Insulin and TOR signal in parallel through FOXO and S6K to promote epithelial wound healing.

The TOR and Insulin/IGF signalling (IIS) network controls growth, metabolism and ageing. Although reducing TOR or insulin signalling can be beneficial for ageing, it can be detrimental for wound healing, but the reasons for this difference are unknown. Here we show that IIS is activated in the cells surrounding an epidermal wound in Drosophila melanogaster larvae, resulting in PI3K activation and redistribution of the transcription factor FOXO. Insulin and TOR signalling are independently necessary for normal wound healing, with FOXO and S6K as their respective effectors. IIS is specifically required in cells surrounding the wound, and the effect is independent of glycogen metabolism. Insulin signalling is needed for the efficient assembly of an actomyosin cable around the wound, and constitutively active myosin II regulatory light chain suppresses the effects of reduced IIS. These findings may have implications for the role of insulin signalling and FOXO activation in diabetic wound healing.

Cell segregation in the vertebrate hindbrain: a matter of boundaries.

Segregating cells into compartments during embryonic development is essential for growth and pattern formation. In the developing hindbrain, boundaries separate molecularly, physically and neuroanatomically distinct segments called rhombomeres. After rhombomeric cells have acquired their identity, interhombomeric boundaries restrict cell intermingling between adjacent rhombomeres and act as signaling centers to pattern the surrounding tissue. Several works have stressed the relevance of Eph/ephrin signaling in rhombomeric cell sorting. Recent data have unveiled the role of this pathway in the assembly of actomyosin cables as an important mechanism for keeping cells from different rhombomeres segregated. In this Review, we will provide a short summary of recent evidences gathered in different systems suggesting that physical actomyosin barriers can be a general mechanism for tissue separation. We will discuss current evidences supporting a model where cell-cell signaling pathways, such as Eph/ephrin, govern compartmental cell sorting through modulation of the actomyosin cytoskeleton and cell adhesive properties to prevent cell intermingling.

Septation of infectious hyphae is critical for appressoria formation and virulence in the smut fungus Ustilago maydis.

Differentiation of hyphae into specialized infection structures, known as appressoria, is a common feature of plant pathogenic fungi that penetrate the plant cuticle. Appressorium formation in U. maydis is triggered by environmental signals but the molecular mechanism of this hyphal differentiation is largely unknown. Infectious hyphae grow on the leaf surface by inserting regularly spaced retraction septa at the distal end of the tip cell leaving empty sections of collapsed hyphae behind. Here we show that formation of retraction septa is critical for appressorium formation and virulence in U. maydis. We demonstrate that the diaphanous-related formin Drf1 is necessary for actomyosin ring formation during septation of infectious hyphae. Drf1 acts as an effector of a Cdc42 GTPase signaling module, which also consists of the Cdc42-specific guanine nucleotide exchange factor Don1 and the Ste20-like kinase Don3. Deletion of drf1, don1 or don3 abolished formation of retraction septa resulting in reduced virulence. Appressorium formation in these mutants was not completely blocked but infection structures were found only at the tip of short filaments indicating that retraction septa are necessary for appressorium formation in extended infectious hyphae. In addition, appressoria of drf1 mutants penetrated the plant tissue less frequently.

Cortical forces in cell shape changes and tissue morphogenesis.

Cortical forces drive a variety of cell shape changes and cell movements during tissue morphogenesis. While the molecular components underlying these forces have been largely identified, how they assemble and spatially and temporally organize at cell surfaces to promote cell shape changes in developing tissues are open questions. We present here different key aspects of cortical forces: their physical nature, some rules governing their emergence, and how their deployment at cell surfaces drives important morphogenetic movements in epithelia. We review a wide range of literature combining genetic/molecular, biophysical and modeling approaches, which explore essential features of cortical force generation and transmission in tissues.

Calyculin A-induced neurite retraction is critically dependent on actomyosin activation but not on polymerization state of microtubules.

Calyculin A (CL-A), a toxin isolated from the marine sponge Discodermia calyx, is a strong inhibitor of protein phosphatase 1 (PP1) and 2A (PP2A). Although CL-A is known to induce rapid neurite retraction in developing neurons, the cytoskeletal dynamics of this retraction have remained unclear. Here, we investigated the cytoskeletal dynamics during CL-A-induced neurite retraction in cultured rat hippocampal neurons, using fluorescence microscopy as well as polarized light microscopy, which can visualize the polymerization state of the cytoskeleton in living cells. We observed that MTs were bent while maintaining their polymerization state during the neurite retraction. In addition, we also found that CL-A still induced neurite retraction when MTs were depolymerized by nocodazole or stabilized by paclitaxel. These results imply a mechanism other than depolymerization of MTs for CL-A-induced neurite retraction. Our pharmacological studies showed that blebbistatin and cytochalasin D, an inhibitor of myosin II and a depolymerizer of actin, strongly inhibited CL-A-induced neurite retraction. Based on all these findings, we propose that CL-A generates strong contractile forces by actomyosin to induce rapid neurite retraction independently from MT depolymerization.

Actomyosin regulatory properties of yeast tropomyosin are dependent upon N-terminal modification.

The yeast tropomyosin 1 gene (TPM1) encodes the major isoform of the two tropomyosins (Tm) found in yeast. The gene has been expressed in E. coli and the protein purified. The gene product (yTm1) is a 199-amino acid protein that has a low affinity for actin compared to the native yTm1 purified from yeast. Mass spectrometry shows that the native protein is acetylated while the recombinant protein is not. A series of yTm1 N-terminal constructs were made with either an Ala-Ser dipeptide extension previously shown to restore actin binding to skeletal muscle Tm or the natural extension found in fibroblast Tm 5a/b. All constructs bound actin tightly and showed similar CD spectra and thermal stability. All constructs induced cooperativity in the equilibrium binding of myosin subfragment 1, to actin but the binding curves differed significantly between the constructs. The apparent cooperative unit size (n) and closed/open equilibrium (K(T)) were determined using a fluorescence titration technique [Maytum et al. (1998) Biophys. J. 74, A347]. The data could be accounted for by changes in K(T) (0.1-1) with no change in n. Values of n were approximately twice the structural unit size (5 actin sites). The presence of yTm on actin had little effect upon the overall affinity of S1 for actin despite showing an ability to regulate the acto-myosin interaction. These results show that the short yTm can aid our understanding of actomyosin regulation and that the N-terminus of Tm has a major influence upon its regulatory properties.

Force transmission in skeletal muscle: from actomyosin to external tendons.

The actual path of force transmission in skeletal muscle from actomyosin interaction to tension at the tendinous insertion site is poorly understood. Within the muscle cell, endo- and exosarcomeric cytoskeletal proteins create series and parallel connections between contractile proteins resulting in a meshwork across which force can be transmitted in practically any direction with respect to the fiber axis. At the surface membrane, connections between the intermediate filament system, dystrophin, and specialized membrane complexes provide the route of force transmission to the extracellular matrix material. Finally, parallel and series connections between muscle fibers allow radial and longitudinal forces to converge on the connective tissue matrix. This complex pathway will certainly be the subject of future studies in muscle biology, biomechanics, and physiology.

Ethanol-induced smooth and skeletal muscle myopathy: use of animal studies.

This article reviews the effects of ethanol on skeletal and smooth muscle. A brief summary of its clinical effects is provided, with a rationale for the use of suitable animal models to study ethanol-induced myotoxicity. Practical details are given for the animal feeding techniques to examine the chronic effects of ethanol toxicity. Information on acute ethanol dosage experiments are also provided. Our results have indicated that ethanol causes net loss of both skeletal and smooth muscle protein and an effect on protein synthesis and/or degradation was implicated. The theoretical and practical basis of measuring protein synthesis in intact laboratory animals is reviewed. However, there are no reliable methods for measuring rates of protein breakdown in vivo. The combined results of our studies indicated that disturbances in protein synthesis were causal mechanisms for ethanol-induced myo-dysfunction. Acute ethanol exposure was largely characterised by reductions in fractional rates of skeletal and smooth muscle contractile protein synthesis. The dominant characteristics of chronic treatments were loss of skeletal and smooth muscle proteins and RNA. Further laboratory animal studies will eventually elucidate the molecular mechanisms of these changes and provide valuable information on the regulation of protein mass.

Inhibitory factor of actomyosin ATPase in the cardiac conduction system.

ATPase activation and superprecipitation of natural actomyosin in the cardiac conduction system (specialized myocardium) and ventricle were compared. The ATPase activity and superprecipitation of natural actomyosin from the specialized myocardium were much lower than those from the ventricle. The sensitivity of natural actomyosin ATPase to calcium was higher in the specialized myocardium than in the ventricle, whereas the maximum activity of the specialized myocardium was lower than that of the ventricle. The Ca2+-desensitized actomyosin of the specialized myocardium presented scarcely any superprecipitation. Addition of the 0-40% ammonium sulfate fraction (mainly containing myosin and actin) of natural actomyosin from the specialized myocardium to ventricular natural actomyosin inhibited superprecipitation of the latter. On the other hand, addition of the 40-60% ammonium sulfate fraction (mainly containing troponins and tropomyosin) of natural actomyosin from the specialized myocardium enhanced superprecipitation of ventricular natural actomyosin. These results suggested that the specialized myocardium contains some factor(s) that inhibits actin-myosin interaction.

[Amplitude of the tension developed by glycerinated muscle fibers during rigidity depends on the nature of the structural reorganizations in F-actin induced by formation of the actomyosin complex]. / Amplituda napriazheniia, razvivaemogo glitserinizirovannym myshechnym voloknom pri rigorizatsii, zavisit ot kharaktera strukturnykh perestroek F-aktina, indutsirovannykh obrazovaniem aktomiozinovogo kompleksa.

Dependence between the amplitude of tension, developed by glycerinated muscle fibers during rigidity, and the character of structural changes in F-actin, induced by the formation of actomyosin complex, was studied by polarized microfluorimetry and tensiometry. It is shown that during rigidity the anisotropy of intrinsic tryptophan residues as well as of rhodamine phalloidin bound to F-actin, and amplitude of tension depend on pH (6-8) and ionic strength (mu = 0.07 M-0.14 M) of solution. Greater changes in polarized fluorescence and in amplitude of tension were registered during rigidity in solutions with low ionic strength (mu = 0.07 M) and pH 8. It suggested that the amplitude of muscle fibre tension depends on the relative quantity of actin monomers, being in the "switched on" state.

Rates of synthesis of actomyosin in atria and ventricles of the perfused working rat heart.

Actomyosin from hearts perfused in vitro with [U-14C] phenylalanine and the remaining plasma amino acids was purified by standard techniques. The rate of actomyosin synthesis was expressed relative to actomyosin protein, to total protein and to total RNA. The rate of atrial total protein synthesis was twice the ventricular rate correlating with the RNA/protein ratios of the compartments. The actomyosin contents relative to total protein were not significantly different in atria and ventricles. The rate of actomyosin synthesis in atria was 2 to 3 times greater than in the ventricles when expressed relative to actomyosin or total protein. Synthesis rates of actomyosin expressed relative to total RNA were similar in the two compartments. These results suggest that the rate of turnover of actomyosin in the atria is 2 to 3 times the ventricular rate and that the greater rate of synthesis of total protein synthesis in atria is reflected in the rate of synthesis of specific intramyocytic proteins.

[Metabolism of skeletal muscle proteins in experimental hypokinesia in rats]. / Metabolizm belkov skeletnykh myshts pri éksperimental'noi gipokinezii u krys.

Using a radioindicator method, the metabolism of sarcoplasmic and myofibrillar proteins was studied in vivo at different stages of hypokinesia in rats. It was shown that the true muscle atrophy and the lowered content of both protein fractions during the first two weeks are due to sharp inhibition of sarcoplasmic protein biosynthesis as well as to deceleration of biosynthesis and acceleration of degradation of actomyosin. In hypokinesia the muscle mass does not increase within 3-8 weeks largely due to the acceleration of degradation of the de novo synthesized components of contractile proteins. In early hypokinesia a stressory mechanism plays an essential role in the pathogenesis of protein metabolism disturbances.

Microfilaments and intermediate filaments in epithelial cells transformed by murine sarcoma or leukemia viruses.

The murine epithelial cell line MMC-E was used to study changes in the cytoskeletal organization associated with viral transformation of epithelial cells by two different viruses. The cells were transformed with Moloney mouse sarcoma virus (MSV) or murine leukemia virus (MuLV). The expression of actin, myosin and of intermediate filament proteins in the cells was then studied. In MMC-E cells actin and myosin were organized as belt-like structures at the edges of the border cells of the cell islands and also circumferentially in the cells inside the islands. The major change after transformation was the decrease of the actomyosin containing belt extending from cell to cell at the borders of the cell islands. Both MMC-E cells and the MSV-transformed cells contained keratin as a juxtanuclear granular aggregate whereas the MuLV-transformed cells showed bright fibrillar arrays of keratin. Both virus-transformed cell lines showed enhanced vimentin-specific fluorescence and analysis of their cytoskeletal polypeptides confirmed the result. Similar molecular forms of keratin polypeptides were seen in all cells by immunoblotting. Viral transformation of MMC-E epithelial cells thus leads to different changes in their cytoskeletal organization depending on the transforming viral or cellular gene.

Synthesis of adult myosin light chains by embryonic muscle cultures.

Myosin light chain synthesis has been analyzed in cultures of fast and slow muscles from chicken and quail embryos. Synthesis was assayed by [35S]methionine incorporation and two-dimensional electrophoresis of total cell extracts. Our results show that differentiated cultures of embryonic anterior latissimus dorsi and pectoral muscles synthesize proteins that comigrate on two-dimensional gels with the five myosin light chains of adult fast (pectoral) and slow (anterior latissimus dorsi) muscle. Partial proteolytic digestion and peptide analyses further confirm the identity of these proteins as adult light chains. Cultures of dividing myoblasts do not synthesize any of these fiber type isozymes, and synthesis of the isozymes is initiated at myoblast fusion. Also, myogenic clones drived from single myoblasts differentiate to synthesize these five myosin light chains, indicating that individual myoblasts have the potential to express the synthesis of all fiber type light chain isozymes. We conclude that the primary events in muscle differentiation include the initiation of synthesis of the entire set of adult fast and slow myosin light chain isozymes. The developmental and physiological implications of these results for the establishment of fiber type specificity are discussed.

Evidence for actin transformation during the contraction-relaxation cycle of cytoplasmic actomyosin: cycle blockade by phalloidin injection.

1) The injection of a mushroom drug, Phalloidin (750 microgram -1 mg/ml), into the endoplasmic channel of Physarum veins induces an irreversible blockade of the intrinsic contraction-relaxation automaticity of the ectoplasmic tube wall, as measured by tensiometrical methods. 2) The morphological responses to Phalloidin injection include an increase and condensation of cytoplasmic actomyosin sheets bordering the plasmalemma invaginations within the ectoplasmic tube and a more pronounced dense layer of "groundplasm" in the cell cortex. This is in accordance with experiments with other cells as well as with Physarum. 3) The addition of marker particles to the injection solution revealed that the injected substances can be brought into direct contact with the contractile substrate, before newly formed membranes separate off the injection fluid. 4) Since Phalloidin irreversibly transforms oligomeric actin into a filamentous "Phalloidin-actin complex" and because this transformation does not hinder the actin in activating myosin ATPase, it is concluded that the contraction-relaxation cycle of cytoplasmic actomyosin in Physarum involves actin transformations. If these transformations are hindered, e.g. by Phalloidin, one stage and thereby the whole cycle is sustained which results in a blockade of the intrinsic contraction automaticity. 5) The functional importance of actin transformations in the congraction physiology of cytoplasmic actomyosins and cell motility phenomena is discussed.

Morphological and biochemical differentiation in RSV transformed chick embryo myoblasts.

Chick embryo myoblasts have been transformed with a temperature sensitive mutant of Rous Sarcoma virus (RSV ts68). At permissive temperature (36 degrees C) it is shown that transformed myoblasts have lost their ability to form myotubes as well as to express the biochemical markers of myogenic differentiation. Upon a shift to the non-permissive temperature (41 degrees C), the normal program of differentiation is restored; myotubes are formed which express muscle specific proteins.

The translation in vitro of mRNA from developing cysts of Artemia salina.

Successive stages in the development of the brine shrimp cyst were used as a model for studying differentiation at the level of mRNA transcription and translation. The poly (A)-containing mRNA from dormant cysts and free-swimming larvae (nauplii) was found to be efficiently translated in a wheat-germ cell-free system, and electrophoretic patterns of translation products in vitro resembled those of the endogenous proteins extracted from the equivalent developmental stages. Each stage, however, exhibits a characteristic protein pattern. Two low-molecular-weight proteins prominent in the cyst disappeared almost completely in the nauplius stage, whereas the proportion of actin increased 3-fold. Parallel patterns were observed upon translation in vitro of the respective mRNA preparations. The percentage of the acidic protein, tubulin, decreased somewhat during development.