muscle cell types where it is not usually expressed. Our recent results indicate that there are distinct TRIM72/MG53 regulated cellular signaling events that control the membrane repair response in multiple cell types. This observation led us to screen for novel TRIM family proteins that may be able to mediate membrane repair in neuronal cells. We found that TRIM2 transfected cells show an increased capacity for membrane resealing following multi‐photon laser injury and that knockdown of TRIM2 decreases membrane repair. Because TRIM2 is highly expressed in the nervous system and was previously shown to regulate neurofilaments (NFL), the objective of this study is to prove that TRIM2 protects against membrane damage in neurons through regulation of neurofilament ubiquitination and that disruption of TRIM2 or NFL leads to compromised membrane repair and neuron cell death. Through confocal microscopy, we have shown that TRIM2 co‐localizes with NFL in neurons. Following injury to the cell this association is altered, leading to changes in the subcellular localization of TRIM2 and an apparent reorganization of the neurofilament cytoskeleton. Given the extensive expression of TRIM2 in the nervous system, it may represent a good candidate for targeting membrane repair in the treatment of a variety of neurodegenerative diseases.Support or Funding InformationAmerican Heart Association, National Institutes of Health, National Institute of Neurological Disorders and Stroke (NINDS)‐sponsored T32, and Jain FoundationThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Rapid and dynamic arginylation of the leading edge β-actin is required for cell migration.

β-actin plays key roles in cell migration. Our previous work demonstrated that β-actin in migratory non-muscle cells is N-terminally arginylated and that this arginylation is required for normal lamellipodia extension. Here, we examined the function of β-actin arginylation in cell migration. We found that arginylated β-actin is concentrated at the leading edge of lamellipodia and that this enrichment is abolished after serum starvation as well as in contact-inhibited cells in confluent cultures, suggesting that arginylated β-actin at the cell leading edge is coupled to active migration. Arginylated actin levels exhibit dynamic changes in response to cell stimuli, lowered after serum starvation and dramatically elevating within minutes after cell stimulation by readdition of serum or lysophosphatidic acid. These dynamic changes require active translation and are not seen in confluent contact-inhibited cell cultures. Microinjection of arginylated actin antibodies into cells severely and specifically inhibits their migration rates. Together, these data strongly suggest that arginylation of β-actin is a tightly regulated dynamic process that occurs at the leading edge of locomoting cells in response to stimuli and is integral to the signaling network that regulates cell migration.

Developmental dosing with a MEK inhibitor (PD0325901) rescues myopathic features of the muscle-specific but not limb-specific Nf1 knockout mouse

Neurofibromatosis Type 1 (NF1) is a common autosomal dominant genetic disorder While NF1 is primarily associated with predisposition for tumor formation, muscle weakness has emerged as having a significant impact on quality of life. NF1 inactivation is linked with a canonical upregulation Ras-MEK-ERK signaling. This in this study we tested the capacity of the small molecule MEK inhibitor PD0325901 to influence the intramyocellular lipid accumulation associated with NF1 deficiency. Established murine models of tissue specific Nf1 deletion in skeletal muscle (Nf1MyoD−/−) and limb mesenchyme (Nf1Prx1−/−) were tested.Developmental PD0325901 dosing of dams pregnant with Nf1MyoD−/− progeny rescued the phenotype of day 3 pups including body weight and lipid accumulation by Oil Red O staining. In contrast, PD0325901 treatment of 4 week old Nf1Prx1−/− mice for 8 weeks had no impact on body weight, muscle wet weight, activity, or intramyocellular lipid. Examination of day 3 Nf1Prx1−/− pups showed differences between the two tissue-specific knockout strains, with lipid staining greatest in Nf1MyoD−/− mice, and fibrosis higher in Nf1Prx1−/− mice.These data show that a MEK/ERK dependent mechanism underlies NF1 muscle metabolism during development. However, crosstalk from Nf1-deficient non-muscle mesenchymal cells may impact upon muscle metabolism and fibrosis in neonatal and mature myofibers.

Interacting-heads motif has been conserved as a mechanism of myosin II inhibition since before the origin of animals

Electron microscope studies have shown that the switched-off state of myosin II in muscle involves intramolecular interaction between the two heads of myosin and between one head and the tail. The interaction, seen in both myosin filaments and isolated molecules, inhibits activity by blocking actin-binding and ATPase sites on myosin. This interacting-heads motif is highly conserved, occurring in invertebrates and vertebrates, in striated, smooth, and nonmuscle myosin IIs, and in myosins regulated by both Ca2+ binding and regulatory light-chain phosphorylation. Our goal was to determine how early this motif arose by studying the structure of inhibited myosin II molecules from primitive animals and from earlier, unicellular species that predate animals. Myosin II from Cnidaria (sea anemones, jellyfish), the most primitive animals with muscles, and Porifera (sponges), the most primitive of all animals (lacking muscle tissue) showed the same interacting-heads structure as myosins from higher animals, confirming the early origin of the motif. The social amoeba Dictyostelium discoideum showed a similar, but modified, version of the motif, while the amoeba Acanthamoeba castellanii and fission yeast (Schizosaccharomyces pombe) showed no head-head interaction, consistent with the different sequences and regulatory mechanisms of these myosins compared with animal myosin IIs. Our results suggest that head-head/head-tail interactions have been conserved, with slight modifications, as a mechanism for regulating myosin II activity from the emergence of the first animals and before. The early origins of these interactions highlight their importance in generating the inhibited (relaxed) state of myosin in muscle and nonmuscle cells.

Structural and Biomechanical Changes During Platelet-Driven Clot Contraction

Blood clot contraction plays an important role in prevention of bleeding and in thrombotic disorders. We found that activated platelets bend and shorten individual fibrin fibers via their filopodia that undergo sequential extension and retraction, as if pulling hand-over-hand. Platelets also induce compaction of fibrin fibers into platelet-attached agglomerates. As a result of simultaneous pulling on multiple, closely-set fibrin fibers, platelets pull themselves closer to each other and form secondary clusters larger than the initial aggregates. Contracting platelets actively remodel a fibrin network by increasing its density followed by enhanced clot stiffness. Kinetic analysis of the time course of structural and mechanical transitions revealed a multiphasic behavior with at least three distinct phases that differ in duration and rate constants. All the observed changes were reduced or abrogated in the presence of specific inhibitors of non-muscle myosin IIA (blebbistatin) and the platelet integrin αIIbβIII (abciximab), indicating that actomyosin-driven cell contractility and integrin-fibrin mediated platelet-fibrin interactions are crucial for contraction of blood clots. Finally, blood clot contraction was found to be a spatially non-uniform process with faster compression of the clot edge and a delayed deformation of the clot interior. Altogether, the results provide a quantitative structural basis for the mechanobiology of clot contraction at various spatial scales from a single cell/single fiber level up to the network and macroscopic levels. Our results obtained on platelet-induced contraction of the filamentous fibrin network are of fundamental importance because they provide a foundation for understanding dynamic and complex biomechanical interplay between non-muscle cells and fibrous extracellular matrices of various compositions.

A Novel Kinase Activity of Calponin

Calponin is an actin filament-associated regulatory protein expressed in both smooth muscle and many types of non-muscle cells. An established function of calponin is an inhibitory regulator of myosin ATPase in modulating smooth muscle contractility and cell motility (Liu and Jin, Gene 585:143-53, 2016). Three isoforms of calponin are present in vertebrates encoded by homologous genes CNN1, CNN2, and CNN3 with conserved structures but diverged in the C-terminal region. We recently reported that a monoclonal antibody (mAb) CP3 raised against calponin 1 has a specific cross reaction to carbonate anhydrase 3 (Feng and Jin, Front Physiol 7:597, 2016), implicating a structural similarity between calponin and metabolic enzymes. In the present study, we developed and characterized another mAb, 1H8, raised by immunization of calponin 2. Western blotting and mass spectrometry identification demonstrated that 1H8 cross-reacts to creatine kinases M and B. Analysis of calponin 2 using a commercial creatine kinase assay kit demonstrated that calponin 2 has a kinase-like activity. The sub-molecular location of the 1H8 epitope is mapped using engineered calponin isoforms and fragments. Two-dimensional gel electrophoresis, Western blotting and mass spectrometry identification are applied to study the presence of 1H8 epitope-like structures in other metabolic enzymes. The results are analyzed together with sequence database search to identify the molecular structures that give calponin the kinase-like properties. Functional significance of the kinase-like structures and activity of calponin is being investigated using calponin knockout smooth muscle tissues and fibroblasts. While extensive research has been done for the functions of calponin in regulating contractility and cell motility, the novel kinase-like structures and activities of calponin opens a new direction to understand its physiological and pathological functions.

Integrated treatment with hyperthermia and chemotherapy for non-muscle invasive bladder cancer

Oncology-applied hyperthermia is a very old form of therapy. In recent years hyperthermia has been investigated with the aim of improving the treatment for non-muscle invasive bladder cancer to prevent relapse and disease progression, in association with mitomycin-C, a well-known chemotherapeutic agent, to enhance its effect. Target patients are those with non-muscle invasive transitional cell carcinoma, showing medium (Ta-T1, G1-2, multifocal, diameter >3 cm) or high (T1, G3, multifocal or rapidly relapsing, CIS) risk for recurrence or progression. The treatment may be prophylactic following tumor eradication, or ablative when tumor cannot be otherwise eradicated. Several studies have shown the benefits of thermochemotherapy with lower risk for relapse than other treatment options, and 66-80% complete responses following ablative treatment. This association of treatments has a synergic therapeutic effect, higher than administering hyperthermia and drug therapy as single treatment.

Actin-Myosin Force Generation and Symmetry Breaking in the Model Contractile Fiber

Myosin-powered force generation in nonmuscle cells underlies many cell biological processes and is based on contraction of random actin arrays. One of the most prominent examples of such arrays is ...

Cross-linkers both drive and brake cytoskeletal remodeling and furrowing in cytokinesis.

Cell shape changes such as cytokinesis are driven by the actomyosin contractile cytoskeleton. The molecular rearrangements that bring about contractility in nonmuscle cells are currently debated. Specifically, both filament sliding by myosin motors, as well as cytoskeletal cross-linking by myosins and nonmotor cross-linkers, are thought to promote contractility. Here we examined how the abundance of motor and nonmotor cross-linkers affects the speed of cytokinetic furrowing. We built a minimal model to simulate contractile dynamics in the Caenorhabditis elegans zygote cytokinetic ring. This model predicted that intermediate levels of nonmotor cross-linkers are ideal for contractility; in vivo, intermediate levels of the scaffold protein anillin allowed maximal contraction speed. Our model also demonstrated a nonlinear relationship between the abundance of motor ensembles and contraction speed. In vivo, thorough depletion of nonmuscle myosin II delayed furrow initiation, slowed F-actin alignment, and reduced maximum contraction speed, but partial depletion allowed faster-than-expected kinetics. Thus, cytokinetic ring closure is promoted by moderate levels of both motor and nonmotor cross-linkers but attenuated by an over-abundance of motor and nonmotor cross-linkers. Together, our findings extend the growing appreciation for the roles of cross-linkers in cytokinesis and reveal that they not only drive but also brake cytoskeletal remodeling.

Regulation of Skeletal Muscle Plasticity by Protein Arginine Methyltransferases and Their Potential Roles in Neuromuscular Disorders.

Protein arginine methyltransferases (PRMTs) are a family of enzymes that catalyze the methylation of arginine residues on target proteins, thereby mediating a diverse set of intracellular functions that are indispensable for survival. Indeed, full-body knockouts of specific PRMTs are lethal and PRMT dysregulation has been implicated in the most prevalent chronic disorders, such as cancers and cardiovascular disease (CVD). PRMTs are now emerging as important mediators of skeletal muscle phenotype and plasticity. Since their first description in muscle in 2002, a number of studies employing wide varieties of experimental models support the hypothesis that PRMTs regulate multiple aspects of skeletal muscle biology, including development and regeneration, glucose metabolism, as well as oxidative metabolism. Furthermore, investigations in non-muscle cell types strongly suggest that proteins, such as peroxisome proliferator-activated receptor-γ coactivator-1α, E2F transcription factor 1, receptor interacting protein 140, and the tumor suppressor protein p53, are putative downstream targets of PRMTs that regulate muscle phenotype determination and remodeling. Recent studies demonstrating that PRMT function is dysregulated in Duchenne muscular dystrophy (DMD), spinal muscular atrophy (SMA), and amyotrophic lateral sclerosis (ALS) suggests that altering PRMT expression and/or activity may have therapeutic value for neuromuscular disorders (NMDs). This review summarizes our understanding of PRMT biology in skeletal muscle, and identifies uncharted areas that warrant further investigation in this rapidly expanding field of research.

UNC-45a promotes myosin folding and stress fiber assembly.

Contractile actomyosin bundles, stress fibers, are crucial for adhesion, morphogenesis, and mechanosensing in nonmuscle cells. However, the mechanisms by which nonmuscle myosin II (NM-II) is recruited to those structures and assembled into functional bipolar filaments have remained elusive. We report that UNC-45a is a dynamic component of actin stress fibers and functions as a myosin chaperone in vivo. UNC-45a knockout cells display severe defects in stress fiber assembly and consequent abnormalities in cell morphogenesis, polarity, and migration. Experiments combining structured-illumination microscopy, gradient centrifugation, and proteasome inhibition approaches revealed that a large fraction of NM-II and myosin-1c molecules fail to fold in the absence of UNC-45a. The remaining properly folded NM-II molecules display defects in forming functional bipolar filaments. The C-terminal UNC-45/Cro1/She4p domain of UNC-45a is critical for NM-II folding, whereas the N-terminal tetratricopeptide repeat domain contributes to the assembly of functional stress fibers. Thus, UNC-45a promotes generation of contractile actomyosin bundles through synchronized NM-II folding and filament-assembly activities.

FAK tyrosine phosphorylation is regulated by AMPK and controls metabolism in human skeletal muscle

Aims/hypothesisInsulin-mediated signals and AMP-activated protein kinase (AMPK)-mediated signals are activated in response to physiological conditions that represent energy abundance and shortage, respectively. Focal adhesion kinase (FAK) is implicated in insulin signalling and cancer progression in various non-muscle cell types and plays a regulatory role during skeletal muscle differentiation. The role of FAK in skeletal muscle in relation to insulin stimulation or AMPK activation is unknown. We examined the effects of insulin or AMPK activation on FAK phosphorylation in human skeletal muscle and the direct role of FAK on glucose and lipid metabolism. We hypothesised that insulin treatment and AMPK activation would have opposing effects on FAK phosphorylation and that gene silencing of FAK would alter metabolism.MethodsHuman muscle was treated with insulin or the AMPK-activating compound 5-aminoimadazole-4-carboxamide ribonucleotide (AICAR) to determine FAK phosphorylation and glucose transport. Primary human skeletal muscle cells were used to study the effects of insulin or AICAR treatment on FAK signalling during serum starvation, as well as to determine the metabolic consequences of silencing the FAK gene, PTK2.ResultsAMPK activation reduced tyrosine phosphorylation of FAK in skeletal muscle. AICAR reduced p-FAKY397 in isolated human skeletal muscle and cultured myotubes. Insulin stimulation did not alter FAK phosphorylation. Serum starvation increased AMPK activation, as demonstrated by increased p-ACCS222, concomitant with reduced p-FAKY397. FAK signalling was reduced owing to serum starvation and AICAR treatment as demonstrated by reduced p-paxillinY118. Silencing PTK2 in primary human skeletal muscle cells increased palmitate oxidation and reduced glycogen synthesis.Conclusions/interpretationAMPK regulates FAK signalling in skeletal muscle. Moreover, siRNA-mediated FAK knockdown enhances lipid oxidation while impairing glycogen synthesis in skeletal muscle. Further exploration of the interaction between AMPK and FAK may lead to novel therapeutic strategies for diabetes and other chronic conditions associated with an altered metabolic homeostasis.