Phenotypes induced by NM causing α-skeletal muscle actin mutants in fibroblasts, Sol 8 myoblasts and myotubes

<p>Abstract</p> <p>Background</p> <p>Nemaline myopathy is a neuromuscular disorder characterized by the presence of nemaline bodies in patient muscles. 20% of the cases are associated with α-skeletal muscle actin mutations. We previously showed that actin mutations can cause four different biochemical phenotypes and that expression of NM associated actin mutants in fibroblasts, myoblasts and myotubes induces a range of cellular defects.</p> <p>Findings</p> <p>We conducted the same biochemical experiments for twelve new actin mutants associated with nemaline myopathy. We observed folding and polymerization defects. Immunostainings of these and eight other mutants in transfected cells revealed typical cellular defects such as nemaline rods or aggregates, decreased incorporation in F-actin structures, membrane blebbing, the formation of thickened actin fibres and cell membrane blebbing in myotubes.</p> <p>Conclusion</p> <p>Our results confirm that NM associated α-actin mutations induce a range of defects at the biochemical level as well as in cultured fibroblasts and muscle cells.</p

Network-based identification of feedback modules that control RhoA activity and cell migration

Cancer cell migration enables metastatic spread causing most cancer deaths. Rho-family GTPases control cell migration, but being embedded in a highly interconnected feedback network, the control of their dynamical behavior during cell migration remains elusive. To address this question, we reconstructed the Rho-family GTPases signaling network involved in cell migration, and developed a Boolean network model to analyze the different states and emergent rewiring of the Rho-family GTPases signaling network at protrusions and during extracellular matrix-dependent cell migration. Extensive simulations and experimental validations revealed that the bursts of RhoA activity induced at protrusions by EGF are regulated by a negative-feedback module composed of Src, FAK, and CSK. Interestingly, perturbing this module interfered with cyclic Rho activation and extracellular matrix-dependent migration, suggesting that CSK inhibition can be a novel and effective intervention strategy for blocking extracellular matrix-dependent cancer cell migration, while Src inhibition might fail, depending on the genetic background of cells. Thus, this study provides new insights into the mechanisms that regulate the intricate activation states of Rho-family GTPases during extracellular matrix-dependent migration, revealing potential new targets for interfering with extracellular matrix-dependent cancer cell migratio

Alpha-actin mutations in congenital myopathies: identification of cell death as a new cellular Nemaline Myopathy phenotype and induction of similar cytoskeletal defects by mutants associated with different myopathies

The most abundant protein in eukaryotes is the cytoskeletal protein actin, of which six isoforms exist. Mutations in α-skeletal muscle actin, main actin isoform in skeletal muscle, are associated with three congenital myopathies : nemaline myopathy (NM), the core myopathies (CCD) and congenital fibre type disproportion (CFTD), all heterogeneous diseases characterized by muscle weakness and hypotonia. Nemaline myopathy is characterized by nemaline rods in patient muscle. However, there is no correlation between severity and rod occurrence. Previous research biochemically characterized 19 actin mutants, and could reproduce rods in fibroblasts. There was no correlation between biochemical and cellular phenotype. Here we biochemically characterized new NM associated actin mutants and expressed them in fibroblasts, Sol8 myoblasts and differentiating Sol8 muscle cells. Some of the mutants behaved as WT in the biochemical analysis, but we could find a cellular defect for all mutants, i.e. the induction of rods or aggregates in fibroblasts and myoblasts, a reduced incorporation in fibroblast stress fibers shown by diffuse cytoplasmic myc-actin staining, cell membrane blebbing in muscle cells and formation of thickened actin fibers in myotubes. None of the NM causing actin mutants induce all of the cellular phenotypes, but some induce multiple defects. However, in a second set of mutants we observed a common phenotype: induction of a calpain involved, caspase independent cell death with apoptotic features, via release of endoG and AIF, indicating other processes than the rod formation could underlie the pathogenesis of NM, and that nemaline rods are secondary to this pathogenesis. CCD and CFTD are characterized by core lesions and a difference in fibre size respectively. Biochemical characterization of CCD and CFTD causing actin mutants showed they behave as WT actin. We expressed them in fibroblasts, myoblasts and differentiating myotubes and found for two of the CFTD causing mutants similar phenotypes as for NM mutants: induction of rods in fibroblasts and thickened fibers in myotubes. These results show that the molecular mechanisms behind NM, CCD and CFTD caused by actin mutations could be (partly) related. I hypothesize on a central role for Ca2+ homeostasis via Ca2+ sensitivity and Ca2+ signaling in this pathogenesis

Systems biology embedded target validation: improving efficacy in drug discovery

The pharmaceutical industry is faced with a range of challenges with the ever-escalating costs of drug development and a drying out of drug pipelines. By harnessing advances in -omics technologies and moving away from the standard, reductionist model of drug discovery, there is significant potential to reduce costs and improve efficacy. Embedding systems biology approaches in drug discovery, which seek to investigate underlying molecular mechanisms of potential drug targets in a network context, will reduce attrition rates by earlier target validation and the introduction of novel targets into the currently stagnant market. Systems biology approaches also have the potential to assist in the design of multidrug treatments and repositioning of existing drugs, while stratifying patients to give a greater personalization of medical treatment.European Commission - Seventh Framework Programme (FP7)Science Foundation IrelandCASy

alpha-Skeletal Muscle Actin Mutants Causing Different Congenital Myopathies Induce Similar Cytoskeletal Defects in Cell Line Cultures

Central core disease (CCD), congenital fibre type disproportion (CFTD), and nemaline myopathy (NM) are earlyonset clinically heterogeneous congenital myopathies, characterized by generalized Muscle weakness and hypotonia. All three diseases are associated with (alpha-skeletal muscle actin mutations. We biochemically characterized the CCD and CFTD causing actin mutants and show that all Mutants fold correctly and are stable. Expression studies in fibroblasts, myoblasts, and myotubes show that these mutants incorporate in filamentous structures. However they do not intercalate between the nascent z-lines it) differentiating muscle cell Cultures. We also show that the distribution of mitochondria and of the ryanodine receptors, and calcium release properties from ryanodine receptors, are unchanged in myotubes expressing the CCD causing mutants. CFTD causing mutants induce partly similar phenotypes as NM associated ones, such as rods and thickened actin fibers in cell Culture. Our results, Suggest that molecular mechanisms behind CFTD and NM may be partly related. Cell Motil. Cytoskeleton 66: 179-192, 2009. (C) 2009 Wiley-Liss, Inc

alpha-Skeletal muscle actin nemaline myopathy mutants cause cell death in cultured muscle cells.

Nemaline myopathy is a neuromuscular disorder, characterized by muscle weakness and hypotonia and is, in 20% of the cases, caused by mutations in the gene encoding alpha-skeletal muscle actin, ACTA1. It is a heterogeneous disease with various clinical phenotypes and severities. In patients the ultrastructure of muscle cells is often disturbed by nemaline rods and it is thought this is the cause for muscle weakness. To search for possible defects during muscle cell differentiation we expressed alpha-actin mutants in myoblasts and allowed these cells to differentiate into myotubes. Surprisingly, we observed two striking new phenotypes in differentiating myoblasts: rounding up of cells and bleb formation, two features reminiscent of apoptosis. Indeed expression of these mutants induced cell death with apoptotic features in muscle cell culture, using AIF and endonuclease G, in a caspase-independent but calpain-dependent pathway. This is the first report on a common cellular defect induced by NM causing actin mutants, independent of their biochemical phenotypes or rod and aggregate formation capacity. These data suggest that lack of type II fibers or atrophy observed in nemaline myopathy patients may be also due to an increased number of dying muscle cells

Robustness and evolvability of the human signaling network.

Biological systems are known to be both robust and evolvable to internal and external perturbations, but what causes these apparently contradictory properties? We used Boolean network modeling and attractor landscape analysis to investigate the evolvability and robustness of the human signaling network. Our results show that the human signaling network can be divided into an evolvable core where perturbations change the attractor landscape in state space, and a robust neighbor where perturbations have no effect on the attractor landscape. Using chemical inhibition and overexpression of nodes, we validated that perturbations affect the evolvable core more strongly than the robust neighbor. We also found that the evolvable core has a distinct network structure, which is enriched in feedback loops, and features a higher degree of scale-freeness and longer path lengths connecting the nodes. In addition, the genes with high evolvability scores are associated with evolvability-related properties such as rapid evolvability, low species broadness, and immunity whereas the genes with high robustness scores are associated with robustness-related properties such as slow evolvability, high species broadness, and oncogenes. Intriguingly, US Food and Drug Administration-approved drug targets have high evolvability scores whereas experimental drug targets have high robustness scores

Robustness and Evolvability of the Human Signaling Network

Biological systems are known to be both robust and evolvable to internal and external perturbations, but what causes these apparently contradictory properties? We used Boolean network modeling and attractor landscape analysis to investigate the evolvability and robustness of the human signaling network. Our results show that the human signaling network can be divided into an evolvable core where perturbations change the attractor landscape in state space, and a robust neighbor where perturbations have no effect on the attractor landscape. Using chemical inhibition and overexpression of nodes, we validated that perturbations affect the evolvable core more strongly than the robust neighbor. We also found that the evolvable core has a distinct network structure, which is enriched in feedback loops, and features a higher degree of scale-freeness and longer path lengths connecting the nodes. In addition, the genes with high evolvability scores are associated with evolvability-related properties such as rapid evolvability, low species broadness, and immunity whereas the genes with high robustness scores are associated with robustness-related properties such as slow evolvability, high species broadness, and oncogenes. Intriguingly, US Food and Drug Administration-approved drug targets have high evolvability scores whereas experimental drug targets have high robustness scores.Science Foundation IrelandNational Research Foundation of KoreaKorean Governmen

Topological characteristics of the evolvable core and robust neighbor sub-network.

<p>(<b>A</b>) Number of self-loops. (<b>B</b>) Number of two-node feedbacks. (<b>C</b>) Number of three-node feedbacks of the original network, evolvable core sub-network, random-deletion sub-network, robust neighbor sub-network, and random-selection sub-network. (<b>D</b>) Degree heterogeneity of the original network, evolvable core sub-network, and random-deletion sub-network. (<b>E</b>) Degree distribution of the original network and evolvable core sub-network. (<b>F</b>) The ratio of robust neighbor links to the whole links for the low-degree, middle-degree, and high-degree nodes, respectively. (<b>G</b>) Characteristic path length of the original network, evolvable core sub-network, and random-deletion sub-network. (<b>H</b>) Number of connected components of the robust neighbor and random-selection sub-network. (<b>I</b>) Characteristic path lengths of the robust neighbor and random-selection sub-network. Error bars denote the standard errors of the average values.</p